PO.IM01.04 · 免疫学
利用SHP2靶向信号传导以强化抗肿瘤免疫应答
Harnessing SHP2-targeted signaling to reinforce anti-tumor immune responses
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：SHP2是一种非受体蛋白酪氨酸磷酸酶，通过PD-1调节免疫检查点信号传导以及致癌性RAS/MAPK通路，使其成为整合抗癌治疗的一个有前景的靶点。虽然SHP2抑制剂主要因其肿瘤内在效应而受到研究，但其免疫调节潜力仍不明确。因此，我们研究了SHP2抑制是否能同时解除PD-1介导的T细胞抑制并抑制肿瘤内在的MAPK信号传导，这是一种特别针对RAS/MAPK信号驱动肿瘤的双重作用机制。
方法：使用原代小鼠T细胞和人Jurkat T细胞系评估免疫细胞应答。通过流式细胞术表征T细胞活化，并使用WST-1和CellTiter-Glo实验量化细胞活力。通过LDH释放实验评估T细胞介导的对癌细胞的细胞毒作用。进行qPCR分析与免疫活化相关的基因表达谱。为评估对致癌信号的影响，通过蛋白质印迹法测量RAS/MAPK通路活性。为进行体内评估，荷MC38-OVA同基因肿瘤的小鼠接受口服SHP2抑制剂TNO155（10或20 mg/kg，每日两次，持续14天）。
结果：使用TNO155（1 nM-10 μM）对SHP2进行药理学抑制不影响T细胞活力，表明其适用于体外免疫学分析。尽管其细胞毒性极小，SHP2抑制显著调节了T细胞活化。流式细胞术分析显示活化标志物（包括CD69和Granzyme B表达）上调，同时通过LDH释放实验测得的T细胞介导的对肿瘤细胞的细胞毒作用增强（在1 μM TNO155时细胞毒作用增加20.9%；P<0.05）。在荷同基因肿瘤的免疫健全小鼠中，口服给予TNO155（口服，每日两次）在第14天使肿瘤体积较载体处理对照显著减小（肿瘤生长抑制：39.6%（10 mg/kg；P<0.05）和62.4%（20 mg/kg；P<0.01））。免疫组化和流式细胞术分析检测到CD8⁺肿瘤浸润淋巴细胞的浸润，qPCR显示肿瘤中IFNgamma反应性基因（包括Cd274，约2.2倍诱导；P<0.001）的表达升高。值得注意的是，肿瘤内CD8⁺ T细胞表现出增强的效应功能，Granzyme B⁺细胞显著增加（对照：4.46±1.86%；TNO155：18.94±12.27；P<0.05），耗竭标志物TIM-3和LAG-3减少。
结论：这些发现表明SHP2抑制增强抗肿瘤免疫应答，并支持其作为癌症免疫治疗中双功能治疗策略的潜力。本研究为SHP2靶向方法在精准肿瘤学中的转化开发提供了坚实的临床前基础。
查看英文原文 English abstract
Background: SHP2, a non-receptor protein tyrosine phosphatase, regulates both immune checkpoint signaling via PD-1 and oncogenic RAS/MAPK pathways, making it a promising target for integrated anti-cancer therapy. While SHP2 inhibitors have been primarily investigated for their tumor-intrinsic effects, their immunomodulatory potential remains unclear. Therefore, we investigated whether SHP2 inhibition can simultaneously relieve PD-1-mediated T cell suppression and inhibit tumor-intrinsic MAPK signaling, a dual-action mechanism particularly for tumors driven by RAS/MAPK signaling.
Methods: Primary murine T cells and human Jurkat T cell lines were used to assess the immune cell responses. Flow cytometry was performed to characterize T cell activation, and cell viability was quantified using WST-1 and CellTiter-Glo assays. T cell-mediated cytotoxicity against cancer cells was evaluated by the LDH release assay. qPCR was conducted to analyze the gene expression profiles related to immune activation. To assess the impact on oncogenic signaling, RAS/MAPK pathway activity was measured by western blotting. For in vivo evaluation, mice bearing MC38-OVA syngeneic tumors received oral SHP2 inhibitor TNO155 (10 or 20 mg/kg, BID for 14 days).
Results: Pharmacological inhibition of SHP2 using TNO155 (1 nM-10 µM) did not affect T cell viability, indicating its suitability for in vitro immunological analysis. Despite its minimal cytotoxicity, SHP2 inhibition significantly modulated T cell activation. Flow cytometric analysis revealed the upregulation of activation markers, including CD69 and Granzyme B expression, alongside enhanced T cell-mediated cytotoxicity against tumor cells, as measured by LDH release assay (increase of 20.9% cytotoxicity at 1 µM TNO155; P < 0.05). In syngeneic tumor-bearing immunocompetent mice, oral administration of TNO155 (p.o. BID) led to a significant reduction in tumor volume compared to vehicle-treated controls (tumor growth inhibition: 39.6% (10 mg/kg; P < 0.05) and 62.4% (20 mg/kg; P < 0.01) on day 14. Immunohistochemical and flow cytometric analyses detected the infiltration of CD8⁺ tumor-infiltrating lymphocytes, and qPCR revealed elevated expression of IFNgamma-responsive genes, including Cd274 (~2.2-fold induction; P < 0.001 ) in tumors. Notably, intratumoral CD8⁺ T cells exhibited enhanced effector function, with a marked increase in Granzyme B⁺ cells (control: 4.46 ± 1.86%; TNO155: 18.94 ± 12.27; P < 0.05 ) and a decrease in exhaustion markers TIM-3 and LAG-3.
Conclusions: These findings demonstrate that SHP2 inhibition enhances anti-tumor immune responses and supports its potential as a dual-function therapeutic strategy in cancer immunotherapy. This study provides a robust preclinical foundation for the translational development of SHP2-targeted approaches in precision oncology.
利益披露 Disclosure
B. Tsevegjav, None..
H. Tsukamoto, None..
O. Kikuchi, None.