PD-L1-directed CAR oncolytic herpesviruses reprogram tumor-associated macrophages to attack resistant tumors. Mus musculus

For subcutaneous tumor models,5E5 B16-F10 cells were inoculated subcutaneously into the right flank of C57BL/6 or nude mice and allowed to establish for seven days. Once the tumors reached the indicated volume,they were injected intratumorally with CAR-oHSV or control virus at a dose of 3E7 PFU,followed by repeated injections on day 0,2,4 and 6 after the initial treatment. Tumors were collected and analyzed on day 15. The tumors were excised and homogenized using a gentleMACS tissue dissociator with a Tumor Dissociation Kit for mice.The cell suspensions were stained with 7AAD and APC/Cyanine7-labeled anti-mouse CD45 antibodies.Subsequently, fluorescence-activated cell sorting (FACS) was employed to isolate CD45 positive cells, ensuring a cell viability exceeded 90%. These sorted cells were then prepared for single-cell RNA sequencing (scRNA-seq).