Spatial transcriptomic analysis of HLA related immune signatures in metastatic cutaneous melanoma patients.

Transcriptomic expression of specific gene signatures within spatial and immune from lymph node metastases in patients with metastatic melanoma.<br><br>In the study presented here, a cohort of 55 metastatic melanoma patients was used to acquire expression profiles of specific HLA-related immune signatures for spatial expression analysis in the intersection of immune and tumor segments within the skin tumors and lymph node metastasis.<br><br>The Whole Transcriptome Atlas RNA probe kit (NantoString Technologies, Seattle, WA, USA) was used for NanoString GeoMx Digital Spatial profiling analysis of skin tumors and lymph node metastasis of metastatic melanoma patients. Formalin-fixed, paraffin-embedded (FFPE) sections of tumor microarrays of 3,5 µm thickness were deparaffinized, and the antigens retrieved, following the manufacturer's instruction (MAN-10150-01, NanoString Technologies).<br><br>In situ hybridization of RNA probes was performed overnight following the manufacturer's instruction (MAN-10150-01, NanoString Technologies).<br><br>Fluorophore-tagged antibodies against CD45, S100B/PMEL17, and SYTO13 nuclear dye (Human Melanoma TME morphology kit, NanoString Technologies) were used for staining to enable detection of the regions of interest (ROIs).<br><br>The arrays were scanned using the GeoMx instrument. 3-channel immunofluorescent digital images were created of S100B/PMEL17 signal representing tumor cells, CD45 signal representing leukocytes and SYTO13 signal representing nuclei. Based on the digital image, regions of interes (ROI) were selected.<br><br>Immune segments were selected from the ROIs. RNA-specific probes coupled with photocleavable oligonucleotides were then collected of the iROIs and subsequently sequenced in a next-generation sequencing pipeline. Gene counts were normalized according the the third-quartile protocol using the GeoMX DSP GEOMX-0061 Version 3.0.0.113.<br><br>RAW files include separate patient RAW counts for studied genes. Normalized matrixes include normalized counts using 3rd quartile normalization method. <br>