Extracellular galectin 4 drives immune evasion and promotes T-cell apoptosis in pancreatic cancer [scRNA-Seq]

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell produced protein deposited into the ECM of PDAC tumors and detected high circulating levels of gal 4 in PDAC patients. In orthotopic transplantation experiments we observed increased infiltration of T-cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. Furthermore, by performing single-cell RNA-sequencing we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression was associated with higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T-cells and antigen presenting dendritic cells in tumors with reduced gal 4 expression. Using a co-culture system, we observed that extracellular gal 4 induced apoptosis in T-cells by binding CD3, resulting in anergy or induction of apoptosis. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC. Single cell RNA-seq analysis of two mouse pancreatic tumours collected 6.5 weeks after orthotopic transplantation of tumour organoid lines (Gal4KD vs scramble control). The two tumour organoid lines were derived from the mT5 line described in Boj et al, 2015 (PMID: 25557080). A tumour from the Galectin 4 shRNA knock-down organoid line (Gal4KD) was compared to a tumour from the scramble control organoid line. Epcam-/Cd45-/Pdpn+ fibroblastic cells and Epcam-/Cd45+/Pdpn- immune cells were sorted into different tubes by flow cytometry, and ~5*10^4 immune cells were pooled with ~5*10^4 immune cells from the same tumour. A total of 7500 cells per sample was used.

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）以富含细胞外基质（extracellular matrix, ECM）沉积为特征，该特征会影响疾病的病理生理学进程。本研究鉴定出半乳糖凝集素4（galectin 4, gal 4）是由癌细胞产生并沉积于PDAC肿瘤细胞外基质中的蛋白，且在PDAC患者外周血中检测到高浓度的gal 4。在原位移植（orthotopic transplantation）实验中，我们观察到在移植了gal 4表达下调的癌细胞的免疫健全小鼠（immunocompetent mice）体内，T细胞浸润增多且小鼠生存期延长。而在免疫缺陷RAG1基因敲除（RAG1-/-）小鼠中未观察到生存期延长现象，这表明该效应由适应性免疫系统（adaptive immune system）介导。
进一步通过单细胞RNA测序（single-cell RNA-sequencing），我们发现移植瘤内的髓系细胞群（myeloid compartment）及癌症相关成纤维细胞（cancer-associated fibroblast, CAF）亚型发生了改变。gal 4表达下调与肌成纤维样CAFs比例升高、炎症型CAFs数量减少相关。我们还发现，在gal 4表达下调的肿瘤中，M1型巨噬细胞（M1 macrophages）、T细胞及抗原呈递树突状细胞（dendritic cells）的比例更高。利用共培养体系（co-culture system），我们观察到细胞外gal 4可通过结合CD3诱导T细胞凋亡（apoptosis），引发T细胞免疫无能（anergy）或凋亡。因此，本研究证实gal 4参与了免疫逃逸（immune evasion）过程，并将gal 4确定为克服PDAC免疫抑制的极具潜力的药物靶点（drug target）。
本数据集为两株小鼠胰腺肿瘤的单细胞RNA测序分析数据，这两株肿瘤均源自肿瘤类器官系的原位移植，于移植后6.5周采集（Gal4KD组 vs scramble对照组）。这两株肿瘤类器官系均衍生自Boj等于2015年报道的mT5细胞系（PMID: 25557080）。本数据集将半乳糖凝集素4短发夹RNA敲低类器官系（Gal4KD）来源的肿瘤与scramble对照类器官系来源的肿瘤进行比对。通过流式细胞术分选出Epcam-/Cd45-/Pdpn+成纤维细胞及Epcam-/Cd45+/Pdpn-免疫细胞并分别置于不同管中，将约5×10^4个免疫细胞与同一肿瘤中分离得到的约5×10^4个免疫细胞混合。每个样本最终使用7500个细胞进行测序。