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Updated: May 16, 2025
A Spheroid Killing Assay by CAR T Cells
Published on: December 12, 2018
A High-Throughput, Three-Dimensional Multiple Myeloma Model Recapitulating Tumor-Stroma Interactions for CAR-Immune
Sudjit Luanpitpong1,2,3, Montira Janan1,2,3, Jirarat Poohadsuan1
1Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Background:
Multiple myeloma (MM) is characterized by an excessive proliferation of clonal plasma cells in the bone marrow (BM). Components in BM niche contribute to the immunosuppressive tumor microenvironment (TME), but three-dimensional (3D) MM models that recreate the complex TME and enable high-throughput cytotoxicity assay of chimeric antigen receptor (CAR)-engineered immune cells are still lacking.
Methods:
Stable, luciferase (Luc)-labeled target MM cells were generated using Luc/RFP dual reporter system to track MM growth. 3D spheroids were formed in a 96-well plate in the presence or absence of cancer-associated fibroblast (CAF)-like stromal cells activated by MM-derived conditioned medium and the cytotoxicity of CAR-immune cells, which were represented by third-generation anti-CD138 CAR-NK-92 cells, was evaluated by luciferase assay using a multimode microplate reader. Immune cell infiltration was visualized under a fluorescence microscope by using multiple fluorescent dyes.
Results:
We first showed that luciferase assay provides a relatively simple and robust means to specifically monitor Luc-labeled tumor cell growth in a coculture system, allowing the high-throughput assessment of CAR-immune cytotoxicity. Through this assay, we demonstrated that CAF-like stromal cells impaired NK cell effector function in 2D culture and 3D spheroids, likely via paracrine signaling and physical barrier function. Importantly, we showed that 3D spheroids consisting of MM cells and CAF-like stromal cells provide a more comprehensive, physiologically relevant immuno-oncology model. Our established model could also be used to investigate the trafficking and infiltration of immune cells into the core of spheroids. Herein, we showed that CAR incorporation did improve the ability of NK cells to infiltrate 3D spheroids.
Conclusion:
Our established 3D spheroid model, which partially recapitulates the complex TME with immunosuppressive environment, is suitable for high-throughput screening of CAR-immune cytotoxicity and could be important in accelerating immuno-oncology drug discovery for MM since there is a pressing need to establish innovative CAR-immune cells.
