The bidirectional crosstalk between macrophages and cancer cells via CX3CR1- and CCR2-signaling : fundamental for lung cancer growth and metastasis

Recent studies indicate that tumor-associated macrophages with an M2 phenotype can play a critical role in tumor growth and metastasis. However, the regulatory pathways remain poorly characterized. Using macrophage-depleted mice, the present study confirms a major contribution of macrophages to primary lung cancer growth and metastatic spread. Furthermore, the data demonstrates that M2-polarized macrophages play a critical role in establishing the vessel structure and microvasculature associated with tumors. Depletion of macrophages restricts the expression of matrix metalloproteinases and vascular endothelial growth factor and thus results in an overall regressive tumor microenvironment. Co-culture experiments of macrophages with lung carcinoma cells revealed cancer-cell-mediated M2-polarization and subsequent M2-macrophage-induced proliferation, migration and CCR2/CX3CR1 expression on carcinoma cells. Importantly, adenocarcinoma cells initiate a vicious circle with an abundant secretion of tumor-associated macrophage-attracting CCL2/CX3CL1, and then recruited macrophages polarize into M2, enhance CCL2/CX3CL1 secretion and induce CCR2/CX3CR1 expression of cancer cells with the functional consequence of enhanced cancer cell proliferation and migration. The co-culture-induced cytokines interleukin-10, interleukin-6, interleukin-1beta, and interleukin-23, CCL1, macrophage-colony stimulating factor, tumor necrosis factor alpha, macrophage inflammatory proteins 1alpha and 1beta, granulocyte-colony stimulating factor and soluble intercellular adhesion molecule 1 may play a crucial role in this cycle. Inhibition of CCR2/CX3CR1 signaling on either lung cancer cells or macrophages attenuates co-culture induced proliferation and migration. In conclusion, lung adenocarcinoma cells hijack CCR2/CX3CR1+ macrophages to support their own proliferation and migration. Consistent with these observations, both CX3CR1-/- and CCR2-/- mice had reduced primary tumor size, lung metastasis and numbers of M2-polarized macrophages compared to WT mice. With the absence of CCR2+ or CX3CR1+ macrophages induction of CCR2/CX3CR1 on cancer cells also failed in vivo. These results support a model whereby TAMs play a critical, chemokine dependent role in the regulation of lung cancer progression. Furthermore, studies with CX3CR1-/- and CCR2-/- reveal a similar reduction of tumor-associated neoangiogenesis with in particular targeted microvasculature and moreover a similar restriction of matrix metalloproteinases and vascular endothelial growth factor expression compared to total macrophage-depleted mice. Consequently, this study indicates CCR2 and CX3CR1 as promising drug targets to inhibit cancer progression and metastasis by directly manipulating the tumor microenvironment. The enhanced concentration of CCR2 and CX3CR1 expression in the tumor microenvironment provides ideal conditions for this purpose. Finally, not only macrophage infiltration but also the ratio of CCR2+ macrophages within human lung adenocarcinoma and squamous cell carcinoma correlate with lung cancer stage and metastasis. These observations confirm the human relevance of TAM-associated CCR2/CX3CR1-signaling cascades. Altogether, these results provide a mechanism for the regulation of lung tumor growth and metastasis by tumor-associated macrophages, and targeting M2-macrophage expressed receptors provides a new strategy for the development of lung cancer therapeutics