Targeting Melanin Heterogeneity in Metastatic Melanoma: A Dual‐Tumour Mouse Melanoma Model

The combination of melanin-targeted radionuclide therapy (TRT) and immunotherapy offers potential in overcoming melanoma resistance to conventional therapies. Studying the potential abscopal effect induced by TRT is essential to evaluate such combination. We develop here a preclinical murine model comprising a target (pigmented) and non-target (non-pigmented) tumour to study the abscopal effect induced by melanin-TRT in melanoma. Murine melanoma cell lines were tested: two pigmented (B16-F10 and B16-OVA) and one non-pigmented (B16-G4F), inoculated in C57BL/6 mice to assess pigmentation levels and immune infiltration. Heterogeneous tumour growth and repigmentation of the B16-G4F tumour led us to develop a non-pigmented cell line (B16-OVAmTYR-/-) by tyrosinase invalidation using CRISPR/Cas9. A dual-tumour model comprising the B16-OVA tumour and the B16-OVAmTYR tumour was evaluated in terms of tumour growth, pigmentation, and immune infiltrate. The B16-OVA model displayed homogeneous tumour growth, pigmentation and high immune infiltrate (CD8+ T cells p < 0.001; CD4+ T cells p < 0.05, regulatory T cells p < 0.001). The new B16-OVAmTYR-/- cell line ensured a consistent genetic background for comparative studies. The B16-OVAmTYR-/- maintained a non-pigmented phenotype without repigmentation (no melanin expression) and demonstrated similar tumour growth characteristics to its pigmented counterpart (DT = 2.4 ± 0.5 days). Establishing a dual-tumour model using both B16-OVA and B16-OVAmTYR-/- cell lines enabled concurrent study of pigmented and non-pigmented tumours in a single host, closely mirroring clinical scenarios of metastatic melanoma. We have successfully developed a new dual-tumour pigmented and non-pigmented mouse melanoma model mimicking clinical observations to study the abscopal effect in metastatic melanoma.