Targeting EGFL6 Protein Halts Growth and Spread of Ovarian Cancer

Dr. Ronald J. Buckanovich, professor of hematology/oncology and gynecologic oncology at the University of Michigan Medical School, and his colleagues have identified a protein that help ovarian cancer cells multiply and spread to other organs.  When he and his coworkers inhibited this protein with an antibody they were able to stop the spread of ovarian cancer cold.

The EGFL6 or epidermal growth factor like 6 precursor protein, which is also known as MAEG, maps to human Xp22 chromosome.  The EGFL6 protein is expressed primarily in fetal tissues and during early development (see Yeung G., et al., (1999) Genomics 62, 304307; and Buchner G., et al., (2000) Genomics 65, 1623).  The expression of MAEG has also been detected in several tissues, including the dermis of the trunk, hair follicles, and the mesenchyme of the cranio-facial region (see Buchner G., and others, (2000) Mech. Dev. 98, 179182).  EGFL6 protein has been proposed as a possible biomarker in ovarian cancer (Buckanovich R. J., and others, (2007) J. Clin. Oncol. 25, 852861).

In this paper, which appeared in Cancer Research, Buckanovich and others amplified the expression of EGFL6 in ovarian cancer cells.  Increased EGFL6 expression stimulated cancer growth some two-three times.  This effect was observed in cultured ovarian cancer cells and in a mouse model of ovarian cancer.  Conversely, elimination of EGFL6 greatly reduced ovarian cancer growth, decreasing the rate of growth some four-fold.

EGFL6 specifically acts in cancer stem cells.  To review, in tumors, not all cancer cells are the same.  Inside malignant tumors or even among circulating cancerous cells (as in the case of leukemia) there are usually a variety of different types of cancer cells.  The stem cell theory of cancer proposes that among cancerous cells, a small population among them act as stem cells that reproduce themselves and sustain the cancer.  Cancer stem cells, therefore, are like normal stem cells that renew and sustain our organs and tissues.  Therefore, cancer cells that are not stem cells can certainly adversely affect health, but they cannot sustain the cancer long-term.  Therefore, cancer stem cells fuel the growth and spread of cancers and also are often resistant to chemotherapy and radiation treatments.

Further experiments by Buckanovich and his colleagues showed that EGFL6 cause cancer stem cells to divide asymmetrically so that the one of the daughter cells remains a cancer stem cell while the other daughter cell is a cancer cell that can affect the patient but cannot sustain the cancer. This asymmetric cell division also generates a good deal of diversity among cancer cells.

Buckanovich noted: “What this means is that the stem cell population remains stable.  But the daughter cells, which can have a burst of growth, multiply, and allow the cancer to grow.”.

EGFL6 does more than just promote cancer cell proliferation.  EGFL6 is also a promoter of cancer stem cell migration.  When blood vessels were engineered to express EGFL6, tumor metastasis (spread) was even more robust.

Treatment of tumor-afflicted mice with an antibody that specifically binds to EGFL6 and inactivates it caused a 35% reduction in cancer stem cells and significantly reduced overall tumor growth.  Additionally, the antibody also prevented tumor metastasis.

Buckanovich thinks that targeting EGFL6 might be a potential therapy for women with stage 3 ovarian cancer.  Such a treatment might control the growth and spread of ovarian cancers.

Dr. Buckanovich added: “The bigger implication is for women at high risk of ovarian cancer.  These patients could be treated before cancer develops, potentially blocking cancer from developing or preventing it from spreading.  If cancer did develop, it could be diagnosed at an early stage, which would improve patient outcomes.”.

The next step for Buckanovich and his colleagues is developing an antibody that can properly work in human cancer patients.

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.


Radio Interview About my New Book

I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Stem Cell-Promoting Gene Also Promotes the Growth of Head and Neck Cancer

Nanog is a very funny name for a gene, but the Nanog gene is an essential part of the cellular machinery that keeps embryonic stem cells from differentiating and maintains them in a pluripotent state. Unfortunately, Nanog also has other roles if it is mis-expressed and that includes in the genesis of cancers of the head and neck.

Nanog function during development
Nanog function during development

This study emerged from work done by researchers at the Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital, and Richard J. Solove Research Institute or OSUCCC-James. Since Nanog has been studied in some depth, understanding Nanog activity might provide vital clues in the design of targeted drugs and reagents for treating particular cancers.

“This study defines a signaling axis that is essential for head and neck cancer progression, and our findings show that this axis may be disrupted at three key steps,” said Quintin Pan, associate professor of otolaryngology at OSUCCC-James and principal investigator in this research effort. “Targeted drugs that are designed to inhibit any or all of these three steps might greatly improve the treatment of head and neck cancer.”

What kind of signaling axis is Dr. Pan referring to? An enzyme called protein kinase C-epsilon or PKC-epsilon can place phosphate groups on the Nanog protein. Phosphate groups are negatively charged and are also quite bulky. Attaching such chemical groups to a protein can effectively change its structure and function. In the case of Nanog, phosphorylation of stabilizes it and activates it.

Phosphorylated Nanog proteins can bind together to form a dimer, which attracts a third protein to it; p300. This third protein, p300, in combination with the paired Nanog proteins acts as a potent activator of gene expression of particular genes, in particular a gene called Bmi1. When expressed at high levels, Bmi1 stimulates the proliferation of cells in an uncontrolled fashion.

Bmi1 - Nanog interaction

“Our work shows that the PKCepsilon/Nanog/Bmi1 signaling axis is essential to promote head and neck cancer,” Pan said. “And it provides initial evidence that the development of inhibitors that block critical points in this axis might yield a potent collection of targeted anti-cancer therapeutics that could be valuable for the treatment of head and neck cancer.”

Stem Cell Gene Provides Target for Cancer Treatment

A gene called SALL4 encodes a zinc finger transcription factor protein that helps stem cells maintain their undifferentiated state and continue dividing. Cells tend to only express SALL4 during embryonic development, but in almost all cases of acute myeloid leukemia, and in 10-30% of liver, gastric, ovarian, endometrial, and breast cancers, SALL4 is re-expressed. This is solid evidence that SALL4 plays a central role in tumor formation.

Harvard Stem Cell Institute (HSCI)-affiliated labs in Singapore and Boston have shown that knocking out the SALL4 gene in mouse tumors leads to a cessation of tumor growth. Additionally, designing small molecules that inhibit SALL4 activity also treat the cancer and cause cessation of tumor growth and shrinkage of the tumor.

“Our paper is about liver cancer, but it is likely true about lung cancer, breast cancer, ovarian cancer, many, many cancers,” said HSCI Blood Diseases Program leader Daniel Tenen, who also directs a laboratory at the Cancer Science Institute of Singapore (CSI Singapore). “SALL4 is a marker, so if we had a small molecule drug blocking SALL4 function, we could also predict which patients would be responsive.”

Studying the therapeutic potential of a transcription factor is unusual in the field of cancer research. Transcription factors are typically avoided because of the difficulty of developing drugs that safely interfere with genetic targets. Most cancer researchers focus their attention on kinases (enzymes that attach phosphate groups to other molecules).

However, inquiry into the basic biology of the SALL4 gene by HSCI researchers has shown that there is another way to interfere with its activity in cancer cells. The SALL4 protein turns off a tumor suppressor gene, and this causes the cell to divide uncontrollably. By targeting the SALL4 protein with synthetic molecules that inhibit its activity, they could halt the growth of the tumors.

“The pharmaceutical companies decided that if it is not a kinase, and it is not a cell surface molecule, then it is ‘undruggable,'” said Tenen. “To me, if you say anything in ‘undoable,’ you are limiting yourself as a biomedical scientist.”

Earlier this year, Tenen’s co-author, HSCI-affiliated faculty member Li Chau, assistant professor of pathology at Harvard Medical School and Brigham and Woman’s Hospital, published a report that synthetic SALL4 inhibitors have treatment potential in leukemia cells.

Chai took blood samples from patients with acute myeloid leukemia, and treated the leukemia cells with this synthetic inhibitor and then transplanted that blood back into the leukemic mice. The cancer showed gradual regression.

“I am excited about being on the front line of this new drug development,” said Chai. “As a physician-scientist, if I can find a new class of drug that has very low toxicity to normal tissues, my patients can have a better quality of life.”

Chai and Tenen are working with HSCI Executive Committee member Lee Rubin from the Harvard Institute of Chemistry and Biology, and James Bradner from the Dana Farber Cancer Institute (another HSCI-affiliated faculty member), to help them with the drug development part of their project. Demonstrating the potential of SALL4-interfering compounds is labor intensive, but might also be efficacious for the treatment of other cancers.

“I think as academics, we seek to engage drug companies because they can do these types of things better than we can,” said Tenen. “But, also as an academic, I want to go after the important biologic targets that are not being sought after by the typical drug company – because if we do not, who will?”

The Nooks and Crannies in Bone Marrow that Nurture Stem Cells

Stems cells in our bodies often require a specific environment to maximize their survival and efficiency. These specialized locations that nurture stem cells is called a stem cell niche. Finding the right niche for a stem cell population can go a long way toward growing more stem cells in culture and increasing their potency.

To that end, a recent discovery has identified the distinct niches that exist in bone marrow for hematopoietic stem cells (HSCs), which form the blood cells in our bodies.

A research team from Washington university School of Medicine in St. Louis has shown that stem niches in bone marrow can be targeted, which may potentially improve bone marrow transplants and cancer chemotherapy. Drugs that support particular niches could encourage stem cells to establish themselves in the bone marrow, which would greatly increase the success rate of bone marrow transplants. Alternatively, tumor cells are known to hide in stem cell niches, and if drugs could disrupt such niches, then the tumor cells would be driven from the niches and become more susceptible to chemotherapeutic agents.

Daniel Link, the Alan A. and Edith L. Wolff Professor of Medicine at Washington University, said, “Our results offer hope for targeting these niches to treat specific cancers or to impress the success of stem cell transplants. Already, we and others are leading clinical trials to evaluate whether it is possible to disrupt these niches in patients with leukemia or multiple myeloma.”

Working in mice, Link and his colleagues deleted a gene called CXCL12, only in “candidate niche stromal cell populations.” CXCL12 which encodes a receptor protein known to be crucial for maintaining HSC function, including retaining HSCs in the bone marrow, controlling  HSCs activity, and repopulating the bone marrow with HSCs after injury.

CXCL12 crystal structure
CXCL12 crystal structure

CXCL12 signaling pathways

In bone marrow, HSCs are surrounded by a whole host of cells, and it is difficult to precisely identify which type of cells serve as the niche cells. These bone marrow cells are known collectively as “stroma,” but there are several different types of cells in stroma. Cells that have been implicated in the HSC niche include endosteal osteoblasts (osteoblasts are bone-making cells and the endosteum in the layer of connective tissue that lines the inner cavity of the bone), perivascular stromal cells (cells that hang out around blood vessels), CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–positive mesenchymal progenitors. Basically, there are a lot of cells in the stroma and figuring out which one is the HSC niche is a big deal.

bone marrow stromal cells

When HSCs divide, they form two cells, one of which replaces the HSC that just divided and a new cells called a hematopoietic progenitor cell (HPC), which can divide and differentiate into either a lymphoid progenitor or a myeloid progenitor. The lymphoid progenitor differentiates into either a B or T lymphocyte and the myeloid progenitor differentiates into a red blood cell, or other types of white blood cells (neutrophil, basophil, macrophage, platelet or eosinophil). As the cells become more differentiated, they lose their capacity to divide.

HSC differentiation

Deleting CXCL12 from mineralizing osteoblasts (bone making cells) did nothing to the HSCs or those cells that form lymphocytes (lymphoid progenitors). Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, causes mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the bloodstream, and loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from blood vessel cells causes a modest loss of long-term repopulating activity. Deletion of Cxcl12 from nestin-negative mesenchymal progenitors causes a marked loss of HSCs, long-term repopulating activity, and lymphoid progenitors. All of these data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow. Also, the expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Link summarized his results this way: “What we found was rather surprising. There’s not just one niche for developing blood cells in the bone marrow. There’s a distinct niche for stem cells, which have the ability to become any blood cell in the body, and a separate niche for infection-fighting cells that are destined to become T cells and B cells.”

These data provide the foundation for future investigations whether disrupting these niches can improve the effectiveness of cancer chemotherapy.

In a phase 2 study at Washington University, led by oncologist Geoffrey Uy, assistant professor of medicine, Link and his team are evaluating whether the drug G-CSF (granulocyte colony stimulating growth factor) can alter the stem cell niche in patients with acute lymphoblastic leukemia and whose disease is resistant to chemotherapy or has recurred. The FDA approved this drug more than 20 years ago to stimulate the production of white blood cells in patients undergoing chemotherapy, who have often weakened immune systems and are prone to infections.

Uy and his colleagues want to evaluate G-CSF if it is given prior to chemotherapy. Patients enrolled at the Siteman Cancer Center will receive G-CSF for five days before starting chemotherapy, and the investigators will determine whether it can disrupt the protective environment of the bone marrow and make cancer cells more sensitive to chemotherapy.

This trial is ongoing, and the results are not yet in, but Link’s work has received a welcome corroboration of his work. A companion paper was published in the same issue of Nature by Sean Morrison, the director of the Children’s Medical Center Research Institute at the University of Texas Southwestern Medical Center in Dallas. Morrison and his team used similar methods as Link and his colleagues and came to very similar conclusions.

Link said, “There’s a lot of interest right now in trying to understand these niches. Both of these studies add new information that will be important as we move forward. Next, we hope to understand how stem cells niches can be manipulated to help patients undergoing stem cell transplants.”

Georgetown Team Discovers New Type of Stem Cell

A research group a Georgetown Lombardi Comprehensive Cancer Center has developed a new and powerful stem cell in the laboratory that grows in sheets and has many characteristics desirable for regenerative medicine.

The senior author of this paper, Richard Schlegel, M.D., Ph.D., chairman of the department of pathology at Georgetown Lombardi, a part of Georgetown University Medical Center, said of these new stem cells: “These seem to be exactly the kind of cells that we need to make regenerative medicine a reality.”

The results of his lab’s research has been published in the November 19 online early edition of the Proceedings of the National Academy of Sciences (PNAS). In this publication, they report that their new stem-like cells do not express the same genes as embryonic stem cells and induced pluripotent stem cells (iPSCs). Thus, they do not produce tumors when injected into laboratory animals. Also, these cells are stable, since they differentiate into the cell types desired by researchers.

This publication is a continuation of a study published in December 2011, when Schlegel and his colleagues invented a laboratory technique that could maintain both normal and cancer cells alive indefinitely. Previously such a technique did not exist and it was simply not possible to keep such cells alive in the laboratory indefinitely.

Schlegel and others showed that if they added two different substances to their cells in culture – fibroblast feeder cells and a chemical that inhibits the Rho kinase – they could push the cells to assume a kind of stem-like state. While in this stem cell-like state, the cells would stay alive indefinitely. Once the feeder cells and the inhibitor were withdrawn, the cells reverted back to their original state. In this paper, Schlegel and his team called these laboratory-derived cells “conditionally reprogrammed cells” or CRCs. See Liu X et al. Am J Pathol. 2012 Feb;180(2):599-607.

Could CRCs be used for personalized medicine? A follow-up study suggested that they could. Published in the New England Journal of Medicine in September 2012, they found a patient who had a 20-year history of recurrent respiratory “papillomatosis” (a type of tumor) that had invaded the lung tissue in both lungs. The tumor was difficult to treat and slow-growing, but it stubbornly resisted treatment. Schlegel and his team made CRCs from this patient’s normal and tumorous lung tissue. By utilizing this technique, they discovered that the tumor cells were infected the same virus that causes warts; the human papillomavirus. They then used these cultured tumor CRCs to determine which cancer drug would work the best. They identified a drug called vorinostat as the best candidate, and 3 months after starting treatment, the tumors stopped growing and the prognosis looked substantially better for this patient (see Yuan H, et al. N Engl J Med. 2012 Sep 27;367(13):1220-7).

Of this paper, Schlegel said, “Our first clinical application utilizing this technique represents a powerful example of individualized medicine. It will take an army of researchers and solid science to figure out if this technique will be the advance we need to usher in a new era of personalized medicine.”

The present study is study was published in PNAS compared CRCs to embryonic stem cells and iPSCs. Both embryonic stem cells and iPSCs have been investigated for use in regenerative medicine, but both cells have the drawback to potentially producing tumors when injected into mice and “it is difficult to control what kind of cells these cells differentiate into,” Schlegel says. “You may want them to be a lung cell, but they could form a skin cell instead.”

In contrast, if lung cells are treated to make lung-specific CRCs, they can be expanded in culture to make a huge quantity of lung-specific cells, but when these conditions are withdrawn, the lung-specific CRCs will revert to mature lung cells. This transformation is rather rapid, since the cells become CRCs within three days of adding the inhibitor and the feeder cells. Once the cells lose their stem-like properties and potentially can be safely implanted into tissue.

A comparison of gene expression patterns from CRCs and embryonic stem cells (ESCs) or iPSCs showed that CRCs do not overexpress the same critical genes that embryonic stem cells and iPSCs express. “Because they don’t express those genes, they don’t form tumors and they are lineage committed, unlike the other cells,” Schlegel says. “That shows us that CRCs are a different kind of stem-like cell.”

In this study, Schlegel’s team used cervical cells and made CRCs from them. However, then they placed the cervical cell-derived CRCs on a three-dimensional platform, they grew into a canal-like structure that looked startlingly like a cervix. A very similar result was seen with cells extracted from the trachea. When the trachea-derived CRCs were grown on a 3-D platform, they begin to look like a trachea.

If and when use of CRCs are perfected for the clinic, which will require considerably more work, they have the potential to be used in a wide variety of novel ways. “Perhaps they could be used more broadly for chemosensitivity, as we demonstrated in the NEJM study, for regenerative medicine to replace organ tissue that is damaged, for diabetes — we could remove remaining islet ells in the pancreas, expand them, and implant them back into the pancreas —and to treat the many storage diseases caused by lack of liver enzymes. In those cases, we can take liver cells out, expand them and insert normal genes in them, and put them back in patients,” Schlegel says.

Schlegel added: “The potential of these cells are vast, and exciting research to help define their ability is ongoing.”

Some Mesenchymal Stem Cells Inhibit Tumor Growth But Other Types of Mesenchymal Stem Cells Enhance Tumor Growth and Metastasis

Mesenchymal stem cells (MSCs) have the ability to home to growing tumors, and for this reason, many researchers have examined the possibility of using MSCs to treat various types of cancers. However, there is a genuine safety concern with using MSCs in cancer patients, because in laboratory animals, MSCs can form blood vessels that help tumors grow and spread. Consider the following publications:

1. Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F 3rd (2011) Concise review: Dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth? Stem Cells 29: 11–19.
2. Kidd S, Spaeth E, Klopp A, Andreeff M, Hall B, et al. (2008) The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe. Cytotherapy 10: 657–667.
3. Coffelt SB, Marini FC, Watson K, Zwezdaryk KJ, Dembinski JL, et al. (2009) The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells. Proc Natl Acad Sci U S A 106: 3806–3811.
4. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, et al. (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557–563.

Because MSCs are multipotental (that is they differentiate into several different adult tissues), they can serve the tumor as a source of blood vessels that augment tumor metastasis and growth. However, several pre-clinical studies with genetically engineered MSCs that deliver chemotherapuetic agents to tumors have proven quite successful (see Waterman RS, Betancourt AM (2012) The role of mesenchymal stem cells in the tumor microenvironment: InTech). So what are we to believe? After MSCs good or bad as tumor treatments?

In 2010, Aline M. Betancourt and colleagues at Tulane University, New Orleans, Louisiana defined two distinct MSC subypes in a MSC population. They referred to these subtypes as MSC1 and MSC2. When challenged with molecules normally found in invading microorganisms, MSC1 populations tend to promote the immune response, where as MSC2 populations tend to suppress the immune response. This simple priming experiment provided a way to distinguish between the two MSC subtypes, but it also gave stem cell scientists a reason why experiments with MSCs tend to give conflicting results in different laboratories – because the two labs were probably working with populations that consisted of different MSC subtypes. See Waterman RS, Tomchuck SL, Henkle SL, Betancourt AM (2010) A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype. PLoS ONE 5(4): e10088. doi:10.1371/journal.pone.0010088.

With this in mind, Betancourt and co-workers examined the ability of the distinct MSC subtypes to interact with cancers. When grown in culture with several different types of tumor-causing cell lines, they discovered that MSC1 do not support tumor growth but MSC2 robustly support tumors growth. MSC2 also increased the ability of the tumors to invade other tissues and migrate in culture whereas MSC1 supported neither tumor invasion nor tumor migration.

Other features were different as well. For example, MSC1 recruited a completely different cadre of white blood cells to the tumor when compared to MSC2. Also, the molecules deposited in the vicinity of the tumor by MSC1 and MSC2 differed greatly. By providing a bed of molecules upon which tumors cell like to move and grow, MSC2s promoted tumor cell activity, but the materials laid down by MSC1 were not nearly as attractive to the tumor cells.

These show why MSCs can promote the growth of particular tumors in some experiments but not others. Furthermore, it shows that there is a relatively simple test to separate these two MSC subtypes. All further pre-clinical experiments with MSCs, should account for these distinct MSC subtypes and determine if one MSC subtype is a better candidate for an anticancer treatment regime than the other.

See Waterman RS, Henkle SL, Betancourt AM (2012) Mesenchymal Stem Cell 1 (MSC1)-Based Therapy Attenuates Tumor Growth Whereas MSC2-Treatment Promotes Tumor Growth and Metastasis. PLoS ONE 7(9): e45590. doi:10.1371/journal.pone.0045590.

Reducing the Incidence of a Deadly Side Effect of Bone Marrow Transplants in Mice

Bone marrow transplants save the lives of leukemia, but they have one risky drawback and that is “graft-versus-host disease.” Graft-versus-host disease (GVHD) results when immune cells in the donor’s bone marrow attack the tissues and cells of the recipient’s body as foreign. Almost half of bone marrow transplant recipients develop graft-versus-host disease (GVHD), the main organs affected are the skin, liver and gut. Obviously, finding a way to quell or even prevent GVHD would be a boon for bone marrow transplantations.

By utilizing a mouse model, researchers at Washington University School of Medicine in St. Louis have managed to reduce the risk of GVHD from bone marrow transplants. Since bone marrow transplants are the only available curative treatment when leukemia returns, decreasing the risk of GVHD is the first step to improving the prognosis of leukemia patients.

The main strategy behind decreasing the effects of GVHD is to direct immune cells from the donor’s bone marrow away from healthy tissue and lead them to their intended purpose, which is to kill cancer cells.

“This is the first example of reducing graft-versus-host disease not by killing the T-cells, but simply by altering how they circulate and traffic,” says John F. DiPersio, MD, PhD, the Virginia E. and Sam J. Golman Professor of Medicine at Barnes-Jewish Hospital and Washington University School of Medicine. “Donor T-cells do good things in terms of eliminating the recipient’s leukemia, but they can also attack normal tissues leading to death in a number of patients. The goal is to minimize graft-versus-host disease, while maintaining the therapeutic graft-versus-leukemia effect.”

By working in a mouse model, Jaebok Choi, PhD, research assistant professor of medicine, showed that if he eliminated or blocked a particular protein known as the interferon gamma receptor on donor T-cells, these cell were unable to migrate to critical organs such as the intestines. However, these same T-cells were still capable of killing leukemia cells.

“The fact that blocking the interferon gamma receptor can redirect donor T-cells away from the gastrointestinal tract, at least in mice, is very exciting because graft-versus-host disease in the gut results in most of the deaths after stem cell transplant,” DiPersio says. “People can tolerate graft-versus-host disease of the skin. But in the GI tract, it causes relentless diarrhea and severe infections due to gut bacteria leaking into the blood, which can result in severe toxicity, reduction in the quality of life or even death in some patients.”

Interferon gamma has, for some time, been known to play a vital role in inflammation. The signal transduction pathway that works downstream of the receptor is just now being better understood. It is this signal transduction pathway downstream of the receptor that is responsible for activating the T-cells so that they cause GVHD. The signaling cascade initiated when interferon gamma binds its receptor activates molecules known as JAK kinases, followed by another protein called “STAT,” and finally a protein called CXCR3. CXCR3 mediates the trafficking of donor T-cells to the GI tract and other target organs.

Deleting the interferon gamma receptor from donor T-cells steers them away from target organs. This, however, leads to a second question: “Could the same result be observed by inhibiting some of the other molecules that act downstream of the interferon gamma receptor?” To address this question, Choi knocked out CXCR3 and discovered that such a knock out reduced graft-versus-host disease, but did not completely wipe it out.

“There are probably additional downstream targets of interferon gamma receptor signaling other than JAKs, STATs and CXCR3 that are responsible for T-cell trafficking to the GI tract and other target organs,” DiPersio says. “We’re trying to figure out what those are.”

This worked beautifully in mice, but could it work in humans? To make these data more relevant to human biology, Choi and DiPersio used drugs known to block JAK kinases in human cells. These drugs are presently approved by the Food and Drug Administration to treat myelofibrosis, which is a pre-leukemic condition in which bone marrow is replaced with fibrous tissue. Ruxolitinib and pacritinib are two such drugs and Choi and DiPersio showed that treating mice with either of these two drugs could mimic the protective effect of deleting the interferon gamma receptor. The JAK inhibitors definitely redirect the donor T-cells away from target organs and reducing graft-versus-host disease in leukemic mice. Unfortunately, they have yet to determine if these drugs preserve the anti-leukemia effect of these T-cells.

“The proof-of-principle behind these experiments is the exciting part,” DiPersio says. “If you can change where the T-cells go as opposed to killing them, you prevent the life-threatening complications and maintain the clinical benefit of the transplant.”

Mesenchymal Stem Cells Are Used In Tumor-Targeted Gene Therapy

My apologies to my readers. I have been in Seattle, Washington for the past week at the Free Methodist National Bible Quizzing Tournament for this week, and I have not had a chance to blog at all. Nevertheless, I have time now. In case you are interested, my team made it to the Senior Teen Veteran Division by winning their subdivision, and then during the double elimination portion of the tournament, they were eliminated in the third round. My quizzers quizzed gallantly, but they happened to quiz the first place and second place teams at the beginning and both teams were quizzing particularly well. They did not go down without a fight, but they were simply out-jumped by the extremely talented quizzers from Winona Lake, IN and Rainer Avenue, Seattle, WA. Oh well; someone has to lose.

I have a paper in my hot little (well not so little) hands that is from C.J. Bruns’ lab at the University of Munich in Munich, Germany. In this paper, Bruns and his German and American collaborators review the use of mesenchymal stem cells (MSCs) as vehicles to specifically deliver toxic genes to tumor cells. These experiments are still preliminary, but with the proper refinements, they might lead to clinical trials for cancers that are difficult to treat with more traditional methods.

MSCs are found in many different locations throughout the body. They are most easily isolated from bone marrow and fat, and they can also be cultured and grown to larger numbers in the laboratory for limited periods of time. When isolated from bone marrow, MSCs appear as a subpopulation of cells that adhere to the plastic tissue culture dishes.

Because of the ease of their isolation and manipulation, researchers have used MSCs to introduce genes into tumor cells. There are several advantages that make MSCs a very attractive cell for such a venture. First, MCSs do not activate the immune system when they are introduced into another body. Secondly, they seem to home to tumors and provide them with a kind of scaffold upon which the tumor cells grow. Thus MSCs and tumors cells form a kind of natural partnership. This means that introduced tumor cells will readily integrate into a growing tumor. Imaging studies that implanted labeled MSCs have borne this out. For example, when implanted into mice with melanomas that have spread to the lung, the transplanted MSCs, after eight days, surrounded the lung tumors (Gao, et al., Oncogene 2010 29(19):2784-94). A similar experiment with tumors in the pancreas also showed similar results (MSCs surrounded the tumors) after three days (Beckermann et al., Br J Cancer 2008 99(4):622-31). Third, given the tendency of MSCs to home to tissue damage and heal it, introduced MSCs and endogenous MSCs tend to view the growing and invading tumor and one big wound that constantly required healing. Thus the interaction between the tumor and the MSCs gets even cozier. For all these reasons, MSCs are very good vehicles for tumor-targeted gene therapy.

One of the first experiments that utilized MSCs for tumor-targeted gene therapy (TTGT) was described by Studeny and colleagues (Studeny M, et al., Cancer Res. 2002 Jul 1;62(13):3603-8). In this paper, MSCs were engineered to express a protein called interferon-beta in order to treat melanomas in mice. Those mice that received intravenous injections of the engineered MSCs showed reduced tumor growth and increased times of survival. Interferon beta (INF-B) is a member of a large group of secreted proteins called interferons. There are three main classes of interferons and the type of receptor bound by the interferon determines which class it belongs to. Type 1 interferons (INFs) are used to treat patients with blood-based tumors (leukemias) or solid tumors. Type 1 INFs prevent tumor growth, staunch the tendencies of tumors to induce the growth of new blood vessels into the tumor mass, and also induce cell death within the cells of the tumor. Clinically, those patients who suffer from recurrent melanomas receive treatments with recombinant IFN-α2b. Thus, getting cells that express INF-B into the tumor could kill of the tumor cells and shut the tumor down.

Since Studeny’s pioneering work, several different studies have used MSCs from bone marrow (Studeny et al., J Natl Cancer Inst 2004 96(21): 1593-603; Loebinger, et al., Cancer Res 2009 69(10):4134-42; Nakamizo, et al., Cancer Res 2005 65(8): 3307-18; Hakkarainen, et al., Hum Gene Ther 2007 18(7):627-41), fat (Grisendi et al., Cancer Res 2010 70(9) 3718-29; Zolochevska et al., Stem Cells Dev 2012 21(7):1112-23), and umbilical cord (Kim, et al., Stem Cells 2010 28(12):2217-28) have been used in experiments like it. Also, several different types of genes other than INF-B have been engineered into MSCs and used to shrink tumors in laboratory animals. These engineered MSCs have also been used to treat melanomas (Studeny M, et al., Cancer Res. 2002 Jul 1;62(13):3603-8; Studeny et al., J Natl Cancer Inst 2004 96(21): 1593-603), breast cancers (Eliopoulos et al., Cancer Res 2008 68(12): 4810-8), lung cancers (Loebinger, et al., Cancer Res 2009 69(10):4134-42; Xim et al., Mol Med 2009 15(9-10):321-7), cervical (Grisendi et al., Cancer Res 2010 70(9) 3718-29) and prostate cancers (Zolochevska et al., Stem Cells Dev 2012 21(7):1112-23), soft tumors (Xiang, et al., Cytotherapy 2009 11(5):516-26), and various types of brain tumors (Gu, et al., Cancer Lett 2010 291(2): 256-62; Miletic, et al., Mol Ther 2007 15(7): Amano, et al., Int J Oncol 35(6):1265-70). The genes with which the MSCs have been engineered include TRAIL (TNF-related apoptosis-inducing ligand), which encodes a protein that causes cells to die (Studeny et al., J Natl Cancer Inst 2004 96(21): 1593-603; Grisendi et al., Cancer Res 2010 70(9) 3718-29; Kim, et al., Stem Cells 2010 28(12):2217-28), PEDF (Pigment epithelium-derived factor), a protein that prevents the growth of blood vessels (Zolochevska et al., Stem Cells Dev 2012 21(7):1112-2), IL-12, a gene that encodes a protein that makes tumors recognizable by the immune system (Eliopoulos et al., Cancer Res 2008 68(12): 4810-8), HSK-Tk, a viral gene that makes tumors susceptible to anti-viral drugs (Gu, et al., Cancer Lett 2010 291(2): 256-62; Uchibori, et al., J Gene Med 2009 11(5):373-81; Miletic, et al., Mol Ther 2007 15(7): Amano, et al., Int J Oncol 35(6):1265-70), and iNOS, a gene that encodes a protein that makes nitric oxide; a toxic molecule (Xiang, et al., Cytotherapy 2009 11(5):516-26). All of these strategies have had some successes in treating artificially induced tumors in laboratory animals.

The main focus of this paper is the use of genetically engineered MSCs to treat tumors of the digestive system. When it comes to tumors found outside the digestive system, the data seem to suggest that MSC-based gene therapies are rather successful. However, when it comes to tumors in the digestive system, the data are unclear, since the experiments show conflicting results. There are some indications that cultured rodent MSCs have the ability to form tumorous growths in extended culture (Miura, et al., Stem Cells 2006 24(4):1095-103; Li, et al., Cancer Res 2007 67(22):10889-98; Qin, et al., Cloning Stem Cells 2009 11(3):445-52). Secondly, implantation of extensively cultured rodent MSCs into the bodies of living rodents leads to the formation of soft tumors (Tolar, et al., Stem Cells 2007 25(2):371-9). Therefore, some risks might accompany MSC-based treatments. In contrast to these concerns, which largely stem from experiments in rodents, the thousands of patients who have had MSC treatments have not experienced cancers as a result of them (Le Blanc, et al., Lancet 2008 371(9624):1579-86).

Animal studies of MSC transplantations into laboratory rodents afflicted with digestive tumors have shown that MSCs stimulate tumor growth, and, in other experiments, inhibit tumor growth. The biology of tumors in the digestive tract is more complicated than other tumors, and, therefore, the results of experiments with MSCs vary from laboratory to laboratory. For example, Qiao and others gave mice with defective immune systems injections of liver cancer cells plus human MSCs that had been engineered to grow continuously in culture. The injected MSCs were able to inhibit tumor growth (Qiao, et al., Cell Res 2008 18(4):333-40). However, work from Bruns’ own lab showed that infused MSCs promoted the growth of pancreatic cancers (Zischek, et al., Ann Surg 2009 250(5):747-53; Conrad, et al., Ann Surg 2011 253(3):566-71) and liver cancers (Niess, et al., Ann Surg 2011 254(5):767-74). Li and his co-workers also showed that human MSC infusions could inhibit the invasion and metastasis of liver cancers in culture. Invasion assays in cultured usually consist of tumor cells grown on a layer of cells and the ability of the tumor cells to penetrate this layer of cells and grow on either side of them. Li and others showed that co-culturing MSCs with liver cancer cells prevented the liver cells from penetrating and invading the cell layer. However, when the same cells were infused into laboratory animals, the MSCs enhanced tumor grow (Li, et al., Cancer Sci 2010 101(12):2546-53). Li and his colleagues found very similar results with cancers of the esophagus (Tian, et al., J Cell Physiol. 2011 226(7):1860-7).

With respect to genetically engineered MSCs, the results are not as equivocal.

A scientist named You and his fellow scientists transplanted stomach cancer cells into mice with hMSCs that had been engineered with a suicide gene called cytosine deaminase (CD). These mice were then given a drug called 5-fluorouracil (5-FU), which kills cells that express CD, and this resulted in a pronounced inhibition of tumor growth (You MH, et al., 2009 J Gastroenterol Hepatol 24:1393–1400). In another experiment by Kidd and others showed that hMSCs engineered with IFN-B or without IFN-B were both found to suppress tumor growth of pancreatic cancers (Kidd S, et al., 2010 Cytotherapy 12:615–625). Additionally, Bruns’ lab also showed that infusion of MSCs in animals with pancreatic cancer strongly promoted tumor growth and increased the tendency of the tumor to spread. However, if the MSCs were engineered to express HSV-Tk, the MSCs substantially inhibited pancreatic tumor growth and prevented the spread of the tumor after the mice we treated with ganciclovir, a drug that kills cells that express HSV-Tk (Zischek C, et al., 2009 Ann Surg 250:747–753).

From all these data, it seems that there is reason to be optimistic that such a treatment strategy might work in humans. However, given the ability of MSCs to stimulate the growth of tumors in animal models is reason for concern, However, with the proper controls and safety regulations in place, anti-cancer treatments with genetically engineered MSCs could be one of the clinical trials we will see in the near future.

Engineering Blood Cells to Fight Melanoma

University of California, Los Angeles (UCLA) scientists have successfully completed a proof-of-principle experiment in mice that shows that blood cells can be re-engineered to become melanoma fighting immune cells.

Senior author on this study, Jerome Zack, who is also a scientist with UCLA’s Jonsson Comprehensive Cancer Center and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, noted that genetic engineering techniques can remodel the blood cells of the mouse so that they form cancer-killing T-cells that seek out the tumor and destroy it. Zack stated: “We knew from previous studies that we could generate engineered T-cells, but would they work to fight cancer in a relevant model of human disease, such as melanoma. We found with this study that they do work in a human model to fight cancer, and it’s a pretty exciting finding.”

White blood cells come in several different varieties, but one group of white blood cells is the “lymphocytes,” which play an exceedingly central role in adaptive immunity. There are two main types of lymphocytes; B-lymphocytes, also known as B-cells and T lymphocytes or T-cells. T-cells receive their name from an organ that sits over the top part of the heart called the thymus. Once T-cells are born, they migrate to the thymus where they undergo a complex maturation process. Once they are released from the thymus into the peripheral circulation, they are ready to serve the immune system. T-cells differ from B-cells in that they possess a surface protein called the “T-cell receptor.” The T-cell receptor recognized foreign substances or “antigens” that are bound to the surfaces of cells. When the T-cell binds to this antigen, it becomes activated and begins to divide and initiates the formation of an immune response against this antigen.

In this experiment, Zack and his co-workers used a T-cell receptor that they had isolated from a cancer patient. This particular T-cell receptor recognized an antigen that is specific to melanomas. The UCLA group then used genetic engineering techniques to place the T-cell receptor gene into the blood-making stem cells in the bone marrow of laboratory mice. After re-introducing these engineered blood-making stem cells into the experimental mice. Next, Zack and his colleagues transplanted a small piece of human thymus into the experimental mice. This gave the mice a place to allow the newly made T-cells to mature.

After approximately six weeks, engineered blood stem cells had formed a large population of mature, melanoma-specific T-cells that were able to target the particular cancer cells. To demonstrate this, the experimental mice were then implanted with two types of melanoma, one that expressed the antigen complex recognized by the T-cell receptor introduced into the bone marrow stem cells, and another tumor that did not. The engineered cells specifically went after the melanoma that expressed the particular antigen, but they left the other tumor alone. Of the nine nude mice used in this study, four animals showed complete elimination of the antigen-expressing melanomas, and the other five showed a marked decrease in the size of the tumors. The immune response against the tumors was determined not only by measuring physical tumor size, but by monitoring the cancer’s metabolic activity using Positron Emission Tomography (PET), which measures how much energy the cancer is “eating” to drive its growth.

Zach noted: “We were very happy to see that four tumors were completely gone and the rest had regressed, both by measuring their size and actually seeing their metabolic activity through PET.” Zack said.

This approach has the advantage of engineering only a few cells that can produce a veritable army of cancer-fighting T-cells. Furthermore, these cells can exist in the circulating blood in low numbers, but if they detect the melanoma antigen, they can replicate and expand their numbers quickly and home to the tumor where they will fight it. Other advantages of this strategy are that the function of the engineered T-cells is not long-lasting in most cases. More engineered T-cells ultimately are needed to sustain a response, but some of these cells will probably become “memory cells.” Memory cells are inactive cells that remember the infection they recently fought, but can be reactivated if they encounter the antigen once again. This suggests that “fresh” cancer-killing cells could be easily generated when needed, perhaps protecting against cancer recurrence later.

The team would like to test this approach in clinical trials. One possible approach would be to engineer both the circulating T-cells and the blood stem cells that give rise to T-cells. The peripheral T-cells would serve as the front line cancer fighters, while the blood stem cells are creating a second wave of warriors to take up the battle as the front line T-cells are losing function. Zack also said that he hopes that this technique could adapted for protocols the battle other cancers like breast and prostate cancers.

Neural stem cells hunt down brain tumors

Brain tumors are one of the biggest bummers in medicine.  Brain tumors are typically inoperable and because the drugs that are used to treat cancers must cross the blood-brain barrier, this greatly limits the available drugs that can be used to treat these tumors.  Now, research from lab in Germany indicates that our own brain-specific stem cells might actively seek out and destroy particular types of brain cancers.

Scientists from the Max Delbrück Center for Molecular Medicine (MDC) in Berlin, Germany, have demonstrated how the brain’s own stem cells and precursor cells control the growth of particular brain tumors called glioblastomas.  Glioblastomas are among the most common and aggressive of all brain tumors.  They typically form in adults in their mid-fifties or early sixties.  The causes for their development are not yet known, but researchers have reasonably good reasons to believe that misdirected neural stem cells / precursor cells mutate into cancer cells and form glioblastomas.

Several years ago researchers from the MDC and Charité (Berlin) showed that normal stem cell/ precursor cells of the brain can attack glioblastomas.  Apparently, the tumor itself entices these stem cells to migrate over relatively long distances from the stem cell niches of the brain.  How?

These used cell culture and other techniques to show that neural stem cells and neural precursor cells release a protein that belongs to the family of the BMP proteins (bone morphogenetic protein).  BMPs received their names because they are able to induce bone and cartilage tissue formation, but they are quite active in the brain.  Neural stem cells release BMP-7 in the brain in the vicinity of the glioblastoma cells. The protein influences a small population of cancer cells, the so-called tumor stem cells.  These tumor stem cells seem to be the actual cause for the continuous tumor self-renewal in the brain, but a fraction of these cells is sufficient to form new tumors again even after surgery. BMP-7 induces signaling in the tumor stem cells, causing them to differentiate, which makes them tumor stem cells no longer.

However, the activity of stem cells in the brain and thus of the body’s own protective mechanism against glioblastomas diminishes with increasing age. This could explain why the tumors usually develop in older adults and not in children and young people.

These discoveries could lead to new concepts in the therapy of glioblastomas.  “Normal cancer cells” can be destroyed using conventional therapies (surgery, radiation, chemotherapy), which are seldom successful in tumor stem cells.  The aim is therefore to develop therapy concepts to destroy these tumor stem cells.  Findings from the mouse experiments could point to a new approach: reprogramming tumor stem cells into less harmful cells, which could then be destroyed with a therapy.