Cisplatin resistance driver claspin is a target for immunotherapy in urothelial carcinoma

Abstract

Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

Keywords: Cisplatin resistance; Claspin; Immunotherapy; Urothelial carcinoma.

Fig. 1

Establishment and characterization of a CR subline. A Schematic summary of the culture strategy for establishing a CR cell line. UM-UC-3 cells were cultured in medium containing CDDP at 0.5 μmol/L. The concentration of CDDP was increased over a period of approximately 6 months. B Sensitivity of WT and CR cells to CDDP. The cells were cultured in medium containing CDDP at several concentrations for 48 h. Cell viability was assessed by a WST-8 assay. Each value is the mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. C ALDEFLUOR assay. WT UM-UC-3 and CR cells were stained with BODIPY®-aminoacetaldehyde and analyzed by FACS. The ALDH^high-positive gate was defined by a sample treated with the ALDH1 inhibitor DEAB. Numerical data indicate ALDH^high rates. D Sphere-formation assay. The sphere-forming ability of WT UM-UC-3 and CR cells was assessed. The cells were seeded at 1.0 × 10³, 10², 10¹, and 10⁰ cells/well in an ultra-low attachment plate and cultured for 1 week. Sphere-forming wells were counted. Stem cell frequency was analyzed using the ELDA web site. CI, confidence interval. Differences of the estimated frequencies of CSCs/CICs were analyzed by a chi-square test

Fig. 2

CLSPN is overexpressed in CR cells. A CAGE analysis of WT UM-UC-3 cells vs. CR cells. Gene expression in CR cells was assessed by CAGE analysis. The mRNA expression of WT and CR cells was analyzed in duplicate and fold-change was plotted in a scatter plot (left panel). The CLSPN plot is indicated. To narrow down candidate genes, (1) CR overexpressed genes, (2) expression in normal organs < 2 (transcripts per million), and (3) HLA-A2-bound peptides were used as filters. Finally, CLSPN and its coding antigenic peptide SLLNQPKAV were identified. B qRT-PCR of CLSPN in normal organs. CLSPN expression in normal organs was assessed by qRT-PCR. Each value is the relative mean ± SD. C qRT-PCR of CLSPN in WT UM-UC-3 and CR cells. CLSPN expression in WT UM-UC-3 and CR cells was assessed by qRT-PCR. Each value is the relative mean ± SD. D Western blot analysis of WT UM-UC-3 and CR cells. CLSPN and MDR1 expression in WT UM-UC-3 and CR cells was assessed by western blot analysis. β-Actin was used as an internal positive control

Fig. 3

CLSPN has a role in CDDP resistance and its expression is induced by CDDP. A Resistance to CDDP. Resistance to CDDP was assessed by a WST-8 assay. WT UM-UC-3 and CR cells were cultured in medium containing serial concentrations of CDDP for 48 h, and then cell viability was examined by a WST-8 assay. Each value is the relative mean ± SD. B CLSPN mRNA induction by CDDP treatment. WT UM-UC-3 and CR cells were cultured in medium containing serial concentrations of CDDP for 48 h, and then CLSPN expression in normal organs was assessed by qRT-PCR. Each value is the relative mean ± SD

Fig. 4

Induction and characterization of a CLSPN peptide-specific CTL clone. A A schematic summary of CTL induction. PBMCs were obtained from an HLA-A*02:01-positive donor and CD8⁺ T cells were then isolated. CD8⁺ T cells were stimulated with HLA-A*02:01⁺ aAPCs (K562 cells transduced with CD80, CD83, and HLA-A*02:01 cDNAs) pulsed with CLSPN peptide (SLLNQPKAV) at day 1, 8, 15, 22, and 29. At day 34 the CD8⁺ T cells were assessed by an ELISPOT assay and FACS. B CTL cloning strategy. Stimulated CD8⁺ T cells were analyzed by an IFNγ ELISPOT assay and FACS with CLSPN tetramer staining. For the IFNγ ELISPOT assay, T2 cells pulsed with CLSPN peptide and non-pulsed cells were used as targets. For FACS analysis, HIV-tetramer was used as a negative control. CLSPN tetramer-positive cells were single cell sorted and cultured. Cells grew in seven wells and one clone (yc3) showed reactivity to CLSPN peptide according to an IFNγ ELISPOT assay and FACS. C CLSPN peptide-specific CTL clone recognizes CR cells. The CLSPN peptide-specific CTL yc3 clone was evaluated for reactivity to WT UM-UC-3 and CR cells by an IFNγ ELISPOT assay. CLSPN peptide-pulsed T2 cells were used as a positive control and peptide non-pulsed T2 cells were used as a negative control. Each value is the mean ± SD. D CLSPN peptide-specific CTL clone recognizes CDDP-treated WT cells. The CLSPN peptide-specific CTL yc3 clone was evaluated for reactivity to WT UM-UC-3 cells and CDDP-treated WT cells by an IFNγ ELISPOT assay. WT cells were cultured in CDDP-containing medium (5 μM) for 48 h, and then used for assay. T2 cells were used as a negative control. Each value is the mean ± SD. E CLSPN knockdown by siRNA decreases the CTL response. WT UM-UC-3 cells were transfected with CLSPN siRNA, and 48 h later, the cells were evaluated by an IFNγ ELISPOT assay with the CLSPN peptide-specific CTL yc3 clone. T2 cells were used as a negative control. Each value is the mean ± SD

Fig. 5

Cytotoxicity assay using the CLSPN peptide-specific CTL clone. A Cytotoxicity of the yc3 clone for CLSPN peptide-pulsed targets. The cytotoxicity of the yc3 clone was assessed by an LDH release assay. Target cells were co-cultured with the yc3 clone for 8 h at different effector/target ratios. HIV peptide-pulsed T2 cells, peptide non-pulsed T2 cells, and K562 cells were used as negative controls. Each value is the mean ± SD. B Cytotoxicity of the yc3 clone to WT UM-UC-3 and CR cells. The cytotoxicity of the yc3 clone was assessed by an LDH release assay. WT UM-UC-3 and CR cells were used as targets. Target cells were co-cultured with the yc3 clone for 8 h at different effector/target ratios. Each value is the mean ± SD