TY - JOUR
T1 - Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments
AU - Goddard, Erica T.
AU - Hill, Ryan C.
AU - Barrett, Alexander
AU - Betts, Courtney
AU - Guo, Qiuchen
AU - Maller, Ori
AU - Borges, Virginia F.
AU - Hansen, Kirk C.
AU - Schedin, Pepper
N1 - Funding Information:
The authors would like to acknowledge Jacob Fischer for assisting with experimental metastasis studies, Hadley Holden for performing special stains, and Weston Anderson for providing editorial review of the manuscript. Monika Dzieciatkowska assisted with QconCAT generation and validation. Finally, the work included in this manuscript includes funding from NIH/NCI NRSA F31CA186524 (to ETG), NIH/NCATS Colorado CTSI UL1 TR001082 for proteomic support, NIH/NCI R33CA183685 (to KH), DOD BC123567 (to PS), BC123567P1 (to KH), and NIH/NCI 5R01CA169175 (to VB and PS). The authors declare no competing financial interests.
Publisher Copyright:
© 2016 The Authors
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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.
AB - 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.
KW - Breast cancer
KW - Extracellular matrix
KW - Liver
KW - Liver metastasis
KW - Mammary gland
KW - Mass spectrometry proteomics
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U2 - 10.1016/j.biocel.2016.10.014
DO - 10.1016/j.biocel.2016.10.014
M3 - Article
C2 - 27771439
AN - SCOPUS:85002125744
SN - 1357-2725
VL - 81
SP - 223
EP - 232
JO - International Journal of Biochemistry and Cell Biology
JF - International Journal of Biochemistry and Cell Biology
ER -