B-ALL GPR65 expression influences susceptibility to CAR-T therapy through a VEGF and macrophage-dependent mechanism [scRNA-Seq]
Chimeric antigen receptor (CAR)-T cell therapy has shown promise in treating CD19+ hematological tumors, but some patients fail to respond. Here we used an immune-competent mouse model of B cell acute lymphoblastic leukemia (B-ALL) to assess clonal tumor populations with distinct responses to CAR-T cell therapy. We identified GPR65 as a determinant of tumor responsiveness to CAR-T cell therapy, with high GPR65 expression associated with a complete response. GPR65 KO tumors were resistant to CAR-T treatment in vivo. This was associated with increased tumor VEGFA expression driven by FOXO1 activation and expanded host macrophages. Either depletion of host macrophages or deletion of VEGFA from GPR65 KO tumors restored responsiveness to CAR-T cell treatment. Anti-VEGFa therapy, in combination with CD19-CAR-T, prolongs the survival of GPR65-KO tumor-bearing mice in pre-clinical models. Our results indicate that GPR65 may be a useful biomarker for tumor responsiveness to CAR-T cell therapy and further suggest VEGFA or host macrophages as therapeutic targets to improve CAR-T efficacy. Overall design: B6 mice were injected with CR or GPR65 KO tumor cells (i.v.) followed by treatment 7 days later with 10 million activated CAR-T cells or mock PBS injection (retro-orbital). Spleens were harvested from untreated mice at day 7 and from CAR-T treated mice at day 11 after tumor engraftment. Processed spleens were stained and sorted to isolate CR or GPR65 KO tumor cells (RFP+), and the host immune cell compartment (RFP–Thy1.1–CD45.2+). Tumor and host cells from untreated mice were mixed at a 1:1 ratio. Tumor and host cells from treated mice were run independently. Each sample contained a total of 200,000 cells. Sorted cells were counted using AO/PI stain on the Luna following dual fluorescence cell sorter and mixed in a 1:1 proportion for 5' single cell RNA seq processing. 6000 cells were targeted from each sample using the 10X Chromium Next GEM Single Cell 5' Reagent kits v2 (Dual Index). Cells were partitioned into droplets and barcoded using the Chromium Controller using Chip K. Reverse transcription, cDNA amplification, TCR amplification, fragmentation, adapter and sample index ligation were all done following the 10X CG000331 Rev C protocol. cDNA and libraries were quantified using the High Sense DNA chip on the Agilent Bioanalyzer. Libraries were pooled and sequenced on the NovaSeq 6000 using paired end reads as recommended by 10X genomics