Brain cancer: Hunger for amino acids makes it more aggressive

To fuel phases of fast and aggressive growth, tumors need higher-than-normal amounts of energy and the molecular building blocks needed to build new cellular components.

Cancer cells therefore consume a lot of sugar (glucose A number of tumors are also able to catabolize the amino acid glutamine, an important building block of proteins. A key enzyme in amino acid decomposition is isocitrate dehydrogenase (IDH). Several years ago, scientists discovered mutations in the gene coding for IDH in numerous types of brain cancer. Very malignant brain tumors called primary glioblastomas carry an intact IDH gene, whereas those that grow more slowly usually have a defective form.

“The study of the IDH gene currently is one of the most important diagnostic criteria for differentiating glioblastomas from other brain cancers that grow more slowly,” says Dr. Bernhard Radlwimmer from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). “We wanted to find out what spurs the aggressive growth of glioblastomas.” In collaboration with scientists from other institutes including Heidelberg University Hospital, Dr. Martje Tönjes and Dr. Sebastian Barbus from Radlwimmer’s team compared gene activity profiles from several hundred brain tumors. They aimed to find out whether either altered or intact IDH show further, specific genetic characteristics that might help explain the aggressiveness of the disease.
The researchers found a significant difference between the two groups in the highly increased activity of the gene for the BCAT1 enzyme, which in normal brain tissue is responsible for breaking down so-called branched-chain amino acids. However, Radlwimmer’s team discovered, only those tumor cells whose IDH gene is not mutated produce BCAT1. “This is not surprising, because as IDH breaks down amino acids, it produces ketoglutarate – a molecule which BCAT1 needs. This explains why BCAT1 is produced only in tumor cells carrying intact IDH. The two enzymes seem to form a kind of functional unit in amino acid catabolism,” says Bernhard Radlwimmer.
Glioblastomas are particularly dreaded because they aggressively invade the healthy brain tissue that surrounds them. When the researchers used a pharmacological substance to block BCAT1′s effects, the tumor cells lost their invasive capacity. In addition, the cells released less of the glutamate neurotransmitter. High glutamate release is responsible for severe neurological symptoms such as epileptic seizures, which are frequently associated with the disease. When transferred to mice, glioblastoma cells in which the BCAT1 gene had been blocked no longer grew into tumors.
“Altogether, we can see that overexpression of BCAT1 contributes to the aggressiveness of glioblastoma cells,” Radlwimmer says. The study suggests that the two enzymes, BCAT1 and IDH, cooperate in the decomposition of branched-chain amino acids. These protein building blocks appear to act as a “food source” that increases the cancer cells’ aggressiveness. Branched-chain amino acids also play a significant role in metabolic diseases such as diabetes. This is the first time that scientists have been able to show the role of these amino acids in the growth of malignant tumors.
“The good news,” sums up Radlwimmer, “is that we have found another target for therapies in BCAT1. In collaboration with Bayer Healthcare, we have already started searching for agents that might be specifically directed against this enzyme.” The researchers also plan to investigate whether BCAT1 expression may serve as an additional marker to diagnose the malignancy of brain cancer.
 

Printing artificial bone

Researchers working to design new materials that are durable, lightweight and environmentally sustainable are increasingly looking to natural composites, such as bone, for inspiration: Bone is strong and tough because its two constituent materials, soft collagen protein and stiff hydroxyapatite mineral, are arranged in complex hierarchical patterns that change at every scale of the composite, from the micro up to the macro.

While researchers have come up with hierarchical structures in the design of new materials, going from a computer model to the production of physical artifacts has been a persistent challenge. This is because the hierarchical structures that give natural composites their strength are self-assembled through electrochemical reactions, a process not easily replicated in the lab.
Now researchers at MIT have developed an approach that allows them to turn their designs into reality. In just a few hours, they can move directly from a multiscale computer model of a synthetic material to the creation of physical samples.
In a paper published online June 17 in Advanced Functional Materials, associate professor Markus Buehler of the Department of Civil and Environmental Engineering and co-authors describe their approach. Using computer-optimized designs of soft and stiff polymers placed in geometric patterns that replicate nature’s own patterns, and a 3-D printer that prints with two polymers at once, the team produced samples of synthetic materials that have fracture behavior similar to bone. One of the synthetics is 22 times more fracture-resistant than its strongest constituent material, a feat achieved by altering its hierarchical design.

Two are stronger than one
The collagen in bone is too soft and stretchy to serve as a structural material, and the mineral hydroxyapatite is brittle and prone to fracturing. Yet when the two combine, they form a remarkable composite capable of providing skeletal support for the human body. The hierarchical patterns help bone withstand fracturing by dissipating energy and distributing damage over a larger area, rather than letting the material fail at a single point.
“The geometric patterns we used in the synthetic materials are based on those seen in natural materials like bone or nacre, but also include new designs that do not exist in nature,” says Buehler, who has done extensive research on the molecular structure and fracture behavior of biomaterials. His co-authors are graduate students Leon Dimas and Graham Bratzel, and Ido Eylon of the 3-D printer manufacturer Stratasys. “As engineers we are no longer limited to the natural patterns. We can design our own, which may perform even better than the ones that already exist.”
The researchers created three synthetic composite materials, each of which is one-eighth inch thick and about 5-by-7 inches in size. The first sample simulates the mechanical properties of bone and nacre (also known as mother of pearl). This synthetic has a microscopic pattern that looks like a staggered brick-and-mortar wall: A soft black polymer works as the mortar, and a stiff blue polymer forms the bricks. Another composite simulates the mineral calcite, with an inverted brick-and-mortar pattern featuring soft bricks enclosed in stiff polymer cells. The third composite has a diamond pattern resembling snakeskin. This one was tailored specifically to improve upon one aspect of bone’s ability to shift and spread damage.
A step toward ‘metamaterials’
The team confirmed the accuracy of this approach by putting the samples through a series of tests to see if the new materials fracture in the same way as their computer-simulated counterparts. The samples passed the tests, validating the entire process and proving the efficacy and accuracy of the computer-optimized design. As predicted, the bonelike material proved to be the toughest overall.

“Most importantly, the experiments confirmed the computational prediction of the bonelike specimen exhibiting the largest fracture resistance,” says Dimas, who is the first author of the paper. “And we managed to manufacture a composite with a fracture resistance more than 20 times larger than its strongest constituent.”
According to Buehler, the process could be scaled up to provide a cost-effective means of manufacturing materials that consist of two or more constituents, arranged in patterns of any variation imaginable and tailored for specific functions in different parts of a structure. He hopes that eventually entire buildings might be printed with optimized materials that incorporate electrical circuits, plumbing and energy harvesting. “The possibilities seem endless, as we are just beginning to push the limits of the kind of geometric features and material combinations we can print,” Buehler says

Polymers key to oral protein-based drugs

For protein-based drugs such as insulin to be taken orally rather than injected, bioengineers need to find a way to shuttle them safely through the stomach to the small intestine where they can be absorbed and distributed by the bloodstream.

Progress has been slow, but in a new study, researchers report an important technological advance: They show that a “bioadhesive” coating significantly increased the intestinal uptake of polymer nanoparticles in rats and that the nanoparticles were delivered to tissues around the body in a way that could potentially be controlled.
“The results of these studies provide strong support for the use of bioadhesive polymers to enhance nano- and microparticle uptake from the small intestine for oral drug delivery,” wrote the researchers in the Journal of Controlled Release, led by corresponding author Edith Mathiowitz, professor of medical science at Brown University.
Mathiowitz, who teaches in Brown’s Department of Molecular Pharmacology, Physiology, and Biotechnology, has been working for more than a decade to develop bioadhesive coatings that can get nanoparticles to stick to the mucosal lining of the intestine so that they will be taken up into its epithelial cells and transferred into the bloodstream. The idea is that protein-based medicines would be carried in the nanoparticles.
In the new study, which appeared online June 21, Mathiowitz put one of her most promising coatings, a chemical called PBMAD, to the test both on the lab bench and in animal models. Mathiowitz and her colleagues have applied for a patent related to the work, which would be assigned to Brown University.
In prior experiments, Mathiowitz and her group have shown not only that PBMAD has bioadhesive properties, but also that it withstands the acidic environment of the stomach and then dissolves in the higher pH of the small intestine.
Adhere, absorb, arrive
The newly published results focused on the question of how many particles, whether coated with PBMAD or not, would be taken up by the intestine and distributed to tissues. For easier tracking throughout the body, Mathiowitz’s team purposely used experimental and control particles made of materials that the body would not break down. Because they were “non-erodible” the particles did not carry any medicine.
The researchers used particles about 500 nanometers in diameter made of two different materials: polystyrene, which adheres pretty well to the intestine’s mucosal lining, and another plastic called PMMA, that does not. They coated some of the PMMA particles in PBMAD, to see if the bioadhesive coating could get PMMA particles to stick more reliably to the intestine and then get absorbed.
First the team, including authors Joshua Reineke of Wayne State University and Daniel Cho of Brown, performed basic benchtop tests to see how well each kind of particles adhered. The PBMAD-coated particles proved to have the strongest stickiness to intestinal tissue, binding more than twice as strongly as the uncoated PMMA particles and about 1.5 times as strongly as the polystyrene particles.
The main experiment, however, involved injecting doses of the different particles into the intestines of rats to see whether they would be absorbed and where those that were taken up could be found five hours later. Some rats got a dose of the polystyrene particles, some got the uncoated PMMA and some got the PBMAD-coated PMMA particles.
Measurements showed that the rats absorbed 66.9 percent of the PBMAD-coated particles, 45.8 percent of the polystyrene particles and only 1.9 percent of the uncoated PMMA partcles.
Meanwhile, the different particles had very different distribution profiles around the body. More than 80 percent of the polystyrene particles that were absorbed went to the liver and another 10 percent went to the kidneys. The PMMA particles, coated or not, found their way to a much wider variety of tissues, although in different distributions. For example, the PBMAD-coated particles were much more likely to reach the heart, while the uncoated ones were much more likely to reach the brain.
Pharmaceutical potential
The apparent fact that the differing surface properties of the similarly sized particles had such distinct distributions in the rats’ tissues after the same five-hour period suggests that scientists could learn to tune particles to reach specific parts of the body, essentially targeting doses of medicines taken orally, Mathiowitz said.
“The distribution in the body can be somehow controlled with the type of polymer that you use,” she said.
For now, she and her group have been working hard to determine the biophysics of how the PBMAD-coated particles are taken up by the intestines. More work also needs to be done, for instance to demonstrate actual delivery of protein-based medicines in sufficient quantity to tissues where they are needed.
But Mathiowitz said the new results give her considerable confidence.
“What this means now is that if I coat bioerodible nanoparticles correctly, I can enhance their uptake,” she said. “Bioerodible nanoparticles are what we would ultimately like to use to deliver proteins. The question we address in this paper is how much can we deliver. The numbers we saw make the goal more feasible.”
Another frontier for the delivery of nanoparticles is devising a safe method to make nanoparticles, Mathiowitz said, but, ”we have already developed safe and reproducible methods to encapsulate proteins in tiny nanoparticles without compromising their biological activity.”
In addition to Reineke, Cho, and Mathiowitz, other authors on the paper are Yu-Ting Liu Dingle, Stacia Furtado, Bryan Laulicht, Danya Lavin, and Peter M. Cheifetz, all of Brown University during the research.