Developing Bioinspired Three-Dimensional Models of Brain Cancer to Evaluate Tumor-Homing Neural Stem Cell Therapy

Abstract

Engineered neural stem cells (NSCs) have recently emerged as a promising therapy. Acting as a tumor-homing drug-delivery system, NSCs migrate through brain tissue to seek out primary and invasive tumor foci. NSCs can deliver therapeutic agents, such as TNFα-related apoptosis-inducing ligand, directly to the tumor and suppress glioblastoma (GBM) in murine models. While the mainstays for evaluating NSC migration and efficacy have been two-dimensional chemotaxis assays and mouse models, these low-throughput and small-scale systems limit our ability to implant and track these cells for human translation. To circumvent these challenges, we developed a three-dimensional culture system using a matrix of poly-l-lactic acid 6100 microfibers suspended in agar. These bioinspired brain matrices were used to model tumor growth, NSC migration, and efficacy of NSC therapy at small and human scale. Kinetic fluorescent imaging confirmed growth of tumors in both small and human-sized bioinspired brain matrix. Tumors proliferated 50-fold and 3-fold for GBM and human metastatic breast cancer, respectively, over 7 days. We next explored the impact of tumor location on NSC migration. When NSCs were implanted 2 mm lateral from the tumor foci, NSCs colocalized with the GBM within 7 days. In models of multifocal disease, NSCs were found to colocalize with multiple tumors, preferentially migrating to tumor foci closest to the site of NSC implantation. Lastly, therapeutic NSCs were implanted at increasing distances (0, 2, 5, or 10 mm) laterally from GBM foci to investigate the effects of distance on NSC efficacy. Serial imaging showed reduced fluorescence at tumor sites, implicating GBM apoptosis across all distances. NSCs coinjected with tumor induced a near-complete response in <10 days, while NSCs implanted 10 mm laterally from the tumor induced a near-complete response by day 30. Lastly, GBM foci were established in each hemisphere of the model and control or therapeutic NSCs were implanted adjacent to tumor cells in the right hemisphere. Kinetic imaging showed that NSC therapy attenuated progression of GBM foci, while GBM cells treated with control NSC expanded rapidly over 21 days. In conclusion, we developed a new bioinspired model that supports growth of human brain cancer cells and enables rapid tracking of NSC therapy. Impact statement Tumor-homing and tumor-killing-engineered neural stem cell (NSC) therapies have shown immense promise in both preclinical and clinical trials. However, as cell therapies continue to evolve, cost-effective and high-throughput screening assays are needed to assess the proliferation, migration, and efficacy of these cells. In this study, we developed a bioinspired brain matrix for the evaluation of engineered NSCs. Importantly, this matrix is easy to fabricate, scalable, and allows for sterile real-time, noninvasive imaging using our custom bioreactor. We then utilized the bioinspired brain matrix system to answer key questions around the tumor-homing migration and efficacy of engineered NSC therapies that are challenging to address with traditional models.

Keywords: 3D cell culture; bioreactor; neural stem cell.