Female breast cancer was the most prevalent cancer worldwide in 2020, according to the Global Cancer Observatory. As a prophylactic measure or as a treatment, mastectomy and lumpectomy are often performed at women. Following these surgeries, women normally do a breast reconstruction to minimize the impact on their physical appearance and, hence, on their mental health, associated with self-image issues. Nowadays, breast reconstruction is based on autologous tissues or implants, which both have disadvantages, such as volume loss over time or capsular contracture, respectively. Tissue engineering and regenerative medicine can bring better solutions and overcome these current limitations. Even though more knowledge needs to be acquired, the combination of biomaterial scaffolds and autologous cells appears to be a promising approach for breast reconstruction. With the growth and improvement of additive manufacturing, three dimensional (3D) printing has been demonstrating a lot of potential to produce complex scaffolds with high resolution. Natural and synthetic materials have been studied in this context and seeded mainly with adipose derived stem cells (ADSCs) since they have a high capability of differentiation. The scaffold must mimic the environment of the extracellular matrix (ECM) of the native tissue, being a structural support for cells to adhere, proliferate and migrate. Hydrogels (e.g., gelatin, alginate, collagen, and fibrin) have been a biomaterial widely studied for this purpose since their matrix resembles the natural ECM of the native tissues. A powerful tool that can be used in parallel with experimental techniques is finite element (FE) modeling, which can aid the measurement of mechanical properties of either breast tissues or scaffolds. FE models may help in the simulation of the whole breast or scaffold under different conditions, predicting what might happen in real life. Therefore, this review gives an overall summary concerning the human breast, specifically its mechanical properties using experimental and FE analysis, and the tissue engineering approaches to regenerate this particular tissue, along with FE models.

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A review of bioengineering techniques applied to breast tissue: Mechanical properties, tissue engineering and finite element analysis

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type–specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type–specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

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

Transforming growth factor (TGF)-β is a multifunctional cytokine that contributes to epithelial–mesenchymal transition, tumor development, and metastasis. The tumor microenvironment experiences dynamic changes in composition and mechanical properties, including alignment of matrix components and variations in the stiffness of the stroma, during cancer progression and recent studies have started to elucidate how these changes govern TGFβ signaling. This chapter describes the impact of matrix mechanical properties and cell-generated forces on the activation of TGFβ by cancer-associated fibroblasts and tumor cells. It further reports TGFβ-induced signaling cascades that respond to biophysical cues and that regulate epithelial–mesenchymal transition and tumor progression. Finally, it highlights approaches for targeting biophysical properties of the tumor microenvironment and mechanoresponsive signaling molecules as strategies to block TGFβ-induced tumor progression and metastasis. 

Biophysical Regulation of TGFβ Signaling in the Tumor Microenvironment

Modeling human breast tissue architecture is essential to study the pathophysiological conditions of the breast. We report that normal mammary epithelial cells grown in human breast extracellular matrix (ECM) hydrogel formed acini structurally similar to those of human and pig mammary tissues. Type I, II, III and V collagens were commonly identified in human, pig, and mouse breast ECM. Mammary epithelial cells formed acini on certain types or combinations of the four collagens at normal levels of breast tissue elasticity. Comparison of the collagen species in mouse normal breast and breast tumor ECM revealed common and distinct sets of collagens within the two types of tissues. Elevated expression of collagen type I alpha 1 chain (Col1a1) was found in mouse and human breast cancers. Collagen type XXV alpha 1 chain (Col25a1) was identified in mouse breast tumors but not in normal breast tissues. Our data provide strategies for modeling human breast pathophysiological structures and functions using native tissue-derived hydrogels and offer insight into the potential contributions of different collagen types in breast cancer development.

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Identification of the Collagen Types Essential for Mammalian Breast Acinar Structures

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.

Chandler R. Keller

Papers

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