1. Introduction

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults, and despite standard-of-care multimodality therapy, including maximal safe resection, radiotherapy, and chemotherapy, the prognosis remains almost universally fatal with a mean overall survival of 14 to 20 months [1]. Since the 2005 pivotal phase III trial by Stupp et al. [1], which established the role of concurrent chemoradiation with temozolomide followed by adjuvant temozolomide for patients with newly diagnosed glioblastoma, no chemotherapies investigated in late-phase clinical trials have significantly improved upon this foundational approach. The U.S. Food and Drug Administration (FDA) has approved the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab for treatment of recurrent glioblastoma on the basis of two phase II studies showing a progression-free survival benefit. However, two phase III clinical trials evaluating its role in the treatment of newly diagnosed disease did not demonstrate an overall survival benefit when bevacizumab was added to standard therapy ^[2][3][4][2,3,4]. The FDA also has approved tumor-treating fields therapy (TTF), which consists of low-intensity, alternating electric fields applied to the scalp for most of the day, for use in recurrent (2011) and newly diagnosed (2015) glioblastoma, although widespread adoption of TTF has been limited by methodological concerns about the generalizability of the data from prior studies of it [5]. Thus, there are currently no effective therapies for glioblastoma. In this review, we discuss the innate mechanisms of treatment resistance common to all glioblastomas before characterizing the various mechanisms of resistance to conventional treatments, targeted therapies, and immunotherapy.

2. General Mechanisms of Treatment Resistance

2.1. The Blood–Brain and Blood–Brain–Tumor Barriers

Figure 1). These transporters actively mediate the efflux of xenobiotics such as cytotoxic or targeted therapeutic agents out of the brain parenchyma ^[9][10][11]. Attempts to modulate these efflux pumps have largely been unsuccessful ^[12][13].

In glioblastoma and other high-grade intracranial neoplasms, the BBB is heterogeneously disrupted to form the blood–tumor–brain barrier, which is characterized by abnormal pericyte distribution, reduced tight junctions, the loss of astrocytic endfeet, and increased permeability to circulating immune cells ^[14][15]. This heterogeneity of tumoral vasculature creates regional niches of variable permeability to blood, oxygen, nutrients, and drugs. Recent work has further extended our understanding of a glioblastoma tumor’s centrally necrotic, hypoxic core, and less-hypoxic periphery ^[16]. Indeed, hypoxic glioblastoma cells secrete VEGF-A via exosomes to promote the proliferation of endothelial cells with downregulated expression of key junctional proteins such as claudin-5 and occludin ^[17][18].

Glioma stem cells (GSCs), which are pluripotent, slowly dividing, and therapy-resistant cells residing in the perivascular hypoxic niches of the brain, have been recognized for their importance in resisting cytotoxic therapies ^[19][20]. GSCs are not only intrinsically resistant to therapy but also exert substantial effects on neighboring cells within the microenvironment to maintain their populations ^[21]. In particular, glioblastoma pericytes derive from GSCs via trans-differentiation and contribute to the integrity of the BBB via the overexpression of proteins such as bone marrow and X-linked (BMX) non-receptor tyrosine kinase (

Figure 1), which activate signaling through signal transducer and activator or transcription 3 (STAT3) to maintain the self-renewal capability of the GSCs occupying perivascular niches ^[14][22]. Indeed, in an orthotopic xenograft glioblastoma model, Zhou et al. ^[23] found that pericyte coverage not only correlated with the prognosis of patients with glioblastoma but also that inhibition of BMX with ibrutinib selectively disrupted the permeability of the blood–brain–tumor barrier and enhanced delivery of chemotherapy (e.g., etoposide) that ordinarily penetrates the BBB poorly, thus prolonging mouse survival ^[24].

The two most important signaling pathways involved in the formation of the BBB and the regulation of its integrity are the Wingless-related integration site (Wnt) and Sonic hedgehog (Shh) pathways. During normal embryonic development and in adulthood, Shh secreted by astrocytes binds to the patched-1 (Ptch-1) protein on endothelial cells or pericytes to activate the smoothened (Smo) protein. This leads to downstream transcriptional activation of genes bound by the Gli family of transcription factors, such as SOX-18 and TAL-1, which increase transendothelial resistance and decrease permeability by enhancing claudin expression ^[25][26][27]. Shh signaling also contributes to the central nervous system’s immune privilege by decreasing the expression of the intercellular adhesion molecule ICAM-1 and the secretion of chemokines such as CXCL8/interleukin (IL)-9 and CCL2/MCP-1 by endothelial cells. Similarly, Wnt/β-catenin signaling in endothelial cells contributes to the regulation of the BBB. The endothelial G-protein coupled receptor Gpr124 is one such crucial coactivator of Wnt7a and Wnt7b-stimulated canonical signaling via the binding of Frizzled receptor and Lrp coreceptor. Gpr124 upregulates claudin-5 expression, decreases platelet-derived growth factor receptor (PDGFR)-B expression, and increases pericyte coverage ^[28]. Recently, Griveau et al. ^[29] extended the understanding of glioma cellular phenotypes vis-à-vis the tumoral microenvironment, demonstrating in a mouse model that Olig2+/Wnt7+ glioma cells—analogous to the oligodendrocytes comprising the leading edge of glioblastoma tumors—invaded the brain parenchyma via co-option of blood vessels by single-cells, while Olig2-/Wnt7- glioma cells—analogous to proneural glioblastoma cells—proliferated in the perivascular niche and expressed abundant VEGF-C and VEGFR-1/2/3 to form dense tumor collections with leaky vasculature. Importantly, anti-angiogenic therapy (i.e., VEGF inhibition) led to selective enrichment of the Olig2+/Wnt7+ cells, indicating a mechanism through which glioblastoma cells may ultimately overcome prolonged anti-angiogenic therapy.

Strategies to breach the BBB and improve drug delivery have therefore focused on mechanical disruption (i.e., osmotic disruption) and invasive local delivery (e.g., convection-enhanced delivery), and these strategies have been limited by either unacceptable toxicity or inefficacy ^[30][31][32]. Focused ultrasound is a relatively new modality which transiently renders the BBB permeable to allow for improved drug delivery with a more favorable adverse effect profile, and clinical study is underway ^[33]. Continued efforts to improve drug delivery via nanoparticle- or peptide-based drug-carrying methods are ongoing, and further study of pharmacological inhibition of Wnt/Shh signaling and pericyte function is warranted ^[34][35].

2.2. Intra- and Intertumoral Heterogeneity

Perhaps the most important and challenging barrier to establishing effective treatments for glioblastoma is tumoral heterogeneity, which encompasses a vast spectrum of molecular, genetic, cellular, temporal, spatial, and evolutionary diversity and prevents the use of any single universal therapeutic approach. The Cancer Genome Atlas (TCGA) Research Network originally used an extensive characterization of the genomic alterations in glioblastoma to identify three critical signaling pathways in the disease—p53, retinoblastoma (Rb), and receptor tyrosine kinase/Ras/PI3K—and subsequent groups have built upon these data to formulate classification schemes with prognostic importance. Verhaak et al. ^[36][37] used factor analysis and consensus clustering of data from TCGA to define 4 glioblastoma subtypes on transcriptional grounds: classical, mesenchymal, proneural, and neural. The classical subtype is characterized by the gain of chromosome 7 and loss of chromosome 10, epidermal growth factor receptor (EGFR) amplification, and cyclin dependent kinase inhibitor 2A (CDKN2A) homozygous deletion with high-level upregulation of Notch (NOTCH1, NOTCH3, JAG1, LFNG)- and Shh (SMO, GAS1, GLI2)-related signaling with downregulation of mitogen-activated protein kinase (MAPK) and pro-apoptotic proteins such as cleaved caspase 7 and 9, Bid, and Bak. The mesenchymal subtype is characterized by mutations in NF1, phosphatase, and tensin-homolog protein (PTEN), and the nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) signaling pathway (e.g., TRADD, RELB, and TNFRSF1A) with increased MAPK and decreased mTOR signaling. The proneural subtype is characterized by IDH1 mutations, PDGFRA amplification, TP53 mutation, PI3K signaling, and high expression levels of oligodendrocytic developmental genes (e.g.,

OLIG2

NKDX2-2, and unique genomic hypermethylation-designated glioma-CpG island methylator phenotype or G-CIMP+) ^[38][39][40]. The genuine existence of the neural subtype is controversial, as subsequent studies by Wang et al. ^[41] and Gill et al. ^[42] have suggested that sampling of non-neoplastic cells at the infiltrative margins of the tumor account for the transcriptional profile was observed. Whole exome and transcriptional sequencing and proteomic profiling have further refined our understanding of the molecular subtypes. Various EGFR alterations (e.g., gene deletions or fusions) have also been detected, signifying the sheer genetic complexity of glioblastoma.

A model of cellular states and genetic diversity in glioblastoma recently postulated by Neftel et al. ^[43] integrated single-cell RNA sequencing and bulk genomic/transcriptomic and single-cell lineage tracing to demonstrate that sets of genes—designated “meta-modules”and encompassing mesenchymal, astrocytic, oligodendroglial, stem cell, and neural progenitor cell programs—recurred at high rates between tumors despite substantial intratumoral heterogeneity. Cellular populations isolated on the basis of these meta-modules generally expressed only 1 meta-module, and 15% of cells expressed 2, suggesting a hybrid subtype. Importantly, multiple cellular states were found to coexist within each tumor, each partially dictated by genetic mutations such as EGFR or PDGFRA.