GSM9669736: protein32; Homo sapiens; OTHER
We retrospectively collected metastatic tissue samples from six breast cancer patients with one or more metastatic lesions. The samples included two bone metastases, two brain metastases, one pericardial metastasis, and one chest wall metastasis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections (5 μm) were mounted on standard microscope slides and processed for spatial proteogenomics analysis using the NanoString GeoMx Digital Spatial Profiler (DSP). To enable tissue compartmentalization and spatial segmentation, sections were stained with a panel of three fluorescently labeled morphological markers: anti-pancytokeratin (PanCK-AF532) to identify epithelial tumor cells, anti-CD45 (CD45-AF594) to delineate immune cell populations, and Syto13 nucleic acid stain to visualize nuclei and define cellular boundaries. Staining was performed according to standard immunofluorescence protocols with overnight incubation at 4°C. DNA barcodes corresponding to RNA and protein targets were photocleaved and collected from each segmented AOI using sequential UV light exposure. Collected barcodes were processed using the GeoMx library preparation protocol with unique dual indexing (i7/i5) for each AOI to enable multiplexed analysis.