在高度定量的肝癌小鼠模式中藉由解析腫瘤微環境來增進T細胞療法

Abstract

肝細胞癌（HCC）為目前最盛行的原發性肝癌，佔其中的80-90％。而病人可以依照巴塞隆納肝癌分級來決定治療方式，目前隨著癌症免疫學的發展，與免疫檢查點抑制劑（ICB）出現，近期在臨床用藥治療上結合標靶治療與免疫檢查點抑制劑的使用有良好的反應。目前Lenvatinib（標靶治療）與Pembrolizumab（免疫檢查點抑制劑）的合併使用已經核准為臨床的一線用藥，ORR可達到44.8％，但仍有接近60%的肝癌病人對於此合併治療反應不佳，而目前對於其中的機制仍然不清楚。因此，為了解決此問題，我們利用尾部靜脈高壓注射技術建立高度定量的原發性肝癌小鼠模型，以研究抗原特異性T細胞與腫瘤微環境（TME）之間的交互作用。此模式透過共同表達致癌基因（NRAS）及小片段的螢光素酶作為高度定量腫瘤生長的標的，另外利用CRISPR剔除腫瘤抑制基因（PTEN-p53 / Cas9）。我們觀察到CD4 +和CD8 + T細胞能進入小腫瘤，但會被大腫瘤排斥。此外，透過送入特異性抗原Ｔ細胞能有效清除腫瘤，然而在晚期卻無法控制腫瘤生長。因此，藉由探討腫瘤微環境與腫瘤抗原特異性T細胞的浸潤和功能之相互關係，希望能有效增強肝癌的免疫療法。

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and accounts for 80-90% cases. Along with the development of cancer immunology, targeting several pathways yields exciting positive results when combining with immune checkpoint blockades (ICB). Surprisingly, the combinatory therapy with lenvatinib (multi-kinase inhibitor) pembrolizumab (anti-PD-1) has synergistic effect and achieves the objective response rate as high as 44.8%. Nevertheless, there remain about 60 % HCC patients who do not respond to the treatment. Therefore, understanding the mechanisms of combinatory therapy against HCC remains a crucial issue. To solve this issue, we first developed a highly quantitative spontaneous HCC mouse model through hydrodynamic injection (HDI) to study the interaction between antigen-specific T cells and the tumor immune microenvironment (TIME). The HDI-HCC model was generated through the insertion and overexpression of oncogene (NRAS) tagged by the reporter (HiBiT) with transposase and the knockout of tumor suppressor genes (PTEN-p53/Cas9) by CRISPR. The expression level of HiBiT in mouse serum correlated with the tumor size. We found that cell populations of TIME exhibit different phenotypes along with cancer development. We observed the infiltration of CD4 + and CD8+ T in small tumor but their exclusion from large tumors. In contrast, the Ly6C/G+ myeloid cells (probably myeloid-derived suppressor cells) were present in both small and large hepatic tumors. Furthermore, adoptive transfer of tumor-specific CD8+ T cells effectively suppressed tumor at early stage but fail to do so at a late stage. In conclusion, my study discovers distinct phenotypes of T cell infiltration in tumors between early (small) and advanced (large) HCCs, and their different treatment responses to adoptive T cell therapy. Understanding the mechanisms regulating TIME and the infiltration and function of tumor antigen-specific T cells will be crucial to optimize anti-tumor immunotherapy.