The VEGF-C treatment significantly extended the life span of the mice with glioblastoma brain cancer


The brain is a sort of fortress, equipped with barriers designed to keep out dangerous pathogens. But protection comes at a cost:

These barriers interfere with the immune system when faced with dire threats such glioblastoma, a deadly brain tumor for which there are few effective treatments.

Yale researchers have found a novel way to circumvent the brain’s natural defenses when they’re counterproductive by slipping immune system rescuers through the fortresses’ drainage system, they report Jan. 15 in the journal Nature.

“People had thought there was very little the immune system could do to combat brain tumors,” said senior corresponding author Akiko Iwasaki. “There has been no way for glioblastoma patients to benefit from immunotherapy.”

Iwasaki is the Waldemar Von Zedtwitz Professor of Immunobiology and professor of molecular, cellular, and developmental biology and an investigator for the Howard Hughes Medical Institute.

While the brain itself has no direct way for disposing of cellular waste, tiny vessels lining the interior of the skull collect tissue waste and dispose of it through the body’s lymphatic system, which filters toxins and waste from the body. It is this disposal system that researchers exploited in the new study.

While the brain itself has no direct way for disposing of cellular waste, tiny vessels lining the interior of the skull collect tissue waste and dispose of it through the body’s lymphatic system, which filters toxins and waste from the body. It is this disposal system that researchers exploited in the new study.

These vessels form shortly after birth, spurred in part by the gene known as vascular endothelial growth factor C, or VEGF-C.

Yale’s Jean-Leon Thomas, associate professor of neurology at Yale and senior co-corresponding author of the paper, wondered whether VEGF-C might increase immune response if lymphatic drainage was increased. And lead author Eric Song, a student working in Iwasaki’s lab, wanted to see if VEGF-C could specifically be used to increase the immune system’s surveillance of glioblastoma tumors. Together, the team investigated whether introducing VEGF-C through this drainage system would specifically target brain tumors.

The team introduced VEGF C into the cerebrospinal fluid of mice with glioblastoma and observed an increased level of T cell response to tumors in the brain. When combined with immune system checkpoint inhibitors commonly used in immunotherapy, the VEGF-C treatment significantly extended survival of the mice. In other words, the introduction of VEGF-C, in conjunction with cancer immunotherapy drugs, was apparently sufficient to target brain tumors.

“These results are remarkable,” Iwasaki said.

“We would like to bring this treatment to glioblastoma patients. The prognosis with current therapies of surgery and chemotherapy is still so bleak.”

Funding: The study was primarily funded by the Howard Hughes Medical Institute and the National Institutes of Health.

Other Yale authors are Tianyang Mao, Huiping Dong, Ligia Simoes,Braga Boisserand, and Marcus Bosenberg. Salli Antila and Kari Alitalo of the University of Helsinki are also authors.A

Introduction: Glioblastoma Multiforme

Glioblastoma multiforme is characterized by poor prognosis, low survival rates, and extremely limited opportunities for therapy. Malignant gliomas are the third leading cause of cancer death for people aged between 15 to 34, accounting for 2.5% of the global cancer death toll. Among gliomas, glioblastoma multiforme represents the 50%, with a maximum incidence in patients aged more than 65 years [1,2,3,4].

Due to the absence of effective surgical and medical treatments currently available for glioblastoma, an early diagnosis coupled with an accurate tumor classification is of key importance to select a personalized treatment [5,6].

Gliomas are tumors with neuroectodermal origin, showing a considerable variability in age of onset, grade of severity, histological features, and ability to progress, as well as to metastasize [7,8].

According to the WHO classification, astrocytomas are histologically and clinically classified into four types: Pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme.

Pilocytic astrocytoma and diffuse astrocytoma are characterized by a relatively low growth rate, while for anaplastic astrocytoma and glioblastoma multiforme by common uncontrolled proliferation, diffuse tissue penetration, and neurodegeneration [9,10,11].

In turn, glioblastomas are classified into three subtypes, depending on the status of the IDH gene mutation: Primary glioblastomas (IDH-wild-type), secondary glioblastomas (IDH-mutant), and unclassified glioblastomas (NOS) [12].

It is important to note that unclassified glioblastomas (NOS) do not belong to a specific glioblastoma category, given their diagnostic and genetic heterogeneity; for that reason, they cannot be classified within any other group [7].

Diagnosis and Treatment of Glioblastoma

One of the main problems of glioblastoma management is related to the lack of effective diagnostic strategies. Currently, the main diagnostic methods for the detection of gliomas rely on neurological tests and neuroimaging methods, performed when the disease is already at an advanced stage [13,14].

Late diagnosis of glioblastoma is mainly caused by the slow dissemination process typical of brain tumors, which allows structures to gradually adapt to both compression and deformation caused by the tumor mass.

For this reason, even in the case of pronounced morphological signs of tumor penetration into brain tissue, clinical manifestations may be completely absent [15]. However, a major drawback comes in patients which make use of antiangiogenic drugs or chemo-radiotherapy, that can significantly deceive the results coming from neuroimaging analyses, thus making the follow-up even more difficult [16,17,18,19].

A typical treatment for glioblastoma involves surgical resection of the tumor mass, followed by radiotherapy and chemotherapy treatments. However, such therapies are often proved to be ineffective, given the high rate of relapse, general tumor resistance appearance over time, coupled with a serious neurological deterioration of the patient [20].

Regardless its radicality, the surgical resection of glioblastomas is often inadequate, given the frequent residual presence of microscopic foci, leading to relapse or even recurrence of the disease [21,22].

This is mainly due to their infiltrative growth, as well as their high proliferative abilities. However, numerous studies have highlighted the importance of maximizing tumor removal to increase life expectancy of glioblastoma multiforme (GBM) patients.

In fact, it is of key importance to remove the tumor mass up to the borders with the healthy surrounding tissue, in order to have a beneficial effect on patient survival rate [23,24]. However, even radical tumor resection is not conclusive since it is often followed by a relapse of the disease [25,26]. These findings explain why glioblastoma is not a surgically treatable disease [27,28,29,30,31,32].

More recently, based on the results obtained with other tumors [33,34], new treatments based on nitric oxide-releasing HIV protease inhibitors have been administered to glioblastoma patients [35,36]. Other studies have shown that despite the high vascularization of glioblastoma, treatments with the anti-VEGF bevacizumab do not significantly improve patients’ overall survival [37]. Finally, although several clinical trials have characterized the usage of carmustine wafers implants (Gliadel, generated 20 years ago) following tumor resection as adjuvant therapy, their clinical application has remained low [38,39].

Localization of Glioblastoma

Glioblastoma development occurs in the trans-barrier space of the blood-brain barrier (BBB), which prevents the translocation of polarized and/or high-molecular-weight substances from the bloodstream towards the brain [20]. Disturbances in BBB function linked with glioma malignancies are often observed, significantly affecting peripheral blood detectable levels of tumor biomarkers [40].

The rapid growth of glioblastoma cells creates areas of local hypoxia, which triggers the process of angiogenesis [41]. In addition to enhanced angiogenesis, changes in the expression of proteins of the aquaporin family in the components of the BBB have been linked with the tumor progression of the glioblastoma [42,43,44,45].

During tumor-induced angiogenesis, neo-formed vases show an abnormal structure, lacking the specific barrier function of normal BBB blood vessels. Surprisingly, this effect is stronger in those high-grade gliomas lacking almost totally the BBB barrier functionality, and weaker in diffuse gliomas and low-grade gliomas [46,47,48].

Noteworthy, all gliomas, including glioblastoma, show intact BBB areas, especially at the periphery of the tumor, representing one of the main obstacles against their response to drug treatments [49,50].

Characteristic and Carcinogenesis of Glioblastoma

During the past 20 years, an increase in the number of patients diagnosed with glioblastoma has been observed. This increase can be attributed both to the improvement of diagnostic investigations for brain tumors, and to an actual higher GBM incidence due to various occupational and environmental risk factors which may increase the incidence of all tumors, including glioblastomas [51,52,53,54,55,56].

Even though it is difficult to make a correlation between brain tumors and the exposure to environmental or lifestyle factors, numerous risk factors have been described to predispose to gliomas and glioblastomas [57]. Several studies have demonstrated that the gut microbiota is also correlated with tumor development [58,59,60].

Recently, different reports have described the existence of a so called “gut-brain axis”, showing that the dysregulation of the gut microbiota may lead to the alterations of several processes predisposing to the development of a number of nervous system diseases, including cancer [61,62].

Moreover, individual genetic background may be linked to the prognosis of patients. Generally, Asian glioblastoma patients survive longer compared with Caucasian, African, or Latin American patients [63].

Glioblastoma is more common in adult patients, mainly affecting the cerebral hemispheres; much less common in children and, as rule, localized in the region of the brain stem [64]. Radiographic contrast enhancement brain tissues studies have revealed significant infiltrates of tumor cells outside the contrasted tumor.

This observation provides incontrovertible evidence that a clinically significant tumor burden also exists outside the tumor volume, thereby supporting the classification of GBM as a whole brain disease [65].

Histologically indistinguishable grade IV gliomas, affecting heterogeneous age groups, are a consequence of the accumulation of several genetic mutations affecting tumor development [66]. In accordance with the assessment of defined genetic parameters, GBM can be classified into primary or secondary.

Approximately 90% of all cases of GBM are primary and occur in elderly patients (Table 1), in which tumor progression is more rapid due to the higher accumulation of gene mutations compared with young individuals. Generally, patients affected by primary GBM experience complication and consequently die 9–12 months after the diagnosis.

In contrast, secondary GBMs develop from primary astrocytomas, bearing a lower degree of malignancy and typical of younger patients (<45 years). At the same time, there is a gradual increase in the rate of tumor cells’ proliferation, angiogenesis, drug resistance, and other parameters, which leads to an increased severity [66] (Figure 1).

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Object name is cells-08-00863-g001.jpg
Molecular alterations responsible for glioblastoma carcinogenesis [66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85].

Characteristics of primary and secondary glioblastoma [71,72,73,74,75,76,77,78,79,80,81,82,83,84,85].

Status/FeaturePrimary GlioblastomaSecondary Glioblastoma
Positive status mutation of IDH gene<5%~80%
Preceding cancer diseaseNot identified; detected for the first time (de novo)Diffuse astrocytoma; anaplastic astrocytoma
The percentage of all detected glioblastoma90%<10%
The average age of diagnosis6244
Sex ratio (M:W)1.42:11.05:1
Median overall survival
surgical treatment and radiotherapy9.9 months24 months
surgical treatment, radiotherapy and chemotherapy15 months31 months
LocalizationSupratentorialPredominantly frontal
TERT promoter mutation72%26%
TP53 mutation27%81%
ATRX mutationRarely71%
EGFR mutation35%Rarely
PTEN mutation25%Rarely

An important aspect that should be taken into account in the diagnosis of GBM is its high internal heterogeneity, which is characteristic of both newly detected and recurrent tumors [86,87]. Evaluation of the internal heterogeneity of GBM, using imaging techniques such as MRI, may give important prognostic information [88,89].

The ability of dividing into subgroups GBM tumor cells within the same tumor mass, based on their spatial and temporal variability, needs to be developed [90]. The heterogeneous nature of this tumor makes it difficult to identify and validate potential biomarkers [91].

For example, morphologically different glioblastoma cells exhibit different in vitro invasion, as well as cell migration abilities, depending on the nature of the surrounding microenvironment [92]. Furthermore, the complex crosstalk between tumor cells and the microenvironment may enhance tumor growth and reduce the chances of successful drug therapy [5,93,94].

In several tumors, including glioblastoma, it was demonstrated that the alteration of the extracellular matrix (ECM) composition and the over-expression of proteolytic enzymes, due to genetic and epigenetic modifications, are responsible for a more aggressive tumor phenotype and a worse prognosis [95,96,97,98,99,100].

It is assumed that many of the invasive signs of gliomas depend on their pathological metabolism, which may either promote tumor cells invasion or create an environment in which glioma cells might gain growth advantage over normal cells [101,102].

Perspective Biomarkers

An ideal tumor marker should be easily accessible for analysis, be detected by the simplest analytical method, and be able to provide accurate information about both the presence of the disease and its severity.

The ideal marker should have 100% sensitivity and specificity, sufficient half-life for detection, the ability to dynamically reflect the tumor load, and its analysis should be economically acceptable for introduction into routine practice [103,104].

However, clinical biomarkers and their respective analysis do not have to be “ideal” in order to be clinically useful for diagnostic purposes. Glioblastoma is usually clinically characterized and diagnosed with diverse physico-chemical analyses, through the use of both tissue and circulating biomarkers [3,105,106].

However, most biomarkers lack either sensitivity or specificity. Several studies have proven that screening for certain nucleic acids may have higher specificity in glioblastomas, compared with individual proteins analysis. In the same manner, low-molecular weight metabolites and lipids have shown a low specificity for systemic diagnostic tasks [3,107,108,109]. However, conflicting results were generated on this matter.

Glioblastoma characterization based on tumor genetic properties is widely accepted. In fact, since 2016, brain tumors, including glioblastomas, are internationally classified based on their molecular genetic properties, as well as the histological features linked with these properties [7].

In general, a series of mutations of DNA and de-regulation of non-coding RNA have been characterized for gliomas; the frequency of their occurrence is different and correlates with the type of brain tumor [110].

Evaluation of genetic mutations in glioma cells by genotyping circulating tumor nucleic acids allows the classification of specific tumors and the definition of the prognosis and tumor burden. Importantly, circulating tumor nucleic acid analysis enables the selection and evaluation of patients’ therapeutic efficacy window [7,111].

Currently, the assessment of genetic parameters in biopsy specimens from glioma patients is of key importance for the formulation of a refined diagnosis and the choice of the best treatment strategy [7,112]. In addition, circulating nucleic acids found in blood and other biological fluids, either free or associated with extracellular vesicles, might be used as markers for brain tumors’ early diagnosis and classification [31,111].

In particular, a number of clinically significant glioma genetic biomarkers are currently analyzed as routine practice. Among these biomarkers, the most representative are: IDH1/2 mutation status, MGMT promoter methylation, 1p/19q co-deletion, and ATRX loss [7,75,113,114].

Widely diffused molecular methods for the analysis of these genetic biomarkers and the identification of nucleic acid mutations include: Direct sequencing, high-resolution melting (HRM), immunohistochemistry, droplet digital PCR (ddPCR), and several others [110,115,116,117].

Through the use of these advanced techniques, it is nowadays possible to classify histologically indistinguishable GBM by the presence/absence of genetic mutations, which has an important therapeutic, prognostic, and experimental value [64,118]. The issues with the analysis of nucleic acids in peripheral blood are similar with the problems encountered when searching for high-molecular polar central nervous system (CNS)-derived compounds. In particular, low concentration, low abundance of the compounds in the systemic circulation, coupled with their weak penetration thought the BBB, make brain tumor-derived nucleic acids difficult to be identified as circulating molecules [31,119,120,121,122,123,124].



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