Exploring the Potential of PEG-Heparin Hydrogels to Support Long-Term Ex Vivo Culture of Patient-Derived Breast Explant Tissues.

Breast cancer is a complex, highly heterogenous, and dynamic disease and the leading cause of cancer-related death in women worldwide. Evaluation of the heterogeneity of breast cancer and its various subtypes is crucial to identify novel treatment strategies that can overcome the limitations of currently available options. Explant cultures of human mammary tissue have been known to provide important insights for the study of breast cancer structure and phenotype as they include the context of the surrounding microenvironment, allowing for the comprehensive exploration of patient heterogeneity. However, the major limitation of currently available techniques remains the short-term viability of the tissue owing to loss of structural integrity. Here, an ex vivo culture model using star-shaped poly(ethylene glycol) and maleimide-functionalized heparin (PEG-HM) hydrogels to provide structural support to the explant cultures is presented. The mechanical support allows the culture of the human mammary tissue for up to 3 weeks and prevent disintegration of the cellular structures including the epithelium and surrounding stromal tissue. Further, maintenance of epithelial phenotype and hormonal receptors is observed for up to 2 weeks of culture which makes them relevant for testing therapeutic interventions. Through this study, the importance of donor-to-donor variability and intra-patient tissue heterogeneity is reiterated.

Label-free isolation and cultivation of patient-matched human mammary epithelial and stromal cells from normal breast tissue.

Breast cancer is primarily derived from mammary epithelial cells, the main cell type in human mammary glands. The majority of knowledge gained thus far around breast cancer has come from research using immortalized epithelial cell lines. The use of primary cells derived from breast tissue can be used in research to provide more biological relevance representative of the heterogeneous nature of breast cancer development and metastasis in its natural microenvironment. However, the successful isolation and propagation of human primary mammary gland cells can be costly and difficult due to their complex in vivo microenvironment and sensitivity when isolated. Here, we present a gentle isolation method for viable human mammary epithelial cells (hMECs) and donor-matched human mammary fibroblasts (hMFbs) from human mammary gland tissue. We isolated, expanded and passaged the hMECs and hMFbs in vitro and characterized cultures using cell-specific markers. A total of four primary cell lines were isolated and established from normal breast tissue and characterized through various markers, including pan cytokeratin (panCK), CK14, CD44, CD31, fibronectin and vimentin by immunofluorescence. To determine functional potential for subsequent studies, epithelial cells were examined via Matrigel® assays to assess spheroid development. Both cell type cultures expressed lineage specific markers with hMECs but not hMFbs forming spheroid structures in 3D Matrigel® assays. Our analyses confirm the successful isolation of two different cell phenotypes from normal breast tissues. This robust technique provides an inexpensive and accessible approach for mammary cell isolation.

Photocrosslinkable liver extracellular matrix hydrogels for the generation of 3D liver microenvironment models.

Liver extracellular matrix (ECM)-based hydrogels have gained considerable interest as biomimetic 3D cell culture environments to investigate the mechanisms of liver pathology, metabolism, and toxicity. The preparation of current liver ECM hydrogels, however, is based on time-consuming thermal gelation and limits the control of mechanical properties. In this study, we used detergent-based protocols to produce decellularized porcine liver ECM, which in turn were solubilized and functionalized with methacrylic anhydride to generate photocrosslinkable methacrylated liver ECM (LivMA) hydrogels. Firstly, we explored the efficacy of two protocols to decellularize porcine liver tissue using varying combinations of commonly used chemical agents such as Triton X-100, Sodium Dodecyl Sulphate (SDS) and Ammonium hydroxide. Then, we demonstrated successful formation of stable, reproducible LivMA hydrogels from both the protocols by photocrosslinking. The LivMA hydrogels obtained from the two decellularization protocols showed distinct mechanical properties. The compressive modulus of the hydrogels was directly dependent on the hydrogel concentration, thereby demonstrating the tuneability of mechanical properties of these hydrogels. Immortalized Human Hepatocytes cells were encapsulated in the LivMA hydrogels and cytocompatibility of the hydrogels was demonstrated after one week of culture. In summary, the LivMA hydrogel system provides a simple, photocrosslinkable platform, which can potentially be used to simulate healthy versus damaged liver for liver disease research, drug studies and cancer metastasis modelling.

Stromal fibroblasts regulate microvascular-like network architecture in a bioengineered breast tumour angiogenesis model.

The plasticity of the tumour microenvironment is a key contributor to cancer development and progression. Here, we present a bioengineered breast tumour angiogenesis model comprised of mammary derived epithelial, endothelial and fibroblast cells, to dissect the mechanisms of cancer-associated fibroblasts (CAFs) on microvascular-like network formation and epithelial spheroid morphology. Primary patient-derived mammary endothelial cells, normal breast fibroblasts (NBF, patient matched) and CAFs were cultured within three-dimensional (3D) semi-synthetic hydrogels where CAFs promoted an increase in the density and morphology of the microvascular-like network. The mammary microenvironment also increased the number of MCF-10a epithelial spheroids when compared with a non-mammary microenvironment, and a malignant mammary microenvironment resulted in further morphological differences in the epithelial spheroids. The morphological changes observed following interactions between breast CAFs and endothelial cells, highlight the plasticity of the malignant stroma in tumour vascularisation. Our in vitro bioengineered breast cancer microenvironment provides a robust model to study cell-cell and cell-matrix interactions. Statement of Significance In recent years there has been an increase in the sophistication of 3D culture models, however less attention has been paid to the cell source utilised. In this study, we describe the influence of a normal and malignant stromal microenvironment on vessel-like behaviour in a 3D model. Using a semi-synthetic hydrogel, we studied the effects of mammary-derived cancer-associated fibroblasts and normal fibroblasts on human umbilical vein endothelial cells or human mammary microvascular endothelial cells. An increase in vessel-like network and epithelial cell density was seen in a mammary versus non-mammary microenvironment. This study highlights the importance of using tissue-specific endothelial cells in cancer research and demonstrates the microenvironmental impact of fibroblasts on endothelial and epithelial growth and morphology.

Three-Dimensional In Vitro Hydro- and Cryogel-Based Cell-Culture Models for the Study of Breast-Cancer Metastasis to Bone.

Bone is the most common site for breast-cancer invasion and metastasis, and it causes severe morbidity and mortality. A greater understanding of the mechanisms leading to bone-specific metastasis could improve therapeutic strategies and thus improve patient survival. While three-dimensional in vitro culture models provide valuable tools to investigate distinct heterocellular and environmental interactions, sophisticated organ-specific metastasis models are lacking. Previous models used to investigate breast-to-bone metastasis have relied on 2.5D or singular-scaffold methods, constraining the in situ mimicry of in vitro models. Glycosaminoglycan-based gels have demonstrated outstanding potential for tumor-engineering applications. Here, we developed advanced biphasic in vitro microenvironments that mimic breast-tumor tissue (MCF-7 and MDA-MB-231 in a hydrogel) spatially separated with a mineralized bone construct (human primary osteoblasts in a cryogel). These models allow distinct advantages over former models due to the ability to observe and manipulate cellular migration towards a bone construct. The gels allow for the binding of adhesion-mediating peptides and controlled release of signaling molecules. Moreover, mechanical and architectural properties can be tuned to manipulate cell function. These results demonstrate the utility of these biomimetic microenvironment models to investigate heterotypic cell⁻cell and cell⁻matrix communications in cancer migration to bone.

Targeting Insulin-Like Growth Factor-I and Extracellular Matrix Interactions in Melanoma Progression.

Insulin-like growth factor (IGF)-I binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation and migration of skin keratinocytes and fibroblasts. Although evidence exists for the role of individual components of the complex (IGF-I, IGFBP-3 and VN), the cellular functions stimulated by these proteins together as a complex remains un-investigated in melanoma cells. We report here that the IGF-I:IGFBP-3:VN trimeric complex stimulates a dose-dependent increase in the proliferation and migration of WM35 and Sk-MEL28 melanoma cells. In 3D Matrigel™ and hydrogel cultures, both cell lines formed primary tumor-like spheroids, which increased in size in a dose-dependent manner in response to the trimeric complex. Furthermore, we reveal IGFBP-3:VN protein complexes in malignant melanoma and squamous cell carcinoma patient tissues, where the IGFBP-3:VN complex was seen to be predominantly tumor cell-associated. Peptide antagonists designed to target the binding of IGF-I:IGFBP-3 to VN were demonstrated to inhibit IGF-I:IGFBP-3:VN-stimulated cell migration, invasion and 3D tumor cell growth of melanoma cells. Overall, this study provides new data on IGF:ECM interactions in skin malignancies and demonstrates the potential usefulness of a growth factor:ECM-disrupting strategy for abrogating tumor progression.