Recent advances in single-cell transcriptomics have greatly improved knowledge of complex transcriptional programs, rapidly expanding our knowledge of cellular phenotypes and functions within the tumour microenvironment and immune system. Several new single-cell technologies have been developed over recent years that have enabled expanded understanding of the mechanistic cells and biological pathways targeted by immunotherapies such as immune checkpoint inhibitors, which are now routinely used in patient management with high-risk early-stage or advanced melanoma. These technologies have method-specific strengths, weaknesses and capabilities which need to be considered when utilising them to answer translational research questions. Here, we provide guidance for the implementation of single-cell transcriptomic analysis platforms by reviewing the currently available experimental and analysis workflows. We then highlight the use of these technologies to dissect the tumour microenvironment in the context of cancer patients treated with immunotherapy. The strategic use of single-cell analytics in clinical settings are discussed and potential future opportunities are explored with a focus on their use to rationalise the design of novel immunotherapeutic drug therapies that will ultimately lead to improved cancer patient outcomes.

Melanoma is known as one of the most immunogenic tumours and is often characterised by high mutation burden, neoantigen load and immune infiltrate. The application of immunotherapies has led to impressive improvements in the clinical outcomes of advanced stage melanoma patients. The standard of care immunotherapies leverage the host immunological influence on tumour cells, which entail complex interactions among the tumour, stroma, and immune cells at the tumour microenvironmental level. However, not all cancer patients can achieve a long-term durable response to immunotherapy, and a significant proportion of patients develops resistance and still die from their disease. Owing to the multi-faceted problems of tumour and microenvironmental heterogeneity, identifying the key factors underlying tumour progression and immunotherapy resistance poses a great challenge. In this review, we outline the main challenges to current cancer immunotherapy research posed by tumour heterogeneity and microenvironment complexities including genomic and transcriptomic variability, selective outgrowth of tumour subpopulations, spatial and temporal tumour heterogeneity and the dynamic state of host immunity and microenvironment orchestration. We also highlight the opportunities to dissect tumour heterogeneity using single-cell sequencing and spatial platforms. Integrative analyses of large-scale datasets will enable in-depth exploration of biological questions, which facilitates the clinical application of translational research.

The biological underpinnings of therapeutic resistance to immune checkpoint inhibitors (ICI) in adolescent and young adult (AYA) melanoma patients are incompletely understood. Here, we characterize the immunogenomic profile and spatial architecture of the tumor microenvironment (TME) in AYA (aged ≤ 30 years) and older adult (aged 31–84 years) patients with melanoma, to determine the AYA-specific features associated with ICI treatment outcomes. We identify two ICI-resistant spatiotypes in AYA patients with melanoma showing stroma-infiltrating lymphocytes (SILs) that are distinct from the adult TME. The SIL^high subtype was enriched in regulatory T cells in the peritumoral space and showed upregulated expression of immune checkpoint molecules, while the SIL^low subtype showed a lack of immune activation. We establish a young immunosuppressive melanoma score that can predict ICI responsiveness in AYA patients and propose personalized therapeutic strategies for the ICI-resistant subgroups. These findings highlight the distinct immunogenomic profile of AYA patients, and individualized TME features in ICI-resistant AYA melanoma that require patient-specific treatment strategies.

Cancer comprises malignant cells surrounded by the tumor microenvironment (TME), a dynamic ecosystem composed of heterogeneous cell populations that exert unique influences on tumor development. The immune community within the TME plays a substantial role in tumorigenesis and tumor evolution. The innate and adaptive immune cells “talk” to the tumor through ligand–receptor interactions and signaling molecules, forming a complex communication network to influence the cellular and molecular basis of cancer. Such intricate intratumoral immune composition and interactions foster the application of immunotherapies, which empower the immune system against cancer to elicit durable long-term responses in cancer patients. Single-cell technologies have allowed for the dissection and characterization of the TME to an unprecedented level, while recent advancements in bioinformatics tools have expanded the horizon and depth of high-dimensional single-cell data analysis. This review will unravel the intertwined networks between malignancy and immunity, explore the utilization of computational tools for a deeper understanding of tumor–immune communications, and discuss the application of these approaches to aid in diagnosis or treatment decision making in the clinical setting, as well as the current challenges faced by the researchers with their potential future improvements.