A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

Karsten Eichholz,Lawrence Corey,Alvason Zhenhua Li,Jia Zhu,Kurt Diem,Michael Claus Jensen

doi:10.1038/s41598-021-92196-x

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.

Highlights

Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases
CAR T cells were co-cultured with γ-irradiated CD19-expressing TM-LCL lymphoblastoid cells for 12 days and purity was assessed by flow cytometry (~ 90%) (Fig. 2 suppl.)
To validate the CAR FMC63 26ZZ probes, anti-CD19 CD8 CAR T cells or Jurkat cells were processed for cytospins and stained for CAR mRNA (Fig. 1B)

Summary

Introduction

Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. CAR T cell performance, proliferation and persistence in preclinical in vivo studies and clinical application are mainly studied by flow cytometry, quantitative PCR, and for preclinical cancer mouse models only, whole body bioluminescence imaging of the tumor burden. These techniques are favorable to measure CAR T cell phenotype, functionality and expansion with high statistical power and specificity and can assess global anti-tumor efficacy, the location of the CAR T cells in the tissue with respect to the tumor cells cannot be determined. RNA in situ hybridization (RNA ISH) approaches have been successfully applied to track CAR T cells in patients with g lioblastoma[4], in a case study in a patient with B-ALL, who died 5 days post-anti-CD19 CAR T cell infusion[5] and in clinical samples from the ZUMA-1 trial that tested the anti-CD19 CAR Axicabtagene ciloleucel[6]

Methods

Results

Conclusion