Stem Cells Lurk in Tumors and Can Resist Treatment

Regenerative medicine seeks to train stem cells to transform into nearly any kind of cell type. Unfortunately, this ability that makes stem cells so useful also is cause for concern in cancer treatments. Malignant tumors contain resident stem cells, which prompts worries among cancer experts that the cells’ transformative powers help cancers escape treatment.

Data from new research shows that the threat posed by cancer stem cells is more prevalent than previously thought. Until now, stem cells had been identified only in aggressive, fast-growing tumors. However, a mouse study at Washington University School of Medicine in St. Louis has revealed that slow-growing tumors also have treatment-resistant stem cells.

Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen
Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen

In mice, low-grade brain cancer stem cells were less sensitive to anticancer drugs. When compared to healthy stem cells, tumor-based stem cells from brain tumors, revealed the reasons behind their resistance to treatments, which points to new therapeutic strategies.

“At the very least, we’re going to have to use different drugs and different, likely higher dosages to make sure we kill these tumor stem cells,” said senior author David H. Gutmann, MD, PhD, the Donald O. Schnuck Family Professor of Neurology.  Their data were published in the March 12 edition of Cell Reports.

First author Yi-Hsien Chen, who is a senior postdoctoral research associate in Gutmann’s laboratory, used a mouse model of neurofibromatosis type 1 (NF1), which forms low-grade brain tumors, to identify cancer stem cells and demonstrate that they could form tumors when transplanted into normal, cancer-free mice.

Neurofibromatosis type I is caused by mutations in the NF1 genes, and such mutations affect about 1 in every 2,500 babies. Neurofibromatosis type I can cause an array of physical problems, including brain tumors, impaired vision, learning disabilities, behavioral problems, heart defects and bone deformities.

In children with NF1 mutations, the most common brain tumor is optic gliomas. Treatment for NF1-related optic gliomas usually includes drugs that inhibit a cell growth pathway originally identified by Gutmann. In laboratory tests conducted as part of the new research, it took 10 times the dosage of these drugs to kill the low-grade cancer stem cells.

Compared with healthy stem cells from the brain, cancer stem cells made multiple copies of a protein called Abcg1 that helps those cells survive stress.

“This protein blocks a signal from inside the cells that should make them more vulnerable to treatment,” Gutmann explained. “If we can identify a drug that disables this protein, it would make some cancer stem cells easier to kill.”

Even though these laboratory mice were bred to model NF1 optic gliomas, Gutmann and others said that their findings could be applied more broadly to other brain tumors.

“Because stem cells haven’t differentiated into specialized cells, they can easily activate genes to turn on new developmental programs that allow the cells to survive cancer treatments,” said Gutmann, who directs the Washington University Neurofibromatosis Center. “Based on these new findings, we will have to develop additional strategies to keep these tumors from evading our best treatments.”

Destroying Brain Tumors With Engineered Stem Cells

Khalid Shah from the Harvard Stem Cell Institute (HSCI) has designed a new way to genetically engineered stem cells to secrete tumor-killing toxins. In collaboration with colleagues from the HSCI and Massachusetts General Hospital, Shah and his team have shown that their toxin-secreting stem cells can be used to eradicate cancer cells in the brains of mice after the main tumor has been removed.

Shah and his coworkers used the toxin produced by a bacterium called “Pseudomonas.” This soil bacterium secretes a powerful exotoxin that blocks protein synthesis in cells. This Pseudomonas exotoxin or PE has the capability to kill tumor cells. Unfortunately, this powerful toxin will also kill any other cell it comes into contact with. Therefore, the administration of PE is a very delicate and precise enterprise. Therefore, Shah and his collaborators modified PE so that it bound to specific receptors that are found on the surfaces of particular brain tumors. This way. the toxin was only taken up by cells that expressed these particular receptors. Shah and others also engineered the stem cells to harbor a mutation in the gene that encodes the target for PE (EF-2) that renders this target protein resistant to the effects of PE (for those who are interested, a G‐to‐A transition in the first nucleotide of
codon 717 that is known to confer toxin resistance).

Next, Shah and others embedded the toxin-secreting stem cells into biodegradable hydrogels that were implanted at the site of the tumor. This way, the toxin-secreting cells are near the site of the tumor and only secrete the toxin where it is needed. Any toxin that escapes from this site undergoes rapid degradation, which prevents it from causing any systemic side effects or toxicities.

Shah and his colleagues are pursuing FDA approval to bring this and other stem cell-based technologies to clinical trials.

“A few years ago recognized hat stem cells could be used to continuously deliver these therapeutic toxins in the brain, but first we needed to genetically engineer the stem cells that could resist being killed themselves by the toxins,” said Shah.

Shah continued: “Cancer-killing toxins have been used with great success in a variety of blood cancers, but they didn’t work as well in solid tumors because the cancers aren’t as accessible and the toxins have a short half-life.”

“We tested these stem cells in a clinically relevant mouse model of brain cancer, where you resect the tumors [from patients] and implant the stem cells encapsulated in a gel into the resection cavity. After doing all of the molecular analysis and imaging to track the inhibition of protein synthesis within brain tumors, we do see the toxins kill the cancer cells and eventually prolong the survival in animal models of resected brain tumors.”

Shah next plans to combine his toxin-secreting stem cells with several different therapeutic stem cells developed by his team to further enhance their positive results in mouse models of glioblastoma, which is the most common brain tumor in human adults. Shah hopes that he will bring these therapies into clinical trials within the next five years.

Using Fat Stem Cells to Treat a Deadly Cancer

Johns Hopkins University researchers have reported the successful use of stem cells derived from human body fat to deliver biological treatments directly to the brains of mice suffering from the most common and aggressive form of brain tumor. Such treatments significantly extended the lives of these cancer-stricken animals.

These experiments offer proof-of-principle that such a technique would work in human patients after surgical removal of brain cancers called glioblastomas. This technique provides a way to find and destroy any remaining cancer cells in those areas of the brain that are difficult to reach. Glioblastoma cells represent a challenge for cancer treatments, since they are quite sprightly, and can migrate across the entire brain, hide out and establish new tumors. Consequently, the cure rates for glioblastoma are notoriously low.

In the mouse experiments conducted by the Johns Hopkins group, investigators used mesenchymal stromal cells (MSCs) from fat tissue. Fat-based MSCs have a mysterious ability to sniff out cancer and other damaged cells. After genetically modifying the MSCs so that they secreted a protein called bone morphogenetic protein 4 (BMP4), these MSCs were injected into the brains of mice that suffered from glioblastomas. BMP-4 is a small, secreted protein that plays essential regulatory roles in embryonic development, but also has a demonstrated tumor suppression function.

Study leader Alfredo Quinones-Hinojosa, M.D., a professor of neurosurgery, oncology and neuroscience at the Johns Hopkins University School of Medicine and his colleagues published the results of this experiment in the journal Clinical Cancer Research. According to their results, those mice that were treated with the BMP-4-secreting fat-based MSCs had significantly less tumor growth and spread. In general the cancers in these animals were less aggressive and had fewer migratory cancer cells compared to mice that didn’t get the treatment. Also, the stem cell-treated mice survived significantly longer (an average of 76 days, compared to 52 days in the untreated mice).

“These modified mesenchymal stem cells are like a Trojan horse, in that they successfully make it to the tumor without being detected and then release their therapeutic contents to attack the cancer cells.”

Standard treatments for glioblastoma include chemotherapy, radiation and surgery. Unfortunately, even a combination of all three rarely leads to more than 18 months of survival after diagnosis. Discovering new ways to seek and destroy straggling glioblastoma cells that other treatments can’t get is a long-sought goal, says Quinones-Hinojosa. However, he also cautions that years of additional studies are needed before human trials of fat-derived MSC therapies could begin.

Quinones-Hinojosa also treated brain cancer patients at Johns Hopkins Kimmel Cancer Center, and he and his co-workers were greatly encouraged that the genetically-engineered stem cells let loose into the brain in his experiments did not transform themselves into new tumors.

These latest findings build on research published in March 2013 by Quinones-Hinojosa and his team, which demonstrated that harvesting MSCs from fat was much less invasive and less expensive than getting them from bone marrow (PLoS One, March 2013).

Ideally, he says, if MSCs work as a cancer treatment, a patient with a glioblastoma would have some adipose tissue (fat) removed from any number of locations in the body a short time before surgery. Afterwards, these fat-derived MSCs would be isolated and manipulated in the laboratory so that they would secrete BMP4. Then, after surgeons removed whatever parts of the brain tumor they could get to, they would deposit these BMP-secreting cells into the brain in the hopes that they would seek out and destroy the left-over cancer cells.


Engineered Neural Stem Cells Deliver Anti-Cancer Drug to the Brain

Irinotecan is an anticancer drug that was approved for use in 1996. It is a modified version of the plant alkaloid camptothecin, and even though it shows significant activity against brain tumors in culture, but in a living body, this drug poorly penetrates the blood-brain barrier. Therefore irinotecan usually does not accumulate to appreciable levels in the brain and is typically not used to treat brain tumors.

That could change, however, if a new strategy published in paper by Marianne Metz and her colleagues from the laboratory of Karen Aboody at the Beckman Research Institute at the City of Hope in Duarte, California, in collaboration with colleagues from several other laboratories.

In this paper, Metz and her co-workers genetically engineered neural stem cells to express enzymes called “carboxylesterases.” These carboxyesterase enzymes convert irinotecan, which is an inactive metabolite, to the active form, which is known as “SN-38.” The efficient conversion of irinotecan to SN-38 in the brain greatly accelerates the therapeutic activity of this drug in the brain. Also, the constant conversion of irinotecan to another molecule accelerates the transport of irinotecan past the blood brain barrier.

To test this strategy. Metz and others grew the engineered neural stem cells in culture and measured their ability to make carboxylesterases in culture, and their ability to convert irinotecan into SN-38 in culture.  In both cases, the engineered neural stem cells made a boat-load of carboxylesterase and converted irinotecan into SN-38 in spades.  More importantly, the genetically engineered neural stem cells behaved exactly as they did before, which shows that the genetic manipulation of these cells did not change their properties.

Next, Metz others tested the ability of the engineered neural stem cells to kill human brain tumor cells in culture in the presence of irinotecan.  Once again, the genetically engineered neural stem cells effectively killed human brain tumor cells in culture in a irinotecan-concentration-dependent manner.  When these genetically engineered neural stem cells were injected into the brains of mice with brain tumors, intravenous administration of irinotecan produced high levels of SN-38 in the brain.  This shows that these cells have the capacity to increase the production of SN-38 in the brain.

This strategy is similar to other strategies that been used in various clinical trials, but because neural stem cells have a tendency to move into brain tumor tissue and surround it, they represent an efficient and effective way to deliver anticancer drugs to brain tumors.  Also, since the particular neural stem cell line used in this experiment (HB1.F3.CD) does not cause tumors and is also not recognized as foreign by the immune system, it is a particularly attractive stem cell line for such an anti-tumor strategy.

Stem Cell-Conventional Treatment Combo Offers New Hope in Fighting Deadly Brain Cancer

A new type of treatment that combines neural stem cells with conventional cancer fighting therapies shows promise in animal studies for the most common and deadliest form of adult brain cancer — glioblastoma multiforme (GBM). The details are revealed in a groundbreaking study led by Maciej Lesniak, M.D., that appeared in the journal STEM CELLS Translational Medicine.

“In this work, we describe a highly innovative gene therapy approach, which is being developed along with the NIH and the FDA. Specifically, our group has developed an allogeneic neural stem cell line that is a carrier for a virus that can selectively infect and break down cancer cells,” explained Dr. Lesniak, the University of Chicago’s director of neurosurgical oncology and neuro-oncology research at the Brain Tumor Center.

The stem cell line used is a neural stem cell line called HB1.F3 NSC. The US Food and Drug Administration has recently approved this cell line for use in a phase I human clinical trial.

Glioblastoma multiforme remains fatal despite intensive treatment with surgery, radiation and chemotherapy. Cancer-killing viruses have been used in clinical trials to treat those tumors that resist treatment with other therapies and infiltrate throughout the brain. Unfortunately, according to Lesniak, this therapy was subject to some “major drawbacks.”

“When you inject a virus into a tumor alone (without a carrier, like NSC), the virus stays at the site of the injection, and does not spread. Moreover, our immune system clears it. By using NSCs, we can achieve a widespread distribution of the virus throughout the tumor mass, since the NSC travel. Also, they act like a stealth fighter, hiding the virus from the immune system.” Lesniak and his co-workers used NSCs loaded with a novel oncolytic adenovirus. This virus selectively targets glioblastoma multiforme in combination with chemo-radiotherapy. Using this strategy, Lesniak’s team was able to overcome the limitations associated with anticancer viral therapies.

Using mice that had glioblastoma multiforme, the research team showed that their neural stem cell line, which is derived from human fetal cells, significantly increased the median survival time of the mice beyond conventional treatments alone. The addition of chemo-radiotherapy further enhanced the benefits of this novel stem cell-based gene therapy approach.

“Our study argues in favor of using stem cells for delivery of oncolytic viruses along with multimodal chemo-radiotherapy for the treatment of patients with glioblastoma multiforme, and this is something that we believe warrants further clinical investigation,” Dr. Lesniak concluded.

Lesniak’s team is completing final FDA-directed studies. He expects to start a human clinical trial, in which a novel oncolytic virus will be delivered via NSCs to patients with newly diagnosed glioblastoma multiforme, early in 2014.

Treatment of glioblastoma multiforme depends on novel therapies,” said Anthony Atala, M.D., Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “This study establishes that a combination of conventional and gene therapies may be most effective and suggests a protocol for a future clinical investigation.”

Making New Neurons When You Need Them

Western societies are aging societies, and the incidence of dementias, Alzheimer’s disease, and other diseases of the aged are on the rise. Treatments for these conditions are largely supportive, but being able to make new neurons to replace the ones that have died is almost certainly where it’s at.

At INSERM and CEA in Marseille, France, researchers have shown that chemicals that block the activity of a growth factor called TGF-beta improves the generation of new neurons in aged mice. These findings have spurred new investigations into compounds that can enable new neuron production in order to mitigate the symptoms of neurodegenerative diseases. Such treatments could also restore the cognitive abilities of those who have suffered neuron loss as a result of radiation therapy or a stroke.

The brain forms new neurons regularly to maintain our cognitive abilities, but aging or radiation therapy to treat tumors can greatly perturb this function. Radiation therapy is the adjunctive therapy of choice for brain tumors in children and adults.

Various studies suggest that the reduction in our cache of neurons contributes to cognitive decline. For example, exposure of mice to 15 Grays of radiation is accompanied by disruption to the olfactory memory and reduction in neuron production. A similar event occurs as a result of aging, but in human patients undergoing radiation treatment, cognitive decline is accelerated and seems to result from the death of neurons.

How then, can we preserve the cache of neurons in our brains? The first step is to determine the factors responsible for the decline is neuron production. In contrast to contemporary theory, neither heavy doses of radiation nor aging causes completely destruction of the neural stem cells that can replenish neurons. Even after doses of radiation and aging, neuron stem cell activity remains highly localized in the subventricular zone (a paired brain structure located in the outer walls of the lateral ventricles), but they do not work properly.

Subventricular Zone
Subventricular Zone

Experiments at the INSERM and CEA strongly suggest that in response to aging and high doses of radiation, the brain makes high levels of a signaling molecule called TGF-beta, and this signaling molecule pushes neural stem cell populations into dormancy. This dormancy also increases the susceptibility of neural stem cells into apoptosis.

Marc-Andre Mouthon, one of the main authors of this research, explained his results in this manner: “Our study concluded that although neurogenesis is reduced in aging and after a high dose of radiation, many stem cells survive for several months, retaining their ‘stem’ characteristics.”

Part two of this project showed that blocking TGFbeta with drugs restored the production of new neurons in aging or irradiated mice.

Thus targeted therapies that block TGFbeta in the brains of older patients or cancer patients who have undergone high dose radiation for a brain tumor might reduce the impact of brain lesions caused by such events in elderly patients who show distinct signs of cognitive decline.