Role of Cancer Microenvironment in Metastasis: Focus on Colon Cancer

Colorectal cancer arises mostly from dysplastic adenomatous polyps. As with other types of cancers, it involves a multistep process characterized by the inactivation of a variety of genes that suppress tumours (e.g.: APC, DCC, p53) and repair DNA (e.g.:hMSH2, hMSLH1) and the simultaneous activation of proto-oncogenes (e.g.: K-ras, N-Ras and c-Myc). This confers a selective growth advantage to colonic epithelial cells and triggers the transformation of the normal epithelium into pre-cancerous adenomatous polyps and subsequently leads to local invasion and metastasis. There are several distinct neoplastic syndromes in which individuals display a marked predisposition to the development of colorectal cancer. In particular, genetic lesions in the familial form of adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPC) are now well delineated and their study has contributed enormously to our understanding of the molecular pathology of sporadic colorectal cancer [2]. Beyond the intrinsic genetic alterations that affect the colon cancer cells, a dynamic interaction occurs between cancer cells and the host stromal microenvironment to support cancerous growth and dissemination [3, 4]. The stroma is constituted mainly of cellular elements such as fibroblasts and non-cellular elements such as the extracellular matrix (ECM). These stromal elements act in a synergistic cross-talk with cancer cells from the primary sites to sustain cancer growth and metastasis. The importance of the stroma in cancer is highlighted by histological observations indicating that some types of neoplasms such as pancreatic cancer are composed mostly of stromal cells [5, 6]. We will first review the influence played by the stroma on the development of the primary neoplasm and thereafter on the formation of metastases.

Following their detachment from the primary neoplasm, cancer cells enter or intravasate into the existing or newly-formed blood or lymphatic vessels to disseminate. The process of intravasation is not very well understood in the case of colon cancer. Several molecular steps involving matrix metalloproteinases and interaction between cancer cells and endothelial adhesion molecules have been described. For example, studies in patients with familial adenomatous polyposis, indicate that urokinase plasminogen activator (u-PA) levels increase during the normal epithelia transition to dysplastic lesions, thus implicating u-PA in the early stages of tumour development [81, 82]. In fact, the u-PA system plays a determinant role in the intravasation of colorectal cancers [83]. Following its binding to receptor (u-PAR), u-PA activates plasminogen and other proteases such as matrix metalloproteinases-2 (MMP-2) and MMP-9. In turn, this contributes to breakdown the ECM, a step that favors intravasation. A recent study has shown that u-PAR gene expression is induced downstream of Src-mediated activation of the AP-1 promoter and by c-Jun phosphorylation at Ser73 and Ser63. Src activity increases during the transition from benign colonic polyps to primary colon cancers and to colon cancer metastases [84]. In addition, Src activity is associated with an increased invasion in vitro of colon cancer cells [85]. Moreover, an elevated Src activity is a poor prognostic factor in colorectal cancer patients [86]. Hence, it is possible that Src by regulating the u-PA/u-PAR system may contribute to ECM breakdown, thereby enabling the access of the detached cancer cells to the vessels and favoring their intravasation. By degrading the ECM, motile colon cancer cells create their path to the vasculature. Moreover, the ability of cancer cells to survive in the presence of broken ECM fragments constitutes an inherent property of metastatic cells. Accordingly, selection for anoikis-resistant cells increases the in vivo metastatic capacity of melanoma cells in mouse model systems [87]. In the case of colon cancer cells, this might possibly be due to the fact that they overexpress focal adhesion kinase (FAK), suggesting that they can by-pass integrin signaling and thereby survive in the absence of contact with the ECM [88]. Reciprocally, specific experimental deletion of FAK in a mouse model of skin tumour is associated with the induction of cell death and the inhibition of tumour progression [89]. FAK may contribute to confer survival by activating survival signal pathways, such as ERK or AKT [90, 91]. Following their survival and migration in the degraded ECM, cancer cells gain access to the blood vessel. At this step, it seems that microvessel diameter is the dominant parameter underlying colon cancer intravasation via passive entry [92, 93]. Once again, the cancer cells must acquire further capacity to survive the process of intravasation. Indeed, a large number of non-metastatic cells undergo fragmentation when they interact with blood vessels, whereas metastatic cells demonstrate an increased survival during this process [94].

As described in the previous sections, colon cancer cells can also disseminate via the lymphatic vessels, which is supported by the close correlation existing between liver metastasis and lymphangiogenesis. The mechanisms by which lymphangiogenesis contributes to colon cancer and cancer metastasis in general are still unclear. It is possible that VEGF-C stimulated lymphatic endothelial cells secrete chemotactic or mitogenic factors that will attract cancer cells to the newly growing lymph vessels enabling their adhesion and intravasation on and through the lymphatic endothelium [72]. In this context, expression of CXCR4 and CCR7 by human breast cancer cells facilitate their migration to the lymph nodes [95]. Once they have interacted with the endothelium of the lymphangiogenic vessels, the entry of cancer cells in these vessels will be greatly facilitated by the fact that they are leaky and tortuous (Fig. 4). As mentioned earlier, colorectal cancer cells express the CXCR5 chemokine receptor and both lymph endothelial cells and the liver express its ligand BCA-1/CXCL13 [79]. It is, then, possible that cancer cells may be directed via lymphangiogenesis to both lymph nodes and liver.

As described above, tumour cells enter the blood circulation directly or indirectly via the lymphatic system. In the case of colon cancer cells, they use the hepatic–portal circulatory system to enter the liver. Interestingly, a large number of cancer cells can be detected in the blood of cancer patients. However, the number of circulating cancer cells do not correlate with the level of metastasis. This suggests that circulation is deleterious for most cancer cells. In fact, several experimental evidences have shown that, only 0.01% of metastatic clonal cancer cells injected into the circulation are able to generate metastases. A major question is then to understand what the fate of cancer cells that enter the circulation is. This question has been addressed in reviews by Ann Chambers, Patrick Mehlen and their colleagues [91, 96]. They report that death of solitary cells in the circulation occurs mostly through tumour immune surveillance or destruction by mechanical stresses. In fact, studies using radio-labeled or fluorescent-labeled cancer cell lines injected into the circulation and intravital videomicroscopy support the hypothesis that solitary cancer cells in the circulation, or soon after extravasation, are sensitive to apoptosis and die [97‐99]. In line with this concept, there is a tenfold decrease in the number of apoptotic cancer cells that are found at the secondary site 1 hour following the injection of cells rendered resistant to apoptosis via overexpression of Bcl2 [98]. As reported above, cancer cell death might occur as a result of two major mechanisms: cell destruction by mechanical stress and cell destruction by the immune system.

The mechanical stress imposed by the circulation is a major stress that cancer cells must survive to form metastases. In fact, mechanical destruction of circulating cancer cells is the first line of defence in the host microenvironment that acts against hematogenous cancer dissemination [100]. Cancer cells circulating in the blood are submitted to strong mechanical forces caused by blood flow. This is especially important in narrow capillaries and within the microvasculature of contracting skeletal and heart muscle, in which sphere-to-cylinder shape transformation is lethal to most cancer cells [101‐103]. Moreover, NO and ROS are known to trigger apoptosis of cancer cells, and their production is activated by mechanical forces including shear and pulsatile stresses and contraction of the endothelium [91]. The production of these oxyradicals is required for apoptosis of intravenously injected melanoma cells or colorectal cancer cells in hepatic sinusoids [104, 105]. Cancer cells have developed several ways to survive the mechanical stresses associated with circulation. Notably, they express high levels of stress proteins such as HSP70. Moreover, they associate with platelets and with themselves in order to form a shield that protects them against external aggression [106, 107]. In addition, shear stress can induce pathways in the cancer cells that involve activation of integrins leading to adhesion of cancer cells to endothelial cells, thereby allowing them to escape anoikis. Nevertheless, these surviving processes are effective to only a small percentage of circulatory cancer cells since only 0.01% of these form metastases. In summary, the balanced response of tumour cells to mechanical destruction and adhesion initiation by circulating stress is an important mechanism that regulates the spread of hematogenous cancer.

Immunosurveillance is also involved in detecting and eliminating circulating tumour cells [108]. Accordingly, interleukin-2 (IL-2) is effective in treating patients with metastatic melanoma and kidney cancers, and its anti-tumoural activity is closely related to its ability to expand and activate specific subsets of immune cells, such as T cells and natural killer (NK) cells [109]. Other cytokines, such as IL-12 and IL-18, stimulate NK cells to suppress metastasis [110]. Mechanisms of tumour-cell killing by NK cells seem to be mediated either by the NKG2/perforin or Trail pathways [111, 112]. Circulating cancer cells have developed several mechanisms to escape immunosurveillance. In some cases, they can interact with platelets and fibrinogen to form clots that protect them against immune cells and facilitate their arrest and their adhesion on the endothelium [113, 114]. Furthermore, cancer cells may use inflammation-associated mechanisms to further protect themselves against apoptosis. For example acute-phase glycoproteins reach abnormally high levels in patients with cancer, which correlate with the extent of disease. It has been shown that these glycoproteins, synthesized by the liver in response to an inflammatory stimulus, may act as ‘non-specific blocking factors’ protecting tumours against the host’s immunological attack [115]. Intriguingly, there is no evidence that human immunodeficiencies are associated with increased development of metastasis from solid tumours. Hence, it is still difficult to understand the specific contribution of tumour-cell death induced by the immune system in limiting metastasis. Nevertheless, manipulating the immune system to induce tumour regression still has a great therapeutic interest.

The tumour microenvironment encountered by the invading cancer cells is a critical component of metastasis and should be permissive for the metastatic growth of incoming tumour cells [116]. Notably, the adhesive interactions between the cancer cells and the endothelial cells of the target organs are determining components of metastasis. These interactions determine the arrest of the circulatory cells in the capillaries and initiate the cascade of events that culminate in extravasation or diapedesis of the cancer cells in the colonized organs. The extravasation of circulating tumour cells in the host organ requires successive adhesive interactions between endothelial cells and their ligands or counter-receptors present on the cancer cells [117]. Typically, the colon cancer cell/endothelial cell interactions in the liver imply first a selectin-mediated initial attachment and rolling of the circulating cancer cells on the endothelium. The rolling cancer cells then become activated by locally released chemokines present at the surface of endothelial cells. This triggers the activation of integrins from the cancer cells allowing their firmer adhesion to members of the Ig-CAM family such as ICAM and VCAM, initiating the transendothelial migration and extravasation processes [118] (Fig. 5). Interestingly, the expression of endothelial adhesion receptors may be induced by the cancer cells via a paracrine pathway. This is supported by studies showing that culture medium supernatants of cancer cells can trigger the expression of E-selectin on endothelial cells suggesting that cancer cells may release cytokines such as TNF-α, IL-β or INF-γ that will directly activate endothelial cells to express E-selectin and by extension P-selectin, ICAM-2 or VCAM [119, 120]. On the other hand, other studies show that cancer cells may initiate the expression of endothelial adhesion molecules in more indirect ways. In particular, highly metastatic human colorectal and mouse lung carcinoma cells, on their entry into the hepatic microcirculation, initiate a rapid host inflammatory response by inducing TNF-α production in resident Kupffer cells. In turn, this event triggers the expression of E-selectin and VCAM by endothelial cells and enhances the binding and extravasation of the cancer cells across sinusoidal endothelial cells [121, 122]. Moreover, the kinetics of the host inflammatory response are tumour-type specific and rely on interactions with hepatic Kupffer cells, which supports the concept that the process is local and liver-specific. In this sense, the process of E-selectin- and other endothelial adhesion receptor-mediated metastasis appears to be local. In particular, increased hepatic local metastasis of B16F1 melanoma cells is observed following the administration of exogenous IL-1α, which induces the expression of vascular adhesion receptor expression, including E-selectin, VCAM-1 and ICAM-1 enabling cell arrest in terminal portal venules [123]. Moreover, syngenic colon adenocarcinoma C-26 cells trigger an endogenous inflammatory cascade upon entry into the liver. The cascade is absent in TNFR1-deficient mice, which is associated with reduced formation of liver metastases [124]. Overall, these findings indicate that, at least in the case of the formation of colorectal metastases in the liver, the process results from a local remodeling of the hepatic microenvironment that enables adhesion and extravasation of the cancer cells. In corollary, these results further suggest that hepatic remodeling involving E-selectin-mediated adhesion is an important component of the hepatic homing of colon cancer metastases.

The binding of cancer cells to E-selectin requires a molecular scaffold composed of oligosaccharides that are borne by a carrier protein and possibly a lipid on cancer cells. This macromolecular scaffolding complex is known as the selectin counter-receptor and is essential for cell binding [120]. We previously reported that Death receptor 3 (DR3) is a typical sialylated-a/-x protein that binds E-selectin and transmits its signal within cancer cells [125]. Other E-selectin ligands expressed by cancer cells involve ESL-1 and CD44 [107]. Interestingly, the binding of DR3 to E-selectin triggers the reciprocal activation of E-selectin, which induces a forward signaling in endothelial cells that contributes to increase endothelial permeability enabling transendothelial migration of cancer cells [126].

Since the adhesion of colon cancer cells to liver endothelium requires the presence of endothelial selectins and their appropriate receptors on the cancer cells, the degree of expression of selectins on the vascular wall and the presence of the appropriate ligand on cancer cells are determinant for their adhesion and extravasation into a specific organ. Along these lines, the homing of B16F10 melanoma cells to the liver requires the expression of E-selectin ligands by melanoma cells and the expression of soluble E-selectin in the liver [127]. On the other hand, endothelial selectins may be expressed differentially in blood vessels from different tissues. For example, LPS and TNFα strongly induce the expression of P-selectin on endothelial cells of the leptomeninges, but at a weaker level on some blood vessels of the brain parenchyma [128]. Based on these findings, it can be proposed that the differential specific interactions of selectins expressed by endothelial cells of potential target organs and their ligands expressed on cancer cells are major determinants that underlie metastasis and organ-specific distribution of metastases. Along these lines, lung is a secondary metastatic site for colon cancer and lung endothelial cells also express E-selectin upon appropriate stimulation [129, 130]. As mentioned previously in this section, it is clear that adhesion molecules other than E-selectin and its ligands are also involved in the early adhesion and extravasation of cancer cells. Notably, direct integrin (α2, α6, β1, β4)-mediated cell adhesion to the ECM in the space of Disse of liver sinusoids is required for the successful formation of liver metastases by colon cancer cells [131]. In addition, αvβ5 integrin expressed by metastatic colon cancer cells is required to enable their adhesion to an appropriate counter-receptor on hepatic microvessels [132]. Moreover, ICAM1 has been shown to dictate the binding of tumour cells to the pulmonary endothelial cells during metastasis [133].

Once circulating cancer cells have gained access to the secondary site, their subsequent growth will depend on the compatibility of the cancer cells (seed) with the “soil” that they encounter in the new organ. Their growth will then be tightly regulated by molecular interactions between cancer cells and the new environment. Most cancer cells will die by apoptosis initiated by an adverse environment [99]. Only a small subset of them initiate cell division to form micrometastases, and only a small proportion of these micrometastases become vascularized and grow to form macroscopic metastases. In the case of colon cancer cells, it has been proposed that they release soluble CD44 which acts as a decoy receptor impairing the interaction of colon cancer cells with its hyaluronate ligand within the ECM, thereby conferring resistance to apoptosis [134]. On the other hand, several lines of evidence indicate that ECM, by modulating the expression of growth factors and growth-factor receptors in colon cancer cells, also modulates the proliferation of these cancer cells in the liver. For example, hepatocyte-derived ECM, in particular heparin proteoglycan, stimulate the proliferation of colon cancer cell lines via induction of autocrine growth factors and their receptors [135]. Another factor that may limit the proliferation of all the incoming cancer cells at the secondary site is the fact that the latter is poorly vascularized at the onset, being devoid of angiogenesis. This will yield dormant micrometastases that may eventually develop in macrometastases following appropriate remodeling of the microenvironment. Incidentally, dormant micrometastases that overexpress Ras will later be more prone to develop metastases that depend or not on angiogenesis. Moreover, the expression of the EGF receptor coupled to the expression of growth factors like TGF-α in the tissue are among the well-known molecular factors that influence the ability of colon and other cancer cells to grow in the liver [136, 137].

During the last 10 years, cancer researches have focussed on the role of chemokines in cancer progression. Chemokines are chemotactic cytokines that cause the directed migration of leukocytes along a chemical gradient leading to the “homing” of leukocytes to specific organs [138]. These chemokines are induced by inflammatory cytokines, growth factors and pathogenic stimuli. Immune cells, endothelial cells and cancer cells express chemokine receptors and respond to chemokine gradients. Many cancers have a complex chemokine network that influence the immune-cell infiltration of the primary and secondary tumour, as well as tumour cell growth, survival, migration and angiogenesis [139]. It is now well accepted that chemokines and their receptors influence the metastatic potential and site-specific spread of tumour cells [140]. For example, breast cancer cells and primary breast tumours express patterns of chemokine receptors that “match” chemokines specifically expressed in organs to which these cancers commonly metastasize, namely the lymph nodes, lung, bone marrow and the liver. Furthermore, blocking one of the chemokine receptors was found to inhibit metastasis of breast cancer cells in experimental animal models [95]. Similarly, the chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases in the liver [141].

As described in earlier sections of this review, progression of solid tumours involves transition of epithelial transformed cells into mesenchymal cells (EMT), a process by which cancer cells acquire a more invasive and metastatic phenotype. Subsequently, the disseminated mesenchymal cancer cells must undergo the reverse transition, MET, at the metastatic site to allow micrometastases to give rise to a secondary neoplasm corresponding to the primary one. In this regard, cancer cells from the secondary site re-express markers of epithelial cells such as E-cadherin [142]. Since initiation of tumour growth at the secondary site is the rate-limiting step in metastasis, this suggests that the ability of mesenchymal cells to undergo MET in the appropriate microenvironments, is a key feature of metastasis [143]. Curiously, despite the importance of MET, little is known of the mechanisms that are involved. In the case of colon cancer cell metastasis, it may be speculated that the liver microenvironment, especially a subpopulation of CAFs, is involved in altering the microenvironment that will contribute to activate the MET process. This will be achieved by promoting the cross-talk between autocrine/paracrine growth factors and cell adhesion molecules expressed by cancer cells and stromal cells. For instance, CAFs taken from liver of patients having colon metastases overexpress COX2 and TGF-β2 and they enhance the proliferation of colon cancer cells in in vitro co-culture systems [144].]. It is possible that the role of CAFs in colorectal metastasis is modulated through the production of IL-8, a chemokine related to invasion and angiogenesis [145, 146]. In addition, a recent study shows that the kinase profile of colorectal cells greatly differs whether they are taken from the primary or the secondary hepatic metastatic sites, which further indicates that the hepatic microenvironment has remodeled the signaling network of the colon cancer cells [147].

In summary, the ability of cancer cells to grow in a specific site depends on features that are inherent to the cancer cell and to the invaded organ, and on the active interplay between these factors. Much remains to be learned about the detailed molecular interactions between cancer cells and specific secondary site, and probably many organ-specific growth factor pathways remain to be identified. However, there is strong evidence indicating that these interactions are important in determining the survival and growing potential of a cancer cell in a specific organ.

The journey of colon cancer cells from the primary site to the secondary sites is paved by obstacles that render the process destructive and ineffective (Fig. 6). Yet, some cells succeed and traverse all these impediments to generate metastases that will become fatal. In each case, the molecular events that lead to the massive destruction of the cancer cells or allow their survival during their journey is governed by an intricate interacting cross-talk between the cancer cells and its microenvironment. We have briefly reviewed some of these molecular interactions that characterize the journey of colon cancer cells to the liver. Much remains to be elucidated to fully understand the whole process. Nevertheless, little by little our knowledge increases, which contributes to improve our therapeutic intervention. The discovery that the anti-VEGF monoclonal antibody bevacuzimab increases the survival of colon cancer patient by impairing angiogenesis is one the best example. In fact, any therapy that interferes with the interaction of cancer cells with its microenvironment has promising clinical outcome. For example, one may envision targeting CAFs, EMT or MET as therapeutic purposes. Targeting the growth of metastases at the secondary site is particularly attractive since this event occurs often before the clinical detection of the primary neoplasm. In this context, components of the TGFβ pathway, a major player of EMT, are considered to be important new therapeutic targets. In line with this, new inhibitors of this pathway are already being tested in pre-clinical and clinical trials. Notably, four main strategies have been designed for disrupting TGFβ signaling: inhibition or sequestration of TGFβ, inhibition of TGFβ receptor kinase activity, inhibition of SMAD signaling downstream of TGFβ and restoration of anti-tumour immunity upon TGFβ inhibition [148]. In this regard, an antisense-based therapy against TGF-β2 is presently in a phase I/II study investigating treatment of colorectal cancer [149]. Moreover, approaches to increase immunosurveillance are under investigation and should lead to important therapeutic breakthroughs. Accordingly, interesting results have been obtained in mice using dendritic cell vaccination [150]. In our opinion, we believe that the next few years will be more fruitful in identifying new therapeutic targets in this direction.