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TGF-β1 INDUCED SOX18 ELEVATION PROMOTES HEPATOCELLULAR CARCINOMA PROGRESSION AND METASTASIS THROUGH TRANSCRIPTIONALLY UPREGULATING PD-L1 AND CXCL12

Date
May 19, 2024

Background and aims: Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive tumor microenvironment, which contributes to tumor progression, metastasis and immunotherapy resistance. Thus, identification of HCC-intrinsic factors regulating immunosuppressive microenvironment is urgently needed. Here, we aimed to elucidate the role of SYR-Related High-Mobility Group Box 18 (SOX18) in inducing immunosuppression and to validate novel combination strategies for SOX18-mediated HCC progression and metastasis. Methods: The role of SOX18 in HCC was investigated in orthotopic allografts and diethylinitrosamine/carbon tetrachloride (DEN/CCL4) induced spontaneous models by using murine cell lines, adeno-associated virus 8 and hepatocyte-specific knockin and knockout mice. The immune cellular composition in HCC microenvironment was evaluated by flow cytometry and immunofluorescence. Results: SOX18 overexpression promoted the infiltration of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) while diminishing cytotoxic CD8+ T cells to facilitate HCC progression and metastasis in cell-derived allografts and chemically-induced HCC models. Mechanistically, transforming growth factor-beta 1 (TGF-β1) upregulated SOX18 expression by activating the Smad2/3 complex. SOX18 transactivated the expression of chemokine (C-X-C motif) ligand 12 and programmed death ligand 1 (PD-L1) to induce the immunosuppressive microenvironment. CXCL12 knockdown significantly attenuated SOX18-induced TAMs and Tregs accumulation and HCC dissemination. Antagonism of CXCR4, the cognate receptor of CXCL12, with AMD3100 or selective knockout of CXCR4 in TAMs or Tregs likewise abolished SOX18-mediated effects. TGFβR1 inhibitor Vactosertib or CXCR4 inhibitor AMD3100 in combination with anti-PD-L1 dramatically inhibited SOX18-mediated HCC progression and metastasis. Conclusions: SOX18 promoted the accumulation of immunosuppressive TAMs and Tregs in microenvironment by transactivating CXCL12 and PD-L1. CXCR4 inhibitor or TGFβR1 inhibitor in synergy with anti-PD-L1 represented a promising combination strategy to suppress HCC progression and metastasis.
Figure 1. SOX18 transactivated the expression of CXCL12 and CD274. (A) Venn diagram illustrating the overlapping differentially-expressed genes after SOX18 manipulation. (B-C) Heatmaps and density plot of ChIP-seq signals for SOX18 and input in TSS region. (D) ChIP-seq-derived de novo motif logo of SOX18. (E) Gene track view of SOX18 ChIP-seq tags in the <i>CD274</i> and <i>CXCL12</i> promoter. (F) The correlation between SOX18 and CXCL12 or CD274. (G) Immunoblotting and qPCR. (H-I) Dual-luciferase reporter assay. (J) ChIP assays. (K) The protein levels of SOX18 and PD-L1. (L-O) C57BL/6 mice were orthotopically inoculated with indicated cells. Representative images (L), overall survival (M), H&E staining (N) and the incidence and quantification of lung colonization (O). (P-Q) Tumor-infiltrating CD8<sup>+</sup> T cells by FCM and immunofluorescence. (R) IHC staining. (S) Correlation between the expression of SOX18 and PD-L1. (T) Kaplan-Meier analysis.

Figure 1. SOX18 transactivated the expression of CXCL12 and CD274. (A) Venn diagram illustrating the overlapping differentially-expressed genes after SOX18 manipulation. (B-C) Heatmaps and density plot of ChIP-seq signals for SOX18 and input in TSS region. (D) ChIP-seq-derived de novo motif logo of SOX18. (E) Gene track view of SOX18 ChIP-seq tags in the CD274 and CXCL12 promoter. (F) The correlation between SOX18 and CXCL12 or CD274. (G) Immunoblotting and qPCR. (H-I) Dual-luciferase reporter assay. (J) ChIP assays. (K) The protein levels of SOX18 and PD-L1. (L-O) C57BL/6 mice were orthotopically inoculated with indicated cells. Representative images (L), overall survival (M), H&E staining (N) and the incidence and quantification of lung colonization (O). (P-Q) Tumor-infiltrating CD8+ T cells by FCM and immunofluorescence. (R) IHC staining. (S) Correlation between the expression of SOX18 and PD-L1. (T) Kaplan-Meier analysis.

Figure 2. Combination of CXCR4 inhibitor with PD-L1 antibody dramatically inhibited SOX18-mediated HCC metastasis. (A-G) C57BL/6 mice orthotopically implanted with Hepa1-6-SOX18 cells were administrated with CXCR4 inhibitor, PD-L1 antibody and the combination therapy. Representative image (A) at the experimental endpoint, the dynamic curve of bioluminescent signals (B), the OS (C) of different groups, the quantification and incidence (D) of lung-colonizing foci and characteristic H&E-stained pulmonary tissues (E) were shown. (F-G) Tumor-infiltrating immune cells were evaluated by FCM (F) and IF (G) analysis. (H) Schematic overview of the experimental schedule. (I) Representative image of whole-liver morphology and H&E staining and the statistical results from DEN/CCl<sub>4</sub>-induced HCC models receiving the indicated treatments. (J) Tumor-infiltrating immune cells were evaluated by FCM. (K) A schematic diagram.

Figure 2. Combination of CXCR4 inhibitor with PD-L1 antibody dramatically inhibited SOX18-mediated HCC metastasis. (A-G) C57BL/6 mice orthotopically implanted with Hepa1-6-SOX18 cells were administrated with CXCR4 inhibitor, PD-L1 antibody and the combination therapy. Representative image (A) at the experimental endpoint, the dynamic curve of bioluminescent signals (B), the OS (C) of different groups, the quantification and incidence (D) of lung-colonizing foci and characteristic H&E-stained pulmonary tissues (E) were shown. (F-G) Tumor-infiltrating immune cells were evaluated by FCM (F) and IF (G) analysis. (H) Schematic overview of the experimental schedule. (I) Representative image of whole-liver morphology and H&E staining and the statistical results from DEN/CCl4-induced HCC models receiving the indicated treatments. (J) Tumor-infiltrating immune cells were evaluated by FCM. (K) A schematic diagram.

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