The extracellular matrix (ECM), a major component of the tumor microenvironment, promotes local invasion to drive metastasis. Here, we describe a method to study whole-tissue ECM effects from disease states associated with metastasis on tumor cell phenotypes and identify the individual ECM proteins and signaling pathways that are driving these effects. We show that decellularized ECM from tumor-bearing and obese mammary glands drives TNBC cell invasion. Proteomics of the ECM from the obese mammary gland led us to identify full-length collagen VI as a novel driver of TNBC cell invasion whose abundance in tumor stroma increases with body mass index in human TNBC patients. Last, we describe the mechanism by which collagen VI contributes to TNBC cell invasion via NG2-EGFR cross-talk and MAPK signaling. Overall, these studies demonstrate the value of decellularized ECM scaffolds obtained from tissues to identify novel functions of the ECM.

Triple-negative breast cancer (TNBC) is the most aggressive and deadly subtype of breast cancer, accounting for 30,000 cases annually in the US. While there are several clinical trials ongoing to identify new agents to treat TNBC, the majority of TNBC patients are treated with anthracycline- or taxane-based chemotherapies in the neoadjuvant setting, followed by surgical resection and adjuvant chemotherapy. While many patients respond well to this approach, as many as 25% will suffer local or metastatic recurrence within five years. Understanding the mechanisms that drive recurrence after chemotherapy treatment is critical to improving survival for patients with TNBC. It is well-established that the extracellular matrix, which provides structure and support to tissues, is a major driver of tumor growth, local invasion and dissemination of cancer cells to distant metastatic sites. In the present study, we show that decellularized extracellular matrix (dECM) obtained from chemotherapy-treated mice increases motility of treatment-naïve breast cancer cells compared to vehicle-treated dECM. Tandem-mass-tag proteomics revealed that anthracycline- and taxane-based chemotherapies induce drug-specific changes in tumor ECM composition. The basement membrane protein collagen IV was significantly upregulated in the ECM of chemotherapy-treated mice and patients treated with neoadjuvant chemotherapy. Collagen IV drove invasion via activation of Src and focal adhesion kinase signaling downstream of integrin α1 and α2, and inhibition of collagen IV-driven signaling decreased motility in chemotherapy-treated dECM. These studies provide a novel mechanism by which chemotherapy may induce metastasis via its effects on ECM composition.

Triple-negative breast cancer (TNBC) is the most aggressive and deadly subtype of breast cancer, accounting for 30,000 cases annually in the US. While there are several clinical trials ongoing to identify new agents to treat TNBC, the majority of TNBC patients are treated with anthracycline- or taxane-based chemotherapies in the neoadjuvant setting, followed by surgical resection and adjuvant chemotherapy. While many patients respond well to this approach, as many as 25% will suffer local or metastatic recurrence within five years. Understanding the mechanisms that drive recurrence after chemotherapy treatment is critical to improving survival for patients with TNBC. It is well-established that the extracellular matrix, which provides structure and support to tissues, is a major driver of tumor growth, local invasion and dissemination of cancer cells to distant metastatic sites. In the present study, we show that decellularized extracellular matrix (dECM) obtained from chemotherapy-treated mice increases invasion of treatment-naïve breast cancer cells compared to vehicle-treated dECM. Using tandem-mass-tag proteomics, we further demonstrate that anthracycline- and taxane-based chemotherapies induce drug-specific changes in tumor ECM composition. We identify the basement membrane protein collagen IV as significantly upregulated in the ECM of chemotherapy-treated mice and patients treated with neoadjuvant chemotherapy. We show that collagen IV drives invasion via Src/FAK signaling and that inhibiting collagen IV-driven signaling decreases invasion in chemotherapy-treated dECM. These studies provide a novel mechanism by which chemotherapy may induce metastasis via effects on ECM composition.One Sentence SummaryChemotherapy alters the extracellular matrix of breast tumors leading to increased invasion of residual cancer cells.

Supplementary Data from Chemotherapy-Induced Collagen IV Drives Cancer Cell Motility through Activation of Src and Focal Adhesion Kinase

Supplementary Data from Chemotherapy-Induced Collagen IV Drives Cancer Cell Motility through Activation of Src and Focal Adhesion Kinase