Applying high-dimensional single-cell technologies to the analysis of cancer immunotherapy

Abstract

Advances in molecular biology, microfluidics and bioinformatics have empowered the study of thousands or even millions of individual cells from malignant tumours at the single-cell level of resolution. This high-dimensional, multi-faceted characterization of the genomic, transcriptomic, epigenomic and proteomic features of the tumour and/or the associated immune and stromal cells enables the dissection of tumour heterogeneity, the complex interactions between tumour cells and their microenvironment, and the details of the evolutionary trajectory of each tumour. Single-cell transcriptomics, the ability to track individual T cell clones through paired sequencing of the T cell receptor genes and high-dimensional single-cell spatial analysis are all areas of particular relevance to immuno-oncology. Multidimensional biomarker signatures will increasingly be crucial to guiding clinical decision-making in each patient with cancer. High-dimensional single-cell technologies are likely to provide the resolution and richness of data required to generate such clinically relevant signatures in immuno-oncology. In this Perspective, we describe advances made using transformative single-cell analysis technologies, especially in relation to clinical response and resistance to immunotherapy, and discuss the growing utility of single-cell approaches for answering important research questions.

Fig. 1 |. Workflow for single-cell analysis in immuno-oncology.

The tumour microenvironment (TME) is often complex and contains myriad cell types and states that can be disaggregated, with varying degrees of success, into a single-cell suspension. Cells can then be processed without further manipulation, or specific cell subsets can be isolated for downstream analysis. Single-cell analysis — most commonly by single-cell RNA sequencing (scRNA-seq) — enables the assessment of tumour, immune and/or stromal cells to yield high-dimensional information on tumour heterogeneity and an atlas of the immune and/or stromal microenvironment in relation to clinical stage, disease subtype and tumour location. Sampling before and after immunotherapy, especially if patients can be stratified based on their response, facilitates a detailed dissection of the mechanisms underlying response and resistance. The ability to track immune cell populations, specifically T cells through paired T cell receptor (TCR) α and β subunit sequencing, enables the detailed characterization of T cell clones involved in mediating response. These findings can be applied to a wider selection of patients, with the goals of improving prognostication and optimal treatment selection in the face of an ever-expanding range of therapeutic options, and enabling therapeutic monitoring through the identification of cellular populations or markers that can be monitored using high-throughput conventional technologies. Findings from within the TME might also be linked to changes in the peripheral circulation, which are more amenable to regular monitoring.

Fig. 2 |. General biospecimen framework for incorporating single-cell analyses in clinical trials involving immunotherapies.

a | Discovery efforts for a limited subset of patients. In-depth characterization of pre-treatment, on-treatment and post-treatment (resistance) tumour biopsies and peripheral immune cells using single-cell and bulk approaches enables the discovery of biological determinants of therapeutic response and resistance. These analyses are technically challenging and expensive, and so would only be performed in a limited number of patients. b | Validation using conventional technologies for a larger set of patients. For a larger set of clinical trial patients, more conventional sample collection (such as frozen or formalin-fixed paraffin-embedded (FFPE) tumour tissue at baseline, peripheral blood cells before and during treatment) is feasible. These samples can be used to validate proposed biological determinants of immunotherapy response and resistance (from the ‘discovery’ efforts depicted in part a) using standardized assays. PBMCs, peripheral blood mononuclear cells; Hi-Dim, high-dimensional; IHC, immunohistochemistry; OCT, optimal cutting temperature compound; scRNA-seq, single-cell RNA sequencing; TCR, T cell receptor.