title: GSM8940679: ROI.062.CD163; Homo sapiens; OTHER
library_construction_protocol: mRNA expression in FFPE tissues was measured using the GeoMx Human Whole Transcriptome Atlas (NanoString, GMX-RNA-NGS HuWTA-4), which targets 18,000 protein-coding genes via UV-cleavable oligonucleotide barcodes. Probe hybridization and immunofluorescent staining were performed according to the GeoMx FFPE slide preparation protocol. Briefly, the slide was baked overnight at 37°C, followed by deparaffinization, rehydration, target retrieval, and proteinase K (0.1 μg/mL) treatment. After post-fixation, in situ probe hybridization was performed overnight. Morphology markers used for immunofluorescent staining included: pan-cytokeratin (pan-CK; Novus Biologicals, NBP2-33200AF532, AF532, 1:40), CD45 (NBP2-34528AF594, AF594, 1:20), CD163 (Abcam, ab218294, AF647, 1:100), and SYTO 13 nuclear stain (NanoString, 121300303, FITC, 1:10). Slides were scanned at 20x magnification using the GeoMx Digital Spatial Profiler. Regions of interest (ROIs) were selected by a pathologist based on morphology marker staining, guided by H&E staining to exclude necrotic areas and big vessels. ROIs were subdivided into areas of interest (AOIs), and only those containing ≥50 cells based on manual counting were included. Four AOI types were collected: (1) panCK+ cancer epithelium, (2) CD163+ monocytes/macrophages, (3) CD45+ CD163- immune cells, and (4) stroma (panCK- CD163- CD45-). Oligonucleotide barcodes from AOIs were collected into 96-well plates via UV ablation. Subsequent PCR amplification, pooling, quality control, and sequencing were conducted following the GeoMx DSP-NGS protocol. Libraries were purified using two rounds of Ampure XP magnetic beads (1.2:1 bead-to-library ratio) with the HighPrep PCR Clean-up System (MAGBIO, AC-60500), then sequenced on an Illumina NovaSeq PE50 flow cell at a depth of 100x/μm².