GeoMX spatial transcriptomics of diffuse pleural mesothelioma: impact of CDKN2A deletion
GeoMX spatial transcriptomics of diffuse pleural mesothelioma: impact of CDKN2A deletion
Spatial transcriptomics was performed using the GeoMx® Digital Spatial Profiler (DSP) platform (NanoString Technologies) on 5 μm FFPE tumor tissue sections. To enable spatial selection, slides were stained with the GeoMx Solid Tumor TME Morphology Kit, which includes antibodies targeting cytokeratin (CK) and a nuclear stain (SYTO-13).  Areas of interest (AOIs) were selected by two pathologists, who identified regions representative of tumor heterogeneity based on tissue morphology and Pan-CK expression. In biphasic tumors, both epithelioid and sarcomatoid regions were selected. Mixed regions were segmented to distinguish between epithelioid and sarcomatoid components.    Slides were hybridized with the GeoMx® Cancer Transcriptome Atlas panel, following the manufacturer's protocol. After AOI selection and UV photo-cleavage of the barcoded probes, the released GeoMx RNA probes were collected and sequenced. Hybridized RNA probes were collected from each selected AOI, and next-generation sequencing (NGS) libraries were prepared using the GeoMx® Seq Code Pack (NanoString Technologies). Libraries were pooled based on AOI size, purified using AMPure XP beads (Beckman Coulter, Brea, California, USA), and resuspended in a volume proportional to the number of pooled AOIs. The quality and concentration of the library pools were assessed using Bioanalyzer (Agilent).