ZEB2 is a Master Switch Controlling the Tumor-Associated Macrophage Program [in vivo]. ZEB2 is a Master Switch Controlling the Tumor-Associated Macrophage Program [in vivo]

Tumor-associated macrophages (TAMs) are major players in regulating the immunosuppressive tumor environment, and their abundance is highly correlated with poor clinical outcomes. Here, we constructed a TAM regulatory network by integrating scRNA-seq data across human solid tumors with a dedicated CRISPR knockout screen. Using a deep generative model capable of learning a local representation for each candidate regulator, we constructed a gene perturbation network that linked individual target genes with prototypical functional modules in TAMs. We identified non-redundant pathways regulating distinct TAM functions, showing modular circuitry. For instance, the complement module is repressed by Stat6, Zeb2, Gpnmb, and Spp1, while the Tgfbr1-Smad2/4 pathway induces this program in TAMs. Importantly, we identified Zeb2 as the master regulator of pro-tumor functions in TAMs, orchestrating the suppression of type I interferon response and antigen presentation alongside the activation of immune suppression programs. Genetic ablation of Zeb2 reprogrammed TAMs identity on chromatin, RNA, and protein levels. In human tumors with high macrophage content, ZEB2 expression was associated with poor prognosis in solid cancers, including lung and bladder. Functional macrophage coculturing assays defined Zeb2 as a critical regulator of TAM immunosuppression activity by inhibiting T cell proliferation and activation. Selective in vivo targeting of Zeb2 in macrophages using a CpGsiRNAZeb2 DNA hybrid reprogrammed TAMs and mobilized systematic anti-tumoral T cell responses, achieving complete tumor clearance as a monotherapy. Overall, our study generated a detailed genetic roadmap of TAM gene circuits and identified ZEB2 as a master switch of TAMs with potential therapeutic implications for macrophage-based immunotherapies. Overall design: WT C57BL/6 mice were s.c. implanted with MC38 cells at the flanks. When tumors reached 80-100 mm^3, mice either received PBS or CpG-siRNA conjugates via intratumoral injections. Tumor-bearing mice were sacrificed at indicated timepoints and tumor-infiltrating leukocytes were extracted and single-cell suspensions were prepared for flow cytometry antibody staining and single-cell FACS index sorting into 384-well-plates. Single-cell RNAseq libraries were prepared with SPID-seq protocol (Yofe et al., 2023) and sequenced with Illumina Novaseq.

肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）是调控免疫抑制性肿瘤微环境的核心细胞类群，其浸润丰度与不良临床预后显著相关。本研究通过整合人类实体瘤的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据与专属的CRISPR基因敲除筛选（CRISPR knockout screen），构建了TAMs的调控网络。本研究采用可学习每个候选调控因子局部表征的深度生成模型，构建了将单个靶基因与TAMs内典型功能模块相连接的基因扰动网络。本研究鉴定出调控TAMs不同功能的非冗余通路，展现出模块化的调控环路。例如，补体模块可被Stat6、Zeb2、Gpnmb及Spp1抑制，而Tgfbr1-Smad2/4通路可在TAMs中激活该程序。尤为重要的是，本研究鉴定出Zeb2是TAMs促肿瘤功能的核心调控因子，其可协同调控I型干扰素应答与抗原呈递的抑制，同时激活免疫抑制程序。Zeb2的基因敲除可在染色质、RNA及蛋白质水平上重编程TAMs的细胞身份。在巨噬细胞浸润丰度较高的人类肿瘤中，ZEB2的表达与肺癌、膀胱癌等实体瘤的不良预后显著相关。巨噬细胞共培养功能实验证实，Zeb2可通过抑制T细胞的增殖与活化，成为调控TAMs免疫抑制活性的关键因子。采用CpG-siRNA-Zeb2 DNA杂交体对巨噬细胞内的Zeb2进行体内靶向干预，可重编程TAMs并激活系统性抗肿瘤T细胞应答，作为单一疗法即可实现肿瘤的完全清除。综上，本研究绘制了TAMs基因调控环路的详细遗传图谱，并鉴定出ZEB2作为TAMs的核心调控开关，为基于巨噬细胞的免疫治疗提供了潜在的治疗价值。
整体实验设计：将野生型（wild type, WT）C57BL/6小鼠于背部皮下（subcutaneous, s.c.）接种MC38肿瘤细胞。当肿瘤体积达到80~100 mm³时，对小鼠分别进行瘤内注射PBS或CpG-siRNA偶联物。于指定时间点处死荷瘤小鼠，提取肿瘤浸润白细胞并制备单细胞悬液，用于流式细胞术抗体染色及单细胞流式指数分选至384孔板中。采用SPID-seq测序技术（Yofe等，2023）制备单细胞RNA测序文库，并通过Illumina Novaseq测序平台完成测序。