Introduction: Melanoma, a highly metastatic skin cancer, exhibits variations in prognosis and response to therapy depending on the site of metastasis. A spectrum of transcriptional states ranging from highly differentiated to undifferentiated phenotypes has been described. These observations raise the question of whether organ-specific microenvironments shape tumour cell state dynamics and clonal selection during progression and in response to therapy.
Material and Methods: To address this, C57BL/6 mice were injected subcutaneously, intravenously, or intracranially with a common pool of luciferase-expressing YUMMER1.7PV1 murine melanoma cells transduced with a SPLINTR (Single-cell Profiling and Lineage Tracing) mCherry barcode library to generate subcutaneous, lung, and brain tumours. Mice bearing subcutaneous and lung tumours were subsequently treated with anti-CTLA4 and anti-PD1 immunotherapy. Subcutaneous and lung tumours were collected at baseline and at defined response or non-response stages following treatment, whereas brain tumours were analysed only at baseline. Tumours were then processed for downstream clonal and single-cell RNA sequencing analyses.
Results: A single distinct dominant clone was observed in subcutaneous and lung tumours of both responders and non-responders, with no overlap in clonal dominance across treatment conditions. Importantly, the dominant surviving clones differed between subcutaneous and lung tumours, suggesting that each microenvironment imposes distinct selective pressures. Despite this clonal divergence, individual clones were capable of expressing multiple transcriptional states. Conversely, similar transcriptional states could be sustained by distinct clones, indicating a high degree of cellular plasticity.
Conclusion: Overall, our findings demonstrate that melanoma progression is governed by an interplay between clonal selection, cellular plasticity, and organ-specific microenvironmental constraints. These results highlight that both clonal identity and dynamic cell state transitions are critical determinants of metastatic fitness and therapeutic resistance across different anatomical sites.