Potential Prostate Cancer Marker Discovered

Images from the desorption electrospray ionization mass spectrometry analysis of prostate tissue samples are shown next to stained slides of the same samples. The images show that cholesterol sulfate is present in cancerous tissue and precancerous legions called high grade prostatic intraepithelial neoplasia, or PIN. A Purdue University-led research team discovered that cholesterol sulfate is a potential marker for prostate cancer. (Credit: Demian Ifa/Purdue Center for Analytical Instrumentation Development)

Studies by a Purdue University-led team have revealed a potential marker for prostate cancer that could be the starting point for less invasive testing and improved diagnosis of the disease.

The team used a new analysis technique to create a profile of the lipids, or fats, found in prostate tissue and discovered a molecular compound that appears to be useful in identifying cancerous and precancerous tissue. The profile revealed that cholesterol sulfate is a compound that is absent in healthy prostate tissue, but is a major fat found in prostate cancer tumors.
Graham Cooks, Purdue's Henry Bohn Hass Distinguished Professor of Chemistry, and Timothy Ratliff, the Robert Wallace Miller Director of the Purdue Center for Cancer Research, led the team.
"It was surprising to find a single compound that is distinctly present in cancerous tissue and not present in healthy tissue," said Cooks, who is co-director of Purdue's Center for Analytical Instrumentation Development. "We've been able to differentiate cancerous from healthy tissue using this new method in the past, but the difference was in the amounts of the same chemical compounds found in healthy tissue. There was no single differentiator of which one could say if it was present there was cancerous tissue."
Ratliff said this characteristic makes the compound a potential marker for the disease, which could lead to new blood or urine tests to screen for prostate cancer.
"Aside from skin cancer, prostate cancer is the most common cancer in men and is the second leading cause of cancer-related deaths," Ratliff said. "Unfortunately, the current screening test has a significant number of false positives because it uses a marker that is present with other non-cancerous conditions. As a result, many men have unnecessary biopsies, which are invasive, expensive and have the potential to cause infection. This new compound appears to be highly specific to prostate cancer cells, which would mean very few false positives."
The current prostate cancer test screens for a protein called prostate-specific antigen, or PSA, that is produced by the cells of the prostate. Elevated levels of PSA in the blood can signify prostate cancer, but non-cancerous conditions such as an enlarged or inflamed prostate also cause an increase in its levels, he said.
The findings of the study, which was funded by the Purdue University Center for Cancer Research and the National Institutes of Health, were published in the journal Analytical Chemistry.
The study was performed in collaboration with physician scientists from Indiana University School of Medicine, who co-authored the paper. They also provided the tissue samples and pathological analysis of the samples to check the new technique's results.
The team used a mass spectrometry analysis technique developed by Cooks and coworkers called desorption electrospray ionization, or DESI, to measure and compare the chemical characteristics of 68 samples of normal and cancerous prostate tissue.
Mass spectrometry works by first turning molecules into ions, or electrically charged versions of themselves, so that they can be identified by their mass. Conventional mass spectrometry requires chemical separations, manipulations of samples and containment in a vacuum chamber for ionization and analysis. The DESI technique eliminates these requirements by performing the ionization step in the air or directly on surfaces outside of the mass spectrometers, making the process much simpler, faster and more applicable to medical examination or surgical settings.
Cooks' research team also has developed software that turns the distribution and intensity of selected ions within a sample into a computer-generated image, much like what would be seen from a stained slide under the microscope. This chemical map of the sample can precisely show the location of cancerous tissue and the borders of tumors, Cooks said.
Livia Eberlin, co-author of the paper and a graduate student in Cooks' group, said the study showed promise in detecting precancerous lesions, as well.
"The DESI examination was able to distinguish a precancerous lesion in a small area of a sample made up of mostly healthy tissue," Eberlin said. "By evaluating the difference in the chemistry of cells, this technique can detect differences in diseased tissue that are otherwise indistinguishable. It could provide a new tool for pathologists to complement microscopic examination."
The team also plans to study differences in the chemistry of different types of prostate cancer tumors to see if there is a way to identify which are aggressive and which are not, she said.
Ratliff said the inability to tell the difference between aggressive and nonaggressive forms of prostate cancer causes problems in its treatment.
"A nonaggressive form of prostate cancer can be very slow to progress, and sometimes it is in the best interest of the patient not to go through rigorous treatments that reduce one's quality of life," he said. "The tests currently used to determine the probability that the cancer is an aggressive form are not very accurate, and about 30 percent of patients are misdiagnosed as having an aggressive form."
Additional co-authors of the paper include graduate students Allison Dill and Anthony Costa, and post doctoral researcher Demian Ifa from Purdue's Department of Chemistry and the Center for Analytical Instrumentation Development; Dr. Liang Cheng from the Indiana University School of Medicine Department of Pathology and Laboratory Medicine; and Dr. Timothy Masterson and Dr. Michael Koch from the Indiana University School of Medicine Department of Urology.
The team is already in the process of performing larger studies and plans to investigate the biological processes responsible for the expression of cholesterol sulfate in cancerous tissue.

Of Bugs and Brains: Gut Bacteria Affect Multiple Sclerosis

In the absence of bacteria in the intestines, pro-inflammatory Th17 cells do not develop in either the gut or the central nervous system; and animals do not develop disease (top panel). When animals are colonized with symbiotic segmented filamentous bacteria, Th17 cell differentiation is induced in the gut. Th17 cells promote experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. In this way, non-pathogenic bacteria of the microbiota promote disease by shaping the immune response in both the gut and the brain

Biologists at the California Institute of Technology (Caltech) have demonstrated a connection between multiple sclerosis (MS) -- an autoimmune disorder that affects the brain and spinal cord -- and gut bacteria.
The work -- led by Sarkis K. Mazmanian, an assistant professor of biology at Caltech, and postdoctoral scholar Yun Kyung Lee -- appears online the week of July 19-23 in the Proceedings of the National Academy of Sciences.
Multiple sclerosis results from the progressive deterioration of the protective fatty myelin sheath surrounding nerve cells. The loss of myelin hinders nerve cells from communicating with one another, leading to a host of neurological symptoms including loss of sensation, muscle spasms and weakness, fatigue, and pain. Multiple sclerosis is estimated to affect about half a million people in the United States alone, with rates of diagnosis rapidly increasing. There is currently no cure for MS.
Although the cause of MS is unknown, microorganisms seem to play some sort of role. "In the literature from clinical studies, there are papers showing that microbes affect MS," Mazmanian says. "For example, the disease gets worse after viral infections, and bacterial infections cause an increase in MS symptoms."
On the other hand, he concedes, "it seems counterintuitive that a microbe would be involved in a disease of the central nervous system, because these are sterile tissues."
And yet, as Mazmanian found when he began examining the multiple sclerosis literature, the suggestion of a link between bacteria and the disease is more than anecdotal. Notably, back in 1993, Caltech biochemist Leroy Hood -- who was then at the University of Washington -- published a paper describing a genetically engineered strain of mouse that developed a lab-induced form of multiple sclerosis known as experimental autoimmune encephalomyelitis, or EAE.
When Hood's animals were housed at Caltech, they developed the disease. But, oddly, when the mice were shipped to a cleaner biotech facility -- where their resident gut bacterial populations were reduced -- they didn't get sick. The question was, why? At the time, Mazmanian says, "the authors speculated that some environmental component was modulating MS in these animals." Just what that environmental component was, however, remained a mystery for almost two decades.
But Mazmanian -- whose laboratory examines the relationships between gut microbes, both harmful and helpful, and the immune systems of their mammalian hosts -- had a hunch that intestinal bacteria were the key. "As we gained an appreciation for how profoundly the gut microbiota can affect the immune system, we decided to ask if symbiotic bacteria are the missing variable in these mice with MS," he says.
To find out, Mazmanian and his colleagues tried to induce MS in animals that were completely devoid of the microbes that normally inhabit the digestive system. "Lo and behold, these sterile animals did not get sick," he says.
Then the researchers decided to see what would happen if bacteria were reintroduced to the germ-free mice. But not just any bacteria. They inoculated mice with one specific organism, an unculturable bug from a group known as segmented filamentous bacteria. In prior studies, these bacteria had been shown to lead to intestinal inflammation and, more intriguingly, to induce in the gut the appearance of a particular immune-system cell known as Th17. Th17 cells are a type of T helper cell -- cells that help activate and direct other immune system cells. Furthermore, Th17 cells induce the inflammatory cascade that leads to multiple sclerosis in animals.
"The question was, if this organism is inducing Th17 cells in the gut, will it be able to do so in the brain and central nervous system?" Mazmanian says. "Furthermore, with that one organism, can we restore to sterile animals the entire inflammatory response normally seen in animals with hundreds of species of gut bacteria?"
The answer? Yes on all counts. Giving the formerly germ-free mice a dose of one species of segmented filamentous bacteria induced Th17 not only in the gut but in the central nervous system and brain -- and caused the formerly healthy mice to become ill with MS-like symptoms.
"It definitely shows that gut microbes have a strong role in MS, because the genetics of the animals were the same. In fact, everything was the same except for the presence of those otherwise benign bacteria, which are clearly playing a role in shaping the immune system," Mazmanian says. "This study shows for the first time that specific intestinal bacteria have a significant role in affecting the nervous system during MS -- and they do so from the gut, an anatomical location very, very far from the brain."
Mazmanian and his colleagues don't, however, suggest that gut bacteria are the direct cause of multiple sclerosis, which is known to be genetically linked. Rather, the bacteria may be helping to shape the immune system's inflammatory response, thus creating conditions that could allow the disease to develop. Indeed, multiple sclerosis also has a strong environmental component; identical twins, who possess the same genome and share all of their genes, only have a 25 percent chance of sharing the disease. "We would like to suggest that gut bacteria may be the missing environmental component," he says.
For their part, Th17 cells are needed for the immune system to properly combat infection. Problems only arise when the cells are activated in the absence of infection -- just as disease can arise, Mazmanian and others suspect, when the species composition of gut bacteria become imbalanced, say, by changes in diet, because of improved hygiene (which kills off the beneficial bacteria as well as the dangerous ones), or because of stress or antibiotic use. One impact of the dysregulation of normal gut bacterial populations -- a phenomenon dubbed "dysbiosis" -- may be the rising rate of multiple sclerosis seen in recent years in more hygienic societies.
"As we live cleaner, we're not just changing our exposure to infectious agents, but we're changing our relationship with the entire microbial world, both around and inside us, and we may be altering the balance between pro- and anti-inflammatory bacteria," leading to diseases like MS, Mazmanian says. "Perhaps treatments for diseases such as multiple sclerosis may someday include probiotic bacteria that can restore normal immune function in the gut… and the brain."
The work was supported by funding from the California Institute of Technology, the Weston Havens Foundation, and the Edward Mallinckrodt, Jr. Foundation.

Nanoparticles Shrink Tumors in Mice

The application of nanotechnology in the field of drug delivery has attracted much attention in recent years. In cancer research, nanotechnology holds great promise for the development of targeted, localized delivery of anticancer drugs, in which only cancer cells are affected.

A dorsal view of a mouse showing accumulation of nanoparticles in a tumor four hours after intravenous administration. Bright fluorescence is observed predominantly in the tumor.

Such targeted-therapy methods would represent a major advance over current chemotherapy, in which anticancer drugs are distributed throughout the body, attacking healthy cells along with cancer cells and causing a number of adverse side effects.
By carrying out comprehensive studies on mice with human tumors, UCLA scientists have obtained results that move the research one step closer to this goal. In a paper published July 8 in the journal Small, researchers at UCLA's California NanoSystems Institute and Jonsson Comprehensive Cancer Center demonstrate that mesoporous silica nanoparticles (MSNs), tiny particles with thousands of pores, can store and deliver chemotherapeutic drugs in vivo and effectively suppress tumors in mice.
The researchers also showed that MSNs accumulate almost exclusively in tumors after administration and that the nanoparticles are excreted from the body after they have delivered their chemotherapeutic drugs.
The study was conducted jointly in the laboratories of Fuyu Tamanoi, a UCLA professor of microbiology, immunology and molecular genetics and director of the signal transduction and therapeutics program at UCLA's Jonsson Comprehensive Cancer Center, and Jeffrey Zink, a UCLA professor of chemistry and biochemistry. Tamanoi and Zink are researchers at the California NanoSystems Institute (CNSI) and are two of the co-directors of the CNSI's Nano Machine Center for Targeted Delivery and On-Demand Release. The lead investigator on the research is Jie Lu, a postdoctoral fellow in Tamanoi's lab. Monty Liong and Zongxi Li, researchers from Zink's lab, also contributed to this work.
In the study, researchers found that MSNs circulate in the bloodstream for extended periods of time and accumulate predominantly in tumors. The tumor accumulation could be further improved by attaching a targeting moiety to MSNs, the researchers said.
The treatment of mice with camptothecin-loaded MSNs led to shrinkage and regression of xenograft tumors. By the end of the treatment, the mice were essentially tumor free, and acute and long-term toxicity of MSNs to the mice was negligible. Mice with breast cancer were used in this study, but the researchers have recently obtained similar results using mice with human pancreatic cancer.
"Our present study shows, for the first time, that MSNs are effective for anticancer drug delivery and that the capacity for tumor suppression is significant," Tamanoi said.
"Two properties of these nanoparticles are important," Lu said. "First, their ability to accumulate in tumors is excellent. They appear to evade the surveillance mechanism that normally removes materials foreign to the body. Second, most of the nanoparticles that were injected into the mice were excreted out through urine and feces within four days. The latter results are quite interesting and might explain the low toxicity observed in the biocompatabilty experiments we conducted."
Researchers at the Nano Machine Center for Targeted Delivery and On-Demand Release are modifying MSNs -- which are easily modifiable -- so that the nanoparticles can be equipped with nanomachines. For example, nanovalves are being attached at the opening of the pores to control the release of anticancer drugs. In addition, the interior of the pores is being modified so that the light-induced release of anticancer drugs can be achieved.
"We can modify both the particles themselves and also the attachments on the particles in a wide variety of ways, which makes this material particularly attractive for engineering drug-delivery vehicles," Zink said.
The team is now planning future research that involves testing MSNs in a variety of animal-model systems and carrying out extensive studies on the safety of MSNs.
"Comprehensive investigation with practical dosages which are adequate and suitable for in vivo delivery of anticancer drugs is needed before MSNs can reach clinics as a drug-delivery system," Tamanoi said.
The research received support from National Institutes of Health and the National Science Foundation. In addition, NanoPacific Holdings Inc. provided critical support for the animal experiments.