Directed movement toward, translocation along, penetration into and exit from vascular networks by breast cancer cells in 3D.

We developed a computer-assisted platform using laser scanning confocal microscopy to 3D reconstruct in real-time interactions between metastatic breast cancer cells and human umbilical vein endothelial cells (HUVECs). We demonstrate that MB-231 cancer cells migrate toward HUVEC networks, facilitated by filopodia, migrate along the network surfaces, penetrate into and migrate within the HUVEC networks, exit and continue migrating along network surfaces. The system is highly amenable to 3D reconstruction and computational analyses, and assessments of the effects of potential anti-metastasis monoclonal antibodies and other drugs. We demonstrate that an anti-RHAMM antibody blocks filopodium formation and all of the behaviors that we found take place between MB-231 cells and HUVEC networks.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.