Site-specific metastasis formation: Chemokines as regulators of tumor cell adhesion,motility and invasion

The metastatic spread of tumors is a well-coordinated process in which different types of cancers tend to form metastases in defined organs. The formation of site-specific metastases requires full compatibility between the intrinsic properties of the tumor cells and the tumor microenvironment. It was recently found that chemokines which are expressed in specific loci promote the adhesion, migration and invasion of tumor cells that express the corresponding receptor(s). Of the different members of the family, the CXCL12 chemokine and its cognate CXCR4 receptor are the prototypes of this process, although other members of the family (e.g. CCR7 and CCR10) also play a role in determination of the metastatic spread. This commentary addresses the fundamental roles of chemokines and their receptors in site-specific metastasis, with emphasis on CXCL12-CXCR4. The article also describes some of the efforts that were performed thus far in order to identify the intracellular components involved in this process. The focus is put on the roles played by proteins that regulate adhesion and migration of tumor cells in response to CXCL12, including mainly focal adhesion kinase (FAK), Pyk2/RAFTK and members of the Rho family of GTPases (RhoA, Rac, Cdc42). This is followed by discussion of open questions that need to be addressed in future research, and of the potential therapeutic implications of the findings that are available to date in this field.Key words: adhesion, cancer, chemokines, CXCL12, CXCR4, invasion, metastasis, migrationThe metastatic spread of tumors is not random. While certain types of cancer preferentially metastasize to particular organs, others “favor” different remote sites for metastasis formation. To form metastasis, tumor cells have to successfully complete a defined set of events that requires adequate properties of the tumor cells and of the target organs.^1–4 Of the different steps involved in this complex process, major emphasis was recently given to the events occurring at the target site, in which tumor cell arrest is followed by extravasation through the vessel wall, migration and invasiveness in the host organ, and finally by tumor cell proliferation and angiogenesis. Together, these steps enable the “seeding” of the cancer cells in distinct target organs and formation of site-specific metastases.^1–4Therefore, to succeed in the metastatic process, appropriate tumor cell properties need to join forces with a tumor-supporting microenvironment at the target site. In line with the “Seed and Soil” theory postulated by Stephaen Paget more than a century ago, an ample number of studies now indicate that full compatibility between the tumor cells and their surrounding milieu at the target organ is essential for site-specific metastasis formation.^1–3The extensive research that was performed on the metastatic process has led to identification of pivotal microenvironmental factors that may dictate the failure or success of the metastatic cascade. In this context, the focus was put on members of the chemokine superfamily. Chemokines are low molecular weight proteins whose activities are exerted primarily in the immunological context, where they induce the migration of leukocytes in response to chemotactic gradients. Chemokines regulate leukocyte homing to lymphoid organs in the course of normal hematopoiesis (homeostatic chemokines) or promote the extravasation of leukocytes to damaged/infected sites in inflammation (inflammatory chemokines). As such, chemokines that are released at specific sites either constitutively (usually homeostatic chemokines) or inducibly (mainly inflammatory chemokines), promote the adhesive properties of leukocytes that express the corresponding receptors, and activate the motility apparatus of such cells. Due to their chemotactic properties, the chemokines are viewed as indispensable regulators of leukocyte homing to specific sites in the body, and of the immune integrity of the host.^5–8Concurrently with the identification of the roles played by chemokines in immunity and inflammation, it became evident that specific chemokines are expressed in organs that are preferential sites of metastasis formation. These observations raised the possibility that chemokines that are present at specific organs promote the adhesion and migration of tumor cells that express the corresponding receptor(s), by that supporting tumor cell invasion and the establishment of metastases at these specific loci. In such a case, the chemokines may promote the “seeding” process of cancer cells in specific target organs and thereafter may also increase their ability to propagate at these sites. Such activities of the chemokines may thus contribute to site-specific metastasis formation, and may constitute an important determinant of the metastatic cascade.Along this rationale, the study by Zlotnik and his colleagues was the first to establish a firm link between chemokines and site-specific metastasis formation.⁹ In their study, the researchers focused on the chemokine CXCL12 which is expressed in lymph nodes, liver, lungs and bone, organs which constitute preferential metastatic sites in breast cancer. The study has demonstrated that breast tumor cells express CXCR4, the corresponding receptor for CXCL12 and that the chemokine induced migration and invasion properties in the tumor cells. Furthermore, by interfering with the intact activity of the CXCL12-CXCR4 axis, the authors have shown that formation of metastases in preferred organs was significantly inhibited, indicating that this chemokine and its receptor significantly dictate the specificity of the metastatic spread of breast tumor cells.In the years that followed, the above study was pursued by an extraordinarily high number of investigations, together establishing a new concept in the field of site-specific metastasis formation. Our current understanding of the metastatic process suggests a very important role to the chemokine-chemokine receptor axis, whereby chemokines that are produced at specific organs increase the adhesive, migratory and invasive properties of tumor cells that have reached these sites and express the corresponding receptor(s), by that promoting site-specific metastasis formation.^10–16The initial observations that were made on the contribution of the CXCL12-CXCR4 pair in breast cancer were soon followed by the demonstration that CXCR4 is expressed by almost all cancer types, suggesting that the CXCL12-CXCR4 pair may be involved in site-specific metastasis formation in a large number of malignant diseases. Alongside with the CXCL12-CXCR4 axis, other chemokines and their receptors were implied in organ-specific metastasis: it was suggested that CCR7 expression by tumor cells facilitates lymph node infiltration by the cancer cells and that CCR10 is involved in skin-directed metastasis by melanoma cells.^10–13The possibility that chemokines and their receptors contribute to site-specific metastasis has led to an extensive research in many cancer types. Not only that the expression of CXCR4 was detected in a large number of cancer types, in many of the cases CXCL12 induced adhesion, migration and invasion by the tumor cells. These in vitro findings suggested that CXCL12 promotes tumor cell activities that are required for the completion of steps that are essential for disease progression, leading to formation of metastases at specific organs. Indeed, such roles were confirmed for the CXCL12-CXCR4 pair in several of the tumor cell systems that were analyzed, by studies in animal model systems showing that impairment or induction of the activities of this axis significantly affected metastasis formation. Finally, the roles of CXCL12-CXCR4 in elevating site-specific establishment of metastases were substantiated in specific malignant diseases by clinical studies, correlating the degree of CXCR4 expression and its specific pattern of intracellular localization with formation of metastases at remote and specific organs in cancer patients. Together, these findings identified a key determinant that dictates the ability of specific tumor cells to establish metastases in a well-coordinated and site-directed process.^10–16However, as attractive as the role of the CXCL12-CXCR4 pair in organ-specific metastasis may be, several ambiguities still remain. The major difficulties emerge from the tumor-wide nature of CXCR4 expression, and from the implications of this phenomenon. The many different cancer types that express CXCR4 and adhere/migrate/invade in response to CXCL12 do not necessarily share the same metastatic pattern in cancer patients. Some form metastases in all or part of the organs that are enriched with CXCL12, while others establish metastases in other loci, in which CXCL12 is not a predominant constituent of the tissue. Obviously, it is possible that the CXCL12-CXCR4 pair acts alongside with other chemokine-chemokine receptor pairs, and that the end result is dictated by the equilibrium that exists in the target organs between different members of the family, acting on tumor cells that express the corresponding receptor/s. Therefore, the final consequence may reflect the activities of several chemokines and their receptors, including for example CCR7, CCR10 and/or CXCR7, the recently identified receptor for CXCL12.^16,17 However, based on the multifactorial nature of malignant diseases, it is quite obvious that chemokines and their receptors are not the only determinants of site-specific metastasis formation, and that intrinsic properties of the tumor cells other than chemokine receptors come into play, by responding to additional microenvironmental stimuli at specific body targets (Fig. 1).Open in a separate windowFigure 1Chemokines and additional microenvironmental factors in site-specific metastasis. Chemokines are important contributors to site-specific metastasis formation and they constitute a part of a very complex microenvironment that interacts with tumor cells that have reached the potential metastatic sites. In this respect, only a full compatibility between the chemokine/additional microenvironmental factor (one or more) and the corresponding properties of the tumor cells would support a successful and full-blown metastatic process. (A) The microenvironment counterpart of the process: successful establishment of metastasis is dictated by factors that are expressed at the microenvironment of the potential metastatic site(s), chemokines and others. (#1) Full compatibility between the factors that are found at the potential metastatic site (chemokines such as CXCL12 and others) and receptors that are expressed by the tumor cells supports the successful establishment of metastases. (#2, #3) The lack of essential chemokine/s, or of other tumor-supporting factor(s) reduces the efficacy of metastasis formation, or leads to failure of this process, despite the fact that receptors for both are expressed by the tumor cells. (B) The tumor counterpart of the process: successful establishment of metastasis is dictated by the array of receptors that are expressed by the tumor cells. Although the potential metastatic site is enriched with the essential tumor-promoting chemokines/additional microenvironmental factors, the metastatic process can not reach its maximal potential or fails, because the tumor cells lack the expression of the required receptor(s) for these factors (or express non-functional receptors). (C) The microenvironment at the potential metastatic site is diverse and complex, including a large array of pro-malignancy factors [chemokine(s) and other(s)] whose activities complement each other, therefore together amplifying the metastatic process. (#1) A microenvironment enriched with all the potential tumor-supporting factors can interact with tumor cells that express functional receptors for all these factors, together leading to the most intensified levels of metastasis. (#2, #3, #4) A microenvironment that lacks one or more of the essential factors would not enable the formation of full-blown metastatic process, although the tumor cells express the required receptors.The take home message is therefore that the roles played by chemokine receptors and their ligands in dictating the metastatic spread of tumors are important, but that they are one of several determinants that are involved in the site-specific metastatic process. The implications are mainly at the therapeutic arena, where the inhibition of the CXCL12-CXCR4 pair, for example, may prove inefficient in preventing the establishment of metastases. In addition, when the CXCL12-CXCR4 pair is considered as a test case, we need to clearly characterize the levels at which this pair acts, in order to identify potential targets for inhibition. One approach would be to inhibit the expression of CXCL12 and/or of CXCR4, by that taking the risk of impairing immune activities that are absolutely dependent on this pair. An alternative attitude would be to down-regulate the factors that induce CXCR4 expression by the tumor cells and/or the intracellular mechanisms leading to CXCL12-induced adhesive, migratory and invasive properties in tumor cells.The latter approach is challenging because it demands precise understanding of the mechanisms that regulate the expression of CXCR4 and of the signaling pathways it induces in tumor cells, together providing the correct conditions for successful “seeding” of the tumor cells at the target organ. Although a number of studies have identified factors that induce CXCR4 expression by tumor cells,^10–16 this avenue of research is just at its beginning. Furthermore, the molecular mechanisms that are induced by CXCL12 via CXCR4 are far from being elucidated, and only in a limited number of cases there is initial understanding of the intracellular mediators involved in tumor cell adhesion, migration and invasion in response to this chemokine.Thus far, many of the different studies that addressed the mechanisms involved in CXCL12-induced adhesion/migration/invasion show that actin filaments are polymerized following stimulation by the chemokine. Beyond this point, most of the investigations were sporadic and have analyzed several potential regulators of adhesion and migration. These included integrins, focal adhesion kinase (FAK), Pyk2/RAFTK, paxillin, Vav and members of the Rho family of GTPases, including RhoA, Rac1 and Cdc42. The number of studies that looked in depth into these issues, and directly associated the activation of these elements with functional regulation of chemokine-induced tumor cell invasion, through increased adhesion and migration, is surprisingly small. This is mainly so when one considers the importance of this field of research and its potential therapeutic implications.However, a more intensive and relatively informative research was performed on the mechanisms involved in CXCL12-CXCR4-induced adhesion/migration/invasion in two of the malignant diseases, breast cancer^9,18–23 and melanoma.^24–27 The studies in these two systems have provided initial insights into the complex net of interactions that exists between the different proteins that coordinate processes of cell adhesion and motility in response to CXCL12. In breast cancer, the different studies provided evidence to the direct involvement of FAK, Pyk2 and phosphatidylinositol 3 kinase (PI3K) in CXCL12-induced migration of the tumor cells.^18–20 In parallel, the studies by the group of Ganju have shown that CXCL12 upregulated the phosphorylation of a large number of proteins that are involved in formation of focal adhesions and in tumor cell motility: FAK, Pyk2, paxillin, Crk and Crk-L.^18,19 This group has also shown that PI3K was directly associated with the tyrosine phosphatase SHP2 and that SHP2 had an important role in the regulation of tumor cell chemotaxis. Combined with their observations regarding the roles of Cbl in control of motility, the authors suggested that stimulation of breast tumor cells by CXCL12 leads to migration processes that require FAK, Pyk2, PI3K, Cbl and SHP2. Furthermore, the authors suggested that Cbl, SHP2 and PI3K form a multimeric complex in response to CXCL12 stimulation, and that this complex is important for tumor cell motility.¹⁹ Since CXCL12 was also found to up-regulate matrix metalloproteinases (MMP) 2 and 9 in breast tumor cells,¹⁹ it is possible that the chemokine leads to increased tumor cell migration which is accompanied with matrix degradation, together supporting site-specific invasion and metastasis formation.In parallel to the studies that were done in breast cancer, major efforts were put by Texido and his colleagues to decipher the mechanisms involved in CXCL12-induced metastasis formation by melanoma cells. This group has provided a sequential and well-designed series of studies on the mechanisms contributing to CXCL12-induced adhesion and migration of the tumor cells. These investigations focused mainly on the activation of RhoA, Rac1 and Cdc42, GTPases that control the dynamics of the actin cytoskeleton and serve as major regulators of cell motility. The authors have shown that CXCL12 triggered in melanoma cells the activation of RhoA, Rac1 and Cdc42, however only RhoA and Rac1 were directly involved in melanoma cell invasion in response to CXCL12.²⁴ CXCL12-induced activation of RhoA and Rac1 has led to up-regulation of MT1-MMP expression, then giving rise to processing of pro-MMP-2 to mature MMP-2.^24,25 Therefore, similar to the findings in breast cancer, the activation of melanoma cells by CXCL12 can increase cell motility and in parallel promote degradation of the extracellular matrix. Together, these two processes can give rise to increased invasion by the tumor cells, which is potently induced by the chemokine.Further analyses by the same group have shed light on the mechanisms that activate RhoA and Rac in melanoma cells. Their findings demonstrated that stimulation of melanoma cells by CXCL12 triggered the activation of Jak, being an upstream event leading to Vav stimulation.²⁵ The activation by CXCL12 induced the phosphorylation of Vav1 and Vav2, and Vav1 phosphorylation correlated with increased quantities of Rac, and to a lesser extent of RhoA. Moreover, interference with Vav1 and Vav2 expression in the cells impaired substantially the activation of Rac and RhoA in response to CXCL12 in the melanoma cells and inhibited tumor cell invasion.²⁵Together, these findings indicate that activation of Jak leads directly or indirectly to Vav activation. Thereafter, Vav-induced activation of RhoA and Rac follows, promoting not only tumor cell motility but also degradation of basement membranes through the activation of MT1-MMP and than of MMP-2.^24,25 This complex chain of events is tightly controlled and careful investigation of the RhoA-mediated pathway indicated that it is oppositely regulated by specific Gα proteins: The stimulation of melanoma cells by CXCL12 has led to coupling of Gα_i to CXCR4, followed by Vav-RhoA activation and stimulation of tumor cell invasion; On the other hand, activation of Gα₁₃ by different measures gave rise to p190RhoGAP-mediated inactivation of RhoA, and to impairment of invasion,²⁶ in a process not involving Vav regulation. Similar indications were also found for Gα₁₂, where the expression of a constitutively active variant of this G protein induced defective RhoA activation.²⁶ Therefore, these findings indicate that activated Gα₁₃ and Gα₁₂ trigger similar RhoA-related functional responses in melanoma cells, and play important roles in regulating the metastatic spread in melanoma.The above findings suggest that it may be possible to inhibit the establishment of site-specific metastasis by targeting well-defined components that regulate tumor cell adhesion/migration/invasion in response to chemokines. Such selected intracellular molecules actually act as balancing factors, and their levels of expression and/or activities may dictate, at least to some extent the success or failure of metastasis formation. Accordingly, using again the CXCL12-CXCR4 axis as a test case, it was shown that breast tumor cell treatment by the tumor suppressor Slit has led to inhibition of breast cancer adhesion, chemotaxis and chemoinvasion.¹⁸ The activity of Slit was mediated by repression of FAK and Pyk2 phosphorylation, inhibition of PI3K and MAPK activation and reduced activities of MMP-2 and MMP-9.¹⁸ Also in breast cancer, it was found that the bisphosphonate Zoledronic acid (ZOL) inhibited the chemotaxis of tumor cells to CXCL12, in a process mediated by inhibition of RhoA and decreased expression of CXCR4.²¹ Based on their results, that authors suggested that the ZOL-induced reduction in RhoA activation has led to disorganization of the actin cytoskeleton, that was accompanied by a loss of stress fibers. These experiments in breast cancer manifest the potential strength of maneuvers that inhibit determinants that are required for completion of tumor cell invasion in processes triggered by chemokines via their receptors.Another example for the potential use of approaches that are based on key molecules in chemokine-induced processes and of their potential use for limitation of metastasis formation was provided in melanoma cells. In this case, the researchers addressed the possibility that inhibition of stimuli that activate Gα₁₃ in melanoma cells may reduce CXCL12-induced RhoA activation, and thus may limit the invasive properties of the tumor cells. Indeed, in this system the expression of a constitutively active form of Gα₁₃ (Gα₁₃QL) in melanoma cells has led to inhibition of RhoA activation in the tumor cells, as well as to inefficient formation of stress fibers and reduced generation of focal contacts. Importantly, although the over-expression of Gα₁₃QL in the tumor cells did not affect the formation of primary tumors, it did lead to a substantial inhibition in lung metastasis formation and to prolonged survival of the mice.²⁶ Together, the findings on Slit and ZOL in breast cancer, and on Gα₁₃QL in melanoma illustrate the potential therapeutic applicability of approaches that target specific components that act as determinants of adhesion, migration and invasion by tumor cells.The therapeutic potential of such approaches requires a very precise identification of the mechanisms involved in the ability of chemokines to increase adhesive, migratory and invasive properties in tumor cells. Moreover, the current information available in this field suggests that the processes are complex and probably are tumor cell- and/or tumor type-specific. To give one example, RhoA was found to be important in activation of melanoma cell migration and invasion, and possibly also in breast cancer.^21,24,25 However, such roles for RhoA are not trivial, since the regulatory mechanisms mediated by RhoA and signaling by members of the G12 family of heterotrimeric G proteins may differ in a variety of tumor cell types. Specifically, it was found that G12 proteins play in breast cancer opposite roles to those described in melanoma cells: Gα₁₂ and Gα₁₃ promoted breast tumor cell invasion, and Gα₁₂ signaling was required for metastasis²³ (but not for formation of primary tumors). Taken together with recent studies in prostate cancer, glioblastoma and Jurkat T cells, and with studies suggesting a role for G12-mediated signaling in RhoA activation,^23,28–30 it is possible that the activities of this family of Rho GTPases and of heterotrimeric G proteins depend on the cellular milieu of the tumor cells, and therefore are tumor cell- and/or tumor type-specific. However, one should also consider the fact that the mechanisms may depend on the experimental system used (some of the above did not include stimulation by CXCL12 or any other chemokine) and whether stimulation by a chemokine was included and not, further emphasizing the need for more intensive research in this respect.Overall, the above information illustrates the importance of chemokines and their receptors in site-specific metastatic dissemination and calls for improved characterization of the mechanisms by which they induce adhesion, motility and invasion by tumor cells, leading to site-specific metastasis. Therapeutic approaches that aim at the inhibition of specific intracellular molecular elements require very definite identification of the events occurring downstream of receptor triggering by the chemokine. It is essential to identify the full cascade of events that takes place and to keep in mind that the pathways may be different, or even opposite, in various tumor cells and in different malignancies. It is also very important to take the research one step further, and to elucidate by direct experimental means the roles of specific targets, and of their inhibitors in in vivo model tumor systems. It is also essential to determine whether in cancer patients there are associations between such elements and disease course and progression.To conclude, the design of therapeutic approaches aimed at inhibition of chemokine-induced metastatic spread and localization depends ultimately on integrated and multidisciplinary research that is based on extensive and thorough experimentations. Only such combined efforts may lead to improved understanding of basic mechanisms taking place in chemokine-mediated processes of site-specific metastasis, and to the development of better therapeutic means in the future.     

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL‐6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP‐1 secretion was reduced by CCL15/CCR3. In conclusion, this in‐vitro study identifies chemokine receptor‐ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL‐6 and HGF and reducing the secretion of TIMP‐1.     

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and Results

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.     

CXCL12-stimulated lymphocytes produce secondary stimulants that affect the surrounding cell chemotaxis

Chemotactic factors locally secreted from tissues regulate leukocyte migration via cell membrane receptors that induce intracellular signals. It has been suggested that neutrophils stimulated by bacterial peptides secrete a secondary stimulant that enhances the chemotactic cell migration of the surrounding cells. This paracrine mechanism contributes to chemokine-dependent neutrophil migration, however, it has not yet been extensively studied in lymphocytes. In this study, we provide evidence that lymphocytes stimulated by the chemokine, CXCL12, affect the CXCR4-independent chemotactic response of the surrounding cells. We found that CXCR4-expressing lymphocytes or the conditioned medium from CXCL12-stimulated cells promoted CXCR4-deficient cell chemotaxis. In contrast, the conditioned medium from CXCL12-stimulated cells suppressed CCR7 ligand-dependent directionality and the cell migration speed of CXCR4-deficient cells. These results suggest that paracrine factors from CXCL12-stimulated cells navigate surrounding cells to CXCL12 by controlling the responsiveness to CCR7 ligand chemokines and CXCL12.     

Migration deficit in monocyte-macrophages in human ovarian cancer

Purpose To examine the migration responses of monocyte/macrophages (MO/MA) expressing complementary receptors to chemokines produced in the tumor environment of epithelial ovarian cancer (EOC). Methods We examined the expression of the chemokine receptors, CCR1, CCR5, and CXCR4, on EOC associated ascitic and blood MO/MA; their response to complementary chemokines in a MO/MA migration assay and the F-actin content in an actin polymerization assay. A validated cDNA microarray assay was then utilized to examine alterations in pathway genes that can be identified with cell migration. Results Ascitic and EOC blood MO/MA express CCR1, CCR5 and CXCR4, but differently. Cell surface expression levels for CCR1 and CCR5 were higher in ascites than that of normal blood in contrast to CXCR4 levels in ascitic MO/MA which were lower. EOC associated ascitic or blood MO/MA failed to migrate in response to the CC ligand RANTES and to the CXCR4 reactive chemokine, SDF1 (CXCL12). Ascitic and most EOC blood MO/MA also behaved differently from normal blood MO in the polymerization/depolymerization assay. A cDNA gene analysis of purified ascitic MO/MA demonstrated that a number of genes involved with chemokine production, focal adhesion, actin cytoskeletal function and leukocyte transendothelial migration were down-regulated in the ascitic MO/MA when compared to normal blood MO. Moreover, PBMC cDNA from EOC patients’ blood also showed gene profiles similar to that of ascitic MO/MA. Conclusions Defective migration and polymerization/depolymerization activity of MO/MA from EOC patients and a significant down-regulation of critical pathway genes suggest that other mechanisms might be involved in the accumulation of systemically derived MO at the tumor site of EOC patients. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells

Background

The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.

Methodology/Principal Findings

We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.

Conclusions/Significance

When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.     

IFN-producing cells respond to CXCR3 ligands in the presence of CXCL12 and secrete inflammatory chemokines upon activation

Human natural IFN-producing cells (IPC) circulate in the blood and cluster in chronically inflamed lymph nodes around high endothelial venules (HEV). Although L-selectin, CXCR4, and CCR7 are recognized as critical IPC homing mediators, the role of CXCR3 is unclear, since IPC do not respond to CXCR3 ligands in vitro. In this study, we show that migration of murine and human IPC to CXCR3 ligands in vitro requires engagement of CXCR4 by CXCL12. We also demonstrate that CXCL12 is present in human HEV in vivo. Moreover, after interaction with pathogenic stimuli, murine and human IPC secrete high levels of inflammatory chemokines. Thus, IPC migration into inflamed lymph nodes may be initially mediated by L-selectin, CXCL12, and CXCR3 ligands. Upon pathogen encounter, IPC positioning within the lymph node may be further directed by CCR7 and IPC secretion of inflammatory chemokines may attract other IPC, promoting cluster formation in lymph nodes.     

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model

Introduction

Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer.

Methods

Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4.

Results

In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. Conclusion: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.     

The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

CXCR4/CXCL12 在肿瘤中的研究进展

研究表明趋化因子及其受体在胚胎发育、干细胞迁移以及各种免疫反应中发挥重要作用,是许多生理及病理过程中细胞运动的重要因素。趋化因子受体CXCR4是一个由352个氨基酸构成的、7次跨膜的G蛋白偶联受体。趋化因子CXCL12为其特异性受体。研究发现,CXCR4/CXCL12在多种肿瘤中都有表达,在肿瘤的生长、血管生成、转移等方面发挥着重要作用。与正常组织相比,肿瘤组织及转移灶CXCR4高表达。因此,对CXCR4/CXCL12轴在肿瘤病生理中的作用机制进行进一步研究,很可能为肿瘤的治疗及对肿瘤转移的预防提供一个新的思路。我们现在就对其在肿瘤病生理中的作用做一综述。     

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11–12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4°C, and co-internalization with their receptors at 37°C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.     

PI3Kγ activation by CXCL12 regulates tumor cell adhesion and invasion

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kγ regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.     

Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions

Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.     

CXC chemokine ligand 12 (stromal cell-derived factor 1 alpha) and CXCR4-dependent migration of CTLs toward melanoma cells in organotypic culture

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1alpha) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.