Journal: NPJ precision oncology

Article Title: High risk clear cell renal cell carcinoma microenvironments contain protumour immunophenotypes lacking specific immune checkpoints.

doi: 10.1038/s41698-023-00441-5

Figure Lengend Snippet: Fig. 1 Schematic of patient characteristics and experiment workflow. The patient characteristics (a) of the six ccRCC patients (n = 3 LG and n = 3 HG) include: patient LG_2 with a vena cava thrombus (VCT) for which we collected primary tumour microenvironment (TME) and thrombi separately but processed in the one capture array for ST-seq; patient HG_1 that we collected and processed tissues from para-TME (pTME) and TME; and patient HG_3 that we collected tissues from pTME and TME. For this experimental workflow (b), ten tissue regions were sampled from pTME, TME and VCT that excluded fibrotic and necrotic regions. ST-seq was completed using 10x Genomics Visium Gene Expression microarrayed glass slides with unique spatially barcoded ST-spots that captured the mRNA released from the overlaying thin ccRCC tissue sections. Annotation of immune ST-spots was completed with data integration of six published single-cell RNA-sequencing (scRNA-seq) datasets. Further immune cell sub-typing was completed with a scRNA and T-cell receptor (TCR) sequencing dataset. Integrated analysis was completed on CD8+ T cells, TAM and monocytes.

Article Snippet: In brief, the Visium ST-seq method (CG000239 Rev D, 2020 October, 10x Genomics, USA) involved the use of microarrayed glass slides with 55 μm spots (or ST-spots) containing oligonucleotides with a sequence of deoxythymine (oligo-dT) and unique spatial barcodes printed within capture arrays.

Techniques: Gene Expression, RNA Sequencing, Sequencing