Cabozantinib於肝細胞癌中誘導細胞焦亡與免疫調控的機制探討

Abstract

Cabozantinib是一種多靶點酪胺酸激酶抑制劑，已獲准用於晚期肝細胞癌的二線治療。雖然其抗血管新生與細胞抗增殖作用廣為人知，但其免疫調控機制仍未被完全理解。因此，本研究旨在探討cabozantinib的免疫調控機制，著重於其在抑制HCC細胞分泌的白血病抑制因子，以及對骨髓來源抑制性細胞的抑制與誘導細胞焦亡現象中所扮演的角色。本研究使用源自Nlrp3敲除小鼠的骨髓來源抑制性細胞及Gsdmd敲除的HCC細胞，分別評估NLRP3與GSDMD在cabozantinib誘導細胞焦亡中的參與。並以Gsdmd敲除的Hepa1-6細胞在免疫健全小鼠中建立腫瘤模型，以評估腫瘤細胞GSDMD與cabozantinib活體治療效果及免疫調控作用之間的關聯。此外，本研究也使用LIF敲落或過度表現的HCC細胞株建立小鼠模型，以評估腫瘤細胞LIF表現與腫瘤生長及治療反應之間的關聯。流式細胞儀分析、單細胞RNA 定序與磷酸化蛋白質體學分析則用於鑑定相關細胞族群、蛋白質活化及基因表現變化。 研究結果顯示，cabozantinib可降低腫瘤細胞的LIF分泌，而外源性LIF及腫瘤細胞LIF表現量的操作，則會改變MDSCs的誘導比例與免疫抑制功能。操縱腫瘤細胞LIF表現量在小鼠腫瘤生長及cabozantinib治療後呈現不同的組內反應，暗示LIF表現可能與腫瘤生長及治療反應相關。我們同時發現，cabozantinib能在HCC細胞與MDSCs中誘發細胞焦亡相關現象。在源自Nlrp3敲除小鼠的MDSCs中，cabozantinib所引起的細胞死亡、NLRP3–ASC鄰近化及GSDMD裂解均受到抑制。在活體模型中，cabozantinib的抗腫瘤與免疫調控效應在帶有野生型腫瘤的小鼠中存在顯著差異，而在帶有Gsdmd敲除腫瘤的小鼠中並沒有，提示腫瘤細胞GSDMD可能與cabozantinib的治療反應相關。整體而言，這些結果支持NLRP3–CASP1–GSDMD相關焦亡訊號可能參與cabozantinib的抗腫瘤與免疫調控作用。 綜合我們的發現，暗示cabozantinib處理後的細胞焦亡相關變化可能與免疫活化有關，並為後續評估cabozantinib與免疫治療在HCC中的聯合使用提供了臨床前觀察依據。

Cabozantinib is a multi-target tyrosine kinase inhibitor approved for second-line treatment of advanced hepatocellular carcinoma (HCC). While its antiangiogenic and antiproliferative effects are well known, its immunomodulatory mechanisms remain incompletely understood. Here, we aimed to investigate the novel immunomodulatory mechanism of cabozantinib, focusing on its role in suppressing the secretion of leukemia inhibitory factor (LIF) by HCC cells, as well as inhibiting myeloid-derived suppressor cells (MDSCs) and inducing pyroptosis. Cabozantinib-induced pyroptosis was evaluated in HCC cell lines and MDSCs. The involvement of NLRP3 and GSDMD in cabozantinib-induced pyroptosis was evaluated using MDSCs derived from Nlrp3 knockout mice and Gsdmd knockout HCC cells, respectively. Gsdmd knockout Hepa1-6 cells were also used to establish tumors in immunocompetent mice to assess the association between tumor cell GSDMD and the in vivo antitumor and immunomodulatory responses to cabozantinib. In addition, mouse models established using LIF knockdown or LIF overexpressing HCC cells were used to evaluate the associations of tumor cell LIF expression with tumor growth and treatment response. Flow cytometry, single-cell RNA sequencing, and phosphoproteomic analyses were performed to characterize relevant cell populations and changes in protein activation and gene expression. Our results showed that cabozantinib reduced tumor-cell LIF secretion, while exogenous LIF and manipulation of tumor-cell LIF expression altered MDSC induction and immunosuppressive activity. Mouse models with different tumor-cell LIF levels showed distinct within-group patterns of tumor growth and response to cabozantinib, suggesting a possible association between LIF expression and treatment response. Cabozantinib also induced features consistent with pyroptosis in HCC cells and MDSCs. In Nlrp3-knockout MDSCs, cabozantinib-associated cell death, NLRP3–ASC proximity, and GSDMD cleavage were attenuated. In vivo, the antitumor and immunomodulatory effects of cabozantinib were statistically significant in mice bearing wild-type, but not Gsdmd-knockout, tumors, suggesting a possible association between tumor-cell GSDMD and treatment response. Collectively, these findings support the involvement of NLRP3–CASP1–GSDMD-associated pyroptotic signaling in the antitumor and immunomodulatory effects of cabozantinib. These findings suggest a possible association between cabozantinib-associated pyroptotic changes and immune activation and provide preclinical observations supporting further evaluation of cabozantinib combined with immunotherapy in HCC.