UTD vs HER2.eSPR macrophage treatment in MC38.HER2 TIL

Here, exploiting myeloid antibody-dependent cellular phagocytosis biology and phagocytosis checkpoint blockade, we report an enhanced synthetic phagocytosis receptor (eSPR) that integrates FcRγ-driven phagocytic chimeric antigen receptors (CARs) with built-in secreted CD47 blockers. To assess modulation of the tumor immune microenvironment by eSPR macrophages, untreated (UTD) and eSPR-macrophage-treated tumor tissues were extracted and processed. CD45⁺ tumor-infiltrating leukocytes were sorted and analyzed by single-cell RNA sequencing. Subcutaneous MC38.HER2 tumor models were generated via s.c. inoculation. Mice were randomized on day 7. UTD or HER2.eSPR macrophages (4×10^⁶ cells/mouse) were intratumorally injected on day 8. Mice were euthanized on day 11, and tumors were excised. Tumor tissue was weighed, chopped into ~1 mm pieces, and digested in RPMI supplemented with 2 mg/mL Collagenase I, 2 mg/mL Hyaluronidase, and 25 μg/mL DNase I (3 mL per 0.2 g tumor). Digestion was carried out at 37°C with vigorous shaking for 45 minutes. The reaction was quenched, passed through a 60-μm mesh, washed twice with PBS, stained with anti-CD45 antibody, and live CD45⁺ cells were sorted using a BD Aria III.