Abstract 2253: Proteases and cytokines in the transition from preinvasive DCIS to invasive breast carcinoma

Abstract

Abstract The transition from preinvasive ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) involves alterations in proteolytic pathways of both epithelial and stromal cells. Infiltration of macrophages, angiogenesis and secretion of chemotactic molecules by DCIS and surrounding stromal cells also facilitate this transition. We have developed a 3D coculture model, MAME (mammary architecture and microenvironment engineering), to study the role of proteases and cytokines in the progression from pre-invasive DCIS to IDC. MAME-A recapitulates the architecture of normal human breast tissue with the epithelial and stromal components growing in overlays of basement membrane and stromal collagen, respectively. In MAME-B, the stromal collagen is eliminated. The cells used in our studies were human MCF10.DCIS cells, WS-12Ti human breast tumor-associated fibroblasts, U937 human monocytes and human umbilical vein endothelial cells (HUVEC). We analyzed proteolysis through live cell imaging of the degradation of quenched fluorescent matrix proteins [DQ-collagen I™ mixed with collagen I and DQ-collagen IV™ mixed with reconstituted basement membrane (rBM)]. DCIS cells in MAME-A cocultures with fibroblasts exhibited invasiveness at 24 days, yet at only 16 days in MAME-B cocultures. Expression and secretion of cytokines, especially IL-6, were elevated in these cocultures. The invasiveness of the DCIS cells in the cocultures with fibroblasts could be reduced by addition of neutralizing antibody to IL-6. Addition of either monocytes or endothelial cells reduced the time at which invasiveness occurred to as early as 4 days in MAME-B cocultures. Addition of monocytes increased secretion of monocyte chemoattractant protein-1 (MCP-1), IL-8 and GRO. The combination of a neutralizing antibody to hMCP-1 and a receptor-blocking antibody to hCXCR2 (a receptor for IL-8 and GRO) reduced invasiveness more effectively than did either of the antibodies used alone. When HUVEC were grown in MAME-B cocultures with DCIS or DCIS and fibroblasts, the HUVEC grew into a network of tubules with distinct lumens over a period of 4 days. In 3D and 4D reconstructions of these same cocultures, DCIS cells could be seen to migrate toward the HUVEC tubules and invade into their lumens. These phenomena occurred in parallel with increases in proteolysis and in expression and secretion of the cytokines MCP-1, IL-8, RANTES and GRO. The MAME models provide a system for the study of proteolytic and cytokine pathways and the interactions between these pathways that are associated with the transition from DCIS to invasive ductal carcinoma. This model could be used as an in vitro tool for screening drugs that target these pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2253.