目的：本研究利用单细胞RNA测序分析技术（scRNA-seq）初步探索乳腺癌4T1细胞表达淋巴细胞抗原6复合物E（Ly6E） 调控肿瘤免疫微环境（TIME）的潜在机制。方法：利用CRISPR-Cas9技术构建Ly6e基因敲除小鼠乳腺癌4T1细胞（Ly6E-KO），观 察其和野生型细胞（Ly6E-WT）体外增殖和体内生长能力的差异。流式细胞术分选两类肿瘤组织中的CD45阳性细胞，再进行scRNAseq分析。对于测序结果，首先利用Seurat软件包筛选差异表达基因谱，根据标记基因进行注释；再利用Cellchat和Monocle2软件 分析细胞互作关系和特定免疫细胞的演化轨迹。结果：与Ly6E-WT 相比，Ly6E-KO体外增殖能力无显著差异，但体内生长能 力显著降低（P<0.001）。ScRNA-seq分析显示，Ly6E-KO移植瘤浸润DC比例显著高于Ly6E-WT，且DC处于更加活跃的增殖和分 裂状态；三种DC亚群（pDC、cDC1和cDC3）与T细胞均存在显著的互作关系。同时发现，Ly6E-KO肿瘤中浸润T淋巴细胞增殖、活 化及效应相关的基因表达水平均升高，提示Ly6E-KO具有更强抗肿瘤能力。最后，本研究对人类乳腺癌队列的scRNA-seq数据进 行了分析，进一步证实了上述发现。结论：肿瘤细胞Ly6e基因敲除后可通过增加TIME中DC活化及浸润，继而增强机体抗肿瘤免 疫应答。

Objective: To preliminarily explore the potential mechanisms by which lymphocyte antigen 6 complex, locus E (Ly6E) regulates tumor immune microenvironment (TIME) in breast cancer 4T1 cells using single-cell RNA transcription sequencing technology (scRNA-seq). Methods: A mouse breast cancer 4T1 cell line with Ly6e gene knockout (Ly6E-KO) was constructed by CRISPR-Cas9 technology. The in vitro proliferation and in vivo growth capabilities of Ly6E-KO cells were compared to those of the wide type 4T1 cells (Ly6E-WT). CD45-positive cells from both types of tumor tissues were sorted by flow cytometry and analyzed using scRNA-seq. The sequencing results were first screened for differentially expressed gene profiles using the Seurat software package and annotated based on marker genes. Then, Cellchat and Monocle2 software were used to analyze the cell-to-cell interactions and the evolutionary trajectories of specific immune cells. Results: Compared with Ly6E-WT, Ly6E-KO cells showed no significant difference in in vitro proliferation capability but demonstrated significantly reduced in vivo growth capability (PConclusion: Ly6e gene knockout in tumor cells can enhance the anti-tumor immune response by increasing DC activation and infiltration in TIME.

福建省科技创新联合基金重点项目（No. 2021Y9036）