背景和目的：食管鳞状细胞癌作为消化道常见恶性肿瘤，早期隐匿起病和恶性程度高，导致食管鳞癌患者预后差。食管鳞癌的发生历经多阶段病理过程，挖掘其组织微环境中重要分子标志物的时间-空间特征，有助于理解食管上皮细胞演变为食管鳞癌的连续过程。本研究目的在于探究驱动食管鳞癌发生潜在功能基因，并解析其表达和功能调控机制，为进一步理解食管鳞癌组织内复杂空间信号传导通路。

方法：本研究通过4-NQO诱导的小鼠食管鳞癌发生模型模拟人食管鳞癌演变连续过程。基于多色免疫荧光对正常、炎症、低级别上皮瘤变、高级别上皮瘤变和食管鳞癌阶段样本对髓系细胞和T细胞特异性标志物染色，得到小鼠食管鳞癌发生多阶段免疫微环境构成组分。通过RT-qPCR在mRNA水平，Western blot和免疫组织化学染色在蛋白质水平检测细胞的表达。在体外使用细胞活力检测、细胞平板克隆形成实验、transwell迁移和侵袭实验，在体内采用免疫缺陷鼠皮下成瘤、鼠尾静脉注射构建转移模型，验证食管鳞癌细胞的增殖、转移和侵袭能力。通过使用细胞免疫荧光染色和免疫共沉淀的方式在机制上验证细胞内蛋白的共定位和结合能力，使用细胞成分分离分析蛋白在细胞内的分布情况，并使用染色质免疫共沉淀的方法验证了转录因子与预测的下游基因启动子区域DNA的结合。并通过免疫组织化学法对小鼠食管鳞癌发生组织样品染色，验证了体外实验的基因表达调控关系。

结果：我们在小鼠食管癌变多阶段组织的髓系细胞时空分布分析发现一群CD11c表达的上皮细胞，在食管鳞癌发生过程中增加且表达升高。通过对人食管癌变多阶段样本富集分析发现CD11c编码基因ITGAX与抗原提呈相关通路和EMT通路相关。我们通过体外细胞实验和小鼠体内模型验证CD11c促进食管鳞癌细胞的增殖、迁移与侵袭能力。我们发现CD11c可以提高MHC II类分子mRNA水平并抑制抗原提呈通路关键基因STAT3表达从而影响抗原提呈通路，同时体外共培养发现CD11c促进了食管鳞癌刺激CD4+ T细胞分泌免疫抑制性细胞因子IL-10的能力。除此之外，CD11c促进食管鳞癌EMT。我们发现CD11c促进EMT和抑制STAT3的表达是通过上调SMAD3的磷酸化实现。对小鼠肺转移模型转移灶进行免疫组织化学染色同样显示CD11c增加p-SMAD3，并下调了肿瘤STAT3蛋白水平。机制上，我们发现SMAD3、TGFBR1和CD11c在人食管上皮细胞膜上存在共定位。膜蛋白CD11c结合SMAD3，增加SMAD3与其活化受体I型TGFBR1的结合并磷酸化活化SMAD3，随后SMAD3入核增多。ChIP-qPCR结果显示p-SMAD3在不同食管细胞系中与STAT3启动子的结合。在小鼠食管多阶段癌变组织样本中，我们同样发现食管上皮STAT3蛋白水平随食管病变阶段逐渐降低。最后，在对人食管多阶段癌变过程ITGAX基因拷贝数的改变和ITGAX启动子区甲基化水平，我们发现这种ITGAX阳性上皮细胞克隆扩增可能来自于ITGAX基因拷贝数增加和ITGAX启动子区甲基化水平降低。

结论：表达CD11c的上皮细胞在食管鳞癌发生过程中数量增加且CD11c表达量升高。ITGAX编码膜蛋白CD11c增加SMAD3的膜募集，促进与其活化受体I型结合随后磷酸化活化并且入核增多。随后，p-SMAD结合于下游靶基因STAT3启动子区域发挥转录调控作用。CD11c通过p-SMAD3的增加，上调食管鳞癌免疫抑制性抗原提呈过程和EMT，从而发挥抑制性免疫免疫调节作用和促进食管鳞癌转移，参与食管鳞癌的进展。

Background&Purpose: Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor of the digestive tract, characterized by early insidious onset and high malignancy, leading to poor prognosis in ESCC patients. The occurrence of ESCC undergoes multiple stages of histological processes. Thus, an urge need for exploring the temporal-spatial characteristics of important lineage markers in the tissue microenvironment helps understanding the continuous process of esophageal epithelial cell evolution into ESCC. The purpose of this study is to investigate potential functional genes involved in ESCC development, and elucidate the underlying functional regulatory mechanisms, in order to further understand the complex spatial signal transduction pathways within ESCC tissues during tumorigenesis.

Methods: A mouse model of ESCC tumorigenesis induced by 4-NQO was applied as an analogue of the continuous process of ESCC evolution in humans. Using multiplexed immunofluorescence (mIF) staining, qualified lineage markers for epithelium, myeloid cells and T cells were detected in serial tissues representing normal, inflammatory, low-grade epithelial dysplasia, high-grade epithelial dysplasia, and ESCC stages, to characterize the immune microenvironment components during the mouse multistages ESCC development. Expression of genes was detected at the mRNA level using real-time quantitative PCR (RT-qPCR), and at the protein level using Western blot and immunohistochemistry (IHC) in ESCC cells and tissues. To validate the proliferation, migration, and invasion capabilities of ESCC cells, cell viability assays, colony formation assays, transwell migration and invasion assays were conducted in vitro, while subcutaneous tumor formation and tail vein injection in immunodeficient mouse models were used to establish a cell derived xenograft and metastasis models in vivo. Mechanisms involving protein co-localization and protein mutually interaction within cells were verified using cell immunofluorescence (IF) and co-immunoprecipitation (co-IP), protein distribution within cells was analyzed through cell component separation, and chromatin immunoprecipitation (ChIP) was used to validate the binding of transcription factors to predicted downstream gene promoter regions. Immunohistochemistry staining of STAT3 was performed on samples from mouse ESCC tumorigenesis panels to validate the gene expression regulatory relationships of CD11c observed in vitro experiments.

Results: Analysis of the temporal-spatial distribution of myeloid cells panel in the multistage tissues from mouse esophageal cancer revealed a group of epithelial cells expressing CD11c, which increased and showed elevated expression during ESCC development. Enrichment analysis of samples representing multiple stages of human esophageal cancer revealed that ITGAX-gene that encodes CD11c-is associated with the antigen presentation (AP) related pathway and Epithelial-mesenchymal transition (EMT). Through in vitro cell experiments and in vivo mouse models, we validated that CD11c promotes the proliferation, migration, and invasion capabilities of ESCC cells. We found that CD11c can increase the mRNA levels of MHC class II molecules and suppress the expression of STAT3, the key gene of the AP pathway. Co-culture of ESCC cells and CD4+ T cells experiments showed that CD11c promotes the immunomodulatory AP ability of ESCC, thus stimulating immunosuppressive cytokine IL-10-producing CD4+ T cells. Additionally, CD11c promotes EMT in ESCC. We discovered that CD11c promotes EMT and inhibits STAT3 expression through upregulation of phosphorylated SMAD3. Immunohistochemical staining of metastatic lesions in mouse lung metastasis model also indicated increased p-SMAD3 levels and decreased protein levels of tumor STAT3. Mechanistically, we found co-localization of SMAD3, TGFBR1, and CD11c on the membrane of human esophageal epithelial cells. Membrane protein CD11c binds to SMAD3, following upregulation of the binding with its activating receptor TGFBR1 and thus activating of SMAD3 through phosphorylation, leading to increased nuclear translocation of SMAD3. ChIP-qPCR results showed binding of p-SMAD3 to the STAT3 promoter in different esophageal cell lines. In our former mouse multistage esophageal cancer tissues, we also observed a gradual decrease in the protein level of STAT3 in esophageal epithelium. Finally, by analyzing changes in ITGAX gene copy number and ITGAX promoter region methylation levels during the human multistage esophageal cancer progression, we found that the clonal expansion of CD11c-expressing epithelial cells may result from increased ITGAX gene copy numbers and decreased ITGAX promoter region methylation levels.

Conclusion: CD11c expressing epithelial cells expand and show elevated CD11c expression level during ESCC development. CD11c, the membrane protein ITGAX encode, enhances membrane recruitment of SMAD3 and promote its binding to the receptor type I TGFBR1, leading to subsequent SMAD3 phosphorylation. After nuclear translocation, p-SMAD3 binds tothe promoter region of SMAD3 downstream target gene STAT3 to exert transcriptional regulation. CD11c upregulates immunosuppressive antigen presentation processes and EMT in ESCC through increasing p-SMAD3, playing roles in immunosuppressive immune regulation and promoting ESCC metastasis, thus contributing to the progression of ESCC.