Canadian researchers develop a new technology for the detection of the deadliest form of brain cancer

Gliomas are malignant primary brain tumors, among which glioblastoma has the worst prognosis. According to the most recent WHO guidelines, it is classified as a grade IV diffuse astrocytoma. Glioblastoma is a particularly aggressive type of cancer affecting the glial cells, in particular astrocytes, which have a supporting role in CNS. Furthermore, it represents about 45.2% of all malignant CNS tumors with an incidence of 5–6 cases per 100,000 people. Glioblastoma is characterized by uncontrolled proliferation, angiogenesis, invasiveness, and necrosis, and it can develop de novo or through the malignant progression of lower-grade astrocytomas. Numerous risk factors leading to the development of glioblastoma have been identified as genetic factors and environmental factors including exposure to therapeutic ionizing radiation, pesticides, and smoking. In the last years, there has been a progressive upsurge in brain tumors incidence; in particular, this increase was most significant in the over 65 age group, and higher in men than women. The manifestations of a brain tumor depend mainly on its location and size that can lead to compression, infiltration of healthy tissue, increasing intracranial pressure, and cerebral edema.

It is generally accepted that the degree of angiogenesis is correlated to the malignancy of the tumour. Therefore, assessment of the rate of tumour angiogenesis using specific biomarkers is potentially useful clinical information for determining the severity of the disease and selecting appropriate treatment modality. Currently, there is a limited number of targeting ligands that have been utilised to non-invasively assess the degree of tumour angiogenesis. Previously Insulin-like growth factor binding protein-7 (IGFBP7) was discovered as a highly upregulated selective biomarker of GBM vessels. The IGFBP7, also known as IGFBP-related protein 1, mac25, TAF and angiomodulin, is a 31 kDa secreted protein that shares structural homology with a family of IGFBP-related proteins, which includes IGFBP1-6. In contrast to IGFBP1- 6 that displays high affinity for insulin-like growth factor (IGF), IGFBP7 is classified in the family subgroup showing low affinity for IGF. Several studies demonstrated that IGFBP7 is overexpressed in tumour blood vessels with little or no expression in normal blood vessels nor in the non-malignant angiogenic placental tissues. IGFBP7 accumulates in the basement membrane of the tumour endothelium, where it can bind to collagen type II, IV and V, heparin sulphate proteoglycans and other cytokines, Insights into mechanisms of IGFBP7 expression in the glioblastoma environment have come from in vitro experiments, which demonstrated that TGF-b1 secreted from glioblastoma tumour cells stimulate IGFBP7 production in human brain endothelial cells (HBECs). IGFBP7 appears to increase the formation of capillary like tubes by HBEC, suggesting a pro-angiogenic activity. Therefore, IGFBP7 is a selective and abundantly expressed biomarker of human GBM vessels that could be exploited for selective targeting of tumour vessels for imaging and therapeutic applications.

To this note, Canadian scientists from the NRC led by Dr. Abedelnasser Abulrob, a professor in the department of Cellular and molecular medicine at University of Ottawa developed a novel anti-IGFBP7 single-domain antibody (sdAb) that specifically designed to detect tumour vessels in brain cancer. The study (Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies) is published in British Journal of Cancer.

The sdAbs are small (13–15 kD) targeting molecules derived from the variable regions of heavy-chain antibodies from the camelid species. In contrast to IgG antibodies, sdAbs have fast pharmacokinetics due to small size, low nanomolar affinities when isolated from an immune library and can be easily engineered into a variety of antibody constructs.

The researchers in the study demonstrated that the anti-IGFBP7 sdAb developed bind specifically to both human and mouse GBM vessels in brain tissue sections and to tumour vessels after systemic injection in a mouse model of orthotopic glioblastoma. They also showed, anti-IGFBP7 sdAb engineered to conjugate to PEGylated superparamagnetic nanoparticles (NPs) (T2-reducing MRI contrast agents) functionalised with the near-infrared probe Cy5.5 for optical detection, to the vessels of orthotopic brain tumour in mice, created high tumour-contralateral side signal-to-noise ratio and selective tumour accumulation compared with other organs In prospective in vivo optical imaging. The study suggests that anti-IGFBP7 sdAb can be used to target appropriate contrast agents to abnormal tumour vasculature for non-invasive assessment of brain tumour angiogenesis using various imaging modalities, explained Dr. Abulrob.

The research goal was to generate novel targeting moieties against brain tumour vasculature useful in brain tumour imaging. IGFBP7 was confirmed in their study as a selective vascular target that is overexpressed in both human GBM and in an orthotopic mouse model of GBM. The anti-IGFBP7 sdAb was then raised and assessed for use as molecular imaging agent/diagnostic for GBM. The sdAb format was chosen due to its small size (15 kDa), fast clearance kinetics and moderate affinity for its targets, characteristics advantageous in molecular imaging applications. After panning an immunised library for binders, an sdAb with moderate affinity (Kd in low nanomolar range) for the IGFBP7 target was selected. In contrast to control sdAb, anti- IGFBP7 sdAb fluorescently tagged for detection was found to bind specifically to both human and mouse GBM tumour vessels ex vivo and to brain tumour vessels in mice after injection in vivo, creating high signal-to-noise ratio in orthotopic brain tumours easily detectable by in vivo optical imaging. In addition, we show that the anti-IGFBP7 sdAb could be utilised to target bimodal optical-MRI contrast agent based on Fe3O4 PEGylated NPs labelled with Cy5.5. Using in vivo optical imaging, brain tumour selective targeting in mice was demonstrated for the anti-IGFBP7 sdAb-PEGylated-NPs- Cy5.5, in contrast to a non-targeted-PEGylated-NPs-Cy5.5. The results of the study suggest that anti-IGFBP7 sdAbs and anti- IGFBP7 sdAb-PEGylated-NPs are both promising molecular imaging agents for targeting brain tumour vessels with potential for clinical translation.

Other biomarkers such as VEGFR2, another target highly expressed on tumour endothelial cells and previously exploited for developing molecular imaging strategies for tumours. The targeting moiety, 64Cu-labelled vascular endothelial growth factor 121 (VEGF121) has been developed for non-invasive PET imaging of VEGFR expression in small animals. Similar to anti-IGFBP7 sdAb, VEGF121 has a low nanomolar affinity and fast clearance from the body due to its small size (25 kDa) . In pre-clinical PET studies, VEGFR expression was shown to be heterogeneous in GBM xenografts depending on the size of the tumour. Another well-studied marker of GBM tumour vessels explored for non-invasive imaging of tumour vessels is the avb3 integrin, against which several RGD containing peptide binders were developed. In a recent study in which [18F]galacto-RGD peptide was used for PET imaging assessment of avb3 integrin expression in patients with GBM, it was demonstrated that GBMs had very heterogeneous tracer uptake, which, however, correlated well with immunohistochemically determined avb3 expression. “In contrast to IGFBP7, avb3 integrin expression is also associated with nontumour angiogenic vessels and tumour cells” said Dr. Abulrob, he then added “Anti-IGFBP7 sdAb have advantages compared with other predominantly peptide-based strategies, including high specificity to the target, while retaining appropriate pharmacokinetic characteristic for imaging applications, including PET; further studies comparing anti-IGFBP7 sdAb with other brain tumour-vessel targeting molecular imaging agents in PET studies are warranted. In addition to GBMs, high vascular expression of IGFBP7 has been shown in some peripheral tumours, including oesophagus, lung and stomach cancers”.

“An added benefit of the anti-IGFBP7 sdAb-targeted bimodal NPs developed in this study is the possibility of using MRI to non-invasively assess tumour angiogenesis. MRI is a widely available modality with high spatial resolution for in vivo imaging” notes Dr. Abulrob. In the study, anti-IGFBP7 sdAb PEGylated NPs displayed excellent and specific targeting to brain tumours compared with non-targeted PEGylated NPs. The anti-IGFBP7 sdAb-targeted PEGylated NPs also displayed a greater level of targeting compared with the anti- IGFBP7 sdAb alone.

For clinical use, a targeted NP that can achieve a high signal to background ratio, followed by complete elimination from the body would be ideal. Despite many therapeutic advances in the field, most patients with GBM still have poor prognosis, with a life expectancy of 12–15 months. In contrast, low-grade gliomas (WHO grade II) show little or no neo-vascularisation and much better prognosis, with a median survival ranging from 4–16.7 years depending on the age and histology of the patient. However, malignant transformation is always a risk for low-grade tumours, such that 13–86% of tumours initially diagnosed as low grade recur at a higher histological grade. The malignancy of the tumour is highly correlated to the degree of angiogenesis and the progression from low to higher tumour grades is commonly referred to as an ‘angiogenic switch’. It is reasonable to argue that treatments should be applied early, either when the angiogenic switch occurs or to prevent the angiogenic switch from happening in the first place. To achieve information on such a switch, however, there is a need for accurate markers of tumour angiogenesis. As lower-grade glioma tissue sections do not express IGFBP7, anti- IGFBP7 sdAb may feasibly represent a useful follow-up molecular imaging tool in determining when the ‘angiogenic switch’ has been turned on for low-grade gliomas. By providing information on the degree of tumour angiogenesis and related clinical aggressiveness, the anti-IGFBP7 sdAb could also be used to assess/monitor efficacy of anti-angiogenic or other anti-tumour treatments, and thus improve the clinical management of brain tumours.

The high expression and accessible nature of the IGFBP7 target in GBM vessels, combined with virtually non-existent expression in normal vessels, in combination with demonstrated versatility and good in vivo targeting characteristics of anti-IGFBP7 sdAb, indicate that linking of this antibody to the appropriate optical, PET or MRI contrast agents could enable in vivo imaging assessment of the degree of angiogenesis in gliomas.

According to Dr. Abulrob “The benefit of using a molecular imaging agent is that it helps neurosurgeons see more accurately where the high-grade cancer is within the brain, in real time. In treating cancer, we are trying to improve survival by tailoring treatments to each individual patient. This technique provides on-the-spot information to help surgeons tailor the operation according to the location, size and grade of the tumour. We know that patients who have near total removal of their tumour have better outcomes, so we are optimistic that, in the long term, these new data will help to increase survival times for glioma patients.” According to the authors their study offer intriguing possibilities for exploring nanoparticle ‘targeting’ of the tumor vessel microenvironment. They also demonstrate exciting new potential to develop targeted nanoparticles as platforms for early cancer detection and therapy.