Ovarian Cancer Stem Cells with High ROR1 Expression Serve as a New Prophylactic Vaccine for Ovarian Cancer

Abstract

Tumor vaccines offer a number of advantages for cancer treatment. In the study, the vaccination with cancer stem cells (CSCs) with high expression of the type I receptor tyrosine kinase-like orphan receptor (ROR1) was evaluated in a murine model for the vaccine's immunogenicity and protective efficacy against epithelial ovarian carcinoma (EOC). CD117⁺CD44⁺ CSCs were isolated from human EOC HO8910 cell line using a magnetic-activated cell sorting system; murine ID8 EOC suspension sphere cells, which are collectively known as cancer stem-like cells, were acquired from serum-free suspension sphere-forming culture. Mice were subcutaneously immunized with the repeat cycles of freezing and thawing whole HO8910 CD117⁺CD44⁺ CSCs and ID8 cancer stem-like cells, respectively, followed by a challenge with HO8910 or ID8 cells at one week after final vaccination. The results showed that the CSC vaccination significantly induced immunity against EOC growth and markedly prolonged the survival of EOC-bearing mice in the prophylactic setting compared with non-CSC vaccination. Flow cytometry showed significantly increased immunocyte cytotoxicities and remarkably reduced CSC counts in the CSC-vaccinated mice. Moreover, the protective efficacy against EOC was decreased when the ROR1 expression was downregulated by shRNA in CSC vaccines. The findings from the study suggest that CSC vaccines with high ROR1 expression were highly effective in triggering immunity against EOC in vaccinated mice and may serve as an effective vaccine for EOC immunoprophylaxis.

Figure 1

Evaluation of CSC vaccination evoking antitumor efficacy in a mouse model. (a, b) Tumor size images on day 45 from the HO8910 tumor-bearing nude mice or on day 80 from the ID8 tumor-bearing mice after mice were initially vaccinated s.c. with 2 × 10⁵ different inactivated vaccines three times with a 10-day interval, followed by a challenge with 1 × 10⁶ HO8910 or 2 × 10⁶ ID8 cells at one week after final vaccination. (c, d) Kinetic quantification of tumor sizes by measuring two perpendicular tumor diameters using calipers. (e, f) Tumor-free mice. All the data represent the mean ± SD (n = 6 per group; representative images). Statistically significant differences between the experiment group and the normal group were indicated.

Figure 2

Analysis of CD44⁺CD117⁺ cell and ALDH-positive cell subsets. (a) Representative FCM plots of HO8910 CD44⁺CD117⁺ double-positive cells were accounted in various groups. (b) Quantification of CD44⁺CD117⁺ cells. (c) FCM plot of 1 × 10⁶ ovarian cancer tissue cells using the ALDEFLUOR assay. The sorting gates were established based on DEAB-stained controls. DEAB were used to establish the baseline fluorescence of these cells (R1) and to define the ALDEFLUOR-positive cell region (R2). (d) Quantitative analysis of ALDH-positive cell subset. (e) Western blot analysis of ALDH expression in ovarian cancer tissues derived from vaccinated mice. (f) Quantification of ALDH expression. Statistically significant differences were indicated.

Figure 3

Detection of the cytolytic activities of NK, CDC, and ADCC in the vaccinated mice. The NK cytotoxic activity (target cells: YAC-1) was analyzed by FCM in the HO8910 CSC vaccine-immunized mice (a) and the ID8 sphere vaccine-immunized mice (c). (b, f) Quantification of NK cytotoxic activity. (d) The splenocyte cytotoxicity (target cells: ID8) was analyzed by FCM in the ID8 sphere vaccine-immunized mice. (g) Quantification of the splenocyte cytotoxicity. (e) FCM analysis of the CDC activity. (h) Quantification of CDC activity in ID8 vaccines. (i) FCM analysis of the CDC activity in the HO8910 CSC vaccine-immunized mice. (j) Quantification of CDC activity in HO8910 vaccines. (k) FCM analysis of the ADCC activity. (l) Quantification of ADCC activity in ID8 vaccines. All the data represent the mean ± SD (n = 12). Statistically significant differences between the different groups were indicated.

Figure 4

The levels of IFN-γ, anti-ROR1 antibody, and TGF-β1 as measured by ELISA. (a, c) Serum IFN-γ level in the different vaccinated groups. (b, d) Serum TGF-β1 level in the different vaccinated groups. (e, f) Anti-ROR1 antibody level in the second (serum 1 : 40 dilution) and the third (serum 1 : 320 dilution) immunizations. Data are represented as the mean ± SEM (n = 12). Statistically significant differences were indicated.

Figure 5

Vaccination-associated predominating antigen expressions and effects of ROR1 knockdown on immunogenicity of CSC vaccination. (a) The mRNA expressions of ROR-1, MUC-1, and NY-ESO-1 in HO8910 CSCs and HO8910 non-CSCs detected by qRT-PCR. (b) ROR-1 expression in the different cells detected by qRT-PCR. (c–g) ROR-1 expression in the different cells analyzed by Western blotting and quantification of analysis. (h) Serum IFN-γ level in shROR1 HO8910 CSC-vaccinated mice. (i) Anti-ROR1 antibody level in the shROR1 HO8910 CSC-vaccinated mice (serum 1 : 320 dilution). (j) Serum TGF-β1 level in shROR1 HO8910 CSC-vaccinated mice. (k) Serum IFN-γ level in shROR1 ID8 sphere-vaccinated mice. (l) Anti-ROR1 antibody level in the shROR1 ID8 sphere-vaccinated mice (serum 1 : 160 dilution). (m) Serum TGF-β1 level in shROR1 ID8 sphere-vaccinated mice. (n) NK cytotoxicity in shROR1-ID8 sphere-vaccinated mice. (o) Quantification of NK cytotoxicity. (p) Splenocyte cytotoxicity in shROR1-ID8 sphere-vaccinated mice. (q) Quantification of splenocyte cytotoxicity. (r) CDC activity in shROR1-ID8 sphere-vaccinated mice. (s) Quantification of CDC activity. Statistically significant differences between the different groups were indicated.

Figure 6

The shROR1 CSC vaccination decreased the antitumor ability. (a) Tumor size images on day 48 from the HO8910 tumor-bearing nude mice immunized initially with the 2 × 10⁵ shROR1 HO8910 CSC and other control vaccines. (b) Tumor size images on day 80 from the ID8 tumor-bearing mice immunized with the 2 × 10⁵ shROR1 ID8 cancer stem-like cell and other control vaccines. A total of three times, with an interval of 10 days between the immunizations, followed by a challenge with 1 × 10⁶ HO8910 cells (a) or 2 × 10⁶ ID8 cells (b) at one week after final vaccination. (c, d) Kinetic quantification of tumor sizes. (e, f) Tumor-free mice. All the data represent the mean ± SD (n = 6 per group; representative images). Statistically significant differences between the different groups were indicated.