Related Experiment Video
Updated: Jan 8, 2026
Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice
Published on: February 24, 2023
Investigating genetic modifications to enhance L1CAM-CAR T cell migration in solid tumors in a 3D bioprinted
Lena Andersch1,2,3, Laura Grunewald2,3, Maria Stecklum4
1German Cancer Research Center (DKFZ), Heidelberg, Germany.
Introduction:
Effective CAR T cell infiltration into solid tumors remains a major barrier to therapy success. Despite their clinical potential, few studies have evaluated phenotypes of CAR T cells successfully invading the tumor mass following infusion. Phenotypic information would enrich our understanding of the mechanisms governing CAR T cell migration into solid tumors. Here we implemented an in vitro strategy to identify genes driving L1CAM-CAR T cell migration into a 3D tumor mass.
Methods:
L1CAM-CAR T cells were separated into 2 groups by their capability to infiltrate (or not) a 3D bioprinted neuroblastoma model. Single-cell and bulk RNA sequencing was performed, and infiltrating CAR T cells were compared to noninfiltrating cells to seek genetic drivers of CAR T cell migration. CRISPR/Cas9 technology was used to generate modified L1CAM-CAR T cells.
Results:
Tumor-infiltrating L1CAM-CAR T cells expressed lower levels of the selectin P ligand (SELPLG) glycoprotein and higher levels of the T cell-specific adaptor protein, SH2D2A. Functional characterization of L1CAM-CAR T cells genetically modified to enforce these characteristics demonstrated that neither trait negatively impacted L1CAM-CAR T cell cytotoxicity, activation and cytokine release upon coculture with neuroblastoma target cells. Transgenic SH2D2A expression did not improve CAR T cell migration in an endothelial transmembrane assay. SELPLG knockout benefited CAR T cell in vitro trans-endothelial migration, but did not enhance anti-tumor efficacy in an immunodeficient mouse model.
Discussion:
Our findings reveal a key limitation of murine xenograft models, which are widely used as the gold standard for preclinical CAR T cell testing. The lack of conservation between the human and murine SELPLG proteins likely accounts for the discrepancy between enhanced in vitro migration of SELPLG-deficient L1CAM-CAR T cells and their lack of improved efficacy in the mouse model. This underscores the need for more predictive human-relevant models to better preclinically evaluate CAR T cell function.
Insights
CAR T cell infiltration into solid tumors is challenging. This study identified SELPLG and SH2D2A as key genes influencing L1CAM-CAR T cell migration, highlighting limitations in current mouse models for CAR T cell therapy research.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Effective CAR T cell infiltration into solid tumors is a significant hurdle for cancer immunotherapy.
- Understanding the molecular mechanisms of CAR T cell migration is crucial for improving therapeutic outcomes.
Purpose of the Study:
- To identify genetic drivers of L1CAM-CAR T cell migration into a 3D tumor mass.
- To investigate the role of specific genes in CAR T cell infiltration and anti-tumor efficacy.
Main Methods:
- Utilized a 3D bioprinted neuroblastoma model to separate infiltrating and non-infiltrating L1CAM-CAR T cells.
- Performed single-cell and bulk RNA sequencing to identify differential gene expression.
- Employed CRISPR/Cas9 technology to genetically modify CAR T cells for functional validation.
Main Results:
- Tumor-infiltrating L1CAM-CAR T cells showed lower selectin P ligand (SELPLG) and higher SH2D2A expression.
- Neither SELPLG nor SH2D2A modification negatively impacted CAR T cell cytotoxicity, activation, or cytokine release.
- SELPLG knockout enhanced in vitro migration but not in vivo anti-tumor efficacy in a mouse model.
Conclusions:
- SELPLG and SH2D2A are key regulators of CAR T cell migration into solid tumors.
- Current murine xenograft models may not accurately predict CAR T cell efficacy due to species-specific protein differences.
- Development of human-relevant models is needed for better preclinical evaluation of CAR T cell therapies.
