The origin and phenotypic plasticity of TAM (M1/M2)

The origin of macrophages can be traced back to either local tissue-resident macrophages, which possess a self-renewal ability, or blood monocytes, which subsequently transform into macrophages [27]. Macrophages within the TME exhibit notable plasticity and heterogeneity. There are two types of macrophages: M1-type and M2-type. M1-type macrophages have pro-inflammatory, immune-activating, and anti-tumor properties. M2-type macrophages express immunosuppressive molecules, indicating poor prognosis in CRC and a high concentration in liver metastases [28,29].

Immunosuppressive roles and pro-angiogenic effects

M2-type TAMs promote immune suppression through multiple mechanisms: directly inhibiting CD8⁺ cytotoxic T lymphocytes (CD8⁺ T cells) cell activity and reducing their infiltration, promoting Treg expansion by secreting interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), and exacerbating immunosuppression via MerTK (c-mer proto-oncogene tyrosine kinase)-mediated phagocytosis [30,31]. Concurrently, TAMs help new blood vessels to form by releasing factors like vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). These cells begin to interact directly with vascular endothelial cells early in the metastatic process [11,32,33], and also participate in remodeling the extracellular matrix by secreting enzymes such as matrix metalloproteinases, which is essentially a pre-metastatic preparation [34]. This advanced prognostic role makes TAMs a highly promising biomarker, as they can be tracked in future clinical trials for their density and phenotype, thereby helping predict a patient’s response to chemotherapy or immunotherapy.

Mediation of drug resistance

TAMs are closely related to chemotherapy resistance. In general, M2-polarized TAMs create an immunosuppressive state in cancer cells by releasing protective factors such as IL-10 and TGF-β [32,35,36]. Metabolic imbalances in the tumor microenvironment often drive this immunosuppressive M2 state. Usually, the accumulation of lactic acid leads to a localized accumulation in the tumor, which further weakens the antitumor response [37,38]. However, this mechanism is reversible. Some studies have demonstrated improved drug resistance through intervention in M2 subpopulations [34,39].

The origin and phenotypic diversity of CAFs

CAFs primarily originate from tissue-resident fibroblasts (also known as quiescent fibroblasts) [40], mesenchymal stem cells (MSCs) [41], and adipocytes [42]. Tissue-resident fibroblasts are a primary source of CAFs. Resident fibroblasts in different tumor tissues are sequentially recruited and activated by continuous stimuli that stimulate various modulators, such as TGF-β and platelet-derived growth factor (PDGF) signaling pathways, thereby creating a specific environment conducive to tumor growth [43]. Interestingly, the liver’s unique immune environment may allow hepatic stellate cells (HSCs) to serve as an additional source of CAFs [44]. CAFs are not static players; they interact with other components of the tumor microenvironment and dynamically evolve during tumor progression in response to signals from the surrounding microenvironment [45].

The Role of CAFs in cytokine secretion, immunoregulation, and drug delivery impairment

CAFs have crucial roles in managing immune cells in tumors. This adaptability enables them to perform critical functions. CAFs directly influence innate immune cells, as well as adaptive immune cells [46]. Also, they can promote an immunosuppressive environment by driving immune checkpoint molecule expression and remodeling the extracellular matrix, roles that indirectly shape immune cell recruitment and function [47]. CAFs can encourage immune cells to take part in the development and growth of cancer by releasing molecules like cytokines, chemokines, TGF-β, and collagen [47,48]. CAFs can make it hard for tumor-killing cells and therapeutic agents to infiltrate and exit the tumor tissue [49].

Classification of MDSC

MDSCs are a group of immature myeloid cells that include different types of cells. There are two main types of MDSCs: granulocytic or polymorphonuclear (PMN-MDSC) and monocytic (M-MDSC) [50]. PMN-MDSCs significantly increase in SMAD family member 4 (SMAD4)-deficient liver metastases via the C-C motif chemokine ligand 15 (CCL15)/C-C motif chemokine receptor 1 (CCR1) and CCL9/CCR1 axes, with single-cell sequencing revealing their strongest immunosuppressive activity [51,52]; Monocytic myeloid-derived suppressor cells (M-MDSCs) are closely associated with immune checkpoint inhibitor resistance [53].

Immunosuppression mechanism

MDSCs directly damage CD8⁺ T cells, inducing T cell death [54,55], while promoting the expansion of regulatory T cells (Tregs) [54,56]. MDSC also blocks T cell activation by secreting metabolites such as arginase 1 (ARG1) and reactive oxygen species (ROS) [57]. In addition to targeting T cells, MDSCs also enhance tumor invasiveness by participating in epithelial-mesenchymal transition (EMT) and chemotherapy resistance (often via the interleukin-23 (IL-23)/signal transducer and activator of transcription 3 (STAT3) pathway) [58]. Its immunosuppressive effects are exerted through synergistic interactions with TAMs, creating a constitutive microenvironment locally in tumor suppression [51,59]. This inhibitory function is also further enhanced by the hypoxia-inducible factor-1 alpha (HIF-1 α) signaling pathway under hypoxic conditions, a common feature of tumors [60].

Role in CRLM progress

MDSCs infiltrate liver metastases at an early stage, and they promote tumor colonization and immune escape by inhibiting natural killer cell and T-cell function [61,62], especially in SMAD4-deficient models, where CCR1⁺ Granulocytic myeloid-derived suppressor cells (G-MDSCs) infiltration is significantly associated with T-cell dysfunction [52]. MDSC accumulation has also been found to be strongly associated with poor patient prognosis [63,64].

Immune therapy resistance

MDSCs also significantly promote resistance to immunotherapy: elevated peripheral blood MDSC levels before anti-programmed cell death protein 1 (anti-PD-1) treatment predict poor response [65]; they attenuate PD-1/programmed cell death ligand 1 (PD-L1) blockade by recruiting Tregs and depleting CD8⁺ T cells, and synergistically maintain the “cold tumor” phenotype through interactions with tumor exosomes [66].

The liver’s “native” cells

In CRLM, the liver-resident macrophage population, Kupffer cells (KCs), exhibit dual roles: on one hand, cytokines secreted by CRC cells can induce KCs to polarize toward an M2 phenotype, promoting metastasis by secreting pro-inflammatory factors such as TGF-β [67,68]. On the other hand, under normal conditions, KCs resist metastasis by phagocytosing circulating tumor cells. Still, tumor-derived small extracellular vesicles (EVs) can suppress their antitumor activity by interfering with the apoptotic protease activating factor 1 (APAF1)-dependent DNA damage response [69,70].

In CRLM, HSCs are activated and transformed into CAFs, which promote fibrosis in the local tumor microenvironment through the secretion of collagen (e.g., Col1α1) and Timp1. This change provides structural support for metastasis [71,72].

The unique role of CRLM in seeding and growth

Activated KCs further stimulate HSCs by releasing TGF-β1. For instance, Lipopolysaccharide-stimulated KCs upregulate CCL2 and Timp1 expression in HSCs while inhibiting MMP1 [72]. In turn, type I collagen secreted by HSCs activates the TGF-β1 signaling pathway via the DDR1 receptor. These changes in the hepatic pro-local immune microenvironment lead to the survival of tumor cells and maintenance of stem cell properties [73].

Regarding intercellular communication and microenvironment regulation, tumor-derived EVs simultaneously target KCs and HSCs: DNA-carrying EVs activate the DNA damage response in KCs. At the same time, lipid metabolites (e.g., PA/OA) promote fibrosis progression via KC-HSC co-culture models [74]. Proinflammatory factors released by KCs, such as IL-6 and Tumor necrosis factor-alpha (TNF-α), synergize with HSC-mediated ECM deposition to construct an immunosuppressive microenvironment, shielding metastatic foci from immune attack [75,76].

Beyond their individual roles, KCs and HSCs engage in a synergistic crosstalk that critically shapes the metastatic niche. This KC-HSC axis operates as a vicious cycle: Activated KCs release profibrotic and pro-inflammatory factors (e.g., TGF-β1, PDGF, IL-6, TNF-α) that are potent activators of quiescent HSCs [77]. Once activated, HSCs undergo a transformation into myofibroblast-like cells, which excessively deposit and remodel the ECM (e.g., collagen I, III), creating a stiff, fibrotic stroma that provides structural support for invading cancer cells [78]. Furthermore, activated HSCs themselves secrete a plethora of factors (e.g., CCL2, hepatocyte growth factor (HGF)) that can, in turn, reinforce the pro-tumorigenic polarization of KCs and recruit additional immunosuppressive cells [79]. This reciprocal activation loop between KCs and HSCs establishes a perpetually activated, fibrotic, and immunosuppressive microenvironment that is highly conducive to the survival and outgrowth of metastatic colonies.

Nutritional supply

The progression of liver metastases from colorectal cancer relies on two distinct nutrient acquisition patterns: some metastatic lesions form new blood vessels through “budding angiogenesis” to supply oxygen and nutrients, which is the primary target for anti-angiogenic therapies (such as anti-VEGF antibodies) [80]. While others directly “hijack” existing hepatic vessels and attach to them for nutrient acquisition. Such metastases exhibit poor responsiveness to conventional anti-angiogenic therapies, limiting treatment efficacy [80,81].

Metastasis

Neovascularization plays a pivotal role in metastasis. In animal models, days 7–9 post-metastasis represent a critical window for neovascularization, directly influencing metastasis survival and expansion [32]. Sialylated immunoglobulin G (sialylated IgG) has been shown to help cancer cells move and spread in the body, including to the liver. It does this by activating a specific pathway in the cells, while anti-sialylated IgG antibodies effectively block this process [82]. Endothelial cell-associated angiogenesis is also associated with metabolic reprogramming (e.g., ketohexokinase-A (KHK-A) upregulation), further accelerating metastatic lesion growth [83].

Immune cell recruitment

Indeed, the process of local tumor angiogenesis does not directly attract immune cells to the tumor site. It is the concomitant microenvironmental changes that occur, such as elevated IL-10 levels and altered metabolic activity, that play a significant role, and these changes lead to a decrease in microenvironmental immune function. For example, IL-10, a key pro-metastatic factor, drives both the overexpression of PD-L1 by tumor cells and a reduction in the number of infiltrating CD8⁺ T cells and an overall weakening of the antitumor immune response, which ultimately results in liver metastases that are less responsive to immunotherapy [84].

T cells

The functional landscape of T and NK cells in CRLM paints a complex picture of local immune suppression. While the presence of specific tissue-resident memory T cells (Trm, marked by CD103⁺ and CD69⁺) is linked to better patient outcomes [85], effective CD8⁺ T cells are often scarce. Their recruitment is finely tuned by chemokine signals like C-X-C motif chemokine receptor 3 [86,87], and their function is readily suppressed by microenvironmental factors such as IL-10 [84,88]. This dysfunction is reflected in a series of aberrant checkpoint interactions: loss of activating receptors such as natural killer group 2 member D (NKG2D) [89], significant expression of PD-L1 on tumor cells [90], and inhibitory CD155-PD-1 binding [88], and it is often the continual occurrence of these changes that renders T cells dysfunctional. Therefore, reversal of this immunosuppression by interventional strategies (e.g., injection of IL-15) will allow some of the T-cell function to be restored by a mechanism that involves restoration of the expression and function of the activating receptor CD226 [88].

NK cells

NK cell infiltration patterns in metastatic lesions: highly cytotoxic CD49a⁺ NK cells recruit via CXCR3 and co-localize with macrophages [91], whereas liver-resident CXCR6⁺ NK cells decrease in number with downregulated T-bet expression [89] and increased granzyme K⁺ quiescent subpopulations [92]. Their functional impairment is primarily characterized by reduced effectiveness, driven by key mechanisms including decreased activity of receptors (NKG2D) and increased activity of receptors that block it [89,92]. Additionally, hepatocyte-derived fibrinogen-like protein 1 directly suppresses NK function via lymphocyte-activation gene 3 (LAG-3) [93]. Immune checkpoints such as HERV-H LTR-associating 2 and LAG-3 further exacerbate functional suppression [93,94].

B cells

B cells have been less extensively characterized in current studies. Still, single-cell RNA sequencing reveals a significant reduction in activated B cells within liver metastases, suggesting their potential involvement in immune regulatory defects [95].

Components of ECM

The ECM is mainly composed of dense matrix proteins secreted by tumor-associated cells (e.g., CAFs), of which collagen is a significant component. Its abnormal accumulation in CRLM is closely associated with drug resistance and metastasis [96]. For example, increased ECM deposition is associated with bevacizumab treatment resistance, possibly mediated through activation of fatty acid oxidation (FAO) [96]. The ECM is not only a passive scaffold but also changes dynamically during metastasis in response to evolving microenvironmental conditions. For example, TIMP1 secreted by tumor cells from EVs regulates the ECM, causing it to remodel and driving the progression of liver metastasis [97].

Physical properties

The physical properties of the extracellular matrix (ECM), especially stiffness, not only form a more robust structural support for tumor metastasis, but are also associated with immune escape. This stiffness stems from abnormal matrix deposition and cross-linking [98]. Atomic force microscopy (AFM) measurements have shown that ECM stiffness is significantly elevated in sorafenib-resistant patients [99]. Importantly, the assessment of tissue stiffness is no longer confined to ex vivo techniques. Clinical non-invasive imaging modalities, particularly magnetic resonance elastography (MRE) and ultrasound-based transient elastography (TE, e.g., FibroScan), can quantitatively measure liver stiffness in patients [100]. These techniques have been widely used to stage liver fibrosis. Emerging evidence suggests that MRE-derived stiffness parameters can also reflect the fibrotic and desmoplastic components of the TME in liver metastases, providing a potential imaging biomarker for predicting treatment response and patient prognosis [101]. Changes in the stiffness of the ECM influence the structure of tumor metastases [102,103], while enhancing metastatic potential by altering cell adhesion and pseudopod formation [103].

Effects on the immune microenvironment

The ECM actively steers cancer cell behavior by reshaping the physical and biochemical properties of the microenvironment. Increased matrix stiffness, for instance, provides structural support for cell adhesion while simultaneously fostering an immunosuppressive milieu that aids cancer cells in colonizing the liver and surviving there [104]. High collagen levels don’t just provide a scaffold; they directly stimulate tumor cell proliferation and enhance survival at metastatic sites, partly by protecting against anoikis—a form of cell death that usually occurs after cells lose adhesion [105].

The impact of drug permeation

The extracellular matrix acts as a dense physical barrier limiting drug entry. When matrix proteins such as collagen are over-deposited and cross-linked, the resulting rigidity impedes drug penetration, a phenomenon that has been well documented during bevacizumab therapy [96]. In addition to physical barriers, ECM actively activates tumor cells through pathways such as the DDR1 signaling pathway, which induces a multidrug-resistant phenotype to aid cancer cell survival [106].

Soluble factors usually play a mediating role. For example, transforming growth factor-β (TGF-β), which induces EMT through activation of mothers against decapentaplegic homolog (SMAD) signaling pathways (e.g., SMAD2/SMAD4), enhances the invasiveness of the primary tumor cells [107], which in turn mediates the passage of primary tumor cells across the basement membrane and thus reaches the distal organs and invades the liver (e.g., [108,109]). It induces TGF-β1 proteins, which usually further promote metastatic formation and angiogenesis after colonization of tumor cells [110]. Here are many examples of the same, such as IL-6 promoting extracellular matrix remodeling by upregulating tissue inhibitor of metalloproteinases 1 (TIMP1) expression through activation of the STAT3 pathway [111].

Intercellular communication

Colorectal cancer metastasis to the liver, which is highly dependent on intercellular communication, alters the immune microenvironment of the distant organ before metastasis occurs, with EVs, especially exosomes, acting as key messengers [112]. These nanoscale carriers transport a diverse cargo of bioactive molecules, including miRNAs, cyclic RNAs, and proteins, thereby facilitating an ongoing dialog between cancer cells, local mesenchymal stromal cells, and the broader liver environment [113–115]. This exchange ultimately boosts the cancer’s invasiveness and its ability to adapt to and thrive in the distant liver tissue [114,116]. A clear example of this is seen when exosomes originating from the primary colorectal tumor travel to the liver. Upon arrival, they activate hepatic stellate cells residing in the liver and deliver specific molecules (e.g., cyclic RNA), which are the “seeds” that reconfigure the local microenvironment, thus effectively creating a suitable “pre-metastatic niche” for the invading cancer cells. The “pre-metastatic niche” is effectively developed for the invading cancer cells [112,117,118].

Pre-metastatic niche formation

PMN formation is the basis for subsequent immunosuppression and angiogenesis. Specific exosome components, such as HSPC111, can upregulate liver stellate cells, inducing immunosuppression and stromal remodeling to promote liver metastasis directly [119]. Furthermore, exosomes enhance the liver’s “readiness” for cancer cells by modulating the phenotypes of immune cells (e.g., macrophages and neutrophils) and promoting vascular alterations [116,119].

Drug resistance

Regarding drug resistance, exosomes enhance tumor survival and metastatic propensity by mediating interactions between tumor cells and stromal cells (e.g., fibroblasts and immune cells), a process closely linked to drug-resistant metastasis [120].

As mentioned earlier, macrophages play a crucial role in promoting neovascularization and immunosuppression, with macrophage polarization being a key component. A recent study focused on reprogramming these cells, specifically converting tumor-promoting M2-type TAMs into cancer-fighting M1-type TAMs. The study demonstrated that targeting the tripartite motif-containing 26 (TRIM26) gene can effectively alter the polarization status of tumor-associated macrophages, thereby inhibiting metastatic spread. This approach may offer a promising strategy for initiating macrophage therapy, as opposed to traditional targeted therapies [146]. The real complexity, however, lies in the diversity of these macrophages. Not only do they change location and behavior over time and space within metastatic foci, but they also contribute to cancer progression through multiple redundant mechanisms, such as the secretion of immunosuppressive extracellular vesicles. Several agents targeting TAMs, such as CSF1R inhibitors (e.g., pexidartinib (PLX3397)) and CD40 agonists, are under clinical investigation to modulate the immunosuppressive TME in various cancers, though their efficacy in CRLM specifically requires further validation [147]. This high degree of heterogeneity underscores the need to develop more precisely targeted strategies [148–150].

In the previous Mechanisms section, we described that CAFs promote the metastatic process by secreting ECM components such as hyaluronic acid (HA), collagen, and cytokines. Therefore, inhibiting the activation of CAFs, such as through the targeted inhibition of the HAS2 enzyme, may provide a viable strategy to suppress the formation of a fibrotic microenvironment [151]. However, the “cross-talk” between CAFs and tumor cells shows bidirectional regulation (e.g., activation of hepatic stellate cells), and such targeting is often ineffective. The impact of targeting CAFs on the repair function of normal tissues needs to be explored in depth in subsequent studies [152].

The ECM barrier in the TME of liver metastases impedes drug penetration, thus limiting the application of anti-angiogenic drugs in CRLM [153]. The development of novel materials for drug delivery is imperative [154]. The efficacy of anti-angiogenic therapy may also be influenced by the histopathological growth pattern of the metastases, with desmoplastic HGPs potentially showing different vascular dependency compared to replacement HGPs [25].

Since ECM remodeling may disrupt standard tissue architecture, and the dynamic changes in ECM components increase targeting difficulty [152,153], specific enzyme preparations (such as hyaluronidase) that degrade HA can still reduce CAF infiltration and metastatic activity. For example, in a fatty liver model, inhibiting HA synthesis significantly reduced metastasis [151].

Blocking key factors (such as TGF-β and CCL2) can reverse immunosuppression [116,130]. For example, exosome microRNA-1246 secreted by CRC plays a crucial role in inducing HSC activation and reprogramming the TME. Blocking such vesicles may represent a direction for future development [116]. However, it is also necessary to consider that factor networks possess redundancy and compensatory mechanisms; single-factor blockade may yield limited therapeutic effects, necessitating more multidimensional inhibition [115].

There is no doubt that tumor-derived EVs play a crucial role in constructing the pre-metastatic microenvironment. Consequently, research has naturally focused on blocking the release of these EVs or intercepting their signaling. Targeting the specific lncRNAs they carry, for example, has emerged as an effective way to inhibit metastasis [117,135,148]. However, a significant challenge is the diversity of EVs themselves, and we still need to clarify their different subtypes and functions more clearly to target them precisely. In addition, the inability to precisely target these tiny vesicles is also a significant challenge for the future [135]. The primary challenges include the heterogeneity of EVs, the difficulty in selectively inhibiting tumor-derived EVs without disrupting physiological intercellular communication, and the lack of efficient delivery systems for EV-targeting agents.

Conventional chemotherapy often falls short because the TME is a complex barrier, characterized by low oxygen levels and an influx of immune-suppressive cells that protect the tumor [155,156]. We must contend with the TME’s physical defenses. Its dense extracellular matrix and high interstitial fluid pressure create a formidable blockade that severely limits how well any drug can penetrate the tumor core [153,154]. A more effective approach involves combining standard chemotherapeutic agents with treatments that specifically target the TME. For instance, a recent study demonstrated that pairing oxaliplatin (OXA) with a specific modulator, such as puerarin, can not only enhance its cytotoxic efficacy but also counteract its potential to foster metastasis by inhibiting chemotherapy-induced EMT [157].

The combination of targeted and immunotherapy effectively enhances immune cell function [158] while leveraging novel local drug delivery and tumor penetration capabilities to disrupt protective barriers within the TME, such as stromal fibrosis [159]. Changes that occur in the entire immune microenvironment of the metastatic tumor may explain why this combination therapy is effective. Recent studies have shown that targeting TAMs or CAFs in combination with PD-1 inhibition is effective. However, the highly tolerant immune microenvironment of CRLM, characterized by the liver’s immune-privileged nature, remains a challenge that must be overcome [135,139].

These combination strategies provide insights into enhancing therapeutic efficacy through targeted delivery—such as modulating the liver fibrosis microenvironment—and overcoming the immune microenvironment of liver metastases may represent a significant direction for future treatments [154]. Simultaneously, targeting the TME may impair normal hepatocyte function, such as HA inhibitors disrupting matrix homeostasis [151].

Nanotechnology offers a promising platform to overcome the physical and biological barriers of the TME. Smart nanocarriers (e.g., liposomes, polymeric nanoparticles) can be engineered to achieve specific targeting of TME components and controlled drug release. For instance, mannose-decorated nanoparticles have been developed to specifically deliver drugs to TAMs via mannose receptors, effectively repolarizing M2-TAMs to the tumoricidal M1 phenotype in preclinical models of CRLM [160]. Similarly, peptide-modified nanoparticles targeting fibroblast activation protein (FAP) on CAFs can co-deliver chemotherapeutic agents and CAF-inhibiting drugs (e.g., losartan), simultaneously killing cancer cells and alleviating stromal desmoplasia to enhance drug penetration [161]. Furthermore, enzyme-responsive nanoparticles that degrade upon encountering MMPs in the TME can achieve site-specific release of anti-angiogenic or immunomodulatory agents, minimizing off-target effects [162]. Although most studies are in the preclinical stage, these nanodelivery systems represent a cutting-edge translational approach to precisely modulate the TME of CRLM.