Establishing a 3D Tumor Spheroid Co-Culture Containing TAMs for the Future Evaluation of CAR-M Polarization

• Magdalena Zdanowicz, Applied Molecular Biology & Biotechnology, University of Delaware

• Catherine Fromen, Chemical and Biomolecular Engineering, University of Delaware
• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Lung cancer remains a leading cause of death in the United States. Recent developments, such as Chimeric Antigen Receptor T cell (CAR-T) therapy, have made advances in the treatment of cancer. However, CAR-T cell therapy is limited in its ability to target solid tumors, including lung cancer, due to the inability of T cells to infiltrate solid tumors. To overcome these challenges, CARs can be applied to macrophages, immune cells that are naturally able to infiltrate solid tumors. However, macrophages are polarized by the tumor microenvironment (TME) towards an anti-inflammatory tumor-associated macrophage (TAM) phenotype, which might render a CAR macrophage (CAR-M) therapeutic ineffective. To study the effect of the TME on the polarization of TAMs and CAR-Ms in lung cancer, an in vitro tumor spheroid model was formed by co-culturing lung carcinoma cells (A549) and human monocyte derived macrophages (differentiated THP-1s) to assess the efficacy and polarization of CAR-Ms in the TME. First, tumor spheroids ranging in size from 3,750 to 25,000 A549 cells were seeded in an InSphero Akura ultralow attachment microplate. The spheroids consisting of 7,500 cells best retained their shape for the full eight days of the experiment; thus this spheroid size was chosen for a follow-on experiment with bystander A549 macrophages. It was found that macrophage composition did not cause a significant difference in diameter, circularity, or cell death in the spheroid over the 6 day follow-on experiment. Future directions include extending the experiment to 12 days and the application of CAR-Ms to the tumor spheroid to determine both the CAR-Ms’ effect on the spheroid and the TME’s effect on the phenotype of the CAR-Ms. This platform will enable testing of the next generation of cancer immunotherapies.