Abstract
BackgroundTreatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres.ResultsHere, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems.ConclusionWe developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.
Highlights
Treatment of cancers has largely benefited from the development of immunotherapy
2 dimensional (2D) in vitro systems involve a mixture of Chimeric Antigen Receptor (CAR) T cells and target cancer cell lines as monolayers to assess the functionality and specificity of these effector cells
Birgersdotter and colleagues demonstrated that a Hodgkin lymphoma (HL) cell line only acquires a gene expression profile that is similar to primary tumor samples if grown in 3 dimensional (3D) [12]
Summary
Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. 2D in vitro systems involve a mixture of CAR T cells and target cancer cell lines as monolayers to assess the functionality and specificity of these effector cells These strategies are important and vital steps of the preclinical development, they do not take complex morphology and three-dimensional (3D) organization of the cancer cells into consideration [8]. Cancer cells cultured in 3D systems, referred to as spheroids, acquire new phenotypic traits through changes in gene expression profile [9] which might influence the recognition by redirected effector cells These can be modifications in regulatory receptor expression, as well as physical constraints on the actin cytoskeleton, which will result in changing the T-cell recognition mechanism of the cognate target [10, 11]. It has been proposed that spheroids present numerous similarities with in vivo models [17,18,19] and are valuable tools to assess CAR efficacy more critically [20]
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