Characteristics of CD133-Sustained Chemoresistant Cancer Stem-Like Cells in Human Ovarian Carcinoma

Abstract

Cancer stem cells (CSCs) are considered to be the origin of ovarian cancer (OC) development, recurrence, and chemoresistance. We investigated changes in expression levels of the CSC biomarker, cluster of differentiation 133 (CD133), from primary OC cell lines to induction of CSC-spheres in an attempt to explore the mechanisms related to modulation of stemness, drug resistance, and tumorigenesis in CSCs, thus facilitating the search for new therapeutics for OC. The effect of CD133 overexpression on the induction of CSC properties was evaluated by sphere-forming assays, RT-qPCR, flow cytometry, cell viability assays, and in vivo xenograft experiments. Moreover, the potential signaling molecules that participate in CD133 maintenance of stemness were screened by RNA-sequencing. CD133 expression was upregulated during OCSC induction and chemotherapeutic drug treatment over time, which increased the expressions of stemness-related markers (SOX2, OCT4, and Nanog). CD133 overexpression also promoted tumorigenesis in NOD/SCID mice. Several signalings were controlled by CD133 spheres, including extracellular matrix receptor interactions, chemokine signaling, and Wnt signaling, all of which promote cell survival and cell cycle progression. Our findings suggest that CD133 possesses the ability to maintain functional stemness and tumorigenesis of OCSCs by promoting cell survival signaling and may serve as a potential target for stem cell-targeted therapy of OC.

Keywords: CD133; cancer stem cell (CSC); chemoresistance; ovarian cancer (OC); sphere-forming assay.

Figure 1

Sphere-forming ability and expression of stemness-related markers of human ovarian cancer (OC) cell lines. (A) Optical microscopic images showed morphological changes and sphere formation of SKOV3, OVCAR3, HTB75, and IGROV1 cells at days 0, 4, and 16. Individual scale bars are shown. OC-sphere cells collected at each time point for (B) cell surface expressions of cluster of differentiation 44 (CD44) and CD133 were assessed using flow cytometry; (C) expression of the stemness-related markers Sox2, Oct4, and Nanog on day 16 were assessed using RT-qPCR; and (D) sphere cell numbers. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Pretreatment with cisplatin (CDDP) combined with paclitaxel (PTX) synergistically enhanced the sphere-forming ability, cluster of differentiation 133 (CD133) expression, and chemoresistant capacities of cancer stem cell (CSC)-like SKOV3 spheres. SKOV3 parental cells (SKOV3-P) were pretreated with 1 μM CDDP alone, 1 nM PTX alone, or their combination for 3, 5, and 9 days, and then cells were collected for 4-day sphere-forming and cell surface CD133 expression assays in (A,B). CD44+CD133⁻ sphere as well as CD44+CD133+ sphere populations were sorted for cell viability assay comparisons among Untreated (C), CDDP-treated (D), PTX-treated (E), and CDDP/PTX-treated (F) after 12, 24, 36, and 48 h of drug treatment. Presented data were acquired from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Cluster of differentiation 133 (CD133) expression is essential for sustaining cancer stem cell (CSC) traits and drug-resistance capacities. Effects of CD133 transduction and overexpression in (A) sphere-forming ability in (C) and morphological changes in CD133 non-expressing SKOV3 cells following 4, 8, 12, and 16 days of sphere cell induction in (B). Effects of CD133 overexpression with 1.2 μM cisplatin (CDDP) alone, 1.4 nM paclitaxel (PTX) alone, or their combination were evaluated by cell viability assays among untreated in (D), CDDP-treated in (E), PTX-treated in (F), and CDDP+PTX-treated in (G) after 12, 24, 36, and 48 h of drug treatment. Data shown were acquired from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Cancer stem cell (CSC)-like SKOV3 spheres transduced with cluster of differentiation 133 (CD133) promoted tumor growth in a xenograft mice model. SKOV3 spheres with CD133 transduction (SP-CD133), SKOV3 spheres with mock-transduction (SP-Mock), and parental SKOV3 (SKOV3-P) cells were subcutaneously (s.c.) implanted into NOD/SCID mice. (A) Detailed treatment schedule of the in vivo study is shown. (B) Tumor volumes (cm³) during the subsequent period (n = 6 per group; * p < 0.05; *** p < 0.001). (C) Body weights during the subsequent period. (D) Gross images of dissected tumors at the study endpoint. (E) Mean tumor volumes (cm³) plotted in a bar graph, and mean tumor weights (mg) expressed in line plots from each mouse group at the study endpoint (n = 6 per group; * p < 0.05; *** p < 0.001).

Figure 5

Cell proliferation and stemness related markers are overexpressed in SKOV3 spheres with cluster of differentiation 133 (CD133) transduction (SP-CD133) tumors. Tumors were harvested from mice bearing SP-CD133, SKOV3 spheres with mock-transduction (SP-Mock), and parental SKOV3 (SKOV3-P) subcutaneous (s.c.) xenografts. (A) Formalin-fixed, paraffin-embedded tumor sections were consecutively cut and stained for human CD44, Ki67, CD133, Nanog, and Oct4. Images were taken using a camera (DP22) and microscope (BX50) under 400× or 200× magnification. Individual scale bars are shown. (B) An average H-scores for each marker and comparison between groups are shown (* p < 0.05; ** p < 0.01).

Figure 6

Overexpression of cluster of differentiation 133 (CD133) in SKOV3 spheres with CD133 transduction (SP-CD133) induced the reduction of extracellular matrix (ECM) receptor interactions/focal adhesion and promoted cell cycle progression. (A) The heatmap of the differentially expressed genes (DEGs) extracted from the three SKOV3-related modules including SP-CD133, SKOV3 spheres with mock-transduction (SP-Mock), and parental SKOV3 (SKOV3-P) cells. (B) KEGG pathway enrichment analysis of all DEGs. (C) Simplified diagram of key factors/pathways modulated by CD133. (D) Representative RT-qPCR analysis comparing expression levels of ITGA6, ITGB8, PIK3CA, FOXO3, RBL2, FasL, Bcl2, GPCR, CREB, LRP5, and TCF. Data shown were acquired from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.

Figure 6

Overexpression of cluster of differentiation 133 (CD133) in SKOV3 spheres with CD133 transduction (SP-CD133) induced the reduction of extracellular matrix (ECM) receptor interactions/focal adhesion and promoted cell cycle progression. (A) The heatmap of the differentially expressed genes (DEGs) extracted from the three SKOV3-related modules including SP-CD133, SKOV3 spheres with mock-transduction (SP-Mock), and parental SKOV3 (SKOV3-P) cells. (B) KEGG pathway enrichment analysis of all DEGs. (C) Simplified diagram of key factors/pathways modulated by CD133. (D) Representative RT-qPCR analysis comparing expression levels of ITGA6, ITGB8, PIK3CA, FOXO3, RBL2, FasL, Bcl2, GPCR, CREB, LRP5, and TCF. Data shown were acquired from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.