## 4 Discussion
demonstrated that GPRC5A was expressed by bladder stem cells, and its depletion led to a reduction in self-renewal and invasiveness, suggesting that GPRC5A might not only serve as a surface biomarker but could also play a role in the stemness phenotype in GBM [63]. To elucidate the involvement of GPRC5A in self-renewal signaling, a sphere formation assay [64] could be used to assess its role in GBM stem cell expansion and growth in vitro. GPRC5A can be selectively knocked down or knocked out using siRNA or CRISPR/Cas9 technology to determine how the loss of GPRC5A affects stem cell properties and tumor growth in vitro and in vivo. Interestingly, at least two stemness subtypes have been characterized in GBM: proneural (PN) and mesenchymal (MES), based on molecular and phenotypic differences [65; 66]. Mesenchymal GBM stem cells (MES-GSCs) are more aggressive, exhibiting higher proliferation rates, greater invasiveness, increased angiogenesis, and enhanced therapy resistance compared to proneural GBM stem cells (PN-GSCs) [67; 68]. Primary PN-GSCs often recur as MES-like tumors after treatment. This phenomenon can be explained by two possibilities: (i) environmental pressure exerted by therapy pushes PN-GSCs to transition into MES-GSCs in response to environmental threats, and (ii) the presence of rare MES-GSC clones among PN-GSCs that proliferate in response to environmental cues. Our results indicate that GPRC5A displayed a mesenchymal stem cell-like phenotype with significant expressions of CD44, VEGFA, uPAR, and CXCR4. Interestingly, [PERSON] et al. demonstrated that the overexpression of CXCR4 diminished the PN phenotype in favor of an MES phenotype in both patient data and in vivo studies, resulting in poorer outcomes for glioblastoma [69]. This transition from the PN to the MES subtype, called the proneval-mesenchymal transition (PMT), has been observed as an overall shift between subtypes and represents one of the key features influencing GBM aggressiveness. The PMT often increases disease severity by promoting stemness, invasiveness, and therapy resistance [70; 71]. Our results show a differential expression of GPRC5A between the PN and MES subtypes and an association with CXCR4 expression, suggesting that GPRC5A expression can serve as an indicator of disease severity. Additional investigations are required to clarify whether GPRC5A plays a role in PMT and to determine if its deletion disrupts the transition to a mesenchymal state. For example, functional assays, such as transwell migration, and Matrigel invasion assays can be conducted to evaluate the impact of GPRC5A on cell motility and invasiveness, which are often associated with mesenchymal features. Moreover, to evaluate if GPRC5A is important for PMT, the gene expression profiles can be analyzed by RNA-sequencing comparing high or low GPRC5A expression in GSCs, and specific genes or pathways influenced by GPRC5A during the transition from proneval to mesenchymal states can be identified. The GBM microenvironment is enriched with immune and stromal cells that play critical roles in various aspects of GBM tumorigenesis. Immune inflammation in GBM involves a complex interplay of pro- and anti-tumorigenic roles. For example, NK dim cells are actively engaged in immune surveillance by directly eliminating tumor cells without prior sensitization [72]. In GBM, NK cells are highly infiltrated in tumor tissue, and their presence is associated with better outcomes [73]. Additionally, NK cells can efficiently target undifferentiated GSCs expressing high levels of CD44, compared to those with low CD44 expression and differentiated cells [74]. Despite their cytotoxicity, NK cells can also contribute to GBM aggressiveness by promoting GSC differentiation by releasing IFNy and TNF\(\alpha\), which leads to resistance to NK cell-mediated killing [75]. Similarly, mast cells can exert anti-tumor effects by inhibiting glioma cell proliferation, migration, self-renewal, and stemness properties.