Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells

Abstract

Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

Identification of the Collagen Types Essential for Mammalian Breast Acinar Structures

“…Thus, understanding the structural and compositional properties of human tissue ECM is crucial for biomedical and bioengineering research. We previously reported the microstructures of decellularized human breast tissue ECM and its hydrogel [ 6 ]. In this study, we compared the proteomic compositions of normal human breast tissue ECM (HB-ECM) and human breast tissue matrix gel (HB-TMG) extracted from the breast ECM in order to verify the preservation of the native ECM proteins in HB-TMG for close mimicry of breast tissue microenvironment and biologically relevant tissue cultures.…”

“…In this study, we compared the proteomic compositions of normal human breast tissue ECM (HB-ECM) and human breast tissue matrix gel (HB-TMG) extracted from the breast ECM in order to verify the preservation of the native ECM proteins in HB-TMG for close mimicry of breast tissue microenvironment and biologically relevant tissue cultures. The preparation of HB-ECM and HB-TMG as well as liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis of the samples were performed as we previously reported [ 6 , 7 , 8 ], with modifications, and have been described in the methods.…”

“…We previously reported that MCF10A human normal mammary epithelial cells formed acini structures when grown on top of ECM protein hydrogel extracted from pig breast tissue ECM (PB-ECM) or HB-ECM [ 5 , 6 , 7 ]. Since human breast acini are surrounded by intralobular stroma [ 12 ], we inspected the acini formation capacities of MCF10A cells embedded in HB-TMG (10,000 cells/uL of gel) at a concentration of 2 mg/mL, which is optimal for the gel to polymerize with an elastic modulus similar to that of normal human breast tissue ECM [ 6 ]. After 7–10 days of culture under optimal conditions, the cells formed clustered acinar structures ( Figure 2 i) resembling those seen in the lobules of human or pig mature normal breast tissues ( Figure 2 ii,iii).…”

“…We previously showed that polymerized Col I, Matrigel, and mammalian breast tissue ECM-derived protein hydrogel exhibited distinct microstructural features and mechanical properties [ 5 , 6 , 7 ]. Mammary epithelial cells grown on the different matrices expressed distinct receptors in response to the matrix protein ligands and displayed unique phenotypes and responses to therapeutic drugs [ 5 , 7 , 8 , 9 ].…”

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment

“…Tumor-derived dECM has previously been used as a substrate for in vitro cell culture studies and cancer modeling. In some reports, it has been processed as hydrogels and further as inks for the printing and development of 3D bioengineered structures ( 34 , 59 ). In other reports, tumor-derived dECM has been used as a 3D scaffold for cell-seeding purposes ( 48 , 49 , 60 – 63 ).…”