Adipose-derived stem cells functionally reverse radiation fibrosis and secrete extracellular vesicles.

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1 Adipose-derived stem cells functionally reverse radiation fibrosis and secrete extracellular vesicles. ABSTRACT Background: Radiation fibrosis (RF), the adverse excess scarring of tissue, affects up to 70% of cancer patients after radiotherapy and may lead to organ failure. As radiation induces fibroblast accumulation and vascular damage, RF is often associated with ischemia. Adipose-derived stem cells (ADSCs) have been shown to be effective in wound healing and tissue regeneration. Extracellular vesicles (EVs), mediators of intercellular communication, have been demonstrated to stimulate tissue repair but not yet been found to be secreted by ADSCs. It is hypothesized that ADSCs can functionally reverse RF and RF-associated ischemia, and their therapeutic effect involves the secretion of EVs. Methods: Non-irradiated and irradiated mice were treated with mouse ADSCs and leg contracture was measured as a functional indicator of radiation fibrosis. Mouse ADSC-derived EVs were isolated by differential ultracentrifugation and their protein and mirna content were characterized. As a preliminary step prior to characterizing RF-associated ischemia, an automated and unbiased image analysis protocol was established to quantify angiogenesis. Results: ADSC treatment improved leg contracture of the irradiated murine hind limb. EVs were successfully isolated from murine ADSCs, as shown by expression of common EV markers. CellProfiler was validated as an image analysis tool for characterizing murine vasculature. Conclusions: This project achieved the first aim of an ongoing study. ADSC transplantation was shown to functionally reverse RF and secrete EVs as a potential therapeutic mechanism. As the number of cancer survivors rises worldwide, the prevalence of RF will increase as well. Understanding the therapeutic mechanisms of ADSCs provides insight into the development of novel clinical treatments for reversing RF. Keywords: Adipose-derived stem cells; extracellular vesicles; radiation; fibrosis; vasculature INTRODUCTION Radiation Fibrosis (RF) Radiation fibrosis (RF), the excess scarring of normal tissue, affects up to 70% of patients after radiotherapy 1,2. RF may lead to severe functional impairments and organ failure, which significantly decreases the quality and duration of life for cancer survivors 1. The pathogenesis is RF is complex and dynamic, involving changes due to inflammation, mutagenesis, and fibrogenesis. Clinical and molecular features of RF include the loss of vascularity and tissue elasticity, endothelial cell damage, fibroblast accumulation, excess collagen deposition, induration (the hardening of a normally soft tissue or organ), and organ damage 1. Adipose-Derived Stem Cells (ADSCs) Stem cells are characterized by their ability to self-renew and differentiate along multiple lineage pathways 3. Mesenchymal stromal cells (MSCs) are non-hematopoietic mesodermal cells that promote angiogenesis, inhibit apoptosis, and exert anti-inflammatory effects on damaged tissue 4.

2 They can differentiate into mesodermal lineage cells (osteoblasts, chondrocytes, and adipocytes) and non-mesodermal lineage cells (neurons, cardiomyocytes, and ectodermal skin) 4 6. In addition, MSCs are radiation insensitive due to their ability to recognize and repair DNA damage, activate pro-survival pathways, and evade apoptosis 7. This allows MSCs to be a potential preventative and regenerative therapy for radiation injury. MSCs have been isolated from various organ sites including bone marrow, skin, and skeletal muscle 8. Due to a limited amount of tissue, however, only a small number of cells can be harvested from these sources. Recent research in progressive osseous heteroplasia, lipomas and liposarcomas, and obesity has revealed that adipose tissue is an abundant and accessible source of a subtype of MSCs, namely adipose-derived stem cells (ADSCs) 3,4,9,10. Human ADSCs can be harvested by liposuction, a minimally invasive procedure that poses low risk of donor-site morbidity 3,8. In vitro, ADSCs display a fast cell doubling time of 2 4 days depending on the culture medium and passage number 3. Various clinical trials have demonstrated the regenerative capacity of ADSCs for wound healing and soft tissue, musculoskeletal, cardiovascular, and nervous system regeneration 5. Cai et al. showed that silencing hepatocyte growth factor expression in ADSCs reduced endothelial cell proliferation, impaired pro-angiogenic effects in vitro, and decreased reperfusion of ischemic tissue 5,11. Extracellular Vesicles (EVs) Extracellular vesicles (EVs) function to transfer proteins, lipids, mrnas and mirnas to neighbouring and adjacent cells leading to coordinative function 12,13. This project focuses on two types of EVs: exosomes and microvesicles. Exosomes are nanosized, intraluminal EVs originating from multivesicular endosomes with a diameter of nm 13. They are secreted through the fusion of multivesicular bodies with the plasma membrane 14,15. Microvesicles are EVs that bud directly from the plasma membrane and are larger than exosomes with a diameter of nm 13,15. Common biomarkers of EVs include flotillin-1 (lipid raft that serves as organizing centre for signalling molecule assembly) and mir-16 (mirna involved in regulation of angiogenesis, cell differentiation, and apoptosis) 13, It is well known that a primary mechanism by which MSCs exert their therapeutic effect is through paracrine and endocrine signaling 13,19,20. MSCs are prolific producers of EVs. As such, it is tempting to hypothesize that the secretion of EVs, though their role as mediators of intercellular communication, is a method by which MSCs exert their therapeutic effect. For example, EVs have been shown to control a diverse set of fundamental cellular and biological functions, such as stem cell maintenance, blood coagulation, and immune surveillance 15,21. In addition, EVs have been shown to suppress inflammation in a manner that produces immunological tolerance 21,22. Studies have demonstrated that MSC-derived EVs stimulate tissue repair in models of wound healing, acute kidney injury, myocardial ischemia-reperfusion injury, and skeletal muscle injury 12,14,19,20. Although research on the long-term outcomes of MSC treatment is lacking, EVs have been shown to be natural tissue-specific drug delivery vehicles that modulate the immune response 13,23. They are biocompatible, immunologically inert if derived from MSCs, can be 2

3 autologous, and can cross the blood-brain barrier 13. Lai et al. showed that administration of human MSC-derived EVs to an immunocompromised murine myocardial infarction/reperfusion injury model gave rise to tissue regeneration in the absence of adverse effects 20,23. Taken together, EVs may serve as a mechanism behind MSC-induced tissue repair. However, it is not well understood if ADSC transplantation is an effective treatment for the reversal of RF, an adverse outcome of radiotherapy. In addition, although MSCs secrete EVs, it is unknown if ADSCs produce these signalling vesicles as well. It is hypothesized that ADSCs can functionally reverse RF and RF-associated ischemia, and that ADSC-derived EVs can be successfully isolated. The therapeutic effect of ADSCs will be shown by measuring leg contracture, an observable functional change due to RF. ADSC-derived EV mirna and protein content will also be characterized. Lastly, an automated and unbiased image analysis protocol will be established to quantify vasculature as a preliminary step prior to conducting future experiments. This project serves to achieve the first aim of an ongoing study. MATERIALS AND METHODS Animals All animal protocols were performed with [REDACTED] approval. Adult female C3H/HeJ mice (Jax Mice, Bar Harbour, Maine, USA) were used, and were age- and litter-matched in all experiments. For the leg contracture study, the left hind limb of all mice was given 40 Gy of ionizing radiation (IR). For the CellProfiler validation study, tissue was irradiated at 40 Gy. Leg contracture In order to determine the role of ADSCs in the reversal of RF, mice (n = 7 per group) were injected with 1x10 6 murine ADSCs (3x) at 4 weeks post-ir. Functional changes in leg contracture, the ratio of extension of the non-irradiated (LC) and radiated (LR) hind limb, was assessed twice a week for a total of 16 weeks (Figure 1; 9 weeks were recorded). To measure leg contracture, the anesthetized mouse was placed on a horizontal surface and held stationary at the hip. A weight was attached to the foot which extended the leg along a ruler and allowed the length of the hind limb to be measured. In addition, the health status of the hind limb was monitored twice a week. Each leg was designated a score from 0 3; a higher score was given to more severe injuries. If a mouse reached a score of 3, it would be deemed moribund, euthanized, and removed from the experiment. Although no mice in this study were removed based on this criteria, one mouse from the vehicle-treated group was removed as an outlier. 3

4 Figure 1. Leg contracture measurement model. The mouse was laid on a horizontal surface and was held at the hip. The hind limb was extended along a ruler with a weight attached to its foot. Leg contracture was measured as a ratio of extension length of the irradiated vs. non-irradiated control hind limb. ADSC isolation and cell cultures Mice (n = 4) were sacrificed by inhalation of carbon dioxide and adipose tissue of the abdominal cavity was dissected. Equal volumes of collagenase was added to the minced adipose tissue and gently rotated for 45 min at 37 C. Dulbecco s modified Eagle medium (DMEM; H16 low glucose; Gibco, Waltham, MA, USA) supplemented with 5% EV-free fetal bovine serum (FBS) was added to dilute the collegenase. FBS was pre-depleted of EVs by ultracentrifugation at 120,000g for 16 h at 4 C. The sample was then centrifuged to separate the stromal vascular fraction (SVF), containing ADSCs, from stromal cells. The SVF pellet was filtered, suspended in media with EV-free FBS, and incubated for12 hours at 37 C/5% CO2. The plastic-adherent cell population was then washed with large volumes of PBS to remove red blood cells. ADSCs were cultured in vitro in DMEM H16 low glucose media with 5% FBS. When the adherent cell monolayer reached 80% confluency, the cells were trypsinized (2.5x trypsin-edta), resuspended in DMEM containing 5% FBS, and maintained at 37 C/5% CO2. Culture supernatant was collected every 2 3 days when the cells were passaged twice. After the second passage, supernatant was collected 4 times. Supernatant from the last two collections was used for the further studies. 4

5 Purification and characterization of ADSC-derived EVs (ADSC-EVs) ADSC-EVs were isolated by differential centrifugation and ultracentrifugation as described by Théry et al. (Figure 2) 24. Ultracentrifugation was done using a SW 32 TI swinging bucket rotor. Recovered ADSC culture supernatant was centrifuged at 2000 g (20 min) to remove living cells, dead cells, and cell debris. This was followed by ultracentrifugation at 10,000 g (30 min) to separate the pellet and supernatant fraction. The pellet, containing microvesicles, was ultracentrifuged at 10,000 g (30 min) in PBS to remove contaminating particles and excess serum proteins. The supernatant was ultracentrifuged at 200,000 g (2 h) to isolate for the exosome fraction. The pellet, containing exosomes, was ultracentrifuged in PBS at 200,000 g (2 h) to remove contaminating particles and excess serum proteins. The supernatant was collected to serve as an EV-free negative control. The washed and purified microvesicle and exosome pellets were suspended in 100 µl of PBS and stored in -80 C for further analysis. Negative control media (DMEM H16 low glucose media supplemented with 5% EV-free FBS) underwent the same centrifugation and ultracentrifugation steps. Figure 2. Flow diagram of microvesicle and exosome isolation via differential centrifugation (Cent.) and ultracentrifugation (UCent.) using adipose-derived stem cell (ADSC) culture supernatant (blue) and extracellular vesicle (EV)-free negative control media (orange). ADSC-derived fractions are colour coded as blue and negative control media-derived fractions are colour coded as orange. If EVs are successfully isolated, ADSC-derived exosome and microvesicle fractions should be enriched in protein and mirna biomarkers. ADSC-derived supernatant and negative control media-derived fractions should not. 5

6 mirna analysis Before isolating mirna, 700 µl of Qiazol was added to prevent degradation and 10 µl of 0.1 mm cel-mir-39 was added as a spike-in control. Total mirna was isolated from ADSC-EVs using the RNeasy Micro Kit (Qiagen, Toronto, ON, Canada) as per manufacturer s protocol. mirna was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Burlington, ON, Canada) according to manufacturer s instructions. Real time PCR was conducted using the TaqMan Universal PCR Master Mix, No AmpErase UNG and primer for hsa-mir-16 (000391) (Applied Biosystems, Burlington, ON, Canada). Supernatant and EV-free negative control media-derived fractions served as the negative controls. Protein analysis EVs were lysed in 100 µl of RIPA lysis buffer with protease inhibitor. Protein content was quantified by a Bradford protein assay (Bio-Rad, Mississauga, ON, Canada) and standardized to 2 µg. Next, proteins were separated by 4-12% gradient SDS-PAGE and transferred to a PVDF membrane that was pre-treated with methanol. The membrane was blocked using 5% milk in 1:1000 TBST (10x TBS, 200mM Tris, 1.5M NaCl) for 1 h at room temperature and incubated with primary antibodies (mouse anti-flotillin antibody, Abcam, Toronto, ON, Canada) overnight at 4 C. After vigorous washing in TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signalling Technology, Danvers, MA, USA) for 45 min. Labelled proteins of interest were detected and visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) with ImageLab software (Bio-Rad, Mississauga, ON, Canada). Band quantification was done using ImageJ. EVfree negative control media-derived, supernatant, and ADSC lysate fractions serve as the negative controls. Vasculature quantification As radiation induces endothelial cell death and vascular damage, ischemia is often associated with RF. As a preliminary step prior to characterizing the angiogenic potential of ADSCs on RFassociated ischemia in future experiments, this project aimed to establish an automated and unbiased protocol to quantify vasculature. Vascular density and lumen area were assessed using immunohistochemically-stained tissue sections from untreated non-irradiated and irradiated tissue previously prepared by the laboratory. Vascular endothelial cells were specifically selected using anti-cd31 (expressed in endothelial precursor cells) as the primary antibody and 3,3 - diaminobenzidine (DAB; stains nucleic acids) as a chromogen. Slides were counterstained with hematoxylin (stains nucleic acids). In order to establish a suitable method to quantify vasculature, CellProfiler was chosen. CellProfiler (Cambridge, MA, USA), an open-source cell image analysis software, identified vasculature based on size, shape and stain intensity in order to quantify vasculature density and diameter 25. Vessel density was calculated as the number of objects identified per tissue area. The main CellProfiler interface and the CellProfiler workflow diagram is shown in Figure 3. 6

7 A B Figure 3. A) CellProfiler main interface with an analysis pipeline on the left B) CellProfiler workflow diagram, categorizing pipeline modules by their function in chronological order. The CellProfiler pipeline consisted of various modules that led to object identification and measurement. For example, the EnhanceOrSuppressFeatures module improved identification of objects by enhancing image features. As vessels have a donut-like shape, this module enhanced the intensity of the endothelial cell boundary relative to the lighter interior and the rest of the image. For the IdentifyPrimaryObjects module, which identified bright objects against a dark background, the threshold size of objects was This range was chosen in accordance with validated images. A Per Object threshold strategy was used due to the varying background intensity of the images. An Otsu threshold method were chosen due to its compatibility with images that have a varied percentage covered by foreground. Lastly, clumped objects were distinguished by a modular two-step approach: 1) recognition and separation of objects and 2) identification of separated objects. The FilterObjects module removed objects that fall outside the accepted range of intensity (min, max 0.85, 1.0). This range was determined based on measurements from the MeasureObjectIntensity module and pixel intensities of the output image. In order to visualize the objects identified by CellProfiler, the OverlayOutlines module was implemented into the pipeline. Vasculature identified by 7

8 Leg Contracture Percentage (L/R) CellProfiler for 6 representative sections was validated against manual quantification in a blinded fashion. Statistical analysis Statistical analysis was carried out using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA) and Microsoft Excel. One-way ANOVA was performed to determine the effect of ADSC treatment on leg contracture and the effect of radiation of vessel density and vessel lumen area. When a significant effect was identified one-way ANOVA, unpaired t-tests were conducted to compare differences between treatment groups. P < 0.05 was considered as significant. RESULTS ADSCs therapeutically reversed hind limb RF Leg contracture was measured as the change in extension length between the high-dose irradiated and non-irradiated hind limb as a functional indicator of radiation fibrosis. ADSCtreated mice showed a significant improvement in leg contracture between left and right hind limb compared to control mice. This significance was observed in week 8 through week 16, the last week of observation (Figure 4) *** ** *** ** *** ** * * * Week Control ADSC Figure 4. ADSC treatment significantly improved leg contracture, a functional indicator of radiation fibrosis, for all time points (8 16 weeks post-adsc treatment) (n = 7; Student s t test; *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < vs. control) EVs were successfully isolated from ADSCs To examine if ADSCs secrete EVs, successive centrifugation and ultracentrifugation was conducted using ADSC culture supernatant. EV protein and mirna content was then characterized. 8

9 Relative Band Density Total EV proteins were extracted from ADSC-EVs and assessed by Western blot (Figure 5). Three negative controls were used: DMEM with 5% EV-free FBS (negative control EV-free media), EV-free supernatant from the ultracentrifugation spin that pelleted down the exosome fraction, and ADSC lysate. Although all other samples were standardized to 2 µg of protein, one sample contained a maximized volume of exosome-derived proteins (exosome max fraction) which served as our positive control. Primary antibody against flotillin-1 was used. A kda Flotillin-1 B Exosome Microvesicle Supernatant Exosome (max) Cell lysate Fraction Derived from ADSC culture supernatant Derived from EV-free media (control) ADSC Figure 5. Western blot analysis of C3H/HeJ ADSC-EVs showing expression of flotillin-1 in ADSC-derived EVs. A) After incubation HRP-tagged secondary antibodies, the membrane was imaged using the ECL chemiluminescence system. ADSC-derived exosome and microvesicle fractions (blue) showed positive flotillin-1 expression. Correspondingly, supernatant and negative control mediaderived (orange) fractions do not show flotillin-1 expression. B) Band intensity for anti-flotillin-1, relative to the ADSC-derived microvesicle band, was analyzed using ImageJ software. Not surprisingly, the ADSC-derived exosome (max) fraction showed the greatest flotillin-1 expression. The ADSC-derived exosome fraction showed greater flotillin-1 expression, possibly because this biomarker is more exosome-specific. 9

10 mir-16 mirna Western blot analysis showed expression of flotillin-1 in both ADSC-derived microvesicle and exosomes. Using ImageJ, the ADSC-derived exosome (max) fraction showed the greatest flotillin-1 expression. Compared to ADSC-derived microvesicles, a 5.6-fold increased expression of flotillin-1 in ADSC-derived exosomes was also observed. Negligible flotillin-1 was observed in the negative control media-derived EV fractions and the ADSC- and control media-derived supernatant fractions. Although a band was present in the ADSC lysate lane, it was extremely faint compared to the ADSC-derived exosome and microvesicle fractions. Total EV mirna was isolated from ADSC-derived EVs, reverse transcribed, and characterized using RT-PCR. Two negative controls were used: DMEM with 5% EV-free FBS (EV-free negative control media) and EV-free supernatant obtained from the exosome-pelleting ultracentrifugation step. mir-16 levels in exosome and microvesicle fractions were determined by reverse transcription and RT-PCR Exosome Microvesicle Supernatant Fraction Derived from ADSC culture supernatant Derived from EV-free media (control) Figure 6. To further characterize the EVs, mirna expression, specifically mir-16, was determined by reverse transcription and RT-PCR. Both the ADSC-derived exosome and microvesicle fractions showed a positive reaction for mir-16. Although mir-16 expression may not appear dissimilar, the exosome and microvesicle fraction had a difference of 1 C(t) value, indicating a 2-fold difference in mir-16 expression. Surprisingly, we see expression of mir-16 in the negative control media-derived fractions but there is a 7 C(t) value difference compared to the ADSC-derived EV fractions, equally a 128-fold difference. This may be due to residual EVs present in the negative control media. Combined with the Western blot, these results suggest that the centrifugation and ultracentrifugation protocol enriches for ADSC-derived EVs. 10

11 ADSC-derived EV fractions obtained 7 cycle threshold C(t) values compared to that of the negative control media-derived fractions (Figure 6). This equals a 128-fold increase in mir-16 expression. As the C(t) value is the number of cycles required by a fluorescent signal to exceed the background threshold and be able to be detected, these strong positive reactions in the ADSC-derived exosome and microvesicle fractions indicate an increased level of target amplicon in the reverse-transcribed sample compared to negative control media-derived fractions 26. In addition, with a 1 C(t) value difference, the ADSC-derived exosome fraction had a 2-fold greater mir-16 expression compared to ADSC-derived microvesicles. No significant difference in vessel density or lumen area between non-irradiated and irradiated tissue CellProfiler was chosen to serve as an unbiased and automated method for quantifying vasculature. Due to heterogeneity in the characteristics of the vessels, the software pipeline was adjusted to reflect differences in shape, stain intensity, and size. Figure 7 shows images produced from the IdentifyPrimaryObjects and OutlineObjects modules in CellProfiler. Vessel quantification and vessel area measurements were based on objects identified from this module. 11

12 A B C DAB Hematoxylin D Figure 7. Images are extracted from the IdentifyPrimaryObjects and OutlineObjects modules of CellProfiler A) CD31-stained tissue section with DAB (brown) and hematoxylin (purple) as chromogens B) Objects identified by CellProfiler as vasculature highlighted in colour. Colours are assigned arbitrarily. C) Objects identified as non-vasculature highlighted in colour. Colours are assigned arbitrarily. D) Objects identified by CellProfiler as vasculature outlined in red, as extracted from the OutlineObjects module. An overestimation of vessel size can be observed, one of the limitations of the image analysis software. Vessel density and vessel lumen area in untreated non-irradiated and irradiated tissue were measured (Figure 8). The vessel density of control, non-irradiated tissue was 1.77 x 10-5 ± 0.20 x 10-5 vessels/unit 2 and the average vessel area was ± units 2. The vessel density of irradiated tissue was 111% greater than that of the control tissue. In addition, the average vessel lumen in irradiated tissue was 102% greater than the average vessel area of control tissue. However, differences in vessel density and area were not significant. Vessel density between control and irradiated tissue within different ranges of vessel lumen areas was also compared. 12

13 Vessel Density (a.u.) Vessel Density (objects/a.u. 2 ) Vessel Area (unit 2 ) Although there was an increasing trend in vessel density across smaller vessel area ranges in irradiated mice compared to control, no significance was found. A B C Control Group IR 0 Control Group IR Vessel lumen area (a.u.) IR Control Figure 8. No significant difference in vessel density, vessel lumen area, or vessel density across different vessel lumen area ranges were found in irradiated mouse tissue compared to non-irradiated tissue. A) Nonsignificant difference in vessel density in control and irradiated tissue (P > 0.05 vs. control, unpaired t-test). B) Non-significant difference in average vessel area in control and irradiated tissue (P > 0.05 vs. control, unpaired t- test) C) Non-significant difference in vessel density separated by vessel lumen areas (P > 0.05 vs. control, unpaired t-test). a.u.: arbitrary unit 13

14 CellProfiler is validated as a tool for vasculature quantification In order to validate its use for this study, objects identified by CellProfiler as vessels were compared against a manual count of 6 representative images in a blinded fashion. A sensitivity (true positive rate) of 79%, a specificity (true negative rate) of 68%, and a false positive rate of 0.32 were determined. DISCUSSION ADSCs are a valuable stem cell source for clinical use, considering their genetic stability in vitro and adipogenic, osteogeneic, neurogenic and chrondrogenic differentiation potential 27. ADSCs are therapeutically effective in treating in soft tissue degeneration, neurodegenerative diseases, and osteoarthritis 27. Our lab has previously shown a therapeutic functional effect of ADSCs on RF. 2 This result was confirmed in the current study, as ADSC treatment significantly improved leg contracture of the irradiated hind limb, a functional indicator of RF. However, the mechanism of action of ADSCs in reversing RF remains to be understood. Recently, EVs have emerged as essential mediators of cell-cell communication to regulate a vast range of biological processes 13. They are involved in the pathobiology of numerous diseases, including cancer, infectious diseases, and neurodegenerative disorders 13. Further, EVs have a therapeutic potential for treating myocardial ischemia/reperfusion injury, cardiovascular disease, and arthritis 12,20,22. EVs are released by various cell types during cell activation and growth, including MSCs. The current study demonstrated that EVs can be successfully isolated from ADSCs, a subtype of MSCs, based on expression of highly conserved biomarkers (flotillin-1 and mir-16). ADSC-derived exosomes showed greater flotillin-1 expression compared to ADSCderived microvesicles, possibly because it is a more exosome-specific marker. Although the ADSC lysate fraction showed a flotillin-1 signal, it was extremely faint compared to ADSCderived fractions. Negative control-media derived and supernatant fractions displayed mir-16 expression but this may have been due to incomplete depletion of EVs in the FBS. It is also important to consider that these fractions showed a 7 C(t) value difference, equalling a 128-fold decrease in mir-16 expression compared to ADSC-derived fractions. Taken together, protein and mirna characterization suggests that the centrifugation and ultracentrifugation protocol was effective in enriching for ADSC-derived EVs. Further characterization of ADSC-derived EVs is needed, using microvesicle and/or exosome-specific markers. ADSCs and EVs promote angiogenesis and neovascularization by the secretion of paracrine factors 27. As EVs may serve as a signalling mechanism for ADSCs in reversing RF, its role in promoting blood vessel formation will be investigated. CellProfiler experiments were done in order to optimize an automated and unbiased vessel quantification method using untreated mouse tissue. Differences in vessel density and vessel area between non-irradiated and irradiated tissue were insignificant. However, the increasing trend of vessel density in irradiated tissue may be due to molecular, cellular, and tissue changes induced by irradiation. Ionizing radiation promotes angiogenesis through the increased expression of pro-angiogenic factors including vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), hypoxia inducible factor-1 alpha (HIF- 1α), and basic fibroblast growth factor (bfgf) 28. Compared to the manual count, CellProfiler s results showed moderate specificity, low sensitivity, and a high false positive rate. Although the 14

15 use of CellProfiler for quantifying and characterizing vasculature in tissue sections did not seem favourable, this result may have been due to sources of error in the software or cell staining. As this project has achieved only the first steps of an ongoing study, further experiments will be conducted over the next year. EVs secreted by ADSCs will be further characterized. CD63, CD81, and CD73 will serve as protein markers of Western blot analysis while mir-39, mir-126, and mir199a will serve as mirna targets for RT-PCR 18,29. In addition, the therapeutic role of ADSC-derived EVs on angiogenesis and fibrosis will be investigated. The in vitro effect of ADSC-derived EVs on vasculature will be investigated using human umbilical vein endothelial cells (HUVEC). A cell proliferation assay, based on 5-bromo-2 -deoxyuridine (BrdU) incorporation, and an endothelial cell tube formation assay will be conducted. Since blood vessel formation required degradation of the basement membrane, a matrix invasion assay will also be performed. Lastly, in vivo and in vitro experiments that examine the effect of ADSC-derived EVs on fibrosis will be done. In vivo radiation fibrosis will be modelled similar to the leg contracture assay but in combination with ADSC-EV treatment. Due to the complexity of the in vivo fibrosis process, a set of in vitro assays are needed to represent key events that occur within the fibrotic pathway. First, changes in rate of cell division and proliferation will be monitored through a BrdU incorporation proliferation assay. Myofibroblast differentiation is often associated with the pathology of lung, liver, and kidney fibrosis as it results in aberrant architectural remodelling 30. Myofibroblast activation will be visualized by Western blot using α- SMA 30,31. Lastly, collagen production by activated myofibroblasts will be investigated by colorimetric measurement of hydroxyproline content as well as by Western blot using collagen- 1, fibronectin, and PAI Human fibroblasts will be used in these in vitro assays. Taken together, this study demonstrated that ADSCs can reverse RF. EVs secreted by ADSCs may serve as a mechanism of action. In addition, CellProfiler serves as a validated tool for vasculature quantification, but protocol optimization is needed. RF functionally impairs the body, possibly resulting in organ failure and a diminished quality of life. As cancer survivorship increases and the prevalence of RF rises as well, understanding the therapeutic mechanisms of ADSCs and EVs can provide insights into the development of novel clinical therapies for this adverse condition. ACKNOWLEDGEMENTS Thank you to [REDACTED] for providing the opportunity for me participate in this fascinating project and for his invaluable support in shaping me to be a better science researcher and communicator. In addition, thank you to [REDACTED] for generously providing the time, mentorship, and patience in teaching me about the intricacies of scientific research and its techniques. I would also like to thank [REDACTED] and [REDACTED] for their expertise and contributions to this project. This study was funded by the [REDACTED] and the [REDACTED]. The authors declare no conflict of interest. REFERENCES 1. O Sullivan, B. & Levin, W. Late radiation-related fibrosis: pathogenesis, manifestations, and current management. Semin. Radiat. Oncol. 13, (2003). 15

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