Anti-HER2/neu TCR-T Cells in Action: Linking Transcriptional Signatures, Secretomics, and In Vivo Tumor Suppression

1. TCR T cell Cytotoxicity In Vitro: Measured the ability of engineered TCR-T cells to kill tumor cells using an LDH release assay after an 18-hour co-culture. 2. Single-cell Analysis: Used single-cell RNA sequencing (BD Rhapsody™ platform) to profile the gene expression of TCR-T cells after they interacted with tumor cells (18-hour co-culture). 2.1. Sample Staining: labeled each sample with a unique barcode, and pooled them for sequencing. 2.2. Library Prep: Created sequencing libraries from the pooled cells using a targeted panel for oncology/immune genes (BD Rhapsody™ Onco-BC Panel HS). 2.3. Data Processing: Processed raw sequencing data to generate a gene expression matrix for each cell, correcting errors and demultiplexing samples. 2.4. Data Analysis: Analyzed the gene expression data to identify T cell clusters and find differentially expressed genes. 3. Cytokine Quantification: Prepared co-cultures of T cells and tumor cells and collected the conditioned media to measure the levels of multiple cytokines in the conditioned media using LegendPlex assay. 4. TCR T cell Cytotoxicity In Vivo: Tested the ability of engineered TCR-T cells to control tumor growth in immunodeficient mice with human tumor xenografts over a 45-day period.