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| Item Type: | Article |
|---|---|
| Title: | Investigating genetic modifications to enhance L1CAM-CAR T cell migration in solid tumors in a 3D bioprinted neuroblastoma model |
| Creators Name: | Andersch, Lena, Grunewald, Laura, Stecklum, Maria, Klironomos, Filippos, Haase, Kerstin, Hollek, Viola, Lam, Tobias, Jung, Beate Anahita, Winkler, Anika, Schwiebert, Silke, Astrahantseff, Kathy, Launspach, Michael, Jens, Marvin, Henssen, Anton, Kloke, Lutz, Blüthgen, Nils, Eggert, Angelika, Schulte, Johannes H., Anders, Kathleen and Künkele, Annette |
| Abstract: | 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. |
| Keywords: | Migration, CAR T Cells, Neuroblastoma, SELPLG, SH2D2A, Animals, Mice |
| Source: | Frontiers in Immunology |
| ISSN: | 1664-3224 |
| Publisher: | Frontiers Media SA |
| Volume: | 16 |
| Page Range: | 1677361 |
| Date: | 27 November 2025 |
| Official Publication: | https://doi.org/10.3389/fimmu.2025.1677361 |
| PubMed: | View item in PubMed |
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