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BackgroundThe heterogeneous and dynamic tumor microenvironment has significant impact on cancer cell proliferation, invasion, drug response, and is probably associated with entering dormancy and recurrence. However, these complex settings are hard to recapitulate in vitro.MethodsIn this study, we mimic different restriction forces that tumor cells are exposed to using a physiologically relevant 3D model with tunable mechanical stiffness.ResultsBreast cancer MDA-MB-231, colon cancer HCT-116 and pancreatic cancer CFPAC cells embedded in the stiffer gels exhibit a changed morphology and cluster formation, prolonged doubling time, and a slower metabolism rate, recapitulating the pathway from competency to dormancy. Altering environmental restriction allows them to re-enter and exit dormant conditions and change their sensitivities to drugs such as paclitaxol and gemcitabine. Cells surviving drug treatments can still regain competent growth and form tumors in vivo.ConclusionWe have successfully developed an in vitro 3D model to mimic the effects of matrix restriction on tumor cells and this high throughput model can be used to study tumor cellular functions and their drug responses in their different states. This all in one platform may aid effective drug development.

Development of a physiologically relevant 3D model system for cancer research and drug development is a current challenge. We have adopted a 3D culture system based on a transglutaminase-crosslinked gelatin gel (Col-Tgel) to mimic the tumor 3D microenvironment. The system has several unique advantages over other alternatives including presenting cell-matrix interaction sites from collagen-derived peptides, geometry-initiated multicellular tumor spheroids, and metabolic gradients in the tumor microenvironment. Also it provides a controllable wide spectrum of gel stiffness for mechanical signals, and technical compatibility with imaging based screening due to its transparent properties. In addition, the Col-Tgel provides a cure-in-situ delivery vehicle for tumor xenograft formation in animals enhancing tumor cell uptake rate. Overall, this distinctive 3D system could offer a platform to more accurately mimic in vivo situations to study tumor formation and progression both in vitro and in vivo.

Efficiency of cell-based tissue engineering and regenerative medicine has been limited by inadequate cellular responses to injury because of aging and poor controllability of cellular interactions. Since cell progression is under a tight epigenetic regulation, epigenetic modulators such as 5-azacytidine (5-Aza-CR) have been utilized to facilitate reprogramming and development of somatic cells in 2-dimensional (2-D) settings. Nonetheless, progression of a specific tissue lineage toward the terminal phenotype is dependent not only on the genomic potential, but also on the microenvironment cues that are beyond the capability of 2-D approaches. In this study, we investigated the combined effects of matrices of variable rigidities and the treatment with the epigenetic modulator 5-Aza-CR on reprogramming adipose-derived stromal cells (ADSCs) into myoblast-like cells by utilizing tunable transglutaminase cross-linked gelatin (Col-Tgel) in vitro and in vivo. Our experiments demonstrated that cellular plasticity and trans-differentiation were significantly enhanced when ADSCs were treated with an effective dose of 5-Aza-CR (1.25 to 12.5 ng) in the optimal myogenic matrix (15 ± 5 kPa Col-Tgel). Our findings suggest that both physical signals and chemical milieu are critical for the regulation of cellular responses.

Tg-Gel as an injectable functional bone graft may enable the use of minimally invasive surgical procedures to treat irregular-shaped bone defects. Furthermore, this novel approach is capable of incorporating and controlling the release of therapeutic agents that may advance the science of tissue regeneration.