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Updated: Nov 1, 2025
Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level
Published on: March 14, 2025
A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue
Karsten Eichholz1, Alvason Zhenhua Li1, Kurt Diem2
1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, MS E3-300, Seattle, WA, 98190, USA.
Abstract:
Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.
Insights
Researchers developed a new CAR RNA FISH histo-cytometry platform to analyze chimeric antigen receptor (CAR) T cell distribution and function in preclinical cancer models. This tool quantifies in vivo effectiveness by linking gene expression to proximity to tumor cells.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Chimeric antigen receptor (CAR) T cells show promise in cancer therapy and infectious disease treatment.
- Effective CAR T cell therapy relies on successful trafficking, persistence, and in vivo functional activity.
- Assessing CAR T cell performance in preclinical models is crucial for therapeutic development.
Purpose of the Study:
- To introduce a novel CAR RNA FISH histo-cytometry platform for high-resolution analysis of CAR T cells.
- To quantitate the spatial distribution and in vivo functional activity of CAR T cells within preclinical models.
- To enable a detailed assessment of CAR T cell effectiveness in relation to target cells.
Main Methods:
- Development of a CAR RNA FISH histo-cytometry platform.
- Integration with a random reaction seed image analysis algorithm.
- Single-cell resolution analysis of CAR T cell populations in preclinical models.
Main Results:
- The platform successfully quantitated spatial distribution and in vivo functional activity of CAR T cells.
- In situ analysis revealed heterogeneous effector gene expression in CAR T cells.
- Effector gene expression correlated with proximity to tumor cells, indicating functional status.
Conclusions:
- The CAR RNA FISH histo-cytometry platform provides a powerful tool for studying genetically engineered immune cells.
- This method allows for high statistical power assessment of in situ immune cell functions and interactions.
- The platform is valuable for preclinical evaluation of CAR T cell-based therapies.
