Dissecting tumor-immune microenvironment in response and resistance to immune checkpoint blockade in metastatic melanoma

Abstract

Although checkpoint inhibitor therapies have shown promise in improving progression-free and overall survival in metastatic melanoma patients, individual responses vary significantly, and the mechanisms underlying these differences remain unclear. To identify new factors driving response, as well as intrinsic and acquired resistance to immune checkpoint blockade (ICB), we employed single-nucleus RNA-sequencing (snRNA-seq) on biopsies from 51 metastatic melanoma patients undergoing ICB. Our dataset includes 30 pre-treatment samples (19 from patients with durable clinical benefit and 11 from those without) and 21 post-treatment samples (15 from patients with durable clinical benefit and 6 from those without). These samples were acquired either prospectively or retrospectively. Given the important role of the tumor immune microenvironment in modulating immunotherapy response, we aim to comprehensively characterize the immune landscape in these melanoma patients to identify key tumor-infiltrating immune cells, their distinct states, and cellular interactions that correlate with clinical outcomes. We characterized the single-nucleus transcriptomes of immune cells from metastatic melanoma patients before or after ICB treatment. In pre-treatment samples, T follicular helper (Tfh) like cells, naïve CD4 T cells, memory B cells, plasmablast-like B cells, and CLEC9A+ type 1 conventional dendritic cells (cDC1) were more enriched in responders to ICB. Analysis of tumor-infiltrating myeloid cells revealed diverse phenotypes of tumor-associated macrophages (TAMs) with distinct functions. Among the TAM subsets, we identified one TAM subset associated with angiogenesis and hypoxia phenotypes that was significantly more abundant in non-responders prior to therapy. Finally, our correlation analyses between tumor transcriptional profiles and immune cell frequencies suggested two distinct tumor microenvironments (TMEs) potentially associated with ICB response. We observed that the frequencies of different lymphocyte and dendritic cell populations were highly correlated. In addition, we observed that the frequencies of most TAM subsets were tightly and inversely correlated with lymphocyte infiltration in non-lymph node metastasis samples, suggesting different tumor immune microenvironments in lymph node and non-lymph node metastases. Overall, this study provides a systematic view of the highly heterogeneous immune landscapes of metastatic melanoma in response to ICB treatment. Our results highlight key immune cell types and interactions related to clinical outcomes, laying the foundation for the development of novel diagnostic biomarkers and therapeutic strategies for melanoma.