SCCA1/SERPINB3 suppresses antitumor immunity and blunts therapy-induced T cell responses via STAT-dependent chemokine production
Liyun Chen, Victoria Shi, Songyan Wang, Lulu Sun, Rebecca Freeman, Jasmine Yang, Matthew J. Inkman, Subhajit Ghosh, Fiona Ruiz, Kay Jayachandran, Yi Huang, Jingqin Luo, Jin Zhang, Pippa Cosper, Clifford J. Luke, Catherine S. Spina, Perry W. Grigsby, Julie K. Schwarz, Stephanie Markovina
Liyun Chen, Victoria Shi, Songyan Wang, Lulu Sun, Rebecca Freeman, Jasmine Yang, Matthew J. Inkman, Subhajit Ghosh, Fiona Ruiz, Kay Jayachandran, Yi Huang, Jingqin Luo, Jin Zhang, Pippa Cosper, Clifford J. Luke, Catherine S. Spina, Perry W. Grigsby, Julie K. Schwarz, Stephanie Markovina
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Research Article Oncology

SCCA1/SERPINB3 suppresses antitumor immunity and blunts therapy-induced T cell responses via STAT-dependent chemokine production

Abstract

Patients with cancer who have high serum levels of squamous cell carcinoma antigen 1 (SCCA1, now referred to as SERPINB3) commonly experience treatment resistance and have a poor prognosis. Despite being a clinical biomarker, the modulation of SERPINB3 in tumor immunity is poorly understood. We found positive correlations of SERPINB3 with CXCL1, CXCL8 (CXCL8/9), S100A8, and S100A9 (S100A8/A9) myeloid cell infiltration through RNA-Seq analysis of human primary cervical tumors. Induction of SERPINB3 resulted in increased CXCL1/8 and S100A8/A9 expression, which promoted monocyte and myeloid-derived suppressor cell (MDSC) migration in vitro. In mouse models, Serpinb3a tumors showed increased MDSC and tumor-associated macrophage (TAM) infiltration, contributing to T cell inhibition, and this was further augmented upon radiation. Intratumoral knockdown (KD) of Serpinb3a resulted in tumor growth inhibition and reduced CXCL1 and S100A8/A expression and MDSC and M2 macrophage infiltration. These changes led to enhanced cytotoxic T cell function and sensitized tumors to radiotherapy (RT). We further revealed that SERPINB3 promoted STAT-dependent expression of chemokines, whereby inhibition of STAT activation by ruxolitinib or siRNA abrogated CXCL1/8 and S100A8/ A9 expression in SERPINB3 cells. Patients with elevated pretreatment SCCA levels and high phosphorylated STAT3 (p-STAT3) had increased intratumoral CD11b+ myeloid cells compared with patients with low SCCA levels and p-STAT3, who had improved overall survival after RT. These findings provide a preclinical rationale for targeting SERPINB3 in tumors to counteract immunosuppression and improve the response to RT.

Authors

Liyun Chen, Victoria Shi, Songyan Wang, Lulu Sun, Rebecca Freeman, Jasmine Yang, Matthew J. Inkman, Subhajit Ghosh, Fiona Ruiz, Kay Jayachandran, Yi Huang, Jingqin Luo, Jin Zhang, Pippa Cosper, Clifford J. Luke, Catherine S. Spina, Perry W. Grigsby, Julie K. Schwarz, Stephanie Markovina

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Figure 6

Targeting SERPINB3 sensitizes tumors to RT and enhances T cell response.

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Targeting SERPINB3 sensitizes tumors to RT and enhances T cell response....
(A) Growth curves of LL2/B3 tumors treated with siNC (red lines) and siB3 (purple lines) with or without RT (sham: solid lines; RT: dotted lines). ***P < 0.001, by 2-way ANOVA. (B) The chemokines CXCL1 and S100A8/A9 in tumor homogenates were assessed by ELISA. Data were normalized to the protein concentration for each tumor homogenate. (C and D) Cumulative data from FACS analysis show the (C) frequencies of immune cell populations including CD11b+Ly6G-Ly6Chi M-MDSCs, CD11b+Ly6G+ PMN-MDSCs, CD11b+Ly6G-F4/80+ TAMs, and CD11b+Ly6G-F4/80+CD163+ M2 macrophages, as well as (D) CD3+CD8+ T cells in total TILs and the ratio of CD3+CD8+ T cells to CD4+CD25+Foxp3+ Tregs. (E) Intracellular cytokine staining for granzyme B and perforin in CD8+ T cells was analyzed by flow cytometry. (F) CellTrace-labeled intratumoral T cells were stimulated with anti-CD3/anti-CD28 antibody for 4 days, and cell proliferation was determined by the dilution of CellTrace. (G) The expression of PD-1 and CTLA-4 was examined by flow cytometry and is shown as MFI. Data in B–G are shown as the mean ± SEM, and each dot represents a biologically independent sample. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test.