T cell phenotypic and receptor mapping across space
In recent years, the field of transcriptomic analyses has been revolutionized by spatial RNA sequencing approaches such as the most prominent Visium platform. Due to the fact that these sequencing techniques are generally based on short RNA reads, the information on tumor mutations as well as the identity of T cell receptor (TCR) sequences is very limited. Here, we developed a pipeline for combining short-read sequencing with long read Nanopore sequencing and spatial TCR sequencing to identify the spatial organization and dynamics of tumor mutations/protein isoform expression and individual TCR clones, respectively. Using long read sequencing of the Visium gene expression library, we were able to spatially map a recurrent driver mutation and an oncogenic fusion that had been predicted by whole exome sequencing of glioma samples. As TCR transcripts are notoriously difficult to detect, due to their low abundance, we implemented an additional hybridization capture and PCR enrichment step. This enrichment resulted in a 100-fold increase of detectable TCR clones, allowing comprehensive analysis of the TCR repertoire in the spatial context. For some TCR clones that were found to be expanded in the tissue, functional reactivity was successfully assessed. Further analyses of correlation with immune niches and spatial pattern of tumor mutations are ongoing. Together, our pipeline advances the understanding of T cell spatial dynamics clinically relevant in cancer and provides a unique opportunity to uncover regulatory checkpoints.