Development of an Enhanced-Throughput Radial Cell Migration Device.

It is often desirable to evaluate the ability of cells to move in an unrestricted manner in multiple directions without chemical gradients. By combining the standard radial migration assay with injection-molded gaskets and a rigid fixture, we have developed a highly reliable and sensitive method for observing and measuring radial cell migration. This method is adapted for use on high-throughput automated imaging systems. The use of injection-molded gaskets enables low-cost replacement of cell-wetted components. Moreover, the design enables secondary placement of attractants and co-cultures. This device and its enhanced throughput permit the use of therapeutic screening to evaluate phenotypic responses, for example, cancer cell migration response due to drugs or chemical signals. This approach is orthogonal to other 2D cell migration applications, such as scratch wound assays, although here we offer a noninvasive, enhanced-throughput device, which currently is not commercially available but is easily constructed. The proposed device is a systematic, reliable, rapid application to monitor phenotypic responses to chemotherapeutic screens, genetic alterations (e.g., RNAi and CRISPR), supplemental regimens, and other approaches, offering a reliable methodology to survey unbiased and noninvasive cell migration.

IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling.

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.