Abstract PD9-09: Proteomic analysis of extracellular matrix helps define drivers of metastatic progression
Authors: Brechbuhl HM , Barrett AS , Kopin E , Gillen A , Hagen J , Johnson L , Finaly-Shultz J , Sartorius CA , Hansen KC , Kabos P 
Published: May 2019

Abstract
Tumor microenvironment (TME) plays a central role in the development of distant metastasis. However, given its complexity, successful targeting of TME will require a detailed understanding of its composition. Cancer-associated fibroblasts represent a large component of TME and are a major contributor to the extracellular matrix (ECM). Previously, we demonstrated the presence of two fibroblast sub-populations (CDCP1pos vs CD146pos fibroblasts) in breast cancer TME, which determine therapeutic response in estrogen receptor (ER) positive breast cancer. We hypothesized that the same fibroblast subtypes would also influence ECM composition and alter the metastatic potential of breast cancer cells. Here, we present the development of a novel fibroblast driven orthotopic model of ER+ breast cancer metastasis, which we combined with an innovative proteomics approach to precisely quantify extracellular matrix proteins.
Results: 1.CDCP1pos fibroblasts promote increased breast cancer cell mobility, invasion and metastasis compared to CD146pos fibroblasts.We demonstrate that CDCP1pos fibroblasts significantly increase the invasion potential of breast cancer cells, when compared to CD146pos fibroblasts. Orthotopic co-implantation of ER+ tumor cells with CDCP1pos fibroblasts into the mammary fat pad of mice more frequently drives distant organ metastases to lung and brain when compared with tumors implanted with CD146pos fibroblasts. 2. Proteomic analysis of ER+ tumors influenced by CDCP1pos fibroblasts revealed known and novel drivers of breast cancer metastasis. Breast cancer cells mixed with CDCP1pos fibroblasts produce a non-uniform collagen orientation to the tumor boarder. Our novel proteomic analysis of TME specific proteins revealed that tumors influenced by CDCP1pos fibroblasts have high expression of many ECM proteins linked to increased risk of breast cancer metastasis, including TNC, FN1, COL5A3, and FBN1 among others. Derived proteomic TME signature accurately predicted lymph node involvement in patients who presented with early stage (T0 and T1) tumors in a cohort of 1,009 breast cancer patients from Cancer Genome Atlas Database. 3. Inhibiting fibroblast production of Tenascin C (TNC) results in decreased breast cancer cell invasion. Our in vitro mixed co-culture models, which contain ER+ breast cancer cells with both fibroblast subtypes, demonstrate that only CDCP1pos fibroblasts produce TNC. Furthermore, in spheroid assays with CDCP1posfibroblasts and breast cancer tumor cells, invasion is inhibited by TNC knockdown. The invasion phenotype can be rescued by addition of EGF, which suggests TNC promotes invasion via EGFR signaling.
Conclusion: Metastatic spread of cancer cells relies heavily on TME alterations and makeup of the extracellular matrix. Our data suggest that fibroblast composition directly influences ECM properties and metastatic potential in breast cancer. Taken together, we believe that a better understanding of ECM composition will lead to a more personalized approach to breast cancer treatment.
Citation Format: Brechbuhl HM, Barrett AS, Kopin E, Gillen A, Hagen J, Johnson L, Finaly-Shultz J, Sartorius CA, Hansen KC, Kabos P. Proteomic analysis of extracellular matrix helps define drivers of metastatic progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-09.