Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.

Optical techniques, such as bioluminescence and fluorescence, are emerging as powerful new modalities for molecular imaging in disease and therapy. Combining innovative molecular biology and chemistry, researchers have developed optical methods for imaging a variety of cellular and molecular processes in vivo, including protein interactions, protein degradation, and protease activity. Whereas optical imaging has been used primarily for research in small-animal models, there are several areas in which optical molecular imaging will translate to clinical medicine. In this review, we summarize recent advances in optical techniques for molecular imaging and the potential impact for clinical medicine.

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. 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. 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.

Bioluminescence imaging (BLI) has emerged as a powerful new method to analyse infectious diseases in animal models. BLI offers real-time monitoring of spatial and temporal progression of infection in the same animal, as opposed to euthanizing a cohort of animals and quantifying colony or plaque forming units at multiple time points. Pathogens or mice are engineered to express genetically encoded luciferase enzymes from bacteria, insects, or the sea pansy. The seminal study showing the feasibility of detecting microbially generated luminescence within a living mouse was published by Contag and colleagues in 1995, using Salmonella typhimurium transformed with the lux operon from Photorhabdus luminescens. Following this, they and others performed many studies of infection by bioluminescent Gram-negative and Gram-positive bacteria. Viruses can also be engineered to encode luciferase. Our laboratory has used bioluminescent reporter viruses to follow HSV and vaccinia pathogenesis; others have used an alphavirus or novirhabdovirus. Recently, even eukaryotic parasites Plasmodium, Leishmania and Toxoplasma have been transformed with luciferase and yielded unique insights into their in vivo behaviour. We expect that both the range of organisms and the molecular events able to be studied by BLI will continue to expand, yielding important insights into mechanisms of pathogenesis.

CXCR7 is a receptor for chemokines including CXCL12 (stromal-derived factor-1), a molecule that promotes tumor growth and metastasis in breast cancer and other malignancies. Building on the recent observation that CXCR7 sequesters CXCL12, we investigated mechanisms for CXCR7-dependent uptake of chemokines. Breast cancer cells expressing CXCR7 accumulated chemokines CXCL12 and CXC11 present at concentrations <1 ng/ml, unlike cells expressing CXCR4. CXCR7-dependent accumulation of chemokines was reduced by inhibitors of clathrin-mediated endocytosis. After CXCR7-mediated internalization, CXCL12 trafficked to lysosomes and was degraded, although levels of CXCR7 remained stable. CXCR7 reduced CXCL12 in the extracellular space, limiting the amounts of chemokine available to acutely stimulate signaling through CXCR4. CXCR7 constitutively internalized and recycled to the cell membrane even in the absence of ligand, and addition of chemokines did not significantly enhance receptor internalization. Chemokines at concentrations less than the Kd values for ligand–receptor binding did not alter levels of CXCR7 at the cell surface. Higher concentrations of chemokine ligands reduced the total cell surface expression of CXCR7 without affecting receptor internalization, indicating that receptor recycling was inhibited. CXCR7-dependent uptake of chemokines and receptor trafficking were regulated by β-arrestin 2. These studies establish mechanisms through which CXCR7 regulates the availability of chemokine ligands in the extracellular space.

Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7-dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small-molecule inhibitor of CXCR7 chemokine limited the growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.