Heterometrus laoticus Venom Attenuates Colorectal Tumor Growth Through Direct Cytotoxicity and Reprogramming of Tumor-Associated Macrophages

Background: Scorpion venoms contain diverse bioactive compounds with potential anti-cancer properties. The venom of Heterometrus laoticus (Black Giant scorpion) has been partially characterized, yet its anti-tumor mechanisms remain poorly understood. We investigated the anti-tumor effects of H. laoticus venom using a murine MC38 colorectal carcinoma model. Methods: A murine MC38 colorectal tumor model was established, and venom was administered intratumorally (22 mg/kg) every 3 days for 28 days. Tumor growth, histopathology, macrophage polarization (immunofluorescence and flow cytometry), serum cytokines, and hepatorenal function were assessed. In vitro studies examined cytotoxicity, apoptosis/necrosis (Annexin V/PI), and gene expression (qRT-PCR) in MC38 cells and bone marrow-derived macrophages (BMDMs). Results: Intratumoral venom significantly inhibited tumor growth from day 19 onward, accompanied by increased apoptosis (cleaved caspase-3+ cells) and enhanced immune cell infiltration. Venom treatment shifted intratumoral macrophage polarization from anti-inflammatory (CD206+) toward pro-inflammatory (CD86+) phenotypes. Systemically, venom attenuated tumor-elevated serum cytokines without hepatorenal toxicity and increased splenic M1 macrophages (F4/80+CD80+) and CD8+ T cells. In vitro, venom exhibited concentration- and time-dependent cytotoxicity, inducing late apoptosis and necrosis in both MC38 cells and BMDMs. Co-culture experiments revealed synergistic necrosis enhancement. Mechanistically, venom upregulated pro-apoptotic (p53, STAT3, CDK-7) and pro-inflammatory (IL-1β, IL-6, iNOS, TNF-α) genes. Conclusions: H. laoticus venom exerts anti-tumor effects through dual mechanisms: direct cytotoxicity and immunomodulation via reprogramming tumor-associated macrophages toward a tumoricidal M1 phenotype. These findings support further investigation of scorpion venom components as potential adjuvants in cancer immunotherapy.