UC San Diego

A fundamental issue in cancer therapeutics that has inhibited treatment progress is the lack of patient-specific models for both discovery and evaluation. Recent advances in sequencing technologies and molecular biology have enabled personalized treatment plans for patients guided by molecular profiling. However, success has been limited by the lack of phenotypic validations of genotype-sourced data. The development of CRISPR screening technologies offers a means of simultaneously identifying and functionally validating potential therapeutic targets to address this, but screens have largely been conducted in 2D cell culture conditions, with applications in more complex models being minimal thus far. As such, we focused on using tissue engineering technologies to construct a more physiologically accurate model, then integrating it with CRISPR screening technologies to enable cancer screening and validation in physiologically relevant systems. We began by developing a 3D-printing technique to engineer tissue constructs from biologically-derived materials and an evacuable poly(vinyl alcohol) (PVA) vascular scaffold. The result was a perfusable tissue construct that could be sustained ex vivo while enabling recapitulation of biological tumors and organs. We then further optimized the system to enable perfusion at physiological flow rates to achieve dense cultures of breast cancer cells, and then successfully applied a large-scale CRISPR screen, the first in a perfused tissue model. Utility of the model was explored further by tuning it to allow for the culture of patient-derived xenograft (PDX) cells, followed by thorough characterization and integration with a CRISPR knockout library containing only therapeutically-actionable drug targets to emulate a point-of-care diagnostics scenario. Results indicated that our engineered systems better represented in vivo biology compared to existing models, and also showed greater reproducibility than PDX mouse models of cancer. Understanding the clear importance of the material environment, we then proceeded to apply this knowledge towards organotypic engineering in a teratoma context, using specific material blends to promote differentiation of pluripotent stem cells towards certain lineages during teratoma development. Overall, our body of work and results highlight the importance and applicability of a multidisciplinary approach, with the integration of tissue and genetic engineering yielding greater avenues for biological discovery.