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. 2024 Jul 9;16(14):2497.
doi: 10.3390/cancers16142497.

Optimizing Ex Vivo CAR-T Cell-Mediated Cytotoxicity Assay through Multimodality Imaging

John G Foulke  1 , Luping Chen  1 , Hyeyoun Chang  1 , Catherine E McManus  1 , Fang Tian  1 , Zhizhan Gu  1
Affiliations

Optimizing Ex Vivo CAR-T Cell-Mediated Cytotoxicity Assay through Multimodality Imaging

John G Foulke et al. Cancers (Basel). .

Abstract

CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With the FDA's initiative to advance alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and inability to automate, among others. To address these limitations, we optimized the assay using multimodality imaging methods, including bioluminescence, impedance tracking, phase contrast, and fluorescence, to track CAR-T cells co-cultured with CD19, CD20, and HER2 luciferase reporter cancer cells in real-time. Additionally, we varied the ratio of CAR-T cells to cancer cells to determine optimal cytotoxicity readouts. Our findings demonstrated that the CAR-T cell group effectively attacked cancer cells, and the optimized assay provided superior temporal and spatial precision measurements of ex vivo CAR-T killing of cancer cells, confirming the reliability, consistency, and high throughput of the optimized assay.

Keywords: CAR-T; CD19; CD20; HER2; cytotoxicity assay; multimodality imaging.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Analysis of common hematologic and solid tumor CAR-T targets in selected cancer cell lines. Heat map of expression levels from the CCLE database for 12 hematological and 26 solid tumor CAR-T targets in 21 cancer lines with luciferase-expressing daughter lines available from ATCC. Values are log2(TPM + 1). Values were gradiently color-coded by the Microsoft Excel software version 2308, with the midpoint (50th percentile) coded as white. Values above the midpoint were coded as red gradients. Values below the midpoint were coded as blue gradients. Note: WIL2-S did not have RNA-seq data within the CCLE database as indicated by N/A.
Figure 2
Figure 2
mRNA Seq analysis of common hematologic and solid tumor CAR-T targets in B lymphoblast and B lymphocyte cancer cell lines. (A) Heat map of RNA-seq data for 2 cancer cell lines and 2 luciferase-expressing daughter cell lines showing expression levels of 12 hematologic tumor CAR-T targets and 26 solid tumor CAR-T targets. RNA sequencing was performed in duplicate by Psomogen. CD19 expression remains consistent pre- and post-luciferase transduction. Values are log2(FPKM + 1). Values were gradiently color-coded by the Microsoft Excel software version 2308, with the midpoint (50th percentile) coded as white. Values above the midpoint were coded as red gradients. Values below the midpoint were coded as blue gradients. (B) Schematic showing CD19-positive WIL2-S-Luc2 and Raji-Luc2, CD20-positive Daudi-Luc2 and Farage-Luc2, and HER2-positive BT-474-Luc2 cells being surrounded and attacked by CD19-, CD20-, and HER2-targeting CAR-T cells, respectively. Created with BioRender.com (accessed on 7 October 2022).
Figure 3
Figure 3
CD19 CAR-T in vitro killing assay of Raji-Luc2 and WIL2-S-Luc2 measured using luminescence and live cell imaging. (A) CD19-positive Raji-Luc2 cells (5 × 10³) or (B) WIL2-S-Luc2 cells (5 × 10³) were seeded into a 96-well plate and were used as target cells for either CD19 CAR-T or mock CAR-T cells (control) from the same donor, which were seeded at various ratios of CAR-T cells to target cells (1:1, 2:1, 5:1, and 10:1). After 24 h of co-culture, Bright-Glo reagent was added to the wells. Luminescence was read within 10 min using a plate reader and was normalized to wells with no CAR-T cells. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05. (C) Raji-Luc2 cells were stained with Vybrant DiO dye, and real-time fluorescent images were captured every 30 min for 24 h during co-culture with either CD19 or mock CAR-T cells. Stained Raji-Luc2 cells (green) from the co-culture experiment were tracked for 6 h and became surrounded by CAR-T cells, resulting in a decrease in fluorescence when treated with CD19 CAR-T cells as compared to co-cultures with mock-CAR-T cells. Scale bar, 300 µm. (D) The larger fields of view of Vybrant DiO stained Raji-Luc2 cells (green) after 6 or 24 h of co-culture with either CD19 or mock CAR-T cells. Scale bar, 1000 µm.
Figure 3
Figure 3
CD19 CAR-T in vitro killing assay of Raji-Luc2 and WIL2-S-Luc2 measured using luminescence and live cell imaging. (A) CD19-positive Raji-Luc2 cells (5 × 10³) or (B) WIL2-S-Luc2 cells (5 × 10³) were seeded into a 96-well plate and were used as target cells for either CD19 CAR-T or mock CAR-T cells (control) from the same donor, which were seeded at various ratios of CAR-T cells to target cells (1:1, 2:1, 5:1, and 10:1). After 24 h of co-culture, Bright-Glo reagent was added to the wells. Luminescence was read within 10 min using a plate reader and was normalized to wells with no CAR-T cells. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05. (C) Raji-Luc2 cells were stained with Vybrant DiO dye, and real-time fluorescent images were captured every 30 min for 24 h during co-culture with either CD19 or mock CAR-T cells. Stained Raji-Luc2 cells (green) from the co-culture experiment were tracked for 6 h and became surrounded by CAR-T cells, resulting in a decrease in fluorescence when treated with CD19 CAR-T cells as compared to co-cultures with mock-CAR-T cells. Scale bar, 300 µm. (D) The larger fields of view of Vybrant DiO stained Raji-Luc2 cells (green) after 6 or 24 h of co-culture with either CD19 or mock CAR-T cells. Scale bar, 1000 µm.
Figure 4
Figure 4
BT-474-Luc2 and WIL2-S-Luc2 used in CAR-T and NK cytotoxicity assays. (A) HER2-positive BT-474-Luc2 cells (5 × 103) were seeded into a 96-well plate, and either HER2 CAR-T or mock CAR-T cells were added at various ratios of CAR-T cells to target cells (1:1, 2:1, 5:1, and 10:1). (B) HER2 CAR-T cells were used to target 2 × 10⁴ HER2-positive BT-474-Luc2 cells at a 10:1 ratio, and cell killing was measured using the xCELLigence system. Mock CAR-T cells from the same donor were used as a control. (C) WIL2-S-Luc2 cells or (D) BT-474-Luc2 cells were co-cultured with NK cells for 16 h at various NK to target cell ratios (1:4, 1:2, 1:1, 2:1, 4:1, 8:1, and 16:1) after which luciferase activity was measured. (E) Antibody-dependent cell-mediated cytotoxicity (ADCC) assay using Rituximab (anti-CD20) with WILS-2-Luc2 cells or (F) Trastuzumab (anti-HER2) monoclonal antibodies with BT474-Luc2 cells. Concentrations of monoclonal antibody or IgG1 control varying from 10 pg to 1 µg/mL were added to co-cultures of primary NK cells and reporter cells at a 2:1 NK/target cell ratio. Luminescence was measured after 4 h of co-culture. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05.
Figure 4
Figure 4
BT-474-Luc2 and WIL2-S-Luc2 used in CAR-T and NK cytotoxicity assays. (A) HER2-positive BT-474-Luc2 cells (5 × 103) were seeded into a 96-well plate, and either HER2 CAR-T or mock CAR-T cells were added at various ratios of CAR-T cells to target cells (1:1, 2:1, 5:1, and 10:1). (B) HER2 CAR-T cells were used to target 2 × 10⁴ HER2-positive BT-474-Luc2 cells at a 10:1 ratio, and cell killing was measured using the xCELLigence system. Mock CAR-T cells from the same donor were used as a control. (C) WIL2-S-Luc2 cells or (D) BT-474-Luc2 cells were co-cultured with NK cells for 16 h at various NK to target cell ratios (1:4, 1:2, 1:1, 2:1, 4:1, 8:1, and 16:1) after which luciferase activity was measured. (E) Antibody-dependent cell-mediated cytotoxicity (ADCC) assay using Rituximab (anti-CD20) with WILS-2-Luc2 cells or (F) Trastuzumab (anti-HER2) monoclonal antibodies with BT474-Luc2 cells. Concentrations of monoclonal antibody or IgG1 control varying from 10 pg to 1 µg/mL were added to co-cultures of primary NK cells and reporter cells at a 2:1 NK/target cell ratio. Luminescence was measured after 4 h of co-culture. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05.
Figure 5
Figure 5
CD20 CAR-T in vitro killing assay of Farage-Luc2 measured using luminescence and live cell imaging. (A) CD20-positive Farage-Luc2 cells (5 × 10³) were seeded into a 96-well plate, and CD20 CAR-T or mock CAR-T cells were added at various ratios of CAR-T to target cells (1:1, 2:1, 5:1, and 10:1). (B) Farage-Luc2 cells (5 × 10³) were co-cultured with CD20 CAR-T cells or mock CAR-T cells in the presence of Incucyte Cytotox red dye. Fluorescent images were captured every hour for 24 h, and red fluorescence was quantified and plotted. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05. (C) Series of images captured during Farage-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red. Scale bar, 300 µm. (D) The larger fields of view of Farage-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red at 24 h. Scale bar, 1000 µm.
Figure 5
Figure 5
CD20 CAR-T in vitro killing assay of Farage-Luc2 measured using luminescence and live cell imaging. (A) CD20-positive Farage-Luc2 cells (5 × 10³) were seeded into a 96-well plate, and CD20 CAR-T or mock CAR-T cells were added at various ratios of CAR-T to target cells (1:1, 2:1, 5:1, and 10:1). (B) Farage-Luc2 cells (5 × 10³) were co-cultured with CD20 CAR-T cells or mock CAR-T cells in the presence of Incucyte Cytotox red dye. Fluorescent images were captured every hour for 24 h, and red fluorescence was quantified and plotted. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05. (C) Series of images captured during Farage-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red. Scale bar, 300 µm. (D) The larger fields of view of Farage-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red at 24 h. Scale bar, 1000 µm.
Figure 6
Figure 6
CD20 CAR-T in vitro killing assay of Daudi-Luc2 measured using luminescence and live cell imaging. (A) CD20-positive Daudi-Luc2 cells (5 × 103) were seeded into a 96-well plate, and CD20 CAR-T or mock CAR-T cells were added at various ratios of CAR-T cells to target cells (1:1, 2:1, 5:1, and 10:1). After 24 h of co-culture, Bright-Glo was added to the wells and luminescence was measured. (B) Daudi-Luc2 cells (5 × 103) were co-cultured with CD20 CAR-T cells or mock CAR-T cells in the presence of Incucyte Cytotox red dye. Fluorescent images were captured every hour for 24 h, and red fluorescence was quantified and plotted. * = significant difference and ns = not significant using an unpaired t test, with a single pooled variance. N = 3 in all experiments. * p < 0.05. (C) Series of images captured during Daudi-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red. Scale bar, 300 µm. (D) The larger fields of view of Daudi-Luc2 cells co-culture with CD20 or mock CAR-T cells in the presence of Cytotox Red at 24 h. Scale bar, 1000 µm.
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