Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is a novel target of lung cancer stem-like cell immunotherapy

Abstract

Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells.

Fig 1. BORIS subfamily 6 expression in lung cancer cell lines.

(A) An image of sphere-cultured SBC5 cells. Magnification, x 400. Bar scale is 100 μm. (B) Expression levels of stem cell-related genes. Expression levels of stem cell-related genes including ABCG2, ALDH1A1, NANOG, POU5F1, SOX2, KLF4 and BMI1 were determined by quantitative RT-PCR. Data are shown as means ± SD. All statistical analyses for data shown in this figure were performed using bilateral Student’s t test. *P-values <0.05. (C) Expression of SOX2 protein in sphere-cultured cells. SOX2 protein expression in sphere-cultured and adherent-cultured SBC5 cells were analyzed by an Western blot. β-Actin was used as a positive control. (D) Expression of the cancer testis antigen BORIS sf6 in adherent-cultured and sphere-cultured lung carcinoma cells. RT-PCR analysis of BORIS subfamily 6 mRNA expression in lung cancer cell lines and primary cancer cells from clinical specimens. Cancer cells were cultured in adherent culture and sphere culture.

Fig 2. BORIS subfamily 6 expression in lung cancer clinical specimens.

BORIS sf6 expression in several lung cancer tissue specimens was examined by RT-PCR. RNAs of tumor cells and non-tumor cells were extracted from formalin-fixed paraffin-embedded (FFPE) samples. GAPDH was used as an internal positive control.

Fig 3. BORIS sf6 protein expression in lung cancer tissues.

(A) Representative images of BORIS sf6^low and BORIS sf6^high case. Original magnification is 100 x. (B) Kaplan-Meier survival estimates were performed according to immunohistochemistry positivity of BORIS sf6. The median survival times of the BORIS sf6^high group (n = 9) and BORIS sf6^low group (n = 43) were 26 weeks and 209 weeks, respectively. The log-rank test revealed a significantly worse prognosis for BORIS sf6^high cases (P = 0.0481). The hazard ratio of BORIS sf6^low cases was 0.4355 (95% confidence interval: 0.1062–0.9906).

Fig 4. Induction of BORIS subfamily 6-specific CTLs and establishment of a CTL clone.

BORIS subfamily 6 peptide-specific cytotoxic T cell (CTL) induction was evaluated by the interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. HLA-A*0201-positive PBMCs were obtained from four healthy donors. Donors A, B and C were HLA-*A0201-positive. Fluorescence-activated cell sorting (FACS) was performed with PE-conjugated BORIS subfamily 6 peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody. Double positive cells were single-cell sorted to establish a CTL clone (total 960 well). Twelve wells showed cell growth and 8 wells were analyzed by tetramer for specificity.

Fig 5. BORIS subfamily 6-specific CTL clone analysis.

(A) ELISPOT assay of BORIS subfamily 6-specific CTL clones 3B9 and 4D2 clones. (B) Tetramer assay of BORIS subfamily 6-specific CLT clones 3B9 and 4D2. CTL clones 3B9 and 4D2 were stained by PE-conjugated BORIS subfamily 6-specific peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody and analyzed. (C) LDH release cytotoxicity assay. Specific cytotoxicity for peptide-pulsed T2 cells was displayed (left panel). HIV peptide-pulsed T2 cells, peptide (-) T2 cells and K562 cells were used as negative controls. Specific cytotoxicity for HLA-A2-positive lung cancer cell lines SBC5 and LC142 was displayed (right panel). Data are shown as means ± SD. All statistical analyses for data shown in this figure were performed using bilateral Student’s t test. *P-values <0.05.

Fig 6. BORIS subfamily 6-specific CTL clone inhibited the sphere-formation of lung cancer cells.

(A) ELISPOT assay of BORIS subfamily 6-specific and non-specific CTL clones. The reactivity for BORIS C34_24(9) peptide were analyzed by ELISPOT assay. HIV peptide and peptide (-) were used as negative controls. (B) Sphere-formation inhibition assay. BORIS C34_24(9) peptide specific CTL clone (3B9) or non-specific CTL clone (5H6) were co-cultured with SBC5 cells in a sphere-forming condition. After 6 days’ culture, the numbers of spheres were counted. Upper panel indicate the numbers of spheres, and data are shown as means ± SD. Lower panel indicates representative images of spheres. Magnification, x 100. Bar scale is 100 μm.