Checkpoint-based immunotherapies, which target cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) pathways, can achieve durable clinical responses in several cancer types including melanoma. In melanoma, combining anti-PD-1 with anti-CTLA-4 has achieved a 65% response rate as compared to anti-PD-1 or anti-CTLA-4 alone with the respective response rate of 43% and 11%. However, a small subset of patients with melanoma has long-term benefit, 30-40% have primary resistance, and another 30% develop acquired resistance. Although a diverse array of malignant and non-malignant cells in a tumour contributes poor response to immunotherapy, cellular profiles and their spatial tumour-immune interactions remain poorly understood. This study aims to determine the molecular and cellular drivers within the tumour and its spatial microenvironment that influence response and resistance to immunotherapy.
Five tumour dissociates and formalin-fixed paraffin-embedded tissue samples were collected from patients with advanced melanoma receiving anti-PD-1 in combination with anti-CTLA-4. Single-cell sequencing and high-plex immunohistochemistry imaging were performed to characterise the tumour ecosystem including immune, malignant and stromal subtypes. Integration of spatial and molecular features was performed using a transfer learning algorithm and neighbourhood anchoring-based approach.
The spatial multi-omics analyses identified finer cell subpopulations in the spatial tissue architecture using thousands of molecular markers and cellular spatial coordinates. While CD8+ memory T cells were enriched in responsive tumours (median = 72.6% vs 38.5%), there was little-to-no difference in the proportion of CD4+ memory T cells in resistant versus responsive tumours. With longitudinal analysis, an immune-striving tumour microenvironment marked by peri-tumour lymphoid aggregates and low infiltration of T cells was identified in the tumour. In addition, the therapy-resistant MITF+SPARCL1+ and CENPF+ melanoma subclones emerged after therapy. Furthermore, the lymphoid aggregate associated B cell signatures (TNFRSF13C, BLK, and CD79A; adjusted p < 0.05) in patients had higher survival rates as compared to those who had minimal or no expression.
The collective findings provide a proof-of-concept for using multimodal analysis to identify cell communities and biological signatures that help to inform patient outcomes. Our work shed lights on the treatment strategies that enhance immunogenicity and T cell priming in the tumour microenvironment, potentially improving immunotherapy outcomes in patients with advanced melanoma who develop treatment resistance.