EVALUATION OF TARGETED CONTRAST AGENTS FOR IN VIVO IMAGING AND DETECTION OF ENDOMETRIOSIS IN MOUSE MODELS By Nazanin Talebloo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry – Doctor of Philosophy 2023 ABSTRACT Endometriosis is a chronic condition that affects about 10% of women during their reproductive years. Yet, no clinically approved agents are available for non-invasive endometriosis detection. This dissertation explores the application of two distinct targeted imaging contrast agents for the non-invasive detection and imaging of endometriosis using magnetic resonance imaging (MRI). Dr. Asgerally (Asgi) Fazleabas and his graduate student, Maria Ariadna Ochoa Bernal, supplied the mouse models of endometriosis and Dr. Christiane Mallett played a critical role in the MR imaging studies (Chapters 2 and 3). Chapter 1 offers a comprehensive introduction about endometriosis. Chapter 2 investigates the utility of a gadolinium-based type I collagen targeting probe (EP-3533) to non-invasively detect endometriotic lesions using MRI. Previously, this probe has been used for the detection and staging of various fibrotic conditions. Recently, endometriosis has been identified as a fibrotic disease. In this study, we evaluate the potential of EP-3533 for detecting endometriosis in two murine models and compare it with a non-binding isomer (EP-3612). For imaging we utilized two GFP-expressing murine models of endometriosis injected intravenously with EP-3533 or EP- 33612. Mice were imaged before and after bolus injection of the probes. The dynamic signal enhancement of MR T1 FLASH images were analyzed, and the relative location of lesions was validated through ex vivo fluorescence imaging. Next, the harvested lesions were stained for collagen and their gadolinium content was quantified by inductively coupled plasma optical emission spectrometry (ICP-OES). We showed that EP-3533 probe increased the signal intensity in T1-weighted images of endometriotic lesions in both models of endometriosis. Such enhancement was not detected in the muscles of the same groups or in endometriotic lesions of mice injected with EP-3612. Consequentially, control tissues had significantly lower gadolinium content compared to the lesions in experimental groups. This study provides evidence for targeting type I collagen in the endometriotic lesions using EP-3533 probe. In Chapter 3 we evaluated cRGD peptide-conjugated nanoparticles (RGD-Cy5.5-MN), to detect lesions using MRI in a mouse model of endometriosis. RGD peptide binds preferentially to the alpha(v)beta3 integrin which has been shown to have expression in endometriotic lesions. We utilized a luciferase-expressing murine suture model of endometriosis. Animals were imaged before, and 24 hours after intravenous injection of RGD-Cy5.5-MN or control nanoparticles (Cy5.5-MN). Next, we performed biodistribution studies and correlative fluorescence microscopy of lesions stained for CD34. Iron content in tissues was quantified using ICP-OES. Our results showed that RGD-Cy5.5- MN targeting of endometriotic lesions caused significantly higher deltaT2* upon accumulation compared to Cy5.5-MN. ICP-OES showed significantly higher iron content in the lesions of the experimental group compared to the control group. Histology showed colocalization of Cy5.5 signal from RGD-Cy5.5-MN with CD34 in the lesions pointing to the targeted accumulation of the probe. This work offers initial proof-of-concept for targeting angiogenesis in the endometriotic lesions which can be useful for potential clinical diagnostic and therapeutic approaches for treating this disease. Chapter 4 summarizes the conclusions drawn from the previous chapters and outlines the future direction of the presented studies. Furthermore, motivated by the fact that endometriosis is a risk factor for ovarian cancer, preliminary results of a project evolving around ovarian cancer are presented. ACKNOWLEDGEMENTS I am immensely grateful to the many individuals who have contributed to the success of this endeavor. However, there is one person who deserves my utmost thanks and recognition above all others—my advisor, Dr. Anna Moore. Her unwavering support, guidance, and mentorship over the course of the last five years have been truly instrumental in shaping my journey. Next, I extend my gratitude to my guidance committee members, Dr. Xuefei Huang, Dr. Erik Shapiro, and Dr. Michael Bachmann, for their continuous presence and support throughout my entire academic journey. I am grateful to the members of the Moore lab for their invaluable contributions to our scientific endeavors. I am deeply grateful to Elizabeth Kenyon, who helped with animal studies, Alan Halim, Dr. Neil Robertson, Shalee Drake, and Bryan Kim for their exceptional support and collaboration as fellow lab members. I would like to express my gratitude to the Precision Health Program (PHP) at Michigan State University and to PHP faculty members, including Dr. Ping Wang, Dr. Ming Chen, Dr. Morteza Mahmoudi, Dr. Lorenzo Sempere, Dr. Sachi Horibata, and Dr. Ali Akbar Ashkarran for providing me with the invaluable opportunity to be part of their exceptional program. I would like to thank Dr. Christiane Mallet for her invaluable assistance with MR imaging studies. Additionally, I would like to express my gratitude to Dr. Asgerally Fazleabas and Ariadna Ochoa Bernal for their support in providing the animal model of endometriosis and offering scientific guidance. I am deeply grateful to my family, especially my parents Fahimeh and Farhad (Hassan), for their support and love throughout my life. I appreciate the support of Farhad, Mohtaram, my uncles, and my grandparents, some of whom are now in a better place and not among us. I would like to extend a special thanks to Ramin for his unwavering support. Finally, I'm grateful for the support of many friends throughout this journey, including Reyhaneh, Alireza, Sarvenaz, Ehsan, Selda, Mohammad K, Amirreza, Iliya, Pegah, Parisa, Pardis, Mostafa, and more. iv TABLE OF CONTENTS CHAPTER 1 ENDOMETRIOSIS: A COMPREHENSIVE REVIEW OF PATHOGENESIS, SYMPTOMATOLOGY, AND DIAGNOSTIC METHODS.....................................................................1 REFERENCES...................................................................................................................................................... 58 CHAPTER 2 DETECTION OF ENDOMETRIOSIS LESIONS USING GD-BASED COLLAGEN I TARGETING PROBE IN MURINE MODELS OF ENDOMETRIOSIS.............82 REFERENCES.....................................................................................................................................................110 APPENDIX............................................................................................................................................................116 CHAPTER 3 IMAGING OF ENDOMETRIOSIS USING RGD-CY5.5-MN PROBE IN A MOUSE MODEL................................................................................................................................................117 REFERENCES.....................................................................................................................................................140 CHAPTER 4 CONCLUSIONS, FUTURE DIRECTIONS, AND DEVELOPMENT OF A NOVEL THERAPY FOR OVARIAN CANCER.....................................................................................145 REFERENCES.....................................................................................................................................................158 v CHAPTER 1 ENDOMETRIOSIS: A COMPREHENSIVE REVIEW OF PATHOGENESIS, SYMPTOMATOLOGY, AND DIAGNOSTIC METHODS 1 1.1 Introduction to Endometriosis Endometriosis is a relatively common, often chronic, hormone-dependent, and inflammatory condition that affects about 6 to 10% of women during their reproductive age, translating to almost 190 million women worldwide 1-4. In this gynecological condition tissue resembling the endometrium (the lining of the uterus) is established in locations outside the uterus, mainly in the pelvic area, including ovaries, ligaments, bladder, intestines, and peritoneal surfaces 5, 6. The classic definition of endometriosis states that ectopic endometrium (lesions) consists of endometrial glands and stroma (Figure 1.1) 7. Endometriosis has various negative impacts on women lives, including fertility problems, chronic pain, and reduced quality of life, causing a heavy financial burden on healthcare systems 8 (discussed in detail in section 1.3). Despite the significant impact that endometriosis has on women, their families, and the economy, there is a lack of public and professional awareness of this disorder 4. It is difficult to determine the precise prevalence and incidence rates of endometriosis in the general population since many cases remain undiagnosed 9. 2 Figure 1.1 Endometriotic implant buried in fibrotic tissue in pelvic peritoneum. Scale bar = 200 μm. Reproduced with permission from Elsevier 7. 1.2 Etiology of Endometriosis Although the etiology of endometriosis is not fully clear, many theories have been proposed to delineate the initiation, propagation, and pathogenesis of this disease, with the retrograde menstruation phenomenon being the most strongly supported by the existing evidence 2, 10, 11. The oldest theory explaining the etiology of endometriosis is Sampson’s theory of retrograde menstruation which is also known as the implantation theory 12-14. This theory originated from observations made in 1920s surgeries, suggesting that during menstruation, viable endometrial cells and tissue fragments reflux into the peritoneal cavity through fallopian tubes. 3 This can lead to implantation, growth, and invasion of endometrial debris onto peritoneal surfaces and organs which is accompanied by adhesion, angiogenesis, fibrosis, scarring, and anatomical distortion 3, 12, 14-16. Retrograde menstruation happens in 76 to 90% of women with patent fallopian tubes 17. However, only a fraction of these women suffers from endometriosis, indicating that retrograde mensuration is required but not sufficient for endometriosis to occur 18. This theory also cannot explain rare cases of endometriosis, such as those found in men 19, 20, premenarchal girls, and in atypical locations like the umbilicus 13. Other theories have been proposed to explain the etiology of endometriosis, including the coelomic metaplasia, lymphatic and vascular metastasis, and stem cell recruitment theory. 4, 10, 20. The coelomic metaplasia theory was first introduced by Meyer and was expanded later by other researchers 21-23. This theory proposes that a committed cell type (such as the peritoneal mesothelium) can transdifferentiate into a different cell type (such as endometrial epithelium) through a process known as metaplasia. It has been suggested that hormonal factors, immunological factors, and endocrine-disrupting chemicals may also play a part in the transformation of these celomic cells to endometrial-like cells 20. Based on this theory atypical transdifferentiation or transformation of extrauterine cells into endometrial cells is the main cause of endometriosis 23, 24. The theory of coelomic metaplasia represents a suitable explanation for cases with the absence of eutopic endometrium and endometrial fragments/cells, such as in cases of men undergoing estrogen therapy for prostate cancer 25. Although there is not enough conclusive evidence to fully support this theory, it can explain how the ovarian endometriomas develop. The mesothelium layer in the ovaries originates from the coelomic epithelium and serves as the covering layer on the surface of the ovary, bearing the potential to undergo metaplasia, transform to another cell type, and penetrate the ovarian cortex, which can ultimately result in the formation of ovarian endometrioma 26-28. In 1927, John Sampson proposed 4 the theory of benign metastasis as another mechanism for the development of endometriosis 29. This theory, also known as the lymphatic spread theory, suggests that lymphatic and hematogenous spread of endometrial cells or tissues during menstruation results in the development of endometriosis 28, 30. Since it is anatomically possible for endometrial cells to transit through the lymphatic system, this theory can explain rare cases of endometriosis in lymph nodes and extrapelvic areas 31, including the spinal canal 32, brain, bone 33, kidneys 34 and lungs 35. The stem cell theory suggests that undifferentiated stem cells with the capacity to self-renew and differentiate into one or more types of specialized cells, can migrate to ectopic sites, and contribute to the development of endometriosis. This theory has two alternatives based on the tissue the stem cells originated from, which are the uterine endometrium or the bone marrow. Several bone marrow–derived stem cell populations, including mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitors have the ability to incorporate themselves into the endometrium and regenerate the endometrial tissue. Based on this theory, the migration of bone marrow–derived stem cells to softer tissues instead of endometrium can lead to the development of endometriosis, which can explain extrapelvic and rare cases of endometriosis in men. The ability of these stem cells in regenerating uterine epithelial cells has been shown to be limited, leading to doubts about their physiological role in the development of ectopic lesions 5, 20, 36. Immune dysregulation as a key factor in the pathogenesis of endometriosis is another concept that has received increasing attention in recent years. Inflammation caused by immune dysregulation can interfere with apoptotic pathways and prevent clearance of endometrial fragments/cells from retrograde menstruation and create a favorable environment for lesions by activation of adhesion, proliferation, and angiogenesis pathways 37. Various studies showed that the aberrant level and concentration of immune cells and inflammatory factors contribute to pain symptoms of the 5 disease 38. Haber et al. used an animal model to show that inhibiting the function of peritoneal macrophages can hinder the growth and formation of endometrial implants 39. Other studies have indicated that peritoneal macrophages in patients with endometriosis produce inflammatory mediators, including interleukin 6 (IL-6) and tumor necrosis factor-α (TNFα) 40, are not fully functional, and have less phagocytic ability 41. Although immune dysregulation can be heterogeneous during different stages of endometriosis and among various patients, examples of common imbalances of the immune system include reduced cytotoxic function of natural killer cells and 41 increased number of regulatory T cells in ectopic lesions 42. Genetic and epigenetic theories have also been investigated to explain the pathogenesis of endometriosis. Familiar clusters of endometrioses have been found in humans and nonhuman primates. However, due to the absence of a clear inheritance pattern and the existence of genetic and epigenetic heterogeneity, relying solely on genetic/epigenetic theories to understand the pathogenesis of endometriosis becomes more challenging (genetic and epigenetic heterogeneity are discussed in section 1.4) 43, 44. Unfortunately, as of today, all mentioned theories remain to be conclusively confirmed and none of them can account for all endometriosis cases by itself 30 which made scientists dependent on a combination of theories to understand the pathogenesis of this disease 30, 45. Figure 1.2 Summarizes the most important theories of endometriosis pathogenesis 44 (DNA methylation, histone modification, and microRNA expression changes are discussed in section 1.4). 6 Figure 1.2 Theories of Endometriosis Pathogenesis. SF-1, steroidogenic factor 1;GATA6, GATA binding protein 6;PGE2, Prostaglandin E2;COX2, cyclooxygenase-2;ESR-2, estrogen receptor alpha; HOXA10, homebox protein A10;PR-B, progesterone receptor isoform B; GATA2, GATA binding protein 2. Reproduced from 44 and under the terms of the CC-BY Creative license Attribution (https://creativecommons.org/licenses/by/4.0/). International Commons 4.0 1.3 Endometriosis Symptoms Endometriosis can be accompanied by a variety of symptoms like pelvic pain, dysmenorrhea (cyclical pain associated with menstruation), dyspareunia (pain during sexual 7 activity), dyschezia (pain during defecation), pain during exercise, urinary dysfunctions, nausea, vomiting, heavy menses, and related fertility problems. Patients with endometriosis can experience one or more of these symptoms or they can be completely asymptomatic, indicating the heterogeneity in endometriosis-related symptoms 2, 46, 47. The adverse effects of endometriosis on quality of life are primarily due to pain, which can lead to decreased sleep quality, activity levels, increased stress, and the development of psychological disorders such as anxiety and depression over the years 48. The perception of pain involves peripheral processes (the conversion of a biochemical signal into a neural signal via activation of nociceptors and transmission to the spinal cord) and central processes (modulation at the spinal level and perception in the brain) which can be influenced by multiple factors, including psychological and physical stress and hormones 46. Endometriosis can cause neuropathic pain, nociceptive pain (including inflammatory pain), or a combination of both 2, 49. Neuropathic pain is caused by a lesion or disease of the somatosensory nervous system, whereas nociceptive pain is caused by actual or threatened damage to non-neural tissue through the activation of nociceptors 50. Patients with deep infiltrating endometriosis (DIE) nodules, which are lesions penetrating deep in tissue and organs, commonly experience neuropathic pain, characterized by severe chronic pelvic pain and hyperalgesia. Hyperalgesia is defined as an increased pain response to a nonpainful stimulus which can be related to nerve injury caused by invasion of lesions, inflammatory stimuli, or an aberrant secretion of cytokines within lesions, around nerve fibers, and in peritoneal fluid 2, 51, 52. It has been shown that in DIE nodules sensory nerve fibers are frequently invaded by endometriotic stromal cells 2. Also, Anaf et al. hypothesized that the high density of nerve structures in DIE nodules may lead to the severe neuropathic pain that characterizes these lesions 53. It has been reported that proinflammatory cytokines, including prostaglandin E2 (PGE2) secreted by endometrial stromal cells and 8 macrophages 52, IL-6 54, and TNFα 55, 56 are elevated in women with endometriosis and can cause nociceptive inflammatory pain 57, 58. Exposure to these proinflammatory substances for a long time can cause peripheral sensitization (increased responsiveness and reduced threshold of nociceptive neurons in the periphery to stimuli) and central sensitization (increased responsiveness of nociceptive neurons in the central nervous system) which can lead to chronic pain and the need for repetitive surgical interventions to remove lesions. Surgical excision of endometriotic lesions may alleviate pain in patients. However, it does not provide pain relief in all cases 52. Unfortunately, in patients with endometriosis invasive interventions may change brain chemistry, increase central sensitization, and increase pain over time 59. Although the mechanism of pain in endometriosis is still poorly understood it is believed that pain that is purely related to a specific function, including dyschezia, dysuria, and dyspenuria are normally a result of localization of the lesions on the related peritoneal organs 60. Cyclical bleeding from lesions, inflammatory response in both lesions and peritoneal cavity, sensory nerve activation, and transitioning of neuronal function into a more sensitized state caused by persistent inflammation can all contribute to endometriosis-related pain 52, 61, 62. Fertility issues represent another important complication related to endometriosis. The prevalence of endometriosis in infertile women ranges from 25% to 50%. However, endometriosis diagnosis is not synonymous with infertility. Pelvic adhesions and mechanical obstruction of the fallopian tubes caused by distorted pelvic anatomy, as well as inflammation-induced endocrine and ovulatory abnormalities, impaired follicle maturation, and altered cell- and hormone-mediated endometrial activities are among proposed reasons for endometriosis-related subfertility and infertility 63-66. 1.4 Heterogeneity of Endometriosis Endometriosis is a heterogeneous and complex disease in its presentation, symptoms, and genetic biosignature which can complicate the disease diagnosis and treatment 4, 67. Superficial 9 peritoneal lesions (SUP), ovarian endometriomas (OMA), and deep infiltrating endometriosis (DIE) are three major phenotypes of the disease 6. Superficial lesions commonly develop on the pelvic peritoneum or organs and can be found in a wide range of colors, including red (red, red- pink, and clear lesions), white (white, yellow-brown, and peritoneal defects), and black (black and blue lesions) with red and white lesions be the hardest to distinguish from normal peritoneum due to their heterogeneous morphology 68, 69. It is believed that multiple shedding in red lesions induces an inflammatory response that encloses an active lesion, and over time, the accumulation of this intraluminal debris can cause the lesion to turn black 70. Ovarian lesions, which are normally found as ovarian cysts, are known as endometriomas. Endometriomas, normally in the form of a pseudocyst, are filled with brown and dense fluid believed to contain non-resorbed blood derived from repeated hemorrhages of the endometriotic cells in the cyst during menstrual cycles 71. Endometriomas present a 2-3-fold increased risk of transformation to epithelial ovarian cancers, with endometrioid adenocarcinoma and clear cell carcinoma being the most common histologic types 72, 73. A DIE lesion is typically a solid nodule, characterized by its ability to penetrate tissue deeper than 5 mm below the peritoneal surface or the surrounding organs, including the intestines and bladder 6, 74. Figure 1.3 represents the most common appearances of endometriotic lesions in women 4. 10 Figure 1.3 Panel A shows minimal endometriosis with four peritoneal endometriotic lesions (white arrows) on the right pelvic side wall. Panel B shows extensive endometriosis with bowel adhesions to the uterus and obliteration of the posterior cul-de-sac. Panel C shows a superficial red peritoneal endometriotic lesion and blood vessels. Panel D shows an endometrioma (chocolate cyst) in the left ovary. Panel E shows a deep bladder nodule with fibrotic content (black arrows, boundaries of the nodule) and red, brown, and black peritoneal endometriotic lesions (white arrows). Reproduced with permission from Zondervan et al. 4. Copyright Massachusetts Medical Society. Endometriosis and infertility are most likely associated, although a cause-and-effect connection has not yet been shown. Endometriosis causes heterogenous reproductive defects ranging from pelvic anatomical defects to dysfunctional immunological and inflammatory 11 environments which leads to infertility or subfertility. Observational studies have shown that the rate of spontaneous pregnancy in women with proven endometriosis is about 30% in moderate and 0% in severe form of the disease 64. Endometriosis is considered to be a genetically complicated and heterogeneous disease with family aggregation, with more than 5-fold risk for first-degree relatives of endometriosis patients 75, and around 50% heritability based on twin studies 76, 77. Even though the increased prevalence of endometriosis among related versus unrelated women strongly suggests the presence of predisposing genetic (heritable) factors, the genetic causes of the disease have not yet been fully identified. This can be due to several reasons, including lack of reproducibility of data, limited number of research subjects, and heterogeneous disease phenotype and/or stage 67, 78, 79. Many candidate gene studies with a focus on potential genes of interest have been conducted with no reproducible data, making them not suitable for use as clinical diagnostic markers 78. Various studies, including those on endometriosis risk loci, suggest that genes involved in perturbations of protein Wnt (WNT) signaling, cell adhesion, cell migration, angiogenesis, inflammation and immune response, steroidogenesis and sex hormone receptorial activity, and metabolism regulation are involved in endometriosis 79, 80. It has been suggested that epigenetic influences, which regulate gene expression at a specific time and in a tissue-specific manner, have an impact on endometriosis. Epigenetic changes, including DNA methylation and histone modification, as well as the regulation of gene expression through the action of double-stranded noncoding RNAs and microRNAs (miRNAs), are believed to alter the expression and function of estrogen and progesterone receptors, aromatase, and nuclear receptors in endometriosis compared to normal endometrium 81. The heterogeneity in epigenetics of similar-looking lesions presents a challenge in endometriosis research, diagnosis, 12 and treatment 82. Environmental factors, aging, diet, and chronic inflammation can impact the epigenome, which includes DNA methylation and histone modifications, and may provide a connection between endometriosis-related molecular changes, lifestyle influences, and environmental factors. DNA methylation is known to be influenced by environmental factors, and its aberrant function could possibly link gene expression changes to endometriosis. However, it is still unclear whether aberrant DNA methylation is the cause or result of the disease 83, 84. Methylation of DNA is initiated and maintained by DNA methyltransferases, the enzymes that catalyze transfer of a methyl group to the 5′ carbon of cytosine in targeted cytosine-phosphate- guanine dinucleotides (CpGs) of the promoter region of genes, causing gene expression silencing 81, 85. DNA methyltransferases have shown different expression levels in endometriotic lesions compared to their normal endometrium or even the eutopic endometrium of the same patients 81. However, interpretation of data from previous DNA methylation studies in endometriosis remains a challenge, since patterns reported in one study are rarely confirmed by others. Since DNA methylation signature can differ among various types of cells, this small overlap of data across various studies can be attributed to the heterogeneous composition of different lesion collected by biopsies from patients 86. Another important epigenetic factor in studying endometriosis is histone modification, which can regulate gene expression patterns by influencing chromatin structure or by regulating binding of chromatin factors 11, 87. Histones can be modified by several enzymatic activities that add or remove specific chemical groups, including acetyl, methyl, phosphoryl, and ubiquitin 88. DNA modifications can alter the structure of chromatin, resulting in changes in the ability of the transcriptional machinery to access the DNA. This, in turn, can affect gene expression through the recruitment and activity of transcription factors, RNA polymerases, and other chromatin-associated proteins 89-93. Examples of these modifications include methylation, 13 demethylation, acetylation, and deacetylation of histone proteins which are correspondingly performed by histone methyltransferase, histone demethylase, histone acetyltransferase, and histone deacetyltransferase 94. The endometrial function appears to be influenced by histone acetylation changes. According to histone acetylation profiles, comparison of normal endometrium with endometriotic stromal cells exhibits hypoacetylation of specific histones in the latter one, particularly in the promoter region of ESR1gene encoding Erα. It has been shown that increased histone deacetylase activity in endometriotic cells results in hypoacetylated promoter regions, which promotes cell cycle induction and proliferation 95. Despite the important role of histone modifications in epigenetics, studies on its mechanisms in relation to endometriosis have been scarce and the results have mostly been ambiguous 79, 95, 96. Colón-Díaz and coworkers showed that various types of endometriotic lesions, and lesions in different locations have different histone deacetylase expression levels, marking the heterogeneity of this epigenetic factor 97. MiRNAs are short, single-stranded, noncoding RNAs that bind to complementary regions of target messenger RNAs (mRNA) and control a wide range of normal and pathological cellular processes through regulating gene expression mainly by blocking translation or controlling mRNA stability and degradation 98. Furthermore, miRNAs can be released into biological fluids, including saliva 99, blood 100, 101, and peritoneal fluids 102, where they can modulate translation in recipient cells. Expression of tissue-specific miRNAs is influenced by endocrine hormones throughout the menstrual cycle. Endometriotic lesions, endometrial biopsies, and stromal and epithelial cells isolated from these tissues, as well as body biofluids have all been examined in attempt to identify miRNA changes in endometriosis, leading to the detection of a substantial number of dysregulated miRNAs 98, 103. MiR-200b is a miRNA that is often found to be downregulated in endometriotic lesions. Downregulation of this miRNA is involved in the epithelial-to-mesenchymal transition 14 (EMT), which is an essential process in endometriosis (the role of EMT in endometriosis and fibrosis is discussed in sections 1.5 and 1.5.1). Furthermore, studies have identified the miR-200 family that have significant regulatory factors related to the disease pathogenesis and are involved in ovarian cancer metastasis 98, 104. Unfortunately, in most of the studies, miRNA expression levels were compared between endometrium (mostly containing epithelial and stromal cells) and lesions with additional surrounding tissues, including peritoneal and ovarian tissues, leading to inconsistent data across various studies 103. Despite studies on this group of epigenetic modulators, it is not fully understood whether miRNA expression differences precede lesion formation or are secondary to lesion establishment and are caused by the surrounding environment of endometrial cells in ectopic locations 100, 103. Also, these studies show contradictive profiles which can be due to the heterogeneous nature of the disease, different miRNA profiles in different lesion types (peritoneal, OMA, and DIE), diversity of the patient population, and experiment design 100, 105-107. According to the study by Braza-Boïls et al., miR-411-3p expression is significantly higher in DIE nodules compared to control eutopic endometrial tissue. However, the expression level of this microRNA in ovarian endometriomas and peritoneal lesions is similar to that of eutopic endometrium 108. To ensure accuracy and comparability of miRNA profiles, it has been suggested that all studies should use a standardized approach for RNA extraction and amplification, and include an endogenous miRNA control 109. Endometriosis is an estrogen-dependent disease, since estradiol, a biologically active estrogen which is secreted by the ovary or locally produced by endometriotic tissue, exacerbates the pathogenic processes (such as growth and inflammation) and symptoms (such as pain) connected with endometriosis 110. Estrogens act via their specific receptors (ERs), including ERa and ERb isoforms and their responsiveness is regulated by ERs distribution, protein function, and expression ratio which are generally different in ectopic and 15 eutopic endometrium of both patients and animal models of endometriosis compared to healthy endometrium 111-113. ERs are involved in the development of endometriosis and mediating its endometrial effects. In the healthy adult endometrium, ERα, as the major mediator of estrogenic activity, is expressed significantly higher than ERβ 114. It was claimed that ERa plays a role in lesion establishment, neoangiogenesis, and proliferation 115. ERβ plays an important role in endometriosis progression by promoting proinflammatory signaling in ectopic lesions and preventing apoptosis to promote their survival 116. Despite conflicting results, the majority of studies claimed higher ERb-to-ERa ratios in endometriotic lesions, partly because of aberrant epigenetic regulation, such as abnormal DNA methylation in the promoter region of ER genes, facilitating the development of endometriosis 114. Based on various studies, the patterns of ER expression profiles are heterogeneous among different patients with endometriosis, among various lesions collected from one patient, or even within different parts of one lesion 117-119. Another dysregulation that normally accompanies endometriosis is the elevation of aromatase expression in ectopic endometrium 120-122. Aromatase is an enzyme that belongs to the cytochrome P450 superfamily and is the key enzyme in increasing the local biosynthesis of estrogen, an essential hormone for the establishment and growth of lesions 95, 123, 124. It is suggested that upregulation of this enzyme is the result of aberrant DNA hypomethylation and subsequent gene silencing prevention 83, 125. Although ectopic endometrium has more aromatase mRNA and enzyme activity than eutopic endometrium, both ectopic and eutopic endometrium of women with endometriosis have elevated levels compared to healthy endometrium with barely detectable aromatase 126, 127. It has been previously shown that aromatase expression can be different among different patients 128 and even within the same OMA cyst wall 129 which contributes to the overall heterogeneity of the disease. 16 In conclusion, due to complex and heterogeneous nature of endometriosis, a more personalized approach with emphasis on understanding the pathogenesis of the disease is required to study, diagnose, and treat this condition. 1.5 Introduction to Fibrosis The formation of fibrotic tissue is characterized by the aberrant buildup of extracellular matrix (ECM) components such as collagen and fibronectin surrounding inflamed or damaged tissue, representing a usual and significant phase of tissue repair in all organs. Fibrosis is not a disease, but rather the result of an imbalanced tissue repair response stimulated by various tissue injuries or inflammatory disorders 130, 131. Despite the fact that each chronic fibrotic disorder has a distinct etiology and clinical presentation, the majority of chronic fibrotic disorders share a sustained or recurrent trigger that maintains the production of growth factors, angiogenic factors, and fibrogenic cytokines, which stimulate the deposition of connective tissue components that damage normal tissue architecture and gradually remodel it 132. There are several cellular components involved in fibrosis development including platelets, macrophages, fibroblasts, myofibroblasts, and epithelial cells. Their complex interplay with various signaling pathways and ECM components including collagen and matrix metalloproteinases (MMPs) underline the development and progression of fibrosis 130, 133, 134. An important concept to understanding fibrosis is the wound healing process - a dynamic process resulting in fibrotic diseases if disrupted or prolonged. Wound healing is characterized by four overlapping, interrelated, precisely synchronized, and sequential phases, including hemostasis, inflammation, proliferation, and tissue remodeling, occurring in response to an injury (insult) 135-137. Early stages of wound healing include hemostasis and activation of inflammatory cells, while the intermediate stage involves the proliferation of fibroblasts, matrix deposition, and angiogenesis, followed by ECM remodeling leading to scar formation and barrier restoration in the later stage. Precise and regulated events 17 during each phase are essential for proper wound healing, and interruptions, aberrancies, or prolongation in the process can lead to delayed wound healing or a non-healing chronic wound. Injury can be a result of infection, autoimmune disease, mechanical damage, and allergic reaction which can disrupt normal tissue architecture and initiate a healing response. After the initial insult, damaged epithelial and endothelial cells release inflammatory mediators including cytokines, chemokines, and growth factors which attract immune cells to the site of injury and initiate a cascade of anti-fibrinolytic coagulation, leading to platelet activation and the formation of blood clot as a temporary barrier 135, 138-140. The coagulation cascade involves a series of proteolytic enzyme activations that ultimately result in the formation of a stable fibrin clot. Platelets exposed to ECM components trigger aggregation, clot formation, hemostasis, and start to release granules containing various molecules, including growth factors and vasoactive mediators. These molecules promote vasodilation and increased blood vessel permeability, which facilitates the influx of inflammatory cells to the site of injury 141, 142. In addition, myofibroblasts, specialized cells that express alpha-smooth muscle actin (αSMA) and help wound contraction, are activated by various factors, such as TGF-β, and recruited to the site of injury. These cells secrete extracellular matrix components, including collagen, and are essential for wound contraction and tissue repair. The immune system has a significant role in repair response and potential consequent fibrosis. In the early phases of wound healing, macrophages and neutrophils are among the first cells to respond to the site of injury and play a crucial role in clearing away debris and dead cells. In addition to their phagocytic activity, these cells produce various cytokines and chemokines that attract other immune cells to the site of injury and promote migration and proliferation of various types of non- immune cells in the wound site and formation of new blood vessels by exerting mitogenic and chemotactic effects on endothelial cells. As the wound healing process progresses, lymphocytes 18 and other immune cells become activated and begin secreting cytokines and growth factors that promote the formation of fibrous tissue, such as TGF-β, interleukin 13 (IL-13), and platelet- derived growth factor (PDGF) 140, 143. These factors further stimulate the activation of macrophages and fibroblasts. Myofibroblasts migrate along the fibrin lattice into the wound and promote wound contraction, which helps bring the edges of the wound closer together. Finally, the wound healing process completes with division and migration of epithelial and endothelial cells over the basal layers, resulting in regeneration of damaged tissue 132, 135. Chronic inflammation and repair processes can lead to the excessive accumulation of ECM components which results in the formation of a permanent fibrotic scar. The net increase or decrease of collagen within the wound is dependent on the balance between the rate of collagen production by myofibroblasts against its degradation and the activity of MMPs and their corresponding inhibitor enzymes (TIMPs) which regulate the equilibrium between its synthesis and catabolism. Normal wound healing and fibrotic disorders share many similarities. However, in fibrotic diseases, tissue remodeling, fibroblast activation, and impaired myofibroblast apoptosis continue as a chronic and uncontrolled process characterized by an exaggerated and prolonged wound healing response, suggesting that fibrotic diseases may result from impaired termination of tissue repair responses 132, 144. 1.5.1 Cellular and Molecular Components of Fibrosis As mentioned in section 1.5 there are multiple cellular and molecular components contributing to fibrosis. Cellular components include various cell types such as fibroblasts, myofibroblasts, immune cells, endothelial cells, epithelial cells, and pericytes. ECM proteins, various cytokines and growth factors, proteases, and integrins are among the important molecular components of fibrosis. Here, a short description of each component is given, which can help to better understand endometriosis-related fibrosis in later sections. Fibroblasts are spindle-like cells with mesenchymal origin that are present in most of the 19 body's tissues and organs, involve in inflammation, angiogenesis, cancer progression, physiological as well as pathological tissue fibrosis in their activated form, and secreting the ECM's structural proteins (e.g., fibrous collagen and elastin), adhesive proteins (e.g., laminin and fibronectin), and ground substance (e.g., glycosaminoglycans, such as hyaluronan and glycoproteins) to maintain hemostatic ECM turnover. These cells, which normally display different morphologies depending on their location, are not terminally differentiated and retain their ability to differentiate into subtypes of fibroblast-like cells. Fibroblasts can be activated by different chemical signals which activate proliferation and differentiation to myofibroblasts 144-148. Recent studies on fibrosis suggest that only certain specialized fibroblastic cells may be responsible for generating myofibroblasts in both normal and pathological conditions 149. Myofibroblasts are a heterogeneous population of cells that produce significant amounts of ECM proteins, and their phenotype is identified by expression of αSMA. They have contractile nature, increased production and secretion of extracellular matrix, and resistance to apoptosis. These cells can originate from several different cell types, including resident fibroblasts, endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT), vascular smooth muscle cells and epithelial cells after epithelial-to-mesenchymal transition (EMT), and circulating bone marrow-derived specialized inflammatory cells (fibrocytes) 144, 146. Activation of myofibroblasts requires neo-expression and integration of αSMA into stress fiber-like bundles and is critically influenced by TGF-β and tissue stiffness. This increases proliferation, migration, and cytokine production of myofibroblasts, which ultimately disrupts the functional integrity of residual tissues due to the production of interstitial matrix. Continues activation of myofibroblast over time leads to accumulation of ECM and collagen overexpression 149, 150. Platelets represent another important cell type in the fibrosis process that circulate in a 20 resting state and become activated upon encountering different stimuli such as inflammation, injury, or bacteria. Activated platelets have been reported to be involved in the initiation and development of fibrosis in both mice and humans by releasing several profibrotic molecules, including PDGF and epidermal growth factor (EGF) and facilitating epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT). Platelets and platelet-derived extracellular vesicles are also the primary sources of circulating TGF-β, which can be released after cell activation. Upon activation, these cells acquire a spherical shape rather than their initial discoid shape which can help them interact with other cells. The activated form can release extracellular vesicles containing various molecules and spread them to body fluids and tissues that are not usually accessible by platelets 134, 151-153. TGF-β is a well-studied and important molecular component in fibrosis, which has three isoforms with similar activity but distinct expression patterns. TGF-β1 is primarily linked to tissue fibrosis and is released from cells in an inactive state, which can be activated by agents including MMPs and integrins. Once activated, TGF-β1 signals via serine/threonine kinase receptors, activating the Smad signaling pathway and modulating the transcription of important target genes. In the Smad pathway, TGF-β ligands bind to cell surface receptors, leading to the activation of Smad proteins. These activated Smad proteins then form complexes with other proteins and translocate to the nucleus of cells where they regulate the expression of genes involved in the development of fibrosis 154, 155. The Wnt/β-catenin signaling pathway is involved in many processes, including development, tissue maintenance, and diseases such as endometriosis. This pathway is partly responsible for regulating the production of ECM components such as collagen and has been found to play a role in development of fibrosis in several organs. Available data suggest that the Wnt signaling pathway also promotes EMT in various diseases 156-159. The Notch 21 signaling pathway is another pathway related to fibrosis in various pathological conditions including endometriosis 160. Multiple studies suggested that this pathway involves the regulation of myofibroblast differentiation, EMT 161, and upregulation of type I collagen promoter activity 162. 1.5.2 Endometriosis and Fibrosis Endometriosis is commonly defined as the presence of endometrial glands and stroma outside of the uterus. In line with this definition, currently, the definitive diagnosis of endometriosis is obtained by analyzing tissue samples from ectopic implants through invasive surgical or laparoscopic procedures and performing subsequent histological examination to confirm the presence of stroma and glands 20. However, this simple definition masks the wide variation and heterogeneity of different aspects of endometriosis, including lesion characteristics (location, size, appearance, and depth of invasion), spectrum of symptoms, and severity of the disease (discussed in section 1.3 and 1.4). In addition, despite the classic definition of endometriosis, only a fraction of lesions consists of both stroma and glands 133. In rectovaginal lesions (rectovaginal nodules) the stroma sometimes appears absent around glandular epithelium 163. Stromal endometriosis, a type of endometriotic lesion containing stroma while lacking glandular components, was reported several times as well 164-167. Pelvic adhesions, which seem to be important in the development of endometriosis-related symptoms, including dyspareunia, chronic pelvic pain, and infertility, do not usually contain endometrial-like components (stroma and glands), but may be associated with the formation of endometriotic lesions, especially deep nodules and endometriomas 133, 168. Interestingly, a study by Zhang et al. in a baboon model of endometriosis showed that as lesions progressed, TGF-β1 and α-smooth muscle actin-positive myofibroblasts increased in the stromal compartment, which correlated with the increasing extent of fibrosis (as demonstrated by Masson trichrome staining). The immunohistochemistry results 22 also revealed that as the lesions become older E-cadherin expression in the epithelial component of the endometriotic lesions decreases, suggesting a progressive EMT. These findings were in line with the hypothesis that repeated tissue injury and repair of lesions leads to EMT and FMT. It has been suggested that these transitions can ultimately promote smooth muscle metaplasia (SMM), a process accounting for universal presence of smooth muscles in or around endometriotic lesions, and fibrosis (Figure 1.4) 158, 169. Figure 1.4 Representative photomicrographs of serial immunohistochemistry analysis of E- cadherin and α-smooth muscle actin (α-SMA), along with Masson trichrome staining in control endometrium and in endometriotic lesions of different ages. (E-cadherin and α-SMA stained in brown, collagen fiber layer stained in blue) Scale bar = 50 μm 169. 23 In this context, Vigano et al. published an opinion article suggesting a need for modification of endometriosis definition and emphasizing the consistent presence of fibrosis and myofibroblasts in endometriotic lesions, indicating their crucial role in the disease's pathogenesis, which can be helpful in the future endometriosis-related research 133. This suggests that while endometriosis is characterized by inherent characteristics such as estrogen dependence, chronic inflammation, neoangiogenesis, neuroangiogenesis, and impaired progesterone responsiveness, strong evidence associates fibrosis with the pathogenesis of endometriosis as a molecular hallmark of the disease 170. Single-cell-RNA sequencing and immunostaining results published by Zhu et al. also suggested endometriosis as a fibrotic disease and showed a significantly higher number of myofibroblasts in ectopic endometrium compared to both eutopic and healthy endometrium 171. Throughout reproductive years, women normally experience about 400 hormone-driven cycles of proliferation, differentiation, and breakdown of the endometrium. During menstruation, the luminal part of endometrium breaks down and sheds, creating an inflammatory response, and leaving the function layer in a wounded state. Within a few days after this process, a healthy endometrium is capable of restoring its integrity without leaving a scar 172. It is important to understand the mechanism underlying this scar-free healing process believed to be similar to classic wound healing, to further investigate fibrosis and understand the differences between healthy endometrium and ectopic lesions 173. The healing process of adult human wounds varies ranging from the (nearly) absence of scars to the development of scars and eventual fibrosis. Within this spectrum, there are several cellular and molecular components that contribute to the healing process, with some being more predominant in scarless wound healing and others in the development of fibrosis. For example, in scar-free wound healing type III collagen and TGF-β3 are dominant and myofibroblasts go through apoptosis at the end of the process while in scarring 24 Figure 1.5 Main cellular types and molecules involved in the development of endometriosis- related fibrosis. IL-6 = interleukin 6; IL-8 = interleukin 8; LOX = lysyl oxidase; MMP = matrix metalloproteinase; PA = plasminogen activator; PAI-1 = plasminogen activator inhibitor-1; ReTIAR = recurrent tissue injury and repair; TGF-β1 = transforming growth factor β 1; TIMP = tissue inhibitor of metalloproteinases; VEGF = vascular endothelial growth factor 134. Reproduced with permission from Elsevier. healing type I collagen and TGF-β1 are dominant and myofibroblasts apoptosis rate is significantly lower 155, 172, 174-177. It has been suggested that due to cyclic bleeding endometriotic lesions can be considered as wounds undergoing repeated tissue injury and repair (ReTIAR), which results in EMT and FMT triggered by stimulating factors (e.g. TGF-β1) secreted from activated platelets, immune cells, or from sensory nerve fibers, ultimately leading to excessive collagen deposition and fibrosis 134, 158. Mustaki et al. hypothesized that tissue stiffness in DIE, in the absence of TGF- β1 signaling, might cause endometrial stromal cells to differentiate into myofibroblasts-like cells and produce type I collagen. This group suggested that in the absence of TGF-β1, an important 25 component of fibrosis process, presence of stiff and dense fibrous tissue (a hallmark of fibrosis) can further activate collagen production and EMT in a rigid and collagenous microenvironment 178. The main molecules and cell types involved in the development of endometriosis-related fibrosis are illustrated in Figure 1.5 which will be briefly explained below. Consequent to the injury phase of ReTIAR and initiation of homeostasis, platelets become activated and release large quantities of TGF-β1 153 and PDGF 179 known to be involved in both FMT 180 and EMT 156. Anti-inflammatory M2 macrophages secrete various chemokines and cytokines, including TGF- β1, IL-6, interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), and lysyl oxidase (LOX) 134. Studies in animal models of endometriosis suggest that infiltration of M2 macrophages increases progressively as endometriotic lesions progress. Activation of these macrophages can induce EMT and FMT in endometriotic cells through TGF- β1/Smad3 signaling pathway, which increases collagen production and cellular contractility (mentioned in section 1.5.1) 181. LOX has a key regulatory role in fibrosis by stabilizing ECM via crosslinking 182 and its expression is increased in cell lines, lesions, and endometrium of women with endometriosis-associated infertility, indicating a potential role in ECM remodeling and fibrotic diseases. However, in a study by Ruiz et al. LOX overexpression only partially induced EMT, and it was implied that additional factors are necessary for a complete remodeling of the extracellular matrix leading to subsequent fibrosis 183. Yan and coworkers suggested that substance P (SP), a neuropeptide secreted by sensory nerves in response to stressful stimuli, can induce EMT and FMT in endometriosis and SMM in endometriotic stromal cells, promoting collagen production and fibrosis in lesions 184, 185. Plasminogen activator inhibitor-1 (PAI-1) has a recognized role in fibrotic diseases by reducing the rate of collagen degradation, while its absence protects various organs from fibrosis. It has been suggested that high levels of PAI-1 in peritoneal 26 fluid of women with endometriosis contributes to the development of peritoneal lesions 186. Alotaibi et al. have shown that PAI-1 expression in DIE and OMA is elevated compared to superficial endometriosis or eutopic endometrium, explaining the higher fibrotic content of DIE, as well as fibrotic and invasive properties of OMA cysts that invade the ovary and are associated with adhesions and fibrosis 187. Also, a study in a mouse model of endometriosis suggested that suppressing PAI-1 reduces both lesion size and its fibrotic content 188. Other important molecules that are suggested to be involved in EMT and endometriosis-related fibrosis are the MMP family of endopeptidases which degrade extracellular matrix components and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). TIMPs have a matrix stabilization role in fibrogenesis, as they suppress the activity of MMPs by binding to their catalytic domains and blocking their enzymatic activity, resulting in increased ECM accumulation 189. These molecular components are secreted from activated macrophages and endometriotic cells, facilitating normal endometrial remodeling during menstruation, and are tightly controlled in the healthy endometrium through various cytokines including IL-8 and TGF-β1. However, abnormal expression of MMPs and TIMPs has been observed in endometriotic lesions 190. Mentioned dysregulations during endometriosis-related ReTIAR, aberrant expression and function of VEGF, aberrant secretion of IL-6 and IL-8 by platelets and macrophages, and continuous presence of activated platelets and immune cells in ectopic endometrium increase expression of αSMA, enhance collagen production and cellular contractility, culminating in fibrosis 131, 158, 169, 191, 192. Over time, this process may result in subsequent adhesion, anatomic distortion, and pelvic pain. This was further confirmed by demonstrating an increase in type I collagen in the endometriotic tissues collecting from patients with severe endometriosis, in comparison to healthy endometrium where type III collagen is the dominant form 193. Matsuzaki et al. showed that disrupting Wnt/β- 27 catenin pathway in stromal endometriotic cells can decrease the expression of α-SMA, type I collagen, connective tissue growth factor and fibronectin mRNAs. This study also demonstrated that targeting the Wnt/β-catenin pathway in a xenograft mouse model of endometriosis can decrease the fibrotic content of endometriotic lesions 194. Various pathways including Wnt/β- catenin and Notch are shown to be involved in endometriosis pathogenesis and the development of endometriosis-related fibrosis. A study by Song et al. indicated that Notch-1 was upregulated in glandular epithelium of endometriotic lesion 195. Another study also suggested that hyperactivation of the ADAM17/Notch signaling pathway leads to an increase in fibrotic content in women with endometriosis 160. Despite an increase in activation of this pathway in ectopic endometrium, the findings of Su et al., demonstrated that the endometrium of women with endometriosis had decreased Notch-1 signaling 196, suggesting that the same pathway or molecule can have different expression and activation levels between ectopic lesions and eutopic endometria. Despite all the aforementioned information, it is still unclear why cyclical inflammation causes fibrosis in ectopic endometrial tissue but not in eutopic endometrium 197. In view of all the above, Guo et al. suggested that fibrogenesis resulting from ReTIAR is a fundamental and intrinsic part of the progression and the major hallmark of the natural history of ectopic endometrium rather than a secondary event 198. 1.5.2.1 Fibrosis and Myofibroblasts in Peritoneal Lesions As of today, various molecular and cellular components of fibrosis have been investigated using samples of endometriotic lesions. Ten peritoneal lesion samples were analyzed for the presence of α-SMA and myofibroblasts, as components involved in the formation of fibrosis using histological techniques. The results showed well-formed smooth muscle bundles and dense type I collagen in these lesions 199. In another study, 21 peritoneal lesions were evaluated for the presence 28 of smooth muscle actin. All lesions displayed different degrees of actin staining positivity, while their smooth muscle content was significantly greater than that of corresponding unaffected pelvic locations 200. In another study, 35 endometriotic lesions were analyzed for α-SMA and showed that 100% of peritoneal lesions stained positive for this marker while unaffected peritoneum showed minimal stained area 201. Barcena de Arellano et al. showed that smooth muscle cells were present in all endometriotic lesion types (n=60 samples) including peritoneal lesions 202. 1.5.2.2 Fibrosis and Myofibroblasts in Ovarian Endometriomas Studies have demonstrated that OMA pseudocysts are usually composed of fibrotic tissue, and in cases where the endometrial lining is absent on the inner surface of the cyst, only fibrotic tissue can be identified. 133. Previously, various groups showed that all ovarian lesion samples from patients with endometriosis stained positive for smooth muscle actin antibody, showing the presence of myofibroblasts 199, 200. Sun-Wei Guo's team found that activation of platelets in cells derived from ovarian endometrioma led to fibrosis by triggering EMT, FMT, and collagen production through releasing TGF-β1 and promoting the TGF-β/Smad pathway 158. In one study, histological analysis revealed that all OMA samples exhibited markers of FMT and stained positive for collagen. These results highlighted similar characteristics of OMA and DIE such as EMT, FMT, SMM, and fibrosis and exhibited common underlying factor as wounds experiencing ReTIAR, while also revealing the differences in the extent of these transformations between the two conditions 203. It has been reported that the incidences of fibrosis were notably higher in the cortex of ovaries with endometriomas compared to contralateral normal ovaries and can ultimately result in depletion of cortex-specific stroma, leading to the structural disruption of follicular nests, reduced ovarian follicle numbers, and fertility problems 204. 1.5.2.3 Fibrosis and Myofibroblasts in Deep Infiltrating Lesions Donnez et al. initially proved that all samples of deep endometriotic rectovaginal nodules 29 (from 65 patients), histologically consisted of sparse stroma and glandular epithelium distributed in substantial fibromuscular tissue 163. This was further confirmed in another study containing 12 rectovaginal nodules and 8 uterosacral lesions which all stained positive for αSMA 200. Itoga et al. have shown that almost all rectovaginal nodules assessed in their study showed presence of fibrosis in fat and connective tissue, as well as, in the endometriotic tissue in severe cases 205. A study in a nude mouse model of endometriosis implanted with human endometrium fragment showed that formation of DIE nodules shares various characteristics with pathological wound healing process including αSMA and collagen expression. These results showed that one week after induction of lesions, the murine fibroblasts surrounding the human endometrium exhibited an elevation of αSMA expression, whereas no expression was seen in the human cells themselves, suggesting that this process highly relied on the response of the surrounding environment 206. It has been suggested that DIE lesions can be resistant to peritoneal fluid suppressor effects, allowing them to invade deeply in tissues. These deep lesions always accompany fibromuscular components 207. In these lesions activation of the Wnt/β-catenin pathway promotes stromal cell proliferation, migration, and collagen contraction, with Wnt signaling also regulating the expression of fibrotic marker genes, including genes encoding for collagen type I, αSMA, and fibronectin 157. It has been reported that various cancer driver mutations, including tumor protein p53 and phosphatase, tensin homolog (PTEN), and AT-rich interaction domain 1A (ARID1A) mutations may arise from significant pressure for progressive fibrogenesis and support its progression in the lesions, making them both cause and consequence of extensive fibrosis around deep lesions. 208. Substance P (SP) is a neuropeptide from tachykinin neuropeptide family that acts as a neurotransmitter and modulator. This neuropeptide is distributed throughout the central and peripheral nervous system and is also produced by lymphocytes, macrophages, neutrophils, and dendritic cells. SP involves 30 in pathological fibrotic conditions, including endometriotic lesions through EMT, FMT, and SMM. This neuropeptide is involved in many other processes, including pain and inflammation 131, 184. Figure 1.6 illustrates the most important components in development of fibrosis in DIE lesions. 31 Figure 1.6 Principal cellular types and molecules implicated in the development of DIE-related fibrosis. DIE, deep infiltrating endometriosis; EMT, epithelial to mesenchymal transition; FMT, fibroblast to myofibroblast transition; IL-6, interleukin 6; IL-8, interleukin 8; MSTN, myostatin; SMM, smooth muscle metaplasia; SP, substance P; S1P, sphingosine-1-phosphate; PAI-1, plasminogen activator inhibitor-1; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor, Wnt, wingless-related integration site 131. Reproduced with permission from Springer Nature via the Copyright Clearance Center. Despite all mentioned hypotheses and pathways participating in endometriosis-related fibrosis, the exact processes leading to this condition are yet to be investigated and clarified with further research. According to recent studies, a more appropriate definition for endometriosis would be a condition that begins with the ectopic implantation of endometrial tissue, which undergoes cyclic bleeding, causing repeated injury and repair, leading to gradual smooth muscle metaplasia, collagen production, and ultimately fibrosis 198. Vigano et al. and Guo et al. provided several reasons for using the term ‘fibrosis’ in the definition of endometriosis, including shifting 32 the research direction toward fibrosis process and myofibroblasts, which provides for developing animal models based on this new definition, gaining a better understanding of lesions development, and investigating new opportunities in the fields of diagnosis, symptom management, and novel therapeutics development 133, 198. 1.6 Introduction to Angiogenesis The vascular system is a closed network of arteries, veins, and capillaries organized into tree-like structures. In this network, endothelial cells create a lining for the lumen of blood vessels, which work with the lymphatic system to control the flow of nutrients, cells, and transportation of fluids and diverse signaling molecules within the vascular system. Although adult endothelial cells rarely proliferate and remain inactive and quiescent, they retain the capability to initiate angiogenesis, which is normally initiated in response to a stimulus like hypoxia. This process is defined as the formation of new blood vessels from pre-existing ones, taking place through the splitting and sprouting pathways. Under physiological conditions, angiogenesis is a crucial biological process for development, wound healing, and function of female reproductive system. It is also a hallmark of various pathological conditions, including tumor growth, metastasis, and rheumatoid arthritis 209-211. In splitting angiogenesis, the endothelial cells are reorganized and remodeled to form new vessels. In sprouting angiogenesis, the binding of growth factors to endothelial cells stimulates them to proliferate, migrate and invade the surrounding matrix towards the stimulus, resulting in the formation of new vessels. Angiogenesis consists of complex and multi-step processes where various cells and ECM components are involved. Generally, angiogenesis consists of four sequential steps, including basement membrane degradation by enzymes, endothelial cell migration and sprouting, endothelial cell proliferation at the migrating tip, and new lumen and basement membrane formation which are followed by blood flow and vessel maturation 212. Normally quiescent endothelial cells form a layer of cells that are surrounded 33 by pericytes, which stabilize the vessels. When angiogenesis is initiated through sprouting pathway, tip cells, a highly motile form of endothelial cell, are attracted by signals from the microenvironment and move towards the source of the signal by breaking down the basal lamina and moving into the extracellular matrix. The tip cells are characterized by their low proliferation activity, high migratory capacity, and their ability to recruit non-vascular cells. Following the tip cells, stalk cells proliferate and form the vascular lumen, ensuring the elongation of the vessel, which ultimately results in merging two sprouts and creating a continuous lumen 213. There are several soluble and membrane-bound factors that help initiation, maintenance, and regulation of angiogenesis, including VEGF, PDGF, fibroblast growth factor (FGF), TGF-β, and angiopoietins. Angiogenic soluble growth factors, such as VEGF, activate dormant endothelial cells in microvessels to secrete MMPs, which break down the basement membrane of the vessel. Angiopoietins prompt perivascular cells to detach from the vessel wall, allowing endothelial cells to migrate and create vascular buds and sprouts. The formation and organization of these sprouts are regulated by Notch signaling, which determines the cellular specialization into tip and stalk cells (the role of Notch signaling in endometriosis-related fibrosis was discussed in sections 1.5.1 and 1.5.2) 214. Vascular endothelial growth factor-A (VEGF-A, generally referred to as VEGF) is the most studied member of the VEGF family which regulates both normal and tumor-related angiogenesis by interacting with cell surface receptors. While other members of VEGF family have been shown to influence the angiogenesis process, their role is less prominent compared to VEGF-A 211. PDFGs are a family of growth factors interacting with their corresponding receptor tyrosine kinases that have a significant role in promoting cellular growth, pericyte recruitment, and modulating cell shape and motility 215. FGFs (acidic and basic FGFs) are heparin-binding protein mitogens that interact with the tyrosine kinase cell surface receptors leading to activation of 34 pathways that can ultimately cause endothelial cells to proliferate, migrate and differentiate by activating tip and stalk cells 211, 215, 216. Membrane-bound factors are a group of membrane proteins that play an important role in angiogenesis. αvβ3 integrin, erythropoietin-producing hepatoma receptor B4 (Eph-B4), and vascular endothelial cadherin (VE-cadherin) are among these membrane-bound proteins that regulate various processes in angiogenesis 211. Integrins are a family of transmembrane glycoprotein receptors that bind to ECM proteins and regulate many fundamental aspects of cell behavior by bi-directional signaling between ECM and intracellular cytoskeleton and play a critical role in promoting cell attachment, migration, and angiogenesis. They are composed of non-covalently associated α and β subunits which form transmembrane heterodimers. Among integrins, αvβ3 is extensively studied for its regulation of angiogenesis through crosstalk with vascular endothelial growth factor receptor 2 (VEGFR2). This integrin is highly expressed on activated endothelial cells, newborn vessels, and some tumor cells but not present in resting endothelial cells or most normal organ systems. Therefore, αvβ3 serves as a marker of angiogenesis and a promising target for anti-angiogenic therapy to treat pathological conditions 217-219. The Eph receptor tyrosine kinases and their corresponding Ephrin ligands comprise a significant signaling system that plays diverse roles in both normal cell physiology and disease pathogenesis. These receptors are a family of transmembrane proteins with a single cytoplasmic kinase domain which are activated by the binding of surface-associated ligands (ephrins) to the extracellular globular domain of the receptor, allowing contact-dependent cell-cell communication with neighboring cells. EphB4 is a transmembrane protein that is a member of the Eph receptor tyrosine kinase family and alongside ephrinB2 as its exclusive ligand play a role in angiogenesis, cell adhesion, migration, and tumor survival 220-222. 1.6.1 Endometriosis and Angiogenesis Angiogenesis plays an important role in the pathogenesis of endometriosis and helps the 35 establishment, survival, and growth of lesions which are exposed to the peritoneal environment and endometrium's angiogenic potential 223. When endometrial tissues shed off from the eutopic uterus and retrograde to the peritoneal cavity, they are subjected to a significant lack of oxygen, resulting in severe hypoxic stress. Even with successful implantation to ovaries or peritoneum, hypoxic stress remains a critical issue because endometrial cells are used to live in a well- oxygenated environment. Under hypoxic conditions, cells undergo epigenetic alterations and develop various survival mechanisms including angiogenesis and inflammation 224. The development of endometriotic lesions involves proliferation, adherence and invasion of endometrial cells into the peritoneum, followed by the establishment of a vascular network to support its nutrient and oxygen requirements 225. Because of that a dense network of blood vessels is present in both the endometriosis lesions and the surrounding tissue which is normally several- folds higher compared to other tissues 226, 227. The crucial role of angiogenesis in endometriosis is highlighted by the fact that the growth and progression of endometriotic lesions are inhibited by antiangiogenic agents and reduced blood supply 223, 228. It has been shown that the angiogenic potential of eutopic endometrium is altered compared to endometrium of women without endometriosis 229. Also, multiple studies reported that different proangiogenic factors including VEGF are elevated in the peritoneal fluid of women with endometriosis 230, 231 which provides a favorable environment for the growth of newly formed lesions. TGF-β plays a crucial role in the development of endometriosis lesions by regulating important cellular functions such as cell adhesion, invasion, and angiogenesis 232. Angiopoietins have been found to be overexpressed in both eutopic and ectopic endometrium of women with endometriosis, suggesting their potential role in promoting the excessive angiogenesis observed in this condition 233. Although endometriosis-associated angiogenesis is a well-known phenomenon, limited information is 36 available regarding the precise mechanism of angiogenesis in this disease 234. In addition to the explained pathway of developing new microvessels from pre-existing adjacent peritoneal ones (sprouting), studies showed that in endometriosis circulating endothelial progenitor cells (EPCs) also help development of microvessels through a process known as vasculogenesis. Vasculogenesis process of new blood vessel formation during embryonic and fetal development through migration and differentiation of angioblastic progenitor cells is now believed to happen in adults in response to specific tissue cytokines, including VEGF and FGF 235. Both pathways are illustrated in Figure 1.7 235. 37 Figure 1.7 Mechanisms of blood vessel formation during endometriosis. (A) Sprouting angiogenesis; Upon activation by pro-angiogenic growth factors, perivascular cells (= blue), such as pericytes and smooth muscle cells, detach from the vascular wall and endothelial cells (= red) release proteases, which degrade their basement membrane (1). This allows them to migrate into the surrounding interstitium, resulting in the formation of capillary buds and sprouts (2). The sprouts further elongate through endothelial cell proliferation, branch and interconnect with each other (3). This leads to the development of blood-perfused microvessels, whose wall is stabilized again by the recruitment of perivascular cells and the production of extracellular matrix compounds (4). (B) Most probably, post-natal vasculogenesis and sprouting angiogenesis occur in parallel in endometriotic lesions. Post-natal vasculogenesis is characterized by the recruitment of circulating EPCs (= orange) from the bone marrow. At sites of hypoxia, they attach, proliferate, and are finally incorporated into the endothelium, where they differentiate into mature endothelial cells. Reproduced by permission of Oxford University Press/Human Reproduction Update 235. 38 1.7 Endometriosis Diagnosis: Current Methods Endometriosis presents challenges in its diagnosis due to various factors, including disease heterogeneity (explained in section 1.4) ranging from different lesion types to various lesion miRNA expression profiles. This, in addition to the limited understanding of endometriosis pathogenesis and inadequate understanding of the disease by healthcare professionals due to insufficient medical training on this subject, adds to the complexity of endometriosis diagnosis. Other factors that complicate diagnosis are a weak correlation between the symptoms and the severity or extent of the disease and lack of any adequate pathognomonic features or biomarkers to define endometriosis 236. Although different countries and healthcare systems may follow different diagnostic protocols, many of the guidelines established by professional organizations state that laparoscopic direct visualization with histologic confirmation is considered the diagnostic standard for endometriosis. These guidelines also recommend medical treatment for patients with suspected endometriosis based on clinical evaluation, without the need for surgical diagnosis. Some experts in the field are promoting a shift toward a clinical diagnosis approach, focusing on patients' symptoms and physical indications, and moving away from the reliance on surgical diagnosis. However, laparoscopy is still being used as a valuable diagnostic tool, especially in cases in which diagnosis is uncertain through non-invasive methods 237. Since performing diagnostic laparoscopy is a costly procedure and involves potential surgical risks, healthcare providers often consider alternative diagnostic techniques before recommending laparoscopy, including full review of patients’ history and symptoms, physical examination, and various imaging exams. The following sections will explore various methods and techniques that are currently employed for the clinical diagnosis of endometriosis. 39 1.7.1 Importance of Medical History, Symptoms, and Physical Examination Taking a complete medical history and carefully acknowledging and evaluating symptoms is typically the initial diagnostic step when endometriosis is suspected in a woman. During history- taking, it is essential to inquire about a family history of endometriosis, as well as previous surgeries that are known to increase the likelihood of local endometriosis, including cesarean delivery. Symptoms associated with endometriosis, including dysmenorrhea, deep dyspareunia, chronic pelvic pain, abdominal pain, cyclic dysuria, fatigue, and fertility problems should be recorded with details. It is also important to note that there is no pathognomonic symptom defining this disease and all above mentioned endometriosis-associated symptoms can represent other pathological conditions, including pelvic inflammatory disease, irritable bowel syndrome, and interstitial cystitis. One of the reasons that delays endometriosis diagnosis is attributing pain symptoms to the normal cyclic bleeding pain that many women experience. Although this is not definite, it may be beneficial to inquire about the timing of pain in relation to the menstrual cycle since primary dysmenorrhea usually begins with menstrual flow and lasts 8-72 hours, whereas endometriosis-related pain can be progressive, cyclic or acyclic and may persist for more than 72 hours 59, 237-239. Performing a physical examination which includes the abdominal, pelvic, and possibly rectovaginal areas can assist in narrowing down the possible diagnoses, ruling out other disorders, and deciding on the necessary imaging technique. During digital pelvic examination and abdominal palpation, fixed retroverted uterus, and fixed adnexal masses can suggest the presence of endometriosis and ovarian endometriomas respectively. However, diagnosing endometriosis solely based on patient history and physical examination is challenging due to the heterogeneous clinical presentations of the disease, the high occurrence of asymptomatic cases, and the absence of a link between symptoms and disease severity 237, 240. 40 1.7.2 Ultrasonography Ultrasonography is the primary investigative imaging technique for suspected endometriosis, enabling the identification of lesions and ovarian cysts. Although transabdominal ultrasound can be used to examine the entire pelvis, it is not an effective method for detecting endometriosis. This is primarily due to the limitations of the transabdominal probe, which cannot detect most deep infiltrating endometriosis (DIE) lesions. Also, the presence of bowel gas and adhesions can impede the evaluation of pelvic organs using this method. Transvaginal ultrasounds (TVUS), and transrectal ultrasounds (TRUS) can give a more detailed image of the anatomy compared to the initial ultrasonography of the pelvis area, especially for organs in proximity to the endovaginal probe, including the uterus, ovaries, and uterosacral ligaments. Experts recommend using endocavitary sonography with a microconvex array probe for comprehensive pelvic examinations. This method enables the evaluation of various pelvic structures and organs, such as the uterus, adnexa, urinary bladder, ureters, and rectum. It is crucial to begin the examination at the vaginal insertion point to avoid missing any pathology, and then gradually and carefully assess the cervix, uterus, and other pelvic structures 241-244. One limitation of TVUS is that its accuracy in lesion detection, especially for deep infiltrating lesions, depends on the experience of the sonographer. Furthermore, ultrasound imaging encounters challenges in identifying lesions involving portions of the intestinal tract, such as the rectosigmoid junction, due to the limited field- of-view of the transvaginal approach, and in detecting lesions in the upper bowel region due to the lower accuracy of transabdominal ultrasound. Variations in the appearance of lesions, superficial, plain lesions, and the distorted anatomy caused by adhesions and fibrosis may cause diagnostic challenges during the comprehensive sonographic assessment of pelvic endometriosis 237, 244, 245. TVUS, like other imaging modalities, cannot accurately detect superficial peritoneal endometriosis, and has been reported to have a significant false-negative rate for detecting such 41 lesions 246, 247. The presence of severe pelvic adhesions, large ovarian cysts, significant uterine retroflexion, and sound-wave blockage caused by obstruction and masses are among other challenges which limit the ability of TVUS in endometriosis diagnosis 248. 1.7.3 Magnetic Resonance Imaging (MRI) Despite being more expensive and less accessible, MRI can be used when ultrasound results are unclear, do not provide conclusive results, and high spatial resolution is needed 246, 249. MRI is a safe and non-invasive method for creating high-quality cross-sectional images of the body using non-ionizing electromagnetic radiation. It utilizes a very strong magnetic field which magnetizes body atom nuclei, generally hydrogen nuclei of water molecules, and radio frequency radiation to map the internal structure and certain aspects of body functions. Different MRI sequences, including T1-weighted and T2-weighted, can enhance the signal from various tissues to create contrast 250. MRI provides morphological information mainly through T1- and T2- weighted sequences and can also provide functional information using contrast agents such as intravenous gadolinium (Gd), and diffusion-weighted imaging. The signal intensity from endometriotic lesions vary according to their composition and appear differently on T1-weighted and T2-weighted images. On T1-weighted images, fat and hemorrhage appear white, while water appears dark. On T2-weighted images, tissues with a high-water content appear bright. Since endometriotic lesions are heterogeneous in presentation, various dedicated MRI protocols which include both T1- and T2-weighted sequences are used to better detect these lesions 248, 251-254. For example, Figure 1.8 represents T1-weighted and T2-weighted MR images of an ill-defined stellate-shaped DIE lesion in rectouterine pouch. This lesion appears hypointense on both T1 and T2-weighted images, and hyperintense on T1-weighted images. Also, multiple hypointense and hyperintense foci were seen within the lesion area respectively on T1- and T2-weighed images, suggesting hemorrhagic foci within the ectopic glands 255. Despite these dedicated MRI protocols, 42 this modality can easily overlook solid masses of endometriotic tissue due to their small and atypical signal character 251. Radiologists should pay more attention while using these dedicated protocols especially in the case of atypical lesions (e.g., stromal lesions without glandular components), which might have different T1- and T2-weighted image characteristics 254. To increase the detection sensitivity of endometriotic lesions with small hemorrhagic foci, it is recommended to conduct an MRI study for the first half of the menstrual cycle. However, the sensitivity of this method may be low due to various factors, including the timing of the MRI examination being in the third or fourth week of the patient's cycle, and nodules no longer being hormone responsive 253. Finding fibrotic lesions can also be challenging due to their relatively low signal intensity on both T1- and T2-weighted images 249. Patient preparation techniques, including proper fasting, urination instructions, and bowel preparation may increase the quality of imaging 256. Figure 1.8 Deep infiltrative endometriosis (DIE) in rectouterine pouch. Axial T1W (A) and T2W axial image (B) depict ill-defined stellate shaped lesion (white arrows) appearing hypointense on both T1W and T2W images. There is also presence of multiple foci seen within the lesion, appearing hyperintense on T1W and hypointense on T2W images, suggestive of hemorrhagic foci within the ectopic glands. Adaptation of original figure 255. 43 Gadolinium is an intravenous contrast agent that is frequently utilized to enhance the signal of MRI scans. Gadolinium accelerates the T1 relaxation of water protons, resulting in brighter MR signals in T1-weighted MRI. While the use of intravenous gadolinium contrast agent is not necessary for the assessment of deep pelvic endometriosis, it can be helpful in identifying atypical adnexal lesions like cysts with mural nodules during MRI evaluation. Fat suppression is commonly used in acquiring MR images to suppress the signal from adipose tissue. This technique is reported to improve the sensitivity of lesion detection and should be performed when contrast-enhanced imaging is used. A factor to consider when utilizing untargeted gadolinium chelates is that chronic endometriosis foci may exhibit inconsistent enhancement on MR images after injection, requiring careful attention to detect all degrees of enhancement 249, 256. 1.7.4 Surgical Diagnosis and Histology Laparoscopy serves a dual purpose in the treatment of endometriosis and can be classified as either diagnostic or operative laparoscopy 257. Diagnostic laparoscopy involves visual diagnosis of endometriotic lesions which is normally followed by collecting biopsies for histological assessment. Generally, the most accurate method to diagnose endometriosis is diagnostic laparoscopy. However, this method has its limitations, costs, and risks which encourage healthcare providers to rely on medical history, physical examination, and imaging studies before performing laparoscopy 258. Many women who undergo a laparoscopic procedure will not be diagnosed with endometriosis, which means that they are subjected to the potential risks of surgery despite not having the disease 246. It has been reported that black, red, small, superficial, and early-stage lesions are amongst the hardest lesions for gynecological surgeons to directly visualize and diagnose. Also, the visual identification of endometriosis during laparoscopy can be challenging when heterogeneous, atypical, and inaccessible lesions are present 69. Operative laparoscopy is a surgical procedure which is used to ablate or remove endometrial implants, adhesions, cysts, and other 44 abnormalities identified during laparoscopy to relieve symptoms and improve fertility in women with endometriosis 246, 259. This method has a higher risk of complications compared to diagnostic laparoscopy, especially in cases requiring extensive dissection. Endometriosis lesion excision provides a histologic diagnosis and potentially obtains a greater depth of treatment compared to ablation. However, it may require greater surgical skill and has a higher risk of injuring adjacent structures. On the other hand, advocates of ablation methods argue that it may achieve better and deeper tissue penetration due to high energy and minimize adhesion formation. Ablation has limitations in adequately determining the full extent of the lesion resected and may cause tissue charring and thermal damage 259. Another main drawback of operative laparoscopy is that about 40-50% of women experience a recurrence of endometriosis within 5 years after surgical removal of the lesions 246. Despite of the value of histological assessment of biopsies in the diagnosis of endometriosis, diagnostic challenges can happen when the glandular and stromal components of lesions have an atypical microscopic appearance. Performing MRI and TVUS can help surgeons to gather useful information about the relative location of lesions and remove them more efficiently 246, 258, 259. 1.8 Endometriosis Diagnosis: Emerging Diagnostic Methods In recent years, great efforts have been made to develop minimally invasive or non- invasive methods to diagnose endometriosis. As explained in sections 1.7.2 through 1.7.4, currently acceptable methods to diagnose endometriosis are either unable to detect various types of lesions or are invasive and can cause further complications, revealing an immediate need for developing new strategies to diagnose all lesion types while reducing potential surgical risks. The prevalence of diagnosed endometriosis in reproductive-age women is about 6 to 10%. However, the true extent of this condition is likely higher due to a significant diagnostic delay of 7 to 15 years 260, 261, further emphasizing the need for extensive research in the field of endometriosis 45 diagnosis. 1.8.1 Imaging, Nanotechnology, and Targeting Endometriotic Lesions Nanocarriers show great potential as vehicles for delivering drugs and imaging agents to targeted locations. They can make insoluble compounds soluble, protect delicate cargo from degradation, extend drug circulation time, and improve drug delivery precision while reducing systemic toxicity through passive or active targeting. Nanotechnology has shown promising advancements in addressing pathological conditions, especially in cancer research, by enabling precise diagnostics, targeted drug delivery, and innovative therapeutic approaches for improved cancer detection, treatment, and management. Although the application of nanocarriers is an emerging field in endometriosis research, there are various studies which utilized them as innovative imaging agents and treatment options 262, 263. Nanomedicines used in endometriosis research include therapeutics for pain treatment 264, antioxidant therapy 265, local therapy 266, photothermal therapy 267, 268, gene therapy 269-271, antiangiogenic therapy 265, 271, and immunotherapy 272. Imaging is an established method to diagnose endometriosis and performing it prior to laparoscopy can guide surgeons to better locate and remove lesions. Even though MRI and ultrasonography already have dedicated untargeted contrast agents, developing targeted imaging contrast agents can help detect lesions through imaging. In recent studies, nanocarriers have been utilized as preclinical imaging agents to better understand the pathogenesis of endometriosis 273, or to visualize lesions using diverse imaging modalities such as fluorescence imaging 268 and MR imaging 252, 274. One approach to better detect and deliver therapeutics to the lesions is by exploiting lesion-specific and/or overexpressed ligands or receptors. In the field of oncology and nanomedicine, there are two main approaches for delivering therapeutics or imaging contrast agents to tumor sites that include passive and active targeting. Passive targeting, which is dependent on physiochemical properties of therapeutics or imaging agents, refers to the 46 accumulation of these agents (e.g., nanoparticles) in tissues solely based on enhanced permeability and retention (EPR) effect, which happens due to tumor leaky vasculature and reduced lymphatic drainage. Active targeting relies on targeting a tumor receptor with a molecule (ligand) that is designed to bind specifically to receptors on tumor cells, which can facilitate the selective delivery of therapeutic or imaging contrast agents to the tumor site for potential treatment or imaging purposes. This promising approach enhances selective accumulation in targeted areas and decrease accumulation in off-target organ, leading to enhanced specificity and reduced systemic toxicity 275. Targeted imaging agents can elucidate the molecular mechanisms responsible for disease and have the potential to enhance image contrast between healthy and pathological tissue and to enhance the specificity and sensitivity of detection. Enhancing the targeted localization of a contrast agent increases its concentration in the region of interest, resulting in a more pronounced contrast between the target area and the surrounding tissues. Also, these contrast agents can significantly reduce background interference across various imaging techniques, such as optical imaging 276-280. Similar to this strategy, various studies have focused on identifying and utilizing specific or overexpressed ligands or receptors in endometriotic lesions for targeted diagnosis or treatment purposes 262. Schwager et al. reported that in vivo delivery of Alexa750 labeled F8 antibody in a mouse model of endometriosis can target endometriotic vasculature and selectively accumulate in lesions. Figure 1.9 shows the near-infrared fluorescence images 24 hours post antibody injection, depicting specific accumulation of F8 antibody in contrast to the negative control antibody (Alexa750 labeled F16) showing no significant accumulation in the lesions. 47 Figure 1.9 In vivo targeting performance of Alexa750 labeled SIP(F8) antibody in the syngeneic mouse model of endometriosis. Note that white circle indicates endometriotic lesion area (A) Near-infrared fluorescence imaging. Endometriosis mice were injected with SIP(F8)- Alexa750 or the negative control antibody SIP(F16)-Alexa750. Near-infrared fluorescence imaging analysis was carried out 24 h after injection. (B) Ex vivo detection of SIP(F8)- Alexa750. To show blood vessels, a double staining with an anti-CD31 antibody was carried out. SIP(F8) is localized around blood vessels in endometriotic lesions. (SPI: in small immunoprotein format), Scale bars = 100 µm. Reproduced with permission of Oxford University Press/Human Reproduction 281. Immunofluorescence staining results from this study confirmed the presence of specific F8 antibody in endometriotic lesions and its colocalization with blood vessels of the lesion 281. Table 1.1 summarizes the most explored endometriosis-specific and/or overexpressed targets. These targets were either used to increase the delivery efficiency of imaging contrast agents or therapeutics utilizing active targeting approach. 48 Table 1.1 Endometriosis-related specific targets, their corresponding ligands, delivery agents, and route of delivery. Target Binding ligand Delivery agent EphB4 TNYL peptide Hollow gold nanospheres (HAuNS) F8 antibody Alternative spliced extra domain A (EDA) of fibronectin Summary Route of delivery IV injection IV injection In a mouse model, EphB4, a marker of high neovascularization, exhibited elevated expression in the endometriotic lesions and congestive uterus, while the expression was negative in the normal uterus. TNYL peptide which has specific affinity for the EphB4 receptor was conjugated to HAuNS and was used to actively target endometriotic lesions. Specific photothermal ablation therapy using TNYL-HAuNS significantly reduced lesion volume 267. Immunohistochemistry and immunofluorescence techniques revealed a robust vascular expression of splice isoforms of fibronectin in human endometriotic lesions. In vivo administration of F8 conjugated with interleukin-10 and a near infrared dye showed selective accumulation of the probe in lesions of syngeneic mouse model as a theranostics agent 281. Antibody-mediated targeted delivery of interleukin-4 in a mouse model of endometriosis helped targeted accumulation of therapeutics and inhibition of lesion growth by impairing adhesion and vascularization 282. CD44 Hyaluronic acid (HA) Polyethylenimine- grafted chitosan oligosaccharide (CSO-PEI) IV injection The theranostic agent was created by conjugating small interfering RNA (siRNA) and a near-infrared dye to the targeted delivery vehicle. The addition of hyaluronic acid (HA) resulted in increased accumulation of the agent, while siRNA effectively reduced the size of lesions in rat model of endometriosis 269. 49 Table 1.1 (cont’d) Chemokine receptor type 4 (CXCR4) Stromal cell- derived factor- 1 (CXCL12) L1 peptide polyplexes CD10 Immunohisto- chemistry PL1 peptide Tenascin C domain C (TNC-C), Fibronectin Extra Domain-B (Fn-EDB) Silver nanoparticles functionalized with synthetic PL1 peptide Low density lipoprotein (LDL) receptors LDE with cholesterol [14C]- oleate LDL-like nanoemulsion, containing lipid nanoparticles (LDE) IV injection CXCR4 is a receptor for CXCL12, expressed in the endothelium of angiogenic vessels, which is upregulated during neoangiogenesis, playing a role in regulating the recruitment of endothelial progenitor cells. Studies have shown that the CXCL12/CXCR4 ligand-receptor pair directly contributes to the development of endometriosis and influences the growth and invasion of ectopic endometrial cells. Administration of CXCR4 receptor-targeted cross-linking peptide L1 was employed to deliver anti-VEGFA siRNA to lesions in a subcutaneous endometriosis rat model, resulting in decreased growth of lesions 271. A combined double-positive expression profile of HOXA11 and CD10, which were expressed in 98% and 91% of endometriosis lesions respectively, was found to be highly sensitive for identifying ectopic endometrial tissue 283, 284. It has been hypothesized that nanocarriers conjugated to CD10 binding ligands can target endometriotic lesions 285. In vitro Nanoparticles conjugated with PL1 IV injection showed specific internalization in 12Z immortalized endometriotic epithelial cells. Targeted nanoparticles loaded with a potent antimitotic drug decreased the viability of endometriotic cells in 2D and 3D cultures 286. Rapidly dividing cells in various proliferative pathological conditions undergo high LDL uptake for membrane synthesis. Radioactive LDE were injected into 14 patients with endometriosis. The overall uptake was lower in lesions compared to endometrium 287. 50 Exploiting overexpressed markers in endometriotic lesions can also help image-guided surgeries and increase accuracy of lesion resection. IRDye 800CW conjugated to bevacizumab, targeting overexpressed VEGF-A in endometriotic lesions, was investigated for its potential use as a targeting molecule in fluorescence-guided surgery of endometriotic lesions (clinicaltrials.gov, NCT02975219). 1.8.2 Biofluids and Circulating Biomarkers In addition to the search for specific and overexpressed receptors in endometriotic lesions as potential biomarkers for diagnosis and treatment of endometriosis (discussed in section 1.8.1), various biomarkers present in blood, urine, and peritoneal fluid have been studied extensively for their potential to screen and diagnose endometriosis noninvasively. These biomarkers include glycoproteins, tumor markers, oxidative stress markers, miRNAs, long non-coding RNAs, adhesion molecules, soluble factors and receptors, matrix metalloproteinases, and immunological/inflammatory markers 260, 288. Serum glycoproteins, commonly utilized in medicine for the detection and evaluation of malignancies, have been extensively studied as diagnostic tools for endometriosis. Over two decades ago, a meta-analysis showed that endometriosis patients, especially those with advanced stages, had increased levels of cancer antigen 125 (CA-125), a high molecular weight glycoprotein and a well-known tumor marker used for ovarian cancer diagnosis, in their serum samples. However, its reliability as a diagnostic biomarker is limited by fluctuations across the menstrual cycle and variations in patients with different lesion types 289-291. Currently, despite its relatively low sensitivity and specificity, CA- 125 remains the only marker widely used in clinical practice as a prognostic marker of endometriosis 292. In addition to CA-125, other glycoproteins, including CA19-9 and carcinoembryonic antigen (CEA) were tested for their potential to diagnose endometriosis, but showed little diagnostic value in detecting the disease 288, 292. Recent studies have shown that 51 endometriosis may be linked to oxidative stress, characterized by the imbalance between reactive oxygen species (ROS) and antioxidants. ROS are byproducts generated during regular oxygen metabolism, regulating cell survival, proliferation, apoptosis, and inflammation. However, excessive ROS production or a compromised antioxidant defense system can lead to accumulation of these highly reactive molecules and cause damage to cellular components. It is believed that in endometriosis the backflow of endometrial fragments to the peritoneal cavity can cause an inflammatory response, which ultimately leads to the production of ROS by activated macrophages and erythrocytes via proinflammatory cytokines. Multiple studies measured oxidative stress markers, including thiol and protein carbonyl levels in blood serum and/or peritoneal fluid of women with endometriosis. Unfortunately, these markers are not specific to this condition and are subject to change in other diseases and conditions. Therefore, using oxidative stress markers alone as a diagnostic tool for endometriosis is unreliable, and further scientific studies are required to determine whether they can be used as diagnostic tests for this condition 292-295. Circulating miRNAs are another type of biomarkers that have recently gained more interest in the field of endometriosis diagnosis. It is well-established that in addition to miRNA obtained from lesions, circulating miRNAs in body fluids can detect various pathological conditions. MiRNAs are stored intracellularly, packed into extracellular vesicles, and released into circulation, where they can be carried to their recipient cells 288, 296. In recent years some studies measured concentrations of various circulating miRNAs in women with endometriosis and compared them with control populations. Some circulating miRNAs including, miR-125b, miR-451, miR-122, miR-145, and let-7 or a panel of these miRNAs showed differential expression levels making them a potential diagnostic tool 98, 102. While sequencing and microarray technology have made it possible to investigate systemic levels of miRNAs and long non-coding RNAs, due to various reasons 52 including heterogeneity of endometriosis there is currently no reliable single or panel of miRNAs that can be used as a biomarker for endometriosis with acceptable sensitivity and specificity 288, 296. 1.9 Animal Models of Endometriosis Animal models provide a valuable tool for pre-clinical research in endometriosis, including assessment and validation of new diagnostic methods and modalities, therapeutics, and potential disease-specific biomarkers. Since ethical considerations prohibit repeated laparoscopies to monitor the progression of the disease and performing controlled experiments on humans, exploiting experimental animal models can help to better understand endometriosis pathogenesis and monitor disease progression 297-299. Since women are rarely diagnosed in the early stages of this disease, animal models can be helpful in better understanding the processes involved in the development of the disease, lesion formation and establishment 300. While endometriosis occurs naturally only in humans and some non-human primates due to menstrual shedding, other animals, especially rodents, can be induced with endometriosis for research purposes. 1.9.1 Induced Models of Endometriosis - Murine Models Rodents, especially mice, are suitable preclinical models in biomedical research. Their smaller size, shorter estrous cycle and gestation, and molecularly homogeneous background allow for the study of larger groups of animals, which enhances the biological and statistical power in experimental research. The ability to do precise genetic manipulation on a mouse and its well- annotated genome enable researchers to better understand molecular mechanisms and pathways underlying the pathogenesis of endometriosis. Additionally, these animals have a well-understood reproductive system, can reproduce quickly, have low housing costs, and do not require large amounts of therapeutics for experimentation, which make them cost-effective candidates for endometriosis studies 1, 301. In contrast with human and non-human primates, rodents do not 53 develop spontaneous endometriosis due to the absence of menstruation, which prevents natural development of endometriosis and requires the artificial induction of the disease in these models 300. This, in addition to the major physiological difference between humans and rodents are the most important disadvantages of using murine models of endometriosis 297. There are two primary categories of murine models of endometriosis which are classified as heterologous and homologous/autologous models. Heterologous (xenograft) models of endometriosis involve introducing human endometrial fragments or cultured cells to immunocompromised rodents using different methods, including intraperitoneal transplantation by suturing fragments to the peritoneal wall or intestines, intraperitoneal injection of fragments or cells, or subcutaneous implantation 302. Human endometrial tissue is usually obtained from menstrual fluid, biopsies from the endometrium of women with or without endometriosis, or even ovarian endometriomas 303. Some studies have induced endometriosis using human cell lines such as immortalized human endometrial epithelial cells 304 or immortalized human endometriotic cells (12z cells) 305. This model enables researchers to study mechanisms involved in endometriosis development and test various therapeutics on human tissues. Despite this advantage, the heterologous model is rarely used to assess the immune system or the effect of immune-regulating therapeutics on endometriosis 297. Homologous models, also known as allograft models, involve implanting small pieces of uterine tissue into the peritoneal wall or injecting tissue from a donor into the peritoneal cavity of a recipient animal of the same species 306. The autologous model is another frequently used model which involves inducing endometriosis in a rodent with an intact immune system (unlike heterologous models) using its own endometrial tissue which can be helpful for better understanding of the role of immune system in the disease pathogenesis 307. In these models, animals can be ovariectomized and receive exogenous estrogen treatment in order to minimize 54 variations in the estrous cycle 300. Researchers have exploited different knockout and transgenic murine models to investigate the functions of the immune system, hormones, and molecular pathways in endometriosis. Genetically engineered animal models offer the opportunity to manipulate specific genes involved in endometriosis, allowing them to study the consequences on disease development and progression 115, 308, 309. Additionally, these models can be designed to express fluorescent proteins or luciferase in specific tissues which helps the early detection of small lesions in animals used in pre-clinical research 310. Figure 1.10 depicts both homologous and heterologous rodent modes of endometriosis which can be induced with either surgical implantation or intraperitoneal injection. Note that the autologous model is similar to the homologous model with the difference that, a fragment of rodent uterine horn is excised and introduced to the same animal (donor and recipient is the same animal). 55 Figure 1.10 Diagram illustrating the different murine models for studying endometriosis. (A) Summary of the homologous-murine models showing the non- and fluorescence models. Note that autologous model is similar to this model with the difference that donor and recipient is the same animal. GFP, green fluorescence protein; Luc, luciferase. (B) Heterologous murine model. HET, human endometrial tissue 300. Reproduced with permission from Elsevier via the Copyright Clearance Center. 1.9.2 Spontaneous Models of Endometriosis - Non-human Primate Models Cynomolgus monkeys, rhesus macaques, and baboons are considered ideal animal models to study endometriosis due to their similar reproductive anatomy to humans, cyclic menstrual patterns, and spontaneous development of the disease with ectopic lesions which are similar in appearance and location to humans. While not all primates develop spontaneous endometriosis, the frequency of occurrence appears to increase with time in captivity, possibly due to fewer 56 pregnancies resulting in more consecutive menstrual cycles and increased exposure to retrograde menstruation 297, 299. Although spontaneous development of endometriosis is the closest model to humans, its low incidence, development, and progression have led to the development of various methods to artificially induce ectopic lesions 300. 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Induction of endometriotic nodules in an experimental baboon model mimicking human deep nodular lesions. Fertility and sterility. 2013;99(3):783-9. e3. 314. D'Hooghe TM, Bambra CS, Suleman MA, Dunselman GA, Evers HL, Koninckx PR. Development of a model of retrograde menstruation in baboons (Papio anubis). Fertil Steril. 1994;62(3):635-8. 81 CHAPTER 2 DETECTION OF ENDOMETRIOSIS LESIONS USING GD-BASED COLLAGEN I TARGETING PROBE IN MURINE MODELS OF ENDOMETRIOSIS 82 Publication Notice The following dissertation chapter describes the non-invasive detection of endometriotic lesions using a Gd-based type I collagen targeting probe in murine models of endometriosis. The following chapter titled " Detection of endometriosis lesions using Gd-based collagen I targeting probe in murine models of endometriosis " has been accepted for publication in Molecular Imaging and Biology (2023). The authors of this article are Nazanin Talebloo, Maria Ariadna Ochoa Bernal, Elizabeth Kenyon, Dr. Christiane Mallett, Dr. Asgerally Fazleabas, and Dr. Anna Moore. I gratefully acknowledge their significant contribution to the research presented in this thesis. 83 2.1 Introduction Endometriosis is a common chronic, inflammatory, and hormone-dependent gynecological condition, which is generally characterized by the presence and growth of ectopic, endometrial- like tissue outside of the uterine cavity. This disease mainly affects about 10% of women during their reproductive years 1, 2. Endometriosis is associated with a variety of symptoms, including pain and related fertility problems which are discussed in detail in Chapter 1. Patients with endometriosis can experience one or more symptoms or they can be completely asymptomatic 3-5. Distinguishing endometriosis from other conditions such as pelvic inflammatory disease, and irritable bowel syndrome is challenging and represents an unmet clinical need. The inability to make this distinction, attributing pain symptoms to the normal cyclic bleeding pain that many women experience, and inadequate understanding of the disease by healthcare professionals due to insufficient medical training on this subject are among the reasons behind the diagnostic delay amongst women suffering from endometriosis which can negatively impact their physical, social, and mental well-being in the long run 6-10. Endometriotic lesions include endometriomas, all forms of peritoneal lesions, and deep infiltrating lesions (DIE). Red lesions have higher levels of vascularization, while white lesions are red lesions which have undergone through inflammation and fibrosis processes over time 11, 12. These lesions can be found mainly in the pelvic area, including ovaries, ligaments, bladder, and peritoneal surfaces which are normally categorized into four stages (minimal to severe) based on the extent of the disease. However, the severity of symptoms does not necessarily correlate with this staging system 1, 13, 14. Multiple endometriosis classification systems exist which are normally governed by complex factors, including color, depth, anatomical location and size of the lesions 15, 16. The heterogeneity of endometriotic lesions and adhesions in their appearance, location, 84 genetics, epigenetics, and symptoms (as previously discussed in Chapter 1), present significant challenges in the diagnosis and treatment of this condition. Diagnostic laparoscopy is the gold standard in endometriosis diagnosis which is normally followed by histological verification. However, since this procedure is invasive, other non- invasive diagnostic methods in conjunction with physical examination and assessment of patient’s medical history are preferable if there is no intention to remove lesions surgically 17, 18. Although developing non-invasive and low-invasive methods to diagnose endometriosis represents a challenge, the diagnostic potential of various genetic tests, biomarkers and imaging techniques have been evaluated 13, 19-23. Imaging techniques that are currently used to diagnose endometriosis include sonography and magnetic resonance imaging (MRI). Ultrasound sonography is the first- line imaging modality in assessment of endometriosis 24, 25. Transvaginal ultrasound (TVUS) can give a more detailed image of the anatomy compared to the initial ultrasonography of pelvis area. Although ultrasound and MRI are two imaging modalities that are commonly utilized in the detection of endometriosis, these modalities, as explained in Chapter 1, are unable to detect various types of lesions, and lesions in specific locations which can cause false negative results 18, 26. These imaging techniques can help complete lesion mapping before operative laparoscopy which can help excision or ablation of lesions 24. Also, due to limitations presented by ultrasound and MRI, patients with negative imaging results must still be subjected to laparoscopy to obtain a definite diagnosis 17. Developing targeted contrast agents for MRI can increase the signal difference between the lesions and surrounding tissues, especially for detection of non-pigmented lesions without intrinsic T1 hyperintense signal 27, 28. Despite several published studies describing the development of targeted contrast agents for imaging of endometriosis, as of today, no clinically approved agents are available 29-31. Availability of such agents would significantly aid in precise 85 diagnosis of this disease including the location of the lesions. Current endometriosis specific or overexpressed targets were discussed in Chapter 1. Ectopic endometriotic lesions are wounds undergoing repeated cycles of tissue injury and repair (ReTIAR) stimulating microenvironment-mediated epithelial–mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), and fibrosis 32-34. The consistent presence of fibrosis in all lesion forms points to endometriosis as a fibrotic condition characterized by excess deposition of collagens, primarily type I collagen (detailed mechanism was discussed in Chapter 1) 9, 33-36. Endometriosis is a complex and heterogeneous disease, with a range of symptoms and genetic variations that can make it challenging to diagnose and treat effectively. However, despite its heterogeneous nature, it has been suggested that the consistent presence of fibrosis in endometriotic lesions can serve as a molecular hallmark of the disease. Importantly, it was previously shown that immunoreactivity toward type I collagen in the ectopic lesion sections collected from a mouse model of endometriosis was elevated as the disease progressed 37. Therefore, the presence of fibrosis in endometriotic lesions makes it an attractive biomarker for targeting by affinity ligands incorporated in contrast agents which can reduce the challenges posed by the heterogeneity of the disease, offer a better understanding of endometriosis pathogenesis, and facilitate the development of novel diagnostic and treatment methods 33, 35. Four main pathogenetic models proposed to explain the presence of myofibroblasts and the development of fibrosis in endometriosis are illustrated in Figure 2.1 35. Figure 2.1 A demonstrates that the presence of ectopic endometrium triggers a response in the local environment surrounding the lesion, leading to the formation of fibrotic inclusions around the affected tissue. Figure 2.1 B shows that activated platelets can promote EMT, FMT, and SMM, resulting in 86 increased cell contractility, collagen production and ultimately to fibrosis, via the release of TGF- β1 and the induction of TGF-β/Smad signaling pathway 32. Figure 2.1 C suggests that larger amounts of retrograde menstrual effluent can cause human peritoneal cells to undergo a process similar to EMT. Peritoneal cells located near endometriotic lesions exhibit a significant upregulation of TGF-β mRNA expression compared to cells found in areas distant from the lesions and some studies have shown that a part of mesenchymal cells can be of peritoneal origin rather than endometrial origin. Exposure of mesothelial cells to TGF-β1 results in increased lactate production and acidification of the local environment which activates the TGF-β ligand, leading to the induction of FMT 38-41. Figure 2.1 D depicts that the increased stiffness of the fibrous tissue in deep infiltrating endometriosis (DIE) can induce EMT. Endometrial stromal cells derived from affected patients have the ability to differentiate into myofibroblasts and produce type I collagen even without TGF-β1 treatment. This increased myofibroblast collagen production can contribute to enhanced stiffness within the matrix, ultimately leading to the progression of a fibrotic environment in DIE lesions over time 42. 87 Figure 2.1 Main pathogenetic models proposed to explain the presence of myofibroblasts and the development of fibrosis in endometriosis. Epithelial to mesenchymal transition, fibroblast-to- myofibroblast transdifferentiation, increased collagen production and ultimately fibrosis have been suggested to be triggered in endometriotic cells by the presence of stimulating factors (e.g. Tranforming Growth Factor (TGF) β1 [B and C], platelets [B] or a stiff tissue matrix [D]). Similar phenomena in other tissues (A, surrounding connective tissue or C, mesothelial barrier) have been also proposed. Reproduced with permission of Oxford University Press/Human Reproduction 35. Previous studies aimed at imaging fibrosis in other pathologies have identified a peptide (GQ1W2H3C4T5T6R7F8P9H10H11Y12C13L14Y15Bip16) directed against human type I collagen using phage display 43. A glycine was introduced at the N-terminus as the original phage-derived peptide had an N-terminal glutamine that underwent cyclization to pyroglutamate 44. Later, in order to introduce the Gd chelates to the peptide, an alanine walk was performed to identify amino acids whose side chains were critical for collagen binding. The alanine walk demonstrated that the systematic replacement of tryptophan, proline, phenylalanine, and the tyrosine closest to the C- terminus with alanine dramatically reduced the collagen binding affinity 45. In Fig 2.2 amino acids 88 W2, C4, F8, P9, C13, and Y15 are amino acids whose side chains are critical for binding 45, 46. After conducting an alanine scan, further investigation was carried out through a lysine walk. In this study, two Gd-DTPA chelates were incorporated using Gd-ITC-DTPA chemistry. One chelate was introduced at the N-terminus, while the second was attached to the ε-NH2 group of lysine. Lysine was introduced at each position where alanine substitution was tolerated. While the chelate was well-tolerated at all tested positions, including the C-terminus, peptide with Arg7 substitution exhibited the highest level of binding. Reduction of the cysteine disulfide markedly reduced collagen binding (<15 % bound), indicating that the cyclic structure was require for efficient binding 44. This type I collagen binding peptide consists of 16 amino acids with 10 of them producing the cyclic part between two cysteines. Biphenylalanine (Bip) at the amidated C terminus of the peptide is believed to increase the type I collagen binding ability (Figure 2.2), with a Kd = 1.8 µM. The peptide was synthesized using conventional solid-phase techniques and functionalized with three Gd(DTPA) moieties through thiourea linkages to improve the sensitivity of the contrast agent (termed EP-3533). Relaxivity of EP-3533 was reported as 16.2 mM-1s−1 at 4.7T MR 43, 45, 47. Also, near the common imaging field of 1.5 T, the relaxivity of EP-3533 is five times higher than Magnevist per Gd atom and 15 times higher per molecule 43. EP-3533 demonstrated successful targeting of fibrotic content in tissues of various pathological conditions using MR imaging at field strengths of 4.7 T 43, 3 T 48, and 1.4 T 49, 50. In previous studies, EP- 3533 has been used for detection and staging of fibrotic lesions in animal models of liver fibrosis 50-53, pulmonary fibrosis 54, cardiac fibrosis 47, and pancreatic cancer 36. Systematically replacing amino acids with their D-amino acid counterparts led to a significant decrease in the activity of the peptide, particularly when cysteine amino acid was substituted. This observation facilitated the design of the non-binding isomer (EP-3612), which exhibited a considerably higher dissociation 89 constant (Kd) of 400 µM when compared to EP-3533 43, 45. Isomer EP-3612 is identical to EP- 3533 with the exception that the chirality of one cysteine residue is inverted (D-Cys) (Figure 2.2). These two peptides have similar relaxivity at a given field/medium. Figure 2.2 Collagen-targeted contrast agents; L-amino acids are designated by one letter code, except where noted; Gd chelates are appended through the N terminus, through branched Lys- Gly residues at the N terminus, and through a Lys side chain within the cyclic portion of the peptide 43. Copyright Wiley-VCH GmbH. Reproduced with permission Caravan, Peter, et al. "Collagen‐targeted MRI contrast agent for molecular imaging of fibrosis." Angewandte Chemie International Edition 46.43 (2007): 8171-8173. In this study we utilized, for the first time, EP-3533 for detection of fibrotic endometriotic lesions using MRI in two murine models of endometriosis. As a result of our studies, we demonstrated that the accumulation of EP-3533 in fibrotic endometriotic lesions in both suture and injection models was significantly higher compared to its accumulation in control tissue (mouse leg skeletal muscle). Also, the specificity of EP-3533 for endometriotic fibrosis was validated by comparison with а control non-binding isomer (EP-3612). This study serves as the first demonstration of the utility of EP-3533 for magnetic resonance imaging of endometriotic lesions. We believe that this agent can be further investigated as a 90 potential vehicle for delivery of therapeutics targeting various signaling cascades that initiate endometriosis development 55-58. 2.2 Results 2.2.1 Dynamic EP-3533 Enhanced Magnetic Resonance Imaging To determine whether the EP-3533 probe could be used for probing endometriotic lesions based on collagen targeting, we performed dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). We expected that in fibrotic lesions with overexpressing collagen, the binding of the contrast agent would lead to a longer signal enhancement and a slower lesion signal washout. As evident from Figures 2.3 a and c, the endometriotic lesions in both suture and injection models appeared relatively homogeneous in the pre-injection T1-weighted MR images. Fifty-five minutes post- EP-3533 injection there was a relatively rapid signal enhancement in the lesion lining (Figures. 2.3 b and d) during the first 6 minutes in both suture and injection models which overall led to an average of 21 ± 4.6% and 16.8 ± 6.2% signal enhancement, respectively (Figures 2.4 a and b). The data from the control muscle tissue from both endometriosis models showed a rapid increase (approximately 5-7% signal enhancement) after the injection followed by the washout that did not show an overall significant enhancement 55 minutes post-injection (3.9 ± 1.5% and 3.3 ± 0.66% for suture and injection models respectively; Figures. 2.4 a and b). Comparison of DCE-MR images of control mice pre- (Figures 2.3 e and g) and post- EP-3612 injection (Figures. 2.3 f and h) showed significantly lower signal enhancement 54 minutes post probe injection in both models (0.2 ± 0.5% and 1.9 ± 2.4% signal enhancement for suture and injection models respectively, Figures 2.5 a and b, compared to the experimental group injected with EP-3533 in both endometriosis models. 91 Figure 2.3 Representative T1-weighted magnetic resonance images of suture and injection mouse models of endometriosis injected with collagen type I targeting probe (EP-3533) and control non-binding probe (EP-3612). a, c) Baseline, before EP-3533 injection and b, d) 55 minutes post EP-3533 injection in suture and injection endometriosis models respectively. e, g) Baseline, before EP-3612 and f, h) 55 minutes post EP-3612 injection in suture and injection endometriosis models respectively. Red dotted ovals: endometriotic lesions. Red arrows point to endometriotic lesion lining. Note the enhancement of lesion lining 55 minutes post injection (red arrows) in the group injected with EP-3533. 92 Figure 2.4 DCE MRI analysis of signal enhancement of endometriotic lesions (blue) and muscle tissue (orange) from mice injected with EP-3533 in a) suture and b) injection models. Percentage of MR signal enhancement on y-axis was normalized to the average of baseline signal. The probe was injected at minute 5 and mice were imaged for 55 minutes. Error bars are standard deviations of signal enhancements averaged within the group of mice for each experimental model. 93 Figure 2.5 DCE MRI analysis of signal enhancement of endometriotic lesions (blue) and muscle tissue (orange) from mice injected with EP-3612 in a) suture and b) injection models. Percentage of MR signal enhancement on y-axis was normalized to the average of baseline signal. The probe was injected at minute 6 and mice were imaged for 54 minutes. Error bars are standard deviations of signal enhancements averaged within the group of mice for each control model group. 94 2.2.2 Lesion Location Validation In order to confirm the presence of the lesions detected with MR imaging, ex vivo fluorescence imaging was performed. After the last MR image acquisition mice were euthanized and the exposed peritoneum was photographed in both suture and injection models (Figures 2.6 a and d; enlarged images are shown in Figures 2.6 b and e). To detect and confirm the presence and location of all GPF-expressing lesions, including smaller lesions that might not be easily visible with the naked eye, we performed fluorescence imaging that detected progesterone-positive tissues including uterine horn and endometriotic lesions (Figures 2.6 c and f). Imaging of excised lesions confirmed GFP expression in both suture and injection models (Figures 2.7 a and b). These findings confirmed the presence and location of the lesions that were identified by MRI. To confirm the presence of endometriotic lesions microscopically, collected lesions induced with both suture and injection methods were stained with H&E showing the typical presence of endometrial glands and stroma (Figures 2.8 a and b). 95 Figure 2.6 Representative ex vivo images of endometriotic lesions. Photographs (a, d) and magnified images of the lesions (b, e) taken after opening the peritoneal wall in suture and injection endometriosis models respectively. Ex vivo fluorescence imaging showing progesterone positive lesions expressing GFP in suture (c) and injection (f) endometriosis models. Green doted oval: endometriotic lesions. Black dotted line: border of the lesions. Note that in the injection model (c) bladder was covered with black paper to better visualize the signal from the lesions. Scale bar= 5mm. 96 a b lesions muscle lesions muscle Figure 2.7 Representative images of excised GFP-expressing lesions from a) suture and b) injection mouse models. a b 100 um 100 um Figure 2.8 Representative Hematoxylin and Eosin (H&E) staining images of lesions obtained from a) the suture model and b) the injection model of endometriosis, demonstrating the presence of glands and stroma. Yellow arrow shows gland and black arrow shows stroma. 97 2.2.3 Histological Assessment of Fibrosis Masson’s trichrome stain is used to assess and visualize the extent of fibrosis in both human biopsies and animal models of human fibrotic disease. Here, we used Masson’s trichrome staining to evaluate the extent of fibrosis in endometriosis mouse models. The results showed a relatively large blue area of the collagen-rich fibrotic regions throughout sections from models induced with either suture (Figures 2.9 a and b) or injection (Figures 2.9 c and d) methods, demonstrating overexpression of collagen as a suitable target for EP-3533 probe. It is noteworthy that most of the lesions especially those induced by the suture method were cystic and filled with liquid and showed collagen expression in their lining. Figure 2.9 Representative images of Masson trichrome staining of endometriotic lesion tissue sections demonstrating the relative extent of fibrosis. a, b) Lesions from suture model. c, d) Lesion from injection model. Blue - collagen fibers, red - cytoplasm, muscle, black - cell nuclei. Magnification bar = 400 µm (a, c); 100 µm (b, d). Lesion in (c) is represented by the yellow dotted circle. Note that the collagen content in the suture model is more pronounced than in the injection model. 98 2.2.4 Quantification of Tissue Gadolinium Content To confirm that the enhancement observed on MR images after the injection of EP-3533 was caused by Gd, we collected tissue samples from both experimental and control groups and analyzed them for Gd content by ICP-OES. The results showed that the Gd concentration in endometriotic lesions of experimental group was 26.7 ± 10.0 and 20.6 ± 7.7µg Gd/g dry tissue in suture and injection models, respectively which was significantly higher than that in skeletal muscle of the same groups (2.2 ± 3.0 and 4.2 ± 1.7µg Gd/g dry tissue for suture and injection models respectively, p< 0.05, Figures 2.10 a and b). Similarly, injection of a non-binding EP- 3612 probe resulted in a low accumulation in the lesions and muscles of both animal models (2 ± 1.6 and 3.2 ± 1.8µg Gd/g dry lesion tissue and 1.44 ± 0.28 and 1.15 ± 0.27µg Gd/g dry muscle tissue in suture and injection models respectively, Figures 2.10 a and b). Our data demonstrate that the only tissues that accumulated a significant amount of Gd were endometriotic lesions from both animal models. a Figure 2.10 ICP-OES analysis of average gadolinium content in lesion and muscle tissues after injection of EP-3533 and EP-3612 probes in a) suture and b) injection models. Gd tissue content was normalized to the dry weight of the tissue. Results are presented as means ±SD. Note that Gd content was significantly higher in endometriotic lesions of mice of both suture and injection models injected with EP-3533 compareд to that in the lesions from the animals injected with EP-3612 or in the muscle tissues (p<0.05). 99 2.3 Discussion A better understanding of endometriosis is needed to successfully develop new non- invasive diagnostic methods 16and effective drugs. While imaging is currently used as a non- invasive method to detect endometriotic lesions 24, it does not allow for specific and reliable diagnosis. The main reason is heterogeneity of endometriotic lesions and the paucity of suitable molecular biomarkers. In our search for a consistent biomarker, we focused on a fibrotic aspect of endometriosis, and investigated whether imaging probes targeting type I collagen in ectopic endometriotic lesions could be used for its detection. Fibrosis is defined by the overexpression of collagen and is considered as one of the hallmarks of the disease 59, 60. In this study a Gd-containing type I collagen binding probe (EP-3533) previously used for imaging fibrosis in multiple animal models 36, 43, 47, 50-54 was evaluated for imaging endometriosis. EP-3612, a non-binding isomer, which only differs from EP-3533 by the chirality of one cysteine residue, thereby significantly reducing its affinity for collagen 43 was used as control. Both EP-3533 and EP-3612 were administered at a dosage of 0.01 mmol/kg. This dosage selection was based on prior studies that effectively utilized EP-3533 to specifically target and evaluate fibrotic conditions. 36, 49, 54. To perform these experiments, two mouse models of endometriosis with lesions induced 2 months prior to imaging were used. Mice were dynamically imaged with MRI T1 FLASH sequence pre- and post-injection of the probes. This was achieved by the acquisition of a series of baseline images without contrast enhancement, followed by a series of images acquired over time during and after the administration of the contrast agent. The rationale behind using the older stage lesion in this proof-of-principle study was based on recent studies demonstrating that the fibrotic component of lesions, especially type I collagen, tends to increase over time and as the disease progresses in both human and baboon models of endometriosis 32, 35, 37. While endometriosis staging systems are complex and multifactorial 15, it is generally observed that higher levels of fibrotic content develop 100 in the later stages of the disease. Since one of the main symptoms of endometriosis is pain, which is often mistaken for menstrual cramps, many patients seek medical advice years after disease initiation, which suggests a need for a probe which can identify established lesions. Here, two endometriosis induction methods, namely the suture method and the injection method, were used. Our goal was to utilize two well-established models commonly employed in preclinical studies 61. By doing so, we aimed to facilitate future investigations, both within our research group and among other interested researchers regarding the use of EP-3533 targeted probe in detecting, imaging, and comprehending the pathogenesis of endometriosis within any of these two endometriotic models of choice. Also, as opposed to the injection model with random lesion locations, which is situation much closer to the clinical scenario, the suture model has the benefit of precise lesion localization, which can be helpful in identifying lesions through imaging in the proof-of-concept studies. It was demonstrated in our studies that detecting lesions in both models using EP-3533 was feasible. Mice in both models received estrogen injections exclusively for a period of 3 days prior to inducing lesions in order to synchronize the uterus. Following the inoculation, the animals were only subjected to normal ovarian hormone levels that are associated with the different stages of the estrous cycle. This mimics the natural hormonal fluctuations experienced by women who are not undergoing any form of suppressive hormone therapy. In both endometriosis models, MR images showed an increase in signal intensity of lesion lining which appeared brighter compared to the signal before injection in group injected with EP-3533. Results from DCE-MRI showed a relatively fast enhancement of normalized signal intensity from the lesion linings after EP-3533 injection in both suture and injection model lesions. This initial signal enhancement can be described as a nonspecific enhancement due to the excess probe in the circulation. In the suture model as the unbound probe clears out gradually and decreases the signal 101 intensity, the specific binding of the probe to its target simultaneously increases the signal. Here we did not observe a significant difference in the signal intensity from minute 5 post-injection to minute 55 which might be because of the same rate of unbound probe washout and specific binding. This can ultimately create an equilibrium which can be seen as a relatively flat line in the MR signal enhancement plot after the completion of both washout and targeted accumulation. As a result, the signal experiences no further significant change and reaches a plateau. In the injection model as the unbound probe clears out gradually and decreases the signal intensity, the specific binding of the probe to its target simultaneously increases. Here the unbound probe washout was higher compared to specific binding which leads to lower final signal enhancement compared to the suture model. This can be due to the fact that there was a higher fibrotic content in the suture model compared to the injection model (Fig. 2.9 c and d) and an overall lower number of lesions for MR analysis in the injection model. The large standard deviation of signal enhancement in both models, especially post probe injection can be attributed to the heterogeneous nature of the lesions in terms of type I collagen content. Regardless, quantitative ICP-OES analysis demonstrated significantly higher Gd content in endometriotic lesions in both models after EP-3533 injection compared to accumulation in muscle tissues or in both lesion and muscle tissues after the injection of EP-3612 non-binding isomer. Histological analysis on harvested endometriotic lesions from these mice showed the presence of a relatively high collagen content, especially in the suture model. This agrees with previous studies showing that collagen content tends to increase in endometriosis over time 37. The majority of animal models used to study endometriosis involve the induction of lesions using endometrial fragments that are not properly separated from the surrounding myometrium. Consequently, in some cases, the myometrium constitutes a significant portion of the resulting lesions, which does not accurately reflect the composition of human 102 endometriotic lesions 62. In both endometriosis models used in this study, the myometrium was included in the endometrial tissue biopsies used for the induction of lesions. However, it was chopped into very fine pieces to produce the lesions. Also, based on Masson's trichrome stained images of lesions (Fig. 2.9), despite the significant presence of collagen in endometriotic lesions, we do not see evidence of distinct myometrial tissue around lesions or collagen within the myometrial tissue and the lack of separation between myometrium and endometrium is unlikely to affect the accumulation of EP-3533 in lesions. We need to acknowledge that besides endometriotic lesions, EP-3533 could non-specifically accumulate in other organs which was observed in previous studies 43, 63. To reduce this effect other collagen type I binding probes such as CM-101 with better pharmacokinetics are being developed and studied 64. Furthermore, 68Ga- labeled version of EP-3533 is currently being evaluated in clinical trials for type I collagen deposition in idiopathic pulmonary fibrosis (NCT05621252), so that clinical translation to the patients with endometriosis could be significantly simplified. Given the impracticality of conducting these experiments on women, this study using murine models of endometriosis can serve as an initial proof of concept. However, for future investigations, a more suitable approach could involve utilizing the baboon model of endometriosis. Baboons are phylogenetically closely related to humans, sharing many ancestral characteristics and closely resembling the reproductive system, lesion size, and location observed in women 65, 66. By employing this model, a comprehensive examination of the efficacy of EP-3533 in detecting endometriosis can be conducted. Also, it is important to note that certain surgeries and laparoscopic procedures may cause adhesions and the development of fibrotic scars 67, 68. Additionally, specific endometriotic lesions, such as cesarean scar endometriosis, may arise due to the presence of scar tissue resulting from prior abdominal surgeries 69. Given that EP-3533 was specifically developed to target type I 103 collagen, a key molecule involved in scar formation, it is essential to gain a comprehensive understanding of the signal characteristics of EP-3533 in fibrotic lesions. To obtain this understanding, it is important to compare the signal characteristics of endometriotic lesions with those of different types of surgery-associated scars found in endometriosis patients or through the utilization of non-human primate models of endometriosis. Evaluating various types of surgical- associated scars is crucial because each type of scar has distinct ratios of type I collagen as the target of EP-3533 70. Also, a patient's clinical history can greatly assist radiologists in differentiating surgically-induced scars from endometriotic lesions, particularly based on the documented anatomical location of previous surgeries 71, 72. Overall, our data demonstrated for the first time that fibrosis, which is considered a pathological feature of endometriosis, can be further investigated as an appropriate target for EP- 3533. After performing initial studies in murine models, we plan to embark on imaging endometriosis in large models such as baboons, with which we have extensive experience and which better recapitulates human disease 32, 58. Concurrently, we are developing image-guided therapies (theranostics) for endometriosis based on EP-3533 targeting aiming to inhibit signaling pathways that cause the disease 55, 57, 58. 2.4 Conclusions Considering the consistent presence of fibrosis in all endometriosis disease forms, we consider it an excellent biomarker, which could be used for diagnostic and/or therapeutic delivery purposes. Here, we evaluated a Gd-based type I collagen targeting probe (EP-3533) as an MR imaging probe and showed its utility for detection of endometriotic lesions in mouse models of endometriosis. 104 2.5 Experimental section 2.5.1 Animal Models of Endometriosis In this study we used two murine models of endometriosis that differ in the way the disease was initiated. For the injection model 8 weeks old female Pgr cre/+ Rosa26 mTmG/mTmG mice (73; n=5) were treated for 3 days with 17b estradiol (E2, Sigma-Aldrich; 1mg/ml in oil, 0.1µg/mouse/day) in order to synchronize the estrus cycle and improve lesion development. After the last E2 injection endometriosis was induced as previously described by us 74. Briefly, the lesions were established by inoculating endometrial tissue into the peritoneal cavity. To access the peritoneal cavity, mice underwent a laparotomy under anesthesia and a midventral incision (1 cm) was performed to expose the uterus and intestine. The left uterine horn was removed, placed in a petri dish with sterile PBS, opened longitudinally and cut into small fragments. The fragments suspended in 0.5 mL sterile PBS were injected into the peritoneal cavity of the same mouse from which the uterus was taken for an autologous implantation, and the abdominal cavity was gently massaged to disperse the tissue. This unique Pgr cre/+ Rosa26 mTmG/mTmG mouse model of induced endometriosis is characterized by the presence of the progesterone receptor (Pgr)-positive cells expressing green fluorescent protein (mGFP) allowing for accurate localization and visualization of the endometriotic lesions using fluorescence imaging 73. In the suture method endometriosis was induced in 7 weeks old female Pgr cre/+ Rosa26 mTmG/+ mice (n=6). The induction of a suture endometriosis model consisted of two steps. During the first step mice were ovariectomized. Briefly, the ovaries were isolated and ligated with sterile absorbable suture. A loop suture was placed between the ovary and the tip of the uterus, and the ovaries were then excised. Wound clips were removed 7-10 days post-surgery and the mice were allowed to recover for 14 days. Following recovery, the ovariectomized mice were treated for 3 105 days with 17b Estradiol (E2, 0.1µg/mouse/day) in order to synchronize the estrus cycle prior to second surgery during which one uterine horn was removed. In order to remove a uterine horn, a midline abdominal incision was made and the caudal end of the uterine horn near the uterotubal junction was cut and ligated with sterile absorbable suture. The removed uterine horn was opened longitudinally, and tissue samples were obtained using a 2 mm dermal biopsy punch. Three biopsies were sutured to the peritoneal wall (3 in each side, total of 6) using a 7-0 braided silk suture. The muscle layer was closed as one layer, then the skin as a second layer. After this procedure, the abdominal incision was closed. A small subcutaneous incision was made at the nape of the neck to create a pocket for an E2 pellet with an expected release concentration of approximately 0.13mg/day to provide a controlled source of hormone that promotes lesion growth in absence of the ovaries. Once the pellet was inserted, a wound clip was placed to close the skin. Wound clips were removed 7-10 days post-surgery. All animal studies were approved by the Institutional Animal Care and Use Committee at Michigan State University and are in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.5.2 Contrast Agent for MR Imaging EP-3533 and EP-3612 contrast agents were purchased from Collagen Medical (Belmont, MA). EP-3612 has an identical structure to EP-3533 except that one of the cysteine moieties is changed from L-Cys in EP-3533 to D-Cys in EP-3612. This change in chirality results in >100- fold loss in collagen affinity for EP-3612, however its relaxivity remains equivalent to EP-3533 36, 43, 47. 2.5.3 Magnetic Resonance Imaging In vivo MRI was performed on both models of endometriosis on average 2 months post endometriosis induction. Previously, it was shown that endometriotic lesions exhibit a gradual 106 increase in fibrotic content and reach a highly fibrotic state in 6 weeks 37. Mice were anesthetized using 1.5% to 3% isoflurane in oxygen, and tail veins were catheterized. Temperature (~35°C) and breathing were monitored and maintained throughout the experiment (SAII Small Animal Instruments, Inc, Stony Brook, NY). For imaging, mice from both groups were injected intravenously with bolus injections of the targeted EP-3533 probe (10 µmol/kg). Control mice in each group were injected with a non-binding EP-3612 probe (10 µmol/kg). Injections were followed by a 100 µL of saline flush over 30 seconds. Imaging continued for 55 minutes post injection with 1- or 2-minute temporal resolution. Images were acquired on a 7 Tesla Biospec 70/30 USR (Bruker, Billerica, MA) using an 86 mm diameter volume transmit coil and 4 channel surface array receive coil (4 × 4 cm). Fat-suppressed T1-weighted FLASH images were acquired for a total acquisition time of 1 hour, with TR=76.2 ms, TE=2.6 ms, FOV=30 × 30 mm, 9 slices, 0.5 mm slice thickness, flip angle 30° and resolution of 200 × 200 × 500 µm. Images were analyzed using Paravision 360 v3.1 software (Bruker). Regions of interest (ROI) were randomly distributed throughout the lesion linings covering most of the lining area. The ROIs of the muscle tissue were used as controls within each group of mice. The percent increase of the signal enhancement was calculated for each time point based on a region of interest and normalized to the baseline. 2.5.4 Ex vivo Fluorescence Optical Imaging After acquiring MR images mice were euthanized and ex vivo fluorescence optical imaging was performed to confirm the location of GPF-expressing lesions in the peritoneal cavity (IVIS Spectrum, Perkin Elmer, Hopkinton, MA). Image analysis was performed using the LivingImage 4.2 software (Perkin Elmer). 107 2.5.5 Histology After ex vivo imaging, lesions and control tissues (muscle) were collected and either embedded into optimal cutting temperature compound (OCT, Sakura Finetek, Torrance, CA) and immediately frozen in liquid nitrogen or fixed in 10% formalin (Fisherbrand, Pittsburg, PA) and embedded in paraffin. OCT-embedded tissues were cut into 10 μm sections, fixed, and stained with Masson’s Trichrome Stain Kit (Polysciences, Warrington, PA) in accordance with the manufacturer’s instructions. Masson’s trichrome stain is used to assess the collagen content of tissues by staining collagen fibers in blue, cell nuclei in black and, both muscle and cytoplasm in red. Slides stained for collagen were analyzed with a digital pathology scanner (Aperio Versa automated slide scanning imaging system, Leica Biosystems Imaging, Deer Park, IL). Paraffin embedded sections were stained with hematoxylin and eosin (H&E; VWR). H&E-stained slides were imaged with light microscopy and analyzed with SPOT 4.0 Advance version software (Diagnostic Instruments, Sterling Heights, MI) for the presence of stroma and glands to validate endometriosis lesions. 2.5.6 ICP-OES Analysis To quantitate gadolinium content in the collected lesions and muscle tissues, samples were dried, weighed, and digested with 69% nitric acid (TraceSELECT™, Fluka, USA). Samples were then diluted further to 4% nitric acid and filtered to obtain a solution with no visible debris. Gadolinium content was determined for each sample by inductively coupled plasma optical emission spectrometry (ICP-OES) using a 710-ES spectrometer (Varian, Palo Alto CA). For all ICP-OES measurements, blank nitric acid, and samples with known Gd concentration as calibration were prepared and tested concurrently with test tissue samples. All measurements were carried out in triplicates and the data were normalized to the dry tissue weight and reported as mean ± SD. 108 2.5.7 Statistical Analysis All data were represented as mean ± SD. Statistical analysis was performed using two- tailed Student’s t test. P<0.05 was considered statistically significant. 109 REFERENCES Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. 1. Nature Reviews Disease Primers. 2018;4(1):9. 2. Malvezzi H, Marengo EB, Podgaec S, Piccinato CdA. Endometriosis: current challenges in modeling a multifactorial disease of unknown etiology. 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J Clin Endocrinol Metab. 2020;105(5):1316-26. 115 APPENDIX Experimental group b e Control group intestines f intestines bladder lesions attached to the peritoneum wall d g bladder intestines bladder intestines lesions h intestines uterine horn intestines intestines lesion a c l e d o m e r u t u S l e d o m n o i t c e j n I leg muscle lesions fat fat Figure 2.11 Representative MR images and corresponding fluorescence images in the GFP channel. a, and b) representative images of the suture model of endometriosis from the experimental group (injected with EP-3533); c, and d) representative images of the injection model of endometriosis from the experimental group (injected with EP-3533); e, and f) representative images of the suture model of endometriosis from the control group (injected with EP-3612); g, and h) representative images of the injection model of endometriosis from the control group (injected with EP-3612). Anatomical structures on MR and fluorescence images are depicted for enhanced visualization of the relative locations of endometriotic lesions in relation to surrounding tissues and organs. The images demonstrate the presence of intestines, muscle tissue, bladder, and fat tissue, offering increased clarity of the spatial relationship between the lesions and adjacent anatomical features. Red and green dotted ovals: endometriotic lesions. 116 IMAGING OF ENDOMETRIOSIS USING RGD-CY5.5-MN PROBE IN A MOUSE CHAPTER 3 MODEL 117 Publication Notice The following dissertation chapter describes the non-invasive imaging and detection of endometriotic lesions using a RGD-Cy5.5-MN probe in a mouse model. The following chapter titled " Imaging of endometriosis using RGD-Cy5.5-MN probe in a mouse model " has been submitted for publication in Nanomaterials journal (2023). The authors of this article are Nazanin Talebloo, Maria Ariadna Ochoa Bernal, Elizabeth Kenyon, Dr. Christiane Mallett, Dr. Asgerally Fazleabas, and Dr. Anna Moore. I would like to express my gratitude for their important contribution to the research presented in this thesis. 118 3.1 Introduction As discussed in Chapter 1, there are multiple theories trying to explain the pathogenesis of endometriosis. Unfortunately, as of today, none of these theories can account for all endometriosis cases by itself, which makes both diagnosis and treatment challenging. Although the exact etiology of this condition is largely unknown, it is believed that a complex interaction of several molecular processes and pathways, including enhanced angiogenesis, promotes the development and progression of endometriotic lesions 1. As explained in Chapter 1, there is limited information describing the precise process of angiogenesis associated with endometriosis. As stated in Chapter 1, various imaging modalities, including MRI and TVUS are being used for detection of endometriotic lesions 2, 3. However, these imaging modalities face some challenges in detection of all lesion types. Developing targeted contrast agents can help diagnose endometriosis through MRI by increasing the signal difference between lesions and their surroundings 4. Various studies investigated overexpressed markers in endometriotic lesions to target the lesions. However, no clinically approved MRI targeted contrast agent is available for endometriosis detection 5, 6. Targeting angiogenesis offers the potential to develop new imaging contrast agents that can help detect lesions using imaging techniques, particularly MRI. As fully discussed in Chapter 1, angiogenesis is a complex process that supports the development of a new vascular system that sprouts from the existing vasculature via the interaction between the cellular matrix, proteolytic enzymes, and cytokines. This cascade plays a crucial role in the progression of endometriosis due in part to the inflammatory response and establishment of lesions which requires new vessel formation which is necessary for delivering nutrients and an oxygen supply to the lesions 7, 8. Angiogenesis is regulated and mediated by integrins, which are members of a family of cell surface adhesion receptors controlling adhesive interactions of vascular cells 9, 10. Alpha(v)beta3 integrin, a receptor 119 for both fibronectin and vitronectin, formed by the dimerization of separate α and β subunits encoded by distinct genes 11, 12 and is significantly upregulated on activated endothelial cells during angiogenesis but not on quiescent endothelial cells 13, 14. Alpha(v) and beta3 integrins which are contributing to cell-cell and cell-matrix interactions and adhesion, communication between cells, and angiogenesis processes 11 have been shown to have an increased expression in endometriotic stromal cells compared to normal endometrial stromal cells 15. When compared to healthy endometrium, the expression of various members of the integrin family, particularly alpha(v)beta3 integrin, are higher in ectopic endometrium tissues (endometriotic lesions), indicating the important role of integrin-associated attachment of lesions 16, 17. Endometrial tissues experience significant hypoxic stress when they separate from the eutopic uterus and shed to the peritoneal cavity (retrograde menstruation). Even with successful implantation to ectopic locations, hypoxic stress continues to be a critical issue since these cells are used to live in a well-oxygenated environment 18. In vitro studies conducted by Lin et al. showed that hypoxia promotes adhesion, migration, and increased mRNA and protein expression of alpha(v) and beta3 integrins in endometrial stromal cells isolated from eutopic endometrium of women with endometriosis 16. This integrin has been proposed as a potential receptor for embryonic attachment and a higher incidence of decreased endometrial alpha(v) and beta3 integrin expression was observed in women with implantation defects 19, 20. Interestingly, it has been shown that the expression of alpha(v)beta3 is decreased in the mid-secretory endometrium of a subset of women with endometriosis with unexplained infertility 21-23. It has been shown that there is an increase in the expression of proangiogenic cysteine-rich angiogenic inducer 61 (CYR61) in endometriotic lesions. It is suggested that proangiogenic function of CYR61 is mediated through the function of integrins 120 such as alpha(v) and beta3, which have been previously implicated in human CYR61-mediated adhesion and angiogenesis and are upregulated in endometriotic lesions 24. RGD peptides (containing an arginine-glycine-aspartic acid sequence) are well-known to bind preferentially to the alpha(v)beta3 and alpha5beta1integrins 25, 26. Targeting angiogenesis and cell adhesion in lesions using imaging probes might increase the chances of reliably detecting endometriosis and we proposed to use cyclic RGD peptide for targeting similar to that in cancer 25, 27, 28. Superparamagnetic iron oxide nanoparticles are biocompatible, biodegradable and have been used for a wide variety of medicinal and biomedical applications such as tumors or vascular imaging, and therapeutics delivery 29-31. Decorating the surface of nanoparticles with ligands like specific peptides can facilitate binding to a biomarker that is selectively overrepresented in targeted cells and reduce off-target accumulation 32. Due to the superparamagnetic nature of the iron core of these nanoparticles they can serve as T2 contrast agents for magnetic resonance imaging (MRI) and their accumulation can be visualized as a darkening of the tissues on T2-weighted MR images 31. Developing nanoparticle-based targeted contrast agents can help both precise diagnosis of endometriosis and delivering potential therapeutics to the lesions, while reducing off-target effects (theranostics). Here, for the first time, c-RGD conjugated, optically active, iron oxide nanoparticles (RGD-Cy5.5-MN) were used to image endometriotic lesions in a murine model of endometriosis using MRI. As a result of our studies, we demonstrated that accumulation of nanoparticles was significantly higher in the lesions of mice injected with RGD-Cy5.5-MN compared to mice injected with unconjugated nanoparticles serving as control (Cy5.5-MN). This study provides the initial proof of utilizing c-RGD conjugated iron oxide nanoparticles to image endometriosis with magnetic resonance imaging in a model of endometriosis in which 121 the pieces of endometrium were sutured in place. The potential of this probe to deliver therapeutics to lesions via targeted delivery should be further investigated. 3.2 Results 3.2.1 Nanoparticle Synthesis and Characterization Iron oxide-based dextran-coated magnetic nanoparticles (MN) were synthesized by co- precipitation of FeCl3.6H2O and FeCl2.4H2O in the presence of dextran solution, functionalized with amine groups (105 amine groups per particle), purified, and conjugated to Cy5.5 near-infrared fluorescent dye resulting in 8 dye molecules per nanoparticle determined by spectrophotometry. The number of cRGD peptides per nanoparticle after conjugation to Cy5.5-MN was 12 as measured by micro-BCA assay (Figure 3.1). The size and zeta potential of the probes was 25 ± 13nm and 15 mv for Cy5.5-MN and 33 ± 8nm and 19 mv for RGD-Cy5.5-MN (PDI: 0.14). 122 Figure 3.1 Synthesis of RGD-Cy5.5-MN. a,b) The synthesis process involves the reaction of the primary amine of cRGD peptide with DSS, followed by precipitation with m-terbutyl ether, and subsequent conjugation with the amine groups on the Cy5.5-conjugated nanoparticles. This sequential procedure results in the formation of RGD-Cy5.5-MN nanoparticles. c) Interaction of RGD-Cy5.5-MN and cell-surface integrins. 123 3.2.2 Magnetic Resonance Imaging of Endometriotic Lesions In vivo MR imaging was performed on animals with endometriotic lesions before and 24 hours after the injection of the probes. Representative images of T2* maps of endometriotic lesion are shown for both experimental and control groups (Fig 3.2) The images qualitatively represent higher decrease in T2* relaxation time from pre-contrast (Fig 3.2 e) to post-contrast (Fig 3.2 g) in the lesions of the RGD-Cy5.5-MN-injected group compared to the lesions of the group injected with Cy5.5-MN (pre-contrast - Fig 3.2 f, post-contrast - Fig 3.2 h). This indicates a higher accumulation of RGD-Cy5.5-MN in the lesions compared to control nanoparticles. Quantitative analysis of the T2* map validated the data showing a significantly larger change in T2* relaxation times in the lesions of animals injected with RGD-Cy5.5-MN compared to the lesions of control group (p<0.02; Fig 3.2 i). There was no significant change in the T2* relaxation times of the muscle tissue before and 24 hours post-injection in both experimental and control groups (0.65 ± 0.6ms vs 1.5 ± 2.6ms respectively, p>0.05). 124 RGD-Cy5.5-MN Cy5.5-MN b d a c t s a r t n o c - e r P t s a r t n o c - t s o P RGD-Cy5.5-MN Cy5.5-MN i f h e g t s a r t n o c - e r P t s a r t n o c - t s o P Figure 3.2 Representative T2* maps of the lesions in a mouse model of endometriosis. a) Pre- contrast and c) post-contrast images of animals injected with RGD-Cy5.5-MN. b) Pre-contrast and d) post-contrast images of animals injected with Cy5.5-MN. e) Enlarged images of lesions captured before and g) after RGD-Cy5.5-MN administration. f) Enlarged images of lesions captured before and h) after Cy5.5-MN administration. Dotted area shows borders of the lesions. i) Quantitative data from the images in e), f), g) and h). 3.2.3 Nanoparticle Biodistribution To demonstrate accumulation of RGD-Cy5.5-MN and Cy5.5-MN in different organs, ex vivo biodistribution studies were performed for both experimental and control group of animals using fluorescence optical imaging (Fig 3.3 a and c). Bioluminescence optical imaging was performed to validate the presence of Cy5.5-labeled nanoparticles in luciferase-expressing endometriotic lesions in both experimental and control groups (Fig 3.3 b and d). Fig 3.4 a shows the quantification of the Cy5.5 signal from both probes accumulated in different organs normalized to the organ surface area. Quantitative analysis demonstrated that accumulation of RGD-Cy5.5- MN in the lesions was significantly higher compared to Cy5.5-MN accumulation (p=0.0008). Furthermore, this accumulation was significantly higher than accumulation in the muscle tissues 125 in both experimental and control groups (p=0.001). Comparing biodistribution to other organs in experimental and control groups we observed significant difference in accumulation of the probes between internal organs such as liver and spleen. This is not unexpected and can be due to expression of alpha(v)beta3 on the cell surface of activated tissue macrophages of the reticuloendothelial system (RES) 33, 34. The changes in the RES uptake were observed in various previous studies investigating RGD-conjugated nanocarriers 33, 35. Despite the increased accumulation of RGD-conjugated nanoparticles in non-targeted tissues, our results show higher lesion to organ Cy5.5 signal in mice injected with the targeted probe compared to control nanoparticles when lesion signal was normalized to the signal from other tissues including muscle, liver, kidney, and heart, demonstrating considerable potential of RGD-Cy5.5-MN for endometriotic lesion targeting (Fig 3.4 b). To corroborate the higher accumulation of RGD-Cy5.5-MN in lesions of experimental mice compared to lesions from mice injected with Cy5.5-MN signified by a T2* relaxivity drop, tissue samples containing endometriotic lesions and muscle tissues from both animal groups were analyzed for iron content by ICP-OES (Fig 3.5). The results showed that the iron content in endometriotic lesions of experimental group (5.9 ± 1.7 µg Fe/g dry tissue) was significantly higher compared to both skeletal muscle of same group (0.52 ± 0.14 µg Fe/g dry tissue) and lesions of control group (1.8 ± 0.22 µg Fe/g dry tissue). There was no significant difference between iron content in control muscle tissues from both experimental and control groups. 126 a b c d Figure 3.3 Ex vivo biodistribution studies. a, c) Biodistribution assessment of the probes in mice injected with RGD-Cy5.5-MN or Cy5.5-MN (respectively experimental and control groups); b), d) Bioluminescence signal from luciferase-expressing lesions in experimental and control groups. Lesions: Le, Muscle: Mu, Liver: Li, Spleen: Sp, Kidney: Ki, Heart: He, and Lung: Lu. 127 Figure 3.4 a) Quantification of ex vivo biodistribution shows significantly higher lesion accumulation of RGD-Cy5.5-MN compared to control group. b) Cy5.5 signal of the lesion normalized to the signal from other organs shows significantly higher ratio when normalized to muscle, kidney and heart. 128 Figure 3.5 ICP-OES analysis of the average iron content in the lesions and muscle tissues after injection of RGD-Cy5.5-MN or Cy5.5-MN. Tissue iron content was normalized to the dry weight of the tissues. Results are presented as means ±SD. Note that endometriotic lesions have significantly higher iron content after RGD-Cy5.5-MN injection (p<0.05) compared to the control probe. 3.2.4 Ex vivo Analysis of RGD-Cy5.5-MN Accumulation and CD34 Expression To further confirm our in vivo MRI results, lesion sections from both experimental and control groups of mice were analyzed with fluorescence microscopy. Accumulation of RGD- Cy5.5-MN in lesions of experimental group (Figure 3.6, top row), was significantly higher compared to accumulation of Cy5.5-MN in the lesions of control group (Figure 3.6, bottom row) which was in line with the MRI findings. Additionally, staining for endothelial marker CD34 36 37 clearly showed colocalization with the Cy5.5 signal suggesting that accumulation of RGD-Cy5.5- MN was specific to overexpression of alpha(v)beta3 integrins 38 and is not solely due to passive targeting (Figure 3.7 a and b). 129 Figure 3.6 Fluorescence microscopy demonstrates relatively higher accumulation of RGD- Cy5.5-MN (top row) in endometriotic lesions compared to Cy5.5-MN (bottom row). Blue – DAPI nuclear stain; red – Cy5.5. Bar = 50 um. a c b 100 um 50 um d e Figure 3.7 Immunofluorescence staining of endometriotic lesion sections from animals injected with RGD-Cy5.5-MN shown with a) low; bar = 100 um and b) high; bar = 50 um magnification. Note that (b) shows the area within the yellow rectangle in (a). Cell nucleus DAPI staining (c), CD34 marker (d, green) and Cy5.5 (e, red) are shown separately in the bottom row. 130 3.3 Discussion Endometriosis is a heterogeneous disease which as of today has no clinically approved biomarkers for noninvasive diagnosis 39. A clinical trial (NCT03376451) designed to analytically validate a cluster of specific biomarkers for endometriosis diagnosis and disease recurrence has yet to publish its results. Although different noninvasive clinical imaging modalities are being used to diagnose endometriosis, they cannot detect all types and sizes of lesions 40. A recent review of the diagnostic accuracy of imaging for endometriosis included 49 studies with 4,807 women and concluded that most studies were of poor methodological quality and none of the imaging methods assessed were able to detect endometriosis with enough accuracy that they could be recommended to replace surgical evaluation 41. Тargeted delivery of contrast agents to endometriotic lesions could potentially resolve this problem and assist in the detection of lesions using various imaging modalities including MRI. One approach for targeted delivery of contrast agents to the lesions could involve developing nanocarriers with ligands targeting specific and/or overexpressed biomarkers in the lesions 42-44. Endometriosis heavily relies on adhesions and angiogenic factors for implantation and survival of lesions 6. Alpha(v)beta3 is a protein from an integrin family that plays an important role in cellular processes including adhesion and angiogenesis. Several studies have already shown the presence of alpha(v)beta3 integrin in endometriotic lesions and suggested that increased adhesive capacity in endometriosis could be caused by integrin dysregulation 16, 45, 46. Alpha(v)beta3 is a type on integrin playing role in different processes including cell adhesion and angiogenesis. Several studies have already shown presence of this integrin in endometriotic lesions and suggested that increased adhesive capacity of endometriosis may be because of integrin dysregulation 16. RGD peptides are shown to bind preferentially to the alpha(v)beta3 integrin25 and immortalized endometriosis derived cell line 47. In this study, we hypothesized that c-RGD conjugation can enhance accumulation of iron oxide nanoparticles in the lesions by targeting 131 alpha(v)beta3 integrin leading to an improvement in lesion detection by MRI. For this purpose, cyclic RGD peptide was conjugated to optically labeled MR biocompatible iron oxide nanoparticles and tested for imaging endometriotic lesions in a mouse model of endometriosis. In our studies we used the model with established lesions and developed vasculature. The reason for that is that one of the main symptoms of endometriosis is pain 48, which is often mistaken by patients for menstrual cramps. Many patients experience this pain for years after the disease initiation before seeking medical advice, and with present older, developed lesions. This disease presentation demonstrates the need for a probe that can identify established lesions with significant angiogenesis. MR imaging and comparison of the average deltaT2* values from experimental and control groups of animals supports our hypothesis showing a significantly larger reduction in the lesions of the animals injected with RGD-Cy5.5-MN. To further corroborate our MRI data, we performed ex vivo biodistribution. Comparing the two groups, a significant difference was observed in several other organs including spleen, liver and muscle which can be attributed to variability of expression of integrins on RES cells 33, 35. Previous studies also showed that the number of RGD molecules on the nanocarrier has an effect on its binding capacity to different cells, organs and its half-life in blood 27, 49. Biodistribution data also showed significantly higher numbers when Cy5.5 signal from nanoparticles accumulated in lesions from the group injected with RGD-Cy5.5-MN when was normalized to the signal from the tissues and organs. ICP-OES results also demonstrated significantly higher iron content in the lesions from the experimental group compared to the control group which supports data acquired from MRI and ex vivo fluorescence imaging. Histological analysis of the lesions from both experimental and control groups showed higher accumulation of RGD-Cy5.5-MN with the majority of the probe accumulating in the areas 132 expressing the angiogenesis marker, CD34. It has been previously shown that CD34+ cells express alpha(v)beta3 integrin 50 which was our target of interest. It is noteworthy that there are various studies quantifying expression of alpha(v)beta3 integrin both in eutopic and ectopic endometrium of human or animal models of endometriosis. However, since not all the data are in agreement a more comprehensive study on the expression of integrins in this disease is needed 16, 45, 46. Considering that endometriosis is a heterogenous disease, type and stage of the lesions can affect the outcome of the imaging studies. Different factors including the type of lesions and the menstrual cycle phase should be considered in a larger cohort of samples in the future studies investigating the role of integrins in endometriosis. Currently, an ongoing clinical trial (NCT05623332) which is scheduled to complete in 2024 is investigating the presence of integrins in endometriotic tissue. This is expected to expand our understanding of endometriosis and could be used for developing a non-invasive imaging method in the future. This trial is also designed to give a better understanding of integrin expression in different phases of menstrual cycle and the potential of different molecules for targeting ectopic lesions utilizing machine learning. This supports our hypothesis that integrins can be a useful target in diagnosis and treatment of endometriosis which should be further investigated. In summary, our data demonstrate that cyclic RGD-conjugated optical/MRI sensitive iron oxide nanoparticles can be used to efficiently target ectopic endometriotic lesions. In this study we chose to use a well-established suture model of endometriosis commonly employed in preclinical studies 51. The primary reason for using this model was based on the fact that it provides the exact location of the lesions so they can be identifiable in our proof-of-concept imaging studies. Based on these studies we plan to validate our findings in the peritoneal injection mouse model 52 133 followed by a non-human primate model 53, 54 where the locations of the lesions are random and closely represent a clinical scenario 55. 3.4 Conclusions Considering that endometriosis is an angiogenesis-dependent disease which strongly requires adhesions and angiogenic factors for its implantation and survival, we evaluated an optical/MRI-sensitive iron oxide nanoparticle conjugated with cyclic RGD peptide to detect lesions in a mouse model of endometriosis. 3.5 Experimental section 3.5.1 Mouse Model of Endometriosis Ten weeks old female Pgr cre/+ Rosa26 Luc/Luc and Pgr cre/+ Rosa26 Luc/+ mice ( 52, n=9) were treated for 3 days with estrogen (E2, Sigma-Aldrich; 1mg/ml in oil, 0.1µg/mouse/day) in order to synchronize the estrus cycle and help lesion growth. After the last E2 injection endometriosis was induced by removing one uterine horn from each mouse. To remove one uterine horn, a midline abdominal incision was made and the caudal end of the uterine horn near the uterotubal junction was cut and ligated with sterile absorbable suture. The removed uterine horn was opened longitudinally, and tissue samples were obtained using a 2 mm dermal biopsy punch. Three biopsies were sutured to the peritoneal wall (3 in each side, total of 6) using a 7-0 braided silk suture. The muscle layer was closed as one layer, then the skin as a second layer. After this procedure, the abdominal incision was closed. Pgr cre/+ Rosa26 Luc/Luc and Pgr cre/+ Rosa26 Luc/+ mice models of induced endometriosis provide an opportunity to visualize the progesterone receptor (Pgr)-positive cells expressing luciferase (Luc) using bioluminescence imaging (BLI) after administration of D-luciferin. In contrast, the Pgr-negative cells in this model do not express Luc, which allows visualization and localization of endometriotic lesions using bioluminescence imaging. 134 All animal studies were approved by the Institutional Animal Care and Use Committee at Michigan State University and are in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 3.5.2 RGD-Cy5.5-MN Synthesis and Characterization Amine functionalized dextran-coated iron oxide nanoparticles (MNs) were synthesized and labeled with a near-infrared fluorescent Cy5.5 dye according to our previously established procedure 56. Briefly, 9 g of Dextan-T10 (Pharmacosmos, Denmark) was dissolved in 30 mL of double- distilled water and stirred in a round bottom flask on ice. 0.65 g of FeCl3·6H2O (Sigma Aldrich, St. Louis, MO) was added while flushing Argon gas into the reaction mixture for an hour. 0.4 g of FeCl2·4H2O (Sigma Aldrich, St. Louis, MO) was added into the mixture and then 15 mL of cold 25% NH4OH (Acros Organics, Morris Plains, NJ) was added to the stirring mixture. The temperature increased to 85 C for 90 minutes to induce the formation of a nanoparticulate colloidal mixture, cooled to room temperature and concentrated to 20 mL using Amicon Ultra centrifugal units (MWCO 30 kDa; Millipore, Ireland). The resulting 20 mL dextran coated magnetic nanoparticles were cross-linked and aminated with subsequent addition and stirring of 35 mL of 5 M NaOH (Thermo Fisher Scientific, Fair Lawn, NJ), 14 mL of concentrated (±)-epichlorohydrin (Sigma Aldrich, St. Louis, MO) for 8 hours and 60 mL of concentrated NH4OH for the next 36 hours. The nanoparticle solution was purified using a dialysis tubing (MWCO 12-14 kDa, Spectrum Chemical Mfg. Corp., Gardena, CA) against water and then concentrated to 20 mL by Amicon Ultra centrifugal units in 20 mM citrate buffer (pH ~8.0).To conjugate MNs to the fluorescencent dye, cynanine5.5 monoreactive NHS ester (Lumiprobe, Hunt Valley, MD) was dissolved in 100 uL of anhydrous dimethyl sulfoxide (Thermo Fisher Scientific, Fair Lawn, NJ) and incubated with MN (10 mg Fe) in 20 mM sodium chloride and sodium citrate buffer (pH ~8.0) overnight. The nanoparticles were purified using Sephadex PD-10 column (Cytiva, United 135 Kingdom) with PBS eluent. c-RGD conjugated nanoparticle (RGD-Cy5.5-MN) was prepared based on a previous published work27. Briefly, to 100 uL of 100 mM suberic acid bis(N- hydroxysuccinimide ester) (DSS, Sigma Aldrich, St. Louis, MO) in dimethylformamide (DMF, Thermo Fisher Scientific, Fair Lawn, NJ) was added diisopropylethylamine (Sigma Aldrich, St. Louis, MO). Next, 40 uL of 1 mM c-RGD peptide (sequence: GSSKGGGCRGDC with disulfide bridge 8-12; fluorescein was attached to the peptide through the use of N-α-Fmoc-N- ε-(5- carboxyfluorescein)-L-lysine ; LifeTein, Hillsborough, NJ) in DMF was added in 5 uL portions to the previous solution and incubated for 1 hour. NHS-activated c-RGD peptide was precipitated with m-terbutyl ether (MTBE, Sigma Aldrich, St. Louis, MO). To the precipitate was added Cy5.5- MNs (5 mg Fe) which was incubated at 25 C overnight and purified from unreacted peptide with a PD-10 column. The size and zeta potential of particles were determined by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, MA). The amount of peptide on MN was quantified by micro-BCA protein assay based on the protocol provided in the kit (Thermo Scientific, Rockford, IL) and iron concentration was determined spectrophotometrically from absorption at 410 nm with iron assay. 3.5.3 Magnetic Resonance Imaging In vivo MRI was performed in the mouse model of endometriosis 4 to 6 months post endometriosis induction. Mice were divided into two groups including experimental (injected with RGD-Cy5.5-MN; n=6) and control group (injected with Cy5.5-MN; n=3). Images were acquired on a 7 Tesla Biospec 70/30 USR (Bruker, Billerica, MA) using an 86 mm diameter volume transmit coil and 4 channel surface array receive coil (4 × 4 cm). Fat-suppressed T2* maps were acquired for quantitative analysis of probe accumulation with the following parameters: multi-gradient echo sequence, TR/TE=1500/3.5 ms, 2 averages, 100 um x100 um resolution, 300 um slice thickness, flip angle 50°, 10 echo images with minimum echo time of 2.62 ms and 4.8 ms spacing, and 136 respiratory gating. Mice were anesthetized using 1.5% to 3%. Temperature (~35°C) and breathing were monitored and maintained throughout the experiment (SAII Small Animal Instruments, Inc, Stony Brook, NY). First, pre-contrast injection images were acquired following by intravenous injection of RGD-Cy5.5-MN or Cy5.5-MN (10 mg Fe/kg) through tail vein injection. Iron oxide contrast agents accumulated in a tissue cause loss of tissue signal on T2* sequences due to the susceptibility effects of the iron oxide core. 24 hours post injection, mice were imaged with the same parameters. Images were analyzed using Paravision 360 v3.2 software (Bruker). The ROIs of the muscle tissue were used as controls within each group of mice. T2* before injection minus T2* after injection (delta-T2*, ms) were used to investigate the accumulation of probes in the lesions. 3.5.4 Ex vivo Bioluminescence Optical Imaging In order to confirm the location of luciferase expressing lesions detected with MR imaging, immediately after the last MR imaging session mice were injected intraperitoneally with IVISbrite D-Luciferin potassium salt in PBS (100 uL of 300 mg/mL, Perkin Elmer, Boston, MA). 10 minutes after injection mice were euthanized and collected tissues were subjected to bioluminescence imaging (BLI) using IVIS Spectrum imaging system (Perkin Elmer). BLI was performed with the following settings: Exposure time, 30 seconds; f number, 8; Binning factor, 2. All images were processed using the Living Image Software (version 4.5.2, Perkin Elmer). 3.5.5 Ex vivo Epifluorescence Optical Imaging To evaluate the biodistribution of nanoparticles, after acquiring bioluminescence optical images ex vivo epifluorescence optical imaging was performed on collected tissues of mice injected with either RGD-Cy5.5-MN or Cy5.5-MN 24 hour prior to ex vivo imaging. The acquisition conditions are summarized as follows: Exposure time, 30 sec; Binning factor, 2; Excitation filter range, 675 nm; Emission filter range, 720 nm; f number, 8. All images were 137 analyzed using the Living Image Software (version 4.5.2, Perkin Elmer). Imaging data were normalized, and quantified signal was expressed as radiant efficiency ([p/s/cm²/sr]/[uW/cm²]). 3.5.6 ICP-OES Analysis To quantitate iron content in the collected lesion and muscle tissues, samples were dried, weighted and digested with 69% nitric acid (TraceSELECT™, Fluka, USA) and stored at room temperature until a dissolution was observed. Samples were then diluted further to 4% nitric acid and filtered to obtain a solution with no visible debris. Iron content was determined for each sample by inductively coupled plasma optical emission spectrometry (ICP-OES) using a 710-ES spectrometer (Varian, Palo Alto, CA). For all ICP-OES measurements, blank nitric acid, and samples with known iron concentration as calibration were prepared from TraceCERT™, Iron (Fe) Standard for ICP (1000 mg/L Fe in nitric acid, Sigma-Aldrich, St. Louis, MO) and tested concurrently with test tissue samples. All measurements were carried out in triplicates and the data were normalized to the dry tissue weight and reported as mean ± SD. 3.5.7 Histology and Immunostaining To analyze endometriotic lesions, excised tissues were embedded in optimal cutting temperature compound (OCT, Sakura Finetek, Torrance, CA) and snap-frozen in liquid nitrogen. OCT-embedded tissues were cut into 10 um sections, fixed with 10% neutral buffered formalin (Fisherbrand, Pittsburg, PA) blocked in a solution of 5% normal goat serum in phosphate-buffered saline and stained for CD34, an endothelial marker, with rat monoclonal anti-mouse CD34 antibody (1:100 dilution; Abcam, Cambridge, MA) primary antibody, followed by Alexa Fluor 594-conjugated goat anti-rat (1:400 dilution; Invitrogen, ), and mounted with a DAPI (4,6- diamidino-2-phenylindole) containing mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) for nuclear staining. To compare nanoparticle accumulation in lesions excised from mice injected with RGD-Cy5.5-MN to mice injected with Cy5.5-MN sections of lesion from 138 both control and experimental group of mice were analyzed for Cy5.5 signal. Tissue sections were analyzed by fluorescence microscopy using a Nikon Eclipse 50i fluorescence microscope (Nikon, Tokyo, Japan), equipped with the necessary filter sets (Chroma Technology Corporation, Bellows Falls, VT). Images were acquired using a charge-coupled device camera with NIRF sensitivity (SPOT 7.4 Slider RTKE; Diagnostic Instruments, Sterling Heights, MI). The images were analyzed using SPOT 5.2 Advance version software (Diagnostic Instruments, Sterling Heights, MI). 3.5.8 Statistical Analysis All data are represented as mean ± SD. Statistical analysis was performed using two-tailed Student’s t test. P<0.05 was considered statistically significant. . 139 REFERENCES Chung MS, Han SJ. 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Induction of endometriotic nodules in an experimental baboon model mimicking human deep nodular lesions. Fertility and sterility. 2013;99(3):783-9. e3. 56. Yigit M, Ghosh S, Kumar M, Petkova V, Kavishwar A, Moore A, et al. Context-dependent differences in miR-10b breast oncogenesis can be targeted for the prevention and arrest of lymph node metastasis. Oncogene. 2013;32(12):1530-8. 144 CHAPTER 4 CONCLUSIONS, FUTURE DIRECTIONS, AND DEVELOPMENT OF A NOVEL THERAPY FOR OVARIAN CANCER 145 4.1 Summary and Future Directions 1. Application of the imaging probes for drug delivery in endometriosis. Targeted delivery methods have been extensively investigated to enhance bioavailability of drugs and imaging agents to specific tissues or cell types while reducing their side effects in off- target tissues. To date, many strategies have been investigated to accomplish this goal with some of them relying on the differences between the cellular and molecular compositions of the targeted tissues and those of the nontargeted ones. One of the most important differences could be extracellular matrix molecules and composition of the tumor microenvironment or cell surface receptors on the tumor cells which can either be specific to the targeted tissue or significantly overexpressed in it 1, 2. Molecules, including peptides, with high affinity to the target tissue, have successfully served as delivery vehicles to direct imaging agents, drug molecules, oligonucleotides, and inorganic nanoparticles to tumors and other tissues 3. In Chapter 2 a targeted delivery approach was employed to transport imaging probes to endometriotic lesions. Namely, a collagen type I targeting peptide EP-3533, which incorporates gadolinium as an MRI contrast agent was used to image endometriotic lesions with fibrotic content in mouse models of endometriosis. Future research can prioritize the conjugation of fluorescent dyes to this peptide, enabling the visualization of probe accumulation through fluorescence imaging techniques. Also, more specific staining, including type I collagen-specific antibodies can be used in ex vivo studies. By utilizing a fluorescent dye-conjugated probe, it becomes possible to observe the distribution and colocalization of type I collagen and the probe within tissues. The application of nanomedicine for endometriosis is still in its early stages, but initial findings suggest that nanoparticle-based approaches could revolutionize the diagnosis and treatment of this condition. Similarities between cancer and endometriosis indicate that principles 146 from cancer nanomedicine can be applied to develop innovative strategies for endometriosis using nanoparticles. However, since causes of endometriosis and the mechanisms of its progression differ of those in cancer, it is crucial to thoroughly investigate and clarify how nanoparticles accumulate in ectopic endometrial tissue after systemic administration. Additionally, it is essential to evaluate the effectiveness of nanoparticle-based platforms for imaging and treating endometriosis in clinically relevant animal models (such as baboons) and human clinical trials. These studies can provide valuable insights into the natural occurrence of endometriosis in humans and non-human primates and assess the true impact of nanotechnologies in the presence of human or non-human primate hormonal cycles 4. In Chapter 3 RGD-Cy5.5-MN was utilized to image endometriotic lesions by targeting Alpha(v)beta3 in the ectopic endometrium. The accumulation of RGD-Cy5.5-MN in the endometriotic lesions was found to be significantly higher compared to the non-targeted Cy5.5- MN, as evidenced by MRI data. These findings were further validated through ICP-OES analysis. Endometriosis targeting RGD-Cy5.5-MN probe can be used for the delivery of different therapeutics, including small interfering RNA (siRNA) to the lesions, and reduce the translation of specific mRNAs. NOTCH1, a transmembrane receptor belonging to the NOTCH family, plays a crucial role in transmitting external signals and facilitating cell-cell interactions. NOTCH signaling pathway is involved in various cellular processes, including cell fate determination, proliferation, survival, immunological regulation, and regulating EMT 5, 6. Recent studies have revealed that NOTCH1 expression is altered in endometriotic lesions, and its aberrant activation contributes to the development of these lesions by influencing cellular functions. Particularly, excessive NOTCH signaling leads to fibrosis, but inhibiting NOTCH cleavage has been shown to reduce fibrosis in ectopic endometrial stromal cells, offering potential therapeutic implications for 147 endometriosis 7. Interestingly, it has been shown that Notch1 can upregulate type I collagen promoter activity, which is the most abundant extracellular protein component in fibrosis 8 (endometriosis-associated fibrosis and the role of type I collagen in this condition were discussed in Chapters 1 and 2). Proposed future research based on the results gathered from Chapters 2 and 3 of this thesis, previous studies published by Dr. Fazleabas group 5, 9, and a review of related literature, will be a collaboration between Dr. Moore and Dr. Fazleabas groups. The proposed study will investigate inhibiting NOTCH1 signaling using RGD-MN delivery of either NOTCH1 siRNA 7, 10 or a synthetic helical peptide (SAHM1) which was previously shown to target tumor cells and result in genome-wide suppression of NOTCH-activated genes 11. Based on our experience in oligonucleotide deliver we will first focus on delivery of NOTCH siRNA. The primary aims of this study will be as follows: 1-Conjugating NOTCH1 siRNA to RGD-MN. 2-Investigating the potential of targeted nanoparticles for delivering NOTCH1 siRNA and inhibiting the NOTCH signaling pathway in animal models of endometriosis. 3-Comparing the outcomes of the experimental group with the control group (RGD-MN conjugated with scrambled siRNA as control). 4-Investigating the impact of NOTCH1 siRNA delivery on lesion formation, disease progression, and subsequent fibrosis, while comparing the outcomes with those observed in the control group. In the second part of this study, we will conjugate EP-3533 peptide (without gadolinium) to the nanoparticles (EP-3533-MN) and design experiments similar to sub-aims 1-4. This can help us compare the potential of RGD-MN (a probe that was designed to target lesions at the earlier stages) 148 to EP-3533-MN (which is proposed to target lesions at the later stages) in successfully inhibiting NOTCH1 and fibrosis formation. 2. Initial studies in drug delivery in ovarian cancer. 4.2 Development of a Novel Therapy for Ovarian Cancer Based on Inhibition of microRNA- 181a 4.2.1 Introduction During my graduate work aimed at investigating imaging in endometriosis, I learned that women diagnosed with endometriosis are at increased risk of developing certain types of ovarian cancer, such as ovarian endometrioid, low-grade serous, and clear-cell adenocarcinoma. However, it is still unclear whether ovarian cancer arises from molecular changes within the endometriosis itself or if it is influenced by the tumor microenvironment present in endometriosis. Furthermore, the exact mechanisms by which the presence of endometriosis affects the prognosis of ovarian cancer remain unknown, although it is likely that the endometriotic microenvironment plays a role in this regard 12. Ovarian cancer is a disease that primarily originates in the ovaries, although in some cases it can develop from other sites outside of the ovary. It is a type of gynecological cancer that poses a serious threat to women's health worldwide, with an overall five-year survival rate as low as 30%. While it was once considered a single entity, ovarian cancer can be further categorized into different histological subtypes with distinct molecular compositions. The most common subtype is epithelial cancer, accounting for approximately 90% of ovarian cancers, including serous, endometrioid, clear-cell, and mucinous carcinomas 13, 14. Motivated by the fact that endometriosis is a risk factor for ovarian cancer, as well as my interest in cancer research and Dr. Moore's expertise in the field of oncology, we have identified an opportunity to embark on a project centered around ovarian cancer. Despite the presence of evidence indicating that the various types 149 of epithelial ovarian cancer are distinct entities, they continue to be managed using a comparable approach involving debulking surgery followed by adjuvant chemotherapy. The standard approach for treating ovarian cancer involves the use of platinum-based chemotherapy, including platinum- based drugs such as e carboplatin or cisplatin. This treatment regimen has remained unchanged for the last twenty years. While many patients initially respond well to these therapies, approximately 80% of women experience a relapse due to the development of platinum resistance. It is crucial to understand the molecular mechanisms underlying this resistance to enhance treatment effectiveness and improve survival rates for patients 15. In an examination of 443 specimens of advanced epithelial ovarian cancer from The Cancer Genome Atlas, it was discovered that an elevated expression of miR-181a was linked to a shorter time of recurrence, suggesting that this specific miRNA may play a substantial role in the development of ovarian cancer 16. It has been shown that the levels of miR-181a were significantly elevated in chemoresistant ovarian cancer tissues compared to chemosensitive tissues 17. The upregulation of miR-181a through ectopic expression has been found to be associated with increased cellular survival, migration, invasion, and development of resistance to platinum-based drugs, as well as with amplified in vivo tumor burden and dissemination 16. Based on the above-mentioned evidence, our hypothesis aims to reduce the expression of miR-181a by delivering inhibiting oligonucleotides to tumors. This will be done by employing dextran-coated iron oxide nanoparticles that serve as vehicles for oligonucleotide delivery. In addition, iron oxide nanoparticles will serve as imaging reporters for monitoring the delivery. Previous research conducted by Dr. Moore's group demonstrated the feasibility of targeting the underglycosylated mucin-1 (uMUC1) antigen overexpressed on various human epithelial cell adenocarcinomas including ovarian cancer using nanoparticles conjugated with a uMUC1-specific 150 peptide (EPPT-MN) 18-21. In this study, as the first step of this ongoing project, we aimed to synthesize a targeted probe carrying antisense oligonucleotides inhibiting miR-181a, characterize the probe, and conduct preliminary in vitro experiments to and assess its potential for targeting underglycosylated mucin-1 expressed on ovarian cancer cells. 4.2.2 Results and Discussion EPPT-MN was successfully synthesized and characterized (number of peptides per particle: 12, size: 69 nm, PDI: 0.22, zeta potential: +15 mv) according to a protocol by Zhao et al. 18. The measurement determined that each particle contained 7 Cy5.5 dye molecules. The Cy5.5- MN, utilized as the control probe for in vitro studies was characterized (size: 62 nm, PDI: 0.18, zeta potential: +13 mv). Immunohistochemistry was performed to evaluate the expression of uMUC1 which revealed that the SKOV3 cell line has higher uMUC1 antigen expression compared to the ES-2 cell line (Figure 4.1). Also, the cell-binding assay exhibited a preferential and concentration-dependent uptake of the EPPT-MN probe by uMUC1-expressing human ovarian cancer cell line (SKOV3) compared to a control probe lacking the EPPT peptide (Figure 4.2). Conversely, control ovarian tumor cells with low uMUC1 expression (ES-2), displayed significantly lower uptake of the EPPT-MN probe. Furthermore, a competitive inhibition assay utilizing free EPPT peptide as a blocking agent confirmed specific binding to SKOV3 cells and no competition with ES-2 cells (Figure 4.3). In preparation for the second step of the synthesis which will include conjugation of anti-miR-181a oligonucleotides, relative expression levels of miR- 181a were quantified in the SKOV3 cell line by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in ovarian cancer cell lines. This experiment showed that SKOV3 cell line has a significantly higher expression of this microRNA compared to A2780 ovarian cancer cell line (kindly provided by our collaborator Dr. Analisa Difeo, University of Michigan). A2780 151 cell line will serve as a control in future experiments due to its relatively low miR-181a expression. To investigate the impact of an antisense oligonucleotide on miR-181a expression, a specific inhibitor sequence targeting miR-181a and a scrambled sequence serving as a control were designed and purchased (Eurogentec). The experimental approach involved transfecting ovarian cancer cell lines with the miRNA inhibitor. Following transfection, the expression levels of miR- 181a were measured using qRT-PCR. The assay demonstrated that the inhibitor sequence effectively reduced the expression of miR-181a in both A2780 (32% inhibition compared to the A2780 transfected with miR-181a scrambled inhibitor) and SKOV3 (79% inhibition compared to the SKOV3 transfected with miR-181a scrambled inhibitor) cell lines, with a particularly notable effect observed in the SKOV3 cell line (Figure 4.4). By doing these experiments we established a suitable model for future in vitro and in vivo experiments. In future experiments, the focus will be on synthesizing and characterizing targeted iron oxide nanoparticles (EPPT-MN) conjugated to miR-181a antisense oligonucleotide (EPPT-MN-anti-miR181a). Next, we will examine the impact of these nanoparticles on viability of ovarian cancer cell lines alone or in combination with cisplatin both in vitro and in vivo. By conducting these experiments, we aim to assess the potential of MN-anti-miR181a, as a miR-181a inhibitor, as a novel therapy for ovarian cancer. 152 a Figure 4.1 Fluorescence microscopy assessing uMUC1 expression in ovarian cancer cell lines. The results indicate higher uMUC1 antigen expression in the SKOV3 cell line compared to the ES-2 cell line. Blue: DAPI, red: Texas Red. Magnification bar = 20 µm. a b Figure 4.2 a) In vitro cell binding assay testing relative accumulation of EPPT-MN or Cy5.5 conjugated control nanoparticles (Cy5.5-MN) in SKOV3 and ES2 cell lines (n = 3). b) Quantitative analysis of cell binding assays showed preferential, concentration dependent uptake of MN-EPPT probe by SKOV3 cells compared to Cy5.5-MN. ES-2 cells exhibited significantly lower uptake of EPPT-MN probe compared to SKOV3 cells (ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). 153 Figure 4.3 a) Competition assay showed dose-dependent accumulation of EPPT-MN which was blocked by free EPPT peptide. b) Quantitative analysis of competitive inhibition assay showed specific binding to SKOV3 cells and virtually no competition with ES-2 cells. … Figure 4.4 qRT-PCR of miR-181a expression in SKOV3 and A2780 ovarian cancer cell lines, transfected with miR-181 inhibitor, corresponding scrambled sequence, or treated with PBS as control. Expression level numbers are relative to A2780-PBS which serves as control (ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). 154 4.2.3 Experimental Section 4.2.3.1 Histology and Immunostaining Previously seeded ES-2 and SKOV3 cells were fixed and incubated with 5% natural goat serum. Subsequently, each cell line was separately incubated with anti-mucin 1 rabbit polyclonal antibody (1:100 dilution; Abcam, Cambridge, MA), with a corresponding fluorescently (Texas Red) labeled IgG secondary antibody (1:200 dilution; Abcam, Cambridge, MA) for visualization of the MUC1 tumor antigen. The cells were counterstained with DAPI-containing mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA) for visualization of cell nuclei. Fluorescent images were observed using a Nikon Eclipse 50i fluorescence microscope (Nikon, Melville, NY), equipped with appropriate fluorescence filter sets. Images were analyzed using SPOT 4.0 Advance version software (Diagnostic Instruments, Sterling Heights, MI. 4.2.3.2 EPPT-Cy5.5-MN Synthesis and Characterization After synthesis and characterization of dextran-coated iron oxide nanoparticles (MN) and conjugation of Cy5.5 dye to particles (described in section 3.5.2), EPPT peptide (NH2-Cys-Tyr- Cys(acm)-Ala-Arg-Glu-Pro-Pro-Thr-Arg-Thr-Phe-Ala-Tyr-Trp-Gly-Lys-CONH2, LifeTein, Hillsborough, NJ) was conjugated to Cy5.5-MN based on the following protocol. N-γ- maleimidobutyryl-oxysuccinimide ester (GMBS) was dissolved in DMSO to make 100mM stock solution and 100uL of GMBS stock solution was well mixed with 4 mg Fe of Cy5.5-MN and 20 μL of sodium chloride/ sodium citrate buffer (pH=8). The mixture was incubated on a rotator overnight at 4°C. Next, GMBS-conjugated Cy5.5-MN were purified using a G-25 Sephadex PD- 10 column which was previously equilibrated with PBS. A stock of 10 μg/uL EPPT peptide was made in distilled water. 60 μL of EPPT solution was mixed with purified GMBS-conjugated Cy5.5-MN and incubated on a rotator overnight at room temperature. Finally, the EPPT-Cy5.5- 155 MN was purified using a G-25 Sephadex PD-10 column. The size and zeta potential of EPPT- Cy5.5-MN were determined by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, MA). The number of Cy5.5 molecules per nanoparticle was determined by spectrophotometry. The amount of peptide on MN was quantified by micro-BCA protein assay based on the protocol provided in the kit (Thermo Scientific, Rockford, IL). 4.2.3.3 In Vitro Cell Binding and Competitive Inhibition Assays Ovarian cancer cell line SKOV3 (expressing uMUC1 tumor antigen) and ES-2 (with low expression of uMUC1 tumor antigen), For assessment of the relative accumulation of experimental (EPPT-Cy5.5-MN) and control (Cy5.5-MN) nanoparticle probes within the ovarian cancer cells, the SKOV3 and ES-2 cell lines were seeded into 96-well plates and incubated with either EPPT- Cy5.5-MN or Cy5.5-MN at 0, 12.5, 25, 50, and 100 μg/ml Fe at 37 °C for 1 h (n = 3). Following the incubation, the accumulation of the probes was evaluated by Cy5.5 fluorescence using an IVIS spectrum imaging system. The Cy5.5 signal was presented as a picogram of iron and normalized to cell number using micro-BCA protein assay. Previously, the number of cells was estimated using a micro-BCA protein assay and an automated cell counter. For competitive inhibition assay, cell lines were seeded into 96-well plates and incubated with EPPT-Cy5.5-MN (100 µg iron/well) for 1.5 h alone or with increasing doses of free EPPT peptide (0–4730 nmol/L) added 1 h before incubation with the probe. After incubation, cells were washed three times with PBS, and fluorescence intensities were read and normalized in a process similar to binding assay. 4.2.3.4 Inhibition of miR-181a using transfection MicroRNA181a antisense oligonucleotides (5’-C*C*A-AAC-UCA-CCG-ACA-GCG- UUG-AAU-GUU*-C*A*C*-U-3’) and the corresponding scrambled oligonucleotides (5’- 156 A*C*C*-A*AG-CAC-ACA-GCU-GUC-UGU-AAU-GUC*-U*C*C*-A-3’) were (purchased from Eurogentec (Liege, Belgium), with "Phosphorothioate linkages" denoted by "*". A2780 (with low expression of mir-181a) and SKOV3 (with high expression of miR-181a) ovarian cancer cell lines. 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