## 3 Results
### GPRC5A Is Strongly Associated with Aggressive Histopathology Features of GBM  
The stroma composition of the tumor microenvironment is crucial for tumor growth and sustainability. We assessed the interaction between GPRC5A and the stroma using ESTIMATE methods and found a positive correlation between GPRC5A expression and the stroma score (Figure 5A). The pathway enrichment analysis (Figure 3A), revealed a strong association between GPRC5A and hypoxia, suggesting that GPRC5A expression might be linked to an aggressive phenotype. To further explore this, we examined the spatial distribution of GPRC5A with the tumor microenvironment. Figure 5: The spatial association between GPRCSA with the stroma microenvironment in GBM. (**A**) Spearman correlation test between stroma score and GPRC5A expression in GBM cohort (n = 166). (**B**) GPRC5A expression assessed in tumor cell tissue (n = 9) compared to pseudopalisading cells (n = 9). Statistical analyses were conducted using an unpaired Student’s \(t\)-test with a significance threshold of **** \(p<0.001\). (**C**) GPRC5A expression was analyzed in various histologic regions of GBM tissue from the Ivy GBM Atlas, including infiltrating tumor (IT, n = 24), leading edge (LE, n = 19), microvascular proliferation (MVP, n = 4), hyperplastic blood vessels (HBV, n = 6), pseudopalisading cells around necrosis (PAN, n = 12), and the perinecrotic zone (PZ, n = 8). The data are presented as the means \(\pm\) SD, and statistical comparisons were made using a one-way ANOVA test to determine the \(p\)-value (**** \(p<0.0001\)). (**D**) GPRC5A expression was assessed in normal tissue compared to the primary and recurrent sites of GBM. The data are presented as the means \(\pm\) SD, and statistical comparisons were made using an unpaired Student’s \(t\)-test. * \(p\) = 0.03, NS: not significant (**E**) Top 10 canonical gene sets from GSEA for high GPRC5A expression versus low expression. (**F**) Spearman correlation bubble plot of various stroma cells associated with GPRC5A expression. CAFs, cancer-associated fibroblasts; P-Fbs, perivascular fibroblasts, M-Fbs, meningeal fibroblasts; SMCs, smooth muscle cells; ECM, extracellular matrix high and low. The significance shows the correlation coefficient with a \(-\)log10 \(p\)-value. A notable characteristic of hypoxia in GBM is the presence of pseudopalisading cells, which exhibit a unique spatial distribution and are recognized as prognostic features [57; 58]. To examine whether GPRC5A is associated with pseudopalisades, we quantified its expression across two distinct histological studies focusing on different tumor regions. In the first analysis, using the GSE2485 dataset, which includes pseudopalisading cells and tumor samples isolated by laser microdissection, we observed significantly higher GPRC5A expression in the pseudopalisading cells compared to the surrounding tumor tissue (\(p<0.0001\)) (Figure 5B). Similarly, analysis of a transcriptomic dataset from the Ivy GBM Atlas revealed a comparable pattern, with significant expression specifically in the pseudopalisading and perinecrotic regions (Anova test \(p<0.0001\)) (Figure 5C). To confirm the association of GPRC5A with aggressive tumor phenotypes, we compared its expression in recurrent GBM versus normal tissue and found it to be significantly higher in the recurrent form (Figure 5D). These results support that GPRC5A is associated with aggressive features in GBM. Previous research by [PERSON] et al. demonstrated that the pseudopalisading area is enriched with an extracellular matrix (ECM) [57]. To test this hypothesis, we performed GSEA analysis and identified several ECM-related signatures, including collagen, integrin, and elastic fiber pathways (Figure 5E). Our results validate that GPRC5A is enriched in pseudopalisades with high ECM signatures. [PERSON] et al. showed that low ECM environments are associated with neurosynaptic pathways, while high ECM environments are characterized by immune and stromal-related signatures [33].