Liver metastasis from colorectal cancer: pathogenetic development, immune landscape of the tumour microenvironment and therapeutic approaches

LSECs perform important physiological and immunological functions, including filtration, endocytosis and antigen presentation [71‐73]. As a selective barrier, LSECs allow for the entry of molecules such as plasma proteins, drugs, small chylomicron remnants, exosomes and smaller viruses (< 200 nm) into the space of Disse; however, they do not allow the entry of cells [74‐76]. The mannose receptor, scavenger receptor and Fc-γ receptor IIb2 efficiently facilitate the clearance and degradation of blood-borne macromolecules by LSECs to perform endocytosis and scavenging functions [77, 78]. Additionally, LSECs exert antigen-presenting functions mediated by the mannose receptor and scavenger receptor, which mainly reshape the immunosuppressive microenvironment in the liver. However, LSECs can dampen effector immune responses. Specifically,, antigen presentation by LSECs mainly induces the differentiation of CD4⁺ T cells into T_reg cells to promote the development of an immune-tolerant TME in the liver [44, 79]. In contrast, LSECs mainly drive a tolerogenic response mediated by an increase in the levels of coinhibitory PD-L1 that interacts with PD-1 to induce CD8⁺ T-cell dysfunction [80].

LSECs play a dual role in advancing tumorigenesis. When disseminated CRC cells enter the sinusoids, they are entrapped by LSECs and are either destroyed due to mechanical stress, phagocytosed by KCs or killed by perforin (PRF)/granzyme (GZM) from NK cells. LSECs and NK cells release interferon-gamma (IFNγ) and nitric oxide (NO) to upregulate Fas and induce apoptosis of cancer cells via the Fas–FasL pathway [81]. Recent studies have highlighted the fact that LSECs are influenced by gut microbiota-modified bile acids to secrete CXCL16, which recruits NKT cells to fight primary and metastatic liver tumours [82]. However, the anticancer proinflammatory response results in the high expression of vascular adhesion factors such as E-selectin, VCAM-1 and ICAM-1 on LSECs, thus leading to the susceptibility of LSECs to adhesion by cancer cells with the help of sialyl Lewis-x, PSGL-1 and ESL-1 [83‐85]. Cancer cells can escape from the destruction of the initial assault through counterreceptor communication, after which they migrate into the space of Disse, where they are protected from the cytotoxic effects of KCs and NK cells [86]. A novel adhesion molecule known as LSECtin mediates the communication between activated T cells and LSECs [87, 88] to inhibit the tumour-killing effects of T cells; in addition, it facilitates adhesion and migration of CRC cells to the liver [89]. In multiple experimental LM models, melittin nanoparticles have been demonstrated to induce the activation of LSECs to reverse the hepatic immunological environment to the activated state, which recruits NK and CD8⁺ T cells and suppresses LM [90].

Due to the fact that LSECs induce a suppressive immune microenvironment in the liver and assist in the growth of disseminated cancer cells, the targeting of LSECs to modulate the hepatic immune microenvironment may be a novel approach to the management of LM in the future. For example, the abundance of beneficial gut organisms that optimise the metabolism and immunity of the liver can be enhanced by modulating the action of LSECs for the effective treatment of CRLM.

KCs, which are the resident macrophages in the liver, serve as a crucial part of the innate immune response, which is the first line of defence of the liver [91]. Localised in the hepatic sinusoid, KCs can recognise all types of antigens (such as immune complexes, senescent cells and cancer cells) from the portal or arterial circulation and exert anti-inflammatory effects to prevent the entry of gut-derived substances into the hepatic sinusoid [92, 93]. In the early stage of CRLM, the adherence of disseminated cancer cells to KCs prompts KCs to capture and phagocytose the cancer cells and release TNF-α, interleukin-1α (IL-1α) and IL-1β, thus reducing the metastasis of colon cancer cells to the liver [94, 95]. The innate receptor Dectin-2 on KCs promotes the phagocytosis and elimination of disseminated CRC cells to resist metastasis [96].

Although KCs mainly play a tumoricidal role in the early stages of metastasis, they also play a vital role in hepatic carcinogenesis [97]. KCs activate and expand FOXP3⁺CD4⁺ T_reg cells through antigen presentation and induce tolerance by upregulating the inhibitory marker PD-L1, thereby resulting in the formation of an immune-tolerant environment to achieve homeostasis [23]. Moreover, HSCs are activated and produce fibronectin induced by KC-derived TGF-β, thus recruiting bone marrow-derived macrophages and neutrophils to form a favourable environment [53]. The corelease of TGF-β, fibronectin, EGF, VEGF and matrix metalloproteinases (MMP-2, MMP-9 and MMP-13) from KCs and HSCs leads to ECM remodelling, angiogenesis and cancer progression [98], which is augmented by the absorption of pancreatic ductal adenocarcinoma (PDAC)-derived exosomes by KCs in a PDAC model [99]. However, CRC-derived exosomal angiopoietin-like protein 1 (ANGPTL1) shuttles to KCs to decrease the expression of MMP-9, which subsequently reduces LM and inhibits vascular leakage mediated via the suppression of the JAK2-STAT3 signalling pathway [100]. In addition, KCs can phagocytose EV-packaged miR-135a-5p, thus mediating immunosuppression and facilitating the development of a premetastatic niche (PMN) in patients with CRLM [101].

It has been reported that a novel immunotherapy strategy by using bacterial genetic modification induces the reprogramming of KCs, which augments the phagocytic ability of cancer cells and strengthens the cytotoxic killing capacity of T cells to suppress LM [102]. An understanding of the functional role of KCs in CRLM may help to identify potential therapeutic targets and to develop novel therapeutic strategies, such as nanoparticle-mediated noncoding RNA-based therapy and bacterial treatment to reprogram the function of KCs. However, further research is required to identify the underlying mechanism and potential for application.

As a resident nonparenchymal liver cell population, HSCs contribute to liver fibrosis and cancer development [67, 103]. HSCs maintain homeostasis in the liver by regulating the ECM, immune tolerance and inflammatory responses; additionally, they play a significant role in the colonisation and metastasis of cancer cells [104‐106].

TGF-β is an important regulator of HSCs in the hepatic microenvironment. Its high expression blocks the initiation of CD4⁺ Th1 cells and weakens cytotoxic responses, thus facilitating LM and leading to a poor prognosis [107, 108]. TGF-β can induce the transformation of HSCs into a fibroblast-like (spindle-like and spread) phenotype (known as activated HSCs [aHSCs]) to promote ECM remodelling [98]. In addition, aHSCs play a vital role in secondary or primary hepatocellular carcinoma [109‐111]. aHSCs can lead to hepatic fibrosis and portal hypertension, thus contributing to hepatocarcinogenesis and metastasis [109]. In a previous study, we demonstrated that CRC-derived exosomal miR-181a-5p facilitates CRLM by activating HSCs [112]. In addition, aHSCs engulf disease-associated lymphocytes, including CD8⁺ T, CD4⁺ T and NK cells, through cell adhesion [113]. As key cells involved in pro-tumour angiogenesis, HSCs have been demonstrated to upregulate fibroblast activation protein alpha (FAPα) and increase CXCL5 secretion, as regulated by cancer cell-secreted fibroblast growth factor-binding protein 1 (FGFBP1). This mechanism stimulates epithelial–mesenchymal transition (EMT) and induces vessel co-option that results in bevacizumab resistance in CRLM models [114]. Therefore, HSCs play an important role in shaping the immune microenvironment of the liver and in inducing resistance to antiangiogenic therapy. The targeting of HSCs expressing specific molecules (such as FAPα) to modulate the immune microenvironment of CRLM represents a beneficial strategy for strengthening the antitumour effects of immune cells and for effectively overcoming drug resistance.

Hepatocytes play an essential role in inducing an immune-tolerant TME, which is required for the implantation of disseminated cancer cells. Hepatocyte-mediated cross-presentation of soluble antigens can induce tolerance of antigen-specific CD8⁺ T cells [115]. After extravasation, disseminated CRC cells can deeply penetrate into the hepatocyte plate, where they proliferate and form metastatic foci. The adhesion of CRC cells to hepatocytes is considered an essential step in the formation of LM [116], which is mediated by integrins [116] or desmosomes [117]. The strongly expressed integrin subunit αvβ5 mediates cell migration and LM in CRC, and its effects are enhanced by hepatocyte-derived heregulin [118]. Hepatocyte-derived SAA can facilitate the development of LM and is highly expressed in patients with CRC. Mechanistically, hepatocytes promote LM by activating IL-6–STAT3 signalling and inducing SAA overexpression, thereby reshaping the hepatic TME to facilitate the formation of a PMN in the liver [119]. Moreover, IGF-I can affect cancer growth and metastasis. The inhibition of IGF-1 released from hepatocytes reduces CRLM in mice [120]. A novel IGF-targeting protein (IGF-Trap) has been demonstrated to markedly block CRLM in experimental models to compensate for the function of the impaired insulin receptor system, thus inducing tumour cell apoptosis and reducing angiogenesis [121].

Altogether, the interplay between resident liver cells and cancer cells contributes to the progression and spread of CRC (Table 1). A better understanding of the communication between CRC cells and the hepatic TME may facilitate the development of new combination therapies for the efficient management of CRLM.

CD4⁺ T cells are essential in the defence against tumours because they regulate the activity of CD8⁺ T cells and influence the outcome of antitumour responses [122]. The classical effector CD4⁺ T helper 1 (Th1) and T helper 2 (Th2) subsets elicit important antitumour immune responses. Specifically, Th1 cells produce cytokines such as IFNγ and TNF-α, thus leading to cell-mediated killing, whereas Th2 cells secrete IL-4, which assists in the activation of humoral immunity [123]. In addition, CD4⁺ T cells can differentiate into new subsets, such as Th9 cells, Th17 cells and FOXP3⁺ T_reg cells. Moreover, the role of Th17 cells in cancer is controversial [124, 125]. The low proportion of Th1 cells and high proportion of Th17 cells in liver metastatic tissue indicate a poor prognosis in patients with CRLM [126], which is consistent with the condition of patients with CRC [127]. Given the immunosuppressive activity of CD4⁺ T cells, we mainly focused on FOXP3⁺CD4⁺ T_reg cells in this review.

One of the characteristics of LM progression is the high infiltration of FOXP3⁺ T_reg cells [128]. The expression of PD-1 on T_reg cells is higher in highly glycolytic LM tissue than in primary cancer tissue; however, it is lower in CD8⁺ T cells, which contributes to resistance to anti-PD-1 treatment [129]. The enrichment of T_reg cells in the TME is responsible for cancer immune evasion [130], which can partly explain the worse prognosis of CRLM. The immunosuppressive mechanism of action of T_reg cells occurs in the following way. (1) Interaction with APCs: Compared with CD28 expressed by naïve T cells, cytotoxic T lymphocyte antigen-4 (CTLA-4) expressed by activated Treg cells has a higher affinity for CD80/86 found on APCs [131, 132]. (2) The use of immune-suppressive metabolites: T_reg cells metabolise ATP to adenosine through CD39 and CD73, and the adenosine–A_2A receptor (A_2AR) interaction inhibits effector T cells [133]. (3) Involvement of cytokines: T_reg cells produce high amounts of CD25 by using IL-2, thus leading to the availability of a low level of IL-2 for activating effector T cells. Additionally, the high levels of TGF-β, IL-10 and IL-35 released from T_reg cells inhibit the activation of effector T cells [134‐136]. Moreover, TGF-β mediates EMT to promote the metastasis of disseminated CRC cells [137, 138]. Therefore, the elimination of the suppressive TME induced by T_reg cells may be a beneficial approach to reviving effector antitumour responses.

CD8⁺ T cells contribute to the clearance of intracellular pathogens and malignant cells and support long-term protective immunity [139, 140]. Based on distinct immune profiles, CD8⁺ T cells can be classified as exhausted (ex) CD8⁺ T cells, effector CD8⁺ T cells (which are also known as cytotoxic T lymphocytes [CTLs]) and memory CD8⁺ T cells [141]. CD8⁺ T cells serve as a useful marker to predict prognosis and therapeutic efficacy in cancer [142].

Upon encountering cancer cells, CD8⁺ T cells are activated by TCR-recognised antigens and rapidly proliferate and differentiate into CTLs to eliminate cancer cells through cell-to-cell contact. After CTLs are conjugated to target cancer cells, they secrete cytotoxic granules and release a cargo of deadly proteins, including PRF, GZM and granulysin, to kill the target cells [143]. A high proportion of CTLs contributes to improved outcomes in CRC [144]. However, to maintain hepatic immune tolerance, effector T cells are induced to undergo anergy, differentiation or apoptosis [145, 146].

CTLs undergoing persistent exposure to cancer antigen signals will gradually lead to the transformation of CD8⁺ T cells to a dysfunctional state, which are known as ex CD8⁺ T cells [147]. Moreover, ex CD8⁺ T cells secrete fewer effective cytokines, including TNF-α, IL-2 and IFNγ [148], thus resulting in failure to induce efficient adaptive tumour-killing effects on disseminated CRC cells. A key hallmark of exhaustion is the high level of inhibitory cell surface receptors, including PD-1 and CTLA-4 [149, 150]. CTLA4⁺CD8⁺ T cells are relatively enriched in primary CRC and LM tissues [151]. Additionally, the high proportion of immunosuppressive regulatory cells in LM, including M2 macrophages, neutrophils and T_reg cells [151], directly or indirectly promotes CD8⁺ T-cell exhaustion [149] (Fig. 3A).

It has been reported that CD8⁺ T cells mediate differentiation into Tim3⁺PD1⁺CD8⁺ T cells by glucocorticoid (GC) signalling in an MC38 colon carcinoma model [152] (Fig. 3B). The progressive upregulation of GC receptor signalling from naïve CD8⁺ T cells to ex CD8⁺ T cells communicates with monocyte–macrophage lineage cells, which impairs the production of immune-effective cytokines, including IL-2, TNF-α and IFN-γ, and promotes the high expression of immunosuppressive checkpoints (such as Tim-3, PD-1 and Lag-3) in CTLs, thus shaping an immunosuppressive TME [152]. Moreover, exosomal circCCAR1 expressed by cancer cells communicates with CD8⁺ T cells to impede the degradation of PD1, thus promoting the exhaustion of CD8⁺ T cells in the liver [153]. In addition, the high expression of MGP in cancer cells from both the primary CRC or LM sites increases intracellular Ca²⁺ to boost NF-κB phosphorylation, which mediates PD-L1 upregulation in CRC cells, thus promoting CD8⁺ T-cell dysfunction [154]. Furthermore, the induction of T cells to differentiate into regulatory cells is mediated by IL-10 release from LSECs, which are prone to activating the regulatory pathway of CD4⁺ T cells to FOXP3⁺CD4⁺ T_reg cells [145].

Recent studies on the apoptosis of CD8⁺ T cells have demonstrated that tumour-associated macrophages (TAMs) induce apoptosis of CD8⁺ T cells and impair cytotoxic functions by reducing the expression of granzyme B and perforin in the liver [155]. The mechanism for this effect involves the fact that activated CD8⁺ T cells experience apoptotic cell death by the Fas–FasL pathway, as mediated by TAMs within the liver, which induces a decrease in activated T cells and transforms the hepatic immune microenvironment in CRLM [25] (Fig. 3C).

Given that cancer-reactive CTLs play a central role in cancer immunity, it is important to reactivate CD8⁺ T cells to suppress the progression and metastasis of CRC. It has been reported that hyper-IL-15, IL-15 and the sushi domain of the IL-15 receptor α chain augment the cytotoxic functions of CD8⁺ T and NK cells, which may be a prospective therapy to reactivate CD8⁺ T cells and recover their anticancer ability to manage CRLM [156].

As multifunctional APCs, macrophages are critical mediators of tumour immunity [157]. Macrophages present exogenous antigens to T cells through MHC-I and MHC-II aided by costimulatory signals, inhibitory signals or other cytokine signals to regulate T-cell activation [158]. Macrophages that infiltrate malignant tissues are known as TAMs.

With inherent plasticity and polarising characteristics, TAMs are conventionally categorised into two subtypes: M1 and M2 macrophages [159, 160]. M1 macrophages suppress cancer growth by releasing cytotoxic reactive oxygen species (ROS), NO and IL-12, which can directly kill cancer cells [161]. However, M2 macrophages induce the formation of an immunosuppressive TME by secreting cytokines, including IL-10, TGF-β, CCL17 and CCL22 [157, 162]. Due to their poor ability to present cancer antigens, M2 macrophages undermine Th1 adaptive immunity [163]. In addition, M2 macrophages produce MMPs to regulate matrix remodelling, thus facilitating the invasion and metastasis of cancer [164]. In CRC, the expanding liver metastatic tumour is rich in TAMs (primarily M2 macrophages) [157, 165], which are recruited through the CCL2/CCR2 chemokine axis to form an immunosuppressive microenvironment [166], which is regulated by the expression of TCF4 in CRC cells to promote LM [167].

TAMs play an important role in CRLM. It has been reported that extracellular matrix glycoprotein spondin 2 (SPON2) reshapes the cytoskeleton and activates integrin β1/PYK2 signalling to promote the migration of TAMs, which increases the infiltration of TAMs and promotes the metastasis of CRC [168]. Furthermore, CRC-derived lipids reshape the metabolism of TAMs with the help of CD36, thus inducing TAM M2 polarisation to drive the development of LM [169]. Moreover, CRC-derived exosomal miRNAs can induce M2 polarisation, thus driving the EMT program, which correspondingly promotes the progression and metastasis of CRC [35, 170]. Additionally, the metastasis-related secreted protein Collagen Triple Helix Repeat Containing 1 (CTHRC1) enhances the infiltration of M2-like macrophages to remodel an immunosuppressive TME in the liver [171]. Mechanistically, CRC-derived CTHRC1 interacts with the TGF-β receptor in macrophages to activate TGF-β signalling to promote CRLM.

With the development of single-cell profiling, TAMs have been classified as C1QC⁺ TAMs, SPP1⁺ TAMs and MRC1⁺CCL18⁺ TAMs [151, 165, 172]. In a previous study, single-cell analysis showed that MRC1⁺CCL18⁺ macrophages and SPP1⁺ macrophages are the predominant M2 cell subsets in liver metastatic tissue [151]. Consistently, the presence of SPP1⁺ macrophages in liver metastatic tissue was reported in a study by Liu et al. Therefore, SPP1⁺ macrophages may be a potential culprit in CRLM. Moreover, MRC1⁺CCL18⁺ macrophages infiltrating liver metastatic tissue exhibit high metabolic activities, thus suggesting that they may promote LM through metabolic pathways [151]. SPP1⁺ macrophages are found in mesenteric lymph nodes with metastasis but not in mesenteric lymph nodes without metastasis, thus indicating that SPP1⁺ macrophages play a role in facilitating the expansion of disseminated cancer cells [165]. Furthermore, it has been reported that in microsatellite-stable (MSS) CRC, SPP1⁺ macrophages and fibroblasts communicate very closely via the ligand‒receptor pathway, which may help to shape an immunosuppressive TME in the liver [173]. However, more studies are required to understand the mechanisms by which different subtypes of macrophages promote LM.

Overall, as the leading tumour-infiltrating immune cells in the TME [174‐176], TAMs play a critical role in the progression and metastasis of CRC. Their high proportion is closely related to a worse prognosis [165, 177]. Specific subsets of macrophages, including MRC1⁺CCL18⁺ and SPP1⁺ macrophages, may serve as potential therapeutic targets for CRLM.

MDSCs are one of the key contributors to the formation of an immunosuppressive TME in the liver [178]. They mediate immune evasion by inducing the production of T_reg cells [179], thus inhibiting NK cell function [180] and impairing the antigen-presenting activity of DCs [181]. In addition, MDSCs facilitate cancer progression and metastasis in a nonimmune manner by producing MMP-9, which is a primary regulator of EMT [182], as well as VEGF, in order to promote TME remodelling and angiogenesis [20]. MDSCs are mainly classified as granulocytic or polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). The phenotypic and molecular features of these subtypes are difficult to identify [183]. Furthermore, the accumulation of MDSCs is one of the most dominant immunological features of CRC and is associated with disease progression and metastasis [184, 185].

MDSCs facilitate the formation of a PMN and the metastatic colonisation of CRC [186, 187]. Clinically, the high expression of CCL15 in patients with CRC results in the recruitment of more CCR1⁺ MDSCs, which is associated with the loss of SMAD4 (which is a TGFβ-relevant transcription factor) and promotes CRLM [188, 189]. In an orthotopic mouse model of CRC, CXCR2-expressing MDSCs are recruited from the circulatory system to the liver by CXCL1 secreted from CRC cells in the premetastatic liver, which facilitates the growth of disseminated CRC and its metastasis to the liver [187]. Mechanistically, sphingosine-1-phosphate receptor 1 (S1PR1)–STAT3 signalling in CRC cells results in the production of IL-6 to induce the activation of S1PR1 and p-STAT3 in MDSCs, thus leading to the formation of a PMN in the liver to promote CRLM [190]. Zeng et al. reported that the overexpression of hepatocyte-derived cell cycle-related kinase (CCRK) increases CXCL1 production to recruit PMN-MDSCs, thereby impairing NKT cell-mediated immunosurveillance, which dramatically promotes the metastasis of CRC cells to the liver [191]. M-MDSC-produced CCL7 binds to CCR2 on micrometastatic cells and stimulates the JAK/STAT3 pathway to activate dormant cells, thereby promoting the progression of CRLM [192]. Moreover, the inhibition of CCL7 may represent a potential strategy for preventing recurrent CRLM.

However, it is difficult to target MDSCs because they do not have a specific phenotype that differs from other mature granulocytes. Therefore, further research is required to identify therapeutic targets.

Under physiological conditions, NK cells are enriched in the liver and contribute to defending against infection and eliminating cancer cells [69]. After NK cells encounter cancer cells and are activated, they release PRF and GZM, thus leading to osmotic lysis and apoptosis of cancer cells [193]. Additionally, NK cells can directly kill target cells via the expression of TNF-related apoptosis-inducing ligand and FasL [194]. NK cells function in tumour immunosurveillance and elicit inflammatory responses by producing cytokines and chemokines [195].

NK cells can eliminate disseminated cancer cells to control metastasis [196]. A high proportion of NK cells indicates a good prognosis in patients with CRLM [197]. However, in the highly glycolytic environment of CRLMs, lactic acid causes the apoptosis of NK cells by downregulating their intracellular pH [198]. In addition, MDSCs attenuate the immunoreaction of NK cells by releasing NO, which interferes with FcR-mediated functions of NK cells, such as antibody-dependent cellular cytotoxicity (ADCC) and cytokine generation [199]. In a previous study on murine models of CRLM, compared with conventional NK (cNK) cells, liver-resident natural killer (LrNK) cells had a high expression of RORα, which is required to maintain LrNK cells but has no impact on cNK cells. The conditional knockout of Rorα aggravated CRLM, thus indicating that RORα is required for LrNK cell-mediated antitumour immunity. However, the RORα agonist SR1078 restrained CRLM [200]. Clinically, LrNK cells are significantly depleted in CRLM due to the accumulation of tumour-derived lactate, thus resulting in mitochondrial dysfunction and apoptosis of NK cells [198]. The targeting of lactate in the TME may restore the tumour-killing effects of NK cells and benefit patients with CRLM. Altogether, LrNK cells exert a great antitumour impact on CRLM and are closely related to prognosis; therefore, they may be qualified as specific therapeutic targets for CRLM.

Dendritic cells are the classical APCs that exert considerable influence in triggering antigen-specific immune responses and inducing immune tolerance [201‐204]. The antigen-presenting function of conventional DCs (cDCs) is important for the antitumour response of effector T cells [205‐207]. Efficient antigen presentation increases the polarisation of CD4⁺ Th1 cells and the activation of CD8⁺ T cells [208, 209].

DCs are heterogeneous and exhibit different characteristics. Compared with plasmacytoid DCs (pDCs), cDCs can more efficiently initiate an immune response against cancer cells [210]. In ICB-treated mouse models of orthotopic pMMR CRLM, the proportion of activated CD8⁺ T cells, CD4⁺ T cells and cDCs is lower in metastatic tumours than in subcutaneous tumours [211]. Liver-derived pDCs have a poor capability to stimulate the proliferation of T cells, thus resulting in anergy of effector T cells and immune suppression to maintain inherent liver tolerogenicity [212, 213]. Moreover, liver-resident regulatory DCs differentiated from bone marrow-derived progenitors secrete high levels of IL-10 but low levels of IL-12, thereby inhibiting effective T-cell function to maintain liver tolerance [214]. A subset of cDCs in CRLM identified as DC3s induces a proinflammatory phenotype and is correlated with a poor prognosis [165]. DC3s may be considered as a promising target for improving the therapeutic outcome of immunotherapy in CRLM. Further investigations are required to elucidate the mechanism by which DC3s promote CRLM.

Similar to TAMs, TANs play a dual role in cancer progression by both promoting and inhibiting the growth and metastasis of cancer [215]. Specifically, TANs facilitate activated T-cell immune reactions by presenting antigens and releasing IL-18 to induce the activation of NK cells [216]. In contrast, TANs release CCL2 and CCL17 to recruit CCR2⁺ M2 macrophages and CCR4⁺ T_reg cells, which shape a suppressive TME in the liver, thus promoting the progression and metastasis of cancer [217]. Additionally, TANs produce MMP-9 and neutrophil elastase to promote the extravasation of cancer cells and drive disseminated cancer cells to metastasise [218].

The accumulation of TANs has been demonstrated to be necessary for the formation of an omental PMN in orthotopic ovarian cancer models [219]. Ovarian cancer induces neutrophils to form NETs, which trap ovarian cancer cells and facilitate their implantation on the omentum [219]. Therefore, NETs can promote the metastasis of ovarian cancer. Several in vivo and in vitro studies on CRC have reported that the formation of NETs is enhanced by cancer-derived IL-8. These NETs can trap CRC cells in the liver and promote their invasive and metastatic capabilities [220‐222]. In addition, anterior gradient-2 (AGR2) released from TANs can promote metastasis in murine models of CRLM. TAN–CRC cell crosstalk between TAN-derived AGR2 and CRC-derived TGF-β1 is considered the primary driver of CRLM [223]. Collectively, TAN is an effective potential target for the treatment of CRLM. However, further investigations are required to explore and develop TAN-based therapeutic strategies for CRLM.

In conclusion, immune cells involved in CRLM shape the susceptible suppressive immune microenvironment for tumour invasion and metastasis in CRC (Table 2). Immunotherapeutic strategies that can reverse the immunosuppressive microenvironment or strengthen effector immunity may be effective against CRLM.

At present, the direct blockage of immune checkpoints to inhibit immune escape is the most well-established immunotherapeutic approach that has demonstrated excellent efficacy in the treatment of several cancers [258‐265]. However, unlike the response of patients with other cancers, that of patients with CRC to immune checkpoint blockade (ICB) depends on the DNA microsatellite instability (MSI) or mismatch repair (MMR) status [266‐268]. ICB agents are effective in patients with mCRC but mostly in those patients with high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) [269‐271]. CRC with MSI-H/dMMR has a high tumour mutational burden that induces tumour-specific neoantigens to alert immune cells and subsequently recruits numerous T cells, which improve sensitivity to ICB [272, 273].

The phase II CheckMate 142 trial demonstrated that patients with mCRC with MSI-H/dMMR who were pretreated with nivolumab, which is a PD-1 immune checkpoint inhibitor, had an objective response rate (ORR) of 31.1% and 12-month PFS and OS rates of 50% and 73%, respectively [270]. Additionally, a subsequent study reported that patients with mCRC with MSI-H/dMMR responded to the combination of nivolumab plus ipilimumab (a CTLA-4 inhibitor) as second-line therapy with a higher ORR of 55%, and the clinical benefit of the combination therapy was better than that of nivolumab monotherapy [274]. A recent investigation showed that the combination of nivolumab and low-dose ipilimumab, which was a first-line treatment, was well tolerated by patients with mCRC with MSI-H/dMMR and provided durable and robust clinical benefit characterised by an ORR of 69% [275], which indicated that ICB agents may serve as new and safe first-line drugs for the treatment of mCRC. The KEYNOTE-164 study showed that pembrolizumab, which is a PD-1 inhibitor, was an effective and safe ICB agent with an ORR of 33% in patients with treatment-refractory mCRC with MSI-H/dMMR [276]. As the first-line standard treatment, pembrolizumab outperforms chemotherapy in terms of PFS (16.5 months versus 8.2 months, respectively) in patients with mCRC with MSI-H/dMMR [277].

However, ICB alone or in combination shows weak outcomes in patients with MSS/mismatch repair-proficient (pMMR) mCRC. A phase II study demonstrated that lenvatinib combined with pembrolizumab resulted in a poor median PFS of 2.3 months with a 50% incidence of treatment-related adverse events in non-MSI-H/pMMR mCRC [278]. In the METIMMOX study, with short-course sequential oxaliplatin-based chemotherapy (FLOX), the addition of nivolumab treatment prolonged PFS by only 1 month compared with chemotherapy alone [279]; however, the therapeutic strategy was not as effective in patients with MSI-H/dMMR CRC. Another study demonstrated that regorafenib plus nivolumab resulted in an encouraging ORR of 21.7% in patients with MSS/pMMR CRC without LM; however, the ORR was remarkably lower in patients with LM [280]. Therefore, ICB treatment of patients with MSS/pMMR CRLM is challenging.

Overall, patients with MSI-H/dMMR CRC exhibited a positive response to ICB, whereas patients with MSS/pMMR CRC exhibited almost no response to ICB (Table 3). As one of the most promising antitumour treatments, ICB-based therapy has great potential for the treatment of CRC. Further investigations are required to verify the efficiency and safety of ICB agents in patients with CRLM, irrespective of their MSI or MMR status.

In adoptive cell therapy, cells with antitumour activity in vivo are isolated, modified and cultured in vitro and infused back into patients for antitumour treatment [281]. As an important branch of immunotherapy, adoptive cell therapy has made significant contributions to advancing the development of immunotherapy [281, 282]. In particular, chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated successful results in the treatment of haematological malignancies and is the only cell product approved by the FDA [283, 284]. Given the effectiveness of CAR-T therapy, new adoptive cell therapies have been developed for the treatment of solid tumours [285]. In CRC, CAR-T therapy remains a major focus of research and has a distinct advantage over other cell therapies (Table 4).

In a small-sample phase I clinical study on patients with CEA⁺ mCRC treated with gradient doses of CAR-T cells, 70% of participants regressed from progressive disease to stable disease without experiencing severe adverse events [286]. Therefore, CAR-T therapy is a safe and hopeful immunotherapeutic option for the effective management of mCRC. A preclinical study demonstrated that human guanylyl cyclase C (GUCY2C)-targeted CAR-T cells triggered T-cell activation, exerted antitumour effects and alleviated CRLM in both syngeneic and human CRC xenograft murine models [287]. Due to the fact that the safety of GUCY2C-targeted CAR-T cells has only been examined in murine models, these cells should be cautiously used in humans because GUCY2C is selectively expressed in intestinal epithelial cells. Furthermore, CYAD-01 (which is an autologous CAR based on natural killer group 2D [NKG2D] with a single generic construct) induces both innate and adaptive immunity to regulate the immunosuppressive TME, thus presenting novel insights into CAR-T-cell therapy [288]. Moreover, the phase I SHRINK trial showed that CYAD-01 in combination with standard chemotherapy elicited partial response (PR) in 25% of participants with resectable mCRC (n = 4) and reduced tumour burden in 60% of participants with refractory mCRC (n = 5) without inducing cumulative toxicity [289].

In the ALLOSHRINK trial, combination therapy with CYAD-101 (a nongene-edited, allogeneic, second-generation NKG2D CAR-T-cell product) and chemotherapy was well tolerated with no evidence of graft-versus-host disease and was reported to be a prospective treatment for patients with incurable mCRC who relapsed after multiple lines of therapy [290]. The KEYNOTE-B79 phase 1b multicentre clinical study was open and recruited patients to assess the effectiveness of CYAD-101 plus pembrolizumab in refractory mCRC [291]. Therefore, CYAD-01 and CYAD-101 CAR-T therapies are novel immunotherapeutic strategies for the effective treatment of mCRC.

In conclusion, appropriate CAR targets are poorly expressed in normal cells but are enriched in cancer cells. Therefore, CAR-T cells can precisely target cancer cells and improve the survival of patients with mCRC. Further preclinical/clinical trials should be conducted to demonstrate the actual effectiveness of CAR-T-cell therapy in solid tumours, including CRC.

Cancer vaccines are prospective therapeutic options in cancer immunotherapy [292‐294]. Unlike ICB and adoptive cell therapy, cancer vaccines exert antitumour effects by introducing tumour antigens into the body, activating immune responses and using the active immune function to kill cancer cells [295, 296]. At present, no products have been approved for developing cancer vaccines against CRC; however, relevant clinical trials are actively ongoing (Table 5).

Various vaccine strategies have been designed, including whole tumour cell-based, protein- or peptide-based and DC-based vaccines [297‐299]. In a randomised clinical trial, patients with colon cancer who received an autologous cancer cell-based vaccine had a significantly longer recurrence-free interval (p = 0.011) and recurrence-free survival (p = 0.032); however, disease-specific survival and OS showed no improvement [300]. Based on this foundation, further clinical studies (Clinicaltrials.gov Identifier: NCT00016133, NCT02448173) have been conducted to examine the protective effects of the autologous cancer vaccine against tumour recurrence after colon cancer surgery [301]. DC-based vaccines are powerful contributors to antigen presentation and the initiation of antitumour immunity [302‐304]; however, the development of DC-based vaccines for CRC is currently at an early stage. In a preclinical murine model, effective immunotherapy using tumour-associated antigen-loaded cDC-based vaccines increased the infiltration of activated effector T cells and inhibited tumour growth [305]. Two related clinical trials (Clinicaltrials.gov Identifier: NCT03730948, NCT02919644) on DC-based vaccines are ongoing to develop strategies for preventing the progression of surgically resected stage I and II hypermutated CRC or curatively resected stage IV CRC. Moreover, peptide-based vaccines have strong specificity and can easily elicit an effective immune response [306, 307]. In a phase I clinical trial, the adenovirus (Ad5)-GUCY2C-PADRE vaccine was efficient in patients with CRC and did not cause grade-3/4 toxic events during the 6-month follow-up period after vaccination [308]. Another phase I study demonstrated that the combination of a single-dose PolyPEPI1018 vaccine and maintenance therapy with fluoropyrimidine and bevacizumab was strongly effective, with 96% of vaccine peptides inducing T-cell responses without causing grade 3 or higher adverse events in patients with mCRC [309].

Based on these encouraging results, cancer vaccines may represent an immunotherapeutic strategy that is not limited by the DNA MSI or MMR status. Cancer vaccines can stimulate immune surveillance to combat initially undetected microscopic lesions and consequently enhance the survival of patients with CRLM.

There are multiple therapeutic options for CRLM; however, they fail to meet the requirements for a disease-free prognosis (Fig. 5). Immunotherapy is an emerging and effective weapon in the fight against CRLM that requires further research to explore its superior potential value.