Tumor Pigmentation Does Not Affect Light-Activated Belzupacap Sarotalocan Treatment but Influences Macrophage Polarization in a Murine Melanoma Model

PURPOSE. Pigmentation in uveal melanoma is associated with increased malignancy and is known as a barrier for photodynamic therapy. We investigated the role of pigmentation in tumor behavior and the response to light -activated Belzupacap sarotalocan (Bel-sar) treatment in a pigmented (wild type) and nonpigmented (tyrosinase knock -out [TYR knock -out]) cell line in vitro and in a murine model. METHODS. The B16F10 (TYR knock -out) was developed using CRISPR/Cas9. After the treatment with light -activated Bel-sar, cytotoxicity and exposure of damage -associated molecular patterns (DAMPs) were measured by flow cytometry. Treated tumor cells were co -cultured with bone marrow -derived macrophages (BMDMs) and dendritic cells (DCs) to assess phagocytosis and activation. Both cell lines were injected subcutaneously in syngeneic C57BL/6 mice. RESULTS. Knock -out of the tyrosinase gene in B16F10 led to loss of pigmentation and immature melanosomes. Pigmented tumors contained more M1 and fewer M2 macrophages compared with amelanotic tumors. Bel-sar treatment induced near complete cell death, accompanied with enhanced exposure of DAMPs in both cell lines, resulting in enhanced phagocytosis of BMDMs and maturation of DCs. Bel-sar treatment induced a shift to M1 macrophages and delayed tumor growth in both in vivo tumor models. Following treatment, especially the pigmented tumors and their draining lymph nodes contained IFN-gamma positive CD8+T cells. CONCLUSIONS. Pigmentation influenced the type of infiltrating macrophages in the tumor, with more M1 macrophages in pigmented tumors. Belzupacap sarotalocan treatment induced immunogenic cell death and tumor growth delay in pigmented as well as in nonpigmented models and stimulated M1 macrophage influx in both models.