Epithelial cells undergoing EMT will lose their apicobasal polarity and adhesion, acquire motile mesenchymal characteristics and become more invasive, which contributes to the onset of CRC metastasis ^[1][75]. Epithelial and mesenchymal cells can be distinguished by specific molecular markers expressed in cells. For instance, epithelial cells express E-cadherin and cytokeratins, while N-cadherin, Snail, Slug and Vimentin are markers of mesenchymal cells ^[2][76]. Cells undergoing the EMT process are distinguished by the loss of E-cadherin expression, a decrease of epithelial cell junctions and cytoskeleton and display a mesenchymal pattern with enhanced cell motility and invasiveness ^[3][77].

TGF-β signaling is an essential regulator of the process of EMT. As reported, TGF-β can induce the EMT process by downregulating the expression of tight junction proteins, resulting in weakened tight junctions, which is the key point for EMT induction of TGF-β signaling ^[4][78]. SMAD4 is demonstrated to downregulate the expression of Claudin 1, which contributes to CRC metastasis ^[5][79]. Most of these observations were made in vitro, although the results of in vivo experiments are more convincing and important. In light of research in human SW480 CRC cells, TGF-β1 can induce Alu RNA expression, the accumulation of which promotes the EMT process, and Alu expression significantly correlates with CRC progression ^[6][80]. TGF-β1 upregulates the expression of C-terminal tensin-like (Cten) and EMT markers, and it promotes the cell motility of the CRC cell lines SW620 and HCT116 ^[7][81]. TGF-β1 can also induce the upregulation of acyl-CoA synthetases 3 (ACSL3) which produces ATP and reduces NADPH, thus sustaining redox homeostasis and mediating the EMT and metastasis of CRC cells ^[8][82]. A functional study indicated that TGF-β can induce SMAD4-dependent EMT followed by apoptosis in HCT-116 and DLD1 CRC cell lines ^[9][83]. As reported in CRC cell assays and murine models, acidosis-induced TGF-β2 activation promotes the formation of lipid droplets, which provides energy for cancer cell metastasis and partially promotes EMT ^[10][84]. SMAD4 in TGF-β signaling is frequently inactivated in human CRC, and SMAD4 codes for a transcription factor central to canonical TGF-β signaling. Therefore, it is generally understood that EMT will not occur in SMAD4-mutant tumors. However, in SMAD4-mutant CRC cell lines and analyses of human CRC transcriptomes, EMT is not categorically precluded. Possible explanations for this may be that SMAD4-mutant tumors escape the tumor-suppressive function of TGF-β or undergo SMAD4-independent EMT ^[11][85]. Moreover, CRC patient tissues exhibited higher GDF-15 expression compared with non-cancerous controls, and in the human CRC cell line LoVo, the overexpression of GDF-15 could upregulate the marker genes of mesenchymal cells. Thus, GDF-15 could lead to EMT and promote CRC cell invasion and migration ^[12][86]. Based on the systematic analysis of samples from seven CRC patients, it was found that some potential EMT biomarkers were enriched in TGF-β/Snail and TNF-α/nuclear factor-κB (NF-κB) pathways, and the integrated pathway may be the main axis connecting cancer cells with their TME during EMT ^[13][87]. In an immunohistochemical study of 48 resected CRC specimens, SMAD4 was positively linked with the expression of Snail-1, Slug and Twist-1, while it was negatively correlated with E-cadherin expression, implying that SMAD4 promotes the process of EMT ^[14][88].

There are also other factors that affect EMT by regulating the TGF-β pathway. For example, atypical protein kinase C-ι (aPKC-ι) knockdown inhibits TGF-β1-induced EMT and cell migration in CRC cells ^[15][89]. Furthermore, in 5-fluorouracil (5-FU)-resistant CRC cell lines, knockdown of transmembrane protein 45A (TMEM45A) attenuated multidrug-resistance-enhanced EMT by suppressing the TGF-β/SMAD signaling pathway ^[16][90]. In studies utilizing cell line experiments and nude mouse models, Numb expression was negatively correlated with TNM stage and lymph node metastasis, and inhibiting Numb expression promoted the EMT process and the invasion of CRC cells induced by TGF-β ^[17][91]. It was found that Paraneoplastic antigen Ma family number 5 (PNMA5) accelerated CRC cell proliferation, invasion and migration in nude mice lung metastasis models, and the knockdown of PNMA5 attenuated TGF-β-induced EMT in CRC cells ^[18][92]. As reported in cell assays and mouse xenograft tumors, beta human chorionic gonadotropin (hCGβ) changed expression of EMT-associated genes, and these changes could be reversed by TGFBR1 and TGFBR2 inhibitors, indicating that hCGβ induces EMT in a manner that depends on the TGF-β pathway ^[19][93].

Angiogenesis in the TME is a pivotal process that promotes tumor development and metastasis ^[20][94]. Newly formed blood vessels can provide oxygen and nutrients to tumor cells as well as allow them to enter into blood circulation and metastasize to distant sites ^[21][95].

First, the TGF-β pathway can regulate tumor metastasis by affecting vascular endothelial growth factor (VEGF). In the CRC HCT116 cell line, the upregulation of VEGF expression caused by the absence of SMAD4 enhanced vascular density and promoted the development of metastasis ^[22][96]. Additionally, SMAD4 overexpression can inhibit CRC growth by inhibiting VEGF-A and VEGF-C expression in the HCT116 cell line and an promote tumor cell apoptosis in HCT116 cells and nude mouse models ^[23][97]. There are primarily two histopathological patterns of vascular changes in CRLM: angiogenic desmoplastic and non-angiogenic replacement ^[24][98]. Overexpression of Runt-Related Transcription Factor-1 (RUNX1) in cancer cells of the replacement lesions, which is mediated by TGF-β1 and thrombospondin 1 (TSP1), enhances cell motility to achieve vessel co-option ^[24][98]. TGF-β expression is increased in the AlCl₃-exposed human CRC cell line HT-29, and this particularly promoted endothelial cell angiogenesis via the induction of VEGF secretion ^[25][99]. In an orthotopic mouse model of liver metastasis, the inhibition of TGF-β-induced protein ig-h3 (TGFBI) suppressed angiogenesis of CRC cells and inhibited the progression of CRLM ^[26][100]. Second, the synergistic effects of TGF-β and other signal cascades can stimulate angiogenesis by accelerating endothelial cell migration and proliferation ^[3][77]. TGF-β can interact with other proteins or pathways to foster angiogenesis in mCRC. The downregulation of platelet-derived growth factor-D (PDGF-D), a downstream signal of TGF-β, inhibited the growth, migration and angiogenesis of CRC cells in vitro and in vivo ^[27][101]. Thrombospondin-4 (THBS4), an ECM protein, plays an essential role in the TME and augments the effects of TGF-β1 on angiogenesis ^[28][29][102,103].

Growing evidence has shown that the TME performs a significant role in tumor initiation, progression and metastasis. The TME comprises non-cancerous cells in the tumor, including cancer-associated fibroblasts (CAFs), endothelial cells, pericytes and different types of immune cells (dendritic cells (DCs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), natural killer (NK) cells, myeloid cells, T cells, B cells, monocytes etc.), as well as non-cellular components, including ECM and soluble products such as collagen, various cytokines, chemokines and other factors that contribute to CRC metastasis ^[30][31][32][104,105,106]. Direct cell-to-cell contact between cancer cells and secretion of cytokines in the TME caused crosstalk, resulting in CRC progression and ultimately metastasis. It was reported that the activity of TGF-β signaling in TME cells such as T cells, macrophages, endothelial cells and fibroblasts improved the organ colonization efficiency of CRC cells, while treating the mice with the TGFBR1-spesific inhibitor LY2157299 inhibited CRC metastasis formation ^[33][42]. Elevated TGF-β expression levels is an important feature in the TME of CRC, and TGF-β signaling can regulate the development of CRC, form the system structure of tumors and inhibit the activity of anti-tumor immune cells, which results in an immunosuppressive microenvironment ^[34][35][36][28,107,108].

Here itwe summarize recent research and find that most of studies focused on CAFs and immune cells such as TAMs, TANs, DCs, T cells, myeloid cells and monocytes. Only a few studies on TGF-β signaling-mediated CRC progression and metastasis were related to collagen (discussed in the CAF section). TGF-β-signaling-related CRC metastasis involving CAFs and immune cells will be further discussed in detail in the following sections.

CAFs are the most numerous cells in the TME, and they affect CRC metastasis by regulating TGF-β signaling directly or indirectly ^[36][37][108,109]. TGF-β is mainly produced by CAFs in CRC, and increased TGF-β promotes T cell exclusion and inhibits the effector phenotype acquisition of type 1 T helper cells (TH1). It has been reported that inhibition of TGF-β enhances the cytotoxic T cell response to tumor cells, thus suppressing liver metastasis ^[38][110]. TGF-β activates CAFs to secrete activin A, a TGF-β family member, which induces colon epithelial cell migration and EMT, resulting in a more metastatic phenotype of CRC ^[39][111]. Wang et al. have recently reported that the activation of C-X-C motif chemokine ligand 12 (CXCL12)/CXCR7 axis drove CRC cells to secrete exosomal miR-146a-5p and miR-155-5p, which could be taken up by CAFs, thus enhancing CAF activation via JAK2-STAT3/NF-κB signaling. CAFs could secrete more inflammatory cytokines, including TGF-β, further promoting EMT and CRC metastasis to the lung in vivo ^[40][112]. ZNF37A, which is upregulated in CRC, is reported to facilitate tumor cell metastasis to the lung and liver via the activation of Thrombospondin Type-1 Domain-Containing protein 4 (THSD4)/TGF-β signaling, and increased TGF-β secretion contributes to transforming fibroblasts to CAFs in the TME, further promoting CRC metastasis ^[41][113]. Integrin αvβ6 secreted by CRC cells induced the expression of TGF-β, thereby converting fibroblasts into CAFs and promoting CRC metastasis through the stromal cell derived factor-1 (SDF-1)/C-X-C motif chemokine receptor type 4 (CXCR4) axis ^[42][114]. Treatment of co-cultured CRC and CAF-like cells with vincristine, which is a chemotherapy drug used widely in mCRC clinical treatment, increased the secretion of TGF-βs, induced EMT and promoted the formation of CAFs, thereby enhancing the invasion and metastasis of CRC ^[43][115]. Interleukin-11 (IL-11) secreted by TGF-β-stimulated CAFs is a TGF-β target gene, and it activated GP130/signal transducer and activator of transcription 3 (STAT3) signaling in CRC cells and promoted the initiation of CRC cells to metastasis ^[33][42]. Endoglin, a TGF-β family coreceptor produced by CAFs, enhanced CRC cell metastasis to the liver in both zebrafish and mouse models ^[44][116]. In addition, it has been demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) secreted by SMAD4-deficient CRC cells promotes fibroblasts to produce BMP2, resulting in CRC cell invasion and metastasis ^[45][117].

Moreover, TGF-β1 can be secreted by tumor cells in metastasis. Neutral endopeptidase (NEP) co-culturing human colon cancer cell line SW620 (derived from metastatic tumors) with normal colon fibroblasts induced a significant increase in expression of TGF-β1 in SW620 cells, and this effect could be reversed by deletion of NEP ^[46][118]. As reported, TGF-β1 promoted the co-migration of colon cancer cells and CAFs, resulting in enhanced liver metastasis and tumor burden ^[47][119]. CAF-derived exosomal microRNA (miR)-17-5p caused CRC cells to secrete TGF-β1 into the TME through RUNX3/MYC/TGF-β1 signaling, which triggered CAFs to release more exosomal miR-17-5p to CRC cells, thus establishing a positive feedback loop for CRC metastasis ^[48][120]. In CRC, fibroblasts could be converted to CAFs via IL-1β/TGF-β1 signaling, and both TGF-β-activated kinase 1 (TAK1) and TGFBR1 inhibitors suppressed CRC metastasis and CAF accumulation ^[49][121]. Two additional studies revealed that CXCR4/TGF-β1 signaling plays an important role in the transformation of mesenchymal stem cells or hepatic stellate cells into CAFs, further promoting CRLM ^[50][51][122,123].

However, CAFs can also suppress CRC progression in some situations. In a genetically modified metastatic CRC mouse model, depletion of alpha smooth muscle actin (αSMA)⁺ CAFs resulted in an increase of forkhead box protein 3 (Foxp3)⁺ regulatory T cells (Tregs) and suppression of CD8⁺ T cells via BMP4/TGF-β1 paracrine signaling, ultimately promoting CRC invasiveness and lymph node metastasis ^[52][124]. A recent study showed that gremlin 1 (GREM1) and the immunoglobulin superfamily contain leucine-rich repeat (ISLR), representing two different types of fibroblast subpopulations that exert opposing roles in the signal transduction of BMP. Neutralization of GREM1 or overexpression of ISLR in fibroblasts could reduce CRC hepatic metastasis ^[53][125].

Furthermore, decreased expression of hyaluronan and proteoglycan link protein-1 (HAPLN1) regulated collagen deposition in CRC via the TGF-β signaling pathway, and increased collagen resulted in TME changes and CRC cell proliferation, migration and invasion ^[54][126].

TAMs, one of the most common immune cells in the TME, have been reported as key contributors to promote tumor metastasis ^[55][56][127,128]. Liu et al. found severe TAM infiltration in tumor tissues of mCRC patients, and TAM-derived TGF-β could activate HIF1α/TRIB3/β-catenin/Wnt signaling to enhance CRC progression ^[57][129]. GDF-15, secreted by macrophages, is a divergent member of the human TGF-β superfamily, and it can increase expression of EMT genes, thereby promoting the invasion and metastasis of CRC via the ERK1/2/c-Fos signaling pathway ^[58][130]. Shimizu et al. found that Kupffer cells, known to be resident hepatic macrophages, released TGF-β1 and promoted liver metastasis of CRC through angiotensin II subtype receptor 1a (AT1a) signaling. Moreover, depletion of Kupffer cells reduced metastatic areas ^[59][131]. It has been proven that TGF-β1 secretion of CRC cells upregulated macrophage expression of Response Gene to Complement 32 (RGC-32) and thus enhanced macrophage migration and promoted tumor progression ^[60][132]. Recently, Chen et al. found that oxaliplatin-based chemotherapy induced TAM recruitment to release TGF-β, which was mediated by CRC-cell-derived CSF1, resulting in programmed cell death-Ligand 1 (PD-L1) upregulation and an immunosuppressive TME. Inhibition of PD-L1 expression in CRC could make cancer cells sensitive to chemotherapy, reduce CRC lung metastasis and increase infiltration of CD8⁺ T cells. Both CSF1R⁺ TAM depletion and TGF-β receptor blockade combined with chemotherapy could inhibit tumor growth significantly ^[61][133]. Through specific differentiation, macrophages can be polarized into two different phenotypes: activated M1-type and alternatively activated M2-type. M1-type macrophages inhibit tumor growth and progression, whereas M2-type macrophages induce the progression and metastasis of tumors in CRC ^[55][62][127,134]. Ma et al. found that M2-type macrophages were positively correlated with infiltrating Foxp3⁺ Tregs in CRC, which may promote the development of CRC via the TGF-β/SMAD signaling pathway ^[63][135]. Cai et al. reported that M2-type macrophages that secreted TGF-β promoted EMT by activating the SMAD2,3-4/Snail/E-cadherin signaling pathway, resulting in CRC lung metastasis ^[64][136]. Zhang et al. revealed that Collagen Triple Helix Repeat Containing 1 (CTHRC1) secreted by CRC cells induced macrophages to the M2-type through activation of TGF-β signaling, further enhancing CRC liver metastasis ^[65][137]. Recently, Li et al. developed a thermosensitive hydrogel called Gel/(regorafenib + NG/LY3200882 (LY)), which could sequentially release regorafenib and LY (a selective TGF-β inhibitor) in tumor cells. Using colorectal tumor-bearing mouse models, they found that Gel/(regorafenib + NG/LY) can effectively inhibit tumor growth and liver metastasis, which was achieved by increasing levels of CD8⁺ T cells, reducing infiltration of TAMs and myeloid-derived suppressor cells and shifting macrophage polarization from M2-type to M1-type in TME ^[66][138].

Apart from CAFs and TAMs, other cellular components, such as TANs, myeloid cells, monocytes, DCs and T cells, in the TME can also affect CRC metastasis through TGF-β signaling. TAN infiltration was demonstrated to be positively correlated with the clinical stage of CRC patients ^[67][139]. Anti-TGF-β treatment attenuated tumor growth, which was mediated by inhibition of PI3K/AKT signaling pathways in TANs and TGF-β/SMAD signaling pathways in CRC cells ^[68][140]. Activation of epithelial NOTCH1 enhanced epithelial TGF-β2 expression and facilitated liver metastasis of CRC through TAN infiltration, which was mediated by TGF-β signaling. Neutrophil depletion led to increased CD8⁺ T cells in both primary tumors and livers and decreased metastasis. In addition, blocking TGF-β signaling in neutrophils can effectively reduce CRC metastasis ^[69][41]. Using mouse xenograft models, Itatani et al. found that a deficiency of SMAD4 in human CRC cells upregulated CCL15 expression, thus recruiting CCR1⁺ myeloid cells and promoting liver metastasis of CRC ^[70][141]. Furthermore, inflammation is an important driver for CRC development and metastasis. CRC cells treated with lipopolysaccharide-stimulated monocyte conditioned medium showed reduced expression of Growth Factor Independence 1 and enhanced EMT and CRC cell metastatic formation, which might have been mediated by TGF-β signaling ^[71][142]. Wang et al. suggested that silencing poly (ADP-ribose) glycohydrolase (PARG) in CT26 cells could suppress liver metastasis of colon carcinoma by suppression of poly (ADP-ribose) polymerase (PARP) and NF-κB and that it could reduce secretion of IL-10 and TGF-β, thus promoting the proliferation and differentiation of DCs and T cells, resulting in inhibition of metastasis by changes in immune function ^[72][143]. Treg and T helper 17 (Th17)-related genes seem to contribute greatly to CRC development and progression. Miteva et al. investigated the expression of Treg and Th17-related genes in CRC tissues and found that Foxp3, IL-10 and TGF-β1 expression was increased in CRC metastases in contrast to IL17A and NOS2. Treg and Th17-related gene expression in both primary tumor and regional lymph nodes might provide a suitable microenvironment for accelerating CRC metastasis ^[73][144]. The mechanism by which other cells in the TME influence T cells via TGF-β signaling directly or indirectly was covered in the previous section ^{[38][52][61][63][66][69][72]}[41,110,124,133,135,138,143].

Most tumors, including CRC, contain a small population of cancer stem cells (CSCs) which are regarded as key contributors to tumor generation, progression, recurrence, metastasis and chemotherapy drug resistance ^[74][75][145,146]. According to recent studies, the TGF-β signaling pathway can affect metastasis of CRC by affecting CSCs in CRC or the stemness of CRC cells.

Mesenchymal stem cells co-cultured with CRC cells showed enhanced invasive ability, which was mediated by increased expression of TGF-β1 and decreased expression of p53, resulting in effective inhibition of CRC metastasis ^[76][147]. Reports suggested that TGF-β could convert Nur77’s role from cancer inhibition to cancer promotion, which is associated with CRC stemness, metastasis and oxaliplatin resistance ^[77][148]. CSCs have specific markers on their surface. CD51, a novel functional marker for colorectal CSCs, could increase the sphere-forming abilities, tumorigenic capacities and migratory potentials of CRC cells, and it may regulate EMT and chemoresistance through TGF-β/SMAD signaling ^[78][149]. In a novel mouse model of CRLM, proteomic analysis revealed that the expression of CRC stem cell markers in CRC cells was elevated compared with the non-metastatic model, and the expression of these markers was regulated negatively by the TGF-β/SMAD4 pathways ^[79][150].

In addition to the four mechanisms mentioned above, TGF-β can also affect the metastasis of CRC through some additional mechanisms. TGF-β regulates matrix metalloproteinase (MMP) expression in cancer cells, while MMPs produced by either cancer cells or stroma cells activate latent TGF-β, together facilitating progression of CRC ^[80][151]. The expression of TGF-β and the podocalyxin-like (PODXL) protein in CRC cells could increase under radiation and then promote ECM deposition, resulting in cell migration and invasiveness ^[81][152]. Bioinformatic analysis and functional characterization indicated that TGF-β and Snail promoted CRC migration by preventing degradation of the non-coding RNA LOC113230-related argininosuccinate synthase 1 (ASS1) ^[82][153]. Reports indicate that cancer epithelial cells show a robust outward apical pole throughout the process of dissemination, which is referred to as tumor spheres with inverted polarity (TSIPs). TSIPs form and propagate via the collective apical budding of hypermethylated CRCs downstream of TGF-β signaling, which could drive the formation of peritoneal metastases ^[83][154]. Moreover, TrkC, which was overexpressed in CRC, could also increase the ability to form tumor spheroids, thus enhancing the metastatic potential of CRC by activation of AKT and suppression of TGF-β signaling ^[84][155]. It has been demonstrated that TGF-β inhibits lymph angiogenesis by inhibiting collagen and calcium-binding EGF domain-1 (CCBE1) expression, and CCBE1 has a pro-tumorigenic role in lymphatic metastasis of CRC ^[85][156]. TGF-β2 could enhance the metastatic potential of human CRC cell lines via upregulating the expression of catalase and controlling H₂O₂ output ^[86][157].