A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments

SIMPLE SUMMARY: Engineered proteins possess a great therapeutic potential, but the development of such therapies is impeded during preclinical studies by the lack of in vitro models that accurately simulate the human physiology. Animal models, on the other hand, also have difficulties predicting human responses, and are ethically concerning. In this study, we employed a hybrid approach where we combined mathematical modeling with 3D in vitro models that mimic aspects of the tumor microenvironment, in order to simulate the delivery of therapeutic proteins targeting cancer cells and to predict the biological activity. By cross-comparing simulated and experimental data from 3D models, we were able to correctly predict the best dose needed to deliver toxic proteins specifically to tumor cells, while leaving the surrounding non-tumor cells untouched. This study shows the potential of combining computational approaches with novel in vitro models to advance the development of protein therapeutics. ABSTRACT: To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm(2)·s(−1) and 2 µm(2) ·s(−1) for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.