Marissa A. Gionet-Gonzales

Breast cancer progression involves extensive remodeling of the tumor extracellular matrix (ECM), resulting in changes in mechanical properties. Tumors are viscoelastic, exhibiting both an initial elastic response to loading and a time-dependent viscous response. While it is well established that tumor elasticity or stiffness is elevated, changes in tissue viscoelasticity during breast cancer progression remain poorly characterized. Here, using a mouse model that recapitulates stages of human breast cancer progression (mouse mammary tumor virus-polyoma middle T antigen, MMTV-PyMT), we show that tumors from both early and late disease time points were significantly more viscoelastic than normal mammary glands. Spatial microindentation revealed that while local stiffness and viscoelasticity are strongly coupled in soft regions of normal tissue, this relationship weakens in late carcinoma. Compared to normal glands, carcinoma tissues also exhibited marked upregulation of glycosaminoglycans (GAGs) and glycoproteins. Selective enzymatic degradation of sulfated GAGs, mucins, or O/N-linked glycans reduced viscoelasticity without altering stiffness, establishing glycoconjugates as major contributors to tumor viscoelasticity. In vitro, increasing matrix viscoelasticity drove non-transformed mammary epithelial cells (MCF10A) toward a malignant phenotype through focal adhesion kinase mechanosignaling. Our work establishes increased viscoelasticity as a defining mechanical feature of breast cancer progression, driven in part by the accumulation of glycoconjugates. These findings provide a fuller picture of tumor mechanical evolution and a benchmark for recapitulating matrix viscoelasticity in engineered models of breast cancer.

There is a critical need to develop non-invasive, non-destructive methods for assessing the quality of engineered constructs prior to implantation. Currently, the composition and maturity of engineered tissues are assessed using destructive, costly, and time-consuming biochemical and mechanical analyses. The goal of this study was to use non-invasive, multimodal imaging to monitor osteogenic differentiation and matrix deposition by human mesenchymal stem/stromal cells (MSCs) during in vitro culture. MSCs were encapsulated in alginate hydrogels and cultured in osteogenic conditions for 4 weeks. Samples were evaluated using fluorescence lifetime imaging (FLIm) and ultrasound backscatter microscopy (UBM) prior to traditional biochemical and mechanical testing. Using linear regression analysis, we identified strong correlations between imaging parameters (e.g., fluorescence lifetime and acoustic attenuation coefficient) and destructive mechanical and biochemical tests to assess the maturation of osteogenically-induced constructs. These data demonstrate the promise of non-destructive label-free imaging techniques to non-invasively ascertain the progression and maturity of tissue engineered bone grafts.

There is a critical need for strategies that effectively enhance cell viability and post-implantation performance in order to advance cell-based therapies. Spheroids, which are dense cellular aggregates, overcome many current limitations with transplanting individual cells. Compared to individual cells, the aggregation of cells into spheroids results in increased cell viability, together with enhanced proangiogenic, anti-inflammatory, and tissue-forming potential. Furthermore, the transplantation of cells using engineered materials enables localized delivery to the target site while providing an opportunity to guide cell fate in situ , resulting in improved therapeutic outcomes compared to systemic or localized injection. Despite promising early results achieved by freely injecting spheroids into damaged tissues, growing evidence demonstrates the advantages of entrapping spheroids within a biomaterial prior to implantation. This review will highlight the basic characteristics and qualities of spheroids, describe the underlying principles for how biomaterials influence spheroid behavior, with an emphasis on hydrogels, and provide examples of synergistic approaches using spheroids and biomaterials for tissue engineering applications.

MicroRNAs (miRNAs) are small RNAs that bind to mRNA targets and regulate their translation. A functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ silica columns or the TRIzol reagent but give out low recovery for small RNAs probably due to their short strand lengths. Herein, we fabricated the titanium dioxide nanofibers using electrospinning to facilitate miRNA extraction and developed the optimal buffer conditions to improve miRNA recovery from biological matrices of cell lysate and serum. We found that our TiO₂ fibers could obtain a recovery of 18.0 ± 3.6% for miRNA fibers while carrying out the extraction in the more complex medium of cell lysate, much higher than the 0.02 ± 0.0001% recovery from the commercial kit. The much improved extraction of miRNAs from our fibers could be originated from the strong coordination between TiO₂ and RNA’s phosphate backbone. In addition, the binding, washing, and elution buffers judiciously developed in the present study can achieve selective extraction of small RNA shorter than 500 nucleotides in length. Our results demonstrate that TiO₂ nanofibers can work as a valuable tool for extraction of miRNAs from biological samples with high recovery. Schematic for extraction of small RNAs using TiO₂ nanofibers