GSM7336885
Formalin fixed paraffin embedded (FFPE) tissue were sectioned at 5 μm thickness and mounted onto Superfrost Plus slides within one month of spatial transcriptomics analysis. Slides were stored at 4 degrees Celsius with desiccator and shipped to NanoString (Seattle, USA) for Technology Access Program (TAP) processing for GeoMx DSP. Pathology review was performed on all the six cases. The tissue sections demonstrated distinct morphological features and were assessed using the cellularity of the tumor cells, nuclear pleomorphisms, presence of mitoses, necrosis, and microvascular proliferation. The five anatomic features proposed by The Ivy Glioblastoma Atlas Project (Ivy GAP) were used to annotate the tumors into regions. These included: Leading edge (LE): the outermost part of tumour with the least tumor cell density and bordering on the normal tissue area; Cellular tumor (CT): the core of the tumor with highest cell density; Infiltrating tumor (IT): the zone interposed between LE and CT with moderate tumour cell density; Microvascular proliferation (MVP): Areas with endothelial proliferation; Pseudopalisading cells around necrosis (PAN): dense tumour cells geographically fenced around core necrosis. Other features recognized are regions of large non-palisaded necrosis (NEC) and normal brain tissue (N). Annotations were performed by two pathologists. After baking the slides for 2 hours for paraffin removal, slides were loaded onto a Leica BOND RX for tissue rehydration, heat-induced epitope retrieval (ER2 for 20 minutes at 100 °C) and proteinase K treatment (0.1 μg/ml for 15 minutes at 37 °C). The tissue sections were then hybridized with the Human WTA probes overnight. Following 2 x 5 min stringent washes (1:1 4x SSC buffer & formamide), the slides were blocked and then incubated morphology marker antibodies: GFAP (488 channel, NBP2-3318AF488, Novus), CD45 (594 channel, 13917BF, Cell Signaling Technologies), and Ki67 (647 channel, 9027BF, Cell Signaling Technologies). Syto83 (532 channel, S11364, Invitrogen) was used as a nuclear stain. Tissue sections were then loaded into the GeoMx platform and Regions of interest (ROIs) were selected guided by the immunofluorescence of the morphology markers antibodies listed above. Segmentation was used in some ROIs used to enrich for Ki67+ and/or CD45+ populations. UV light was then directed by the GeoMx at each AOI and released the RNA ID and UMI-containing oligonucleotide tags from the WTA probes for collection and sequencing preparation. Illumina i5 and i7 dual indexing primers were added to the oligonucleotide tags during PCR to uniquely index each AOI. AMPure XP beads (Beckman Coulter) were used for PCR purification. Library concentration as measured using a Qubit fluorometer (Thermo Fisher Scientific) and quality was assessed using a Bioanalyzer (Agilent). Sequencing was performed on an Illumina NovaSeq 6000 and .fastq files were processed into gene count data for each AOI using the GeoMx® NGS Pipeline.