结直肠癌（Colorectal cancer, CRC）是全球癌症相关死亡的第三大原因，其高复发率、转移率及化疗/免疫治疗耐药性是临床治疗的主要挑战。尽管近年免疫检查点抑制剂在微卫星高度不稳定（Microsatellite instability-high, MSI-H）型CRC中展现出疗效，但大多数患者仍面临治疗响应率低的问题。中国作为CRC高发国家，结直肠癌的发病率和死亡率均呈上升趋势‌，且晚期患者比例较高，亟需突破性治疗策略。

肿瘤干细胞（Cancer stem cells, CSCs）与肿瘤免疫抑制性微环境（Tumor microenvironment, TME）的相互作用是驱动肿瘤进展和耐药的核心机制。其中，肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）作为TME中最丰富的免疫细胞群体，通常通过分泌因子参与CSCs生态位的构建与免疫逃逸调控，促进肿瘤恶性进展，但其分子调控网络尚未完全阐明。

分化抑制因子1（Inhibitor of differentiation 1, ID1）是一种在生物发育过程中调控细胞生长分化的关键转录调节因子。在静息状态下，ID1的表达水平较低，但在增殖分化活跃的组织和细胞中，其表达显著增加。研究发现，ID1在结直肠癌、卵巢癌、胰腺癌、食道癌、胶质母细胞瘤等多种类型的肿瘤组织中的表达水平显著升高，增强肿瘤的耐药性、血管生成能力、干性特征和肿瘤转移能力，并且与肿瘤患者的不良预后密切相关。

本研究利用GEO数据库分析TAMs与正常巨噬细胞的转录组差异，并深入挖掘包含101例CRC患者肿瘤组织芯片数据，我们发现ID1在CRC患者TAMs中显著高表达，且与患者不良预后、淋巴结转移、病理进程密切相关，这提示ID1在维持TAMs促肿瘤表型中的作用。为了探究高表达ID1的TAMs对结肠癌发生发展的作用，我们在免疫健全的C57BL/6小鼠、BALB/c小鼠和免疫缺陷的裸鼠上建立了包括皮下移植瘤模型、结肠癌原位移植瘤模型、脾脏肝转移模型、结肠癌原位肝转移模型、尾静脉肺转移模型等多种小鼠CRC模型，证实高表达ID1的TAMs通过改变分泌组分影响CD8⁺ T细胞和肿瘤细胞两个方面促进结肠癌生长和转移。

我们从结肠癌患者临床样本、整体动物水平、细胞生物学及分子生物学四个层面对具体的调节机制展开了研究。在肿瘤免疫方面，流式细胞术、免疫荧光共聚焦实验证实CD8⁺ T细胞在肿瘤组织中的浸润数目增多，利用小鼠来源和结肠癌患者来源的CD8⁺ T细胞进行T细胞迁移实验，结果表明敲除ID1的TAMs促进CD8⁺ T细胞的迁移能力。在肿瘤固有特性方面，流式细胞术、成球实验、侵袭实验证实高表达ID1的TAMs促进肿瘤的干性特征。在分子生物学层面，我们利用转录组测序、Western Blot、CO-IP、RT-qPCR、Duolink PLA、荧光素酶报告基因等实验进行研究。结果表明，ID1通过直接结合信号转导与转录激活因子1（Signal transducer and activator of transcription 1, STAT1），增强核输出蛋白染色体区域维持蛋白1（Chromosome region maintenance 1, CRM1）对STAT1的募集，促进STAT1的核输出及胞质滞留，从而抑制其转录活性。这一过程导致STAT1靶基因SerpinB2和CCL4的转录显著下调。一方面，SerpinB2的转录和分泌减少，促进肿瘤细胞中干性相关信号通路FAK-YAP信号轴活化，增强肿瘤干性；另一方面，CCL4的转录和分泌减少，抑制CD8⁺ T细胞向肿瘤部位的迁移，进而促进肿瘤免疫逃逸。

为验证ID1的临床转化潜力，本研究利用靶向ID1去泛素化酶USP1的小分子抑制剂ML323，降低了ID1蛋白表达。我们在多种CRC小鼠模型中开展单药治疗和联合治疗实验。结果显示，抑制ID1表达可显著减少CSCs比例，并促进CD8⁺ T细胞向肿瘤部位的募集。更重要的是，ML323与抗CTLA4抗体或5-氟尿嘧啶（5-Fluorouracil, 5-FU）联用可协同抑制肿瘤生长，提示靶向ID1可能突破现有治疗瓶颈。

本研究首次揭示ID1在TAMs中通过STAT1依赖性机制，作为“分子支架”同时调控肿瘤干细胞特性与免疫微环境的重编程，拓展了对ID蛋白家族非经典功能的认识，为转录因子亚细胞定位调控提供了新范式。ID1高表达TAMs可作为预测CRC免疫治疗响应的生物标志物，且ID1抑制剂与现有疗法的联用策略有望实现“一石二鸟”效应——即同步清除CSCs并逆转免疫抑制。除CRC外，ID1维持TAMs促癌表型的分子机制在肝癌及胰腺癌TAMs中同样适用，提示ID1可能成为泛实体瘤治疗的共性靶点。

综上所述，TAMs中高表达的ID1是介导固有免疫反应与获得性免疫反应之间交叉调控的分子桥梁，从诱导肿瘤免疫逃逸和维持肿瘤干性两个维度发挥“双重打击”的作用，加速肿瘤恶性进展。本研究的发现不仅为理解TAMs与CSCs的交互作用提供了理论框架，更通过靶向ID1的临床前证据，强调了靶向ID1可使结肠癌治疗实现“一靶多效”的抗肿瘤效果，为开发兼具“去干细胞化”与“免疫正常化”功能的联合疗法奠定了科学基础。

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide, and its high recurrence rate, metastasis, and resistance to chemotherapy/immunotherapy present significant clinical challenges. Although immune checkpoint inhibitors have shown efficacy in microsatellite instability-high (MSI-H) CRC, most patients still face low response rates. As a high-incidence country for CRC, China has rasing CRC incidence and mortality rates, with a high proportion of patients presenting in the advanced stages, underscoring the need for breakthrough therapeutic strategies.

Research indicates that the persistent presence of cancer stem cells (CSCs) and their interaction with the tumor immune microenvironment (TME) are core mechanisms driving tumor progression and resistance. Tumor-associated macrophages (TAMs), the most abundant immune cell population in the TME, participate in the construction of the CSC niche and immune escape regulation through secretory factors or direct contact, although their molecular regulatory network remains poorly understood.

Inhibitor of differentiation 1 (ID1) is a key transcriptional regulator that modulates cell growth and differentiation during biological development. Under resting conditions, ID1 expression is low but significantly increases in proliferative and differentiating tissues and cells. Studies have shown that ID1 is upregulated in the tumor tissues of various cancers, including CRC, ovarian cancer, pancreatic cancer, esophageal cancer, and glioblastoma, where it enhances drug resistance, angiogenesis, stemness, and metastasis, correlating with poor prognosis.

This study utilized the GEO database to analyze transcriptome differences between TAMs and normal macrophages, and conducted in-depth exploration of tumor tissue microarray data from 101 CRC patients. We found that ID1 was significantly overexpressed in CRC patient TAMs and was closely associated with poor prognosis, lymph node metastasis, and pathological progression, suggesting a role for ID1 in maintaining tumor-promoting properties of TAMs. To investigate the role of high ID1-expressing TAMs in the development of CRC, we established various mouse models, including subcutaneous tumor models, orthotopic colon cancer models, spleen-liver metastasis models, orthotopic-liver metastasis models, and lung metastasis models, in immunocompetent C57BL/6 mice, BALB/c mice, and nude mice. We confirmed that high ID1-expressing TAMs promote colorectal cancer growth and metastasis by altering secretory components that affect both CD8⁺ T cells and tumor cells in two distinct manners.

We conducted comprehensive studies on the regulatory mechanisms of CRC at multiple levels, including clinical samples from colorectal cancer patients, overall animal studies, cellular biology aspect, and molecular biology aspect. In the context of tumor immunology, we utilized flow cytometry and immunofluorescence to confirm that the number of CD8⁺ T cells infiltrating tumor tissues is increased. Through experiments using CD8⁺ T cells derived from both mice and colorectal cancer patients, we demonstrated that knockdown of ID1-expressing TAMs enhances the migratory ability of CD8⁺ T cells. In terms of tumor intrinsic properties, flow cytometry, sphere formation assays, and invasion experiments revealed that ID1-high expressing TAMs promote the stemness characteristics of tumors. At the molecular biology aspect, we employed transcriptome sequencing, Western blotting, co-immunoprecipitation (CO-IP), RT-qPCR, Duolink PLA, and luciferase reporter assays to investigate these mechanisms. The results show that ID1 directly interacts with signal transducer and activator of transcription 1 (STAT1), enhancing the recruitment of chromosome region maintenance 1 (CRM1) to STAT1, promoting STAT1 nuclear export and cytoplasmic retention, thereby inhibiting its transcriptional activity. This process significantly downregulates the expression of STAT1 target genes SerpinB2 and CCL4. On one hand, reduced transcription and secretion of SerpinB2 activate the FAK-YAP signaling axis in tumor cells, enhancing stemness. On the other hand, decreased CCL4 expression inhibits the migration of CD8⁺ T cells to the tumor site, promoting immune escape.

To validate the clinical translational potential of ID1, we used a small molecule inhibitor ML323, which targets the deubiquitinase USP1 to decrease ID1 expression. We performed monotherapy and combination therapy experiments in various CRC mouse models. The results showed that ID1 inhibition significantly reduced CSCs and promoted CD8⁺ T cell recruitment to the tumor site. More importantly, ML323 combined with anti-CTLA-4 antibodies or 5-fluorouracil (5-FU) synergistically inhibited tumor growth, suggesting that targeting ID1 may overcome current treatment bottlenecks.

This study reveals, for the first time, that ID1 in TAMs regulates both tumor stem cell characteristics and immune microenvironment reprogramming through a STAT1-dependent mechanism, expanding the understanding of the non-classical functions of the ID proteins and providing a new paradigm for subcellular localization regulation of transcription factors. ID1 overexpression in TAMs may serve as a biomarker for predicting CRC immunotherapy response, and ID1 inhibitors combined with existing therapies may offer a synergistic effect—simultaneously clearing CSCs and reversing immune suppression. Beyond CRC, the molecular mechanism by which ID1 sustains the protumor phenotype of TAMs is also significantly activated in liver and pancreatic cancer TAMs, suggesting its potential as a common therapeutic target for solid tumors.

In summary, the high expression of ID1 in TAMs is a molecular bridge mediating the cross-regulation between innate and adaptive immune responses, exerting a "dual strike" effect by inducing tumor immune escape and maintaining tumor stemness, thus accelerating tumor malignant progression. The findings of this study not only provide a theoretical framework for understanding TAMs and CSC interactions but also emphasize the preclinical evidence for targeting ID1 to achieve a multifaceted antitumor effect, laying the scientific foundation for developing combination therapies with both "stem cell-depleting" and "immune-normalizing" functions.