Suramin Blocked hCAP18/LL-37-induced Macrophage Recruitment and M2 Polarization to Enhance the Therapeutic Efficacy of 1,25(OH)2D3 against Hepatocellular Carcinoma in Vitro and in Vivo Mouse Model

Huidan ZhangWenjing XieWenliang DuanXueli Yuanyaxin YangQin ChenYiqiang ZhuYuqing Chen^*

• Life Sciences College, Nanjing Normal University, Nanjing, China

Background: 1,25(OH)2D3 supplementation alone does not provide sufficient benefit to hepatocellular carcinoma (HCC) patients in clinical trials. Tumor-associated macrophages (TAMs)-mediated immunosuppression is regarded as a major hurdle for the effectiveness of several treatments. Previous studies revealed that hCAP18/LL-37 was an important factor which directly suppresses the anticancer activity of 1,25(OH)2D3 on HCC cells. However, whether TAMs contribute to the limited clinical efficacy of 1,25(OH)2D3 through hCAP18/LL-37 remains unclear.: Co-culture systems of HCC cells (PLC/PRF-5, Huh7) with THP-1-derived macrophages and co-xenograft mouse models were established. Anticancer activity was evaluated in vitro and in vivo mouse models using standard assays. Mechanistic investigations utilized qRT-PCR, Western blot, flow cytometry, ELISA, and immunohistochemistry. Therapeutic efficacy of 1,25(OH)2D3/suramin combination was assessed in co-xenograft and N-Nitrosodiethylamine (DEN)/Carbon tetrachloride (CCl4)-induced HCC models. Results: 1,25(OH)2D3 (200-500 nM) promoted macrophage recruitment, M2 polarization, Akt/mTOR signal and STAT3 signal activation in HCC/macrophage co-culture systems. This effect was mediated by 1,25(OH)2D3-induced hCAP18/LL-37 overexpression, which facilitated TAM infiltration and M2 reprogramming. Suramin, a potent LL-37 inhibitor, abrogated these immunosuppressive effects by blocking LL-37 internalization, restoring M1 polarization and suppressing Akt/mTOR and STAT3 pathways. Notably, 1,25(OH)2D3/suramin combination therapy synergistically inhibited HCC proliferation, colony formation, and invasion in vitro. In xenograft models and DEN/CCl4-induced HCC models, suramin enhanced 1,25(OH)2D3's efficacy by promoting M1 polarization, increasing intratumoral M1/M2 ratios, reducing tumor growth, and diminishing macroscopic nodules.The 1,25(OH)2D3-LL-37-TAM axis drives immunosuppression in HCC by modulating macrophage phenotypes. While suramin potently disrupts this axis, blocking LL-37-mediated TAMs recruitment and M2 polarization, while promoting antitumor M1 phenotype responses. These findings highlight suramin as as a promising adjunct to 1,25(OH)2D3-based immunotherapy for HCC.