Analysis of T-cell repertoire (TCR) has been proposed as a new approach to better understand the adaptive immune response in cancer diseases. Next generation sequencing of the TCR has proven to be a powerful approach to characterize this repertoire, with potential applications to select and follow patients who benefit from immunomodulatory therapies. However, current TCR sequencing in tumor tissues is based on bulk RNA or DNA extraction, which does not provide neither the functional information at the clonotype level nor the spatial context. Here, we explore a new method which provides the spatial localization of individual T cell clonotypes in tumor samples.
Frozen tumor sections (12 μm) were positioned on a 10x Visium spatial gene expression slide, and mRNA was captured in spatial spots of 55 μm diameter. Each transcript was tagged with a Unique Molecular Identifier (UMI) and a spatial barcode during an in situ reverse transcription. Starting from the generated cDNA, the TRB genes were amplified using a multiplex PCR with a set of primers positioned on the TRBV genes. TRB libraries were sequenced on the Illumina MiSeq platform. Analyzed samples were 3 mm diameter Tru-Cut tumor biopsies collected before and during neoadjuvant treatment of locally advanced breast cancer patients with informed consent.
With our spatial TCR sequencing protocol we identified polyclonal T cell repertoires and localized individual T cell clonotypes on breast tumor slides. We observed distinct predominant clonotypes and accurately determined their spatial distribution before and during treatment.
This proof of concept work demonstrates the feasibility of spatial TCR sequencing in tumor biopsies, providing a new method to explore the spatial organization of T cell clonotypes in the tumor microenvironment, explore T cell functional profiles at the clonal level and follow modifications during tumor development and treatments.
The authors.
Seqalis.
F.P. Duhoux: Non-Financial Interests, Institutional, Advisory Role: Roche, Pfizer, AstraZeneca, Lilly, Novartis, Amgen, Daiichi Sankyo, Pierre Fabre, Gilead sciences, Seattle Genetics, MSD Oncology; Non-Financial Interests, Personal, Other: Amgen, Roche, Teva, Pfizer, Daiichi Sankyo/AstraZeneca. J. Carrasco: Non-Financial Interests, Institutional, Research Grant: AstraZeneca, Roche; Non-Financial Interests, Personal, Advisory Board: Seqalis. All other authors have declared no conflicts of interest.