CXCL12 signaling is a promising therapeutic target in breast cancer and many other common human malignancies, based on numerous studies showing key functions of this chemokine in primary tumor growth and metastasis. While initial efforts focused on CXCR4 , identification of CXCR7 as a second receptor for CXCL12 increased the complexity of this chemokine in cancer biology and as a drug target26, 27
. To understand functions of CXCL12 in cancer and optimize development and application of new chemotherapeutic agents, it is essential to determine interrelated effects of CXCR4 and CXCR7 in vivo
Toward the objective of understanding functions of CXCR7 in living subjects, we developed a new bioluminescence imaging method based on Gaussia
luciferase to quantify chemokine scavenging by CXCR7 in a mouse model of human breast cancer. Using this imaging technique, we established that CXCR7 reduced CXCL12 in primary breast tumors and significantly limited release of this chemokine into the circulation. These data build upon prior in vitro
studies showing that CXCR7 scavenges and degrades chemokine ligands13-15
. Furthermore, this imaging strategy allowed us to analyze effects of pharmacologic inhibition of CXCR7 chemokine scavenging in living mice and compare the extent of inhibition to complete absence of CXCR7+ breast cancer cells in orthotopic tumors. Using in vivo
imaging and ex vivo
quantification of CXCL12-GL levels in serum will inform and direct ongoing efforts to develop molecules and treatment protocols that effectively block CXCR7-dependent chemokine scavenging in vivo
We used bioluminescence imaging to selectively monitor growth of CXCR4+ breast cancer cells in an orthotopic tumor xenograft model of human breast cancer. In this model, we used separate populations of breast cancer cells expressing either CXCR4 or CXCR7. This experimental strategy reproduces primary human breast cancers in which we found these receptors to be expressed on different populations of malignant cells in the same tumor. For studies of tumor growth and metastasis, we also implanted human mammary fibroblasts stably expressing CXCL12-GL, based on prior studies showing carcinoma associated fibroblasts as a key source of this chemokine in primary breast tumors2, 12
. While 231 cells used in these studies do not endogenously express CXCL12 (data not shown), some breast cancer cells do secrete this chemokine. Other cell types in tumors, such as endothelium, also are potential sources of CXCL12 (ref.28
). In these settings, CXCL12-GL secreted by stromal fibroblasts serves as a marker for localization and total amounts of this chemokine originating in tumor xenografts.
Using this mouse model, we discovered that a separate population of CXCR7+ breast cancer cells significantly increased overall growth of CXCR4+ cells, establishing important intercellular interactions between these receptors in cancer. CXCL12 signaling through CXCR4 promotes growth of primary tumors in breast cancer and other malignancies. These effects of CXCR4 occur through mechanisms including proliferation of malignant cells, vasculogenesis, and angiogenesis. Using bioluminescence imaging to selectively monitor CXCR4+ breast cancer cells, we established that CXCR7+ breast cancer cells in orthotopic tumors enhanced overall proliferation of CXCR4+ tumor cells. By comparison, we did not detect any effects of CXCR7-expressing breast cancer cells on recruitment of endothelial progenitor cells or tumor angiogenesis. Collectively, these data show that increased tumor growth was mediated by effects of CXCR7 on CXCR4+ breast cancer cells in the same local tumor microenvironment.
The presence of CXCR7+ breast cancer cells in a primary tumor also enhanced metastasis of CXCR4+ breast cancer cells. This result is consistent with prior studies showing that CXCR7 is required for normal chemotaxis of CXCR4+ cells in response to CXCL12. During migration of primordial germ cells in zebrafish, absence of CXCR7 disrupts normal trafficking of these CXCR4+ cells toward CXCL12 (ref.13
). Similarly, genetic deficiency of CXCR7 prevents appropriate migration of CXCR4+ neurons toward CXCL12 during development22
. Using a microchannel device to precisely pattern cell positions, we have shown that a separate population of CXCR7 cells is essential for migration of CXCR4+ breast cancer cells toward cells producing CXCL12 (ref.11
). Furthermore, computational modeling revealed that formation of chemotactic gradients was critically dependent upon distances and relative positions of cells producing CXCL12, CXCR4+ cells, and CXCR7+ cells, respectively. These data establish that CXCL12 and CXCR7 cells function in a source-sink model of gradient formation for CXCR4-dependent chemotaxis. We propose that CXCR7 cells perform the same function in primary breast tumors, establishing gradients of CXCL12 that increase invasion and intravasation of CXCR4+ breast cancer cells into the circulation to form metastases.
In addition to generating chemotactic gradients, CXCR7 may promote tumor growth by maintaining cell surface levels of CXCR4 during chronic stimulation with CXCL12. When treated for extended time with CXCL12, 231-CXCR7 cells limited internalization of CXCR4 on co-cultured 231-CXCR4 breast cancer cells. These results parallel recent studies in mice showing that genetic deletion of CXCR7 elevates CXCL12 and causes loss of CXCR4 expression in developing neurons22, 23
. By limiting desensitization of CXCR4 to chronic CXCL12, CXCR7 may maintain CXCL12-CXCR4 signaling and promote local growth and metastasis of a separate population of CXCR4+ breast cancer cells.
We established that treatment with a small molecule inhibitor of CXCL12-CXCR7 binding partially blocked chemokine scavenging in breast tumors and increased amounts of chemokine released systemically. Pharmacologic inhibition of CXCR7 chemokine scavenging limited proliferation of CXCR4+ breast cancer cells in orthotopic tumors that also contained CXCR7+ cancer cells. These results may be relevant to human breast cancers comprised of malignant cells expressing CXCR4 or CXCR7. Since CXCR7 is upregulated on blood vessels in many common cancers, compounds that block CXCL12 binding to this receptor also may benefit patients with tumors containing only CXCR4+ cancer cells. While pharmacologic inhibition of CXCR7 limited local growth of CXCR4+ cells in orthotopic primary tumors, treatment with CCX771 had minimal effects on metastasis of these cells. This result may be due to incomplete inhibition of chemokine scavenging. Relative differences in serum levels of CXCL12-GL between mice with or without CXCR7+ breast cancer cells in tumors were greater than differences between mice with CXCR7+ tumor cells treated with CCX771 or vehicle. Potentially, more complete inhibition of CXCR7 chemokine scavenging will be required to inhibit metastasis of CXCR4+ cells. Alternatively, since molecules and pathways other than CXCL12-CXCR4 are required for metastasis, the result also suggests that CXCR7+ cells may promote metastasis of CXCR4+ cells through mechanisms other than chemokine scavenging.
We used human breast cancer cells and mammary fibroblasts for these studies to directly link our mouse model to human breast cancer biology. This selection necessitates use of immunocompromised mice. Immunocompromised mouse models of breast cancer and other malignancies have substantially advanced knowledge of CXCL12, CXCR4, and other chemokine pathways in tumor growth and metastasis29, 30
. An inherent limitation of immunocompromised mice is the inability to investigate effects of specific molecules and receptors on host immune responses to tumor progression at primary and metastatic sites. Further studies in immunocompetent mice or mice with partially humanized immune systems will be required to determine integrated effects of CXCL12, CXCR4, and CXCR7 on host immune responses to breast cancer.
In summary, we developed a new bioluminescence imaging method to analyze levels of CXCL12 in living subjects and used this technology to establish that CXCR7 limits amounts of CXCL12 in primary breast tumors in vivo
. CXCR7+ breast cancer cells promote growth and metastasis of a separate population of CXCR4+ breast cancer cells in a mouse model of human breast cancer, indicating that CXCR7 enhances effects of CXCR4 to increase tumor progression. We note that functions of CXCR7 as a scavenger receptor for CXCL12 do not preclude other potential mechanisms of action reported for this receptor in cancer biology, including direct activation of signaling pathways, resistance to apoptosis, and/or enhanced cell migration31-33
. These studies advance our understanding of intercellular regulation of CXCR4 by CXCR7 in breast cancer. This knowledge is essential for ongoing development and clinical translation of therapies targeting CXCL12 signaling in breast cancer and other malignancies.