Journal: bioRxiv

Article Title: Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of “prime-and-kill” synNotch-CAR-T cells

doi: 10.64898/2026.03.26.713760

Figure Lengend Snippet: ( A–C ) Histograms and empirical cumulative distribution function (ECDF) plots showing distribution of maxUMI CAR values in in vitro E-SYNC cells ( A ), in vitro B-SYNC cells ( B ), and in vivo samples ( C ). In the histograms, the bin width is 1, and the Y-axis is shown on a log 10 scale. In both histograms and ECDF plots, maxUMI CAR values >50 were capped at 50 for visualization. (D) Dot plot showing expression of representative DEGs by sample type (same gene list as in and ), stratified by CAR-expressing versus non-expressing groups. (E) Swarm plot and ECDF plot depicting the distribution of TRM core gene signature scores. Differences between CAR-positive and -negative cells within the brain were assessed using the Kolmogorov–Smirnov test ( P = 4.0 × 10 -5 ). (F) ECDF plots comparing the nCount_RNA and nFeature_RNA values between CAR-expressing and non-expressing cells within the brain-derived CD8 + synNotch-positive population. Differences were assessed using the Kolmogorov–Smirnov test. (G) Schematic overview of the time-course co-culture experiment of B-SYNC cells with the human GBM cell line GBM6 (BCAN + /EphA2 + /IL13Rɑ2 + ). Co-culture was performed using 24-well plates, with each well containing 0.5 × 10 5 GBM cells and 1 × 10 6 CD8 + B-SYNC cells. The data presented are representative of two independent experiments using different donors. Additional data are shown in Fig. S16 . (H) Flow cytometry analysis showing the percentage of cells expressing GFP fused to CAR within the live, singlet, non-tumor (mCherry-negative) population. Corresponding scatter plots are shown in Fig. S16 . (I) RT-qPCR data showing CAR transcript abundance across conditions. Relative expression was calculated using the 2 -ΔΔCq method, with the B-SYNC no-stimulation (no-stim) condition and the RPS18 transcript as references. ( J–K ) Bulk RNA-seq principal component analysis (PCA) plot ( J ) and gene expression heatmap ( K ) illustrating transcriptomic similarities and differences across samples. The heatmap displays scaled expression of 6,885 genes expressed in the B-SYNC no-stim sample at transcripts-per-million (TPM) ≥ 10, sorted by the magnitude of expression changes between no-stim and post-stimulation conditions. ( L ) Representative genes minimally expressed without co-culture but upregulated and sustained after co-culture. The full list of gene expression comparisons is provided in Supplementary Table S13 .

Article Snippet: To prepare the B-SYNC primed sample, 2 × 10 4 cells were cultured for 24 hours on wells coated with Laminin (100 μg/ml, Thermo Fisher Scientific,23017015) and recombinant BCAN (25 μg/ml; R&D Systems, 7188-BC-050 and 4009-BC-050).

Techniques: In Vitro, In Vivo, Expressing, Derivative Assay, Co-Culture Assay, Flow Cytometry, Quantitative RT-PCR, RNA Sequencing, Gene Expression