Cancer cells avoid ferroptosis induced by immune cells via fatty acid binding proteins [ChIP-seq]. Cancer cells avoid ferroptosis induced by immune cells via fatty acid binding proteins [ChIP-seq]

Background Cancer creates an immunosuppressive environment that hampers immune responses, allowing tumors to grow and resist therapy. One way the immune system fights back is by inducing ferroptosis, a type of cell death, in tumor cells through CD8+ T cells. This involves lipid peroxidation and enzymes like lysophosphatidylcholine acyltransferase 3 (Lpcat3), which makes cells more prone to ferroptosis. However, the mechanisms by which cancer cells avoid immunotherapy-mediated ferroptosis are unclear. Our study reveals how cancer cells evade ferroptosis and anti-tumor immunity through the upregulation of fatty acid-binding protein 7 (Fabp7). Methods To explore how cancer cells resist immune cell-mediated ferroptosis, we used a comprehensive range of techniques. We worked with cell lines including PD1-sensitive, PD1-resistant, B16F10, and QPP7 glioblastoma cells, and conducted in vivo studies in syngeneic 129 Sv/Ev, C57BL/6, and conditional knockout mice with Rora deletion specifically in CD8+ T cells, Cd8 cre;Rorafl mice. Methods included mass spectrometry-based lipidomics, targeted lipidomics, Oil Red O staining, Seahorse analysis, quantitative PCR, immunohistochemistry, PPARγ transcription factor assays, ChIP-seq, untargeted lipidomic analysis, ROS assay, ex vivo co-culture of CD8+ T cells with cancer cells, ATAC-seq, RNA-seq, Western blotting, co-immunoprecipitation assay, flow cytometry and Imaging Mass Cytometry. Results PD1-resistant tumors upregulate Fabp7, driving protective metabolic changes that shield cells from ferroptosis and evade anti-tumor immunity. Fabp7 decreases the transcription of ferroptosis-inducing genes like Lpcat3 and increases the transcription of ferroptosis-protective genes such as Bmal1 through epigenetic reprogramming. Lipidomic profiling revealed that Fabp7 increases triglycerides and monounsaturated fatty acids (MUFAs), which impede lipid peroxidation and ROS generation. Fabp7 also improves mitochondrial function and fatty acid oxidation (FAO), enhancing cancer cell survival. Furthermore, cancer cells increase Fabp7 expression in CD8+ T cells, disrupting circadian clock gene expression and triggering apoptosis through p53 stabilization. Clinical trial data revealed that higher FABP7 expression correlates with poorer overall survival and progression-free survival in patients undergoing immunotherapy. Overall design: Chip-seq of histone modification H3K27ac and H3K9ac in Sen, Res, Res_ctrl and Res_shFabp7 cells

研究背景 癌症会构建免疫抑制性微环境，抑制机体免疫应答，进而促进肿瘤生长并使其产生治疗抵抗。机体免疫系统的反击途径之一，是通过CD8+ T细胞在肿瘤细胞中诱导铁死亡（ferroptosis，一种细胞死亡形式）。该过程涉及脂质过氧化以及溶血磷脂酰胆碱酰基转移酶3（lysophosphatidylcholine acyltransferase 3, Lpcat3）等酶类，此类酶可增强细胞对铁死亡的易感性。然而，肿瘤细胞规避免疫治疗介导的铁死亡的具体分子机制尚未阐明。本研究揭示了肿瘤细胞通过上调脂肪酸结合蛋白7（fatty acid-binding protein 7, Fabp7），从而逃避铁死亡与抗肿瘤免疫的具体机制。

研究方法 为探究肿瘤细胞如何抵抗免疫细胞介导的铁死亡，本研究采用了一系列全面的实验技术。我们使用了包括PD1敏感型、PD1耐药型、B16F10及QPP7胶质母细胞瘤细胞在内的多种细胞系，并在同基因129 Sv/Ev、C57BL/6小鼠以及CD8+ T细胞中特异性敲除Rora基因的条件性敲除小鼠（Cd8-Cre;Rorafl小鼠）中开展了体内实验。本研究采用的实验技术包括：基于质谱的脂质组学、靶向脂质组学、油红O染色、海马细胞能量代谢分析、实时定量PCR、免疫组织化学、PPARγ转录因子活性检测、染色质免疫共沉淀测序（ChIP-seq）、非靶向脂质组学分析、活性氧（ROS）检测、CD8+ T细胞与肿瘤细胞的离体共培养、转座酶可及性测序（ATAC-seq）、RNA测序（RNA-seq）、蛋白质免疫印迹、免疫共沉淀实验、流式细胞术以及成像质谱流式细胞术。

研究结果 PD1耐药型肿瘤会上调Fabp7的表达，通过驱动保护性代谢重编程，使肿瘤细胞免于铁死亡并逃避抗肿瘤免疫。Fabp7可通过表观遗传重编程，抑制铁死亡诱导基因（如Lpcat3）的转录，并上调铁死亡保护基因（如Bmal1）的转录。脂质组学分析结果显示，Fabp7可提升肿瘤细胞内甘油三酯与单不饱和脂肪酸（monounsaturated fatty acids, MUFAs）的水平，从而阻碍脂质过氧化与活性氧的产生。Fabp7还可改善线粒体功能与脂肪酸氧化（fatty acid oxidation, FAO）过程，增强肿瘤细胞的存活能力。此外，肿瘤细胞可上调CD8+ T细胞中Fabp7的表达，通过破坏生物钟基因的表达并介导p53蛋白稳定化，诱导CD8+ T细胞凋亡。临床试验数据表明，接受免疫治疗的患者中，FABP7高表达与更差的总生存期及无进展生存期显著相关。

实验整体设计：对Sen、Res、Res_ctrl及Res_shFabp7细胞进行组蛋白修饰H3K27ac与H3K9ac的染色质免疫共沉淀测序（ChIP-seq）。