研究放射線治療對肝細胞癌T細胞療法之影響

Abstract

肝細胞癌（hepatocellular carcinoma, HCC）是一種複雜的惡性腫瘤，在全球範圍內死亡率之高。目前針對晚期不可切除HCC的標準療法包括酪胺酸激酶抑制劑和免疫療法。近年來，過繼細胞療（adoptive cell therapy, ACT）已成為HCC治療中的具有潛力的治療方針。其中，嵌合抗原受體T細胞（CAR-T）或T細胞受體T細胞（TCR-T）最為廣泛使用。現行的臨床試驗正在評估此治療方法，然而有研究表明，單獨使用ACT治療對實體腫瘤的療效可能較低。因此，使用具非侵入性特徵的放射療法（radiotherapy, RT）來增強對腫瘤的免疫反應已成為一種潛在組合療法。然而，這種組合在HCC中的有效性和免疫機制仍不清楚。在此，我們通過流體動力尾靜脈注射（hydrodynamic tail vein injection, HDTVi）致癌質體發展出自發性且可追踪的HCC小鼠模型。我們對HCC小鼠分別施加了8Gy的低劑量全肝照射和20Gy的高劑量局部照射，在HDTVi後五週給予CD8+OT-I T細胞。治療後連續三週測量腫瘤標記HiBiT發現在各組之間沒有顯著差異。然而在8Gy RT組中有一個樣本的HiBiT持平未升高，因此促使我們研究此條件下的免疫細胞分佈。我們觀察到8Gy RT組在早期HCC小鼠之中免疫細胞浸潤略有增加。為了達到更好的腫瘤控制，我們在RT結合ACT再加上anti-VEGF-A。經過一個月的追踪，與RT加anti-VEGF-A組或僅anti-VEGF-A組相比，三重治療可以有效抑制肝腫瘤。儘管這些發現仍須進一步研究，但結果表明8Gy低劑量RT加T細胞療法和抗VEGF-A可能活化抗腫瘤免疫並緩解自發性HCC的腫瘤進展。對於皮下注射肝腫瘤模型，我們使用了從自發性HCC小鼠中分離的原發性肝癌細胞株。治療結果顯示在RT、ACT、或RT加上ACT的組別中有抑制效果的腫瘤幾乎都是體積較小的腫瘤，大腫瘤在治療後幾乎都沒有抑制效果，因此無法看出治療差異。然而，分析治療後的免疫細胞群和T細胞特性可以發現，RT能夠增加CD45+免疫細胞比例與抗原特異性CD8+ T細胞，並且減少腫瘤中的巨噬細胞。TIM-3耗竭標記和KLRG1效應標記在OT-I T細胞上的表達水平增加，表明RT可以有效刺激腫瘤特異性T細胞的抗腫瘤活性。總結來說，通過研究RT後腫瘤抗原特異性OT-I細胞的表型，可以理解RT和過繼T細胞組合療法在HCC中的潛在效應。

Hepatocellular carcinoma (HCC) is a complex malignancy with high mortality globally. Current standard systemic treatments for advanced unresectable HCC include tyrosine kinase inhibitors and immunotherapy. Recently, adoptive cell therapy (ACT) has emerged as a promising treatment for HCC, with chimeric-antigen receptor T cells (CAR-T) or T cell receptor T cells (TCR-T) being typical candidates. Ongoing clinical trials are evaluating this novel treatment, but reports suggest ACT treatments alone may have low efficacy for solid tumors. Therefore, radiotherapy, which is characterized by a non-invasive nature for treating unresectable HCC, has been used to boost the immune response against tumors. However, the effectiveness and underlying immune mechanisms of this combination in HCC remain unclear. Here, we utilized a unique spontaneous and trackable HCC mouse model by hydrodynamics tail vein injection (HDTVi) of oncogenic plasmids. Low-dose whole-liver and a high-dose regional of radiation, 8Gy and 20Gy, respectively, were applied to the tumor-bearing mice, followed by the transfer of CD8+OT-I T cells five weeks after HDTVi. The HiBiT level, an artificial circulating marker for tracking tumor growth, showed no significant difference between the sham, 8Gy, and 20Gy radiation groups three weeks post-treatment. Intriguingly, one sample in the 8Gy radiation group remained at a constant HiBiT level. This prompted an investigation into the immune cell distribution under these conditions. We observed a modest increase in immune cell infiltration in 8Gy radiotherapy groups. To achieve better tumor control, RT plus ACT and anti-VEGF-A were implanted in five-week HCC-bearing mice. The liver tumor was repressed in a triple combination group compared to groups with RT plus anti-VEGF-A or anti-VEGF-A only after one month of tracking. Although preliminary, these findings suggest that 8Gy low-dose radiation plus T cell therapy and anti-VEGF-A likely activate anti-tumor immunity and alleviate tumor progression in spontaneous HCC. For the subcutaneous model, we utilized a primary tumor cell line isolated from spontaneous HCC murine. The treatment, including RT plus ACT, RT alone, and ACT alone, were able to reduce the volume of small tumors (≤ 500mm2). In contrast, they could barely inhibit large tumor progression. After analyzing the tumor immune microenvironment, CD45+ total immune cells and tumor-specific OT-I T cells were significantly increased after RT. TIM-3 exhaustion and KLRG1 effector markers show increased levels of expression on OT-I T cells, suggesting that RT can effectively stimulate the anti-tumor activity of tumor-specific T cells. Collectively, the combinational therapeutic effect of RT and adoptive T cell therapy in HCC can potentially be understood by studying the phenotypes of tumor antigen-specific OT-I cells after RT.