Abstract
Tumor associated macrophages (TAMs) are increasingly recognized as major contributors to the metastatic progression of breast cancer and enriched levels of TAMs often correlate with poor prognosis. Despite our current advances it remains unclear which subset of M2-like macrophages have the highest capacity to enhance the metastatic program and which mechanisms regulate this process. Effective targeting of macrophages that aid cancer progression requires knowledge of the specific mechanisms underlying their pro-metastatic actions, as to avoid the anticipated toxicities from generalized targeting of macrophages. To this end, we set out to understand the relationship between the regulation of tumor secretions by Rho-GTPases, which were previously demonstrated to affect them, macrophage differentiation, and the converse influence of macrophages on cancer cell phenotype. Our data show that IL-4/IL-13 in vitro differentiated M2a macrophages significantly increase migratory and invasive potential of breast cancer cells at a greater rate than M2b or M2c macrophages. Our previous work demonstrated that the Rho-GTPases are potent regulators of macrophage-induced migratory responses; therefore, we examined M2a-mediated responses in RhoA or RhoC knockout breast cancer cell models. We find that both RhoA and RhoC regulate migration and invasion in MDA-MB-231 and SUM-149 cells following stimulation with M2a conditioned media. Secretome analysis of M2a conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Results from our functional assays reveal that M2a TAMs synergistically utilize VEGF and CCL-18 to promote migratory and invasive responses. Lastly, we show that pretreatment with ROCK inhibitors Y-276332 or GSK42986A attenuated VEGF/CCL-18 and M2a-induced migration and invasion. These results support Rho-GTPase signaling regulates downstream responses induced by TAMs, offering a novel approach for the prevention of breast cancer metastasis by anti-RhoA/C therapies.
Highlights
Since the mid-1990s, strategies to detect and treat early breast tumors have greatly improved, reflected in improved survival rates [1]
To examine the effects the diverse M2-like macrophage populations had on breast cancer cells, we first induced in vitro monocyte-to-macrophage polarization by the addition of macrophage colony stimulating factor (M-CSF) to U937 monocyte cells
Primer efficiency for reverse transcriptase-quantitative PCR (RT-qPCR) primers utilized in this study were verified to ensure fidelity, and primer sequences are listed in Supplementary Table 1
Summary
Since the mid-1990s, strategies to detect and treat early breast tumors have greatly improved, reflected in improved survival rates [1]. While some patients can live for years with late-stage metastasis, early diagnosis of metastatic dissemination offers no improvement to 5-year survival rates over diagnosing metastases when symptoms occur, most likely due to our current lack of available therapies designed to target metastases and/or inhibit widespread cancer cell dissemination from a micrometastatic disease stage. An environment of chronic inflammation is presumed to direct macrophage polarization toward an anti-inflammatory, M2-like phenotype [7]. Despite the rapidly growing number of studies which have characterized TAMs, the vast number of secreted factors by cancer cells and other cells of the TME leads to a diverse and transient TAM population that can readily switch between polarization states. Characterizing which M2 population of macrophages (e.g., M2a, M2b, M2c) are responsible for promoting tumorigenic outcomes in breast cancer remains an important, but not yet achieved goal
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