GRIK2 is a target for bladder cancer stem-like cell-targeting immunotherapy

Abstract

Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDH^high clone cells were established. ALDH^high clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDH^high clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDH^high clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

Keywords: Antigen; Bladder cancer; Cancer stem cell; Cytotoxic T lymphocytes; GRIK2.

Fig. 1

Establishment of a bladder CSC/CIC model. A ALDEFLUOR assay of the UM-UC-3 human bladder cancer line cell. Left panel: UM-UC-3 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. The top 0.9% (ALDH^high) and lower 0.9% (ALDH^low) cells were single cell sorted and cultured for more than 1 month. Right panel: Clone cells established from ALDH^high and ALDH^low cells were analyzed by an ALDEFLUOR assay. The ALDH^high-positive gate was defined by a sample treated with DEAB. Percentages indicate the ALDH^high-positive rates. Clone cells derived from ALDH^high cells (H-1, H-6, and H-10) and ALDH^low cells (L-1, L-3, and L-8) were analyzed. B Sphere-formation assay of UM-UC-3 WT, H-10, and L-3 cells. Sphere-forming ability of WT UM-UC-3 cells, ALDH^high cells (H-10), and ALDH^low cells (L-3) 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. C and D Tumor-forming ability of UM-UC-3 H-10 and L-3 cells. BALB/c-nu/nu mice were injected subcutaneously with 1.0 × 10³, 10², 10¹, and 10⁰ H-10 cells or 1.0 × 10⁴, 10³, 10², and 10¹ L-3 cells. The tumor growth curves of mice injected with 1.0 × 10³ H-10 cells and 1.0 × 10³ L-3 cells are shown in Fig. 1C. Data are shown as means ± standard deviation. Statistical analyses of the data were performed using a bilateral Student’s t-test. Tumor incidence and estimated CSC/CIC frequency are summarized in Fig. 1D. 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. E and F. Resistance to cisplatin and radiation. H-10 and L-3 cells were treated with cisplatin (CDDP) or radiation at a serial dose, and cell viability was assessed by a WST-8 assay. Data are shown as means ± standard deviation. Statistical analyses for the data were performed using a bilateral Student’s t-test

Fig. 2

Isolation of HLA ligands expressed in bladder CSCs/CICs. A Schematic summary of HLA ligandome analysis. HLA-class 1 molecules were immunoprecipitated using an anti-HLA-A2 antibody (BB7.2), and HLA ligands were isolated by acid treatment. The HLA ligand landscape was analyzed by mass spectrometry. A summary of peptide length is shown in the right panel. UM-UC-3 H-10, L-3, and WT cells were used. B Summary of the antigenic peptides. WebLogo analysis using 9-mer peptides isolated from H-10, L-3, and WT cells is shown. L: leucine; V: valine. C Venn diagram of antigenic peptides. The diagram indicates the number of peptides isolated from H-10, L-3, and WT cells; 848, 1832, and 1073 peptides were isolated from H-10, L-3, and WT cells, respectively, with 123 H-10 cell-specific peptides

Fig. 3

Gene expression analysis of bladder CSCs/CICs. A Heatmap of CAGE. A summary of gene expression using CAGE is shown. WT, L-1, L-3, L-8, H-1, H-6, and H-10 cells were used. B Volcano plot. Gene expression of ALDH^high clone cells (H-1, H-6, and H-10) and ALDH^low clone cells (L-1, L-3, and L-8) was analyzed by a Volcano plot. Several genes were specifically expressed in ALDH^high clone cells, and only GRIK2 and CFH (indicated in red) were shared with the ALDH^high-specific HLA ligandome data. C. Quantitative real-time PCR. Relative quantities of GRIK2 mRNAs in UM-UC3 cells, H-1, H-6, H-10, L-1, L-3 and L-8 cells were analyzed by qRT-PCR. Each value is the mean relative quantity ± SD

Fig. 4

Immunogenicity of a GRIK2-derived antigenic peptide. A Establishment of a GRIK2 peptide-specific CTL clone. PBMCs derived from an HLA-A2-positive donor were stimulated several times with GRIK2 peptide and analyzed by IFNγ ELISPOT and tetramer assays (left panel). GRIK2 peptide-pulsed T2 cells were used for the IFNγ ELISPOT assay. Peptide non-pulsed T2 cells were used as a negative control. For the tetramer assay, PBMCs were stained with phycoerythrin-labeled GRIK2 peptide-HLA-A2 tetramer and analyzed by FACS. The tetramer-positive cells were single cell sorted. Established CTL clones were evaluated by IFNγ ELISPOT and tetramer assays (right panel) for specificity. B GRIK2 peptide-specific CTL clone recognizes endogenously expressed GRIK2. GRIK2 peptide-specific CTL clones were evaluated for reactivity to GRIK2-overexpressing UM-UC-3 cells by an IFNγ ELISPOT assay. K562 cells were used as a negative control. Data are shown as means ± standard deviation. Statistical analyses for the data were performed using a bilateral Student’s t-test. c GRIK2 peptide-specific CTL clone recognizes H-10 clone cells. GRIK2 peptide-specific CTL clones were evaluated for reactivity to H-10, L-3, and WT cells by an IFNγ ELISPOT assay. K562 cells were used as a negative control. Data are shown as means ± standard deviation. Statistical analyses for the data were performed using a bilateral Student’s t-test

Fig. 5

In vitro immune selection model. A Summary of immune selection model. H-10 clone cells and L-3 clone cells were mixed at a ratio of 1:9. Mixture cells were cultured with (CTL +) or without CTL clone 9G23 (CTL-) for overnight at a effector/target ratio = 5:1 and then cultured for 2 days. Percentages indicate the ALDH^high-positive rates. B and C Resistance to cisplatin and radiation. CTL + cells and CTL—cells were treated with cisplatin (CDDP) or radiation at a serial dose, and cell viability was assessed by a WST-8 assay. Data are shown as means ± standard deviation. Statistical analyses for the data were performed using a bilateral Student’s t-test