Spatial transcriptomic analysis reveals lack of response to PD-1 blockade in recurrent glioblastoma

Immune checkpoint inhibitors have transformed treatment for several cancers, yet clinical trials of programmed cell death protein 1 (PD-1) blockade in glioblastoma (GBM) have consistently failed to show therapeutic benefit. While some studies have reported treatment-related transcriptional changes, particularly in T cells, findings remain limited and inconsistent. The aim of this study was to investigate changes in tumor cells and tumor-associated macrophages (TAMs) after PD-1 blockade in recurrent GBM using spatial transcriptomics.We performed Digital Spatial Profiling (GeoMx, NanoString) on FFPE tumor samples from 26 patients with matched primary and recurrent IDH-wildtype GBM, including 16 patients who received neoadjuvant nivolumab at recurrence. Tumor (SOX2⁺) and TAM (IBA1⁺) segments were selected for targeted spatial analysis. Following quality filtering, transcriptomic profiles were compared between nivolumab-treated and untreated recurrent tumors. 2 mm. cores was extracted from FFPE tissue blocks, to form tumor microarrays, consisting of 40 cores per TMA. 5 um sections were cut of the FFPE TMA tissue blocks, before continuing with the geomx analysis. Spatial transcriptomics libraries where constructed based on the manufactures instructions, utilising the GeoMx Human Whole Transcriptome RNA assay probe set. Following library construction GeoMx® DSP NGS Readout User Manual (MAN-10153) was followed. Sequencing was performed on Illumina Novaseq 6000. BCL files were converted to FASTQ and then subsequently DCC files using NanoString’s GeoMx NGS Pipeline v2.3. Raw counts were normalized using quantile normalization and batch effects were corrected using Combat. normalized.txt.tar.gz: tab-seperated file containing normalized and batch-corrected count matrix of AOIs that were used in this analysis. metadata.csv.tar.gz: comma-seprated file containing matadata of the AOIs used in this analysis. RAW.tar.gz: dcc files (GeoMx output) of all AOIs (i.e., raw data) sampleAnno.xlsx.tar.gz: excel file containing dcc-file annotation Hs_R_NGS_WTA_v1.0.pkc.tar.gz: pkc file containg probe-to-gene conversion