Colorectal cancer (CRC) is one of the most prevalent malignant tumors of the digestive tract and the second leading cause of cancer-related mortality worldwide (1). Both its incidence and mortality rates are rising annually (2), In 2020, it was estimated that there were over 1.9 million new cases of CRC and approximately 930,000 deaths attributed to the disease. The burden of CRC is projected to rise significantly, with an anticipated increase to 3.2 million new cases and 1.6 million deaths by the year 2040. Most of these cases are expected to occur in countries classified as having a high or very high Human Development Index (3), posing a significant challenge to public health. Early-stage CRC is typically managed with surgical resection combined with chemotherapy. However, due to its high malignancy and substantial risks of metastasis and recurrence, traditional treatment outcomes are often unsatisfactory (4). Identifying new therapeutic targets is, therefore, crucial for improving the prognosis of patients with CRC.

Current research has identified that the mechanisms underlying colorectal cancer (CRC) are diverse, including factors such as gut microbiota (5, 6) and epigenetic alterations (7, 8). However, the complex molecular mechanisms remain to be further elucidated. There is substantial evidence indicating that immunoediting plays a significant role in tumors (9, 10). In CRC patients, immune balance is disrupted (11), and the interactions between tumor cells and the tumor microenvironment (TME) facilitate further tumor progression (12, 13). Macrophages, crucial immune components of the TME, play a significant role in recognizing and phagocytosing pathogens as well as necrotic cells, thereby contributing to immune regulation and maintaining tissue homeostasis (14, 15). Under various physiological and pathological conditions, macrophages can differentiate into distinct phenotypes based on their gene expression profiles. These diverse subpopulations coordinate tumor progression, metastasis, and invasion by secreting specific cytokines and chemokines (16). Macrophage heterogeneity offers new insights into the diagnosis, treatment, and prognosis of CRC.

Tumor-associated macrophages (TAMs) are among the most abundant immune cells in tumor tissues. Traditionally, macrophages have been classified into two subtypes: M1 (classically activated or proinflammatory) and M2 (alternatively activated or anti-inflammatory) (17). The M1 subtype is characterized by tumoricidal properties, while the M2 macrophages exhibit anti-inflammatory characteristics that can indirectly promote tumor growth (18, 19). Although this binary classification has been widely utilized in the past, technological advancements have expanded our understanding of the complex state of the TME. Consequently, this dichotomy fails to accurately capture the diversity of macrophage phenotypes present in vivo, particularly within complex TME contexts (20–22). Furthermore, the gene expression profiles associated with these two phenotypes are not entirely oppositional (23). With advances in single-cell RNA sequencing (scRNA-seq), researchers can now identify cellular subpopulations with greater precision. This technology enables the discovery of unique macrophage subpopulations by capturing transcriptomic variations in macrophages under different pathological conditions and disease stages, thereby distinguishing various functional subsets (24, 25). Additionally, the integration of scRNA-seq with spatial transcriptomics has enhanced the ability to detect finer details within heterogeneous cell populations (26, 27). This article focuses on non-classical classifications of TAMs identified through single-cell omics research on colorectal malignancies and explores their potential roles in tumorigenesis and development.

Macrophages are phagocytic cells widely distributed in various human tissues and organs. They play crucial roles in recognizing, phagocytosing, and degrading apoptotic cells, tissue debris, and pathogens, thereby contributing significantly to immune responses. It is currently understood that macrophages within tissues have two primary sources (28): during prenatal development, they originate from yolk sac progenitors or hematopoietic stem cells. These progenitor cells retain proliferative potential and can undergo self-renewal. During organ development, they differentiate into specific tissue subpopulations (29, 30), such as microglia in the central nervous system and Kupffer cells in the liver. This differentiation establishes stable communication and connections with specialized tissue cells (31, 32). In contrast, adult macrophages arise from infiltrating monocytes and are involved in a wide range of physiological and pathological processes within the body. They play central roles in various processes, including inflammation regulation and tissue repair (33). Both types of macrophages can coexist within specific tissues, and their respective abundance reflects the characteristics and history of these tissues (34).

Macrophages are highly plastic cells (35) and the generation of TAM is multifaceted. Chemokines and cytokines, such as monocyte chemoattractant protein-1 (MCP-1) and colony-stimulating factor 1 (CSF1), along with complement cascade products, are the primary determinants of the recruitment and localization of macrophages within tumors. Under the influence of these factors, peripheral blood monocytes are locally recruited and differentiate into macrophages, forming TAMs alongside resident tissue macrophages (36–39). Proinflammatory cytokines produced by tumor cells contribute to the recruitment of immune cells and promote macrophage polarization during the early stages of carcinogenesis. Proper activation of macrophages can exert certain anti-tumor effects by enhancing phagocytosis and cytotoxicity against tumor cells. Additionally, macrophages can induce extracellular killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC) (40).However, it is notable that macrophages in the TME do not appear to exert protective effects; rather, a substantial body of research indicates that they seem to promote tumor progression in the vast majority of circumstances (41, 42).

Based on the functions of TAM, two activation phenotypes, M1 and M2, have been studied extensively. M1 macrophages respond to various stimuli, such as bacteria, interferon-γ (IFN-γ), lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α). They exhibit enhanced antigen presentation and complement-mediated phagocytosis, contributing to the inflammatory response (43, 44). Additionally, M1 macrophages activate or recruit adaptive immune cells and are capable of phagocytosing and killing tumor cell (45). In this stage, M1 macrophages specifically produce cytotoxic substances such as nitric oxide (NO) and reactive oxygen species (ROS), which can damage tumor cells, participate in phagocytosis, and release proinflammatory cytokines. This process further stimulates anti-tumor immunity (46–48). Moreover, tumor cells induce the polarization of macrophages into M2-type TAMs through various mechanisms, including cytokine production, metabolic abnormalities, and hypoxia (49–54). M2 macrophages can be categorized into the subtypes M2a, M2b, M2c, and M2d based on their activation factors (55). Subtypes M2a and M2b primarily exert immunoregulatory functions by supporting T helper cell 2 (Th2)-mediated responses, whereas subtypes M2c and M2d are involved in immune suppression and tissue remodeling (56). TAMs of the M2 phenotype predominantly inhibit tumor immune responses by secreting immunosuppressive factors, thereby disrupting the immune barrier and leading to an imbalance in defense mechanisms. Additionally, they secrete growth factors such as transforming growth factor (TGF) and vascular endothelial growth factor (VEGF), which promote tumor proliferation and extracellular matrix (ECM) remodeling. M2-type TAMs play a significant role in promoting angiogenesis, tissue repair, and tumor progression (47).

In the TME, TAMs typically exhibit an M2 phenotype, which facilitates tumor malignancy either by directly enhancing malignant biological behaviors or by engaging in crosstalk with immune cells present in the TME to promote tumor progression (57). However, increasing evidence suggests that the polarization state of macrophages is far more complex than the simplistic M1/M2 dichotomy.

The elevation of TAMs is associated with poor prognoses in various cancers (16, 58, 59). M2 TAMs can further promote tumor progression (14, 57, 60, 61). This correlation primarily arises from their tumor-promoting characteristics, which include facilitation of angiogenesis, immune suppression, and promotion of cancer cell dissemination. TAMs regulate angiogenesis in the TME by releasing pro-angiogenic factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), chemokines, and antiangiogenic substances such as thrombospondin-1 (TSP-1). Consequently, TAMs play a complex role in vascular formation (42, 62–64). Moreover, TAMs promote tumor progression by suppressing immune responses (65). They achieve this by expressing T cell immune checkpoint ligands that attract regulatory T cells (Tregs) and by producing immunosuppressive molecules such as transforming growth factor (TGF) and interleukin-10 (IL-10), which impair the function of cytotoxic T cells (CTLs) and natural killer (NK) cells (66). TAMs inhibit the recruitment and function of T-cells via cytokines, surface immune checkpoint ligands, and exosomes. They play a crucial role in regulating programmed death receptor-1 (PD-1) and its ligand PD-L1-mediated immunosuppression (67). Additionally, TAMs can suppress T cells infiltration into tumor tissues, thereby promoting tumor growth (39). Furthermore, TAMs secrete matrix metalloproteinase-9 (MMP-9), which induces epithelial-mesenchymal transition (EMT) via the transcription factor Snail, enhancing tumor invasiveness (68). Other studies have shown that TAMs co-localize with tumor cells undergoing EMT (69); they also produce chemokines that facilitate distant tumor metastasis (70). The recruitment of cancer-associated fibroblasts (CAFs) by TAMs is associated with extracellular matrix remodeling, promoting tumor invasion (71, 72). In summary, TAMs exhibit dynamic roles throughout tumor development and engage in complex interactions within the TME.

TAMs exhibit significant heterogeneity not only among different patients with cancer but also within various malignant lesions of the same patient and even within specific tumor sites. The intratumoral heterogeneity of macrophages may pose challenges for treatment, yet it also underscores the complexity of their functions (72–77). Due to its low resolution, traditional bulk RNA sequencing fails to accurately represent subtle variations in cell populations within the TME (78, 79). In contrast, single-cell RNA sequencing (scRNA-seq) allows for a comprehensive and detailed characterization of cellular diversity and heterogeneity at the single-cell level (78), thereby providing a more nuanced perspective on the complexity of TAMs. The traditional M1/M2 classification of macrophages faces certain challenges, primarily for the following reasons: 1. Diversity of Macrophages: Macrophages exhibit a high degree of heterogeneity in vivo, and their functions and phenotypes are influenced by various factors and complex interactions with the microenvironment (80–82). The relatively simple polarization into M1 and M2 macrophages may not fully encompass the intricate spectrum of macrophage subtypes present in the body. For instance, studies have identified other macrophage subtypes, such as SPP1+ TAMs (83–85) and FOLR2+ tissue-resident macrophages (TRMs) (86, 87), which include populations that are significant for prognosis in patients with cancer but do not conform to the classical M1/M2 polarization paradigm. 2. Inaccuracy of Single Markers: The classification of M1 and M2 macrophages often relies on specific markers such as CD68, CD163, and CXCL10. However, the expression of these markers may overlap between different subtypes, leading to inaccuracies in classification; thus, it is essential not to consider these markers in isolation (88–90). Furthermore, variations in the markers used and the experimental conditions across different studies may further impact the classification accuracy. 3. Misunderstanding of the Dynamic Balance: The simplistic classification of macrophages into M1 and M2 types may lead to misconceptions regarding the dynamic balance of macrophages in vivo. Macrophages undergo phenotypic transitions from one subtype to another in response to various stimuli. This dynamic change is crucial for inflammatory responses and immune regulation; however, a binary classification approach fails to accurately capture the true state of macrophages (91, 92). Research has evolved from straightforward and convenient classical M1 and M2 models to more complex models that reflect the diverse functional spectra of macrophages (93, 94). This increasing level of detail necessitates the distinction between the subtypes and functions of TAMs from the perspective of single-cell sequencing.

Ma et al. (94) summarized seven subgroups of TAMs identified in various pan-cancer single-cell studies (23, 94) and classified them based on their functions, signatures, and enriched pathways. These subgroups include interferon-primed TAMs (IFN-TAMs), immune regulatory TAMs (Reg-TAMs), inflammatory cytokine-enriched TAMs (Inflam-TAMs), lipid-associated TAMs (LA-TAMs), pro-angiogenic TAMs (Angio-TAMs), RTM-like TAMs (RTM-TAMs), and proliferating TAMs (Prolif-TAMs). Notably, functional overlaps and shared characteristics are present among these seven subtypes. In subsequent sections, we outline the unique phenotypes and functions of specific TAM subpopulations discovered in single-cell research on CRC. Meanwhile, more concise information can be more readily accessed in Table 1 .

CRC is one of the most prevalent malignant tumors worldwide, with an increasing annual incidence rate and a complex pathogenesis.

Based on the role of TAMs in cancer and their high plasticity, several therapeutic strategies targeting TAMs have been developed. For example, the reduction and depletion of TAMs are influenced by CSF1.Clinically, AMG 820, a fully human anti-CSF1 receptor antibody, inhibits the binding of its ligand CSF1 to interleukin 34 (IL-34), thereby preventing subsequent ligand-mediated receptor activation. This results in a decrease in the accumulation of immunosuppressive TAMs in solid tumors (155). Chemokine ligand 2 (CCL2) recruits monocytes expressing chemokine receptor 2 (CCR2) from the peripheral blood to the tumor site, where they further mature into TAMs (156). Therapeutic blockade of the CCL2/CCR2 axis suppresses the recruitment, infiltration, and M2 polarization of inflammatory monocytes mediated by TAMs. This leads to the reversal of immune suppression within the TME and activates anti-tumor CD8+ T cell responses (157). Clodronate is a chemical agent that induces macrophage depletion and significantly reduces TAMs in the TME, thereby diminishing their pro-angiogenic effects (158). The triggering receptor expressed on myeloid cell 2 (TREM2) represents a potential therapeutic target for modulating immunosuppressive TAMs (159). The use of TREM2 inhibitors and similar tools selectively eliminates immunosuppressive macrophages, restores normal immune responses, and inhibits tumor growth (160). Additionally, TAMs can be repolarized into M1-like macrophages to regain their anti-tumor properties. Toll-like receptors (TLRs) are crucial pathogen-recognition receptors expressed by immune cells. The utilization of TLR agonists is an effective strategy for rapidly activating both innate and adaptive immunity (161). Following TLR-3 stimulation, the upregulation of M1-specific markers, such as major histocompatibility complex class II molecules (MHC II), and co-stimulatory molecules, such as CD86, CD80, and CD40, is observed in M2 macrophages. In contrast, there is a decrease in the expression of M2 markers, including CD206, Tim-3, and proinflammatory cytokines. This phenotypic shift effectively inhibits tumor growth (162). In addition, TAMs exhibit anti-tumor properties that can be leveraged. For instance, using SIRP1α-CD47 inhibitors and anti-MS4A4A antibodies, certain unique TAM subpopulations can be utilized to impede tumor growth or restore T cell-mediated anti-tumor immunity (147, 163). Furthermore, TAMs express PD-1, and the expression level of PD-1 is negatively correlated with the phagocytic capacity of macrophages. Blocking the PD-1/PD-L1 pathway can enhance macrophage-mediated phagocytosis of tumor cells and inhibit tumor progression (164). Macrophages are critical components of the complex interplay between the immune system and tumors; thus, they represent significant therapeutic targets for cancer prevention and treatment. In response to tumor stimuli, TAMs undergo M2-like polarization, promoting tumor growth and severely affecting prognosis. Therefore, the development and application of drugs based on TAMs and the TME are of substantial significance (36).

The article discusses several specific subpopulations of tumor-associated macrophages (TAMs) that have been the focus of various treatment-related studies. One study (165) demonstrated that the knockout of SPP1 significantly inhibited the infiltration of M2 TAMs in xenograft tumors, while RNA aptamer-based blockade of SPP1 markedly suppressed tumor growth and M2 TAM infiltration in mouse models. Additionally, literature reports (166)indicate that a certain compound can downregulate SPP1 both in vitro and in vivo, leading to tumor remission in different mouse models of cancer. Research by Zhang et al. (167) demonstrated that targeting C1Q+ TAMs effectively alleviates immune suppression and enhances the efficacy of immune checkpoint blockade therapy. These findings suggest that specific subpopulations of macrophages may serve as potential targets for therapeutic intervention, thereby presenting new opportunities for cancer treatment.

TAMs, which differentiate from monocytes, play a crucial role in tumor immunity in CRC. The M1/M2 polarization subtypes provide a foundational framework for summarizing certain functions of macrophages; however, an increasing body of research (94, 168) has demonstrated the complexity of TAM functions and phenotypes. The advent of technologies such as single-cell RNA sequencing (scRNA-seq) has further substantiated these findings at a more precise level, emphasizing the necessity of leveraging new techniques to enhance existing molecular identification systems based on previous knowledge. However, given the limited number of single-cell studies focusing on specific TAM subpopulations, the relationships among these unique subtypes still require further investigation. Additionally, some TAM subtypes identified in previous studies are challenging to classify within the existing functional subsets based on their characteristics, transcription factors, enriched pathways, and predicted functions. For instance, in a study by Bao et al. (169), a distinct population of S100A9+ macrophages was identified, leading to an immunosuppressive phenotype cluster described as immune tolerance in CRC at the single-cell level. However, additional information is needed for a more accurate characterization of this subgroup. In another study integrating CRC tissues with adjacent normal tissues (170), seven specific macrophage subpopulations were identified. Among these, IDO1+ TAMs were the only macrophage subtype predominantly present in CRC tissues. It has been observed that IDO1+ macrophages interact with various cell types; however, a clear understanding of these interactions is still lacking. Several large-scale pan-cancer studies utilizing single-cell analyses (171, 172) have identified unique subpopulations. For example, MMP9+ TAMs may play a role in tumor tissue remodeling (173–175), CX3CR1+ TAMs may exhibit unique phagocytic patterns (176, 177), ECM-associated TAMs (ECMMac) involved in extracellular matrix remodeling are upregulated (178, 179), and SPP1AREGMac exhibit distinct distribution within tumor tissues. This evidence further underscores that the polarization of macrophages into M1 and M2 phenotypes is not the sole critical factor in cancer. Therefore, the functional diversity of macrophages should be comprehensively considered. With sufficient research, it may become possible to establish a novel molecular classification system for CRC based on TAM functionality. Such an approach would allow for a more detailed characterization of TAM heterogeneity, thereby facilitating the development of targeted therapies for specific TAM populations in CRC.