# Single-cell mapping of alternative splicing linked to checkpoint immunotherapy response

## TL;DR

This study introduces a method to analyze alternative splicing in single-cell RNA data, linking it to cancer immunotherapy response and tumor antigen generation.

## Contribution

A novel method for detecting alternative splicing in single-cell 5′-RNA-seq data, revealing splicing events linked to immunotherapy response.

## Key findings

- Splicing events specific to cancer or stromal cells and different breast cancer subtypes were identified.
- ESRP1 was validated as a splicing regulator in breast cancers responding to immunotherapy.
- Splicing events correlated with immune activity and patient survival in cancers treated with immunotherapy.

## Abstract

Evidence suggests that alternative RNA splicing (AS) plays a critical role in tumor biology and may contribute to the generation of tumor antigens. Here, we develop a method to detect AS in short-read single-cell 5′-RNA-sequencing data, allowing us to uniquely characterize the heterogeneity and dynamic changes in AS in individual cell types within the tumor microenvironment. We identify numerous splicing events specific to either cancer cells or stromal cell types or for triple-negative versus estrogen receptor-positive breast cancers (BCs). By correlating these splice events with expression of splicing regulators in individual cells, we also identify their potential mediators. For instance, we identify and functionally validate the Epithelial Splicing Regulatory Protein-1 (ESRP1) to drive AS in BCs responding to immune checkpoint blockade (ICB). Prioritization of splicing events based on their likelihood to represent tumor antigens reveals that their aggregated load also correlates with high immune activity in multiple cancers, while also predicting expansion of T cells in BCs receiving ICB and prolonging long-term survival of cancer patients treated with ICB. Collectively, our method provides a framework for analyzing AS in single-cell data and defines a key role for AS in the response to ICB.

Graphical Abstract