Paracrine Activation of STAT3 Drives GM-CSF Expression in Breast Carcinoma Cells, Generating a Symbiotic Signaling Network with Breast Carcinoma-Associated Fibroblasts

Abstract

This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell–cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation of GM-CSF followed by a marked decrease, indicating a tightly regulated control of GM-CSF in CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such as IL-6, IL-6R and IL-8, thereby forming a positive feedback loop. We propose that the tight regulation of GM-CSF in CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell–cell communication network governing the BCa TME.