TNF-a released by melanoma liver metastasis activates AKT and NFKß1 pathways to promote collagen type I secretion by hepatocytes
A total of four human melanoma liver metastasis (MLiM) formalin-fixed paraffin-embedded (FFPE) sections were analyzed using Nanostring GeoMx digital spatial profiling (NGDSP). The FFPE tissues were obtained from patients who underwent surgery and were pathological diagnosed with MLiM. Patients who had received any preoperative therapy were excluded. In each region of interest (ROI), two areas of interest (AOI)s were defined by HepPar1+ (hepatocytes); HMB45+ (melanoma cells) staining. A total of 43 AOIs were carefully reviewed for segments with sequencing saturation < 25%, nuclei fewer than 80, negative probe counts <3, or a surface area of <12,000 squared microns. Background correction and scaling were performed using geometric means and normalization was performed using Q3 averages of housekeeping genes. Genes with expression levels at or lower than limit of quantification (LOQ) in at least 5% of segments were filtered out. A total of 1,811 genes passed quality control and were analyzed. A total of seven ROIs with tumor margin (TM) and 13 ROIs with tumor center (TC) were selected, respectively. TM was defined as the areas on the border of malignant tumor cells close to normal tissue based on pathological features. TC was defined as central tumor tissue further inside from TM. In addition, 14 ROIs were selected at ANH. The ANH areas were defined as an area within 1,200 µm from the TM. Furthermore, nine ROIs were selected at the DNH areas. DNH was defined as an area at least 6 mm away from TM. Differential expressed genes (DEGs, Log2 fold-change >1.5, <-1.5, p < 0.05) were calculated on AOIs selected from MLiM comparing TC vs. TM; ANH vs. DNH; and ANH vs. Melanoma cells (TC+TM). Overall design: Four FFPE tissue samples were analyzed by NanoString GeoMX Digital Spatial Profiling. The regions of interest (ROI) were collected in each tissue samples and segmented in areas of interest (AOI) based on the staining pattern of two fluorescently labeled cell type specific morphological markers: HepPar1+ and HMB45+.