Fig. 1

Differential gene expression analysis in spatially distinct GBM regions. Schematic figure delineating the steps for tissue collection from distinct GBM regions (Core, Rim, and Invasive margin) followed by 5ALA-based FACS isolation of Invasive margin cells into 5ALA + and 5ALA − subpopulations (A). The spatially resolved RNA profile (SPRP) from each unsorted region and sorted cells were interrogated by gene set enrichment analysis (GSEA), Deconvolution algorithm, single-cell gene signature scoring (SC sig-scoring), Exon–intron split analysis (EISA), network inference, and complemented by spatially resolved transcriptomics (stRNA-seq) (A). Representative hematoxylin and eosin-stained images of 5ALA + GBM infiltrative margin, scanned using NanoZoomer, magnification × 40, scale bar 200 μm for patient 28 and 100 μm for patient 37. Top (patient 28): Dashed line boundary demarcates cellular tumor-filling white matter. Individual tumor cells are observed within the cerebral neocortex (black circles), with neurons normally distributed (normally formed cerebral neocortex) (green circles). Microvascular proliferation (dark blue arrow) beyond the margin of the cellular tumor. Representative normal blood vessel (red arrow). Bottom (patient 37): Individual tumor cells are observed within the cerebral neocortex (black circles), with a normal distribution of white matter blood vessels (red circles). Deep white matter diffusely infiltrated by tumor cells at low density (green arrows). Other cells observed within this deep white matter region are oligodendrocytes with macrophages and astrocytes (B). The Inv margin representing the non-enhancing on T1 with gadolinium region located outside the MRI contrast region (C). The two-photon excitation fluorescence image demonstrates a distribution of PpIX across radiologically defined spatially distinct tumor core (Core) (D, Upper panel) and Invasive margin (Inv) (D, Lower panel) regions. Volcano plot representing differential gene expression between 5ALA + and Tumor Core (E), Rim (F), Invasive margin (G), and 5ALA − cells (H). Heatmap showing the normalized enrichment scores (NES) representing significantly enriched hallmarks (padj < 0.05) in a specific GBM region (I). GSEA was performed between a particular region and all other regions. The color represents the value of NES where yellow and black indicate the highest (NES = 3.5) and lowest (NES = 0) NES values, respectively. Only significant NES values are shown (adj. p-values < 0.05). Ki67 immunohistochemistry (IHC) of tumor core—superficial medial (J), anterior medial (K), tumor rim (L), and Invasive margin (M) to estimate the fraction of proliferating cells in spatially distinct regions of GBM. The scale bar indicates 25 µm. CD31-IHC represents the tumor vascularity identifying the number of vascular structures in the Core (N), Rim (O), and Invasive margin (P)