Extracellular Vesicles in Colorectal Cancer: Mediators of tumor aggressiveness

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EXTRACELLULAR VESICLES IN COLORECTAL CANCER – MEDIATORS OF TUMOR AGGRESSIVENESS

Thesis for the degree of Philosophiae Doctor (PhD) by

Tonje Bjørnetrø

Department of Oncology, Akershus University Hospital Faculty of Medicine, University of Oslo

2019

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© Tonje Bjørnetrø, 2019

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-503-7

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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1 Acknowledgments

The work presented in this thesis has been conducted at the Department of Oncology at Akershus University Hospital and partly at the Department of Medical Biochemistry at Oslo University Hospital from August 2015 to February 2019. Financial support from the South- Eastern Norway Regional Health Authority (Helse Sør-Øst) and Akershus University Hospital Internal Strategic Research Funds is gratefully acknowledged.

I am grateful to Anne Hansen Ree, who has been my main supervisor and provided financial support for my PhD. Thank you for giving me the opportunity to be a part of such an exciting project and diverse research group. Thank you for your enthusiasm, positivity and especially for sharing your knowledge of the clinical perspectives. I appreciate that you trusted me with great responsibilities and independence, and that you inspired me with your brilliant writing skills.

I would also like to express my gratitude to my other co-supervisors. Kathrine Røe Redalen, thank you for always being positive and encouraging my research projects. Your admirable ability to collaborate has been essential in this work. Reidun Øvstebø, I am grateful for the support and appreciate you giving me the opportunity to be a part of your research group. Thank you for including me in your research project, guiding and sharing your knowledge, and for always finding time for my questions. My sincere thanks to my former co-supervisor Karianne Risberg Handeland. Your hard work, dedication and motivation inspire me, and your guidance, sense of detail and enthusiasm have been vital for this work. You gave me a flying start, it would not have been possible without you.

I am grateful to all my co-authors for their contribution, provision of biological material, clinical data, and scientific expertise. I would like to thank the present and former members of the Clinical and Molecular Oncology of ColoRectal Cancer research group; Janne S., Linda S., Imon B., Marius B., Siv S., Hanna A., Sebastian M., Kine B., Simer B., Paula B., Lise B., Hanne H., Dawn PB., Erta K., and Christin J. You have made colorectal cancer research exiting.

I appreciate your support and scientific discussions, and your inspirational effort to provide increased knowledge that can be beneficial for the patients. I would also like to thank the present and former members of The Blood Cell Research group; Beate V., Lilly S., Trude A., Kari Bente FH., Hans Christian Aa., Berit SB., Anne-Marie ST., Tine HS., and Marit H. It has been a delight to have someone to discuss EV-research with, but I have also appreciated the non- scientific discussions and all your support.

Finally, I would like to thank my friends and family for always supporting me and believing in me. Thank you for listening to me and reminding me of life outside research/the lab. My dear Michael, I could not have done this without your support, patience and love.

Oslo, February 2019 Tonje Bjørnetrø

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2 Abbreviations

AGO Argonaute AKT Protein Kinase B

APC Adenomatous Polyposis Coli BRAF B-Raf-Proto-oncogene CAIX Carbonic anhydrase 9

cDNA Complementary Deoxyribonucleic acids CEA Carcinoembryonic Antigen

CRC Colorectal Cancer CRT Chemoradiotherapy ECM Extracellular Matrix EV Extracellular Vesicle

EXO Exosome

EMT Epithelial to Mesenchymal Transmission HIF1 Hypoxia Inducible Factor 1

IL-1b Interleukin-1 beta IL-6 Interleukin-6 IL-8 Interleukin-8

IP-10 Interferon Gamma-Induced Protein 10 IPA Ingenuity Pathway Analysis

KRAS Kirsten Rat Sarcoma oncogene LARC Locally Advanced Rectal Cancer MCP-1 Monocyte Chemoattractant Protein-1 MET Mesenchymal to Epithelial Transmission MIP-1b Macrophage Inflammatory Protein-1beta miRNA Micro Ribonucleic Acid

MMP Matrix Metalloproteinase MRI Magnetic Resonance Imaging mRNA Messenger RNA

MVs Microvesicles

NTA Nanoparticle Tracking Analysis OS Overall Survival

O2 Oxygen

PCR Polymerase Chain Reaction PFS Progression Free Survival PI3K Phosphoinositide 3-Kinase

PTEN Phosphatase and Tensin Homologue ROS Reactive Oxygen Species

SAM Significance Analysis of Microarrays SEC Size Exclusion Chromatography TAMS Tumor Associated Macrophages TME Tumor Microenvironment TNF-a Tumor Necrosis Factor alpha TNM Tumor Node Metastasis TRG Tumor-Regression Grade ypTN Histological assesment of TN

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3 List of papers

Paper I

Bjørnetrø T, Redalen KR, Meltzer S, Thusyanthan NS, Samiappan R, Jegerschöld C, Handeland KR and Ree AH. An experimental strategy unveiling exosomal microRNAs 486- 5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer

Journal of Extracellular Vesicles 2019;8(1):1567219

Paper II

Meltzer S, Bjørnetrø T, Lyckander LG, Flatmark K, Dueland S, Samiappan R, Johansen C, Kalanxhi E, Ree AH, Redalen KR. Circulating exosomal miR-141-3p and miR-375 in metastatic progression of rectal cancer

(Submitted)

Paper II has been a part of another thesis (ISBN 978-82-8377-144-2)

Paper III

Bjørnetrø T, Ree AH, Steffensen LA, Brusletto BS, Olstad OK, Trøseid AM, Aass HCD, Haug KBF, Llorente A, Bøe SO, Lång A, Samiappan R, Redalen KR, Øvstebø R. Uptake of circulating extracellular vesicles from rectal cancer patients and the functional responses by human monocytes

(Manuscript)

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4 Introduction

The human body is comprised of a complex network of highly regulated tissues where communication at different levels has evolved to ensure homeostasis. For an organ to function properly, its cells have to communicate with their neighbors on site or more distantly. A cell needs to read the environment and adapt. Deregulation of homeostatic processes results in imbalance that can turn into disease. Abnormal growth of tissue can lead to cancer and ultimately secondary tumors in other organs, known as metastasis.

The blood is an important source of molecules released from cells all over the body. The work of this thesis comprises cell signaling factors enclosed in small packages (extracellular vesicles (EVs)), protecting them from degradation. EVs isolated from biofluids have emerged as an area of clinical interest as a “liquid biopsy” for monitoring of molecular tumor changes as a more convenient alternative to tissue biopsy [1].

The global burden of colorectal cancer (CRC) is expected to increase by 60 %, with 2.2 million new cases and 1.1 million deaths by 2030 [2]. CRC was the third most common cancer type and the second leading cause of cancer deaths worldwide in 2018. The highest colon cancer rates are found in parts of Europe, with Norway ranking first among females [3]. The pattern and trends of CRC incidence and mortality are still rising in low- and middle-income countries which might reflect the adoption to a more western lifestyle [2]. In highly developed countries the trend is stabilizing or decreasing [2]. The reasons for the high prevalence in the Norwegian population are not known, thus it is important to study several aspects of this disease.

Tumor hypoxia (oxygen (O2) deficiency) is recognized as a main mechanism in tumor resistance to therapy [4]. Because of the natural disease course of rectal cancer, frequently comprising heterogeneous primary tumors with predominant hypoxic regions, this specific entity is a unique model to explore the role of EVs as mediators of hypoxia in therapy resistance and metastasis.

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4.1 Cancer 4.1.1 Genetics

The development of cancer is a multistep process involving sequential genetic and epigenetic alterations resulting in cellular properties required for cancer development. Cancer develops as a result of alterations in oncogenes, tumor-suppressor genes and microRNA (miRNA) genes.

Chromosomal rearrangements, mutations, and gene amplification can cause activation of oncogenes that control cell proliferation, apoptosis or both, and increases the cell’s ability to survive or acquire growth advantages [5]. A mutated oncogene is analogous to a car still moving forward without the driver pushing the gas pedal. A mutation in a tumor-suppressor gene reduces the activity of a gene product that normally inhibits proliferation and tumor development. Such inactivation can be caused by chromosomal rearrangements and mutations or epigenetic silencing [6]. A mutated tumor-suppressor gene is analogous to a car that does not stop when the driver pushes the break. miRNA genes encode single RNA strands of approximately 21 to 23 nucleotides, with the function of regulating gene expression. miRNA genes can be up-regulated or down-regulated in human cancers, and function as oncogenes by down-regulating tumor-suppressor genes or as tumor-suppressor genes when down-regulating oncogenes. One miRNA can target several different genes, and the function of the miRNA is target- and tissue specific [5].

4.1.2 The hallmarks of cancer

Hanahan and Weinberg introduced the hallmarks of cancer in year 2000, proposing six fundamental traits acquired and shared by normal cells in the transformation into malignant cancers: self-sufficient proliferation, insensitivity to anti-proliferative signals, evasion of programmed cell death (apoptosis), unlimited replicative potential, sustained angiogenesis, and tissue invasion and metastasis [7]. These alterations in cell physiology allow cancer cells to survive, proliferate, and disseminate. In different tumors, the functions are acquired via distinct mechanisms and at different times during the multistep process of tumorigenesis. In 2011, four additional hallmarks were described, including two emerging ones: deregulation in cellular energetics and avoidance of immune destruction [8]. Among characteristics that make this process possible are genome instability in cancer cells and promotion of tumor progression by cells of the immune system (Figure 1) [7].

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Figure 1. Hallmarks of cancer. The ten hallmarks of cancer presented, as by Hanahan and Weinberg in 2011. Reprinted and adapted from reference [8] with the permission from Elsevier.

Apart from the malignant cells, a tumor recruits and interacts with non-transformed cells and creates the tumor microenvironment (TME). The major cell types found in TME are cells of the immune system, as well endothelial cells, fibroblasts, and pericytes carrying dynamic and often tumor-promoting events such as tumor angiogenesis, proliferation, invasion, and metastasis [9]. Also important is the extracellular matrix (ECM) that provides a scaffold for the cells and also contains growth factors [10]. The intercellular communication is driven by cytokines, chemokines, growth factors, and matrix remodeling enzymes, as well as the extracellular vesicles that have gained huge interest recently [9].

4.1.3 Metastasis

Metastatic spread of the disease is a leading cause of cancer death, with few effective treatment options [11]. The dissemination of carcinoma cells from the primary tumor and the formation of new tumor colonies in distant tissue involve a multistep process with local invasion of the primary tumor cells through the basement membrane, intravasation into the blood vessels or the lymphatic system, followed by extravasation, implantation and tumor cell proliferation at a distant site. Epithelial-to-mesenchymal transition (EMT) represents an important process

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enabling these steps where the cancer cells acquire multiple malignant traits associated with loss of epithelial properties (such as E-cadherin) and gain of mesenchymal properties (increased motility, invasiveness, and ECM degradation) coordinated by master EMT transcription factors such as Snail, Slug and ZEB-1 [12]. In the process of metastasis, the primary tumor cells are also known to induce the formation of microenvironments in distant organs, termed pre- metastatic niches. The formation of pre-metastatic niches results from tumor-secreted factors and extracellular vesicles, which involves induction of vascular permeability^,remodeling of stroma and ECM, and recruitment of immune cells to the target organ [13]. In the metastatic cascade, components such as hypoxia, inflammation and the TME all play a role in expression of malignant and invasive phenotypes [14].

4.2 The tumor microenvironment 4.2.1 Tumor hypoxia

Tissue hypoxia results from an inadequate supply of oxygen that compromises biological functions and tumor hypoxia is associated with tumor propagation, malignant progression, and resistance to therapy [15]. In solid tumors, oxygen delivery is often reduced or abolished (either acutely or chronically) because of structural abnormalities of tumor microvessels, disturbed microcirculation, and increased diffusion distances (Figure 2) [15]. Severe and prolonged hypoxia is toxic to both cancer cells and normal cells. However, in a hypoxic environment, cancer cells undergo genetic and adaptive changes affecting the biology of the tumors with repression of apoptosis by altering pro-survival gene expression, genomic instability, and EMT [4]. Hypoxic cells undergo a shift from aerobic to anaerobic metabolism [16], produce growth factors that induce angiogenesis [17] as well as promoting tissue invasion and metastasis [18], all of which contribute to tumor progression. The molecular mechanisms of response to hypoxia are complex, and a master regulator of the hypoxic response is the transcription factor hypoxia inducible factor 1 (HIF-1) [19]. The subunits HIF-1a and HIF-1b dimerize and bind to hypoxia response elements of target genes and orchestrate the expression of a myriad of genes thought to be critical for adaptation to hypoxia [20]. The stability of the HIF-1a protein is controlled by the oxygen level; under normoxic conditions HIF-1a interacts with the von Hippel-Lindau protein which activates the ubiquitin ligase system and marks HIF-1a for degradation, whereas under low oxygen conditions the dimerization with HIF-1b is stabilized [21].

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Figure 2. Schematic representation of characteristics of tumor tissue and vasculature. The abnormal vasculature network in cancers is the result of an imbalance of pro- and anti-angiogenic signaling within different parts of the tumor. The tumor vasculature has abnormal structure with heterogeneous blood flow that can lead to hypoxia. Blood vessel leakiness leads to extravasation of macromolecules to reach the tumor cells and contributes to a high interstitial pressure in tumors. Tumor cell growth is rapid near the vessels, while deprivation of nutrients and oxygen occurs in tumor cells at or beyond the diffusion limit leading to regions of necrosis. Reprinted from reference [22] with permission from Ivyspring International Publisher (open access).

Tumor hypoxia is associated with poor prognosis and resistance to therapy in cancer, as both radiation and systemically administered cytotoxic drugs depend on oxygen and stable blood flow for efficient eradication of tumor cells [23]. Under hypoxia, oxygen depletion disrupts the intracellular reactive oxygen species (ROS) production, which is essential for destroying cells by therapeutic ionizing radiation. HIF-1 also plays a role in radioresistance by activating genes involved in apoptosis (by activating anti-apoptotic genes and inhibiting pro-apoptotic genes), metabolism (by activating genes related to glycolysis), proliferation, and neovascularization [4]. Furthermore, hypoxic tumor cells do not receive sufficient chemotherapeutic agents because of abnormal vascularization and the distance from the capillaries. HIF-1 regulates drug

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efflux genes such as the multidrug resistance protein-1 (the MDR1) and some miRNAs regulating both chemoresistance and other cellular processes including angiogenesis, apoptosis, proliferation and metastasis [4]. Also, a hypoxic TME supports protumor inflammatory responses and suppresses antitumor responses, thus enhancing the immune tolerance [24].

4.2.2 The immune system with emphasis on monocytes

Immune cells that infiltrate tumors include cells of the innate immune system (including macrophages, neutrophils, mast cells, and myeloid-derived suppressor cells, dendritic cells, and natural killer cells) and adaptive immune cells (T and B lymphocytes) [25]. The immune system play a dual role in cancer, as it can both antagonize and enhance tumor development and progression [8], dictated by the expression of immune mediators and modulators as well as the abundance and activation stage of the different cell types in the TME [25].

Monocytes are derived from hematological precursors in the bone marrow and enter the blood circulation before typically being recruited into tissues throughout the body [26]. Circulating monocytes have a half-life of 1-3 days in the blood [27]. Monocytes show plasticity and can differentiate into different cell types, such as macrophages and dendritic cells, dictated by the tissue microenvironment. In response to infection, they are essential in pathogen clearance and their main immune functions are phagocytosis, antigen presentation, and production of inflammatory mediators [26, 28]. Monocytes have important functions in tissue homeostasis including phagocytosis of apoptotic cells, production of growth factors, and induction of angiogenesis. Tumor-infiltrating monocytes give rise to tumor-associated macrophages (TAMs), which together with T cells, are the most common immune cells in the TME [29]. In general, TAMs can be polarized to the classically activated M1 phenotype, associated with killing responses and tumor suppression, or alternatively activated M2 phenotype that promotes tissue repair and tumor growth, and both are probably present in a mixed pool in the tumor bed in vivo [30]. Cytokines are soluble factors that can be divided into functional classes based on their biological properties; some promote leukocyte growth and differentiation, other pro- or anti-inflammatory properties. Today the term cytokine encompasses interferons, interleukins (IL), the chemokine family, and the tumor necrosis factor family (TNF) among others [31].

Cytokines produced by both the tumor cells, and tumor-associated leukocytes and platelets, may also contribute to the progression of cancer, with many of them induced by hypoxia [32].

Macrophage infiltration is used as an independent prognostic factor in various solid tumors;

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macrophage presence is usually associated with poor prognosis as observed in breast-, prostate- and ovarian cancer [33]. In CRC cases, the role of TAMs is controversial with a few studies showing beneficial associations with TAMs, although recent studies show that TAMs facilitate CRC growth and do not support tumor suppressive activities [34]. Circulating monocyte counts are associated with tumor infiltrating macrophages [35, 36], and the pre-operative monocyte level correlates with disease burden and overall survival in CRC [37, 38].

4.3 Colorectal cancer

4.3.1 Colorectal cancer development and risk factors

CRC is cancer that originates in the large bowel (colon and rectum). In 2016 in Norway, 4343 men and women were diagnosed with CRC, where 1340 (approx. 30%) were located in the rectum. The CRC incidence rates have increased the last decades, but rectal cancer has levelled off since 1990s [39]. Although the increase is not completely understood, CRC has been associated with western lifestyle, with moderately increased risk of increased body mass index, cigarette smoking, low vegetable and fruit consumption, and lack of physical activity [40].

More effective therapies and better disease management have contributed to improved survival rates over the last decades, with an increase of five-year survival from 40% in the late 1970s to almost 70% in 2016. However, patients with metastatic disease have a five-year overall survival of approximately 16%, giving a considerably poor prognosis for these patients [39].

A family history of CRC in first-degree relatives and a history of inflammatory bowel disease are risk factors with biological causes for CRC [40]. CRC typically develops from benign precancerous polyps; fortunately, few of these polyps acquire malignant features and become cancerous. There are two types of polyps; adenomas and sessile serrated polyps. The progression from polyp to cancer is the result of an accumulation of genetic and epigenetic changes. About 5% of the CRC cases are associated with truly inherited mutations, with most tumors appearing sporadically. 65-70% of the sporadic cancers are associated with the chromosomal instability pathway characterized by a cascade of accumulating mutations. This pathway is generally associated with adenomas, with the APC gene being the first mutation, which affects chromosome segregation in dividing cells. A KRAS mutation subsequently follows, leading to cell growth, differentiation, motility, and survival. TP53 is a tumor- suppressor gene mutated in many types of cancer, and is a master regulator of apoptosis and transcription, and over time results in carcinogenesis [41]. The sessile serrated polyps tend to develop by a pathway starting with mutations in the BRAF gene, altering the growth and loss

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of apoptosis. KRAS mutations can occur, but more frequently hypermethylation of the gene promoter region is observed, leading to inhibition of gene transcription affecting many different genes. Microsatellite instability can occur in both polyp types, where genes involved in the DNA mismatch repair functions are affected [41].

4.3.2 Staging of colorectal cancer

Staging of cancer is used to classify patients into different treatment strategies. The guidelines of Tumor-Node-Metastasis (TNM)-staging (Table 1) categorize the cancer according to the size and extent of the primary tumor (T), involvement of regional lymph nodes (N), and confirmed distant metastasis (M) [42], essentially pertaining to any solid tumor type.

Table 1. TNM-staging of CRC. 8^TH edition 2017 [42].

TNM clinical classification T-status

TX Primary tumor cannot be assessed T0 No evidence of primary tumor

Tis Carcinoma in situ: invasion of lamina propria T1 Tumor invades submucosa

T2 Tumor invades muscularis propria

T3 Tumor invades subserosa or into non-peritonealized pericolic or perirectal tissues T4 Tumor directly invades other organs or structures and/or perforates visceral peritoneum T4a Tumor perforates visceral peritoneum

T4b Tumor directly invades other organs or structures N-status NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in 1-3 regional lymph nodes N1a Metastasis in 1 regional lymph node N1b Metastasis in 2-3 regional lymph nodes

N1c Tumor deposit(s) in subserosa, or in non-peritonealized pericolic or perirectal soft tissue without regional lymph node metastasis

N2 Metastasis in 4 or more regional lymph nodes N2a Metastasis in 4-6 regional lymph nodes N2b Metastasis in 7 or more regional lymph nodes

M-status M0 No distant metastasis

M1 Distant metastasis

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4.3.3 Diagnosis and treatment of rectal cancer

Digital rectal examination and endoscopy with biopsy for histopathological confirmation are essential for diagnosis, and tumors located £ 15 cm orally of the anal verge (measured by a rigid scope) are classified as rectal cancer. More orally located tumors are classified as colon cancers [42]. Magnetic resonance imaging (MRI) is the preferred modality for tumor and lymph-node staging in rectal cancer [43]. In addition, a physical examination with full blood count, liver and renal function tests, serum carcinoembryonic antigen (CEA), and computed tomography (CT) scan of the thoracic and abdominal cavities are carried out in order to determine the clinical status of the patients and presence of metastatic disease [42].

Improved preoperative assessment, surgical quality, and use of pelvic radiotherapy have reduced the local recurrence from historically about 25% to about 5-10% [44]. Total mesorectal excision is the recommended surgical technique for rectal cancer, first described in 1982 [45], and involves removal of the primary tumor and all of the mesorectal tissue along the mesorectal fascia [45]. Locally advanced rectal cancer (LARC) is defined as a tumor that grows beyond the rectal wall to such an extent that it is close to or infiltrates surrounding organs or structures [43]. Therefore, neoadjuvant (preoperative) radiotherapy, often with the addition of radiosensitizing chemotherapy, is recommended to shrink and down-stage the tumor in order to enable radical surgery [46].

Similar improvement, has not been observed for distant metastasis, which is the major cause of death in rectal cancer patients [44]. Approximately 25% of patients with CRC have metastasis at time of diagnosis, and about 25% develop metastatic spread during follow up. Metastases to the liver and lungs are the most common locations [47]. Surgical resection of metastasis, either before or after systemic treatment (with chemotherapy and targeted therapy), offers the best chance for a cure. However, most of the patients have more advanced and unresectable metastatic disease, and curative surgery is thus not a realistic goal. For them, systemic treatment prolongs survival [48].

4.3.4 Response evaluation

The tumor response to neoadjuvant treatment is an established prognostic indicator in LARC.

Distant relapse strongly correlates with survival outcomes, and progression-free survival (PFS) and overall survival (OS) are appropriate primary clinical trial endpoints [49].

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OS is defined as time to death by any cause and is considered the most convincing measurement of clinical benefit; however, it requires large patient numbers and long follow-up time. PFS is defined as time until tumor progression by any cause (primary tumor or metastasis) or death.

Since patients usually develops local recurrence or metastatic spread before death, patients PFS is shorter than OS [50]. These long-term endpoints are generally analyzed after years of monitoring, and surrogate endpoints are needed to more rapidly test hypotheses and advance clinical improvement [49]. Histopathological assessment of tumor response to the neoadjuvant therapy is defined as ypTN stage [49]. A pathological complete response is defined as no residual viable tumor at histological examination (ypT0) and serves as an accurate surrogate endpoint for local control [51, 52]. Tumor-regression grade (TRG) is another system for assessment of histological response after neoadjuvant therapy and is based on the presence of residual tumor cells and the extent of fibrosis. In the College of American Pathologist classification, the scale goes from grade 0 where there is no residual tumor (pathological complete response, ypT0, TRG0) to absence of any tumor regression (TGR3) [53]. TRG scoring systems have been criticized for poor reproducibility among pathologists, and do not take into account lymph node assessment [49].

4.4 Extracellular vesicles

4.4.1 Extracellular vesicle biogenesis and nomenclature

Cells secrete membrane-derived vesicles into their milieu. These extracellular vesicles (EVs) serve as vehicles of intercellular communication and transfer of proteins, lipids, and nucleic acids and thereby influence the physiology and pathology of the recipient cells [54]. The secretion of EVs into the extracellular environment appears as an evolutionarily conserved process found in eukaryotic and prokaryotic cells [54]. At least three subgroups of membrane- contained vesicles are released from cells: apoptotic bodies, microvesicles, and exosomes [54, 55]. Apoptotic bodies are released when plasma blebbing occurs during apoptosis. This process is not discussed in this thesis. Microvesicles (MVs) are directly pinched off the plasma membrane and have different sizes, typically ranging from 100-1.000 nm in diameter.

Exosomes are approximately 30-100 nm intraluminal vesicles contained in multivesicular bodies and are released upon fusing with the plasma membrane (Figure 3) [56]. Assessment of an EV to a particular biogenesis pathway is difficult except with the use of live imaging techniques and tracing of the EVs [57]. The overlapping range of size, similar morphology, and variable composition challenges the attempts to classify the subgroups of EVs [58].

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The current available isolation techniques of EVs from conditioned media from cell cultures or biological fluids of the body often recover a heterogeneous mix of vesicles [59]. The term EV is an umbrella term for all vesicle types released from cells, and since consensus has not yet emerged on specific markers of EV subtypes, the use of the term EV is urged [57, 60]. The term exosomes was adopted in the late 1980s for 30-100 nm-sized vesicles of endosomal origin and is the most extensively used term in the literature [61]. In this thesis, the terms that are used mainly reflect the usage in the relevant references, or collectively called EVs. In the articles included in this thesis, the terms that are used reflect the vesicle types intended to be isolated by the method or kit.

Figure 3. Schematic representation extracellular vesicle biogenesis and secretion. Exosomes are

generated within the endosomal system, initially as intraluminal vesicles by budding into early endosomes, generating multivesicular bodies (MVB) that either fuse with lysosomes for degradation or with the plasma membrane with the resulting release of exosomes into the extracellular milieu. Microvesicles arise with outward budding and fission of the plasma membrane mediated by redistribution of phospholipids and contraction of cytoskeletal proteins. Reprinted from reference [56] with permission from Gustafson, Veitch and Fish (open access).

4.4.2 Molecular properties of extracellular vesicles

The lipid-enclosed extracellular vesicles contain a cargo, including nucleic acids (both RNA and DNA), lipids, and functionally active proteins (Figure 4). EVs from different cellular

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origins contain cell-type-specific proteins and a distinct repertoire of molecules essential for their biogenesis, structure, and trafficking [62]. Exosomes typically contain proteins from the endosomal compartment, such as annexin and flotillin that are important in transport and fusion, tetraspanins and integrins involved in cell targeting, Alix and TSG101 involved in biogenesis from multivesicular bodies (MVB), and other proteins involved in lipid metabolism. Many exosomes also contain the cell surface proteins of the major histocompatibility complexes (MHCs) that are involved in antigen binding and presentation [63]. The exosome membrane contains cholesterol, sphingomyelin, ceramide, and lipid rafts associated with several proteins [59]. The main MV protein markers are integrins, selectins and CD40, and their membrane contains larger amounts of cholesterol and phosphatidylserine compared to exosomes [59].

Some of the proteins mentioned above are typically used for the characterization of exosomes but are commonly found on all EVs. An updated version of “Minimal Information for Studies of Extracellular Vesicles 2018” reflects an evolved understanding of the subtypes of EVs, and consequently does not propose markers that could characterize specifically each subtype [57].

Figure 4. Exosome. a) Cryo-electron microscopy of an extracellular vesicle isolated from plasma from a rectal cancer patient (original, unpublished data.) b) Schematic representation of an exosome. The vesicle is surrounded by a phospholipid bilayer and contains different transmembrane-proteins and carries a cargo of proteins, lipids, and nucleic acids that can be distinct from those of the cell of origin. Reprinted from reference [64] with permission from Yang, Li, Zhang and Wang (open access).

Extracellular RNA may be enclosed in EVs, bound in protein complexes, or free in the circulation [54]. Functionally active mRNA in EVs was first described in 2006 [65]; however, EVs have now been shown to contain RNA in different forms: long-non-coding RNAs, miRNAs, and ribosomal RNA among others [66]. miRNAs are short (~22 nucleotides),

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endogenous, non-coding single-stranded RNAs regulating gene expression post- transcriptionally. miRNAs are transcribed as primary miRNA (pri-miRNAs) and follows a two- step process with nuclear and cytoplasmic cleavage events by RNase III endonucleases. The pri-miRNA is processed by Drosha into hairpin precursors (pre-miRNA), that are exported to the cytoplasm and cleaved by Dicer into mature-miRNAs. One strand is associated with argonaute (AGO) proteins and is loaded into the miRNA-inducing silencing complex (miRISC) for mRNA target regulation (Figure 5) [54]. Mature miRNAs originate from the 5′ arm or the 3′ arm of the precursor miRNA and are denoted with a -5p or -3p suffix [67]. Mature functional miRNAs loaded in RISC bind complementary sequences usually at the 3′-untranslated region of mRNAs, promoting mRNA degradation and/or repressing their translation, thus reducing the production of proteins [68]. Multiple miRNAs can target the same mRNA and have profound effects in the target cells regulating the development and progression of various human diseases [68].

The process of miRNA loading into exosomes is not fully understood. The enzyme neutral sphingomyelinase 2 was the first molecule reported to be involved in miRNA secretion into exosomes [69]. Specific binding motifs at the 3´-portion of exosomal miRNA are recognized by individual proteins, such as sumoylated heterogeneous nuclear ribonucleoproteins hnRNPA2B1 and hnRNPA1, and selectively incorporated into exosomes [70]. miRNAs that are preferentially sorted into exosomes have also been shown to have more uridylated than adenylated 3´ end [71]. Taken together, specific sequences in certain miRNAs may guide their incorporation into exosomes. Other modes for sorting include enzymes and proteins in miRISC related pathways. The main components of the miRISC complex is miRNA, mRNA,GW182, and AGO proteins, which form P-bodies that are involved in RNA turnover. These P-bodies fuse with multivesicular bodies, and miRISC co-localizes with the site of exosome biogenesis.

Studies have shown a possible correlation between AGO2 and miRNA-sorting [72-74], summarized in Figure 5. In addition to play a role in the inward budding of endosomes and vesicle sorting, the endosomal sorting complex required for transport (ESCRT) has also been indicated to be involved in the sorting of miRNAs into exosomes. Knock-down of ESCRT genes or blocking of endosomal formation impair miRNA activity [75].

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Figure 5. Schematic representation of miRNA-sorting into exosomes. miRNAs are transcribed as pri- miRNAs and processed to pre-miRNAs that are exported to the cytoplasm where they are digested to mature miRNAs. Four potential sorting mechanisms for mature miRNAs into exosomes are shown: 1) nSMase2-dependent pathway 2) miRNA motif and sumoylated hnRNPs-dependent pathway 3) 3´-miRNA sequence dependent pathway 4) miRISC-related pathways. See text for details. Reprinted from reference [76] with permission from Elsevier (open access).

4.4.3 Uptake mechanisms

EVs induce functional effects in target cells by docking onto recipient cells and initiating signaling pathways or promoting phenotypic changes dependent on the cargo upon internalization [77]. The mode of vesicle interaction with the cell surface and mechanism of transferring vesicle cargo are still incompletely characterized. Tetraspanins, integrins, lipids, and ECM components are involved in target cell specificity [78]. The internalization may be via clathrin-mediated or clathrin-independent endocytosis, such as micropinocytosis, phagocytosis, or endocytosis via caveolae and lipid rafts (Figure 6) [78]. Internalized vesicles may reach multivesicular endosomes targeted to the lysosome or escape digestion and release their content into the cytoplasm of the recipient cell. EVs can also be recycled and released in the extracellular space. EVs that fuse directly with the plasma membrane of recipient cells can release their intraluminal content in the cytoplasm [79]. The fusion with the plasma membrane or endocytic membrane is a key step to support the release of miRNAs and mRNAs in order to regulate gene expression, as well as in the exchange of lipids and transmembrane proteins. The degradative pathway would provide metabolites to the recipient cells (Figure 6) [78].

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Figure 6. Schematic representation of interactions of extracellular vesicles and recipient cell.

Extracellular vesicles can bind to the surface of the cells and initiate intracellular signaling pathways or be internalized by multiple pathways. Inside the cell, they will reach early endosomes and likely mix with endogenous intraluminal vesicles (ILVs). By fusion with the plasma membrane or the membrane of multivesicular endosomes (MVE), extracellular vesicles can release their intraluminal content, however the process is currently poorly understood. Reprinted from reference [78] with permission from Springer Nature.

4.4.4 Function

When first discovered, the primary role of EVs was attributed to a clearance system for unnecessary proteins; however, important roles in cell-to-cell communication have later been demonstrated. The generation of EVs can be induced by many factors, including microbial attack and other stress conditions [80]. Since EVs carry information from their cell of origin, they have emerged into the field of biomarker discovery [63]. In contrast to a single-molecule signal, EVs have the potential to affect several signaling pathways inside the recipient cells.

The release of EVs can promote phenotypic changes and synchronize the physiologic state of the surrounding cells in a paracrine manner [81]. In normal physiological processes, EVs are involved in pregnancy, tissue repair, immune responses, and blood coagulation [77].

EVs are also involved in disease processes, and EVs are potential contributors in tumor development and highly involved in many of the hallmarks of cancer [82]. An elevated level of circulating exosomes has been observed in CRC patients compared to healthy controls, and high levels of exosomes in plasma of patients correlated with parameters of poor prognosis [83]. The distinct mechanisms and signaling pathways that EVs participate in during tumorigenesis, progression, and drug resistance have been studied extensively. In CRC, exosomes derived from tumor cells seems to be implicated in different processes such as modifying the TME, facilitating EMT and cytoskeleton reorganization, promoting tumor

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angiogenesis, and influencing tumor immunity, which result in the formation, invasion, and metastasis of cancer cells (Figure 7) [84].

Figure 7. Multiple roles of exosomes in CRC. Exosomes derived from CRC cells can activate critical oncogenic signaling pathways to promote tumor transformation and progression, invasion, and metastasis as well as therapy resistance. The figure is inspired by reference [84].

Elevated exosomal function has been suggested to occur during the adenoma-carcinoma sequence of CRC [85]. EVs from cancer cells containing oncogenic mRNA or miRNAs and proangiogenic proteins are able to alter epithelial cells with a non-malignant phenotype to gain properties essential for tumor initiation [84]. For example, the KRAS status of the cell dramatically affects the composition of the proteome in exosomes. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including mutant KRAS-protein, that could increase the growth of wild-type KRAS-expressing cells [86]. A recent study showed increased cellular glucose uptake and increased growth in recipient cells after stimulation with exosomes from KRAS mutated cells where functional exosomal glucose transporter 1 (GLUT- 1) contributed to the metabolic changes [87]. Exosomes from mutant KRAS cells have also up- regulated levels of miR-100, suggesting that KRAS could participate in selective loading of miRNAs [88]. Taken together, mutant KRAS influence the composition and function of CRC exosomes.

In the case of EVs derived from hypoxic cancer cells, evidence for the transfer of a hypoxia tolerance phenotype has been described [89]. Under hypoxic conditions, CRC derived

KRASEGFR

Wnt/β-catenin TGF-β MMPs

EMT (Snail, Cadherin, ZEB)

Recipient cell

Transformation

& Progression Metastasis

& Invasion Chemotherapy

resistance CRC

exosome

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exosomes were enriched with Wnt4 mRNA in a HIF-1a dependent manner and increased the nuclear translocation of b-catenin in endothelial cells, which is a common CRC phenotype. The induction of Wnt/b-catenin signaling promoted proliferation and migration of the endothelial cells in vitro. In animal studies, tumor promoting effects of CRC cell-derived exosomes comprised enhanced tumor growth and angiogenesis [90]. EVs may contribute to cancer progression via the intercellular network. Data from in vitro and in vivo studies have shown that EVs increased the proliferation rate and resistance to apoptosis in fibroblasts, epithelial and endothelial cells [91]. CRC-derived MVs, containing miR-92a [92] as well as miR-1246 [93], could promote proliferation, migration, and tube formation of endothelial cells promoting angiogenic activity [92, 93]. A recent study observed that most of the miRNAs sorted into exosomes have tumor-suppressive activity (miR-10a-5p, miR-193a-3p, mir-200b-5p), whereas miRNAs with oncogenic effects (miR-196a/b, miR-181d-5p, miR-155-5p) were retained in the tumor cells, resulting in higher levels of oncogenic miRNAs in the tissue compared to exosomes [94]. Further, elevated levels of miR-193a in plasma exosomes were observed in colon cancer patients compared to healthy controls and circulating levels of miR-193a were elevated in advanced stage colon cancer [94].

The metastatic capability can be transferred via tumor-derived exosomes from cells with high metastatic potential to cells with low metastatic potential [82]. Metastasis-associated miRNAs, including miR-21, miR-192, and miR-221 has been detected in exosomes from CRC cell lines [95]. EVs may also be involved in the metastatic process by carrying molecules involved in EMT [96, 97]. For example, miR-210 contribute to EMT and metastatic potential and is frequently up-regulated in CRC. In an in vitro model of spontaneous metastasis of colon cancer cells, miR-210 was highly expressed in exosomes derived from the adherent cells and taken up by neighboring metastatic cells. The adhesion dynamics of metastatic cells was influenced by exosomal miR-210. Paracrine signaling from the adherent growing cells could sustain the invasive capacity after EMT, inhibiting the mesenchymal to epithelial transition (MET). Taken together, exosomes containing miR-210 mediated cross-talk between primary cancer cells and disseminated cells and might redirect metastatic cells to free new sites of dissemination [98].

In CRC patients, distant metastasis appears mainly in the liver and the lungs and less commonly at other sites such as the bones or the brain. Organ tropism is the proposed phenomenon where some types of cancer cells preferentially home and colonize in specific organs. However, the molecular mechanisms are not well understood [84]. Exosomes have been shown to facilitate

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metastasis by generating a pre-metastatic niche [99, 100]. Exosomes from lung-, liver- and brain-tropic tumor cells homed preferentially to resident cells at their predicted metastatic site, determined by their integrin expression pattern [100]. Tumor-derived exosomes in combination with cytokines and other soluble factors can recruit bone-marrow derived cells to pre-metastatic tissue and contribute in the modulation of the microenvironment for tumor establishment.

Highly metastatic pancreatic ductal adenocarcinoma exosomes induced liver pre-metastatic niche formation in mice and increased the liver metastatic burden, probably through the transfer of macrophage migration inhibitory factor (MIF) [99]. The uptake of exosomes caused transforming growth factor beta (TGF-b) secretion and a fibrotic microenvironment, recruiting bone marrow-derived macrophages [99]. Quantitative proteomics analysis of serum-derived exosomes from patients with primary CRC and healthy controls discovered that exosomes from CRC patients contain several proteins that are typically implicated in the processes for pre- metastatic niche establishment, and suggested to take part in ECM remodeling, cell communication, signal transduction as well as enhancement of vascular permeability and tumor-promoting inflammation [101]. These exosomes significantly increased the invasion and migration capability of CRC cell lines compared to circulating exosomes from healthy volunteers [101].

Chemotherapeutic agents can be transported out of the cells within the vesicles or active efflux mechanisms [102, 103]. EVs also bind monoclonal antibodies in the peripheral circulation reducing its bioavailability and redirecting the drugs away from the cancer cells [104]. The cargo of EVs can also potentiate resistance to chemotherapy [105]. For CRC cells different mechanisms have been documented, such as extracellular disposal of the tumor-suppressor miRNAs miR-145 and miR-34a [106], or increased levels of circulating onco-miRNAs such as miR-196b-5p, which have all been observed to maintain cancer stemness and chemoresistance [107], or transfer of proteins with oncogenic potential converting them to drug resistant phenotypes by increasing the clonogenic capacity [108].

The immunomodulatory role of tumor-derived EVs involves immune cells both in the TME and circulation. The immune cells are educated by the tumor-derived EVs, and the uptake can either result in escape from the immune response or activation of immune suppression [109].

CRC-derived EVs are capable of affecting monocyte/macrophage differentiation with a general trend towards M2 polarization. However, a mixed M1/M2 has been shown and can potentially be explained by several factors, such as timing of the exposure and the source of EVs. This

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points to the functional effects of exposure of EVs from early developing cancer cells being very different to those from metastatic cells [110]. CRC-derived EVs have also been shown to attenuate monocyte differentiation to dendritic cells as well as induce apoptosis of T-cells, circumventing their anti-tumor activity and promoting pro-tumor activities [110].

4.5 Exosomes - colorectal cancer biomarkers

The definition of a biomarker is any measurement variable that is associated with health or disease outcome [111]. Biomarkers can, for instance, serve as an assessment tool for risk stratification and early detection (diagnostic biomarkers), evaluation of disease progression of the disease (prognostic biomarkers), or prediction of treatment response (predictive biomarkers), as well as monitoring of disease recurrence (surveillance biomarkers) [112]. The sensitivity of a biomarker is the ability to detect the disease in a patient where the disease is truly present (i.e. a true positive), whereas specificity is the ability to rule out the disease in a patient with no disease (i.e. a true negative) [113]. To date, RAS and BRAF mutations as well as DNA mismatch repair deficiency are biomarkers measured in the clinical practice for CRC patient risk stratification and guide the choice of therapy [114]. Only a few biomarkers have been proven to be clinically relevant and non-invasive liquid biopsies are an intriguing source for exploring new biomarkers [115].

Currently, available blood-based CRC biomarkers in the clinics include CEA; a circulating protein maker used to monitor response to treatment and tumor recurrence, and carbohydrate antigen 19-9 (CA 19-9); a marker used in therapy monitoring [116]. For surveillance and monitoring of post-resected CRC patients, the sensitivity of CEA ranges from 41% to 97% and specificity from 52% to 100%, depending on threshold-values. Further, an increase in CEA does not occur in 20% of patients with true recurrence (false negatives) [117]. Moreover, high levels of CEA are not specific to CRC, but can also occur in other malignancies [116] and smokers.

EVs have received increased interest as probable diagnostic biomarkers for cancer and other human diseases [1]. miRNAs are attractive blood-based cancer biomarker candidates because of their high abundance and stability in patient samples [118]. Although circulating miRNAs have proved to be promising diagnostic and prognostic biomarkers in CRC, it is plausible that their expression in EVs will add another layer of specificity [115].

Serum exosomal CEA could achieve higher specificity and sensitivity compared to total

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circulating CEA in detecting distant metastasis in CRC patients [119]. One of the first studies to comprehensively study exosomal miRNAs for early detection of CRC showed 16 miRNAs up-regulated in both colon cancer patients and colon cancer cell lines. The serum level of seven miRNAs, let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, were higher in CRC patients, including those with early stage disease, than in healthy controls, and further down-regulated after surgical removal of their primary tumor [120]. Notably, exosomal miR- 1229 showed high sensitivity for early-stage patients (95%) compared to CEA. In addition, for early diagnosis, a panel of four serum exosomal miRNAs, miR-19a-3p, miR-223-3p, miR-92a- 3p, and miR-422a, could successfully differentiate CRC from colon adenoma and healthy control cases [121]. An exosomal miR-17-92a cluster has been identified as a marker of early detection of recurrence of CRC, and the expression of exosomal miR-19a was especially up- regulated in both early and advanced stages of CRC and high expression was identified as a prognostic biomarker [122].

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5 Aims

The overall objective of this thesis was to characterize the role of EVs in tumor hypoxia and thus as mediators of metastasis in colorectal cancer patients, in order to improve the biological insights into cancer aggressiveness.

EVs (exosomes and MVs) are shed from most cells under normal and pathological conditions.

They are released into the surrounding milieu and can be found in almost all body fluids such as plasma, urine, and saliva, as well as in solid tissue. EVs are a rich source of miRNAs, which function in recipient cells by regulating post-transcriptional gene expression. In cancer, the miRNA-profile is often altered and exosomal miRNAs can act as biomarkers. We therefore hypothesized that circulating vesicles have a cargo that carries information about hypoxia, cancer aggressiveness, and treatment resistance. In this project, we aimed to:

1. Isolate and characterize hypoxic exosomes in CRC cell lines and plasma from rectal cancer patients, and investigate their impact on tumor aggressiveness and resistance to therapy, specifically aiming to identify hypoxia-specific exosomal miRNAs (Paper I).

2. Identify plasma exosomal miRNAs associated with tumor aggressiveness and outcome of chemoradiotherapy (CRT) in rectal cancer patients (Paper II).

Tumor-derived EVs are important players in the modulation of immune cell activity. Blood borne monocytes may be influenced by the EV content and further affect end-organ target cells or be part of the communication between tumor cells and TME cells. This phenomenon is fundamental in CRC progression, and therefore the final aim of this thesis was to:

3. Characterize the uptake and response by cultured human monocytes of EVs (both exosomes and MVs) from a CRC cell line and rectal cancer patients’ plasma (Paper III).

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6 Summary of the papers

6.1 Paper I

An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer

In this paper, we used five CRC cell lines exposed to hypoxia as a model system to detect hypoxia-regulated exosomal miRNAs. The cells were incubated in a hypoxic chamber (0.2%

O2) for 24 hours and exosomes were isolated using sequential centrifugation. A cancer specific miRNA panel was used to identify candidate miRNAs. The exosomal miRNAs showed cell- line specific profiles, however, many of the most abundant miRNAs were shared among all the cell lines. Using a set of criteria, the same miRNAs were analyzed in exosomes precipitated from plasma from rectal cancer patients and correlated to baseline disease characteristics.

36 miRNAs were regulated by hypoxia in vitro and based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments as well as the prevalence in patient plasma, six miRNAs were further analyzed in plasma exosomes. Of these, the level of three miRNAs, miR-486-5p, -181a-5p, and -30d-5p, correlated with characteristics of high-risk rectal cancer. Low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumor and lymph node metastases. Additionally, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression.

All three miRNAs have previously been associated with tumorigenesis and have been shown to be regulated by hypoxia and associated with exosomes. In conclusion, our findings suggested that oxygen-sensitive miRNAs in exosomes circulating in plasma of rectal cancer patients may be potential markers for cancer aggressiveness and tumor hypoxia.

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6.2 Paper II

Circulating exosomal miR-141-3p and miR-375 in metastatic progression of rectal cancer

Further focusing on the circulating exosomal miRNA profile of rectal cancer patients, this article included the patients in the first paper in addition to five patients with metastatic disease at time of diagnosis. Exosomes were isolated from plasma (sampled at baseline) using miRCURYÔ Exosome Isolation Kit, and the same miRNA panel as in Paper I was used to identify candidate miRNAs associated with metastasis.

The expression of plasma exosomal miRNAs was correlated with clinical traits of aggressiveness, and CRT outcome. Using the Significance Analysis of Microarrays (SAM) software, we selected miRNAs associated with clinical endpoints. We found candidate miRNAs miR-141-3p, -375, -301a-3p, -423-5p, -431-5p, and -20b-5p associated with metastasis and TRG. Thereafter, we validated a selection of the miRNAs in a larger patient cohort, where two miRNAs were verified in both cohorts. A higher expression of plasma exosomal miR-141-3p and miR-375 was found in patients with liver metastasis at the time of diagnosis compared to patients with localized disease (confined to the pelvic cavity). The miRNAs were also associated with low systemic lymphocyte and monocyte counts, commonly associated with immune evasion of tumors, and also correlated with routine clinical markers of aggressive rectal cancer (high systemic levels of g-glutamyl transferase and lactate dehydrogenase).

Both miR-141-3p and miR-375 are associated with tumor angiogenesis and immune modulation. In conclusion, our findings suggested that these miRNAs may be candidate biomarkers of rectal cancer aggressiveness and treatment resistance.

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6.3 Paper III

Uptake of circulating extracellular vesicles from rectal cancer patients and the functional responses by human monocytes

Exosomes from cancer cells are released into their surrounding milieu, including blood.

Exosomes can encounter and be taken up by immune cells, such as circulating monocytes, where they can influence their differentiation and secretory profile. In this paper, we used the CRC cell line SW480-derived MVs and exosomes, isolated by sequential centrifugation and ultrafiltration, as a model system for uptake- and inhibition studies in cultured human primary monocytes. Plasma EVs from rectal adenocarcinoma and adenoma polyp patients were isolated by size exclusion chromatography (SEC), and also used for uptake-studies. We performed a cytokine assay to explore seven cytokines for assessment of inflammation and an Affymetrix gene array to analyze transcriptional responses in the monocytes.

All EV specimens were taken up by the monocytes and affected the secretion of several pro- inflammatory cytokines. Exosomes were shown to be partly endocytosed by phagocytosis and seemed to be a more potent cytokine inducer than the MVs. EVs from patients did not have the same potency in stimulating monocyte cytokine secretion, which can be due to the heterogeneity in sources for the EV pool. However, plasma EVs from patients with adenoma polyps as compared to invasive cancer, as well as localized (non-metastatic) as compared to metastatic disease, caused differential induction of transcriptional changes in the monocytes and showed different ability to regulate biological functions. Monocytes receiving plasma EVs from patients with metastatic disease showed stronger inflammatory responses.

The findings distinguished EVs in subjects with adenoma polyps from cases with invasive cancer. In conclusion, our findings support that monocytes can be a relevant model system for investigating host responses to circulating EVs in rectal cancer patients.

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7 Methodological considerations

7.1 Study populations and model systems

Figure 8. Overview of the studies; cell lines and rectal cancer patient cohorts (OxyTarget and LARC-RRP) in the respective papers used for isolation of exosomes (exo), microvesicles (MVs), and extracellular vesicles (EVs) from conditioned media or plasma. Analysis of the exosomal micro-RNA (miRNAs) was performed in paper I and II, whereas the uptake of EVs, transcriptional changes, and cytokine release in monocytes were analyzed in paper III.

7.1.1 Colorectal cancer cell lines

In vitro tumor models are important tools for cancer research, providing insight into the molecular mechanisms of tumor growth and metastasis [123]. Well-established CRC cell lines, regularly tested for mycoplasma infection, were used to study the hypoxic miRNA profile of exosomes and EV uptake and response by primary monocytes (Figure 8).

The commercially available CRC cell lines HCT116, LoVo and RKO, as well as the two CRC cell lines HCC2998 and KM20L2 received as a gift from Prof. Kjersti Flatmark, Oslo University Hospital (Oslo, Norway) were used to characterize the miRNA content in hypoxic exosomes in Paper I. The cell line identities have also been validated by short tandem repeat analysis to exclude cell misidentification [124]. In Paper III, the commercially available CRC cell line SW480 was used to characterize the uptake mechanism in cultured primary monocytes.

The HCC2998, HCT116, RKO, KM20L2, and SW480 cell lines were derived from primary colon carcinoma, whereas the LoVo cell line was derived from a metastatic site.

Paper I Paper II Paper III

Cell lines n=5 HCC2998

HCT116 KM20L2 LoVo RKO

Hypoxia Normoxia exo exo

24h

OxyTarget cohort n=24

Cell line n=1 SW480

Monocytes 4h

OxyTarget cohort n=22 Validation cohort

n=64 OxyTarget

n=12 LARC-RRP

n=52

exo Investigation cohort

n=29 OxyTarget

n=29

exo exo MVs

Adenoma polyps n=6

Adeno- cacinoma n=16

Exosomal miRNA Exosomal miRNA

Exosomal miRNA

Uptake Transcriptional changes

Cytokine secretion EVs EVs

Extracellular Vesicles in Colorectal Cancer: Mediators of tumor aggressiveness