The 5th Industrial Revolution: A strategic partnership of combating Glioblastoma with fusion nanotec

知己知彼，百戰不殆 (Sun Tzu , The Art of War)

“If you know both yourself and your enemy, you can win numerous battles without jeopardy”

It has been so long, we were in the battle zone with the brain tumor. Glioblastoma (GBM) is the most common primary brain tumor, highly proliferative and invasive, characterized by remarkable biological heterogeneity and poor response to present treatments. Even when treated aggressively, GBM has the tendency to recur, and this is the main cause of the poor prognosis. The median overall survival of GBM patients with the best currently available therapy is approximately 15 months, and the 5-year survival rate is only 9.8% at present. Right now, the standard treatment for glioblastoma is a combination of surgery, radiation, and chemotherapy. Previously, there are few options when those fail. However, we believe with an as-yet-to-be-determined combination of advanced technology, new bio-compatibility nanomaterials, and various biomolecules, we are cautiously optimistic for the future. Brain Cancer researcher may finally make some headway wins this long intractable battle. The smart ideas for combating brain tumors once considered fantasy are now becoming reality.

The Great Spy: Improving early stage cancer diagnostics with biosensors

Biosensor technology is fuelling an explosion in the amount of cancer prognosis and diagnostics. Since the development of the first oxygen biosensor by Led and Clark in 1962, biosensors have gained enormous attention in recent years in medicine and nanotechnology. Nowadays, biosensors are an emerging field with the potential to revolutionize our understanding of the basis of brain cancer and the treatment of this disease. Just like a spy, this technology aims to get information as early and as much as possible about the capability and potency of our “enemy”. The better we know them, the more chances to design a precise strategy against them.

If a person has a brain tumor in their body, those cells will produce proteins that cancer cells can only produce, known as biomarkers. These proteins circulate through the bloodstream, and sometimes are very low. For brain tumor patients, some of the most promising biomarkers till date include the loss of chromosomes 1p/19q in Oligodendrogliomas and expression of O-6-methylguanine-DNA methyltransferase or epidermal growth factor receptor (EGFR). Other promising biomarkers in glioma research include Glial Fibrillary Acidic Protein, Galectins, Kir potassium channel proteins, angiogenesis, and apoptosis pathway markers. The biosensor is designed to have bio-recognition elements which can detect the biomarker. The interaction between them produces some signal.

Figure 1. (a) Diagram of Biosensor component (b) BRET based on suppressor PTEN conformational changes (c) In vivo pHe mapping in brain tumors

Fernandes, et al. (1) developed an Intramolecular Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which is capable of detecting signal-dependent tumor suppressor PTEN conformational changes in living cells. Similarly to BRET, biosensors based on the principle of FRET also have been developed to visualize the activities of the signaling molecules in glioblastoma. (2) Instead of biomarkers, the environment condition around the tumor could become one alternative for diagnostics and prognostics. For example, in GBM, extracellular pH (pHe) is more acidic than intracellular pH (pHi). Rapidly growing tumor cells possess elevated rates of glucose uptake but reduced rates of oxidative phosphorylation (i.e. Warburg effect), thereby causing acidity in the extracellular space. This acidic pHe is conducive to tumor cell propagation and builds resistance to therapy. In vivo mapping brain tumors utilize shifts of non-exchangeable protons from macrocyclic chelates complexed with paramagnetic thulium (Tm3+) ion. There are many more bio-sensing techniques for early-stage diagnostic. Early detection is difficult, yet new biosensors could change that.

The Magic Bullets: The cure is inside our body itself

Immunotherapy has been long eulogised as the "Magic Bullet" for cancer treatments. However, when it is about the brain, immune-based treatments face some substantial obstacles before they can even reach a tumour. The most significant challenge is the Blood-Brain Barrier (BBB), which keeps the brain and its associated fluid tightly isolated from the circulatory system. This barrier protects the brain from viruses or toxins that may be circulating in the bloodstream but it can also impede the delivery cancer treatments. However, some study has proven that immune system can fight to foreign threats in the brain and the immune cells from other parts of the body can also travel there. GBM express EGFR III which is not expressed by other health cells. These receptors attract researcher to create a peptide vaccine which contains a mutated form of EGFR III which is known as Rindopepimut.(3) Based on reports from Society of Neuro-Oncology annual meeting on November 2015, like for 2 years treatment, 25% of patients who receive this treatment are still alive, in comparison with patients who didn’t underwent this treatment. The good news is this vaccine already has completed the enrollment in a phase III trial testing. In addition to that, dendritic cell is also one of vaccine candidate for treating GBM. Dendritic cell engineered to target cells that express proteins, or antigens, induced by infection with Cytomegalovirus (CMV).

Figure 2. Genetically engineered T cell attacking the tumour

Another type of immune-based treatment is adoptive T cell therapy (T-cell), this T cell is the most radical for several new approaches that recruit the immune system to attack cancers. However there is a problem, it’s difficult to inject the T-cell from donors to the patient because they will recognize this patient cell as ‘non-self’ and start firing off at everything, and the patient will melt down. However, that risk is mostly eliminated when T cells which are harvested from donor’s blood are stripped down with gene editing like by deleting the receptor that T cells normally use to sniff out foreign-looking molecules and adding new DNA (fig. 2). “Bi-specific” Chimeric Antigen receptor (CAR -T cells, relies on T cells collected from the patient that have a strong affinity for CMV-infected cells. And, because approximately 80 percent of glioblastoma cells overexpress a receptor that binds to the HER2 protein, the T cells are also engineered to express a receptor that binds to HER2 (fig. 3). In GBM, sometime the checkpoint inhibitors interfere signals from tumor cells to T cells that, in effect, directly the T cells to stand down. So to overcome this problem, two checkpoint inhibitors, ipilimumab (Yervoy®) and nivolumab (Opdivo®), which targets the CTLA-4 and PD-1 checkpoint protein on T cells, respectively has been developed and come into early-phase trial tests.

Figure 3. Ipilimumab (a)8 and Nivolumb (b) works by binding to and blocking a ‘checkpoint’ protein called CTLA-4 and PD-1 respectively which functions as a brake on the immune system by preventing T-cell activation. When ipilimumab and Nivolumb releases the brake, T cells are free to attack cancer cells.

The Canal Guard: Conquering the CTC

Hemodialysis is widely known for one kidney failure patient’s treatment. Recently, with the help of microfluidic system, many researchers think that hemodialysis concept can be adopted for GBM treatments. Similar to the normal hemodialysis which purpose to remove harmful object in blood, this hemodialysis also captures enemies which escaped and expanded their territory through blood stream, called circulating tumor cells (CTCs). CTCs, was discovered by pathologist Thomas Ashworth in 1869. He noticed that some unusual cells in the blood of a patient who had died of cancer were similar in appearance to those found in the numerous solid tumors present all over the patient’s body. CTCs was shed from the primary tumor, intravasate (entered the bloodstream), translocate to distant tissues, extravasate, adapt to the new microenvironment, and eventually seed, proliferate, and colonize to form metastases (fig. 4). Metastases in vital distant organs are triggering a mechanism that is responsible for the vast majority of cancer-related deaths. Cancer patients have only between 5 and 50 CTCs per teaspoon of blood, so their presence is dwarfed by blood cells.

Figure 4. Schematic view of the metastatic ( CTCs journey) process in blood stream

There are several strategies for capturing CTCs such as size based separation,(4) passive capturing,(5) nano Velcro,(6) etc (fig. 5). Size based separation technique, a microfluidic chip with a spiral channel or micro cavity channel isolates CTCs (roughly 10-20 μm in diameter ) from blood based on their size which tends to be larger than blood cells (approximately 7–12 μm in diameter). However, they have are some limitations such as the small sized tumor can escape from the channel and falsely capture white blood cells. Passive capturing; basically it isolates CTCs indirectly by depleting a blood sample of its other components. In this method, the blood sample is mixed with antibody-coated magnetic microbeads that bind a protein on the surface of white blood cells. Then, the blood sample is pumped into a microfluidic device which equipped with a magnet. Nano Velcro based separation; using capture agent (antibody/aptamer)-coated nanostructured substrates like nanowire, nanopillar, nanofiber, etc. to capture CTCs in a stationary device setting. This technique is enhancing the affinity for CTC capturing. Capturing the CTC is not only for cleaning the blood but also CTC itself is very useful in order to understand of the metastases process, and to search for the advance in the medical approach to cancer.

Figure 5. Various noble nanomaterials for capturing CTCs

The specific mass destroyer machine: Better brain cancer treatments delivery

The soldiers of our immune system fight viral spies, cancerous saboteurs, bacterial terrorists, and parasitic war machines all the time. However, when our soldiers are weak than the cancerous saboteurs in glioblastoma, allowing us to surrender. Modern science allows a good bit of outside interferences. For example, we ship cancer drugs weapons, high-tech surveillance equipment, and the occasional blast of radioactive power to our body. However, these weapons lack efficiency and are insufficient. It’s just like if we drop a bomb on a city block and wipe out enemy insurgents, unknowingly we will also kill innocent civilians and destroy substance infrastructure. We damage the particular thing we aim to protect. Because of this reasons, we need something which is capable of making deadly accurate strikes at a cellular level, a military drone to patrol our inner body.

In the nanotech era, scientists try to solve these problems by creating similar type of DNA origami “DNA robots” which are a useful and promising nano-vehicle for nanomedicine in the future. The DNA nanorobots are machines which can transport “molecular weapon” such as drugs or other molecular payloads to cells, sense signals from cell surfaces for trigger activation, and reconfigure their structure for payload delivery. The DNA nanorobots are built by using DNA origami. The technique involves the folding of long single strands of DNA and DNA templates combined with hundreds of short “staple” DNA. When the long and short DNA strands are mixed and incubated, they will self-assembly to form a shape which is deserved by the designer. The shape of the DNA robot is like an open barrel/clamps shells. It contains 12 sites on the inner side for attaching payload molecules and has two linkers on the outer side for attaching aptamers, short nucleotide strands with special sequences, for recognizing protein signals on the target cell (fig.6b). Therefore, the DNA robots recognize very specific cells and deliver the payload of molecular. Other approaches are by using biocompatible nanoparticle conjugated to chlorotoxin, camptothecin or other drugs, a peptide reported to bind selectively to glioma cells. The nanoparticles allow detection and quantification of the cellular uptake by assistant light or magnet. ![endif]--

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Figure 6. Schema of drug delivery using a. biocompatible nanoparticle b. DNA nanorobot

GBM are very tricky enemies; some patients with glioblastoma produce a certain protein in their tumors that make this tumour uniquely resistant to chemotherapy. To unlock the tumor cell to that chemotherapy, benzylguanine had been used. However just like a bomb, this drug is very harmful to blood cells. Kiem and his research colleagues10 developed a strategy to protect the blood cells from those side effects by engineering the cells in the lab with a gene that shields them to benzylguanine, and then transplanting the cells back into the patient before proceeding with chemotherapy cycles. Because the patients can now receive benzylguanine safely, they can be treated with lower doses of chemotherapy than the standard treatment dose, meaning the treatment is less toxic overall while still attacking the cancer.

In the end, with those all current strategy, there are many reasons to be hopeful about the future of cancer care and research. As Joan Massagué, Director of the Sloan Kettering Institute and a prominent metastasis researcher, said that “Mankind is turning cancer from what we’ve known it to be — the way we’ve related to it in the 20th century as an impossible, obscure disease — into a ‘normalized’ disease. Our relationship with it will be much more like the one we have with infectious diseases, for which we have antibiotics and other treatments.”

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