Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments

Abstract

Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization

“…CNBr cleavage was specific to the C-termini of methionine residues as reported 12, 18 . In the case of CNBr database searches, we also included two additional variable modifications, homoserine (Met to Hse) and homoserine lactone (Met to Hsl).…”

“…Skyline was used for method development and to extract the ratio of endogenous light peptides to heavy internal standards from LC-SRM data for protein quantification as described 26 . LC-MS/MS data was processed as previously described 12 . Limits of detection, quantification, and dynamic range were determined for each peptide as previously described 11 .…”

“…After this extraction an insoluble pellet remains that contains a significant amount of protein based on strong acid hydrolysis and amino acid analysis 11 . It was recently shown that greater than 80% of fibrillar collagen in murine liver and mammary gland resides in the iECM fraction 12 – a fraction excluded from traditional proteomic methods. Additionally, we have shown previously that the use of chemical digestion with cyanogen bromide (CNBr), prior to enzymatic cleavage with trypsin, is both necessary and sufficient to fully solubilize insoluble ECM components, such as fibrillar collagen from the liver and lungs 11, 12 .…”

Mammary extracellular matrix directs differentiation of testicular and embryonic stem cells to form functional mammary glands in vivo

“…Given the wealth of literature showing the unique proteinaceous composition of the mammary ECM, which also alters during distinct developmental stages12202425262728, pinpointing the exact molecules and the orchestration of signaling between the stroma and parenchyma to direct cell behavior is a challenge. ECM isolated from nulliparous rats has been shown to promote the formation of epithelial ducts with bifurcation, while matrix isolated from mid-involuting mammary glands induced cell death.…”

Physical and Chemical Gradients in the Tumor Microenvironment Regulate Tumor Cell Invasion, Migration, and Metastasis

“…Intriguingly, using this approach and relying on analysis that uses species-specific peptides, it was revealed that many of the tumor ECM proteins including a number of abundant laminins (LAMA3, B3, C2) and Sned1 are secreted predominantly by the tumor cells, whereas other ECM components such as tenascin C (TNC) and vitronectin are secreted exclusively by resident fibroblasts and infiltrating stromal cells (Naba et al 2014). Complementing these studies, advanced proteomic approaches have been able to profile the insoluble ECM proteins within the tumor, revealing distinct insoluble protein profiles within a tumor type that not only reflect the tumor stage but also the tumor genotype (Goddard et al 2016;Laklai et al 2016). Data revealed that the most aggressive pancreatic tumors that harbor SMAD4 mutations and that are associated with the shortest patient survival deposited quantities of collagen 12, TNC, and fibronectin (FN) nests surrounding the invading ductal lesions ).…”