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Fig. 2 | Journal of Hematology & Oncology

Fig. 2

From: Targeting the DNA damage response enhances CD70 CAR-T cell therapy for renal carcinoma by activating the cGAS-STING pathway

Fig. 2

Olaparib activation of cGAS-STING pathway is key to promote tumor killing of CAR-T cell. a Olaparib (OLA)- and vehicle (DMSO)-treated tumors were harvested 5 days post-treatment, detecting the phenotype of CAR-T/T cells infiltrating in tumor tissue by flow cytometry. b OLA- and DMSO-treated tumors were harvested 5 days post-treatment, subjected to immunofluorescence (IF) analysis for CD8. (Scale bar: 50 μm) c Statistical analysis of CD8+ T cells in the results of immunofluorescence analysis. d Representative images of the level of γH2AX and cytosolic double-stranded DNA (dsDNA) in 786-0 cells after OLA or DMSO treatment. Scale bar, 10 μm. e Immunoblots of markers in the cGAS-STING pathway including total and phospho (p) STING (S366), total and phospho TBK1 (S172), cGAS, total and phospho IRF3 (S396) in lysates collected from RCC cell lines treated with OLA or DMSO. TBB5 served as a loading control. f Representative images of the level of chemokines CCL5 in 786-0 cells after OLA or DMSO treatment. Scale bar, 10 μm. g The CCL5, CXCL10 and Granzyme B IF staining were performed in tumors from the resected tumors from Fig. 2b. Representative images of staining intensity are shown. (Scale bar, 20 μm). h–j Quantification of tumor sections immunostained for CCL5, CXCL10 and Granzyme B -positive areas quantified for each field (N = 5). k CAR-T- and OLA-treated 786-0shSTING tumors were harvested 5 days post-treatment, quantification of CCL5, CXCL10 and Granzyme B in the results of IF analysis from Additional file 1: Fig. S6f (N = 3). l CAR-T- and OLA-treated 786-0shSTING tumors were harvested 5 days post-treatment, statistical analysis of CD8+ T cells in the results of IF analysis from Additional file 1: Fig. S6g (N = 3). m Model for cGAS-STING pathway activation in response to DDR targeting in RCC. In the proposed model, targeting the DDR protein PARP with the small-molecule inhibitor OLA leads to cytosolic DNA in RCC models. The cytosolic DNA is then recognized by cGAS, which leads to activation of the STING/TBK1/IRF3 pathway. IRF activation leads to increased expression of IFNβ and enhanced expression of the chemokines CXCL10 and CCL5. STING pathway activation and increased chemokine expression lead to the recruitment and secretion of large amounts of Granzyme B by CD8+ CAR-T cells leads to enhanced antitumor immunity in RCC models. All error bars represent SD. In all plots, ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

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