The existence of EOC CSCs might explain why standard chemotherapy may shrink tumors; however, most tumors of this highly lethal gynecologic malignancy re-grow and eventually cause a relapse [28, 29]. Therefore, it is necessary to investigate the molecular mechanisms of EOC CSC chemoresistance. However, cancer investigation in the premise of “cancer as a cell based-disease” focused on finding tumor suppressor genes and oncogenes, while the role of the cancer cell environment was neglected . In our current study, we focused on the microenvironment of EOC CD44+CD117+CSCs and evaluated the characteristic of CD44+CD117+CSCs in the 3D model versus the 2D model.
We firstly demonstrated that the CD44+CD117+cells that were isolated from the SKOV-3 ovarian cell line and were identified by immunofluorescence microscope and the FCM (Figure1) possessed the CSC properties. This is because the CD44+CD117+CSCs had powerful tumorigenic capability in the xenograft mice (Figure2) and had fast growth activity in the 3D model versus in the 2D one when compared with the SKOV-3 cells (Figure3). These data provided authentic evidence that the CD44+CD117+cells were EOC CSCs, and established a foundation for further evaluation of EOC CSC chemoresistance in the 3D model versus the 2D one.
It is known that there have existed significant discrepancies between the efficacy of potential anticancer drugs when they are tested in vitro using cancer cells generally grown in monolayer cells and the clinical application of these drugs they are evaluated in vivo . For this reason, we employed the 3D culture based on the BME scaffold that is more adequate for analyzing the efficacy of anticancer drugs was employed in the present study. Because 5FU, CDDP, CBP, DXT and PTX were standard chemotherapeutic drugs for EOC, we selected these drugs to investigate their effect on the CD44+CD117+CSCs. The adopted IC50 concentration was based on our previous experiments (data no shown here). The results in Figure4 demonstrate the discrepancy in the effects of 5FU, CDDP, CBP and DXT between the 3D model and the 2D one. 5FU, CDDP, CBP and DXT inhibited CD44+CD117 +CSC growth by 50% in the 2D culture with the IC50 concentration; the effects of the drugs on the CD44+CD117+CSCs in the 3D model was significant lower than the effects of the drugs on the monolayer cells in the 2D model; 5FU, CDDP, CBP and DXT inhibited the growth of CD44+CD117 +CSCs by only 34.4%, 40.8%, 34.8% and 21.9%, respectively. The standard 2D cell culture condition drastically differs from that in the 3D one. Our findings may help to explain why the effect of some anticancer drugs have demonstrated valid effects on cancer cells when evaluated in vitro using 2D cell culture system, but the drugs have shown significant discrepancies in the observed efficacy when these drugs are evaluated in vivo .
In the study, we also found that the CD44+117+CSCs grew very slowly in the 2D culture and needed more than 20 days to form large, nonadherent clusters. Surprisingly, the CD44+117+CSCs grew fast in the 3D BME scaffold and formed a distinctive cell clusters on day 2, and developed the tumor spheres on day 4. However, this was not observed in the SKOV-3 cells. The property of fast growth observed in the CD44+117+cells in 3D BME scaffold was consistent with the characteristic of CSCs in vivo, which was reflected in the tumorigenic capability of the xenograft mice (Figure2).
PTX is known to have induced the G1 arrest and early apoptosis of cancer cells in the treatment of ovarian cancer . Consequently, more SKOV-3 cells underwent apoptosis than CD44+117+CSCs in the 3D and 2D culture systems, respectively. However, when the CD44+117+CSCs and SKOV-3 cells were cultured in the 2D environment, the effect of PTX on these cell survival was not as obvious as in the 3D culture (Figure5B). The results suggested that the compared to the 2D culture, the 3D culture reflected the tumor cells in vivo response to PTX therapy more objectively.
Evidence from recent research has shown that the oxygen and nutrient supply in the center of the tumor tissue are not enough to satisfy the need of cells of the tumor tissues, and this hypoxic microenvironment would make it hard for the tumor cells to survive [33, 34]. Thus, the data of survival assay showed that the SKOV-3 cells in 3D BME scaffold were hardly to keep long time and gradual apoptosis compared with the CD44+117+CSCs that were induced to adapt the hypoxic and innutritious environment in the 3D structure. This finding was consistent with CSCs in tumor tissues in vivo .
In this study, we observed differences in the efficacy of the anticancer drugs, the growth activity of the cancer cells, and the survival potency in the 3D and the 2D models; these differences led us to investigating the mechanism of the CD44+117+CSCs’ resistance to chemotherapeutic agents when the CD44+117+CSCs were cultured in the 3D and the 2D environments. Figure6 indicates that the expression of ABCG2 and ABCB1 on the CD44+117+CSCs were more significantly upregulated in 3D environment compared to 2D one in presence of the 5FU, CDDP, CBP and DXT, respectively. According to the CSC hypothesis , CSCs are naturally resistant to chemotherapy through the expression of ABC-transporter that enables a cancer to escape the cytotoxic effects of chemotherapy, which might be one of important mechanism in the development of resistance to chemotherapy. Compared with the SKOV-3 cells, the CD44+117+CSCs not only markedly increased the expression of ABCG2 and ABCB1 in the 2D environment compared with the SKOV-3 cells, but also enhanced their expression in the 3D BME scaffold in contrast to the 2D environment. We analyzed the BME scaffold that formed the elaborate 3D microenvironment that was similar to the tumor in vivo and facilitated the maintenance of the inherent malignant trait of CD44+117+CSCs, and this is maybe one of the reasons that CD44+CD117+CSCs were more resistant to 5FU, CDDP, CBP, DXT and PTX in the 3D environment than in the 2D environment at IC50 concentration. However, the expression of Nanog molecule in the CD44+CD117+CSCs was not the same as the expression of ABCG2 and ABCB1 in the 3D culture system. Because the Nanog molecule is transcriptional determinants and directs the multi-potential differentiation of undifferentiated stem cells [24, 36], it may be not closely associated with the resistance to chemotherapy.