Recent data utilizing specific fate mapping technologies have provided evidence for the embryonic origin of tissue macrophages (26–28). These macrophages possess self-renewal and proliferation capacity. In most normal tissues, TRMs are mainly embryonic macrophages (29), which are necessary for the development of tissues and organs. Notably, solid tumors have requirements similar to those of developing organs and tissues in forming complex structures (30). Thus, macrophages in tumor tissues can also be derived from embryo-derived TRMs (31, 32). These TAMs from embryo-derived TRMs can contribute to the occurrence and development of cancers (33, 34).

In solid tumors, monocyte-derived macrophages (MDMs) are recruited by cytokines and chemokines and then polarized into TAMs, which are universally heterogeneous (13–15). MDMs can also self-renew and proliferate (35). For example, the proliferation of TAMs can be induced in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) in liver cancer tissues (36). These monocyte-derived TAMs can switch from one phenotype to another. They display remarkable plasticity within the TME (37), which can result in distinct subpopulations. Tumor-associated factors, such as tumor hypoxia in the TME, contribute to the heterogeneity of monocyte-derived TAMs. Thus, monocyte-derived TAMs may consist of multiple subpopulations generated through distinct developmental pathways.

In addition, there is also a minor splenic contribution to monocyte-derived TAMs (38, 39). Although the bone marrow is the primary hematopoietic tissue and monocyte reservoir, the spleen is also an identified reservoir of monocytes, which can play a significant role in the inflammatory response (40). Thus, the spleen is also an important extra-medullary site that can continuously supply growing tumors with monocytes (39).

SPP1 ⁺TAM subpopulation is identified by specific expression of a core gene SPP1. SPP1 ⁺ TAM subpopulation also often expresses the following genes such as FN1, IL1RN, and other TAM genes IDO, Mrc1(CD206), PD-L1, PD-L2, CD68 and CD163. Notably, TAM associated CD68 (46) and/or CD206 (47) genes can be found only in the SPP1 ⁺ TAM subpopulation of some solid tumors but not in all solid tumors (48). In addition, SPP1⁺CD206 ⁺ TAM subpopulation also produces epithelial growth factor (EGF) (49).

SPP1 ⁺TAM subpopulation was initially found in colorectal cancers (CRC) (43), and later in lung and breast cancers (50, 51). In human CRCs, SPP1 ⁺ TAM subpopulations not only showed specific expression of SPP1 but also MARCO and VEGFA (43, 52, 53). SPP1 with glycolysis genes (GAPDH, ENO1, LDHA, ALDOA, and TPI1) was also expressed in TAMs in non-small cell lung cancer (NSCLC) (54). In lung squamous cell carcinoma, there also existed subpopulation of SPP1 ⁺TAMs. SPP1 ⁺ macrophages were significantly increased in the tumor microenvironment, which was related to the poor prognosis of patients with lung squamous cell carcinoma. SPP1 ⁺ macrophages (53) were also found in hepatocellular carcinoma. Macrophage subsets that express SPP1, TREM2 and FN1 anti-inflammatory TAMs were found in breast cancer (8, 55, 56). The patients with SPP1 ^low TAMs had the best prognosis for cervical cancer, whereas the worst prognosis appeared in patients with SPP1 ^high TAMs (57). Multi-omics analysis also revealed the distinct clinical significance of SPP1 ⁺ TAMs in cervical cancer (57). SPP1 ⁺, TAM subpopulations could also be detected in rental cancer. and in pancreatic cancer (58).

The core gene signature of C1Q ⁺TAM subpopulation is the expression of C1QA/B/C. C1Q ⁺ TAMs are also characterized by the expression PD-L1, PD-L2, HAVCR2, LGALS9, and CEACAM1 (52). PD-L1 and PD-L2, which are checkpoint molecules, can prevent CD8⁺ and CD4⁺ Th1 immunity (59). C1Q ⁺ TAMs can also highly express TREM2, MERTK, CD80 (43, 57, 60), SLC40A1, GPNMB (43, 52, 61) and other genes in TAMs, such as IDO, Mrc1 (CD206), CD68 and CD163. In addition, this population of macrophages can also produce pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF).

A subpopulation of TAMs expressing C1QA/B/C is present in multiple cancers, such as colorectal cancer (43), NSCLC (62), liver cancer (44), lung cancer (63), rental cancer (60, 64, 65), breast cancer (8, 50), pancreatic cancer (58) and cervical cancer (57). Single-cell and spatial analyses revealed immunosuppressive phenotypes of C1Q ⁺ APOE ⁺ TAMs in CRCs (66). The patients with C1QC^high TAMs had the best prognosis for cervical cancer, whereas the worst prognosis could appear in patients with C1QC^low TAMs (57).

Another TAM subset, characterized by high expression of the core gene FCN1, is also identified in cancer tissues. This subpopulation of macrophages is a precursors of C1Q⁺ TAM (43, 44). They are derived from inflammatory monocytes. Indeed, tumor-enriched FCN1+ monocyte-like cells showed a high similarity to blood CD14⁺ monocytes, representing a monocyte population migrating into tumors and harboring a tumor-specific transcriptional program (43, 45).

FCN1⁺ TAM subpopulation was also identified in human multiple solid cancers such as NSCLC (62), liver (44, 53), breast cancer (8), lung (45), CRCs (43) and pancreatic cancer (58). In human NSCLC, FCN1 ⁺ macrophages, together with typical FYN ⁺ and STAT1 ⁺ macrophages, expressed genes related to increased inflammatory function (62).

This population is characterized by the expression of a core gene CCL18. CCL18 is associated with the immunosuppressive nature of the tumor microenvironment. CCL18 ⁺ TAM subpopulation is also an important element in cancer immune evasion (67). Notably, both CCL18 ⁺ and SPP1 ⁺ macrophages highly express immunosuppressive M2-like genes (62), implying that they are the same type of TAMs. CCL18 ⁺ TAMs should be terminal SPP1⁺ macrophages.

CCL18 ⁺ TAM subpopulations can be found in many tumors such as NSCLC (62), breast cancer (68), CRCs (69, 70) hepatocellular carcinoma (53, 71), thyroid cancer (72) and intrahepatic cholangiocarcinoma (53).

Thus, four TAM subpopulations with different core genes such as C1Q ⁺, SPP1 ⁺, FCN ⁺ and CCL8⁺ TAM have been identified in distinct solid tumors by scRNA-seq data. However, there mainly are two TAM subpopulations in solid tumors, including C1Q ⁺ and SPP1 ⁺ TAMs (43, 57, 73).

Notably, there also exist other classification methods based on single cell RNA sequences. For example, one study separated TAM into seven TAM subsets in tumor tissues, including inhibin beta A chain (INHBA)⁺ TAMs, complement C1q subcomponent subunit C (C1QC)⁺ TAMs, ubiquitin like protein ISG15 (ISG15)⁺ TAMs, NACHT, LRR and PYD domains containing protein 3 (NLRP3)⁺ TAMs, LYVE1⁺ TAMs, and sphingosine- 1-phosphate phosphatase 1 (SPP1)⁺ TAMs (74). TAMs were also separated into five TAM subsets in another study, including transcription factor HES-1 (HES1)⁺, complement component 1q (C1Q)^hi, triggering receptor expressed on myeloid cells 2 (TREM2)⁺, IL4I1⁺ and proliferating TAMs (75). Other also showed seven TAM subsets in tumor tissues, including 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), resident-tissue macrophage-like TAMs (RTM-TAMs) and proliferating TAMs (prolif-TAMs) (76).

The number of SPP1 ⁺ macrophages significantly increases in the tumor microenvironment, which is correlated with poor prognosis (46, 79–85). SPP1 ⁺ TAM subpopulation has multiple roles in tumor development such as tumor metastasis (62, 86–91), angiogenesis (92–94), tumor stem cell activation and immunosuppression (95).

Tumor metastasis includes multiple steps, such as epithelial-to-mesenchymal transition, trans-endothelial migration, extracellular matrix remodeling, and formation of pre-metastatic niches. In the patients with tumors, the enrichment SPP1 ⁺ TAMs was associated with worse overall survival (86, 96). SPP1 ⁺ macrophages from tumor tissues showed high expression of MMP9, MMP12, MMP14, and MMP19, which could contribute to the degradation of the basement membrane for the invasion of tumor cells (82). There had also an interaction between SPP1 and CD44 in SPP1 ⁺ macrophages to promote metastasis. The interaction between SPP1 ⁺ macrophages and tumor epithelial cells activated downstream genes to mediate the activation of NF-κB, PI3K/Akt, VEGF, uPA, and MMPs, which could promote endothelial cell proliferation. The interaction of SPP1 ⁺ macrophages with FAP ⁺ fibroblasts also appeared in colorectal cancer to promote metastasis (97). In SPP1 ⁺ macrophages, the main metabolic pathway was glycolysis, which could promote tumor metastasis via angiogenesis and matrix remodeling (62). SPP1 ⁺ macrophages were predominant in liver metastasis (86) and also potential biomarker for early lymph node metastasis (89). Colorectal cancer metastases in the liver could establish immunosuppressive spatial networking between tumor-associated SPP1 ⁺ macrophages and fibroblasts, which supported colorectal cancer growth in the immunosuppressed metastatic niche in the liver (98). Studies also found that SPP1 ⁺macrophages were metastasis accelerators of colorectal cancer (99). They were often found in mesenteric lymph node (MLN) with metastases. SPP1 ⁺ macrophages also promoted prostate tumor progression by increasing the incidence of prostate intraepithelial neoplasia (100).

For tumor angiogenesis, SPP1⁺ TAMs can be acted as “angiogenic switch.” They produce cytokines, such as VEGFA, platelet-derived growth factor (PDGF) and angiopoietin, to promote angiogenesis (93, 101). SPP1⁺ TAMs with high angiogenesis scores showed the expression of genes related to angiogenesis. There was also a strong enrichment of tumor angiogenesis and tumor vasculature pathways in SPP1 ⁺ TAMs (43). Under the influence of hypoxia, SPP1⁺ TAMs could develop a pro-angiogenic phenotype by directly upregulating angiogenic molecules, such as VEGF-A (102) and angiogenic modulators, such as matrix metalloproteinase (MMP)7 (103). These cells could also interact with endothelial cells to promote angiogenic functions (94).

Cancer stem cells (CSCs) are subsets of tumor cells that play a key role in tumorigenesis. SPP1⁺ TAMs could regulate CSC activation (104–106). For example, SPP1⁺ TAMs promoted tumor growth and proliferation by secreting collagen (107) and insulin-like growth factor-1 (IGF-1) (108).

SPP1 ⁺ TAMs are also implicated in T cell suppression (97, 109), and play a role in immune evasion in the cancers such as colon cancer (52, 62). Upregulation of PD-L1 by SPP1 could mediate macrophage polarization and facilitate immune escape in lung adenocarcinoma (110). A dramatic increase in SPP1 ⁺ TAMs was positively correlated with FAP ⁺ fibroblasts in CRC tissues, which could impair the immunotherapeutic effects (97). The patients with high SPP1 expression achieved less therapeutic benefit from an anti-PD-L1 therapy cohort (97). A tumor immune barrier (TIB) formed by the interaction of SPP1 ⁺ macrophages and cancer-associated fibroblasts (CAFs) was related to immunotherapy efficacy. Disruption of the TIB structure by blocking SPP1 should be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockage (ICB) in HCC (111).

SPP1, an osteopontin, from SPP1 ⁺ macrophages is a multifunctional secreted phosphorylated glycoprotein (112). It is also present in other cells such as osteoblasts, fibroblasts, and in tumor cells (112). The correlations between levels of circulating SPP1 and/or increases in SPP1 expression on tumor cells had poor prognosis in many types of cancer (112). The interaction of SPP1 and FN1 in TAMs with certain integrins promoted tumorigenesis in CRCs (43). SPP1-CD44 signaling in the glioma perivascular niche also enhanced cancer stem cell phenotypes and promoted aggressive tumor growth via the γ-secretase-regulated intracellular domain of CD44 (113). SPP1 was a key gene in the lymph node metastasis and a potential predictor of poor prognosis in head and neck carcinoma (114). In addition, SPP1 is involved in resistance to chemoradiotherapy through the induction of EMT, autophagy, epigenetic alterations, aberrant glucose metabolism and reduction of drug uptake (79, 111, 115).

The main functions of C1Q⁺ TAM subpopulation are immune regulation and immunosuppression (116–119). Indeed, TAMs with high C1Q A/B/C, HLA-DR, APOE, and TREM2 expression have a classically immunosuppressive phenotype (65, 116). These TAMs exert their immunosuppressive effects through surface molecules, cytokines, and metabolites. Surface molecules such as PD-L1, PDL2, CD80, and CD86, and cytokines such as IL-10 from TAMs could induce the differentiation of CD4⁺ T cells to Tregs (iTregs) (120, 121). A metabolite of tryptophan mediated by indoleamine 2, 3-dioxygenase (IDO) in C1Q ⁺ TAMs also played an immunosuppressive function directly or via the aryl hydrocarbon receptor in Treg, NK, and DC cells (122). Arginine depletion in TAMs could cause “arginine starvation” in T cells to inhibit these cells (123). Increased levels of IDO also limited the proliferation of cytotoxic CD8 cells (124). C1Q ⁺ macrophages could effectively suppress T cells (65, 125, 126). In RCC, the presence of C1Q+ macrophages correlated with exhausted T cells, forming a dysfunctional immune circuit (65, 127).

Notably, C1Q ⁺ TAMs are also related to positive responses to ICB therapy in melanoma and lung-carcinoma patients (117–119). Indeed, C1Q⁺ cell density was correlated with inhibitory receptors PD-1 and LAG3 at the CD8⁺ T cell surface (128). These C1Q⁺ macrophages express additional immune checkpoint ligands, such as PD-L1, PDL-2 (127, 128) and others such as CD40L, CTLA4, LAG3, PD-1, and TIGIT (57). Thus, these C1Q ⁺ TAMs may be a beneficial population for clinical applications (86). Notably, patients with C1Q^high and SPP1^low TAMs had the best prognosis, whereas the worst prognosis appeared in the patients with C1Q^low and SPP1^high TAMs (57, 129). In lung cancer, increased CXCL-10 was described in C1Q ⁺ TAM, which was related with an enrichment of the transcription factors IRF1, IRF7, and STAT1 (62). Interestingly, IRF1 was correlated with STAT1, HLA-DR, PD-1, and LAG-3 in metastases of colorectal cancer (130). In addition, C1Q ⁺ TAMs were also involved in phagocytosis and antigen presentation (43).

C1Q ⁺ macrophages to exert their functions also depend on C1Q, a recognition molecule of classical complement pathway, which can bind to immune complexes or other activators in the tumor microenvironment. Indeed, C1Q could regulate human macrophage polarization via interactions with LAIR1 (131), and modulate the cytokine expression while they digested lipid proteins, causing an M2-like polarization (132). C1Q also directly regulated T cell phenotype through internalization, binding to mitochondria, and regulation of mitochondrial metabolism (133, 134). However, it is incompletely clear how it controls immune activation, tolerance and exhaustion. In addition, C1Q could also interact directly with endothelial cells (EC) to promote neoangiogenesis, via still unknown cell surface receptors (128, 135).

FCN1 ⁺ TAM subpopulation comprises inflammatory macrophages. This subpopulation of macrophages is associated with increased inflammatory function (62). Enriched FCN1 ⁺ TAMs were detected in tumor-adjacent tissues. These FCN1 ⁺ TAMs were considered an intermediate stage from monocytes to tumor macrophages (136). They were associated with angiogenesis. Subsequently, these cells can produce C1Q+ and SPP1+ TAM (43). In addition, FCN1 (ficolin-1) from FCN1 ⁺ TAMs can be as a novel macrophage infiltration-associated biomarker for the diagnosis of pediatric inflammatory bowel diseases (137).

The effects of CCL18 ⁺ TAM subpopulation on tumor cells include tumor cell proliferation, migration induction, invasion, EMT, angiogenesis, and lymphangiogenesis (67). CCL18 ⁺ macrophages can be found in solid tumor tissues (53, 71) and in an immunosuppressive state in tumor tissues (70). They have a stronger tumor-promoting role than SPP1 ⁺ macrophages (53). Single-cell spatial transcriptomic analysis also identified highly metabolic CCL18 ⁺ TAMs in colorectal liver metastasis sites.

In the tumor, CCL18 chemokine produced by CCL18 ⁺ TAMs (67) was a marker of neoplastic diseases (67). Notably, elevated CCL18 levels in the serum and tumor are related to a worse prognosis in patients (53). Indeed, CCL18 is associated with immunosuppressive functions and cancer immune evasion. The CCL18 could recruit CD4⁺CD45RA⁺CD25^- naïve T cells into the tumor niche, and then differentiated into Treg cells, as shown in gastric cancer (138) and breast cancer (139). Notably, the importance of CCL18 in neoplastic processes mainly includes a signal transduction from PITPNM3 (one of CCL18 receptors) in CCL18-dependent migration, invasion, and epithelial- EMT cancer cells (67). Studies with human umbilical vein endothelial cells (HUVECs) showed that CCL18 could cause the VEGF-independent migration and tube formation of these cells (140). CCL18 also had an influence on the proliferation of cancer cells, but this effect was dependent on the type of tumor (67). In breast phyllodes tumor, CCL18 also participated in myofibroblast differentiation (141). Thus, CCL18 should be a potential therapeutic target for anti-cancer therapy. However, there is very few studies on the effects on tumor development through blocking the activity of CCL18.

Monocyte subsets that highly expressed FCN1, S100A8,and S100A9 could subsequently produce C1Q ⁺ TAM populations (43). C1Q ⁺ TAMs include pre-mature and mature C1Q ⁺ TAMs. Pre-C1Q ⁺ macrophages possess pro-inflammatory and phagocytic phenotypes based on the TAM differentiation routine. C1Q ⁺ and FCN1 ⁺ macrophages with a pro-inflammatory phenotype could be detected in NSCLC (62). C1Q ⁺ TAMs with high phagocytic activity were also found in tumor tissues (125, 128). These TAMs highly also express genes involved in antigen presentation (43, 60). However, mature C1Q ⁺ macrophages are the predominant contributors to immunosuppression. These C1Q ⁺ macrophages express multiple immunosuppressive genes such as PD-L1, PD-L2, HAVCR2, LGALS9, and CEACAM1 (52). In addition, several different gene signature clusters in C1Q ⁺ TAM subpopulations have been reported, such as C1Q ⁺TAMs with genes C1QA/B/C, SLCO2B1, NRP1, SLAMF8, FCGR1A, MERTK, and SIGLEC1 (43); C1Q ⁺TAMs with genes C1QA/B, APOE, TREM2, GPNMB and SLC40A1 (44), C1Q ⁺TAMs with genes TREM2, CD81, MARCO, APOE, CALR, CD63 and SPP1 (63); and C1Q ⁺TAMs with genes C1QB, APOE, FN1, CD276, TREM2, CHIT1, CCL18, MARCO, CD81 and NRP2 (50). Since C1Q ⁺ TAMs mainly exert immunosuppressive functions, which are related to multiple immune cells, these TAMs with different gene signatures suggest the existence of multiple C1Q ⁺ TAM populations with distinct immunosuppressive functions ( Figure 3 ).

SPP1 ⁺ macrophages not only promote tumorigenesis, but also metastasis and angiogenesis, which are possessed by embryo-derived TRMs (11, 24, 29, 142, 145), implying that this subpopulation is derived from embryo-derived TRMs. Several different gene signature clusters in SPP1 ⁺ TAM subpopulations have been reported, such as SPP1 ⁺TAMs with genes SPP1, BCL6, ADM, MARCO, FN1, AQP9, TIMP1, VEGFA, and IL1RN (57); SPP1 ⁺ TAMs with genes SEPP1,CD68, LYZ, MARCO, APOC1, SPP1 (82), SPP1 ⁺ TAMs with genes SPP1, MARCO, and VEGFA (43, 52); and SPP1 ⁺ TAMs with genes SPP1, AQP9, TNS3, FN1, C15ORF48, PHLDA1, and NDRG1 (43). These TAMs with different clusters suggest that SPP1 ⁺ TAM can be divided into different populations with distinct functions. In addition, SPP1 ⁺macrophages possessed anti-inflammatory phenotypes (62), which played a role in immune suppression and tumor evasion in colon cancer (97, 109).

CCL18 ⁺ macrophages belong to terminal TAMs. Increased immunosuppressive CCL18 ⁺ TAMs with a terminally differentiated state and metabolically energetic phenotype could be found in tumors. CCL18 ⁺ macrophages with high expression of CD163, MARCO, and CSF1R also exhibited stronger tumor-promoting effects than SPP1 ⁺ macrophages (53). These macrophages with an anti-inflammatory phenotype could also induce a worse prognosis (53, 62). The effects of CCL18 on tumor cells were similar to SPP1 TAM (67), implying that this CCL8 ⁺ macrophage subpopulation was originated from SPP1 ⁺ TAMs.