Lauren E. Haydu

Background: Glioblastoma (GBM) remains a devastating cancer with limited treatment options, largely due to its heterogeneity. While supramaximal resection has recently provided survival benefits, therapeutic profiling of different tumor compartments, particularly its infiltrative edge remains largely unexplored. Methods: Here, we leveraged magnetic resonance imaging (MRI)-guided multi-sampling, collecting 2 cores and 2 margins per case, to query GBM heterogeneity. Whole-exome and RNA-seq with drug testing in two patient-derived 3D models were used to reveal similarities and differences in genomic and transcriptomic makeups, cellular compositions, and drug responses across cores and margins. Bioinformatics interrogations further identified response biomarkers. Results: Mutation analysis showed that oncogenes exhibited a higher degree of spatial heterogeneity than tumor suppressor genes, regardless of MRI status. While the mesenchymal transcriptional subtype with extracellular matrix remodeling, stress response, and immune programs were preferentially enriched in enhancing cores, proneural tumors with neurological processes favored non-enhancing margins. Using a 15-drug GBM-targeted panel, ERK (ulixertinib) and PI3K pathway (paxalisib, CC-115) inhibitors showed preferential efficacy in enhancing cores and non-enhancing margins, respectively. The anti-apoptosis, pan-Bcl2 agent navitoclax and the epigenetic drug trotabresib represented the most effective, tumor-wide monotherapies. Importantly, drug combinations generally outperformed single agents across all regions. Conclusions: This work demonstrates the regional heterogeneity of therapeutic vulnerabilities in GBM ex vivo, showing various drugs with tumor-wide or MRI-enhancement informed activity. These findings offer preclinical bases of numerous monotherapies and drug combinations for future clinical trial design.

Body mass index values by patient ID for all RNASeq cohorts.

Supplementary Table 1: Baseline characteristics for patients included in the integrated gene set enrichment analysis (Figure 2-3) by cohort; Supplementary Table 6: The most frequent somatic alterations (alts) in patients with regionally metastatic melanoma by body mass index from The Cancer Genome Atlas cohort; Supplemental Table 7: Integrated gene set enrichment analysis for pathways associated with fatty acid metabolism by body mass index; Supplemental Table 8: Gene expression of selected genes of interest involved in fatty acid metabolism.

Beta-diversity analysis stratified by sex based on Bray-Curtis dissimilarity represents microbiome samples with metastatic melanoma in terms of the two top principal components (explaining around 17% of variance) obtained from the principal coordinate analysis. Red dots are from overweight/obese (OW/OB) BMI patients, and blue dots are from normal (NL) BMI patients. Shading inside the dots indicates the female gender. This is a subgroup analysis of Figure 5B.

Immune cell analysis by BMI. A. Immunohistochemistry (IHC) analysis for CD8-, CD45RO-, FOXP3-, CD68-, GZMB-, PD-1-, LAG-3-, and CD3-positive cells in overweight/obese (OW/OB) verse normal (NL) tumors as defined by body mass index from the MD Anderson Cancer Center (MDA) cohort. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis for PD-L1-, CD45RO-, FOXP3-, EOMES-, GZMB-, PD-1-, TBET-, and TBET:FOXP3-positive cells in OW/OB verse NL tumors as defined by body mass index from the Gide cohort. Line represents median +/- interquartile range; each dot represents a single tumor.

Integrated gene set enrichment analysis of subgroups by body mass index BMI. This figure compares gene expression between overweight/obese (OW/OB) and normal (NL) BMI patients with metastatic melanoma across subgroups by sex, cohort, and tissue site. Supplementary Figure 2 presents a dotplot of genes differentially up- or downregulated in OW/OB patients versus NL BMI patients by subgroups. Red indicates upregulation in OW/OB verse NL.

Immune cell analysis by BMI and sex. A. Immunohistochemistry (IHC) analysis of the MDA cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis of the Gide cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor.

Differential gene expression analysis for each subgroup.