肿瘤细胞治疗研究
英文原题：PGE2-mediated NK cell reprogramming drives acquired immunotherapy resistance in lung adenocarcinoma.
肿瘤来源的PGE2是肺腺癌获得性PD-1阻断耐药的重要促成因素。治疗性干扰EP2/EP4-cAMP-CREM轴可恢复NK细胞功能并克服获得性耐药。
获得性耐药限制了程序性细胞死亡蛋白-1（PD-1）阻断在肺腺癌中的持久疗效，然而驱动获得性耐药复发的肿瘤内在程序与免疫环路仍缺乏明确界定。本研究的目的是鉴定获得性耐药的肿瘤内在介导因子，并确定它们如何重塑抗肿瘤免疫。
在经 anti-PD-1 治疗的免疫健全小鼠中建立原位生物发光示踪的 Lewis 肺癌（LLC1）肺腺癌模型。通过体内全基因组 CRISPR 功能缺失筛选鉴定肿瘤内在调控因子，并使用可诱导的四环素关闭敲低进行验证。通过肿瘤细胞 Ptgs2 敲低/缺失、给予 16,16-dimethyl PGE2、选择性 EP2/EP4 拮抗剂以及 celecoxib 治疗，探究前列腺素 E2（PGE2）信号通路。通过流式细胞术、免疫荧光、RNA 测序、cAMP 测定、钙流试验、小鼠和人 NK 细胞共培养细胞毒性试验以及 NK 细胞过继转移，分析自然杀伤（NK）细胞功能。使用 celecoxib 评估药物性 PGE2 阻断在体内的治疗潜力。分析公共免疫治疗数据集，以评估 PTGS2 的临床相关性。
原位LLC1模型重现了异质性anti-PD-1应答的关键特征，包括初始退缩后的复发。CRISPR筛选鉴定出Ptgs2是获得性耐药的关键驱动因子。肿瘤来源的PGE2在耐药肿瘤中逐步升高，其基因沉默通过恢复NK细胞浸润和功能克服了耐药。机制上，PGE2通过EP2/EP4受体传导信号，升高cAMP并诱导CREM，从而抑制NK细胞细胞毒性和细胞因子产生。该轴在人NK细胞中得到验证。使用celecoxib药理学抑制cyclooxygenase-2可逆转获得性耐药，而该效应在NK细胞清除后被消除。
BACKGROUND: Acquired resistance limits the durability of programmed cell death protein-1 (PD-1) blockade in lung adenocarcinoma, yet the tumor-intrinsic programs and immune circuits that drive acquired resistance relapse remain poorly defined. The purpose of this study was to identify tumor-intrinsic mediators of acquired resistance and determine how they remodel antitumor immunity. METHODS: An orthotopic bioluminescence-tracked Lewis lung carcinoma (LLC1) lung adenocarcinoma model was established in immunocompetent mice treated with anti-PD-1. Tumor-intrinsic regulators were identified by an in vivo genome-wide CRISPR loss-of-function screen and validated using inducible tetracycline-off knockdown. Prostaglandin E2 (PGE2) signaling was interrogated through tumor-cell Ptgs2 knockdown/deletion, 16,16-dimethyl PGE2 administration, selective EP2/EP4 antagonists, and celecoxib treatment. Natural killer (NK)-cell function was analyzed by flow cytometry, immunofluorescence, RNA sequencing, cAMP measurement, calcium flux assays, mouse and human NK-cell co-culture cytotoxicity assays, and NK-cell adoptive transfer. Celecoxib was used to evaluate the therapeutic potential of pharmacologic PGE2 blockade in vivo. Public immunotherapy datasets were analyzed to assess the clinical relevance of PTGS2. RESULTS: The orthotopic LLC1 model captured key features of heterogeneous anti-PD-1 responses, including relapse after initial regression. The CRISPR screen identified Ptgs2 as a key driver of acquired resistance. Tumor-derived PGE2 progressively increased in resistant tumors, and its genetic silencing overcame resistance by restoring NK-cell infiltration and function. Mechanistically, PGE2 signaled through EP2/EP4 receptors to elevate cAMP and induce CREM, thereby suppressing NK-cell cytotoxicity and cytokine production. This axis was validated in human NK cells. Pharmacologic inhibition of cyclooxygenase-2 with celecoxib reversed acquired resistance, an effect abrogated by NK-cell depletion. CONCLUSIONS: Tumor-derived PGE2 is an important contributor to acquired resistance to PD-1 blockade in lung adenocarcinoma. Therapeutic disruption of the EP2/EP4-cAMP-CREM axis restores NK-cell function and overcomes acquired resistance.
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