The expressions of differential markers in OCSPCs and NOSPCs
The OCSPCs showed higher expressions of CA125, Flt4, AC133, CD34, CD117, and CD146, but lower expressions of CD24, NANOG and OCT3/4 compared to the NOSPCs (Fig. 1a). The expression levels of BMP2 and BMP4 were also higher in the OCSPCs (Fig. 1b). The OCSPCs also highly expressed cell cycle-related genes such as those encoding P21, P27, P53, cyclin D, and Bcl-xL (Fig. 1b). The epithelial-like OCSPCs showed higher expressions of cytokeratin 18 and E-cadherin than the mesenchymal-like OCSPCs (Fig. 1a). In contrast, the mesenchymal-like OCSPCs had higher expressions of AC133, CD117, integrin α2β1, CD146, CXCR4, NANOG and OCT3/4 than the epithelial-like OCSPCs (Fig. 1a).
Both epithelial- and mesenchymal-like OCSPCs originated from mesothelial cells not ovarian cancer cells
Isolated epithelial- and mesenchymal-like OCSPCs in ascites and cancerous tissues were further analyzed to differentiate their origin. The G281T point mutation in exon 8 of TP53 (aspartic acid changed to tyrosine) was detected in the cancerous tissues (Additional file 3: Figure S1). However, only wild-type TP53 of both epithelial- and mesenchymal-like OCSPCs was noted. This indicated that the non-cancerous origin of the OCSPCs. Furthermore, immunocytochemical analysis demonstrated strong expressions of ovarian epithelial cancer cell and mesothelial cell markers, PAX8 and calretinin, in both types of OCSPC (Fig. 2a), implicating that a mesothelial origin of the OCSPCs was most likely.
MS-MLPA profiles of TSG methylation in the OCSPCs and ovarian cancer cells
DNA methylation of TSGs in the OCSPCs and ovarian cancer cells was analyzed by MS-MLPA. The CMI among 40 TSGs was significantly higher in the OCSPCs from ascites than that from tissues (p < 0.001, Additional file 4: Table S3). The gene with the most frequent hypermethylation in the OCSPCs from ascites was CDKN2B (50 %), followed by RASSFIA (44 %) and DLC1 (44 %) (Additional file 5: Table S4). Whereas, the most frequently hypermethylated gene detected in the OCSPCs from cancerous tissues was CCND2 (50 %), followed by CDKN2B (25 %) and DLC1 (25 %) (Additional file 5: Table S4). Among these 40 genes, none was found to be methylated in the NOSPCs. The most frequently hypermethylated genes in the bulk tumors were CDKN2B (63 %), RASSFIA (50 %), and DLC1 (38 %) (Additional file 5: Table S4). In addition, the CMIs of CDKN2B, RASSFIA, DLC1 and CCND2 in the OCSPCs from ascites were significantly higher than those in the OCSPCs from tissues (p = 0.001) or bulk tumor cells (p = 0.038).
Expression levels of CDKN2B, RASSFIA, DLC1 and CCND2 genes in the OCSPCs and ovarian cancerous tissues
To clarify whether the methylation status was correlated with the expression levels of CCND2, RASSF1A, DLC1 and CDKN2B, the mRNA levels of these genes were quantified by QRT-PCR. The mRNA levels of CCND2 (0.374 ± 0.433 vs. 0.733 ± 0.583, p = 0.012, student’s t test) and CDKN2B (0.143 ± 0.048 vs. 1.172 ± 0.740, p < 0.001, student’s t test), but not RASSF1A (1.635 ± 0.433 vs. 2.150 ± 1.630, p = 0.229, student’s t test) and DLC1 (1.269 ± 0.502 vs. 1.985 ± 1.099, p = 0.085, student’s t test), were significantly lower in the OCSPCs from ascites than those from bulk tumor tissues. When stratifying the OCSPCs by origin, the mRNA levels of CCND2 (0.074 ± 0.06 vs. 0.733 ± 0.583, p < 0.001, student’s t test), RASSF1A (0.553 ± 0.164 vs. 2.150 ± 1.630, p < 0.001, student t test) and CDKN2B (0.153 ± 0.056 vs. 1.172 ± 0.740, p < 0.001, student’s t test), but not DLC1 (1.471 ± 0.573 vs. 1.985 ± 1.099, p = 0.374, student t test) were significantly lower in the epithelial-like OCSPCs from ascites than those from bulk tumor tissues. Whereas, the mRNA levels of CDKN2B (0.128 ± 0.034 vs. 1.172 ± 0.740, p < 0.001, student’s t test) were significantly lower in the mesenchymal-like OCSPCs from ascites than those from bulk tumor tissues, but CCND2 (0.824 ± 0.325 vs. 0.733 ± 0.583, p = 0.441, student’s t test), RASSF1A (2.987 ± 0.872 vs. 2.150 ± 1.630, p = 0.066, student’s t test) and DLC1 (1.068 ± 0.357 vs. 1.985 ± 1.099, p = 0.051, student’s t test) remained similar in the mesenchymal-like OCSPCs from ascites than those from bulk tumor tissues. The mRNA levels of these four genes were significantly lower in the epithelial-like OCSPCs (0.604 ± 0.588 vs. 1.550 ± 1.280, p = 0.002, student’s t test) than those in the mesenchymal-like OCSPCs.
In addition, the mRNA levels of these four genes were significantly lower in the OCSPCs from ascites (1.038 ± 1.069 vs. 1.212 ± 0.902, p = 0.022, student’s t test) than those in the OCSPCs from tissues or those in the bulk tumor tissues (1.038 ± 1.069 vs. 1.509 ± 1.279, p < 0.001, student’s t test).
Epithelial-like but not mesenchymal-like OCSPCs exhibited epithelial-mesenchymal transition (EMT) and formed tumorspheres
Both epithelial- and mesenchymal-like OCSPCs were able to form non-adherent, aggregated spheroids in low adherent discs supplemented with specific growth factors and could be maintained for at least 7 days (Fig. 2b). Only integrin α2β1 significantly increased in the cells within the tumorsphere compared to those in the original adherent OCSPCs (Figs. 1a, 2c). However, a higher expression of E-cadherin was detected in adherent epithelial-like OCSPCs than in the cells that formed non-adherent OCSPC spheroids (93.7 vs. 75 %) (Figs. 1a, 2c). However, similarly lower levels of E-cadherin were found in the mesenchymal-like non-adherent spheroid cells and the adherent OCSPCs (Figs. 1a, 2c). Furthermore, the glycan cell surface molecules, stage-specific embryonic antigen SSEA3 and SSEA4, were also highly expressed in the non-adherent spheroid cells (Fig. 2c). When the spheroids formed by both types of adherent OCSPCs were cultured for 14 days, the expression of E-cadherin decreased by at least fivefold (17.9 vs. 93.4 %), however vimentin increased by approximately 40-fold (83.1 vs. 2.4 %) in non-adherent spheroid cells compared to those in adherent epithelial-like OCSPCs (Fig. 2d), implicating the occurrence of EMT. E-cadherin remained at a similar lower level in the mesenchymal-like non-adherent spheroid cells and the adherent mesenchymal-like OCSPCs (Fig. 2d). The expressions of the other stemness markers including AC133, CD34, CD117, CXCR4, NANOG and OCT3/4 were also significantly increased in the non-adherent tumorspheres than those in the adherent OCSPCs (Fig. 2d).
Multipotent capability of the OCSPCs to differentiate into various cell lineages
The OCSPCs were further examined to see if they had the potential to produce different types of cells. The results showed that the mesenchymal-like OCSPCs had the capability to differentiate into multiple cell types including myogenic (Fig. 3a), neurogenic (Fig. 3b), adipogenic (Fig. 3c), osteogenic (Fig. 3d), chondrogenic (Fig. 3d), and vascular (Fig. 3e) cells under different conditional culture media.
The OCSPCs promoted tumor growth in vivo
The influence of the OCSPCs on ovarian tumor growth was further evaluated with modified SKOV3 ovarian cancer cells, SKOV3-Luc cells carrying the luciferase reporter. The representative chemiluminescent images of SKOV3-Luc tumor growth in the various groups are shown in Fig. 4a. The mice that received SKOV3-Luc cells with epithelial-like OCSPCs demonstrated a consistent increase in luciferase activity compared with SKOV3-Luc cells alone, SKOV3-Luc cells with mesenchymal-like OCSPCs, and the SKOV3-Luc cells with NOSPCs (Fig. 4b). On day 39 after xenograft, the mean tumor sizes were largest in the mice that received SKOV3 cells with the epithelial-like OCSPCs compared with the other groups (Fig. 4c). These data suggest that epithelial-like OCSPCs can promote ovarian tumor growth. Beside, the histological analysis of these tumors was performed to reveal that all these tumors were similar to those in the histopathology of high grade serous adenocarcinoma of human ovarian cancer.
5-aza-2-dC changed the expression profiles and reactivated TSGs in the OCSPCs
To investigate the impact of demethylation on the tumorigenic promoting activity of OCSPCs, representative marker expressions were analyzed in cells treated with 5-aza-2-dC. The OCSPCs treated with 5-aza-2-dC had higher levels of CD24, CD105, and CXCR4, but lower levels of AC133, CD34, CD90, CD117, EGFR, integrin α2β1, CD146, FLT4, NANOG, and OCT3/4 compared to the cells without treatment (Fig. 5a). In addition, the CMI among the 40 TSGs tested was significantly lower in the epithelial-like OCSPCs after 5-aza-2-dC treatment compared to those without treatment (p < 0.001, Additional file 6: Table S5). However, the demethylating effect of 5-aza-2-dC was not detected in the mesenchymal-like OCSPCs, although demethylation (Fig. 5b) and increased RNA levels (Fig. 5c) of DLC1, CCND2, RASSF1A, and CDKN2B genes were identified in both the epithelial- and mesenchymal-like OCSPCs on day 4 or day 6 post-exposure to 5-aza-2-dC.
5-aza-2-dC altered self-renewal, growth- and stemness-related gene expressions in the OCSPCs and reduced the niche potential of OCSPCs for tumorigenicity of ovarian cancer
Neither mesenchymal- nor epithelial-like progenitor cells derived from malignant ascites had significant morphological changes upon 5-aza-2-dC treatment (Fig. 5d). Kinetic analysis demonstrated that the growth rates of the mesenchymal- or epithelial-like progenitor cells were reduced when treated with 5-aza-2-dC (Fig. 6a). The expressions of the stem-like cell surface markers including AC133, CD34, CD117, EGFR, integrin α2β1, Flt4, and CD146 in the OCSPCs were decreased upon 5-aza-2-dC treatment (Fig. 5a). In addition, both the epithelial- and mesenchymal-like OCSPCs showed significantly reduced levels of TWIST1, HIF-1α, MDR1, and ABCG2 on treatment with 5-aza-2-dC (Fig. 6b). The higher expression levels of MDR1 and ABCG2 efflux pumps may reflect increased drug resistance.
Exposure to 5-aza-2-dC also resulted in twofold decrease in the number of in vitro tumor spheres formed by the OCSPCs (Fig. 6c). The in vivo animal experiments revealed that the mice receiving SKOV3-Luc plus 5-aza-2-dC-treated epithelial-like OCSPCs demonstrated a consistent decrease in luciferase activity compared to the mice receiving SKOV3-Luc plus epithelial-like OCSPCs without 5-aza-2-dC for up to 39 days (Fig. 4a, b). However, 5-aza-2-dC treatment of the mesenchymal-like OCSPCs did not affect their influence on the growth of SKOV3-Luc tumor cells in the xenograft mice. In addition, 5-aza-2-dC treatment reduced the ability of the epithelial-like OCSPCs to promote tumorigenicity of SKOV3-Luc cancer cells and inhibited sphere formation of these OCSPCs. However, 5-aza-2-dC treatment also suppressed the tumorigenicity of SKOV3 in a mouse model [29].
5-aza-2-dC altered the expressions of genes related to EMT and drug resistance in the OCSPCs
To correlate the demethylating effect of 5-aza-2-dC with the expression levels of EMT- and drug resistance-related genes, tumors isolated from the xenograft mice were analyzed. The RNA levels of drug resistance-related genes MDR1 and ABCG2, and hypoxia-related genes HIF-1α and HIF-2α were reduced in tumors derived from SKOV3 with 5-aza-2-dC-treated epithelial-like OCSPCs compared to SKOV3 with epithelial-like OCSPCs without treatment, or with mesenchymal-like OCSPCs or normal progenitor cells (Fig. 6d). Sustained downregulation of key regulatory genes including TWIST, Slug and Snail, which promote the process of EMT, was decreased in the tumors generated by SKOV3 with 5-aza-2-dC-treated epithelial-like OCSPCs (Fig. 6d). These data support the hypothesis that epigenetic demethylating stem-like cells in the niche of ovarian cancer may reduce the tumorigenicity of epithelial ovarian cancer, possibly through the ablation of the EMT.