Chemokines have been reported to be elevated in the plasma of breast cancer patients, compared to healthy individuals [46
], and may serve as prognostic indicators for disease spread and relapse [48
]. The inflammatory microenvironment rich in chemokines is thought to be ideal for tumor cell development and growth. Although breast cancer cells and cell lines are known to secrete various pro-inflammatory chemokines including CCL2, CCL5 and CXCL2 (34, 46–48), relatively little is known about the effect of mammary tumors on the expression of these chemokines and their receptors by T lymphocytes. In this study, we report that CCL2 and CCL5 were upregulated in both splenic and tumor-infiltrating T cells and that CXCL2 is overexpressed by splenic T lymphocytes of tumor-bearing mice but not normal controls. We also demonstrate that the chemokine receptors, CCR1, -2 and -3 are upregulated in tumor bearers’ T lymphocytes at both mRNA and protein levels, while CXCR2 is expressed at the mRNA level. Furthermore, treatment of T lymphocytes with rmCCL2 increased the expression of CCL5, CXCL2, CCR1, -2 and -5.
The pro-inflammatory and pro-angiogenic chemokine, CCL2, has been shown to be highly expressed by breast cancer cells at primary tumor sites [35
] and is known to contribute towards tumor growth. We have previously demonstrated that this chemokine is also secreted by splenic and tumor-infiltrating T lymphocytes of mammary tumor-bearing mice and that this induced secretion is mediated by tumor-derived phosphatidyl serine and GM-CSF. In this study, we further explored these findings and found that CCL2 is expressed primarily by the CD8+
subset of T lymphocytes in our tumor model. Since CCL2 is known to affect tumor growth directly via its pro-angiogenic activity and indirectly by attracting monocytes that secrete tumor-promoting factors [50
], secretion of CCL2 by T cells may affect tumor growth in a similar manner. Our previous studies have shown that increased production of CCL2 may also play a role in compromised T cell effector function, as exposure of the T cells to recombinant CCL2 resulted in the decreased secretion of IFN-γ [19
]. Thus, the increased levels of CCL2 may contribute towards tumor progression by inhibiting anti-tumor effects of T lymphocytes, as well as by promoting angiogenesis.
The expression of CCL5 by tumor cells has been correlated with tumor progression in several types of cancer, including breast cancer [12
]. The DA-3 mammary tumor cells used in this study expressed low levels of CCL5, which does not account for the levels observed in circulation. We therefore investigated for other sources of this chemokine. In this report, we have shown that splenic T lymphocytes, as well as tumor-infiltrating CD8+
T cells, express CCL5 and that the receptors for CCL5, CCR1 and CCR3 are expressed at high levels in TILs and T cells of tumor bearers, compared to normal mice. Two possible explanations may be made for the role of differential expression of CCL5 and CCL5 receptors in tumor bearers’ T cells in our study. It is possible that tumor derived CCL5 is responsible for lymphocyte infiltration into tumors to help establish anti-tumor immunity or conversely, CCL5 may recruit T cells which promote tumor growth. Fischer et al. [52
] showed that CCL5 produced by Reed Sternberg cells is one mechanism by which CCR3+
mast cells can be attracted into the tumor tissue in Hodgkin’s lymphoma. Furthermore, Robinson et al. showed that CCR1+
monocytes are attracted to a mammary tumor expressing CCL5, and that treatment with a CCR1 receptor antagonist resulted in decreased tumor growth [28
]. Likewise, the interaction of mammary tumor cells and lymphocytes may play an important role in tumor growth. In contrast, Mellado et al. have shown that the binding of CCL5 to CCR5 promotes cell death of tumor infiltrating cells via activation of a cytochrome c-dependent pathway [53
]. Okita et al. have found that gastric tumor cells acquire an invasive potential through interaction with peripheral blood mononuclear cells and that CCL5 plays a role in this interaction [54
]. Contrary to this, studies by Jayasinghe et al., investigating the role of CCL5 in tumor progression, found that tumor-derived CCL5 does not contribute to breast cancer progression and pointed towards a role for host-derived chemokines in the progress of the disease [18
]. Future studies in our laboratory will investigate the role of T lymphocyte-derived CCL5 in mammary tumor progression.
The CXC chemokines are important regulators of tumor growth and metastasis, as they play an extensive role in angiogenesis. Production of CXCL2 is associated with macrophages, endothelial cells, epithelial cells and tumor cells [55
]. Tumor growth has been linked to increased angiogenesis due to the interaction of CXCL2 with endothelial cell expressed CXCR2 [57
]; in addition, IL-8, which is the human analogue of mouse CXCL2, was found to act as a direct autocrine growth factor for malignant melanoma [58
], liver and pancreatic tumors [59
], and for colon carcinoma cells [60
]. In this study, we show that in addition to being produced by the DA-3 mammary tumor cells, splenic T lymphocytes of mammary tumor-bearing mice also secrete CXCL2.
We have demonstrated elevated levels of CCL2 in the circulation of mammary tumor-bearing mice and have shown that T cells and more importantly, tumor-infiltrating T cells, express this chemokine [19
]. In this study we analyzed whether CCL2 plays a role in the induction of other inflammatory chemokines or their receptors. We show for the first time that T cells treated with CCL2 upregulate the gene expression of CCL5 and CXCL2 and the receptors, CCR1, -2, and -3. It is important to note that CCL2 is known to recruit monocytes that differentiate into TAMs within the tumor microenvironment and facilitate tumor progression [61
]. Thus, CCL2 may prove to be a target for immunotherapy in cancer, and is currently being investigated in Phase I clinical trials [63
Enhanced leukocyte infiltration of tumors has been associated with increased tumor vascularity [64
]. The chemokines expressed in our tumor system can exert angiogenic effects either by directly acting on the cancer cells or indirectly through the effector cells they recruit. Soria et al. have shown that co-expression of CCL5 and CCL2 in the same tumor was associated with more advanced stages of breast cancer and have suggested that breast tumors “benefit” from interactions between the two chemokines [12
]. Our studies show that splenic and tumor-infiltrating T lymphocytes of mammary tumor-bearing mice secrete CCL2, CCL5 and CXCL2 and express CCR1, -2, -3 and CXCR2. Other studies suggest that chemokine receptor expression, e.g., the CCL5 receptors CCR 1 and CCR5, contribute to breast tumor development as treatment with CCR1/CCR5 receptor antagonists reduced the volume and weight of the tumors [28
]. Based on these findings, we postulate that T cells of tumor-bearing mice may be promoting tumor growth instead of inhibition due to increased expression of angiogenic chemokines and their receptors. In order to validate a role for these T lymphocyte-derived molecules in mammary tumor progression, we plan to explore the effects of gene silencing on D1-DMBA-3 tumor growth and angiogenesis.