Anti-proliferative effects of mesenchymal stem cells (MSCs) derived from multiple sources on ovarian cancer cell lines: an in-vitro experimental study

Abstract

Mesenchymal stem cells (MSCs) have surfaced as ideal candidates for treatment of different therapeutically challenging diseases however their effect on cancer cells is not well determined. In this study, we investigated the effect of MSCs derived from human bone marrow (BM), adipose tissue (AT), and umbilical cord derived MSCs (UC-MSCs) on ovarian cancer.Measurements of ovarian tumor marker proteins were computed by ELISA. Proliferative, apoptosis and anti-inflammatory effects of the MSCs were measured by Flow cytometry (FCM). MMPs expression was measured by RT-PCR.The co-culture of cancer cell lines OVCAR3, CAOV3, IGROV3 and SKOV3 with the conditioned media of MSCs (CM-MSC) and MSCs showed an increase in cellular apoptosis, along with a reduction in the level of CA-125 and a decline of LDH and beta-hCG. A decrease in CD24 of the cancer cell lines in co-culture with the CM-MSCs showed a reduction of the cancer tumorigenicity. In addition, the invasion and aggressiveness of cancer cell lines was significantly decreased by CM-MSC; this was translated by a decrease in MMP-2, MMP-9, and CA-125 mRNA expression, and an increase in TIMP 1, 2, and 3 mRNA expression. An increase in IL-4 and IL-10 cytokines, and a decrease in GM-CSF, IL-6, and IL-9, were also noted.In conclusion, mesenchymal stem cells derived from different sources and their conditioned media appear to have a major role in inhibition of cancer aggressiveness and might be considered as a potential therapeutic tool in ovarian cancer.

Keywords: Adipose tissue; Bone marrow; Mesenchymal stem cells; Ovarian cancer cell lines; Umbilical cord.

Fig. 1

Morphology and flow cytometry analysis. a-b Morphology of MSC from AD, BM and CB. Scale bar, 100 μm. Morphological observation of OVCAR3, SKOV3, IGROV3 and CAOV3. Scale bar,100 μm. c-f Representative FCM result of MSC markers: CD34, CD45, HLADr, CD14, CD105, CD90, CD73, CD44, and CD24. All MSCs were positive for CD105, CD90, CD44, and CD73, and negative for the leukocyte common antigen CD45, the hematopoietic lineage marker CD34, the cancer marker CD24, and the macrophage markers HLADr and CD14 (Table 1). FCM results of CAOV3, OVCAR3, IGROV3 and SKOV3 in co-culture with MSCs and CM-MSCs. Error bars represent the means ± SD, n = 5; * p = 0.051, **p = 0.005, ***p < 0.001,****p < 0.0001

Fig. 2

Differentiation capacity of MSCs. Image (a) Morphology of MSC from Different sources. Cells were incubated for 3 weeks with adipogenic, osteogenic, and chondrogenic media. Representative images (b) of differentiated cells first row represent day 0 of coloration. Second row shows that the intracellular lipid droplets were confirmed by Oil Red O staining compared to the control cells. The third row, the presence of calcium deposit was visualized by Alizarin Red staining compared to the control. The presence of GAG in the last row was confirmed with Alcian Blue staining and the solid chondrogenic micromass compared to the control

Fig. 3

Expression of Tumor markers. a-d CD44+/CD24- expression in CAOV3, OVCAR3, IGROV3. e-h Cell culture supernatant from co-culture was collected, and undiluted samples were analyzed for detection of CA-125, LDH and beta-hCG. The concentration of secretion is shown in the x-axes. The limits of detection (IU/ml) were CA-125: 0.95, beta-HCG: 0.99, LDH = 0.99. i Real time PCR showed a decrease in Ca-125 levels in CAOV3, SKOV3 and OVCAR3, in mRNA levels. The results were displayed as percentage of controls. Error bars represent the means ± SD, n = 5; * p = 0.051, **p = 0.005, ***p < 0.001,****p < 0.0001

Fig. 4

Apoptosis and cell death. a-e FCM using Annexin V /PI showed an increase in cell death in cancer cell lines. The increase rate was calculated by subtracting value from the control. Error bars represent the means ± SD, n = 5; * p = 0.051, **p = 0.005, ***p < 0.001,****p < 0.0001. f-j DAPI staining revealed an increase in blue fluorescence relative to the co-culture with MSC and/or supernatant. f MSCs control. First row (g): CAOV3 CTR, CAOV3 with ADMSC, CAOV3 with BM-MSC, CAOV3 with UC-MSC, CAOV3 with CM-ADMSC, CAOV3 with CM-BMMSC and CAOV3 with CM-UCMSC. Second row(h): OVCAR3 CTR, OVCAR3 with ADMSC, OVCAR3 with BM-MSC, OVCAR3 with UC-MSC, OVCAR3 with CM-ADMSC, OVCAR3 with CM-BMMSC and OVCAR3 with CM-UCMSC. Third Row (i) IGROV3 CTR, IGROV3 with ADMSC, IGROV3 with BM-MSC, IGROV3 with UC-MSC, IGROV3 with CM-ADMSC, IGROV3 with CM-BMMSC and IGROV3 with CM-UCMSC. Fourth Row (j) SKOV3 CTR, SKOV3 with ADMSC, SKOV3 with BM-MSC, SKOV3 with UC-MSC, SKOV3 with CM-ADMSC, SKOV3 with CM-BMMSC and SKOV3 with CM-UCMSC

Fig. 5

mRNA expression of MMPs and TIMPs. a-d Real time PCR showed a decrease in MMP-2 and MMP-9 mRNA levels, and an increase in TIMP-1, TIMP-2, and TIMP-3 mRNA levels. The results were displayed as percentage of controls, Error bars represent the means ± SD, n = 5; * p = 0.051, **p = 0.005, ***p < 0.001,****p < 0.0001

Fig. 6

Interleukin expression. a-d Cell culture supernatants from co-culture were collected, and undiluted samples were analyzed for the detection of cytokines as indicated in the methods. The cytokine limits of detection (pg/ml) were: tumor necrosis factor (TNF-α) = 0.98, IFN-α = 0.99, granulocyte/macrophage colony stimulating factor (GM-CSF) = 1, and interleukins (IL-4 = 0.99, IL-9 = 0.98, IL-10 = 0.98, IL-17A = 1). The means of 5 independent experiments were performed in triplicate. P-value < 0.0001. Error bars represent the means ± SD, n = 5; * p = 0.051, **p = 0.005, ***p < 0.001,****p < 0.0001