The invasive capacity of GBM is one of the key tumoral features associated with treatment resistance, recurrence, and poor overall survival. The molecular machinery underlying GBM invasiveness comprises an intricate network of signaling pathways and interactions with the extracellular matrix and host cells. Among them, PI3k/Akt, Wnt, Hedgehog, and NFkB play a crucial role in the cellular processes related to invasion. A better understanding of these pathways could potentially help in developing new therapeutic approaches with better outcomes. Nevertheless, despite significant advances made over the last decade on these molecular and cellular mechanisms, they have not been translated into the clinical practice. Moreover, targeting the infiltrative tumor and its significance regarding outcome is still a major clinical challenge. For instance, the pre- and intraoperative methods used to identify the infiltrative tumor are limited when trying to accurately define the tumor boundaries and the burden of tumor cells in the infiltrated parenchyma. Besides, the impact of treating the infiltrative tumor remains unclear. Here we aim to highlight the molecular and clinical hallmarks of invasion in GBM.
In adults, glioblastoma (GBM) is the most common primary tumor in the central nervous system, with an incidence of 4.5 cases per 100,000 inhabitants. The median survival remains 14 months despite highly aggressive standard treatment protocols [
The first stage of glioma cell invasion is detachment from the surrounding tumor tissue, a process that involves cell surface adhesion molecules such as neuronal cell adhesion molecule (NCAM) and cadherins as key players in this process. It had been demonstrated that cadherin instability leads to glioma cell migration [
In addition to NCAMs, intercellular adhesion molecule-1 (ICAM1), a member of the immunoglobulin family of genes and expressed in several cell types, has recently been shown to contribute to glioma cell invasion [
Integrins (ITGs) are another key component of the interface between tumor cells and other cells in the microenvironment and function as receptors that regulate cell adhesion to ECM proteins or cell surface proteins on other stromal cells [
ITGs are expressed by various cell types in the tumor microenvironment including endothelial cells, immune cells, and pericytes and promote tumorigenesis. In particular, ITGs regulate invasion and metastasis by providing the traction necessary for cell migration [
ECM composition plays a critical role in the invasion process and the tumor-associated ECM is intrinsically different from the ECM within the normal parenchyma [
Glioma cells also degrade the surrounding ECM to favor their migration. Proteases, among others, are the enzymes that tumor cells use to perform this activity. Matrix metalloproteinases, such as MMP-2 and MMP-9, are related to the tumor grade and the invasive capacity of glioma [
Epithelial to mesenchymal transition (EMT) is a biochemical process through which the cytoskeleton of polarized epithelial cells is remodelled, and they shift to a nonpolarized mesenchymal phenotype. Extensive evidence suggests that EMT is an essential process for tissue remodelling, wound repair and cancer metastasis. While in an epithelial state cells are held tightly and are anchored to the basement membrane, mesenchymal cells are mainly spindle-shaped and are loosely attached to the ECM through interaction with focal adhesion molecules. Specific transcription factors such as Snail and Slug, the zinc-finger E-box-binding homeobox (ZEB)1/2, and Twist1/2 are considered the main regulators of the EMT process, as they regulate transcription of genes, including N-cadherin, vimentin, and fibronectin that are typically expressed in mesenchymal cells [
It is important to note that the role of cadherin switch as a hallmark of EMT in carcinomas is not well established in GBM, as these tumors are not epithelial in nature. E-cadherin is expressed at very low levels in neural tissues and is found only in a small proportion of aggressive GBM cells. On the other hand, N-cadherin is absent in epithelial tumors before the initiation of EMT, while it is highly expressed in astrocytes and regulates cell polarity and migration, resulting in a less regulated cell movement [
In addition to the master regulators, several cytokines play a role in EMT. In particular, Tumor Necrosis Factor-
Cytoskeletal remodelling is a key process in the formation of invadopodia and lamellipodia that are necessary for tumor cell motility [
Other important factors that regulate acto-myosin complex engagement during EMT include RHO GTPases, among which RHOA promotes formation of actin stress fibres. RAC1 and CDC42, on the other hand, regulate the formation of lamellipodia and filopodia. Following the activation of GTPases, the RHO-associated kinase (ROCK) cooperates with the formin diaphanous 1 (DIA1) to enhance actin polymerization and also induces the phosphorylation of myosin light chain to promote acto-myosin contraction and activation of LIM kinase (LIMK)[
It is important to note that glioma cell motility is not only influenced by the biochemical processes associated with the ECM but also by biophysical properties such as cell density and the rigidity and geometry of the ECM [
Tumor cells integrate with supportive stromal cells that are components of the tumor microenvironment. Stromal cells secrete growth factors and molecules that have the capacity to alter the milieu in which neoplastic cells proliferate. In fact, the microenvironment has been demonstrated to play key regulatory roles in response to therapy and tumor progression [
Nearly a third of GBM mass is composed of glioma-associated macrophages (GAMs). Due to the breakdown of the blood-brain barrier, these GAMs are derived primarily from bone-marrow derived cells and, to a lesser extent, from local resident inflammatory cells [
Extensive body of literature suggests that GAMs are not simple passenger cells in the tumor microenvironment as they play a key role in regulating tumor growth and invasion with complex interactions with many other cell types [
Large-scale genetic analyses have demonstrated that multiple signaling networks are employed by GBM cells to promote tumor growth and invasion. The most comprehensively studied pathways involved in GBM invasion include
Many of the signal transduction pathways that regulate the tumor microenvironment, including Ras/Raf/MAPK, Raf/JNK, Rho/Rac/PAK, and PI3K/Akt/mTOR, are convergent downstream signaling pathways of RTKs, implicating their role in GBM invasiveness and aggressiveness [
The Phosphoinositide-3-kinase (PI3K) signaling cascade is one of the main canonical pathways that have been implicated in GBM pathogenesis. This pathway transduces extracellular signals via receptor tyrosine kinases (RTKs) to regulate a series of biological processes such as cellular metabolism, growth, survival, and invasion. The PI3K pathway can be activated through interaction of ligands such as the epidermal growth factor (EGF) and TGFß with their respective RTKs. Induction of PI3K leads to activation of Akt family of kinases that regulate cell growth and survival. Regulation of the PI3K-Akt signaling pathway occurs through the tumor suppressor phosphatase and tensin homolog (PTEN) protein that dephosphorylates and, thereby, inactivates Akt [
Constitutional activation of the PI3K-Akt pathway is implicated in many cancers. In GBM, this pathway is activated by two frequent alterations, an in-frame deletion of amino acids 6–273 in EGFRvIII resulting in a mutant EGFR protein which is present in more than 50% of high grade gliomas and its activation is ligand-independent [
Additionally, the RTK c-Met and its ligand hepatocyte growth factor (HGF)/Scatter factor are overexpressed in gliomas and they have been shown to play a role in cell proliferation, invasion, angiogenesis and survival in several cancers [
In addition to EGFR and cMET, Wang et al. have demonstrated that the RTK Mer (MerTK) is overexpressed in GBM and this is accompanied with increased invasiveness [
WNT signaling pathway is a crucial regulator of proliferation, migration and cell fate in the central nervous system during embryogenesis [
Activation of the canonical WNT pathway leads to disassembly of the transmembrane receptors of the ß-catenin destruction complex, consisting of the GSK3B, AXIN and adenomatous polyposis coli (APC) [
In addition, WNT signaling is a major factor in epithelial-mesenchymal transition (EMT) and tumor invasion. Several studies have demonstrated that WNT pathway activation enhances the motility of cancer cells [
Similar to WNT pathway, the Hedgehog pathway plays a crucial role in the development of the central nervous system. Hedgehog pathway dysfunction during embryogenesis leads to congenital defects such as microcephaly or cyclopia. In many cancers including glioma, the Hedgehog pathway is upregulated and plays a role in tumorigenesis and tumor progression. Generally, Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH) ligands can activate the Hedgehog pathway by binding to the transmembrane protein Patched (PTCH1). Hedgehog pathway activation leads to upregulation of GLI1, PTCH1, cyclin D2 (CCND2), Bcl-2, and VEGF. In addition, Hedgehog pathway modulates the expression of stemness genes, such as NANOG, OCT4, and SOX [
Although GLI1 amplification is relatively rare in GBMs, a novel truncated isoform, tGLI1, has been linked to increased cell motility and tumor invasion in GBM and breast cancer [
Epigenetic modulators may also play a role in hedgehog pathway activation. Bromodomain-containing protein 4 (BRD4) is a critical regulator of GLI1 transcription through direct occupancy of the gene promoter [
NF-
Invasiveness is one of the key features that allow GBM to overcome the current treatment strategies [
Targeting tumor invasion and infiltration is a major clinical challenge. Novel pre- and intraoperative imaging techniques are being developed to accurately assess the extent of parenchymal infiltration in the clinical setting. Besides, new insights into potential therapeutic approaches have been recently reported.
The radiological definition of infiltrated parenchyma remains unclear and the current imaging techniques, summarized in Table
Pre- and intraoperative methods to assess GBM’s invasive capacity.
Preoperative methods | Intraoperative methods |
---|---|
| |
T2/FLAIR hyperintensity | 5-aminolevulinic acid (5-ALA) |
DTI | Fluorescein sodium (Fl-Na) |
DWI (ADC and FA) | |
Perfusion | |
Spectroscopy | Contrast enhanced US |
Quantitative MR | Elastosonography |
Radiomics radiophenotype | |
| Fluorescein |
Fluorothymidine | Indocyanine green |
Fluoroethylthyrosine | Acriflavine hydrochloride |
Tryptophan | |
Methionine |
MRI= Magnetic Resonance Imaging, FLAIR= fluid attenuated inversion recovery, DTI= Diffusion tensor imaging, PET= Positron emission tomography, and iMRI= intraoperative MRI.
GBM-induced T2/FLAIR hyperintensity in the MRI represents the area of peritumoral oedema and tumor-induced alterations in the parenchyma. It is a result of changes in the composition of the ECM and impairment of the blood-brain barrier in a process associated with the expression of endogenous tenascin-C [
It has been widely demonstrated that glioma cells infiltrate the peritumoral T2/FLAIR high signal region beyond the contrast enhancement on the preoperative MRI [
Nevertheless, the distinction of the diffuse nonenhancing tumor invasion from the peritumoral vasogenic oedema can be challenging in the clinical practice [
Moreover, considering GBM’s diffuse infiltration, the burden of tumor cell invasion in the “normal” brain is not yet possible by using imaging techniques [
Radiomic analyses have focused on the invasion-related radio-phenotype applying quantitative volumetric to assess the correlation between specific radiological invasion features and IDH mutation status, outcome, or response to surgery [
Other imaging methods, as Positron Emission Tomography (PET), have been used to assess the parenchymal response to tumor invasion [
Intraoperatively, the tumor infiltrating the adjacent parenchyma maintains the macroscopic aspect of normal or oedematous brain parenchyma. Therefore, it is critical to develop and validate methods to accurately define the boundaries of the infiltrative tumor.
In the last two decades, the 5-aminolevulinic acid (5-ALA), an intermediate metabolite in the porphyrin intracellular pathway that results in the accumulation of fluorescent protoporphyrin IX molecule inside tumor cells, has been used to intraoperatively define the infiltrative tumor [
Moreover, the concordance between 5-ALA fluorescence and intraoperative MRI (iMRI) findings is still poorly understood. For instance, residual contrast enhancement in the iMRI after 5-ALA fluorescence-guided resection can be found in the majority of cases. Histopathological analysis of these regions revealed tumor core or tumor infiltration in 39 and 25% of cases, respectively [
On the other hand, preoperative 18F-fluoroethyl-L-tyrosine (FET)-PET can predict 5-ALA fluorescence [
Fluorescein sodium (Fl-Na) is another marker used in fluorescence-guided surgery. Despite a good correlation of Fl-Na and histopathological [
Intraoperative ultrasound (US) is another intraoperative resource used to assess tumor extension [
Among other techniques described to identify the boundaries of the infiltrative GBM during the surgical resection, intraoperative confocal microscopy is an emerging approach capable of identifying fluorescein-, indocyanine green-, or acriflavine hydrochloride-enhanced differences in cell density and cellular morphology corresponding with the T2 hyper-intensity on MR imaging [
Finally, optical coherence tomography, a real-time tissue microstructure imaging technique based on low-coherence interferometry in the near infra-red range of wavelengths, is another promising tool for assessing the tumor infiltrative margin in gliomas. It provides comprehensive qualitative and quantitative analysis of the tumor and the peritumoral tissue, generating color-coded maps that correlate with the histological findings and help to accurately identify the tumor boundaries [
The current standard of care for patients with GBM involves surgical resection and adjuvant chemo-radiation with temozolomide [
In GBM, tumor cell invasiveness can lead to the infiltration or destruction of surrounding parenchyma resulting in neurological deficits [
Thus far, several studies have shown that resection of the infiltrative portion of the tumor, based on DTI, ADC, or T2/FLAIR abnormalities is associated with longer progression-free survival (PFS) and overall survival (OS) [
Although there is evidence supporting that resection of the infiltrative tumor can result in better outcomes, opposite results highlight the need for further research, as it remains unclear the more appropriate method to identify the areas of the surrounding parenchyma with greater tumor cell density and to distinguish them from the oedematous brain [
Accurate tumor volume definition is critical in conformal or intensity-modulated radiotherapy (IMRT) planning. Analogously to surgical approaches, a subtherapeutic radiation dose within the tumor may result in treatment failure and recurrence, whereas whole-brain dose increments may lead to radiation-induced toxicity [
Despite the infiltrative nature of GBM, radiation planning protocols have evolved from whole brain radiotherapy towards more tailored tumor volume targets, partially based on that the great majority of recurrences arise within 2 cm from the primary site [
On the other hand, the use of DTI-based clinical target volumes (CTV) has been proposed, as they are smaller than the ones based on the T2-hyperintensity, sparing the peritumoral oedema. Besides, this reduction in the CTVs could allow dose escalation [
Other approaches for CTV definition are based on PET findings. For instance, a higher dose coverage of 18F-FET-PET tumor regions is positively correlated with time to progression and PET-based CTVs better-predicted failure sites when compared to MRI-based CTVs [
Overall, current commonly used therapies for GBM, including alkylating agents as Temozolomide (TMZ) and the anti-VEGF compound Bevacizumab, failed in targeting glioma cell invasion. Although TMZ can potentially inhibit invasion in vitro [
Considering the lack of an effective therapeutic approach against GBM invasiveness, further research is warranted to better understand the invasion pathways contributing to glioma cell infiltration and, consequently, to develop new therapeutic agents. An effective therapeutic strategy should target both infiltrative GBM cells and the tumor cell-stroma interaction [
Up to now, no clinically transferable results have been achieved after trying to target some of the mechanisms involved in GBM invasion, including cytoskeleton reorganization and cell motility, cell adhesion, and degradation of ECM [
Current areas of research include several potential targets in glioma cell invasion pathways. Glutamate-mediated infiltration inhibition has been assessed in several Phases I-II trials with promising results. Besides, the role of different tumor cell ion channels and transporters, microtubule-based tumor cell network, microRNA-related invasion, and the mechanisms involved in the interaction between the tumor and the host open potential opportunities for targeted therapy approaches [
The GBM invasiveness capacity is one of the main features contributing to tumor recurrence, treatment resistance, and low survival rates. It results from an intricate combination of several signalling routes, mainly receptor tyrosine kinases and transcriptional pathways and also cellular processes that include cytoskeletal remodelling and interactions with ECM components and host cells (Figure
The authors declare that they have no conflicts of interest.
Carlos Velásquez and Sheila Mansouri contributed equally to this work.
This work received funding from Instituto de Salud Carlos III, grant PI17/01399.