Chemokine biology in cancer

Seminars in Immunology 15 (2003) 49–55

Chemokine biology in cancer Fran Balkwill∗ Cancer Research UK, Translational Oncology Laboratory Barts, The London Charterhouse Square, Queen Mary’s Medical School, London EC1M 6BQ, UK

Abstract Many human cancers possess a complex chemokine network that may influence the extent and phenotype of the leukocyte infiltrate, angiogenesis, tumor cell growth, survival and migration. Restricted expression of chemokine receptors on leukocytes may allow concise control of cell movement and retention at the tumor site. Restricted and specific expression of chemokine receptors on tumor cells may be involved in the characteristic patterns of metastasis , and may promote tumor cell growth and survival. Detailed study of chemokine and chemokine receptor antagonists in experimental cancer models is warranted. Manipulation of the tumor chemokine network could have therapeutic potential in malignant disease. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Cancer; Chemokines; Invasion; Metastasis

1. Introduction Many cancers express an extensive network of chemokines and chemokine receptors [1–3]. These tumors are characterized by disregulated production of chemokines and abnormal chemokine receptor signaling and expression [4,5]. Tumor-associated chemokines are thought to play at least five roles in the biology of primary and metastatic disease: control of the leukocyte infiltrate into the tumor; manipulation of tumor immune response; regulation of angiogenesis; action as autocrine or paracrine growth and survival factors; and control of the movement of tumor cells themselves. All the evidence published so far indicates that these biological activities of chemokines in the tumor microenvironment are more likely to contribute to cancer growth and spread than to any host anti-tumor response. This article will summarize current information on these five areas and discuss how this could be exploited in novel biological therapies of cancer.

2. Chemokines may control the leukocyte infiltrate in cancers Most solid tumors, be they of epithelial, mesothelial or haemopoietic origin, comprise a mixture of malignant and stromal cells. Stromal cells are recruited into the tumor tissue and are integral to growth of primary tumor and spread ∗ Tel.:

+44-20-7882-6106; fax: +44-20-7882-6110. E-mail address: [email protected] (F. Balkwill).

of metastases [6–8]. The predominant stromal cells found in cancers are macrophages, lymphocytes, endothelial cells and fibroblasts, with eosinophils, granulocytes, natural killer cells and B cells also reported in some tumor types. 2.1. Chemokine production in human cancers The presence of leukocytes in solid tumors is related to local production of chemokines by both tumor and stromal cells. CC chemokines are major determinants of macrophage and lymphocyte infiltration, for instance, in human carcinomas of the breast and cervix, sarcomas and gliomas [2,9]. In breast cancer, there is a positive correlation between macrophages, lymph node metastasis and clinical aggressiveness, and the tumor cells have been reported to produce CCL2 (MCP-1) and CCL5 (RANTES) [10,11]. CCL5 production by breast cancer cells is associated with tumor progression, with only minimal chemokine expression found in benign breast disease [12]. Levels of CCL2 expression also correlated with breast cancer progression and macrophage accumulation, but were additionally found to relate to levels of MT1-MMP, the angiogenic factor thymidine phosphorylase, as well as microvessel density [11]. The majority of cases of Hodgkin’s disease are characterized by a complex chemokine network, that includes chemokines attracting Th2 lymphocyes such as CCL17 (TARC), CCL11 (eotaxin), CCL22 (MDC), as well as Th1-attracting chemokines CXCL10 (IP-10), CXCL9 (Mig-1), CCL2, CCL3, CCL5, and CXCL1 (gro/MGSA) [5]. In our laboratory, we have studied the chemokine

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network of epithelial ovarian cancer. Within this tumor microenvironment, the predominant infiltrating cells are macrophages and CD8+ T lymphocytes [8]. CCL2 localized to epithelial areas of the tumor [13] and correlated with the extent of lymphocyte and macrophage infiltration [8]. CCL3 (MIP-1␣), CCL4 (MIP-1␤), and CCL5 (RANTES) were also present in solid ovarian tumors, and localized to tumor infiltrating leukocytes. CCL5 expression correlated with the extent of the CD8+ T lymphocyte infiltrate [8]. Ascites formation is a common occurrence in human epithelial ovarian cancer. mRNA and pico to nanomolar levels of protein for CCL2, CCL3, CCL4, CCL5, CCL8 (MCP-2), and CCL22 were found in the ascites cells and ascitic fluid [14]. Ascitic fluid contained variable numbers of tumor cells, macrophages and CD3+ T lymphocytes that were predominantly CD4+ . A direct correlation was found between CCL5 concentration in ascitic fluid and CD3+ T cell infiltrate [14]. 2.2. Chemokine receptor profiles of infiltrating cells Despite abundant chemokine expression in epithelial tumors, available data suggest that there is limited expression of chemokine receptors on the infiltrating leukocytes. CCR1 was the only CC chemokine receptor consistently expressed within the solid ovarian tumors and this localized to infiltrating CD68+ macrophages and CD8+ lymphocytes [15]. Down regulation of chemokine receptors on tumor-associated leukocytes may be due to high levels of chemokine ligands, or inflammatory cytokines such as TNF-␣ [16]. Altered physiological conditions, such as hypoxia, may also alter chemokine receptor profiles and response to chemokines in the tumor microenvironment [17]. When there are multiple chemokine gradients, restricted expression of chemokine receptors in solid tumors may permit concise control of the extent and movement of the infiltrating leukocytes within the tumor, encouraging their retention within the tumor. The expression of chemokine receptors was markedly different in the ascitic form of ovarian cancer where leukocytes expressed a wide range of chemokine receptors, at levels comparable to those found circulating in the periphery [14]. This may reflect the differences in microenvironment between solid and ‘liquid’ form of this malignancy. In Hodgkin’s disease, CCR3, which binds CCL11, was detected in some, but not all cells of the reactive infiltrate [5]. The expression profile of this chemokine receptor was abnormal: not only T cells but also B cells that do not normally express this receptor, stained positive. The receptor for CXCR3, which binds CXCL10 (IP-10) and CXCL9 (Mig-1), was moderately up-regulated on CD4+ T cells in the tumor. 3. Chemokines contribute to immune suppression in tumors Infiltrating leukocytes may not only contribute to tumor progression by producing MMPs and growth, angiogenic

and immunosuppressive factors, but the profile of the cells attracted by chemokines to the tumor may contribute to an immunosuppressive environment. There is often a prevalence of Th2 cells in tumors and this polarization may be a general strategy to subvert immune responses against tumors [2]. Hodgkin’s disease, for instance, is characterized by constitutive activation of NF␬B and overproduction of Th2 cytokines and chemokines into the tumor, causing an influx of Th2 cells and eosinophils [5]. These reactive cells are thought not only to contribute to proliferation and survival of the malignant cells, but to suppression of cell-mediated immunity. Chronic exposure to high chemokine concentrations in the tumor microenvironment may encourage activated Type II macrophages that release immunosuppressive IL-10 and TGF-␤ [18]. These macrophages may also release CCL2, which could contribute to a Th2 polarized immunity [19] and stimulate a Type II inflammatory response [2]. Tumors are also known to inhibit DC1 migration and function, thus suppressing any specific immune response. In ovarian cancer, there is evidence that tumor cell production of CXCL12 weakens immunity by attracting and protecting CXCR4-expressing preDC2 cells, and altering preDC1 distribution, immunity and stimulation of fibrosis [20]. Another example of a strategy to encourage a Th2 environment in tumors comes from Kaposi’s sarcoma. The viral genome encodes three chemokines (vMIPI, II, and III) that are selective attractants for polarized Th2 cells [21].

4. A balance of angiogenic and angiostatic chemokines exists in tumors Chemokines may also regulate angiogenesis in the epithelial tumor microenvironment. CXC chemokines containing the three amino acid (ELR) motif glutamine-leucine-arginine, such as CXCL8 (IL-8), CXCL1, CXCL5 (ENA-78), CXCL6 (GCP-2), and CXCL7 (NAP-2), promote angiogenesis [22]. They are directly chemotactic for endothelial cells and can stimulate angiogenesis in neo-vascularization experiments in vivo. Elevated levels of CXCL5 were found, for instance, in primary non-small cell lung cancer, NSCLC, and correlated with the vascularity of the tumors [23]. In contrast, CXC chemokines such as CXCL9 and CXCL10, lack the ELR motif are often anti-angiogenic [22]. Levels of CXCL10 in human lung cancers were inversely related to tumor progression [22].

5. Chemokines and tumor cell growth As well as promoting angiogenesis, deregulated chemokines may contribute directly to transformation of tumor cells by acting as growth and survival factors, generally in an autocrine manner. This action of chemokines has been extensively characterized in malignant melanoma. CXCL1 and CXCL8 are constitutively produced by melanoma cells, but

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not by untransformed melanocytes [1,4,24]. Melanoma cells also show elevated levels of the CXCR2 receptor for these chemokines, and autocrine chemokine stimulation enhances survival, proliferation, and tumor cell migration [4,25]. In addition, CXCL8, CXCL1, as well as the CC chemokine CCL20 (MIP-3␣), all stimulate growth of pancreatic tumor cell lines [26,27]. In epithelial ovarian cancer, tumor cells express CXCR4 and its ligand CXCL12 enhances tumor cell proliferation in conditions of sub-optimal growth [28]. This autocrine chemokine stimulation may also have paracrine implications because CXCL12 stimulated the production of TNF-␣ by ovarian tumor cells. Production of TNF-␣ in the tumor microenvironment has been implicated in tumor progression [29]. The ability of tumor cells to proliferate in response to chemokines appears to have been exploited by the human Kaposi’s sarcoma herpes virus, KSHV. The KSHV genome encodes a G-protein coupled receptor that signals constitutively and is structurally similar to CXCR2 [30]. Expression of this receptor is associated with cellular transformation. Cells transfected with a consitutively signalling mutated CXCR2 are similarly transformed [31]. Transgenic mice overexpressing the KSHV-GPCR under the control of the CD2 promoter develop lesions with remarkable similarity to Kaposi’s sarcoma [32].

6. Malignant cells may respond to chemokine gradients Chemokines are central to the normal and pathologic trafficking of leukocytes and it seems that mechanisms utilized in homing of leukocytes may also be used by tumor cells. Restricted and specific expression of chemokine receptors, especially CXCR4 and CCR7, by tumor cells, may be one important step in the development of site specific metastasis. For example, tumor cells from breast, prostate, pancreatic, gastric and ovarian carcinomas, neuroblastoma, glioblastoma, melanoma and some leukaemias, express a limited and highly specific range of chemokine receptors [33–39]. Generally, only one or two of the known chemokine receptors are expressed on each tumor type. In breast, prostate and ovarian cancer, neuroblastoma, melanoma and some forms of leukemia, the respective ligand is strongly expressed at sites of tumor spread. Expression of CCR7 and CCR10 on melanoma cells, for example, was linked to expression of ligands for these receptors at the two major sites of metastasis, skin and lymph nodes [34]. Functional CCR7 and CXCR4 chemokine receptors are found on human breast cancer cells [34]. The ligand for CCR7, CCL21, (SLC) is highly expressed in lymph nodes and in a tissue screen, strong expression of the ligand for CXCR4, CXCL12, (SDF1) was only seen in the target organs for breast cancer metastases. Moreover, breast cancer cells migrated towards tissue extracts from these target organs, and chemotaxis could be partially abrogated by neutralizing antibodies to CXCR4 [34].

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CCR7 is also found on gastric cancer cells, which respond to chemokine ligand, by directed migration in vitro [37]. In biopsies from this disease, 66% of cases contained CCR7 positive tumor cells and there was a significant difference in both lymph node metastasis and lymphatic invasion between CCR7 positive and negative cases. Stepwise regression analysis showed that the most important factor determining lymph node metastasis in gastric cancer was CCR7 expression in the primary tumor. Animal cancer studies provide some experimental proof that cancer cells may co-opt normal mechanisms of leukocyte homing to lymph nodes. When B16 melanoma cells were transduced with a retroviral vector containing cDNA for the chemokine receptor CCR7, metastasis to lymph nodes was increased [40]. However, the process of metastasis is complex and multi-step. There are several stages at which the interaction between tumor cell chemokine receptors and their ligands could be important. For instance, in epithelial ovarian cancer, CXCR4 was the only one of [14] chemokine receptors investigated that was expressed by a panel of ovarian cancer cell lines [33]. The CXCR4 ligand, CXCL12, was present in nanogram quantities in ascitic fluid from ovarian cancer patients. Stimulation of CXCR4 induced a calcium flux and directed migration of the tumor cells. Moreover, CXCR4 was expressed on a subset of cells in primary ovarian tumors, and more strongly expressed by tumor cells in ascites. Our initial conclusion was that this chemokine receptor/ligand interaction could be involved in peritoneal spread of this cancer. However, in agreement with Zou et al. [20], we then found that the CXCR4 ligand, CXCL12, was also strongly expressed by tumor cells in the solid ovarian cancer biopsies [28]. As described above, stimulation of CXCR4-expression ovarian tumor cells with CXCL12 not only promoted invasion of cells through extracellular matrix, but caused production of the pro-inflammatory cytokine TNF-␣ and increased cell proliferation under sub-optimal conditions [28]. Taken together, these chemokine actions may allow tumor cells to grow in distant and less favorable sites, and via the pro-inflammatory cytokine TNF-␣, initiate a cytokine network in the surrounding stroma. However, the significance of high levels of tumor-derived CXCL12 at the site of the primary lesion is not understood. Although this may stimulate tumor growth, it could also serve to retain the malignant cells, rather than encourage metastasis. The multiple autocrine and paracrine actions of CXCL12 on CXCR4-expressing ovarian cancer cells are summarized in Fig. 1.

7. Manipulation of the chemokine network in tumors There are a number of areas where inactivation of chemokines and their receptors could be therapeutic in cancer. A change in the extent and composition of the leukocyte infiltrate could inhibit angiogenesis, survival and spread of tumor cells. Manipulation of some tumor chemokines

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Fig. 1. Multiple actions of the chemokine CXCL12 on CXCR4-expressing ovarian cancer cells.

could also directly affect angiogenesis. Direct inhibition of the action of tumor cell chemokine receptors may prevent or delay lymph node and haematogenous metastases and may decrease survival of tumor cells at the site of the primary or metastatic deposits. However, in spite of extensive preclinical studies of chemokine and chemokine receptor antagonists in inflammatory and infectious disease models [41], there are little published data on such strategies in models of malignant disease. 7.1. Manipulation of the leukocyte infiltrate As described above, the macrophage/lymphocyte balance of the tumor infiltrate appears to be a critical factor in the development of cancer [42]. For instance, overexpression of the cytokine CSF-1 (M-CSF) in a mouse model of mammary cancer increased infiltration of macrophages and accelerated tumor invasion and metastasis [43]. Stromal cell-derived MMP-9, most likely from tumor-associated macrophages, plays a critical role in angiogenesis and growth of ovarian cancer xenografts in nude mice [44]. Direct evidence for the involvement of chemokines in this stromal tumor promotion comes from studies of melanoma cell lines grow-

ing in nude mice. The effects of tumor-derived CCL2 depended on the level of secretion [45]. Low level production of CCL2 was associated with modest macrophage infiltration, tumor formation and neo-vascularization of the tumor mass. High CCL2 secretion led to a massive macrophage infiltration and destruction of tumor cells [45]. Attempts to decrease macrophage infiltration in tumors by neutralizing chemokines/receptors or chemokine antagonists, seem warranted. Another approach is to overexpress certain chemokines in the tumor microenvironment. In the past 10 years, many experimental animal studies have shown that overexpression of chemokines can increase tumor-associated leukocyte numbers and phenotypes, with concomitant stimulation of destructive immune or inflammatory responses. This was first shown by overexpressing CCL2 in experimental tumors and stimulating a destructive macrophage influx [46]. Overexpression of CCL20 (MIP3␣) suppressed tumor growth, by attracting dendritic cells to activate tumor specific cytotoxic T lymphocytes [47]. CCL19 (MIP3␤) overexpression mediated rejection of murine breast tumors in an NK cell and CD4+ T cell-dependent manner [48] and CCL21 (6Ckine) reduced growth of a colon carcinoma cell line in mice using

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a similar mechanism [49]. In contrast to the above results, overexpression of CCL3 in B16 melanoma cells did not stimulate an effective anti-tumor response, but prevented the initiation of metastases [50]. Direct injection of chemokines may achieve the same effect. When CCL21 was injected into axillary lymph node draining experimental lung tumors, there was extensive lymphocyte and DC infiltration into the tumors and enhanced mouse survival [51]. Enhanced production of Th1 cytokines and anti-angiogenic chemokines was noted, accompanied by decreased lymph node and tumor site production of TGF-␤. To date, these delivery approaches have not been translated into clinical trial. 7.2. Chemokines in anti-angiogenic strategies In experimental models using human NSCLC cell lines grown in SCID mice, neutralization of CXCL5 [23] and CXCL8 [52] reduced tumor growth, vascularity and metastasis, but had no direct effect on in vitro cell growth. The ELR+ chemokine CXCL8 was also important in promoting angiogenesis and tumorigenesis in human ovarian cancer xenografts implanted into the peritoneum of nude mice [53]. In this study, mouse survival was inversely associated with CXCL8 expression within the tumor. Overexpression of the ELR− chemokines CXCL9 and CXCL10 inhibited the growth of Burkitt’s lymphoma tumors established in nude mice [54]. Likewise, administration of intra-tumoral CXCL10 inhibited NSCLC tumorigenesis and metastasis in SCID mice [52]. CCL21 (6Ckine) also inhibited growth and angiogenesis of human lung cancer cell lines in SCID mice [55]. Thus, chemokines contribute to the network of proand anti-angiogenic factors in tumors and could be useful targets. 7.3. Chemokine antagonists and metastases Chemokine and chemokine receptor antagonists may also have direct actions on tumor cells. In this respect, there are three published studies of antibodies that neutralize chemokines or chemokine receptors. As described above, B16 melanoma cells transduced with a retroviral vector containing cDNA for the chemokine receptor CCR7, showed increased lymph node metastasis [40]. Lymphatic spread of these CCR7 transfected cells was blocked by neutralizing antibodies to the CCR7 ligand, CCL21 [40]. A majority of non-Hodgkin’s lymphoma cells express CXCR4 and treatment of cells in vitro with neutralizing antibodies to CXCR4 inhibited cell migration, pseudopodia formation and decreased proliferation [56]. In an animal model (NOD/SCID) of human high-grade NHL, CXCR4 neutralization delayed tumor growth, reduced the weight of any tumors that subsequently developed and significantly increased mouse survival [56]. Neutralizing antibodies to CXCR4 also suppressed lymph node metastasis in a xenograft model of a CXCR4-expressing breast cancer [34].

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8. Concluding remarks The chemokine network in human tumors is complex and its role is only partially understood. There is a great deal of information on potential roles of individual chemokines, but more work is needed to define the overall chemokine and chemokine receptor profile of individual tumor types, and the cells within them. The chemokines that are produced by human tumors are part of an even more complex network of inflammatory, immunomodulating and growth promoting cytokines, and little is known about interactions between all these mediators. A common theme is emerging of the involvement of constitutively activated NF-␬B in chemokine disregulation in cancers. Preclinical studies of chemokine antagonists in animal cancer models are warranted, as well as investigations of tumor development and spread in animals deficient in individual chemokines or receptors. Such experiments could provide a rationale for novel approaches to cancer treatment, where manipulation of the chemokine balance could be a useful adjunct to existing biological therapies of cancer.

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