Euphorbia Pekinensis Rupr. sensitizes colorectal cancer to PD-1 blockade by remodeling the tumor microenvironment and enhancing peripheral immunity

Background: Immune checkpoint blockade, such as monoclonal antibodies targeting programmed cell death protein 1 (PD-1), has been a major breakthrough in the treatment of several cancers, but has limited effect in colorectal cancer (CRC), which is a highly prevalent cancer worldwide. Current chemotherapy-based strategies to boost PD-1 response have many limitations. And the role of peripheral immunity in boosting PD-1 response continues to attract attention. Therefore, candidate combinations of PD-1 blockade need to be drugs with multi-targets and multi-modulatory functions. However, it is still unknown whether traditional Chinese medicines with such property can enhance the applicability and efficacy of PD-1 blockade in colorectal cancer.

Methods: Euphorbia Pekinensis extract (EP) was prepared and the constituents were analyzed by HPLC. CRC cells were used for in vitro experiments, including cell viability assay, colony formation assay, flow cytometry for 7-AAD staining, western blotting for caspase 3 and caspase 7, HMGB1 and ATP detection. An orthotopic CT26 mouse model was subsequently used to investigate the combination of EP and PD-1 blockade therapy. Tumor volume and tumor weight were assessed, tumor tissues were subjected to histopathological HE staining and TUNEL staining, and tumor-infiltrating immune cells were evaluated by immunofluorescence staining. RNA-sequencing, target prediction and pathway analysis were further employed to explore the mechanism. Molecular docking and cellular thermal shift assay (CETSA) were utilized to verify the direct target of the core component of EP. And, loss-of-function analysis was carried to confirm the upstream-downstream relationship. Flow cytometry was employed to analyze CD8⁺ T cells in the peripheral blood and spleen.

Results: The main constituents of EP are diterpenoids and flavonoids. EP dramatically suppresses CRC cell growth and exerts its cytotoxic effect by triggering immunogenic cell death in vitro. Moreover, EP synergizes with PD-1 blockade to inhibit tumorigenesis in tumor-bearing mice. Disruption of ISX nuclear localization by helioscopinolide E is a central mechanism of EP-induced apoptosis in CRC cell. Meanwhile, EP activates immune response by upregulating Phox2b to reshape the immune microenvironment. In addition, EP regulates peripheral immunity by regulating the T cell activation and proliferation, and the ratio of CD8⁺ T cells in peripheral blood is drastically increased, thereby enhancing the therapeutic efficacy of anti-PD1 immunotherapy.

Conclusion: EP triggers intra-tumor immunogenic cell death and modulates the immunoregulatory signaling to elicit the tumor immunogenicity. Moreover, EP participates in transcriptional activation of immune response-related pathways. Consequently, multiple stimulating functions of EP on macro- and micro-immune potentiates the anti-tumor effect of PD-1 blockade in CRC.