Abstract PR03: Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles
Authors: Dakhlallah Duaa , Patterson Ivory , Gross Amy C. , Evans Randall , Eubank Tim D. 
Published: May 2015

Macrophage phenotypes are reported to regulate tumor progression, angiogenesis, and metastasis in breast cancer by producing soluble factors modulating these programs. Macrophages also communicate via secreted microvesicles (MVs) which are taken-up by neighboring epithelium. MVs contain mRNAs coding the epigenetic regulating machinery, DNA methyltranferases (DNMTs) and histone deacetylases (HDACs), which augment or silence expression via promoter CpG island methylation. Tie2-expressing monocytes (TEMs) is a subset of monocytes reported to augment tumor angiogenesis and metastasis. Recently, we found that increased levels of colony stimulating factor-1 (CSF1) can expand the TEM population in circulation, enabling an influx into breast tumors. Interestingly, we also found that expansion of TEMs by hypoxia was regulated by HIF-1α; and not HIF-2α, but only once they enter the tumor proper. We hypothesized that MVs secreted from M1 and M2 macrophages or TEMs contain epigenetic regulatory machinery which regulate CpG island methylation and gene expression of tumor suppressor genes (TSGs) and genes driving epithelial-to-mesenchymal transition (EMT).

We differentiated M1, M2, and TEMs in vitro from CD14+ monocytes isolated from peripheral blood. These cell populations were confirmed using flow cytometry for CD68 and CD80 for M1, CD163 for M2, and CD14/Tie2 for TEMs, as well as M1 (IL-6 and TNFα) and M2 (IL-10 and mannose receptor-1) gene expression profiles. After, we collected MVs using high speed centrifugation techniques characterized by flow cytometry and isolated their nucleic acid content. Using qRT-PCR, we found differential presence of mRNAs for DNMTs and HDACs between M1, M2, and TEM MVs. We cultured these MVs with MCF-10A normal mammary epithelial cells or BEAS-2B normal lung epithelial cells (target cells) for 24 hours and demonstrated MV uptake using Syto RNASelect (RNA) and DiIC16(3) (lipid membrane) and confocal microscopy. After, we isolated RNA and DNA from the target cells and analyzed DNMTs, HDACs, and EMT mRNA expression as well as methyl-specific PCR for CpG island methylation in the promoters of EMT genes. We found that MVs from M1 macrophages increased DNMTs mRNA expression compared to MVs produced from M2 and quiescent macrophages (M0) as well as untreated target cells. To the contrary, HDACs mRNA expression in these target cells cultured with M1-derived MVs was abrogated compared to target cells cultured with MVs from M2 and M0 macrophages and untreated target cells. As a result of the differential MV-carrying DNMTs and HDACs mRNA transferred to the target cells, we found significant differences in CpG island promoter methylation and resultant gene expression in a signature of EMT genes, including TWIST, WNT5A, VIM, FOXC2, KRT19, STAT3, SNAI1 BMP1, TGFb, DSP, AKT1, NUDT13, and ZEB1. The regulation of EMT and tumor suppressor gene promoter methylation and gene expression in MDA-MB-231 human breast cancer cells, as well as the disparate regulation of methylation and gene expression patterns on these target cells as well endothelial cells (HUVEC) by MVs collected from CD14+/Tie2+ TEMs is ongoing.

Our current and ongoing work, we establish that M1 and M2 macrophages, and TEMs, secrete MVs containing distinct epigenetic profiles which are taken-up by target cells to regulate promoter methylation and gene expression of TSGs and genes driving EMT. This program of macrophage function may be important in the progression of solid tumors via inhibition of TSGs and activation of a signature of EMT genes in normal epithelial cells as well as to direct the endothelium to support tumor progression.

This abstract is also presented as Poster A31.

Citation Format: Duaa Dakhlallah, Ivory Patterson, Amy C. Gross, Randall Evans, Tim D. Eubank. Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR03. doi:10.1158/1538-7445.CHTME14-PR03