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1.    Introduction
Glioblastoma (GBM) is the most common and deadly primary brain tumor diagnosis in Europe and North America1,2. It is an aggressive disease characterized by rapid tumor growth, potent invasive behavior, and high therapeutic resistance. Due to its severity, the prognosis for GBM is bleak with a median survival of ~15 months and a 5-year survival rate of only 5%3. While there is no cure for GBM, standard treatment entails surgical resection followed by dual therapy irradiation and the adjuvant chemotherapeutic temozolomide (TMZ)4. Years of clinical investigation indicate this regimen can improve patient survival rates5. Unfortunately, this combinatorial therapy does not comprehensively clear all tumor cells6. As a consequence, GBM is notorious for its inevitable recurrence and readministering these interventions provides only modest benefits7. Several features of GBM impair therapeutic efficacy, the first of which is the blood brain barrier (BBB). Drugs that improve tumor sensitivity to TMZ have difficulty penetrating the restrictive BBB, presenting a major challenge for chemotherapeutics8. Further, the vasculature network is leaky in GBM tumors, contributing to insufficient drug delivery across the tumor and decreasing their efficacy9. Also, due to their potent invasive nature, the boundary between tumor cells and healthy cells is poorly delineated, making complete surgical resection virtually impossible10.

Tumor heterogeneity is another key feature of GBM. A single GBM tumor is comprised of diverse cell types harboring a variety of genetic and transcriptomic phenotypes11,12. High levels of heterogeneity mean differing sensitivities to therapies, impairing their universal potency13–15. Based on these pleiomorphic features, there is growing support for the presence of cellular populations that exhibit stem cell-like properties termed “cancer stem cells” (CSCs). Co-existing symbiotically alongside bulk tumor cells, CSCs are thought to strongly contribute to tumor development, drug resistance, and cancer recurrence15–21. Distinct niches or microenvironments house CSC populations and amplify signals for tumor progression and maintain stemness. In addition to intrinsic resistance mechanisms, under therapeutic stress, CSCs and bulk tumor cells can reversibly exit the cell cycle, thereby evading therapies that rely on cell division before reinitiating tumorigenesis22.

As the recognition and importance of CSCs continue to grow, it is imperative to understand their molecular properties, invasive behavior, and interactions with surrounding cells. This review aims to better inform clinicians and scientists entering the complex field of GBM research. Here, we summarize issues related to cellular heterogeneity, tumor microenvironment, and therapeutic resistance while exploring potential therapeutic targets aimed at the eliminating CSCs.

2.    Heterogeneity in glioblastoma
The variety of cell phenotypes present in GBM has been observed since it’s early diagnoses. Due to GBM’s hallmark complexity, it was the first cancer to be fully sequenced by The Cancer Genome Atlas Initiative23 and other –omics data quickly followed24–27. Molecular profiling now accompanies histology in identifying and classifying GBM28. Sequencing showed many differently expressed genes between GBM tumors but also several common alterations now used for diagnosis. These abnormalities include amplification of the endothelial growth factor receptor (EGFR), loss of chromosome 10, amplification of chromosome 7, and mutations of the telomerase (TERT) promoter23,27,29. One significant mutation used for GBM classification is in the isocitrate dehydrogenase (IDH1) gene25. Found in ~9% of patients, this mutation impairs tumor cell metabolism and influences overall survival rate – 1.1 years for wild type IDH1 (IDH-wt) compared to 3.8 years for mutant IDH1 (IDH-mut)30–32. So clinically divergent are IDH-wt and IDH-mut GBMs that the 2021 WHO nomenclature redefined ‘glioblastoma-mutant IDH’ as grade 4 ‘astrocytoma - mutant IDH33.

Using molecular profiling criteria, GBM tumors have been clustered into three subtypes: proneural (PN), proliferative, and mesenchymal (MES)34. Transcriptome and single-cell analysis of multiple GBM tumors by Patel et. al identified that transcript signatures describing these bulk tumor subtypes also exist as geographically separate regions inside a single tumor26. Not only can a whole tumor be classified MES or PN, but regions within that tumor obeyed the same classification and expression signature, often with more than one such region in a single tumor26,35,36. In addition to spatial heterogeneity, tumors can also exhibit temporal heterogeneity, with ~45 - 63% of GBM tumors changing expression signatures during evolution and in response to environmental stimuli37–39. One of the most relevant examples of this change is the PN to MES transition observed in response to radiation therapy40. The MES signature is correlated with decreased therapy sensitivity, leading to an enrichment of MES cells in recurrent GBM which contributes to its intractability40,41.

2.1.    The clonal evolution theory and the cancer stem cell theory
One hypothesis to explain tumor heterogeneity is the clonal evolution theory42,43. In this theory, a single transformed ancestor cell divides to establish and populate the tumor, using its selective growth advantage to outcompete normal cells and pass down cancerous mutations to each new generation. Each cancer cell then possesses the same tumor- initiating mutations that transformed its ancestor while accumulating new, potentially advantageous mutations through genetic instability. Heterogeneity then is the result of this Darwinian process, with naturally selected cells creating individual regions expressing a unique, advantageous genetic and epigenetic profile. This is, however, not the only explanation for the origins of heterogeneity.

In addition to clonal evolution, the cancer stem cell (CSC) theory can explain tumoral heterogeneity (Figure 1). By the clonal evolution theory, each cancer cell harbors the transformative mutations to make it tumorigenic and able to replicate indefinitely44. In contrast, in 1997, Bonnet et al. presented evidence in acute myeloid leukemia that only a small population of tumor cells are capable of recapitulating the tumor18. Specifically, isolated cancer cells expressing the stem cell marker CD34 formed tumors in xenograft transplants while cells lacking this marker did not18. Therefore, they concluded this small malignant population were tumor-initiating cells (TICs) and serve as progenitors for the remaining bulk of cancer cells. This theory contradicts clonal evolution in which all cells possess tumorigenic potential44. In a foundational review, Reya et al. expanded this idea to explain tumor growth mechanics and heterogeneity in all cancers, coining the term ‘cancer stem cell and laying the foundations for the CSC theory45.

Figure 1. Tumor heterogeneity within glioblastoma lesions. Tumor heterogeneity can be explained by the cancer stem cell (CSC) theory. In this hierarchical model, a single transformed stem cell (purple) can self-renew to create another CSC and a rapidly cycling multipotent progenitor (blue/green). These progenitors divide to supply the tumor bulk with differentiated cells (red). Heterogeneity arises from the degrees of differentiation through the tumor. The clonal evolution theory can also be incorporated into this framework through mutational events during CSC self-renewal to create a genetically novel CSC (orange) or by dedifferentiation of a differentiated cell back into a CSC (pink). These populations of CSCs and their descendants can then compete through natural selection of growth advantages. Created with BioRender.

According to this theory, CSCs are a rare and uniquely tumorigenic population of cells with the potential to self-renew and differentiate, similar to conventional stem cells46. Mimicking stem cell behavior in organogenesis, CSCs are slow cycling but generate lineage restricted progenitors47,48 that populate the bulk of the tumor with differentiated cells. In this model, the spectrum of differentiation is the source of tumoral heterogeneity and only CSCs can recapitulate it in a new tumor. Recently, the unidirectionality of this hierarchy has been reevaluated due to evidence that differentiated cells can regain stem-like characteristics via modulation of signaling pathways49,50 caused by intrinsic genetic instability or environmental factors. Numerous studies in varying cancer types have shown differentiated tumor cells adopting CSC characteristics through wingless/nuclearization factor kappa B (Wnt/NF-κB) signaling modulation, growth factor release, and during the epithelial to mesenchymal transition (EMT)51–54. This plasticity of differentiated cells has led some to merge the clonal evolution and CSC models55,56, proposing that CSCs can clonally evolve. The new model suggests CSCs can undergo random genetic mutation during cell division, thereby producing heterogeneous CSC populations. Most importantly, the hierarchy is not locked in one direction and differentiated cells can also accumulate novel mutations before regaining self-renewal and making a CSC distinct from its ancestor (Figure 1). These unique CSCs and their progeny can then follow Darwinian processing described by the clonal evolution model. Despite some controversy57– 62, CSCs, and stemness in general, is recognized as an emerging hallmark of cancer63.

2.2.    Cancer stem cells in glioblastoma
Cancer stem cells were identified in GBM by Singh et al. in 2003 and later termed glioblastoma stem cells (GSCs)64. While no single biomarker has been established to identify these cells, CD133, CD44, SSEA-1, L1CAM, Nestin and others have been used65,66. Satisfying the definition for CSCs, these cells possess self-renewal67,68, differentiate into neuronal, astroglial, oligodendroglial, and even endothelial lineages69–71, and initiate tumorigenesis in xenografts64. Originally, GSCs were believed to arise from transformed neural stem cells (NSCs) as many glioblastomas originate in the compartment housing healthy NSCs, the subventricular zone (SVZ), and they share similar gene expression profiles64,72,73. However, it is now understood NSC transformation is not the only origin for GSCs as differentiated brain cells have been shown to revert to a self-renewing, tumorigenic GSC state74–76. Astrocytes and mature neurons have been induced into GSCs by shRNA knockdown of Nf1 and p5377, expression of Oct4, Klf4, Sox2, c-Myc, and Nanog transcription factors76,78–81, and by environmental cues such as low oxygen availability (hypoxia). Alarmingly, chemotherapies and irradiation have also been shown to trigger dedifferentiation, underscoring the clinical relevance of these populations82–84. While GSCs do lose their stemness in response to differentiation cues like bone morphogenic protein (BMP), cells differentiated from GSCs display incomplete terminal differentiation and more easily reenter the cell cycle or dedifferentiate and form new GSCs, further amplifying heterogeneity85,86 (Figure 2).

Glioblastoma stem cells are crucial in tumor expansion, maintenance, and survival. Through an upregulation of genes involved in migration and extracellular matrix degradation, GSCs are more invasive than their differentiated peers87. In addition to expanding tumor boundaries, deep GSC infiltration makes complete surgical resection difficult10 and as few as 50 GSCs are capable of tumor recurrence88. Equally troublingly, GSCs exhibit strong chemo and radioresistance89,90 with high GSC numbers correlating to decreased therapy response and negative outcomes91,92. Recurrent GBMs are therefore enriched in GSCs90,93. Their DNA repair pathways are upregulated and along with their slow cell cycling, GSCs have ample time to repair therapy-induced DNA lesions, negating their cytotoxic effect90,94. Overall, GSCs are implicated in filling the tumor with bulk differentiated cells47,95 and driving tumor growth and survival. To facilitate this progression, these cells both influence and are influenced by their environment.

Figure 2. Organization and development of glioblastoma stem cells. Glioblastoma stem cells (GSCs) are multipotent and capable of differentiation into multiple cell lineages including both neuronal and glial cells. Pericytes and endothelial cells can also be differentiated from GSCs to form the expanding vasculature in GBM tumors. Glioblastoma stem cells can originate from transformed neural stem cells or from dedifferentiation of differentiated brain cells (top panel). Representation of the sub-populations of GSCs relative to other tumor cell constituents and differentiation status (bottom left panel). The conversion between GSCs and differentiated cells within tumors is regulated by a variety of signaling molecules, oncogenes, and environmental conditions (bottom right panel). Created with BioRender.

3.    The tumor microenvironment and supporting niches
Tumor cells, non-tumor cells, and other biomolecules are constantly interacting within the bulk tumor and surrounding space defined by the tumor microenvironment (TME). Highly specialized zones within TMEs are defined as specific niches. Many cancers, including GBM, contain three major CSC niches: the perivascular region, the hypoxic zone, and the invasive niche96,97 (Figure 3).

Figure 3. Niches that define the glioblastoma tumor microenvironment. Defined regions within the tumor microenvironment are contained within specific niches. Glioblastoma tumors contain three major niches including the perivascular niche (left), the hypoxic niche (center), and the invasive niche (right). A variety of cell types and populations comprising each niche are depicted in the top panels. Corresponding histological features of niche components are displayed below each schematic. Denoted are: a glomeruloid microvascular proliferation (bottom left), pseudopalisading cells circumscribing necrosis (bottom center), leading tumor edge (bottom right). Histology images adapted from online resources98-99. Created with BioRender.

3.1.    The perivascular niche
As a tumor grows, rapid cellular expansion eventually outpaces existing blood vessels’ supply capacity. New blood vessels are created through the process of angiogenesis to deliver the oxygen and other nutrients required to sustain the existing cell mass and fuel further development. Cells lacking adequate blood supply enter hypoxia, a state of oxygen deprivation, which triggers signaling responses that spur angiogenesis100,101. With a lack of available oxygen, the hypoxia inducible factor (HIF1/2) transcription factors are stabilized and upregulate vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), all important angiogenesis agents102. Release of VEGF triggers matrix metalloproteinases (MMP) to remove the reinforcing pericytes coating blood vessels and induce remodeling103. Signaling molecules from the Notch pathway then cause leading endothelial cells to morph into tendrilled endothelial tip cells which extend in the direction of the VEGF signal, recruiting new endothelial cells and pericytes to construct blood vessels104.

Excessive VEGF-triggered angiogenesis can cause chronic vascular hyperplasia (CVH), a condition where excessive endothelial cell recruitment generates crowded, circular bundles of blood vessels termed glomeruloid microvascular proliferations (GMPs). This aberrant vasculature is a common histopathological feature of GBM often used for diagnosis105,106. Importantly, because of its rapid growth, the new vascular structures are poorly assembled and prone to leakage and collapse107,108.

Small numbers of Nestin+/CD133+ GSCs are positioned in perivascular niche adjacent to existing vasculature and interact with endothelial cells to exacerbate angiogenesis while maintaining their stemness109. GSCs produce elevated levels of VEGF which accelerates and worsens angiogenesis110. Correspondingly, tumors generated in nude mice using CD133+ cells show increased vascular growth and a higher number of branching points than CD133- tumors110. The perivascular niche may also be a refuge for GSCs during therapies as DNA repair capacity, and therefore resistance, is elevated in this region111.

3.2.    The hypoxic niche
While hypoxic stress and necrosis may be expected to inhibit tumor growth, the opposite reaction occurs in GBM which develops a hypoxic niche with pseudopalisading tumor cells surrounding and escaping from a central site of hypoxia-induced necrosis112. The extent of necrosis is positively associated with a more severe prognosis113. Hypoxic conditions greatly support GSC stemness through transcription upregulation of stem- promoting genes such as Sox2, Oct4, NANOG, Klf4 and c-Myc while decreasing differentiation signals like BMPs114–116. Mesenchymal GSCs are especially sensitive to HIF2α signaling through CD44 which may explain the severity of the MES signature117. Hypoxia also pushes GSCs to be more resistant to therapeutics118–121. Following cessation of chemotherapeutics, the surviving GSCs repopulate the tumor and can adopt a therapy-resistant state to evade immune attack and preserve GSC stemness93.

In addition to contributing to hypoxic conditions, porous blood vessels produced during cancer- related angiogenesis enable circulating immune cells, predominantly bone marrow-derived monocytes, to enter the brain112,122. After squeezing through leaky vessels, the tumor environment converts these monocytes into tumor-associated macrophages (TAMs)123,124 which promote oncogenesis, tumor proliferation, and contribute to tumor survival125–128. In addition, necrotic cell death causes proinflammatory signals such as interleukin 6 (IL-6), VEGF, and stromal cell derived factor 1 (SDF-
1) to be released in the tumor129,130. These factors promote monocyte polarization into M2 macrophages, imparting immunosuppressive effects and disabling the monocyte’s ability to clear the necrotic debris127,131. Hypoxia itself, through HIF1α activity, improves the immune suppression activity of TAMs and accelerates their polarization132. Release of interleukin 1 (IL-1) by TAMs disrupts the BBB and allows for more immune cell invasion, fueling a vicious cycle of immune cell recruitment, immunosuppression, and tumor development133.

3.3.    The invasive niche
At the edge of the growing tumor is the invasive niche, where the tumor contacts normal brain tissue containing astrocytes, neurons, and extracellular matrix (ECM), among other brain features. Astrocytes comprise 50% of brain tissue134,135, and when the CNS is injured, astrocytes convert from their normal quiescent state into a reactive state through astrogliosis136. Reactive astrocytes upregulate the production of growth factors, including VEGF, cytokines (especially IL-6), and MMPs, which help the brain recover from injury by activating numerous pathways including phosphoinositide-3 kinase/protein kinase B (PI3K/AKT),  Sonic  hedgehog  (Shh),  p53,  and  NF- κB137,138. Cell sorting has shown converted astrocyte populations surrounding and inside GBM regions with a unique astrogliosis-associated transcriptome termed tumor-associated astrocytes (TAA)135,139,140. Within TAAs, the same signaling pathway proteins normally responsible for repair post-injury promote GBM invasion and/or proliferation134,141. In-vitro studies have shown an increase in proliferation and invasion when cancer cells are cultured in astrocyte- conditioned media142–144. TAAs also show high levels of connexin-43 (CX43) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling to enhance cell survival by inhibiting apoptosis and promoting immunosuppression145.

The resident GSC population is an active participant in the invasive niche with Nestin+ cells often found on the leading edge146,147. White matter tracts, which are utilized for migration, express Jagged1, activating Notch signaling and upregulating the transcription factor Sox2148 which creates a stemness-favoring environment that encourages GSC migration along the tracks. Many of the products secreted from TAAs also act to enforce cell stemness. For example, Shh signaling activates the Gli transcription factor which promotes stemness and self-renewal149. Upregulation of IL-6 and STAT3 signaling by TAAs is critical for stem maintenance, while STAT3 knockdown eliminates GSC multipotency and proliferation150,151. Macroscopically, the larger the area GBM invades, the more GSCs escape resection. As GBM can be revived by small colonies of GSCs even after treatment, the expansion of GSCs outside the tumor bulk will allow relapse.

GBM niches are highly complex and have been the focus of much research. Significantly, niches are dynamic in their temporal and spatial properties. As the invasive edge expands outward, new perivascular niches are assembled in its wake and can eventually expand the necrotic region. As niches change during GBM progression, GSCs accumulate and fluctuate, especially when exposed to therapies. The robust nature of GSCs within the different niches are thought to improve tumor cell survival and assist with therapeutic resistance.

4.    Therapy evasion and resistance
4.1.    Ionizing radiation resistance
As a complement to surgical resection for GBM treatment, radiation and chemotherapeutics are employed to preferentially inflict DNA damage to cancer cells. Cancer cells are vulnerable to DNA damage due to their inherent genomic instability, DNA stress from unrestrained proliferation, and mutated damage response proteins152. Ionizing radiation (IR) therapy bombards the tumor area with high energy particles that directly damage the DNA backbone153. Additionally, the high energy particles instantaneously create reactive oxygen species (ROS), such as hydroxyl radicals, inside cells and trigger mitochondria to increase their own ROS production154. These radicals oxidize DNA bases to form double stranded breaks (DSBs), a form of damage monitored by the ataxia telangiectasia mutated (ATM) kinase. Upon encountering DSBs, ATM kinase is phosphorylated and helps facilitate cell cycle arrest at the G1/S phase, utilizing cell cycle checkpoint 2 (CHK2) and p53155. Additionally, ATM acts as a scaffold to recruit repair elements to the DSB with an increase in phosphorylated ATM associated with better DNA repair155. A parallel system using ATM and Rad3- related protein (ATR) and CHK1 acts on single strand breaks (SSBs) in DNA and regulates entrance from G2 into M phase. Both axes pause the cell cycle for DNA damage repair and to prevent apoptosis. While elevated DNA damage fuels genomic instability, once the cancer phenotype is achieved, tumor cells rely more strongly on repair mechanisms for survival156,157. The diverse genetic makeup of GBM means that radioresistance in cells will also be variable in nature.

Its no surprise that GSCs are a main culprit of radioresistance as they possess a greater ability to survive than their more differentiated counterparts. Following IR dosing, GSCs, identified as CD133+ cells, exhibited less apoptosis, higher ATM kinase activity, and an equal tumor-forming capacity as non-irradiated CD133+ cells90,158. Other upregulated repair genes in GSCs includes ATR, CHK1, and Poly ADP ribose polymerase (PARP1)158,159. As a result, it is common following IR therapy to see an enrichment in CD133+ populations in the tumor environment160. Since these cells maintain their differentiation capabilities, they can proceed to repopulate the tumor91,161. This new population often retains radioresistance from their surviving GSC ancestors. Finally, autophagy is suspected to play a role in radioresistance. As a defense mechanism, autophagy is triggered by cell stress such as hypoxia or elevated ROS and markers for autophagy such as LP3 and ATG5 and ATG12 increase in CD133+ cells following IR therapy162.

4.2.    Chemotherapeutic resistance
In addition to IR therapy, GBM cells are often resistant to chemotherapies, including temozolomide (TMZ), the foundational adjuvant chemotherapeutic prescribed for GBM. Capable of penetrating the BBB, TMZ is an alkylating agent that interacts with DNA to preferentially methylate the N3 position of adenine and the N7 and O6 positions of guanine163. Creation of O6-methylguanine, although only 5-10% of the modifications made by TMZ, is the most cytotoxic of TMZ’s effects163. O6- methylguanine leads to a nucleotide mismatch during DNA replication where thymine is incorrectly inserted instead of cytosine. During repeated cycles of mismatch repair, DNA breaks are generated, causing cell cycle arrest at the G2/M transition and eventual apoptosis164.

The most well characterized and arguably most important mechanism of alkylating agent resistance occurs through O6-methylguanine-DNA methyltransferase (MGMT), a mismatch repair protein that repairs the damage caused by TMZ. Unfortunately, MGMT is one of the most common differentially expressed proteins in GBM tumors, meaning that many patients possess some immunity against TMZ treatment. In fact, because of the prevalence of MGMT upregulation, TMZ effect is reduced in ~50% of patients163 and GSCs possess greater MGMT and DDR mechanisms, making them more tolerant to TMZ treatment94,165. Tumors with diminished MGMT show better upfront reaction to TMZ and exhibit longer progression-free survival. Decreased levels of MGMT have been shown to be related to methylation status of the MGMT promoter, with a methylated promoter transcribing less MGMT and an unmethylated promoter more166. This epigenetic marker can be easily identified using methylation-specific PCR and is now considered a useful and accurate predictor of TMZ efficacy167,168.

4.3.    Resistance via senescence
Some cancer cells do not undergo apoptosis in response to therapy-induced DNA damage and instead adopt therapy induced senescence (TIS)169. Senescent cells exit the cell cycle at the G2/M phase and upregulate DNA repair and apoptosis survival signals170. While conventional understanding suggests senescence is permanent, some studies indicate senescent tumor cells can re- enter the cell cycle to become more aggressive171. During senescence, tumor cells secrete cytokines, chemokines, matrix metalloproteinases, and growth factors collectively known as the senescence- associated secretory phenotype (SASP)172 (Figure 4). In addition to fueling proliferation and invasion, the SASP also assists with GSC maintenance by activating Wnt signaling to maintain self- renewal22,173. The SASP can reprogram non- senescent cells to possess stem cell like qualities by releasing factors that modulate the transcription factors Oct4, Sox2, Klf4, and c-Myc, as seen in other cancers22,173–175.

Figure 4. Therapy evasion of glioblastoma cells through senescence. Glioblastoma cells (top left) that resist radio/chemotherapy are enriched for therapy-resistant mesenchymal (MES) signatures. Some assume therapy induced senescence, bypassing normal apoptosis processes. Senescent tumor cells (top middle) over time can recur to give rise to new masses (top right). During senescence, cells secrete cytokines, chemokines, matrix metalloproteinases, and other growth factors known as the senescence-associated secretory phenotype (SASP). These collective signals support stem maintenance by activating the Wnt pathway. Factors from the SASP can also contribute to invasion, migration, and angiogenesis supporting tumor recurrence. Created with BioRender.

5.    Therapeutic avenues and clinical trials
5.1.    Receptor tyrosine kinase signaling as a therapeutic target
Targeting the molecular pathways implicated in GBM pathology is a promising avenue for therapy development (summarized in Table 1). Proteins of interest include receptor tyrosine kinases, stem maintenance factors, and angiogenic markers. Crosstalk among these pathways is also an important consideration since the combination and balance/imbalance of signaling effects drives cell growth, proliferation, stemness, immune evasion, and invasion. Among the most appealing targets are the receptor tyrosine kinases (RTKs) such as EGFR and its downstream effector PI3K/AKT. Malignant amplification of these proteins is common in GBM cells with EGFR mutations occurring in 57.4% of GBM27. Enhanced EGFR signaling in GBM supports proliferation, angiogenesis, invasion, and survival. Small molecule RTK inhibitors that target receptors like EGFR have shown promise in other cancer types along with GBM176,177. Erlotinib is one such FDA approved RTK inhibitor for non-small cell lung cancer where it improved progression-free survival178. In combination with TMZ and IR therapy, Erlotinib showed some improvement in overall survival for newly diagnosed GBM patients, although its use as a monotherapy falls short179,180. Another promising EGFR inhibitor, Gefitinib, showed a decrease in EGFR phosphorylation, but this reduction did not translate to a reduction in tumor severity181. A combination of both Erlotinib and Gefitinib, however, was shown to be ineffective in GBM tissue samples182. Similarly, second- generation EGFR inhibitors Dacomitinib and Afatinib failed to show meaningful improvement as single-agent treatments183,184. A third-generation EGFR inhibitor, AZD9291 (osimertinib), was approved by the FDA for non-small cell lung cancer and is being evaluated for GBM with positive pre- clinical results185–187.

In addition to small molecule inhibitors, monoclonal antibodies against EGFR have also been explored, namely Cetuximab. Although most patients experienced minimal benefit, a small population showed some improvement, though the molecular underpinnings of the improvements are unclear188,189. Combined with standard therapy, the monoclonal antibody Nimotuzumab increased survival and tolerability in Phase II trials of MGMT- methylated GBM190,191. Due to their size, antibody- based therapies have difficulty breaching the BBB with only 0.1-0.2% of circulating antibodies showing BBB penetration192. New engineering approaches to overcome this limitation are themselves a novel field for therapeutic development193–195.

The PI3K/AKT/mTOR pathway is another popular frontier for drug design. Buparlisib (BKM-120) and PX-866 are two pan-PI3K inhibitors that affect multiple PI3K isoforms. Buparlisib induces apoptosis through mitotic disfunction and reduced tumor growth while increasing survival in mice with xenografted GBM tumors196,197. In recent clinical trials, the drug was deemed to be less effective198,199. Buparlisib is still being explored as a combination therapy alongside the VEGF inhibitor, Bevacuzimab200 and the PARP inhibitor Rucaparib201. Similarly, PX-866 demonstrated powerful effects in GBM cells but came up short in clinical trials with a similarly vexing absence of explanation202,203. Another avenue being explored is targeting individual PI3K isoforms, as it has been suggested that each isoform plays stronger roles in certain cancer characteristics204. Individually targeting one isoform may be specific enough to avoid redundancies while maintaining therapeutic efficacy205,206. Downstream from PI3K targets, new mTOR inhibitors are being developed for clinical trials207. They may have value in a combination therapy if monotherapies are unsuccessful. The well- tolerated drug, Perifosine, targets AKT and is under investigation in combination with PI3K and mTOR inhibitors208. Drugs like Temsirolimus and Everolimus that target mTOR suffer from the same theme plaguing most inhibitors of this pathway: monotherapies fail due to insufficient intertumoral drug levels or compensatory signaling209,210.

5.3.    Notch, Wnt and Sonic hedgehog targets
Other potential targets in GSC maintenance include Notch, Wnt, and Shh. Notch signaling is part of neural stem cell compartmentalization, preventing apoptosis and ensuring a stock of stem cells during brain development211. The Notch pathway also has a protective role in GSC self-renewal211. Gamma- secretase inhibitors (GSIs) are used to inhibit activation of the Notch signaling pathway and early reports indicate their effectiveness at depleting GSCs or sensitizing them to radiotherapy212–214. Clinical trials using the GSI MK-0752 for refractory CNS malignancies (including GBM) showed good tolerance and favorable Notch inhibition215–217. Another Notch inhibitor, RO4929097, successfully induced differentiation and decreased self-renewal in GSCs in orthotropic mouse models218. In a Phase 0/1 combinatorial trial of RO4929097 with TMZ and radiotherapy, Notch signaling was diminished and explanted tumors showed decreased CD133+ markers. Gene expression analysis of recurrent tumors in the treatment group revealed adaptive Notch downregulation but the upregulation of mesenchymal genes and angiogenic factors as compensatory mechanisms219. The modulation of Notch  with  MK-0752  affected  PI3K/AKT  while RO4929097 altered VEGF levels220,221, a good indication that targeting several pathways simultaneously may be a productive path forward. Notably, GSIs have the potential to impair Notch regulation in healthy cells as well as in tumor cells222,223. As such, new generations of GSIs are being developed and tested in GBM cell lines224 with the hope of reducing impacts on healthy tissue.

Another upregulated pathway in GBM is the Wnt network, having roles in stem maintenance, therapy evasion, proliferation, and invasion225–227. Due to crosstalk between Wnt and cyclooxygenase-2 (COX-2) pathways, non-steroidal anti- inflammatory drugs (NSAIDs) that inhibit COX-2 also suppress Wnt signaling228,229. Small molecule inhibition with the NSAID celecoxib sensitized GBM tumors to TMZ by regulating the expression of MGMT225. Celecoxib was also effective at sensitizing tumor-derived GSCs to radiotherapy and increased the mean survival rates of mice with xenografted tumors230. In one clinical trial of 28 patients with recurrent GBM, treating with celecoxib and continuous low dose TMZ showed improvements compared to alternating TMZ dosage, with median survival time rising from 11.1 months to 16.8 months231. An additional clinical trial incorporating celecoxib in a multi-drug cocktail accompanied by TMZ showed anti-neoplastic effect, though the effect of celecoxib in this result is unreported232. Moreover, Wnt inhibitors continue to be explored in other solid tumors233.

Sonic hedgehog signaling mediates stem cell differentiation, and its overexpression is implicated in sustaining GSCs, providing therapy resistance, and promoting GBM invasion234–236. The transcriptional effector Gli1 in Shh cross-talks with the PI3K/AKT and VEGF pathways237. Clement et al. sensitized GSCs to TMZ through blockade of Shh using cyclopamine in GBM cell lines149. A dual approach with a Shh pathway inhibitor, NVP-LDE- 225, and a PI3K/mTOR inhibitor, NVP-BEZ-235, diminished proliferation, growth, and stemness of isolated GSCs238, demonstrating the benefits of Shh inhibition. Furthermore, another Shh inhibitor, Vismodegib, is among five drugs under investigation in the NCT Master Match, an ongoing clinical trial for newly diagnosed GBM patients for whom TMZ is excluded239. Vismodegib has also showed promising anti-cancer activity in medulloblastoma clinical trials240.

5.4.    Targeting angiogenesis and DNA repair
Angiogenesis targets such as VEGF are highly appealing to minimize nutrient delivery to GBM tumors. The humanized monoclonal antibody, Bevacuzimab, binds the VEGF ligand to prevent it from interacting with cell-surface receptors241. In a Phase II clinical trial, Bevacuzimab elicited a decrease in tumor size, lessened cerebral edema, and offered longer benefits to patients with recurrent GBM242. In light of this success, the FDA granted Bevacuzimab accelerated approval as both a monotherapy and in combination with the cytotoxic agent Irinotecan243. Full authorization was granted in 2017. Some GBM subtypes may decrease Bevacizumab efficacy in GBM as GSCs persist to enable therapy resistance and invasion244–246. Anti-angiogenesis treatments also enhance delivery of other therapeutics by repairing the leaky vessels needed to transport drugs to the tumor247. For example, the addition of an anti- VEGF agent was shown to improve delivery of immunotherapeutic CAR-T cells to tumors in mice248. This is especially valuable to reach the outer, invasive edges of the tumor249. Importantly, appropriate dosing is needed to balance angiogenesis prevention as over-inhibition will create necrosis through lack of nutrient supply and trigger increased tumor invasion and worse prognosis250,251. Despite potential risks, normalization of the vasculature through anti- angiogenic means continues to be a promising avenue to improve chemotherapy252.

Finally, DNA repair mechanisms are being explored as an approach for most cancer treatments including GBM. By disabling the machinery needed to overcome DNA damage, GBM tumors are sensitized to the cytotoxic effects of alkylating agents such as TMZ and the nitrosureas, lomustine and carmustine. PARP inhibitors have also become a popular sensitizing agent under considerable investigation. These agents disable the base excision repair mechanism that GSCs rely on for single strand break repair and improve radiotherapy effectiveness253,254. Efficacy in xenografts depends on the ability of PARP inhibitors to penetrate the BBB255,256. In clinical trials, the PARP inhibitor ABT-888 failed to overcome TMZ resistant in GBM, but modifications to PARP treatment remains an active area of research257,258. Likewise, MGMT is a main culprit chemoresistance and is a popular DNA repair target being explored for combinatorial treatments. Drug-induced knockdown of MGMT may improve outcomes for patients with unmethylated MGMT. In addition, proteosome inhibitors, such as Bortezomib, can deplete MGMT levels leading to prolonged survival in mouse models with unmethylated MGMT promoters259,260. An initial clinical trial showed adequate tolerance and efficacy in newly diagnosed GBM, though MGMT-methylated patients show a greater effect261.

6.    Conclusions
Many challenges remain as we seek to reduce the burden of GBM in newly diagnosed patients and those experiencing recurrence. Historically recognized hurdles such as heterogeneity and therapy resistance have been reexamined with the discovery of cancer stem cells. The development of the cancer stem cell theory offers putative explanation for tumor initiation and the origin and prevalence of the highly damaging heterogeneity observed in GBM. Through this heterogeneity, therapies are less universally effective. Inclusion of GSCs into the GBM model also offers further explanations for GBMs radiation and chemotherapeutic evasion as GSCs possess improved DNA repair mechanisms, a slower cell cycle, and reversible senescence phenotype, all of which allow them to overcome therapies cytotoxic effects.

As modern paradigms in cancer biology implicate GSCs in sustaining incurable GBMs, research aimed at eliminating this sub-population is imperative. New therapeutic approaches targeting GSCs alongside bulk tumor cells may enhance treatment outcomes. Many clinical trials have been performed and more are currently underway that target pathways critical to GSC function such as RTKs, angiogenesis, DNA repair and the stem- maintenance pathways Notch, Wnt, and Shh. Compared to other cancer types, brain tumors have to navigate the restrictive BBB which limits the delivery of novel drugs, but new drug delivery may facilitate penetrance across the BBB. As a result, therapies that prove effective in eliminating GSCs can likely be easily applied to CSCs in other cancer types.

Finally, new pharmacological discovery tools are needed to close the gap between cellular success stories and those of clinical trials. Better structure- activity-relationship predictive tools are imperative to elevate rational drug design based on native protein structures. The combined use of automated drug screening tools and cryo-Electron Microscopy (EM) structure determination creates a new era in therapeutic development. As many protein structures are now amenable to three-dimensional analysis at high-resolution, the next wave of drug design may include pinpointing protein binding sites to improve inhibitors of signaling pathways or DNA repair. Overall, improved outcomes for GBM patients are expected through the use of new platforms to target GSC-specific properties at the nanoscale. Future research efforts aimed at eliminating GSC self-renewal is a necessary consideration for scientists and clinicians working in the GBM field.

Conflict of interest statement.
The authors have no conflicts of interest to declare.

Funding statement.
The work was funded by the Center for Structural Oncology supported through the Huck Institutes of the Life Sciences at Pennsylvania State University.

Acknowledgements.
The authors thank Maria J. Solares and Liam Kaylor for their insights and help in the initial stages of the manuscript development.

References:

1. Rock K, Mcardle 0, Forde P, et al. A clinical review of treatment outcomes in glioblastoma multiforme-the validation in a non-trial population of the results of a randomised Phase III clinical trial: has a more radical approach improved survival? Br 1 Radio!. 2012;85(1017):e729-e733. doi:10.1259/bjr/83796755

2. Hanif F, Muzaf f or K, Perveen K, Ma!hi SM, Simjee SU. Glioblastoma Multiforme: A Review of its Epidemiology and Pathogenesis through Clinical Presentation and Treatment. Asian Pac J Cancer Prey AMP. 2017;18(1):3-9. doi:10.22034/APJCP.2017.18.1.3

3. Ostrom QT, Gittlemon H, Uao P, et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007-2011. Neuro-Oncot 2014;16(suppl_4):ivl-iv63. doi:10.1093/neuonc/nou223

4. Stupp R, Mason WP, van den Bent Ml, et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N Engl .1 Med. 2005;352(10):987-996. doi:10.1056/NEJMoa043330

5. Schapiro AHV. Neurology and Clinical Neuroscience E-Book. Elsevier Health Sciences; 2006.

6. Kim 1, Lee IH, Cho H.1, et al. Spatiotemporal Evolution of the Primary Glioblastoma Genome. Cancer Cell. 2015;28(3):318-328. doi:10.1016/j.cce11.2015.07.013

7. Weller M, van den Bent M, Hopkins K, et al. EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Once!. 2014;15(9):e395-e403. doi:10.1016/51470-2045(14)70011-7

8. Agarwal 5, Mittapalli RK, Zellmer DM, et al. Active Efflux of Dasatinib from the Brain Limits Efficacy against Murine Glioblastoma: Broad Implications for the Clinical Use of Molecularly Targeted Agents. Mol Cancer ?her. 2012;11(10):2183-2192. doi:10.1158/1535- 7163.MCT-12-0552

9. Overcoming the blood-brain tumor barrier for effective glioblastoma treatment - ClinicalKey. Accessed April 15, 2023. https://www.clinicalkey.cornall/content/playCont ent/1-s2.0- 51368764615000126?returnurl=null&ref errer =n ull

10. Shergalis A, Bankhead A, Luesakul U, Muangsin N, Newhall N. Current Challenges and Opportunities in Treating Glioblastoma. Pharmacol Rev. 2018;70(31:412-445. doi:10.1124/pr.117.014944

11. Della Monica R, Cuomo M, Buonaluto M, et al. MGMT and Whole-Genome DNA Methylation Impacts on Diagnosis, Prognosis and Therapy of Glioblastoma Multiforme. Int J Mol Sci. 2022;23(13):7148. doi:10.3390/Ijms23137148

12. Etcheverry A, Aubry M, de Tayrac M, et al. DNA methylation in glioblastoma: impact on gene expression and clinical outcome. BMC Genomics. 2010;11(1):701. doi:10.1186/1471-2164-11- 701

13. Bonavia R, Inds M del M, Cavenee WK, Furnari FB. Heterogeneity Maintenance in Glioblastoma: A Social Network. Cancer Res. 2011;71(12):4055-4060. doi:10.1158/0008- 5472.CAN-11-0153

14. Porker NR, Khong P, Parkinson JF, Howell VM, Wheeler HR. Molecular Heterogeneity in Glioblastoma: Potential Clinical Implications. Front Oncol. 2015;5. Accessed April 27, 2023. https://www.frontiersin.org/orticles/10.3389/fon c.2015.00055

15. Happier GH, Miller BE. Tumor heterogeneity: biological implications and therapeutic consequences. Cancer Metastasis Rev. 1983;2(1):5-23. doi:10.1007/BF00046903

16. Dick JE. Breast cancer stem cells revealed. Proc Nall Acad SO. 2003;100(71:3547-3549. doi:10.1073/pnas.0830967100

17. O Brien CA, Pollett A, Ga!linger 5, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445(7123):106-110. doi:10.1038/nature05372

18. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Not Med. 1997;3(71:730-737. doi:10.1038/nrn0797-730

19. Bathe E, Clevers H. Cancer stem cells revisited. Nat Med. 2017;23(10):1124-1134. doi:10.1038/nm.4409

20. Ayob AZ, Ramasamy TS. Cancer stem cells as key drivers of tumour progression. J Biomed SO. 2018;25(1):20. doi:10.1186/s12929-018-0426- 4

21. Najaf i M, Farhood B, Mortezaee K. Cancer stem cells (CSCs) in cancer progression and therapy. 1 Cell Physiol. 2019;234(6):8381-8395. doi:10.1002/jcp.27740

22. Milanovic M, Fan DNY, Belenki D, et al. Senescence-associated reprogramming promotes cancer stemness. Nature. 2018;553(76861:96-100. doi:10.1038/nature25167

23. McLendon R, Friedman A, Signer D, et al. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061-1068. doi:10.1038/nature07385

24. Verhaak RGW, Hoadley KA, Purdom E, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDHI, EGFR, and NH. Cancer Cell. 2010;17(1):98-1 10. doi:1 0.10164ccr.2009.12.020

25. Parsons DW, Jones 5, Thong X, et al. An Integrated Genomic Analysis of Human Glioblostoma Multiforme. Science. 2008;321(5897):1807. doi:10.1126/science.1164382

26. Patel AP, Tirosh I, Trombetta 11, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014;344(6190):1396-1401. doi:10.1126/science.1254257

27. Brennan CW, Verhaak RGW, McKenna A, et al. The somatic genomic landscape of glioblastoma. Cell. 2013;155(2):462-477. doi:10.1016/kell.20 I 3.09.034

28. Louis DN, Perry A, Reifenberger G, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Ado Neuropathol (Berl). 2016;131(6):803-820. dol:10.1007/s00401 - 016-1545-1

29. Ohgaki H, Kleihues P. Genetic Pathways to Primary and Secondary Glioblastoma. Am J Pathol. 2007;170(5):1445-1453. doi:10.2353/aipath.2007.070011

30. Han 5, Liu Y, Cai SJ, et al. IDH mutation in glioma: molecular mechanisms and potential therapeutic targets. Br 1 Cancer. 2020;122(11):1580-1589. doi:10.1038/s41416- 020-0814-x

31. Nobusawa 5, Watanabe T, Kleihues P, Ohgaki H. IDH1 Mutations as Molecular Signature and Predictive Factor of Secondary Glioblostomas. Clin Cancer Res. 2009;15(19):6002-6007. doi:10.1158/1078-0432.CCR-09-0715

32. Yan H, Parsons OW, lin G, et al. IDH1 and IDH2 Mutations in Gliomas. N Engl 1 Med. 2009;360(8):765-773. doi:10.1056/NEJM0a0808710

33. Louis DN, Perry A, Wesseling P, et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro-Oncol. 2021;23(8):1231-1251. doi: I 0.1093/neuonc/noab106

34. Phillips HS, Kharbanda 5, Chen R, et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. 2006;9(3):157-173. doi:10.1016/j.ccr.2006.02.019

35. Sottoriva A, Spited I, Piccirillo SGM, et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Nall Acad Sci. 2013;110(10):4009-4014. doi:10.1073/pnas.1 219747110

36. Bergmann N, Delbridge C, Gempt J, et al. The Intratumoral Heterogeneity Reflects the Intertumoral Subtypes of Glioblastoma Multiforme: A Regional Immunohistochemistry Analysis. Front Oncol. 2020;10. Accessed February 20, 2023. https://www.f rontiersin.org/articles/10.3389/f on c.2020.00494

37. Swanton C. Intratumor Heterogeneity: Evolution through Space and Time. Cancer Res. 2012;72(19):4875-4882. dol:10.1158/0008- 5472.CAN-12-2217

38. Becker AP, Sells BE, Hague Si, Chakrovarti A. Tumor Heterogeneity in Glioblastomas: From Light Microscopy to Molecular Pathology. Cancers. 2021;13(4):761. doi:1 0.3390/cancers 3040761

39. Wang 1, Cozzato E, Ladewig E, et al. Clonal evolution of glioblastoma under therapy. Not Genet. 2016;48(7):768-776. doi:10.1038/ng.3590

40. Fedele M, Cerchia L, Pegoraro 5, Sgarra R, Manfioletti G. Proneural-Mesenchymal Transition: Phenotypic Plasticity to Acquire Multitherapy Resistance in Glioblastoma. Int J Mol Sci. 2019;20(11):2746. doi:10.3390/iims20112746

41. Segerman A, Niklasson M, Haglund C, et al. Clonal Variation in Drug and Radiation Response among Glioma-Initiating Cells Is Linked to Proneural-Mesenchymal Transition. Cell Rep. 2016;17(11):2994-3009. dol:1 0.1016/1.celrep.2016.11.056

42. Nowell PC. The Clonal Evolution of Tumor Cell Populations. Science. 1976;194(4260):23-28. doi:10.1 I 26/science.959840

43. Greaves M, Maley CC. Clonal evolution in cancer. Nature. 2012;481(7381):306-313. doi:10.1038/naturel 0762

44. Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in Cancer: Cancer Stem Cells versus Clonal Evolution. Cell. 2009;138(5):822-829. doi:10.1016/i.ce11.2009.08.017

45. Reya T, Morrison Si, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414(6859):105-111. doi:1 0.1038/35102167

46. Clarke MF, Didc JE, Dirks PB, et al. Cancer Stem Cells-Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res. 2006;66(19):9339-9344. doi: I 0.1158/0008-5472.CAN-06-3126

47. Lan X, Jorg DJ, Cavolli FMG, et al. Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy. Nature. 2017;549(7671):227-232. doll 0.1038/nature23666

48. Jackson M, Hassiotou F, Nowak A. Glioblastoma stem-like cells: at the root of tumor recurrence and a therapeutic target. Carcinogenesis. 2015;36(2):177-185. doi:1 0.1093/carcin/bgu243

49. Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006;126(4):663-676. doi:10.10164ce11.2006.07.024

50. Visvader JE, Lindeman GJ. Cancer Stem Cells: Current Status and Evolving Complexities. Cell Stem Cell. 2012;10(6):717-728. doi:10.1016/istem.2012.05.007

51. Schwitalla 5, Fingerle AA, Cammareri P, et al. Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. Cell. 2013;152(1-2):25-38. doll 0.10164ce11.2012.12.012

52. Vermeulen L, De Sousa E Melo F, von der Heijden M, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol. 2010;12(5):468-476. doi:10.1038/ncb2048

53. Pradella D, Naro C, Sette C, Ghigna C. EMT and stermess: flexible processes tuned by alternative splicing in development and cancer progression. Mol Cancer. 2017;16(1):8. doi:10.1186/s12943-016-0579-2

54. Wang S sha, Jiang 1, hang X hua, Tang Y ling. Links between cancer stem cells and epithelial– mesenchymal transition. OncoTargels Ther. 2015;8:2973-2980. doi:10.2147/OTT.591863

55. Kreso A, Dick JE. Evolution of the Cancer Stem Cell Model. Cell Stem Cell. 2014;14(3):275-291. doll 0.10164stem.2014.02.006

56. Wang W, Quan Y, Fu Q, et al. Dynamics between Cancer Cell Subpopulations Reveals a Model Coordinating with Both Hierarchical and Stochastic Concepts. PLOS ONE. 2014;9(1 ):e84654. doi:10.1371/journal.pone.0084654

57. Dirkse A, Golebiewska A, Buder T, et al. Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment. Nat Commun. 2019;10(1):1787. doi:10.1038/s41467-019-09853-z

58. van Niekerk G, Davids LM, Hattingh SM, Engelbrecht AM. Cancer stem cells: A product of clonal evolution? Int 1 Cancer. 2017;140(5):993-999. do4:10.1002/4c.30448

59. Meacham CE, Morrison 5.1. Tumour heterogeneity and cancer cell plasticity. Nature.  2013;501(7467):328-337. doi:1 0.1038/noture12624

60. Hung KF, Yang T, Kao SY. Cancer stem cell theory: Are we moving past the mist? 1 Chin Med Assoc. 2019;82(11):814. doi: I 0.1097PCMA.0000000000000186

61. Yoo MH, Hatfield DL The cancer stem cell theory: Is it correct? Mol Cells. 2008;26(5):514-516.

62. Wang J, Sakariassen P0, Tsinkalovsky 0, et al. CDI 33 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. Int Cancer. 2008; I 22(4):761 -768. doi:10.1002/ijc.23130

63. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov. 2022;12(1):31-46. doi: I 0.1158/2159-8290.CD-21-1059

64. Singh SK, Clarke ID, Terasaki M, et al. Identification of a Cancer Stem Cell in Human Brain Tumors. Cancer Res. 2003;63(18):5821-5828.

65. Brescia P, Richichi C, Pelicci G. Current Strategies for Identification of Glioma Stem Cells: Adequate or Unsatisfactory? J Onto!. 2012;2012:e376894. doi:10.1155/2012/376894

66. Tang X, Zuo C, Fang P, et al. Targeting Glioblostoma Stem Cells: A Review on Blomarkers, Signal Pathways and Targeted Therapy. Front Oncol. 2021;11. Accessed February 20, 2023. https://www.f rontiersin.org/articles/10.3389/f on c.2021.701291

67. Gimple RC, Bhargava 5, Dixit D, Rich JN. Glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer. Genes Dev. 2019;33(11-12):591-609. doi:10.1101/gad.324301.119

68. Lathia JD, Mack SC, Mulkearns-Hubert EE, Valentim CLL, Rich JN. Cancer stem cells in glioblastoma. Genes Der. 2015;29(12):1203-1217. doi:10.1101/god.261982.115

69. Galli R, Binda E, Orf anelli U, et al. Isolation and Characterization of Tumorigenic, Stem-like Neural Precursors from Human Glioblastoma. Cancer Res. 2004;64(19):701 1-7021. doi:10.1 I 58/0008-5472.CAN-04-1364

70. Soda Y, Marumoto T, Friedmann-Morvinski D, et al. Transdifferentiation of glioblastoma cells into vascular endothelial cells. Proc Nail Acod Sci 2011;108(11):4274-4280. doi:10.1073/pnas.1016030108

71. Scully 5, Froncescone R, Faibish M, et al. Transdifferentiation of Glioblastoma Stem-Like Cells into Mural Cells Drives Vasculogenic Mimicry in Glioblastomas. J Neurosci. 2012;32(37):12950-12960. doi:10.1523/1NEUROSCI.2017-12.2012

72. Lee 1H, Lee JE, Kahng JY, et al. Human glioblastoma arises from subventricular zone cells with low-level driver mutations. Nature. 2018;560(7717):243-247. do1:10.1038/s41586- 018-0389-3

73. Jiang Y, Uhrbom L On the origin of gliomo. Ups J Med Sci. 2012;117(2). doi:10.3109/03009734.2012.658976

74. Sofa AR, Saadatzadeh MR, Cohen-Gadol AA, Pollok KE, Bijangi-Vishehsaraei K. Glioblastoma stem cells (GSCs) epigenetic plasticity and interconversion between differentiated non-GSCs and GSCs. Genes Dis. 2015;2(2):152-163. doi:10.1016/tgendis.2015.02.001

75. Olmez I, Shen W, McDonald H, Ozpolat B. Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth. 1 Cell Mol Med. 2015;19(6):1262-1272. doi:10.1111/jcmm.12479

76. Moon 1H, Kwon 5, Jun EK, et al. Nanog-induced dedifferentiation of p53-deficient mouse ostrocytes into brain cancer stem-like cells. Biochem Biophys Res Commun. 2011;412(1):175-181. doi:10.1016/tbbrc.2011.07.070

77. Friedmann-Morvinski D, Bushong EA, Ke E, et al. Dedifferentiation of Neurons and Astrocytes by Oncogenes Can Induce Gliomas In Mice. Science. 2012;338(6110):1080-1084. doi:10.1126/science.1226929

78. Rios AFL, Tirapeili DP da C, Cirino ML de A, Rodrigues AR, Ramos ES, Carlotti CG Jr. Expression of pluripotency-related genes In human glioblastoma. Neuro-Oncol Adv. 2022;4(1 ):vcia 6163. doi:10.1093/noalnl/vdob163

79. Fang X, Yoon JG, Li L, et al. The SOX2 response program in glioblastoma multiforme: on integrated ChIP-seq, expression microarray, and microRNA analysis. BMC Genomics. 2011;12(1):11. doi:10.1186/1471-2164-12-11

80. Kim JB, Sebastian V, Wu G, et al. Oct4- Induced Pluripotency in Adult Neural Stem Cells. Cell. 2009;136(3):411-419. doi:10.10164cell.2009.01.023

81. Wang ML, Chlou SH, Wu CW. Targeting cancer stem cells: emerging role of Nanog transcription factor. OncoTorgels Ther. 2013;6:1207-1220. doi:10.2147/OTT.538114

82. Lee G, Auffinger B, Guo D, et al. Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced H1F Signaling in the Recurrent GBM Model. Mol Cancer Ther. 2016;15(12)13064-3076. doi:10.1158/1535-7163.MCT-15-0675

83. Dahan P, Martinez Gala 1, Delmas C, et al. Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible Involvement in radioresistance. Cell Death Dis. 2014;5(11):e1543-e1543. doi:10.1038/cddis.2014.509

84. Auffinger B, Tobias AL, Han Y, et al. Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy. Cell Death Differ. 2014;21(7):1119-1131. doi:10.1038/cdd.2014.31

85. Piccirillo SGM, Reynolds BA, Zanetti N, et al. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature. 2006;444(7120):761-765. doi:10.1038/nature05349

86. Caren H, Stricker SH, Bulstrode H, et al. Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest. Stem Cell Rep. 2015;5(51:829-842. doi:10.1016/tstemcr.2015.09.014

87. Inoue A, Takahashi H, Harada H, et al. Cancer stem-like cells of glioblastoma characteristically express MMP-13 and display highly invasive activity. Int 1 Oncot 2010;37(5):1121-1131. doi:10.3892/ijo_00000764

88. Suva ML, Rheinbay E, Gillespie SM, et al. Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells. Celt 2014;157(3):580-594. doi:10.10164cell.2014.02.030

89. Eramo A, Ricci-Vitiani L, Zeuner A, et al. Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ. 2006;13(7):1238-1241. doi:10.1038/sj.cdd.4401872

90. Boo 5, Wu 0, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444(7120):756-760. doi:10.1038/nature05236

91. Baumann M, Krause M, Hill R. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer. 2008;8(7):545-554. doi:10.1038/nrc2419

92. Zeppernick F, Ahmadi R, Campos B, et al. Stem Cell Marker CD133 Affects Clinical Outcome in Glioma Patients. Clin Cancer Res. 2008;14(1):123-129. doi:10.1158/1078-0432.CCR-07-0932

93. Chen 1, Li Y, Yu TS, et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature. 2012;488(7412):522-526. doi:10.1038/noturel 1287

94. Liu G, Yuan X, Zang Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer. 2006;5(1):67. doi:10.1186/1476-4598-5-67

95. Couturier CP, Ayyadhury 5, Le PU, et al. Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy. Nat Commun. 2020;11(1):3406. doi:10.1038/s41467-020-17186-5

96. Hambardzumyan D, Bergers G. Glioblastoma: Defining Tumor Niches. Trends Cancer. 2015;1(4):252-265. doi:10.1016/j.trecan.2015.10.009

97. Plaks V, Kong N, Werb Z. The Cancer Stem Cell Niche: How Essential Is the Niche in Regulating Stemness    of    Tumor    Cells?    Cell    Stem    Cell. 2015;16(3):225-238. doi:10.1016/j.stem.2015.02.015

98.    WebPathology. Accessed April 29, 2023. http://webpathology.com

99.    Specimen Detail :: Ivy Glioblastoma Atlas Project. Accessed April 29, 2023. https://glioblastoma.alleninstitute.org/ish/specime n/show/268000655

100.    Krock BL, Skuli N, Simon MC. Hypoxia- Induced Angiogenesis: Good and Evil. Genes Cancer. 2011;2(12):1117-1133. doi:10.1177/1947601911423654

101.    Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003;9(6):677-684. doi:10.1038/nm0603-677

102.    Hardee ME, Zagzag D. Mechanisms of glioma-associated neovascularization. Am J Pathol. 2012;181(4):1126-1141. doi:10.1016/j.ajpath.2012.06.030

103.    Dvorak HF. Tumors: Wounds That Do Not Heal—Redux. Cancer Immunol Res. 2015;3(1):1- 11. doi:10.1158/2326-6066.CIR-14-0209

104.    Rosińska S, Gavard J. Tumor Vessels Fuel the Fire in Glioblastoma. Int J Mol Sci. 2021;22(12):6514. doi:10.3390/ijms22126514

105.    Brat DJ, Van Meir EG. Glomeruloid Microvascular Proliferation Orchestrated by VPF/VEGF: A New World of Angiogenesis Research. Am J Pathol. 2001;158(3):789-796. doi:10.1016/S0002-9440(10)64025-

106.    Birner P, Piribauer M, Fischer I, et al. Vascular Patterns in Glioblastoma Influence Clinical Outcome and Associate with Variable Expression of Angiogenic    Proteins:    Evidence    for    Distinct Angiogenic Subtypes. Brain Pathol. 2003;13(2):133-143.        doi:10.1111/j.1750- 3639.2003.tb00013.x

107.    Bergers G, Song S. The role of pericytes in blood-vessel formation and maintenance. Neuro- Oncol. 2005;7(4):452-464. doi:10.1215/S1152851705000232

108.    Nagy JA, Dvorak AM, Dvorak HF. VEGF-A and the induction of pathological angiogenesis. Annu Rev Pathol. 2007;2:251-275. doi:10.1146/annurev.pathol.2.010506.134925