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

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.

The Composition of Surgical Wound Fluids from Breast Cancer Patients is Affected by Intraoperative Radiotherapy Treatment and Depends on the Molecular Subtype of Breast Cancer

“…Regulates T cell recruitment under homeostatic and inflammatory conditions [5] G-CSF Controls the maturation of neutrophils [8] HGF Induces cancer cell migration and invasion, wound healing, angiogenesis [21] IL-1 beta Inhibits the proliferation of some cancers, amplifies the function of dendritic cells, stimulates the proliferation and differentiation of CD4, CD8 and natural killer (NK) cells, increases antibody production by lymphocytes B, and finally increases the expression of adhesion molecules on vascular endothelium [22] IL-12 Induction of Th1 cells [23,24] IL-13 Induces the activation of M2 macrophages, polarizes M2a macrophages and induces migration and invasion of breast cancer cells [25,26] [27,28] IL-8 Increased the formation of primary and secondary tumor spheres, as well as the cancer stem cell phenotype in BC [29] MCP-1 Increases monocyte mobilization and poor survival and is involved in monocyte polarization to M2-cells [30] MCP-3 Attracts monocytes, dendritic cells (DCs), and activated T lymphocytes, to invasion sites [31] MIF Induces myeloid-derived suppressor cells in tumor microenvironment [32] PDGF-beta Regulates metastasis and vascular remodeling in cancer [33]…”

“…M2 macrophages have also been implicated in tumor growth [42,43], and their presence has been correlated with a poor prognosis [44]. Recent data published by Little et al showed that IL-4/IL-13 polarizes M2a macrophages and induces migration and invasion of breast cancer cells [26]. Based on those findings, we can assume that IORT treatment may be beneficial not only but direct tumor cell killing but also in changing the tumor bed microenvironment, making it less favorable for tumor recurrence due to decreased concentration of tumor-facilitating cytokines.…”

Integration of high-content fluorescence imaging into the metabolic flux assay reveals insights into mitochondrial properties and functions

“…The Rho-like family of Rho-GTPases have been well characterized for their participation in the progression of breast cancer 32,34–36 and the alteration of metabolic phenotype 33 . Therefore, we next applied our imaging and analysis strategy to explore the potential for fragmented mitochondria in breast cancer cells that lack either the Rho-like family of Rho-GTPases RhoA or RhoC, via CRISPR/Cas9 knockout (WT, RhoA KO, RhoC KO) ( Supplementary Fig.…”

“…Finally, application of this strategy yielded insights into the role of Rho-GTPases in mitochondrial dynamics in breast cancer. While the roles of the Rho-like family of Rho-GTPases in breast cancer progression were well characterized, our study is the first to determine their role in the regulation of mitochondrial content, fragmentation, and respiratory capacity 32–36 . Collectively this study enhances the utility of the metabolic flux assay and provides a more complete platform to study mitochondrial biology from multiple dimensions, simultaneously.…”

Development of a high-throughput radial migration device

“…A highly reproducible assay allows for fewer experiments to be performed to arrive at convincing and robust conclusions, saving time and resources. This is particularly useful in the design of high value chemotherapeutics that aim to inhibit cancer metastasis or understand the relationship between genetic mediators of cancer cell migration or invasion; examples can be seen here (15,16).…”