PO.IM02.05 · 免疫学
全基因组CRISPR筛选揭示卵巢癌中肿瘤内在的免疫逃逸机制
Genome-wide CRISPR screening reveals tumor-intrinsic immune evasion mechanism in ovarian cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
卵巢癌是致死率最高的妇科恶性肿瘤。既往研究表明卵巢癌中存在高水平的肿瘤浸润T细胞，可预测免疫治疗获益。然而，诸如KEYNOTE-100等临床试验报告pembrolizumab的总体缓解率仅约10%。这一差异表明卵巢癌存在异质性的、肿瘤内在的免疫逃逸机制。为区分免疫耐药的癌细胞系，我们建立了一个基于报告基因的共培养平台，将预激活的人T细胞与卵巢癌细胞系直接共培养。我们对24个经过实时细胞毒性报告基因改造的卵巢癌细胞系进行了分析，以量化肿瘤细胞死亡。将来自两名健康供者的预激活T细胞与每一株癌细胞系共培养，报告基因读数将该组细胞系分层为11株免疫敏感系和6株免疫耐药系。为揭示耐药的遗传决定因素，我们选取了两株耐药性最强的细胞系进行全基因组CRISPR筛选。将经CRISPR编辑的癌细胞与预激活T细胞共培养后，对sgRNA丰度进行测序，并采用MAGeCK-MLE流程进行分析。聚焦于一致性缺失，我们鉴定出56个基因（P < 0.05；beta < -0.5），其缺失可在两株细胞系中均增强T细胞介导的肿瘤杀伤。其中，我们优先选择ITGA8（integrin alpha8，整合素alpha8）作为有前景的表面靶点。ITGA8与beta1异源二聚化形成alpha8beta1，后者结合细胞外基质配体并激活黏附相关和存活相关的信号轴，包括FAK/Src。鉴于整合素调控免疫细胞向肿瘤的浸润以及TGF-beta信号等通路，我们假设ITGA8维持促存活信号，从而限制T细胞活性。因此，对ITGA8进行药物性或生物性阻断可能提供一种可及于细胞表面的策略，将免疫耐药的卵巢肿瘤转化为免疫敏感性疾病。
查看英文原文 English abstract
Ovarian cancer is the most lethal gynecologic malignancy. Previous studies demonstrated high levels of tumor-infiltrating T cells in ovarian cancer, which predict benefit from immunotherapy. However, clinical trials such as KEYNOTE-100 report only ~10% overall response to pembrolizumab. This discrepancy indicates that ovarian cancers harbor heterogeneous, tumor-intrinsic mechanisms of immune evasion.To distinguish immune-resistant cancer cell lines, we established a reporter-based co-culture platform in which pre-activated human T cells were directly co-cultured with ovarian cancer cell lines. We profiled 24 ovarian cancer cell lines engineered with a real-time cytotoxicity reporter to quantify tumor-cell death. Pre-activated T cells from two healthy donors were co-cultured with each cancer cell line, and reporter readouts stratified the set into 11 immune-sensitive and 6 immune-resistant lines. To uncover genetic determinants of resistance, we selected the two most resistant cell lines for whole-genome CRISPR screening. Following co-culture of CRISPR-edited cancer cells with pre-activated T cells, sgRNA abundances were sequenced and analyzed using the MAGeCK-MLE pipeline. Focusing on concordant depletions, we identified 56 genes (P < 0.05; beta < -0.5) whose loss augments T-cell-mediated tumor killing in both cell lines. Among these, we prioritized ITGA8 (integrin alpha8) as a promising surface target. ITGA8 heterodimerizes with beta1 to form alpha8beta1, which engages extracellular-matrix ligands and activates adhesion- and survival-linked signaling axes, including FAK/Src. Given that integrins regulate immune-cell infiltration into tumors and pathways such as TGF-beta signaling, we hypothesize that ITGA8 sustains pro-survival signaling that constrains T-cell activity. Accordingly, pharmacologic or biologic blockade of ITGA8 may offer a surface-accessible strategy to convert immune-resistant ovarian tumors into immune-sensitive disease.
利益披露 Disclosure
J. Park, None..
J. Lee, None..
H. Kang, None..
J. Lee, None.