(D-133) Engineered basement membrane matrices to study morphogenesis and invasion of mammary epithelium

Introduction:: Basement membrane proteins are required for proper epithelial morphogenesis. Reconstituted basement membrane products, like Matrigel, have typically been used as extracellular matrix (ECM) surrogates for in vitro 3D culture of epithelial tissues, organoids, spheroids and cancer models. Despite its widespread use, Matrigel has several inherent disadvantages that contribute to irreproducible results, including inconsistencies between batches, lack of tunability, and recently, chronic shortages.

To overcome these limitations, we seek to develop a 3D culture system for mammary epithelial morphogenesis culture by conjugating ECM peptide motifs to alginate to create an engineered basement membrane. Our platform can recapitulate mammary acinus morphogenesis from MCF10A cells that are morphologically equivalent to Matrigel cultures using different combinations of ECM protein adhesion motifs (e.g. IKVAV, YIGSR) conjugated to an alginate matrix. Using this system, we can tune the mechanical properties of the matrix (e.g. stiffness and stress relaxation), independent of ligand density and type, and generate both the acinar and tumorigenic phenotypes of MCF10As.

Materials and Methods:: Using carbodiimide chemistry, peptide motifs (IKVAV and YIGSR) were conjugated to alginate. MCF10A cells were encapsulated in alginate matrices, cultured for 14 days, and then analyzed to compare acinus formation versus invasion. We chose substrate stiffnesses 100 Pa and 2000 Pa to represent properties that have been measured in vivo for healthy breast tissue and malignant tumors, respectively. Acinus formation versus invasion was categorized via roundness and polarity markers.

Results, Conclusions, and Discussions:: We observed that acinus formation and the fraction of invasive clusters depended on matrix stiffness, stress relaxation, and ligand concentration. Notably, regardless of stiffness, high peptide concentrations and fast relaxing alginate led to acinus-like cluster development within the IKVAV and YIGSR matrices. In contrast, clusters in slow relaxing alginate matrices with low peptide concentration were more invasive. We will continue to evaluate the distinct contributions of each microenvironmental input in enhancing acinus formation versus driving invasion.



In the future, we plan to optimize this system so that it is easily tunable for different kinds of systems. For example, we plan to culture mammary organoids in this system to see if our matrix can support mammary gland morphogenesis in addition to MCF10A acinar morphogenesis. This would lay the foundation for both basic biological investigations into microenvironmentally-guided morphogenesis and more translational applications in engineered replacement tissues.

These results demonstrate a proof-of-concept that our reductionist matrices are sufficient to induce 3D mammary acinar morphogenesis and are an excellent platform to decipher how cell-matrix interactions lead to phenotypic outcomes.


Acknowledgements (Optional): : T32 Quantitative Mechanobiology Training Grant, NIH, 1T32GM141846


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