Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

Abstract

Background: In ovarian cancer, massive intraperitoneal dissemination is due to exfoliated tumor cells in ascites. Tumor-initiating cells (TICs or cancer stem cells) and cells showing epithelial-mesenchymal-transition (EMT) are particularly implicated. Spontaneous spherical cell aggregates are sometimes observed, but although similar to those formed by TICs in vitro, their significance is unclear.

Methods: Cells freshly isolated from malignant ascites were separated into sphere samples (S-type samples, n=9) and monolayer-forming single-cell suspensions (M-type, n=18). Using western blot, these were then compared for expression of protein markers of EMT, TIC, and of cancer-associated fibroblasts (CAFs).

Results: S-type cells differed significantly from M-type by expressing high levels of E-cadherin and no or little vimentin, integrin-β3 or stem cell transcription factor Oct-4A. By contrast, M-type samples were enriched for CD44, Oct-4A and for CAF markers. Independently of M- and S-type, there was a strong correlation between TIC markers Nanog and EpCAM. The CAF marker α-SMA correlated with clinical stage IV. This is the first report on CAF markers in malignant ascites and on SUMOylation of Oct-4A in ovarian cancer.

Conclusions: In addition to demonstrating potentially high levels of TICs in ascites, the results suggest that the S-type population is the less tumorigenic one. Nanog(high)/EpCAM(high) samples represent a TIC subset which may be either M- or S-type, and which is separate from the CD44(high)/Oct-4A(high) subset observed only in M-type samples. This demonstrates a heterogeneity in TIC populations in vivo which has practical implications for TIC isolation based on cell sorting. The biological heterogeneity will need to be addressed in future therapeutical strategies.

Figure 1

Spheres constitute a separate population. Ascitic cells were isolated from ovarian cancer patient ascites and separated into monolayer-forming single-cell suspensions (M-type samples; M) and spontaneous spheres (S-type samples; S), all as described in Materials and Methods. Lysates of each sample were analysed by western blot, with GAPDH as loading control. As interblot reference, a lysate (one and the same for all experiments) of the SKOV-3 cell line, derived from EOC malignant ascites, was used. Numbers refer to patients. A. Representative images of spheres, 40x magnification. Left: immediately after isolation. Right: Dispersed spheres do not form monolayers in culture, but continue to form spheres like this one and which then detach from the few adherent cells seen in the background. B.Top: Representative example of E-cadherin and vimentin expression in paired M- and S-type samples, each pair from one and the same patient. Below: The data on E-cadherin and vimentin expression levels throughout the whole cohort are summarized in box plots comparing the distribution of relative expression of these proteins in all M (n = 18) and S (n = 9) samples, respectively. Asterisks denote statistically significant differences (Mann–Whitney U test). C. Representative examples of E-cadherin and vimentin expression in paired M- and S-samples from two patients, and artificial spheroids (AS) created in vitro using the M-sample cells. D. Expression of integrin β3 in two paired M- and S-type samples, each pair from one and the same patient.

Figure 2

Presence of cancer-associated fibroblasts in ascites. A representative western blots showing co-expression of PDGFβR and α-SMA in paired M- and S-samples from three patients. In total 9/25 samples showed expression of PDGFβR, all of them M-type. Numbers refers to patients. B. Box plots comparing the distribution of relative expression of α-SMA in M- (n = 17) and S-type (n = 8) samples (left) and in samples representing clinical stage IIIC (n = 17) and IV (n = 8) at diagnosis (right). Asterisks denote statistically significant difference (Independent t-test).

Figure 3

TIC protein marker expression in M- and S-type samples. A. Representative blot showing CD44 expression in M- and S-type samples. Based on all samples, CD44 was associated with M-type (p<0.001; Table 2). Numbers refer to patients. B. The SKOV-3 cell line was used for immunoprecipitation with antibodies to Oct-4 and to SUMO, respectively. Shown here is a representative western blot of three immunoprecipitates made with antibodies to Oct-4, SUMO and mouse IgG, respectively, and which were then probed using anti-Oct-4A. Mouse IgG served as negative control. SUMO-Oct-4A could be detected when probed with anti-SUMO (not shown). The results were confirmed using two other cell lines (not shown). Arrow: native ~40 kDa Oct-4A just above the 25 kDa IgG light chain. IP: immunoprecipitation; WB: western blot probe; m-IgG: mouse IgG.C. Oct-4A and SUMO-Oct-4A in M-type samples and paired samples of M- and S-type. D. Box plots comparing the distribution of relative expression of Oct-4A, SUMO-Oct-4A and Nanog, respectively, in all M (n=18) and S (n=9) samples. Asterisks denote statistically significant differences (Mann–Whitney U test).