Arenobufagin Enhances T-cell Anti-Tumor Immunity in Colorectal Cancer by Modulating HSP90β Accessibility

Background Colorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin(Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. Purpose This study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90β-STAT3-PD-L1 axis activity. Methods We investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits colorectal cancer. Utilizing Target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90p (HSP90β) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. Results We observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90p's accessibility significantly decreasedupon Are binding. We demonstrated a decrease in the activity of the HSP908-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. Conclusion Contrary to previous research focusing on the direct cytotoxicity of Arenobufagin towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Arenobufagin and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Arenobufagin.