Optimizing ex vivo CAR-T cell infiltration and cytotoxicity assays using 2- and 3-D multimodality imaging

Background

CAR-T cell therapy is a revolutionary cancer treatment that is highly efficient in treating liquid tumors and continues to expand in its applications, including which immune cells are engineered, what constructs are utilized, and which cancers are targeted. In such a rapidly evolving field, sensitive, quantitative assays to test the cytotoxicity of CAR cells ex vivo are required to continue improving the efficacy of this treatment. Furthermore, testing infiltration of engineered CAR-T cells into solid tumor models is critically important to gain more specific insight into the mechanisms of how cold tumors become hot with CAR cells infiltrating, targeting and killing cancer cells.

Methods

We developed a single clone reporter cancer cell line, Raji-Luc2, that endogenously expresses high levels of the key CAR-T target antigen CD19. Using CD19 and mock engineered CAR-T cells, we targeted Raji-Luc2 cells and assessed the killing efficacy of the CAR-T cells by a multimodality imaging approach that included a bioluminescence luciferase assay as well as a phase contrast and fluorescence live cell imaging assay. Next, we engineered a dual reporter line, Raji-GFP-Luc2, and demonstrated CAR-T cell infiltration into Raji-GFP-Luc2 spheroids by confocal live imaging in 3-D.

Results

We combined the luciferase assay with live imaging to create a multimodal cytotoxicity assay that provides superior sensitivity as well spatial and temporal resolution compared to existing assays. We found that CD19 CAR-T cells killed cancer cells at more significant levels relative to mock CAR-T control cells by luciferase assay after co-culture with the reporter lines. Furthermore, we developed a CAR-T cell infiltration assay by co-culturing dyed CAR-T cells with Raji-GFP-Luc2 spheroids. By imaging in 3-D and generating time-lapse videos, we show how CAR-T cells infiltrated non-gel-embedded as well as gel-embedded spheroids in real time.

Conclusions

Overall, we show that these engineered cell lines are highly useful for assaying CAR-T cell cytotoxicity as well as infiltration into 3-D solid tumor models such as spheroids and organoids. The quantitative readout of the luciferase assay combined with the dynamic, real-time information provided by multiple live imaging techniques in both 2- and 3-D results in a sensitive, multimodal assay that can be used in a variety of applications. Importantly, visualization of T cell infiltration in 3-D is critical to understanding how CAR cells infiltrate into solid tumors, target, and kill cancer cells under more physiologically relevant conditions so that the efficacy of this treatment can continue to be improved.