目的 肿瘤转移相关蛋白1（Metastasis-associated protein 1,MTA1)在多种人类肿瘤细胞中表达上调，MTA1作为ATP依赖性染色质重塑复合物的一个亚基，能够广泛影响下游基因的表达，因此对肿瘤细胞分泌的细胞因子、趋化因子以及肿瘤微环境具有调控作用。但目前关于肿瘤细胞高表达MTA1对局部微环境免疫状态的调控尚不可知。本文旨在基于结肠癌癌种，阐释肿瘤细胞高表达MTA1对肿瘤微环境中巨噬细胞浸润及功能活化的调控及其对CTL抗肿瘤杀伤效应的影响。

方法 分析TCGA结直肠癌队列转录组数据和TIMER免疫浸润数据库评估MTA1表达水平与趋化因子表达水平、免疫细胞浸润水平的关系，并使用细胞因子抗体芯片检测MTA1高表达对趋化因子表达的调控；qPCR检测巨噬细胞炎性相关分子表达水平；流式细胞术检测巨噬细胞极化表面标志分子、自噬水平、淋巴细胞功能活化及功能耗竭相关分子表达、淋巴细胞凋亡水平、自噬水平、肿瘤细胞凋亡水平、肿瘤细胞、巨噬细胞与淋巴细胞的相互作用。多重荧光免疫组化实验检测结直肠癌组织中的CD8、CD4、CD20、CD206、CD163、CD68、CD86、PD-L1、MTA1分子的表达水平。使用inform2.2.4进行图像分析，R包ISAT进行距离分析，Flow jo软件进行流式细胞实验结果分析， Graphpad8.3进行所有的数据统计学分析。

结果 在肿瘤细胞MTA1表达水平与其趋化效能的评估中，结肠癌细胞高表达MTA1广泛下调单核细胞募集相关的趋化因子表达水平，上调T细胞募集相关细胞因子；诱导巨噬细胞极化方面，提升了以CD206阳性为特征的M2型巨噬细胞极化水平，降低了以CD163阳性为特征的M2型巨噬细胞极化水平，但对M1型巨噬细胞的诱导并无显著下调。在MTA1高表达水平的共培养体系中，巨噬细胞分泌的抗炎因子IL-10和促炎因子iNOS、TNF-α、IL-6相比于阴性对照都显著升高。在MTA1高表达水平的共培养体系中，巨噬细胞分泌促炎因子IL-6显著低于MTA1低表达的两组。MTA1高表达的共培养体系中，淋巴细胞活化水平、衰竭水平和凋亡水平均显著高于对照组和MTA1敲低组，然而MTA1高表达组中，肿瘤细胞与淋巴细胞的相互作用却显著低于对照组和MTA1敲低组，TCR激活后表现出自噬水平提高的这一过程也显著低于对照组和MTA1敲低组。最终体现在杀伤效果上，MTA1高表达水平的共培养体系中杀伤效能显著低于MTA1低水平的共培养体系。与此同时，流式细胞术检测到共培养体系中巨噬细胞的存在能够在降低淋巴细胞的活化标志分子表达、提高功能耗竭的分子表达水平的同时，降低淋巴细胞的凋亡水平，大大提高淋巴细胞对肿瘤细胞的杀伤效能。

在结直肠癌组织切片中，高表达MTA1的组织中巨噬细胞的浸润密度显著降低，CD8+T浸润密度显著升高；在活化类型上，CD206阳性的巨噬细胞相对升高，CD163阳性和CD86阳性的巨噬细胞浸润水平相对降低。在距离分析中CD8+T与CD163+巨噬细胞的细胞间通讯程度更强；CD8+T细胞与肿瘤细胞的距离随着MTA1表达水平升高呈现降低的趋势，体现MTA1高表达肿瘤细胞对CD8+T的募集效果。另外，结直肠癌组织中的PD-L1表达水平与巨噬细胞浸润密度正相关，而与MTA1表达水平不存在明显的相关性。

结论 结直肠癌中MTA1高表达造成肿瘤微环境中巨噬细胞的募集减少，巨噬细胞分泌的炎症因子降低、抗原提呈过程缺失，进而致使T细胞抗肿瘤杀伤效应无法激活、功能抑制的比例上调，从而造成肿瘤微环境中局部免疫抑制，肿瘤细胞免疫逃逸。

Objective Metastasis associated protein 1 (MTA1) is upregulated in a variety of human tumor cells. As a subunit of ATP-dependent chromatin remodeling complex,MTA1 can extensively affect downstream gene expression.Therefore, it has a regulatory effect on cytokines, chemokines and tumor microenvironment secreted by tumor cells.However, the regulation of MTA1 overexpression in tumor cells on the immune state of the local microenvironment is still unknown.The aim of this study was to elucidate the role of MTA1 overexpression in the regulation of macrophage infiltration and functional activation in the tumor microenvironment and the anti-tumor killing effect of CTL based on colon cancer species.

Methods TCGA colorectal cancer cohort transcriptome data and TIMER immune infiltration database were analyzed to evaluate the relationship between MTA1 expression and chemokine expression and immune cell infiltration, and the regulation of MTA1 high expression on chemokine expression was detected using cytokine antibody microarray.The expression levels of inflammation-related molecules in macrophages were detected by qPCR.The polarization surface marker molecules of macrophages, autophagy level, expression of molecules related to functional activation and functional depletion of lymphocytes, apoptosis level of lymphocytes, autophagy level, apoptosis level of tumor cells, interaction between tumor cells, macrophages and lymphocytes were detected by flow cytometry.The expression levels of CD8, CD4, CD20, CD206, CD163, CD68, CD86, PD-L1 and MTA1 in colorectal cancer tissues were detected by multiple fluorescence immunohistochemistry assay.Inform2.2.4 was used for image analysis, R package ISAT was used for distance analysis, Flow Jo software was used for Flow cytometry test results analysis, and Graphpad8.3 was used for all data statistical analysis.

Results In the assessment of the expression level of MTA1 in tumor cells and its chemotactic efficacy, the high expression of MTA1 in colon cancer cells significantly down-regulated the expression level of chemokines related to monocyte recruitment and up-regulated the cytokines related to T cell recruitment.In terms of the induction of macrophage polarization, the polarization level of M2 macrophages characterized by CD206 positive was increased, and the polarization level of M2 macrophages characterized by CD163 positive was decreased, but the induction of M1 macrophages was not significantly down-regulated.In the co-culture system with high expression level of MTA1, the anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines iNOS, TNF-α and IL-6 secreted by macrophages were significantly increased compared with the negative control.In the co-culture system with high expression of MTA1, the secretion of pro-inflammatory cytokine IL-6 from macrophages was significantly lower than that in the two groups with low expression of MTA1.In the co-culture system with high expression of MTA1, the activation, depletion and apoptosis levels of lymphocytes were significantly higher than those in the control group and MTA1 knockdown group. However, the interaction between tumor cells and lymphocytes in the MTA1 high expression group was significantly lower than that in the control group and MTA1 knockdown group.The process of increased autophagy level after TCR activation was also significantly lower than that of the control group and MTA1 knockdown group.In the end, the killing effect of co-culture system with high MTA1 expression level was significantly lower than that of co-culture system with low MTA1 expression level.At the same time, flow cytometry showed that the presence of macrophages in the co-culture system could not only reduce the expression of activation marker molecules of lymphocytes and increase the expression level of functional depletion molecules, but also reduce the apoptotic level of lymphocytes, thus greatly improving the killing efficacy of lymphocytes against tumor cells.

Conclusions MTA1 high expression in colorectal cancer caused by macrophages in tumor microenvironment of raise reduction, reduced secretion of macrophage inflammatory factors and antigen presenting process is missing, then the T cells antitumor effects cannot be activated, inhibition ratio increases, resulting in local immune suppression in the tumor microenvironment, immune escape of tumor cells.