Glioblastoma is an incurable brain cancer that kills most patients less than two years after diagnosis.
The disease is difficult to treat largely because tumors each contain multiple kinds of cells.
The aggressive brain cancer can also vastly differ between patients, so much so that researchers debate whether glioblastoma should be considered a single disease.
A new study may help clarify what drives this important heterogeneity and makes glioblastoma so deadly.
The researchers profiled gene expression in more than 24,000 tumor cells from 20 adult and eight pediatric glioblastoma patients and also analyzed glioblastoma models in the lab.
They found four glioblastoma cellular states, which each has a unique gene expression program and together help account for the large variation in the disease.
The scientists then used the single-cell data to reanalyze glioblastoma data from The Cancer Genome Atlas and identified genetic alterations associated with each of the four states.
The results, published in Cell, also show that the cells are remarkably flexible or plastic—that is, they can switch between the four states.
This shape-shifting ability could help explain why these cancer cells are so difficult to kill with drugs and help inform the development of better therapies for glioblastoma.
The research was led by co-first authors Cyril Neftel, Department of Pathology at Massachusetts General Hospital (MGH) and Broad Institute; Julie Laffy, Weizmann Institute of Science; Mariella Filbin, Department of Pathology at MGH, Broad Institute, and Dana-Farber Institute; Toshiro Hara, Salk Institute for Biological Studies; and co-senior authors Itay Tirosh, Weizmann Institute of Science, and Mario Suvà, Department of Pathology at MGH and Broad Institute.
The state of the cell
Previous studies suggested that glioblastoma samples fall into at least three subtypes: proneural, classical, and mesenchymal, which differ mostly by the genes they express.
Researchers have also shown that glioblastoma tumors are almost always made up of more than one subtype, and that the proportions of different subtypes in a tumor can change over time and during treatment.
However, most of these studies looked at bulk samples, where genetic material from many tumor cells is combined and sequenced. This method provides a rough idea of the subtypes in a tumor and their relative abundance, but not the finer details.
To better understand glioblastoma states at the cellular level, the researchers examined cells using single-cell RNA sequencing, a suite of genomic techniques that reveal individual cells’ gene expression profiles.
“We are placing the individual cellular states and their associated genetic alterations in their developmental context and understanding what cell types are driving the disease,” said Suvà, who is an institute member in the Broad’s Epigenomics Program, a molecular pathologist in MGH’s Department of Pathology, and a member of the Center for Cancer Research at MGH.
He added that this effort is the largest single-cell sequencing study for glioblastoma to date.
The researchers discovered four distinct gene expression programs among the cells, representing four dominant cellular states: neural-progenitor-like, oligodendrocyte-progenitor-like, astrocyte-like, and mesenchymal-like.
All four states resemble normal brain cell types to some extent, but with important differences that make the cells cancerous.
These states also correlate well with the previously-described subtypes in glioblastoma.
The four states were represented in both adult and pediatric tumors, and the majority of tumors contained cells in every state while the remaining minority had cells representing at least two states. However, the proportions of cell states varied among tumors.
To see whether these differences were due to genetics, the tumor’s surrounding microenvironment, or both, the researchers looked for genes that were associated with tumors containing a high percentage of cells in a particular cell state.
They determined that common genetic drivers of glioblastoma (genes including EGFR, PDGFRA, and CDK4) play a role in which state a glioblastoma cell takes and how many cells of that state are in a patient sample.
For one of the states, mesenchymal-like, they also found that the microenvironment played an important role.
Crossing state lines
While most cells seemed to fit clearly into one of the four states, about 15% expressed two programs simultaneously.
“Finding a lot of cells in between two states is strong evidence that those states can transition from one to the other,” Suvà said.
To test this observation, the researchers injected mice with cells derived from a human glioblastoma tumor, with each mouse receiving cells that represented only one state.
They found that the resulting tumors contained not just that cell state, but all the other states that were present in the original tumor, and in about the same proportions.
This suggests the glioblastoma cells can transition from one state to any of the others.
The team came to the same conclusion after using genetic barcoding to follow how individual cells developed and changed in both a mouse glioblastoma model and in mice harboring human glioblastoma cells.
The findings provide clues as to why existing cancer treatments fail to stop glioblastoma growth.
“If you understand that this is a disease with multiple states driving it, each one with a corresponding favorite cancer gene, you understand better why targeting one gene at a time has failed so far, and you can dissect the mechanisms of adaptation and resistance to therapies,” said Suvà.
The researchers now plan to study how current therapies affect each of the four states and search for novel ways to target each state.
Glioblastoma, more commonly known as glioblastoma multiforme (GBM), is the most common and most aggressive type of malignant brain tumor in adults.1
Globally, it has an annual incidence of <10 per 100,000 people.2
Despite all the advances of modern medicine, it remains incurable, with an extremely poor prognosis.
The Public Health England estimates the median survival as 6 months from diagnosis without treatment, close to the worst of any cancer.2
With treatment, median survival time can increase to around 15 months, although for unknown reasons, some patients can survive much longer.
This treatment follows a three-pronged approach, consisting of maximal safe surgical resection, followed by concurrent temozolomide and radiotherapy, followed by temozolomide alone.3
Recurrence is inevitable, most commonly occurring within 1 cm of the surgical resection margin, owing to the highly invasive nature of GBM.
GBM is classified as a grade IV tumor (the most aggressive category according to the WHO criteria),1 and accounts for 55% of all malignant brain tumors.4
Though its cellular origins remain elusive, the astrocyte, a form of glial cell, is a principal candidate.
Primary tumors represent the more aggressive de novo types, whereas the less common secondary tumors develop as a result of progression from a lower-grade glioma.
The term “glioma” encompasses all brain tumors of glial cell origin, with GBM representing the most aggressive type.
As such, the vast majority of glioma research studies GBM, given the dismal prognosis.
Many years of research have led to insufficient improvement in patient prognosis.
In the last two decades, there has only been one major advancement, namely the discovery of temozolomide, an alkylating chemotherapy which now forms part of the standard treatment for primary GBM patients.5,6
The Stupp protocol has certainly helped increase overall survival; however, we may need to think outside the protocol to increase disease-free survival time.
Currently, the only US Food and Drug Administration (FDA)-approved targeted drug for GBM treatment is the anti-VEGF antibody bevacizumab, although strong evidence of its benefits is lacking, and it may only be effective in reducing peritumoral edema. Neither increased temozolomide dosage nor bevacizumab has been shown to improve overall survival.3
There is certainly a need for new forms of GBM treatment.
Consistent with most recurrence occurring within close proximity of the surgical resection margin, increasing the extent of surgical removal has been shown to increase patient survival, though this carries a greater risk of damage to eloquent or other important brain tissue. Even with the greater surgical resection, recurrence is unavoidable.
A recent landmark paper, using data from The Cancer Genome Atlas (TCGA), identified four distinct subtypes of glioblastoma: classical, proneural, neural, and mesenchymal.7
Each subtype has a unique molecular profile of protein expression and genetic mutations, with the mesenchymal subtype representing the majority of primary glioblastoma diagnoses.
However, the findings of this paper have yet to translate into changes in clinical practice, and there is significant overlap between the subtypes.
Traditionally, cancer research has taken a very tumor cell-centric view, typically utilizing drugs to target tumor cells.
A more tumor-centric approach, focusing on the specific mechanisms utilized by invading GBM cells in the context of a complex tumor microenvironment, may yield better approaches to improve patient outcomes.
This review investigates some of the mechanisms underpinning the complex interplay between tumor cells and the microenvironment to stimulate GBM cell invasion.Go to:
Glioblastoma invasion and potential cell origins
Aggressive invasiveness remains a common feature of malignant gliomas, despite high levels of tumor heterogeneity and possible divergent cells of origin.7
In vitro studies comparing central and peripheral cell samples of a GBM tumor mass showed discrepancies in levels of proliferation and invasiveness, with peripheral cells appearing markedly less proliferative but more invasive than their central counterparts.8
Although cells are inherently motile, this motility is typically restricted to specific stages of cell life such as embryonic development or, for example, immune surveillance. This process becomes dysregulated in invading cells.
It is important to distinguish between migration and invasion. Migration is a normal physiological process undertaken by many cells, particularly neural stem cells (NSCs) in the brain, in which they navigate tissue boundaries.
Although tissue disruption can occur in both, invasion is an unscheduled, anatomically inappropriate, and nonphysiological process.
There are clear differences between the invasiveness of other high-grade solid cancers and that of GBMs. While the former commonly intravasate into blood and lymphatic vessels, GBMs rarely intravasate into any vessels.9
Murine models have shown human glioma cells (HuN) navigating along blood vessels, in a region known as the perivascular space.10
This is reminiscent of neural progenitor cells in rodents, which also use this mechanism of vascular scaffolding, particularly in the rostral migratory stream into the olfactory bulb. While resident astrocytes do not readily migrate into central nervous system (CNS) lesion sites, NG2 cells can migrate into these areas.
This is of interest since NG2 expression has been associated with an aggressive molecular signature in GBM.11
The similarities that can be drawn between these cell types improve our understanding by allowing us to draw from our knowledge of normal brain function. In addition, they provide further insight into the potential cells of origin for GBMs.Go to:
Role of glutamate signaling
GBM cells are unusual in their use of glutamate (a neurotransmitter), which acts in both an autocrine and paracrine manner as a growth factor to enhance invasion.
GBM cells release significant amounts of glutamate in vitro, and glutamate is significantly increased in the microenvironment of the tumor in vivo.12
This unusual feature resembles mechanisms seen in developing neurons. While developing neurons use the NMDA receptor, GBMs utilize a specific isoform of the AMPA receptor to respond to glutamate.
This particular AMPA receptor lacks the GluR2 subunit to confer Ca2+ permeability, potentially opening up the possibility of tumor-specific AMPA receptors being a therapeutic target.
Tumors with increased levels of glutamate secretion showed enhanced growth in rodent models.12 Excitotoxicity from elevated glutamate levels may act as a space-vacating agent, by killing nearby neuronal cells to facilitate invasion into surrounding parenchyma. Thus, glutamate release could also be targeted pharmacologically, although such targeting may be very toxic.
More information: Cyril Neftel et al. An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma, Cell (2019). DOI: 10.1016/j.cell.2019.06.024
