Revisiting the immune microenvironment of human colorectal cancer in the era of single-cell and spatial omics

Revisiting the immune microenvironment of human colorectal cancer in the era of single-cell and spatial omics | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 25 August 2025 V1 Latest version Share on Revisiting the immune microenvironment of human colorectal cancer in the era of single-cell and spatial omics Authors : Shuomin Zhang , Qingfeng Fu , Xiaotong Yuan , Sijun Wang , Chao Liu , Chaojun Zhang , Bing Liu , and Gong Yandong 0000-0003-3971-1478 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175613488.82145372/v1 294 views 193 downloads Contents Abstract Abstract Background Dendritic cells Lymphoid B cells Innate lymphoid cells Fibroblasts Conclusion Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Colorectal cancer (CRC) remains a significant global health challenge, ranking among the leading causes of cancer-related morbidity and mortality. The tumor microenvironment (TME)—comprising immune cells, stromal elements, and extracellular matrix—plays a pivotal role in tumorigenesis, progression, and therapeutic resistance. Recent advancements in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have revolutionized our understanding of the CRC TME by allowing high-resolution profiling of cellular heterogeneity and spatial dynamics. These cutting-edge technologies have uncovered novel, previously uncharacterized subsets of immune and stromal cells, all of which significantly relate to immune evasion and malignant progression. This review consolidates recent insights from single-cell and spatial transcriptomic studies, emphasizing the identification of novel cellular subtypes and their roles in modulating the TME. We also discuss the therapeutic implications of these findings, particularly in the development of targeted immunotherapies and combinatorial strategies aimed at improving clinical outcomes for CRC patients. By deepening our understanding of this complex cellular ecosystem, these insights are expected to inform more precise and effective therapeutic approaches for CRC. Revisiting the immune microenvironment of human colorectal cancer in the era of single-cell and spatial omics Shuomin Zhang 1# ; Qingfeng Fu 1# ; Xiaotong Yuan 1# ;Sijun Wang 1# ; Chao Liu 2 ；Chaojun Zhang 1* ; Bing Liu 3* ；Yandong, Gong 3* 1 Chinese PLA General Hospital, Beijing 100080, China 2 Department of Radiation Oncology, Peking University First Hospital, Beijing, 100034, China 3 State Key Laboratory of Experimental Hematology, Haihe Laboratory of Cell Ecosystem, Senior Department of Hematology, Fifth Medical Center, Medical Innovation Research Department, Chinese PLA General Hospital, Beijing 100071, China # These authors contributed equally to this work * Correspondence: Chaojun, Zhang ( [email protected] ), Bing Liu ( [email protected] ) and Yandong Gong ( [email protected] ) Abstract Colorectal cancer (CRC) remains a significant global health challenge, ranking among the leading causes of cancer-related morbidity and mortality. The tumor microenvironment (TME)—comprising immune cells, stromal elements, and extracellular matrix—plays a pivotal role in tumorigenesis, progression, and therapeutic resistance. Recent advancements in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have revolutionized our understanding of the CRC TME by allowing high-resolution profiling of cellular heterogeneity and spatial dynamics. These cutting-edge technologies have uncovered novel, previously uncharacterized subsets of immune and stromal cells, all of which significantly relate to immune evasion and malignant progression. This review consolidates recent insights from single-cell and spatial transcriptomic studies, emphasizing the identification of novel cellular subtypes and their roles in modulating the TME. We also discuss the therapeutic implications of these findings, particularly in the development of targeted immunotherapies and combinatorial strategies aimed at improving clinical outcomes for CRC patients. By deepening our understanding of this complex cellular ecosystem, these insights are expected to inform more precise and effective therapeutic approaches for CRC. Keywords: Colorectal cancer, Single-cell RNA sequencing, Spatial transcriptomics, Tumor microenvironment Background Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths worldwide(1-3). Recent advances in immunotherapy have demonstrated clinical benefit in CRC patients(4-8). However, immune checkpoint blockade (ICB), particularly targeting the programmed death-1 (PD-1) pathway, has shown promising clinical benefit in a subset of patients, only approximately 20% respond favorably to such therapies, primarily those with mismatch repair-deficient (dMMR) or microsatellite instability-high (MSI-H) tumors(9-11). In contrast, the majority of CRC patients, particularly those with microsatellite stable (MSS) tumors, exhibit limited or no response(6, 12). A major contributor to this therapeutic disparity is the tumor microenvironment (TME)-a highly heterogeneous and immunosuppressive niche composed of immune cells, stromal elements, vasculature, extracellular matrix, and soluble factors(13, 14). The intricate nature of the TME in CRC has the potential to attenuate immune responses and curtail the effectiveness of treatment. As such, elucidating the cellular composition, spatial architecture, and functional states of TME components is critical for understanding mechanisms of treatment resistance and identifying novel therapeutic targets in CRC(15). Traditional bulk transcriptomic and flow cytometric analyses have provided valuable insights into the TME; however, these methods average signals across cell populations, obscuring the cellular diversity and functional complexity of individual cell types(16, 17). The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized our ability to deconstruct this complexity by enabling high-resolution analysis of individual cells within the TME(18-22). More recently, spatial transcriptomics (ST) has emerged as a complementary technology that retains spatial context while profiling gene expression, thereby allowing the mapping of cell-cell interactions and spatial gradients of signaling activity within intact tissue sections(15). This review synthesizes recent findings from scRNA-seq and ST studies in CRC, with an emphasis on characterizing previously unrecognized immune and stromal cell subsets, and their spatial interactions and the key signaling pathways driving tumor progression and therapeutic resistance. These insights are reshaping our understanding of CRC pathobiology and providing a foundation for developing innovative therapeutic strategies aimed at reprogramming the TME to improve patient outcomes. Macrophages Macrophages are sentinel immune cells that maintain tissue homeostasis, defend against pathogens, and orchestrate both innate and adaptive immune responses. In physiological conditions, tissue-resident macrophages carry out key functions such as phagocytosis (e.g., C1QA, C1QB, C1QC ), antigen presentation, and immune regulation (e.g., IL1B, NR4A1, NR4A2, NR3A3, BHLHE40 )(23). In the TME, macrophages represent one of the most abundant immune populations and are collectively referred to as tumor-associated macrophages (TAMs). TAMs play a pivotal role in tumor progression by regulating tumor cell proliferation, angiogenesis, immune suppression, and metastatic dissemination(24, 25). Recent scRNA-seq studies have greatly refined our understanding of the macrophage landscape in CRC(26). Rather than conforming to the classical M1 (pro-inflammatory) or M2 (anti-inflammatory) paradigm, TAMs in CRC frequently co-express gene signatures associated with both M1 and M2 phenotypes, reflecting functional and transcriptional heterogeneity driven by local microenvironmental cues. Based on gene expression profiles, functional state, and spatial localization, TAMs can now be classified into distinct subpopulations that contribute differentially to immune regulation and tumor dynamics. Functional Subtypes of TAMs in CRC: Immunosuppressive vs. Inflammatory vs. Anti-tumor SPP1⁺ TAMs are among the most immunosuppressive subsets identified in hypoxic and necrotic regions of CRC by scRNA-seq analysis. They are characterized by the expression of angiogenic and immunomodulatory genes (e.g., VEGFA , VCAN , and CXCL8 )(27), and Their abundance increases progressively from normal to primary tumor to metastatic tissues(28). It is noteworthy that most SPP1⁺ TAMs originate from circulating classical monocytes with pro-inflammatory properties, while a subset derives from tissue-resident macrophages(29). Furthermore, ICB therapy has been shown to reduce both the proportion of monocyte-derived SPP1⁺ TAMs and their output of inflammatory cytokines, further suggesting their role in immune suppression(28). SPP1⁺ TAMs exert their immune-suppressive effects through extensive interactions with other cell types in the TME. Specifically, SPP1 + TAMs activate FOXP3 + regulatory T cells (Tregs) in close proximity at primary and metastatic sites by decondensing chromatin of the MHC-II. SPP1 + TAMs can secrete high levels of IL-10, sustaining their activated state via autocrine feedback. Additionally, transcriptomic profiling reveals that SPP1 + TAMs exhibit elevated expression of genes linked to tumor growth, metastasis, and therapeutic resistance (e.g., SDC2, S100A6, S100A8, S100A10, MT2A, MT1E, MT1F). TCGA and GEO cohorts showed a positive correlation between SPP1 + TAMs and poorer survival outcomes, as well as resistance to immunotherapies in CRC(34, 35). Another immunosuppressive subset, TREM2⁺ TAMs, characterized by high expression of TREM2 and lipid metabolism abnormalities(24). The TREM2 + macrophage are specifically enriched within tumor tissues and highly express genes related to M2 macrophage activation (e.g., MARCO , TREM2 ), immune suppression (e.g., HAVCR2 ), lipid metabolism (e.g., MSR1 , APOC1 , APOE , OLR1 ), and foam cell formation, all of which contribute to tumor progression(30). Interestingly, TREM2 + TAM range from small mononuclear cells to large foam cells or multinuclear giant cells morphologically, reflecting its high degree of heterogeneity. Chronic inflammation is a hallmark of TME. Inflammatory macrophage subsets have also been identified in CRC by scRNA-seq. Specifically, NLRP3⁺ TAMs s was identified with high expression of inflammatory cytokines (e.g., IL-18 , ICAM1 , IDO1 , and iNOS ) and the activation of inflammasomes. These cells are typically found either diffusely distributed throughout the tumor or clustered with neutrophils, suggesting developing inflammatory niches by different mechanism (31). Another notable inflammatory population is the MRC1⁺CCL18⁺ TAM subset, present in both primary CRC and liver metastases. These macrophages upregulate pro-inflammatory mediators (e.g., TNF, IL1B, CCL3, and CCL4), amino acid metabolism genes (resembling Kupffer cells), and M2 macrophage polarization-related genes (e.g., APOE and MARCO )(32). Importantly, this subset interacts preferentially with metastatic tumor cells via the CD47–SIRPA axis, which inhibits their phagocytic capacity. This represents a key immune evasion mechanism employed by CRC metastases. Remarkably, both SPP1⁺ and MRC1⁺CCL18⁺ TAM subsets decline significantly following neoadjuvant chemotherapy (NAC), a shift accompanied by a concurrent increase in effector CD8⁺ T cells, may helping restore effective anti-tumor immunity(32). On the other hand, C1QC⁺ TAMs represent an anti-tumor subset characterized by expression of M1 macrophage polarization-related markers (e.g., CD68 , CD80 , and MAF ) and complement-related genes (e.g., C1QA, TREM2, MERTK , and CD80 ). However, their primary function appears to be the regulation of anti-tumor T cell responses. This population facilitates recruitment and activation of diverse T cell subsets—including CD8⁺ effector T cells, Tregs, and Th1-like cells—via extensive receptor–ligand interactions. Through this immunostimulatory activity, C1QC⁺ TAMs contribute to anti-tumor immunity and have been associated with improved patient outcomes(33). Spatial Organization of TAMs Beyond transcriptomic heterogeneity, the spatial localization of TAMs is closely linked to their functional specialization(34). Under physiological conditions, TAMs exhibit zonated distribution across mucosal layers (31). For example, IL4I1⁺ TAMs are found in the uppermost mucosal layer and retain their phagocytic capacity even in the tumor microenvironment. These cells preferentially localize to fibrotic or apoptotic tumor margins, where they phagocytose tumor cell, correlating with favorable prognosis. Their activity may be modulated by the CD274–CD47 axis. In the middle and lower layers, FOLR2 + TAMs contribute to the formation of the plasma cell niche. Additionally, the LYVE1 + TAM subpopulation located in the submucosa of the colon, surrounding blood vessels, resembles murine perivascular macrophages. The subpopulation specifically expresses IL-10 and LYVE1 and contributes to suppress inflammation and fibrosis(34, 35). Within tumors, TAMs display pronounced spatial divergence. Macrophages at the invasive front often display anti-tumor features, whereas those located deeper in the tumor core adopt immunosuppressive or pro-metastatic functions(25). A ST study using 10x Genomics Visium revealed that immunomodulatory TAMs (Reg-TAMs) localize primarily to stromal and peritumoral regions, regulating immune cell infiltration(15). Conversely, tumor-core TAMs engage in direct cell-cell interactions that promote invasion and metastasis(25). Further supporting this differential localization, transcriptome profiling revealed that CD68⁺ macrophages infiltrate and tightly surround tumor cells, while CD163 + macrophages are preferentially located further from the tumor nests(31). In colorectal liver metastasis, two morphological subclasses have been described: large TAMs (L-TAMs), which display a foam cell-like morphology with high cholesterol metabolism and phagocytic gene expression (e.g., C1QA, C1QB, C1QC, MERTK, MSR1, MRC2); and small TAMs (S-TAMs), which express inflammatory markers. Notably, L-TAMs are particularly associated with poor prognosis, emphasizing the clinical significance of TAM diversity in metastatic CRC(36). Spatial has also facilitated mechanistic insights into TAM function. For example, SPP1⁺ TAMs, initially identified by scRNA-seq, have been found to co-localize with FAP⁺ fibroblasts by ST, which contribute to immune exclusion by constructing fibrotic barriers that physically impede T cell infiltration(37). These interactions are regulated by chemerin, TGF-β, and IL-1, which may underlie resistance to PD-L1 blockade in patients expressing high levels of FAP and SPP1 (24). A recent study employed spatial multi-omics techniques, including imaging mass spectrometry, spatial proteomics, and spatial transcriptomics, to elucidate the spatial TME of metastatic CRC patients receiving immunotherapy. The study revealed that, regardless of microsatellite status, C1QC+ TAMs are more abundant in responders, where C1QC+ TAMs are observed to co-localize with CD4+ T cells and the expression of MHC-II facilitate their interaction. In contrast, in non-responders, cancer-associated fibroblasts can inhibit this interaction(38). These spatial distinctions highlight the importance of context-dependent TAM phenotypes and underscore the complexity of macrophage-mediated regulation within the CRC microenvironment. Therapeutic Targeting of Macrophages in CRC Given their central role in shaping tumor immunity, TAMs represent attractive therapeutic targets in CRC. However, clinical efforts have encountered several challenges. For instance, CSF1R inhibitors, while effective at depleting proliferative, inflammatory macrophages, have shown limited clinical efficacy in CRC. This may be due to their inability to eliminate immunosuppressive subsets such as SPP1⁺ TAMs(29). More promising strategies have focused on intracellular signaling pathways. SHP2, a phosphatase that negatively regulates type I interferon (IFN) signaling, has emerged as a viable target. SHP2 inhibition enhances anti-tumor immune responses and synergizes with immune checkpoint blockade in preclinical CRC models(39). Additionally, dual blockade of TREM2 and PD-1 has demonstrated efficacy in murine CRC models. This approach depletes M2-like TAMs, promotes neutrophil expansion, and reprograms the remaining TAM population toward a pro-inflammatory phenotype. These findings collectively highlight the need for more precise, subset-specific macrophage-targeting strategies in CRC—ideally guided by spatial and single-cell transcriptomic profiling—to overcome immunotherapy resistance and improve clinical outcomes. Figure 1. Dissection of major tumor-associated macrophage subpopulations in the tumor microenvironment using multi-omics approaches. IL4I1+ TRMs, primarily involved in phagocytosis, retain their function in the tumor microenvironment. FOLR2+ TRMs contribute to plasma cell niche formation, while LYVE1+ TRMs, localized around blood vessels, specifically express IL-10 and LYVE1 to regulate inflammation and tissue fibrosis.C1QC+ TAMs are associated with complement activation and enhance antigen presentation to T cells. SPP1+ TAMs, located in the ischemic and necrotic regions of tumors, express inhibitory receptors, induce physical barrier formation, activate suppressive cells, and limit the infiltration and function of cytotoxic cells. NLRP3+ TAMs are macrophages linked to inflammation, contributing to the inflammatory milieu within tumors. TREM2+ TAMs mediate immunosuppression, lipid metabolism through scavenging modified lipoproteins, and foam cell formation, while also promoting Treg activation and complement activation. Dendritic cells Dendritic cells (DCs) are pivotal antigen-presenting cells that play a critical role in inducing and maintaining anti-tumor immunity. Although they constitute a relatively small fraction of immune cells in the TME, they exert substantial immunomodulatory influence. Traditionally, DCs are categorized into classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs)(40-44). cDCs can be further subdivided into CD141 + cDC1s and CD1c + cDC2s. While cDC1s are primarily responsible for activating CD8⁺ cytotoxic T cells, cDC2s are more involved in the activation and polarization of CD4⁺ helper T cells. In contrast, pDCs are primarily involved in antiviral responses and the modulation of other immune cells, such as T cells and cDCs(45-47). In CRC, the distribution of DC subsets is notably altered: pDCs are significantly increased and cDC1s markedly reduced(29). While pDCs contribute to both inflammatory responses and anti-cancer immunity, their immunosuppressive effects are often more pronounced, potentially facilitating tumor cell migration. In contrast, cDC2s in CRC exhibit substantial phenotypic and functional heterogeneity, giving rise to multiple subpopulations with distinct roles(33). For instance, a distinct cDC2 subpopulation found in liver metastatic tissue is characterized by the expression of pro-inflammatory genes (e.g., C1QA, CD68, CD163, CD14, VSIG4, CCL3 , and MAFB ). This subpopulation closely resembles the recently described DC3 population in both human and murine models, and its enrichment is associated with poor prognosis in CRC. Additionally, TIMP1 + cDC2 cells, identified in peritumoral liver metastases and mesenteric lymph nodes, express maturation markers like CCR7 and participate in regulating angiogenesis (e.g., EREG , CREM , and VEGFA )(33). A specialized subset of DCs, termed LAMP3+ DCs, has been identified in both primary CRC lesions and liver metastatic lesions(48). LAMP3 + DCs are defined by high expression of maturation markers (e.g., CD40 , CD80 , CD86 ), regulatory molecules (e.g., PD-L1 ), migration-related genes (e.g., CCR7 ), and low expression of Toll-like receptor (TLR) signaling genes(26). Lineage tracing and transcriptomic profiling have revealed that LAMP3+ DCs originate from both cDC1 and cDC2 populations, with a predominance of cDC1-derived cells(26, 49). LAMP3 + DCs derived from cDC1s are essential in priming naïve CD8⁺ T cells. However, these cells also possess immunosuppressive properties, including the induction of regulatory T cells (Tregs) via BTLA upregulation, contributing to immune tolerance(50). This dual-functionality mirrors the behavior of mregDCs observed in lung cancer, which similarly promote Treg differentiation(49). Recent studies highlight that LAMP3⁺ DCs are core components of a specialized immune triad, comprising CD8⁺ T cells, CXCL13⁺ helper T cells, and regulatory DCs. This spatial organization, driven by IL-15 signaling, correlates with improved response to immune checkpoint blockade and may serve as a predictive marker for immunotherapy efficacy(51). Mechanistically, LAMP3⁺ DCs enhance anti-tumor immunity through IL-12 production and may modulate immune responses via PD-L1 expression, depending on their activation state. Similar immunoregulatory dynamics have been observed in hepatocellular carcinoma, where LAMP3⁺ DCs and CD4⁺ T cell niches support effective CD8⁺ T cell differentiation following PD-1 blockade(52). Furthermore, anti-CD40 therapy has been shown to stimulate the clonal expansion and functional enhancement of these DC subsets, offering a promising avenue for therapeutic intervention(29). Lymphoid T cells T cells are central mediators of anti-tumor immunity and a focal point of immunotherapy research. Over the past decade, the advent of ICIs has revealed the intricate regulation of T cell differentiation, governed by factors such as antigen exposure intensity and duration(53, 54). Nevertheless, strategies to reverse dysfunctional or exhausted T cell states remain elusive. ScRNA-seq and ST now offer unprecedented resolution to map T cell heterogeneity and guide therapeutic innovation(54, 55). Exhausted T Cell T-cell exhaustion is a state of functional impairment that occurs in both chronic infections and tumors and represents a significant challenge to sustaining effective T-cell responses in cancer therapy(56, 57). Exhausted T cells are typically characterized by co-expression of inhibitory receptors (e.g., TIGIT, PD-1, LAG3 and CXCL13) (58) . In CRC, exhausted T cells also exhibit high proliferative activity and express angiogenesis- and immunosuppression-associated genes (e.g., CCL15, CXCL1, CXCL12)(54, 59). Furthermore, these exhausted T cell populations accumulate to a greater extent in MSI-H patients, who tend to have a better response to immunotherapy. Exhaustion is not a binary state but rather a continuum. Specifically, during the development of exhausted T cells, their transcriptional profile shifts from high expression of effector molecules (e.g., IFNG , GZMB , GZMH , and PRF1 ) to the accumulation of exhaustion markers and dysregulated lipid metabolism(54, 55, 59). Given the continuous and heterogeneous nature of the T cell exhaustion process, it is challenging to comprehensively capture their dysfunctional state at a single time point. Recent scRNA-seq analyses introduced the concept of tumor-reactive CD8⁺ T cells (Ttr), which span from precursor exhausted (Texp) to terminally exhausted states(60). It is particularly encouraging that effective immune checkpoint blockade (ICB) therapy can partially reverse the process of Ttr cell exhaustion and reactivate their anti-tumor function. In particular, the baseline infiltration level of this subpopulations can be potential predictors of disease progression and outcome. Notably, recent studies have shown that reversing the reprogramming of lipid metabolism in failing T cells restores their cytotoxicity, and transcription factors such as PPARG and SREBF2 are expected to be potential therapeutic targets(61). In the intestinal mucosa, tissue-resident memory T cells (TRMs), another subset of exhausted T cells, have garnered particular interest. These cells show high expression of tissue-residency markers (e.g., CD69, CD103 ) and immune checkpoint molecule PD-1 (62). Unlike conventional exhausted T cells (CD8 + Tex), TRMs still recover their immune function upon re-encountering the corresponding antigen, thus mediating a rapid immune response. The characteristics of TRMs in colorectal cancer have been associated with favorable clinical prognosis and highly effective therapeutic outcomes(23). Interestingly, a recent study identified a subset of CD8 + T cells expressing GZMK , but not GZMB . These GZMK + CD8 + T cells exhibit minimal or no expression of typical exhaustion markers and have been shown to exert anti-tumor effects in CRC and other cancer types. This underscores the functional and origin-related heterogeneity within exhausted T cell populations and highlights the complexity of T cell-mediated immunity in cancer. Regulatory T Cells Tregs represent a key immunosuppressive population within TME, complementing the role of exhausted T cells in shaping immune evasion. The accumulation of Tregs in the TME has been associated with poor prognosis and reduced sensitivity to treatment. Furthermore, anti-PD-1 therapy has been shown to reduce Treg cell numbers in responsive tumors(63). The development and function of Tregs are primarily governed by the transcription factors FOXP3 and TCF-1 . In murine models of polyposis, deletion of Tcf-1 impairs CD4⁺ T cell polarization and promotes local inflammation, which exacerbates tumor progression in CRC(64). In CRC patients, the majority of Tregs infiltrating the TME originate from peripheral circulation, driven by chemokine receptors such as CCR4 , CCR7 and CCR8 (65). However, a distinct subset arises from local PRDM1⁺ TRM CD4⁺ T cells, highlighting a dual origin(63). Local inflammation within the tumor milieu provides critical cues for Treg activation. Mechanistically, Tregs suppress anti-tumor immunity through multiple pathways. These include the secretion of inhibitory cytokines (e.g., IL-10, TGF-β), and contact-dependent expression of immune checkpoint receptors, both of which contribute to the formation of an immunosuppressive barrier. A spatial and single-cell colocalization analysis identified that tumor cells at the adenoma–carcinoma interface exploit the MDK–SDC4 axis to engage Tregs and promote immune suppression. These molecular interactions facilitate the establishment of a permissive microenvironment for tumor growth(66). Interestingly, another study identified a unique niche, termed the Treg-mregDC-lymphatic niche, which is located around the lymphatic vessels. This niche is composed of mregDCs enriched in immune-modulatory molecules, which activate Tregs via chemotactic cues. Concurrently, this spatial unit impedes the trafficking of tumor antigens to draining mesenteric lymph nodes, thereby blunting the priming of adaptive anti-tumor responses. The presence of such spatially restricted niches illustrates how the localization of immune cells is integral to their function within the TME(67). Tissue-specific heterogeneity of Tregs is also evident when comparing primary CRC tumors with liver metastases. In primary intestinal tumors, both IL10⁺ and CTLA4⁺ Treg subpopulations coexist and exhibit an activated, tissue-resident phenotype. By contrast, metastatic lesions in the liver predominantly harbor only the CTLA4⁺ subset(33). Notably, the CTLA4⁺ Tregs from both sites share considerable TCR repertoire overlap with FOXP3⁺ Tregs circulating in the peripheral blood, indicating potential clonal relationships between tissue-infiltrating and circulating Tregs(66). In addition to classical FOXP3⁺ Tregs, CRC tissues also harbor a subset of FOXP3⁺ EOMES⁺ Tr1-like cells. These unconventional regulatory cells undergo clonal expansion in both primary and metastatic tumors. While they share immunosuppressive functions with conventional Tregs, they are distinguished by the expression of chitinase-like protein 3 (YM1), a defining marker of this subset. Importantly, their accumulation has been associated with advanced disease progression and resistance to immunotherapy, underscoring their potential as novel therapeutic targets in CRC(68). Conventional T cells Compared to Tregs，conventional T cells (Tconvs) demonstrate significantly greater heterogeneity in both gene expression and T cell receptor (TCR) repertoires across various cancer types through scRNA-seq. In CRC, scRNA-seq has identified a distinct subpopulation of Th1-like CD4 + T cells expressing CXCL13 , BHLHE40 and effector molecules such as IFNG and GZMB . These cells are notably enriched in MSI-H patients and have been associated with enhanced responsiveness to ICB, suggesting their potential utility as biomarkers for immunotherapy outcomes. Furthermore, a related subset of CXCL13⁺ Th1-like T cells characterized by high expression of IGFLR1 has been shown to promote the activation of CD4⁺ memory T cells and facilitate robust IFNG production. These findings indicate that IGFLR1 may serve as a promising therapeutic target to potentiate anti-tumor immune responses in CRC(65). Addressing T cell exhaustion and selectively eliminating immunosuppressive T cell subsets remain major obstacles in advancing effective anti-tumor immunity. Although single-cell RNA sequencing has provided unprecedented insights into the transcriptional states of tumor-infiltrating T cells, current spatial transcriptomic technologies have yet to fully resolve their spatial localization, functional states, and interactions within TME(67). Future studies should prioritize mapping the spatially resolved signaling networks and cell-cell interactions that contribute to T cell dysfunction. Such efforts will be critical for the development of precise, TME-aware immunotherapies aimed at restoring the durability and cytotoxic functionality of tumor-reactive T cells within the immunosuppressive landscape of CRC(57). B cells In recent years, ICB and chimeric antigen receptor T-cell (CAR-T) immunotherapy have garnered significant clinical attention, underscoring the critical role of T cells in anti-tumor immunity. Despite their success, these therapies have also highlighted the inherent limitations and vulnerabilities of T cell-mediated responses in combating cancer. Emerging evidence has shed light on the essential synergistic role of tumor-infiltrating B cells and plasma cells, collectively referred to as tumor-infiltrating B cells (TIBs)(69, 70). TIBs have been found to have diverse functions (Figure 2). As antigen-presenting cells (APCs), B cells interact with CD4+ T helper cells via the CD40-CD40LG axis, further activating cytotoxic T lymphocytes (CTLs) to target and eliminate tumor cells(28). This interaction is notably enhanced in treatment-responsive groups, and similar findings have been observed in mouse models. Beyond antigen presentation, TIBs secrete cytokines that promote the formation and maturation of tertiary lymphoid structures (TLSs) in tumors(27). Furthermore, TIBs play a pivotal role in orchestrating the initiation and maintenance of adaptive immunity through chemokine pairs such as CCL19-CCR7, CXCL13-CXCR5, and CCL28-CCR10(71). However, within tumor tissues, a simultaneous decrease in the number of TIBs, their proliferation index, and MHC II expression suggests a compromised antigen-presenting function and diminished immune recruitment, ultimately impairing their anti-tumor efficacy(27). On the other hand, following antigenic stimulation, B cells differentiate into plasma cells (PCs), which clonally expand and secrete IgG or IgA antibodies. These antibodies exert their effects through antibody-dependent cellular cytotoxicity (ADCC). Spatial transcriptomics has revealed significant heterogeneity within plasma cell subpopulations. Specifically, IgG + PCs are predominantly enriched in TLS-positive tumors, whereas IgA + PCs are more prevalent in TLS-negative tumors(71). Additionally, compared to normal tissues, tumor tissues show a higher ratio of IgG + to IgA + PCs(72). Notably, Erbin knockout reduces TGF-β secretion by IgA + PCs, hindering tumor cell migration and inhibiting STAT6-mediated PD1 expression. These findings are consistent with previous research in lung cancer, where Erbin was shown to promote tumor progression(28). In contrast to T cells, B-cell-targeted therapies remain in the early stages of development and face several challenges(73). Using scRNA-seq, researchers have now linked specific TIB clonotypes to distinct phenotypes, shedding light on their active antigen recognition and effector functions. Recent advances increasingly focus on integrating multi-omics technologies to achieve a more precise characterization of B-cell clonotypes, offering valuable insights for the future development of B-cell-based tumor immunotherapies and their potential clinical applications. Figure2. The role of B cells in tumor microenvironment. B cells express various immune-regulatory molecules and participate in anti-tumor immunity. Upon antigen stimulation, B cells proliferate, differentiate, and mature into PCs to exert anti-tumor functions. However, tumor-infiltrating B cells exhibit a reduced anti-tumor immune response, manifested as impaired proliferative capacity, along with compromised antigen presentation and recruitment abilities. Innate lymphoid cells Innate lymphoid cells (ILCs) are an important part of innate immunity, specifically located on the mucosal surface. While ILCs share a common lymphoid origin with B cells and T cells, they are inability to undergo RAG-mediated antigen receptor rearrangement, resulting in a lack of the specificity and diversity characteristic of TCR and BCR receptors(74, 75). Based on the transcriptional profile and cytokines production, ILCs are classified into five groups: natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer (LTi) cells. Their non-redundant roles in regulating intestinal physiology, immune tolerance, and mucosal health have been increasingly recognized (76, 77). ILC3s are the major subset in the intestinal mucosa and exhibit a wide range of homeostatic functions, including coordination of lymphoid tissue development, regulation of commensal bacteria, support of host defenses, modulation of adaptive immune responses, and promotion of tissue repair. However, significant dysregulation of ILC3 occurs in response to a variety of microbial exposures and human diseases, including inflammatory bowel disease (IBD), food allergies, chronic infections, and gastrointestinal cancers(78, 79). During different stages of tumor progression and the various phases of adaptive immune defenses, ILC3s show diverse molecular expression patterns and participate in CRC development (Figure 3). As innate analogs of Th17 cells, ILC3s can produce IL-22, which promotes intestinal epithelial regeneration, activates DNA damage response pathways, and restrains inflammation during early tumorigenesis. While chronic inflammation persists, ILC3s become a driver of cancer progression(75, 78). In addition to participating in inflammation regulation, ILC3s have been reported to regulate microbiota composition and support anti-tumor type 1 immunity through MHC molecules(80). In a DSS-induced colitis mouse model, loss of PD-1 disrupted ILC3 homeostasis, dysregulated gut microbiota composition, reduced STAT3 activation, and reshaped their metabolic programming. ILC3s have also been shown to express several co-stimulatory molecules (eg., OX40L, CTLA4 , and ICOS ), though their relevance in immunotherapy remains unclear(78). ILC3s are also crucial contributors to the formation and maturation of TLS within TME. These cells express TLS-inducing factors (eg., LTA, LTB, TNF ) and MHC II , and selectively localize to the tumor microenvironment, particularly at the invasive edges, as observed in AOM/DSS-induced mouse model. Interestingly, an inverse correlation was found between ILC3 abundance and TLS density, suggesting a complex spatial regulation that warrants further investigation(80, 81). ILC3s exhibit significant plasticity in TME. Emerging evidence suggests that TGF-β signaling can induce the transdifferentiation of intestinal ILC3s into regulatory ILCs (ILCregs), which acquire immunosuppressive functions by secreting cytokines such as interleukin-10 (IL-10), thereby facilitating tumor immune evasion. These ILCregs retain the expression of several ILC-associated surface markers (e.g., Cd25, Il-2r, Sca-1 , and Cd90 ), but notably lack key transcriptional markers characteristic of Tregs (e.g., Cd4 and Foxp3 ). Importantly, depletion of ILCregs has been shown to reverse their pro-tumorigenic effects, underscoring their potential as a therapeutic target. Additionally, in the Apc Min/+ mouse model, tumor-infiltrating ILC3s exhibit a dynamic phenotypic shift during CRC progression. Specifically, these cells gradually increase the expression of ILC1-related genes, while losing their characteristic ILC3 markers. This transition suggests a functional reprogramming of ILC3s in the evolving tumor microenvironment, potentially altering their role from protective to pro-tumorigenic as the disease advances(81). Subpopulations of ILC1-like and ILC2 cells have also been identified in human CRC tissues, which are notably absent in normal colonic mucosa, suggesting a tumor-associated expansion or differentiation of these subsets. Single-cell transcriptomic analysis of an AOM/DSS-induced inflammatory CRC mouse model further revealed that ILC1-like cells undergo a functional shift from an activated phenotype toward an immunosuppressive state as the disease progresses. ILC1-like cells induce T cell dysfunction through the expression of the immune checkpoint TIGIT . Concurrently, ILC2-like cells upregulate multiple genes associated with immune suppression (e.g., Hs3st1 , Ctla2a , Ltb4r1 , Pdcd1 , Tnfrsf18 , and Hes1 ) and promote tumor growth through the activation of the PTGDR2 and secretion of IL-13, indicating a potential role in shaping a tumor-permissive microenvironment(81) Notably, research in lung cancer has highlighted a subset of ILC2s that specifically express granzyme B (GZMB) and play a unique role in tumor progression(82, 83). A recent study also revealed that IL-33-activated ILC2s can induce the formation of TLS in pancreatic cancer. Furthermore, engineered recombinant IL-33 protein is shown to enhance anti-tumor immune responses, offering promising targets for future immunotherapies(84). As research in CRC continues to progress, single-cell and spatial transcriptomics will be essential in identifying novel ILC subsets with therapeutic potential. These technologies will enable the discovery of targetable ILC populations, ultimately paving the way for more precise and effective CRC treatments. Figure3. ILCs participate in tumor progression through multiple mechanisms. Under the stimulation of inflammatory cytokines such as IL-23 and IL-1β, ILC3s secrete inflammatory cytokines that can exert either anti-tumor or pro-tumor effects, depending on the stage of tumor progression. ILC3s also express immune-regulatory molecules, regulating adaptive immunity. Furthermore, under the influence of environmental factors, ILC3s exhibit functional plasticity, undergoing remodeling to various extents in the TME. Fibroblasts Tumor-associated fibroblasts (CAFs), a major stromal component of the TME, play multifaceted roles in CRC progression. These cells contribute to malignancy by promoting tumor proliferation, invasion, and therapeutic resistance through secretion of immunomodulatory factors, direct interaction with neighboring cells, and remodeling of the extracellular matrix (ECM) (Figure 4)(51, 85, 86). Advancements in scRNA-seq, have enabled the identification of canonical CAF markers, including FAP , VCAN , and COL1A2 (87). Recent studies have revealed critical functions of CAFs in promoting tumor cell proliferation through the secretion of a variety of factors, such as TGF-β, NRG1, HGF, GDF15, AREG, PDGFRA, and BMP2 (48). Complementing these transcriptomic studies, spatial transcriptomics has provided crucial insights into the tissue localization and functional specialization of CAF subpopulations. For instance, CXCL12 + fibroblasts, expressing mesenchymal markers (e.g., DCN , SLIT2 , and CXCL12 ), are predominantly located in the lamina propria. Notably, CAFs can recruit CD8 + Effector Memory T Cells through the CXCL12-CXCR4 axis, which could be enhanced by ICB or NAC(88). In addition, CCL2 + fibroblasts, marked by genes like F3 , WNT5A , BMP2 , POSTN , and HSD17B2 , localize near the epithelial layer(28). Additional spatial analyses have identified CAF subsets residing at the luminal margin (LM) and near dilated blood vessels promote angiogenesis and inflammation through the secretion VEGFA and NRG1 , particularly in MSI-H CRC tumors. Additionally, a distinct CAF population enriched at the LM edge, characterized by the expression of MCAM , can modulate the generation of CXCL13 + CD8 T cells through the Notch signaling pathway(48). These treatment responses-related CAFs populations become activated and amplified under the influence of prostaglandin (PG) secreted by adjacent endothelial cells in TME(88). CAFs also play a significant role in promoting T cell dysfunction. Emerging evidence suggests that a subset of MHC + CAFs express antigen-presentation-related genes (e.g., MHC-II , CD74 , TAP1 ), yet lacks the co-stimulatory molecules (e.g., CD40 , CD80 , and CD86 ) essential for full T cell activation. This incomplete antigen presentation may contribute to T cell anergy and immune evasion(27). Moreover, CAFs have been shown to express a range of immune checkpoint ligands that actively induce T cell exhaustion. Notably, these molecules exhibit subtype-specific expression patterns among CAF populations. For instance, the TIGIT–NECTIN2 axis is shared between CAFS1⁺ CAFs and CD8⁺ T cells, while the LGALS9–HAVCR2 signaling pathway is specifically enriched in ecm-myCAF subtypes and GZMB⁺ CD8⁺ T cells(89). Given their immunosuppressive and tumor-supportive roles, CAFs have emerged as promising therapeutic targets in CRC. Current strategies focus on eliminating immunosuppressive CAFs or inhibiting CAF-derived factors in order to reprogram the CAF phenotype toward a more quiescent or tumor-restraining state. However, several challenges remain, including the absence of specific CAF biomarkers and the high degree of functional and spatial heterogeneity across CAF subsets(90). Integrating scRNA-seq and spatial transcriptomics will be critical to resolve specific CAF population functions and guide the development of more precise and effective stromal-targeting therapies. Figure4. Major functions of tumor-infiltrating fibroblasts in TME. ScRNA-seq revealed the diversity of CAF phenotypes, characterized by their involvement in various processes: supporting malignant epithelial growth, angiogenesis, immunosuppression, and participation in the epithelial-mesenchymal transition (EMT) process, contributing to tumor cell invasion. Conclusion Despite substantial progress in targeted therapies and immunotherapies, significant challenges persist in the treatment of CRC, primarily due to the inherent heterogeneity of tumors and the complexity of the TME. These factors continue to limit the effectiveness of current therapeutic strategies and underscore the need for more refined approaches. Over the past decade, the advent of scRNA-seq and spatial transcriptomics has revolutionized our understanding of the TME(91-94), By integrating the high-resolution transcriptomic profiling of scRNA-seq with the spatial contextualization offered by spatial transcriptomics, researchers have been able to precisely localize distinct cell populations and map intercellular interactions within the tumor niche. This dual approach has overcome limitations of earlier methodologies and provided unparalleled insights into the cellular composition, functional heterogeneity, and regulatory dynamics of tumor, immune, and stromal cells. Together, these technologies have significantly accelerated the discovery of novel biomarkers and informed the development of more effective, personalized cancer therapies. Nonetheless, spatial transcriptomics remains an evolving discipline. Its broader adoption has been constrained by high costs and the limited resolution of many existing platforms, which often fall short in capturing the intricate cell–cell interactions within the TME at true single-cell resolution. Recent advances, particularly the release of 10X Visium HD in 2024, represent a major technological leap—enabling unprecedented spatial resolution and transcriptomic depth at the single-cell level. This breakthrough holds transformative potential for spatial biology, as it facilitates the fine-grained dissection of regulatory networks and functional architectures within the tumor microenvironment. In parallel, the incorporation of nuclear recognition-based cell segmentation has markedly improved the delineation of cellular boundaries, allowing for more accurate reconstruction of intercellular communication pathways and spatial tissue organization. As these innovations continue to mature, spatial transcriptomics is poised to become a cornerstone technology in cancer research—crucial for unraveling the mechanisms underlying immune evasion, therapeutic resistance, and for guiding the rational development of next-generation precision immunotherapies. Looking forward, future efforts will not only expand the catalog of cellular diversity in the TME but also aim to capture its temporal dynamics across multiple stages of disease progression. These longitudinal, high-resolution datasets will be essential for building a comprehensive and dynamic atlas of the TME, ultimately guiding the development of precision oncology strategies and uncovering novel therapeutic vulnerabilities in CRC and beyond. Acknowledgements Not applicable. Author contributions YG conceived the idea of compiling this review. SZ prepared the figures. All authors performed the literature search, wrote the manuscript, and approved the submission of the article. Funding This work was supported by the National Key R&D Program of China (2021YFA0805703), the National Natural Science Foundation of China (82370107), and Beijing Nova Program (20230484407). Availability of data and materials Not applicable. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References 1. 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Keywords comparative immunology/evolution human tumor immunity Authors Affiliations Shuomin Zhang 1st Medical Center of Chinese PLA General Hospital View all articles by this author Qingfeng Fu 1st Medical Center of Chinese PLA General Hospital View all articles by this author Xiaotong Yuan 1st Medical Center of Chinese PLA General Hospital View all articles by this author Sijun Wang 1st Medical Center of Chinese PLA General Hospital View all articles by this author Chao Liu Peking University First Hospital View all articles by this author Chaojun Zhang 1st Medical Center of Chinese PLA General Hospital View all articles by this author Bing Liu Chinese PLA General Hospital Fifth Medical Center South Campus View all articles by this author Gong Yandong 0000-0003-3971-1478 [email protected] Chinese PLA General Hospital Fifth Medical Center South Campus View all articles by this author Metrics & Citations Metrics Article Usage 294 views 193 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shuomin Zhang, Qingfeng Fu, Xiaotong Yuan, et al. 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