Anti-tumor effects of PLAC1-specific TCR gene modified T-cells on breast cancer

Abstract

Objective To investigate the anti-tumor effects of placenta-specific 1(PLAC1) -specific T-cell receptors (TCR) gene modified T-cells on breast cancer. Methods CD8+ T-cells were selected from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors with magnetic beads. The TCR gene specific for HLA-A2-restricted PLAC1 were cloned into the lentiviral vector, followed by transduction into human CD8+ T-cells. These CD8+ T-cells transduced with the lentiviral vectors carrying TCR or control empty vector were named TCR-T cells and NC-T cells respectively. The accurately expressing efficiency of PLAC1-specific TCR in CD8+ T-cells was analyzed by flow cytometry. The expressing of PLAC1 and HLA-A2 in breast cancer cell lines MCF-7 and MDA-MB-231 (triple-negative breast cancer cell line) were analyzed by immunofluorescence and flow cytometry. The cytotoxicity and specific release of IFN-γ of TCR-T cells or NC-T cells after co-culture with breast cancer cells with different effector-target ratios (5:1, 10:1 or 20:1) were detected by WST-1 and ELISA assays. The anti-tumor effects of PLAC1-specific TCR gene modified T-cells in vivo were observed by establishing mice models of the subcutaneous human breast cancer. Statistical analysis was performed by one-way analysis of variance and t test. Results The proportion of CD3+ CD8+ T cells isolated from PBMCs was up to (98.89±0.3)% purity, with selection procedure using magnetic beads. The accurately expressing efficiency of PLAC1-specific TCR in TCR-T cells reached (24.58±0.82)%, and NC-T cells did not express PLAC1-specific TCR. Both MCF- 7 and MDA-MB-231 cells were PLAC1 and HLA-A2 serotype positive breast cancer cell lines. The HLA-A2 expression efficiency in MCF-7 and MDAMB-231 cell lines reached (93.04±1.36)% and (98.72±0.12)%, respectively. In a E : T ratio 20 : 1, the efficacy of TCR-T cells-mediated MCF-7 lysis was (51.5±1.37)%, higher than that of the NC-T cells (5.93±2.40)%, t = 15.507, P < 0.01; the efficacy of TCR-T cells-mediated MDA-MB-231 lysis was (44.34±2.20)%, higher than that of the NC-T cells (5.15±2.40)% (t = 10.694, P < 0.01). The TCR-T cells demonstrated an efficient cytotoxicity against MCF-7 and MDA-MB-231 cells when compared with the NC-T cells in a same E : T ratio. In a E : T ratio 20 : 1, the release of IFNγ observed in TCR-T cells co-cultured with MCF- 7 was [ (347.49±4.10) pg/ml], higher than that of in NC-T cells [ (18.14±6.22) pg/ml] (t = -76.638, P < 0.01); the release of IFN-γ observed in TCR-T cells co-cultured with MDA-MB-231 was ( 255.25±6.85 ) pg/ml, higher than that of in NC-T cells [ (14.70±6.38) pg/ml] (t = -44.526, P < 0.01). The xenograft mouse assays revealed that TCR-T cells significantly delayed the tumor progression in mice bearing breast cancer when compared with the NC-T cells and normal saline (P < 0.05), and the mean tumor volume in normal saline group, NC-T cells group and TCR-T cells group reached (5636.96±2879.55) mm3, (5522.12±3391.48) mm3 and (1403.85±1394.31) mm3 respectively on day 35 post-injection, TCR-T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline (F = 0.1813, P < 0.05) or NC-T cells group (F = 0.1307, P < 0.05). Conclusion The PLAC1-specific TCR gene modified T cells possessed efficient anti-tumor effects on breast cancer. The present study demonstrated PLAC1 as a potential target for breast cancer treatment; the usage of PLAC1-specific TCR gene modified T cells was a novel strategy for PLAC1-positive breast cancer treatment. Key words: Breast cancer; PLAC1; TCR; CD8+ T cells; Cancer immunotherapy