Colorectal cysts as a validating tool for CAR therapy

Abstract

Background: 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. 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.

Results: Here, 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.

Conclusion: We 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.

Keywords: CAR T cells; Immunotherapy; Microscopy; Spheroids.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Protocol principle

Fig. 2

Retroviral transduction of Caco-2 and T cells. a Representative FACS flow showing Caco-2 cells retrovirally transduced to express GFP, CD19 or both. b Representative FACS flow showing T cells retrovirally transduced to express the CD19CAR construct

Fig. 3

CD19 is expressed on the surface of Caco-2 cells and do not hinder their ability to form cysts. a BLI killing assay of Caco-2 cells expressing CD19 or not, co-cultured with CD19CAR or Mock T cells (E:T ratio of 1:10). Data represent mean ± S.D. of hexaplicates. Representative data from one of three experiments are shown. Statistics analysis were conducted from timepoints 3 to 7 (2-way ANOVA). b Time lapse of Caco-2 GFP+/CD19+ cysts formation observed with a high-throughput microscope. Scale bar represents 20 μm. c Airyscan micrographs showing the establishment of a basal/apical polarity on a Caco-2 organoid marked by Hoechst 33342, PKC-ζ, E-cadherin and actin. Scale bar represents 10 μm

Fig. 4

CD19CAR T cells demonstrate killing of Caco-2 CD19+ cysts. a Representative confocal micrographs showing specific killing of Caco-2 GFP+/CD19+ cysts mediated by CD19CAR T cells. Cells were marked with Hoechst 33242 and actin. Scale bar represents 10 μm. Yellow arrows outline the presence of T-cells. b BLI killing assay of Caco-2 cysts expressing CD19 or not, co-cultured with CD19CAR or Mock T cells. Data represent mean ± S.D. of hexaplicates. Representative data from one of three experiments are shown. Statistics analysis were conducted from timepoints 3 to 7 (2-way ANOVA). c Total apoptosis signal as measured by Annexin V of Caco-2 cysts expressing CD19 or not, co-cultured with CD19CAR or Mock T cells and imaged by high throughput microscope. Data represent mean ± S.D. of octuplicates. Representative data from one of three experiments are shown. Statistics analysis were conducted at timepoint 232 (1-way ANOVA). d Corresponding time lapse of the killing assay described in C. Scale bars represent 80 μm. Yellow arrows outline the presence of T-cells