Mesenchymal Stem Cell Microvesicles from Adipose Tissue: Unraveling Their Impact on Primary Ovarian Cancer Cells and Their Therapeutic Opportunities

Abstract

Mesenchymal stem cells (MSCs) and their derivatives can be promising tools in oncology including ovarian cancer treatment. This study aimed to determine the effect of HATMSC2-MVs (microvesicles derived from human immortalized mesenchymal stem cells of adipose tissue origin) on the fate and behavior of primary ovarian cancer cells. Human primary ovarian cancer (OvCa) cells were isolated from two sources: post-operative tissue of ovarian cancer and ascitic fluid. The phenotype of cells was characterized using flow cytometry, real-time RT-PCR, and immunofluorescence staining. The effect of HATMSC2-MVs on the biological activity of primary cells was analyzed in 2D (proliferation, migration, and cell survival) and 3D (cell survival) models. We demonstrated that HATMSC2-MVs internalized into primary ovarian cancer cells decrease the metabolic activity and induce the cancer cell death and are leading to decreased migratory activity of tumor cells. The results suggests that the anti-cancer effect of HATMSC2-MVs, with high probability, is contributed by the delivery of molecules that induce cell cycle arrest and apoptosis (p21, tumor suppressor p53, executor caspase 3) and proapoptotic regulators (bad, BIM, Fas, FasL, p27, TRAIL-R1, TRAIL-R2), and their presence has been confirmed by apoptotic protein antibody array. In this study, we demonstrate the ability to inhibit primary OvCa cells growth and apoptosis induction after exposure of OvCa cells on HATMSC2-MVs treatment; however, further studies are needed to clarify their anticancer activities.

Keywords: MSC-microvesicles; extracellular vesicles; mesenchymal stem cells; ovarian cancer; primary ovarian cancer cells.

Figure 1

Characteristics of primary OvCa cells from post-op. of OvCa and ascitic fluid. (a) Box and whiskers plots show the expression of selected markers by cells from all patients. Cells were stained with selected antibodies conjugated with fluorochromes. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each marker. Lines represent the median. The left panel shows plots for cells from high-grade serous OvCa, and the right panel shows plots for cells from other histological types of OvCa. (b) Representative histograms of flow cytometry analysis of high-grade serous OvCa (left panel) and cystadenofibroma (right panel) of OvCa cells from post-operative tumor and ascitic fluid. Blue-filled histograms correspond to cells labeled with defined antibodies, and empty histograms represent the isotype controls. Abbreviations: post-op. of OvCa-post-operative tissue of ovarian cancer.

Figure 2

Characteristics of primary OvCa cells from post-op. of OvCa and ascitic fluid. Box and whiskers plots show 2^−ΔCT of selected mRNA transcripts in cells from all patients. Blue dots represent cells from each analyzed patient. Box and whiskers plots show minimal and maximal values for each transcript. Lines represent the median. The upper panel shows plots for cells from high-grade serous OvCa, and the lower panel shows plots for cells from other histological types of OvCa. ** p < 0.01 and * p < 0.05 calculated vs. each marker by one-way ANOVA. For other histological types of ovarian cancer, there were not significant differences between markers. Abbreviations: post-op. of OvCa-post-operative tissue of ovarian cancer.

Figure 3

Characteristics of primary OvCa cells from post-op. tissues of OvCa and ascitic fluid. Box and whiskers plot shows expression of CD133 marker by cells from all patients. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for CD133 marker. Lines represent the median. Abbreviations: post-op. of OvCa-post-operative tissue of ovarian cancer.

Figure 4

Characteristics of primary OvCa cells from post-op. of OvCa and ascitic fluid. The top panel shows cells from high-grade serous OvCa, bottom panel shows cells from cystadenofibroma. Fluorescence microscopy images: cell nuclei stained with DAPI (blue), selected markers stained with Alexa Fluor 488 (green) or Alexa Fluor 647 (red), F-actin stained with Alexa Fluor 488 phalloidin (green), bars represent 20 µm. Abbreviations: post-op. of OvCa—post-operative tissue of ovarian cancer.

Figure 5

Confocal microscopy imaging of HATMSC2-MVs internalization into primary OvCa cells. Cells treated with HATMSC2-MVs at the ratio 100:1 for 24 h (cell nuclei stained with DAPI in blue, cytoplasmic expression of PDGFRα stained with Alexa Fluor 488 in green, HATMSC2-MVs stained with DiD in red). Bar represents 10 µm. HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 6

Effect of HATMSC2-MVs on the metabolic activity of primary OvCa cells from post-op. tissue and ascitic fluid. Metabolic activity of primary OvCa cells cultured in standard conditions was assessed using an MTT assay on days 0, 1, 2, and 3 following treatment with HATMSC2-MVs at a ratio of 100:1. Untreated cells without MVs served as a control. Box and whiskers plots show the relative absorbance of cells from all patients treated with HATMSC2-MVs. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values of analyzed cells. Lines represent the median. The top panel shows plots for cells from high-grade serous OvCa, and the bottom panel shows plots for cells from other histological types of OvCa. *** p < 0.001, ** p < 0.01, * p < 0.05 calculated vs. day 0 by one-way ANOVA. Abbreviations: post-op. of OvCa—post-operative tissue of ovarian cancer; HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 7

Effect of HATMSC2-MVs on the migration activity of primary OvCa cells from post-op tissues and ascitic fluid. Migration activity of primary OvCa cells cultured in standard conditions was measured using a scratch test following treatment with HATMSC2-MVs at a ratio of 5:1. Untreated cells without MVs served as a control. (a) Representative images of scratch closure (measured between the red lines) by cells from patients of high-grade serous OvCa on 0 h, 8 h, 16 h, and 28 h. The top panel shows control and HATMSC2-MVs treated cells from post-op. tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 200 µm. (b) Representative images of cells from patients with cystadenofibroma on 0 h, 8 h, 16 h, and 28 h. The top panel shows control and HATMSC2-MVs-treated cells from post-operative tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 200 µm. (c) Box and whiskers plots show relative scratch closure for cells from all samples treated with HATMSC2-MVs. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each cell type. Lines represent the median. The left plot shows values for cells from high-grade serous OvCa, and the right plot shows values for cells from other histological types of OvCa. Abbreviations: post-op. of OvCa—post-operative tissue of ovarian cancer. HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 7

Effect of HATMSC2-MVs on the migration activity of primary OvCa cells from post-op tissues and ascitic fluid. Migration activity of primary OvCa cells cultured in standard conditions was measured using a scratch test following treatment with HATMSC2-MVs at a ratio of 5:1. Untreated cells without MVs served as a control. (a) Representative images of scratch closure (measured between the red lines) by cells from patients of high-grade serous OvCa on 0 h, 8 h, 16 h, and 28 h. The top panel shows control and HATMSC2-MVs treated cells from post-op. tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 200 µm. (b) Representative images of cells from patients with cystadenofibroma on 0 h, 8 h, 16 h, and 28 h. The top panel shows control and HATMSC2-MVs-treated cells from post-operative tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 200 µm. (c) Box and whiskers plots show relative scratch closure for cells from all samples treated with HATMSC2-MVs. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each cell type. Lines represent the median. The left plot shows values for cells from high-grade serous OvCa, and the right plot shows values for cells from other histological types of OvCa. Abbreviations: post-op. of OvCa—post-operative tissue of ovarian cancer. HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 8

Effect of HATMSC2-MVs on the survival of primary OvCa cells from post-op tissues and ascitic fluid. Survival of cells following treatment with HATMSC2-MVs at a ratio of 100:1 was assessed by confocal microscopy imaging. Untreated cells without MVs served as a control. Live cells stained with Syto 9 (green), dead cells stained with propidium iodide (red), HAT-MSC2-MVs stained with DiD (violet). (a) Box and whiskers plots show relative ratio of live to dead channel. Whiskers show minimal and maximal values for each cell type. Lines represent the median. Blue dots represent cells from each analyzed patient. (b) Representative confocal microscopy images of cells from high-grade serous OvCa. The top panel shows control and HATMSC2-MVs-treated cells from post-op tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 100 µm. (c) Representative confocal microscopy images of cells from cystadenofibroma. The top panel shows control and HATMSC2-MVs-treated cells from post-operative OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 100 µm. Abbreviation: post-op. of OvCa—post-operative tissue of ovarian cancer. HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 8

Effect of HATMSC2-MVs on the survival of primary OvCa cells from post-op tissues and ascitic fluid. Survival of cells following treatment with HATMSC2-MVs at a ratio of 100:1 was assessed by confocal microscopy imaging. Untreated cells without MVs served as a control. Live cells stained with Syto 9 (green), dead cells stained with propidium iodide (red), HAT-MSC2-MVs stained with DiD (violet). (a) Box and whiskers plots show relative ratio of live to dead channel. Whiskers show minimal and maximal values for each cell type. Lines represent the median. Blue dots represent cells from each analyzed patient. (b) Representative confocal microscopy images of cells from high-grade serous OvCa. The top panel shows control and HATMSC2-MVs-treated cells from post-op tumor of OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 100 µm. (c) Representative confocal microscopy images of cells from cystadenofibroma. The top panel shows control and HATMSC2-MVs-treated cells from post-operative OvCa, and the bottom panel shows cells from ascitic fluid. Bars represent 100 µm. Abbreviation: post-op. of OvCa—post-operative tissue of ovarian cancer. HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 9

Effect of HATMSC2-MVs on the survival of primary OvCa cells isolated from post-op. tissues of OvCa and ascitic fluid. Survival of cells following treatment with HATMSC2-MVs at a ratio of 100:1 was assessed by flow cytometry. Untreated cells without MVs served as a control. (a) Quantification of cell viability after treatment with HATMSC2-MVs for 72 h was determined by the presence of apoptotic and necrotic cells via the double-staining of cells with propidium iodide and Annexin V. The percentages of alive, early apoptotic, late apoptotic, and necrotic cells were determined using Flowing Software 2. (b) Box and whiskers plots show percentage of alive cells, early apoptotic, late apoptotic, and necrotic cells for control cells and cells treated with HATMSC2-MVs. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each cell type. Lines represent the median. Untreated cells served as a control. ** p < 0.01 calculated vs. each respective control by Mann–Whitney test. Abbreviations: A—ascitic fluid, T—ovarian tumor tissue, MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin, ns—not significant.

Figure 10

Characteristics of primary OvCa spheroids derived from post-op tissues of OvCa and ascitic fluid. (a) Box and whiskers plot shows the mean diameter of spheroids. Blue dots represent spheroids derived from one patient. Whiskers show minimal and maximal values for spheroid of each patient. Lines represent the median. (b) Representative images of spheroids. The left panel shows spheroids from high-grade serous OvCa cells, and the right panel shows representative images of spheroids cells from cystadenofibroma. Bars represent 200 µm and 100 µm. (c) Box and whiskers plots show the expression of selected CSCs markers on cells formed spheroids. Cells were stained with selected antibodies conjugated with fluorochromes. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each marker. Lines represent the median. *** p < 0.001, ** p < 0.01, * p < 0.05 calculated vs. each marker by one-way ANOVA. Abbreviations: post-op of OvCa—post-operative ovarian cancer.

Figure 11

Effect of HATMSC2-MVs on the survival of primary OvCa spheroids. Effect of HATMSC2-MVs on the survival of primary OvCa spheroids derived from post-op. tumor and ascitic fluid. Survival of cells test following treatment with HATMSC2-MVs at a ratio of 100:1 was assessed by confocal microscopy imaging. Untreated cells without MVs served as a control. (a) Box and whiskers plots show relative ratio of live to dead channel. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each spheroid. Lines represent the median. (b) Representative confocal microscopy images of spheroids from high-grade serous OvCa and from cystadenofibroma. Live cells stained with Syto 9 (green), dead cells stained with propidium iodide (red), HATMSC2-MVs stained with DiD (violet). Bars represent 100 µm. The top panel shows control and treated spheroids from high-grade serous OvCa and the bottom panel shows spheroids from cystadenofibroma. Abbreviations: post-op. of OvCa-post-operative ovarian cancer; HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 11

Effect of HATMSC2-MVs on the survival of primary OvCa spheroids. Effect of HATMSC2-MVs on the survival of primary OvCa spheroids derived from post-op. tumor and ascitic fluid. Survival of cells test following treatment with HATMSC2-MVs at a ratio of 100:1 was assessed by confocal microscopy imaging. Untreated cells without MVs served as a control. (a) Box and whiskers plots show relative ratio of live to dead channel. Blue dots represent cells from each analyzed patient. Whiskers show minimal and maximal values for each spheroid. Lines represent the median. (b) Representative confocal microscopy images of spheroids from high-grade serous OvCa and from cystadenofibroma. Live cells stained with Syto 9 (green), dead cells stained with propidium iodide (red), HATMSC2-MVs stained with DiD (violet). Bars represent 100 µm. The top panel shows control and treated spheroids from high-grade serous OvCa and the bottom panel shows spheroids from cystadenofibroma. Abbreviations: post-op. of OvCa-post-operative ovarian cancer; HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.

Figure 12

Composition of bioactive factors of the HATMSC2 cells and the HATMSC2—MVs. (a) Scans of representative protein arrays. The signal intensity for each antibody spot is proportional to the relative concentration of the protein in the sample. (b) Heat map of protein levels for the HATMSC2 cells and the HATMSC2-MVs; the magenta and yellow indicate higher and lower expression limits, respectively. Outstanding values (above 100% of positive control) are depicted in blue and magenta. Data are normalized to the internal positive control spots, which are consistent between the arrays and represent 100%. (c) Column graph representing bioactive factors and molecules regulating apoptosis. The data are presented as mean ± SEM values from a duplicate assessment. Abbreviations: HATMSC2-MVs—microvesicles derived from immortalized human mesenchymal stem cells of adipose tissue origin.