Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Schematic representation of the pro-phagocytic module screening strategy. (B) Correlation between log2 fold change and −log10 rho scores for pro-phagocytic CAR variant screens. Data represent 18 biological replicates across six NaSDC lentivirus batches. (C) FACS-based quantification of PC3-PSMA tumor cell phagocytosis by CAR-Ms (n = 4 biological replicates). (D) Schematic representation of the pro-inflammatory module screening strategy. (E) Correlation between log2 fold change and −log10 rho scores for pro-inflammatory CAR variant screens. Data represent 15 biological replicates across five NaSDC lentivirus batches. (F) Relative mRNA expression of TNF , IL6 and IL1B in CAR-Ms cocultured with PSMA positive target tumor cells (n = 4 biological replicates). (G) Schematic representation of the pro-infiltrating module screening strategy. (H) Correlation between log2 fold change and −log10 rho scores for pro-infiltrating CAR variant screens. Data represent 9 biological replicates across three NaSDC lentivirus batches. (I) Relative mRNA expression of MMP family genes in CAR-Ms cocultured with PSMA positive target tumor cells (n = 4 biological replicates). Dashed lines in ( C , F , I ) indicate −log10(0.05) (horizontal) and log2 fold change of truncated CARs (CARΔ) (vertical). Data in (C) are presented as mean ± s.e.m. and analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison test.

Article Snippet: Surface anti-PSMA CAR expression was detected using PE-Labeled Human PSMA (ACROBiosystems), or Biotinylated Human PSMA (ACROBiosystems) followed by Streptavidin/APC (Solarbio) staining.

Techniques: Variant Assay, Expressing, Comparison

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Description of CAR variants used for individual characterization. (B) FACS-based quantification of PC3-PSMA tumor cell phagocytosis by UTD and CAR-Ms (n = 4 biological replicates). (C) Flow cytometric histograms showing surface expression of CD80 and CD86 in UTD and CAR-Ms cocultured with PSMA positive tumor cells. (D) Flow cytometric histograms of reactive oxygen species (ROS) levels in UTD and CAR-Ms cocultured with PSMA positive tumor cells. DCF, 2’-7’dichlorofluorescein. (E) Relative mRNA expression of TNF and IL6 in UTD and CAR-Ms cocultured with PSMA positive tumor cells (n = 4 biological replicates). (F) Relative mRNA expression of MMP11 and MMP13 in UTD and CAR-Ms cocultured with PSMA positive tumor cells (n = 4 biological replicates). Data in ( B) are presented as mean ± s.e.m. and analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison. Data in ( C , D) are representative of three independent experiments. Dashed lines ( C , D ) represent the mean fluorescence intensity (MFI) for UTD macrophages.

Article Snippet: Surface anti-PSMA CAR expression was detected using PE-Labeled Human PSMA (ACROBiosystems), or Biotinylated Human PSMA (ACROBiosystems) followed by Streptavidin/APC (Solarbio) staining.

Techniques: Expressing, Comparison, Fluorescence

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Schematic illustration of the humanized immune system TME model. PC3-PSMA tumors were established bilaterally, followed by PBMC engraftment on day 7. On day 14, 5 × 10 6 UTD or CAR-Ms were injected IT. Tumors were harvested on day 18 for scRNA-seq. SC, subcutaneously. IV, intravenously. IT, intratumorally. (B) Proportions of cell types from scRNA-seq (left) and tSNE-based clustering of all sequenced cells (right). B, B cell. T, T cell. Mac, macrophage. (C) Dimensionality reduction (tSNE) and clustering for macrophages. Data are colored by treatment conditions. (D) Density plots within tSNE maps depicting the distribution of CAR-expressing cells. (E) AUC scores of M1 and M2 gene sets in the indicated macrophage clusters. (F) Feature plots showing expression levels of selected genes. (G) MFI of HLA-ABC and HLA-DR in UTD and CAR-Ms after 24 h coculture with PSMA positive tumor cells (n = 3 biological replicates). (H) AUC scores of T cell cytotoxicity and exhaustion gene sets in the indicated T cell clusters. Unless specified otherwise, data are presented as mean ± s.e.m. and analyzed using the two-tailed Student’s t-test.

Article Snippet: Surface anti-PSMA CAR expression was detected using PE-Labeled Human PSMA (ACROBiosystems), or Biotinylated Human PSMA (ACROBiosystems) followed by Streptavidin/APC (Solarbio) staining.

Techniques: Injection, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

doi: 10.1101/2025.02.16.638489

Figure Lengend Snippet: (A) Specific killing of PC3-PSMA cells by UTD and COMB7 CAR-Ms at varying E/T ratios. Data represent two independent experiments from two human donors. (B) Specific killing of PC3-PSMA cells by UTD, CD247 CAR-Ms or COMB7 CAR-Ms at an E/T ratio of 3/1. Data collected from two human donors. (C-D) Specific killing of HER2 + SKOV3 or GPC3 + HepG2 cells by UTD or COMB7 CAR-Ms at an E/T ratio of 3/1 (n = 6 biological replicates). (E) NCG mice were IP injected with 1 × 10 6 PC3-PSMA cells, followed by treatment with PBS, 5 × 10 6 UTD, or 5 × 10 6 COMB7 CAR-Ms one day later. (F) Overall survival for mice in each treatment group (n = 6 for PBS, n = 5 for UTD and COMB7). (G) Tumor burden measured by bioluminescence (total flux). Data shown are individual values (n = 6 for PBS, n = 5 for UTD and COMB7). Unless specified otherwise, data are presented as mean ± s.e.m. and analyzed using the two-tailed Student’s t-test.

Article Snippet: Surface anti-PSMA CAR expression was detected using PE-Labeled Human PSMA (ACROBiosystems), or Biotinylated Human PSMA (ACROBiosystems) followed by Streptavidin/APC (Solarbio) staining.

Techniques: Injection, Two Tailed Test