Breakthrough work in cancer
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Updated 11/22/2015; 12/17/2015
Sequencing Metastatic Cancers Could Lead to Improved Therapies
- Unravelling the genetic sequences of cancer that has spread to the brain could offer unexpected targets for effective treatment, according to a study (“Genomic characterization of brain metastases and paired primary tumors reveals branched evolution and potential therapeutic targets”) published in Cancer Discovery.
Scientists say they found that the original, or primary, cancer in a patient’s body may have important differences at a genetic level from cancer that has spread to the patient’s brain. This insight could suggest new lines of treatment.
Priscilla Brastianos, M.D., a neurooncologist and director of the Brain Metastasis program at Massachusetts General Hospital, points out that “brain metastases are a devastating complication of cancer. Approximately eight to ten percent of cancer patients will develop brain metastases, and treatment options are limited. Even where treatment is successfully controlling cancer elsewhere in the body, brain metastases often grow rapidly.”
She and her colleagues studied tissue samples from 104 adults with cancer. In collaboration with researchers at the Broad Institute, they analyzed the genetics of biopsies taken from the primary tumor, brain metastases, and normal tissues in each adult. For 20 patients, they also had access to metastases elsewhere in the body.
The team discovered that, in every patient, the brain metastasis and primary tumor shared some of their genetics, but there were also key differences. In 56% of patients, genetic alterations that potentially could be targeted with drugs were found in the brain metastasis but not in the primary tumor.
“We found genetic alterations in brain metastases that could affect treatment decisions in more than half of the patients in our study,” notes Dr. Brastianos. “We could not detect these genetic alterations in the biopsy of the primary tumor. This means that when we rely on analysis of a primary tumor we may miss mutations in the brain metastases that we could potentially target and treat effectively with drugs.”
This study also found that if a patient had more than one brain metastasis, each was genetically similar. The researchers used their findings to map the evolution of a cancer through a patient’s body, and draw up a phylogenetic tree for each patient to demonstrate how the cancer had spread and where each metastasis had come from.
They concluded that brain metastases and the primary tumor share a common genetic ancestor. Once a cancer cell, or clone, has moved from the primary site to the brain, it continues to develop and amass genetic mutations. The genetic similarity of the brain metastases in individual patients suggests that each brain metastasis has developed from a single clone entering the brain.
The genetic changes in brain metastases are independent of any occurring at the same time in the primary tumor, and in metastases elsewhere in the body, the researchers said. Characterization of the genetics of a patient’s primary cancer can be used to optimize treatment decisions, so that drugs that target specific mutations in the cancer can be chosen. However, brain metastases are not routinely biopsied and analyzed.
“When brain metastasis tissue is available as part of clinical care, we are suggesting sequencing and analysis of that sample,” continues Dr. Brastianos. “It may offer more therapeutic opportunities for the patient. Genetic characterization of even a single brain metastasis may be superior to that of the primary tumor or a lymph node biopsy for selection of a targeted treatment.”
Scientists Discover How Cancer Cells Escape Blood Vessels
12/16/2015 – Anne Trafton, MIT News Office http://www.biosciencetechnology.com/news/2015/12/scientists-discover-how-cancer-cells-escape-blood-vessels
A rounded cancer cell (top left) sends out nanotubes connecting with endothelial cells. Genetic material can be injected via these nanotubes, transforming the endothelial cells and making them more hospitable to additional cancer cells. (Image: Sengupta Lab)
Scientists at MIT and Massachusetts General Hospital have discovered how cancer cells latch onto blood vessels and invade tissues to form new tumors — a finding that could help them develop drugs that inhibit this process and prevent cancers from metastasizing.
Cancer cells circulating in the bloodstream can stick to blood vessel walls and construct tiny “bridges” through which they inject genetic material that transforms the endothelial cells lining the blood vessels, making them much more hospitable to additional cancer cells, according to the new study.
The researchers also found that they could greatly reduce metastasis in mice by inhibiting the formation of these nanobridges.
“Endothelial cells line every blood vessel and are the first cells in contact with any blood-borne element. They serve as the gateway into and out of tumors and have been the focus of intense research in vascular and cancer biology. These findings bring these two fields together to add greater insight into control of cancer and metastases,” said Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology, a member of MIT’s Institute for Medical Engineering and Science, and one of the leaders of the research team.
The lead author of the paper, which appears in the Dec. 16 issue of Nature Communications, is Yamicia Connor, a graduate student in the Harvard-MIT Division of Health Sciences and Technology (HST). The paper’s senior author is Shiladitya Sengupta, an assistant professor at HST and at Harvard Medical School.
Building bridges
Metastasis is a multistep process that allows cancer to spread from its original site and form new tumors elsewhere in the body. Certain cancers tend to metastasize to specific locations; for example, lung tumors tend to spread to the brain, and breast tumors to the liver and bone.
To metastasize, tumor cells must first become mobile so they can detach from the initial tumor. Then they break into nearby blood vessels so they can flow through the body, where they become circulating tumor cells (CTCs). These CTCs must then find a spot where they can latch onto the blood vessel walls and penetrate into adjacent tissue to form a new tumor.
Blood vessels are lined with endothelial cells, which are typically resistant to intruders.
“Normal endothelial cells should not enable a cancer cell to invade, but if a cancer cell can connect with an endothelial cell, and inject signals that enable this endothelial cell to be controlled and completely transformed, then it facilitates metastasis,” Sengupta said.
The researchers first spotted tiny bridges between cancer cells and endothelial cells while using electron microscopy to study the interactions between those cell types. They speculated that the cancer cells might be sending some kind of signal to the endothelial cells.
“Once we saw that these structures allowed for a ubiquitous transfer of a lot of different materials, microRNAs were an obvious interesting molecule because they’re able to very broadly control the genome of a cell in ways that we don’t really understand,” Connor said. “That became our focus.”
MicroRNA, discovered in the early 1990s, helps a cell to fine-tune its gene expression. These strands of RNA, about 22 base pairs long, can interfere with messenger RNA, preventing it from being translated into proteins.
In this case, the researchers found, the injected microRNA makes the endothelial cells “sticky.” That is, the cells begin to express proteins on their surfaces that attract other cells to adhere to them. This allows additional CTCs to bind to the same site and penetrate through the vessels into the adjacent tissue, forming a new tumor.
“It’s almost like the cancer cells are cooperating with each other to facilitate migration,” Sengupta said. “You just need maybe 1 percent of the endothelial cells to become sticky, and that’s good enough to facilitate metastasis.”
Non-metastatic cancer cells did not produce these invasive nanobridges when grown on endothelial cells.
Erkki Ruoslahti, a professor of cell, molecular, and developmental biology at the University of California at Santa Barbara, said that the discovery is an important advance in understanding tumor metastasis.
“I found it particularly interesting that the transfer of regulatory macromolecules from tumor cells to endothelial cells via intercellular nanotubes appears to be more effective (at least over relatively short distances) than exosome-mediated transfer, which has received a lot of attention lately,” said Ruoslahti, who was not part of the research team.
Shutting down metastasis
The nanobridges are made from the proteins actin and tubulin, which also form the cytoskeleton that gives cells their structure. The researchers found that they could inhibit the formation of these nanobridges, which are about 300 microns long, by giving low doses of drugs that interfere with actin.
When the researchers gave these drugs to mice with tumors that normally metastasize, the tumors did not spread.
Sengupta’s lab is now trying to figure out the mechanism of nanobridge formation in more detail, with an eye toward developing drugs that act more specifically to inhibit the process.
“If we can first understand how these structures are formed, then we can try to design targeted therapies to inhibit their formation, which could be a promising new area for developing drugs that specifically target metastasis,” Connor said.
Source: Massachusetts Institute of Technology
Inspired by Nature
Researchers are borrowing designs from the natural world to advance biomedicine.
When biomedical engineer Jeff Karp has questions, he looks to animals for answers. In 2009, Karp gathered his team at the Brigham and Women’s Hospital in Boston to brainstorm novel ways to capture circulating tumor cells (CTCs) in the bloodstream. They mulled over the latest microfluidic devices. Then the conversation turned to the New England Aquarium, and to jellyfish.
Scientists have tried to grab cancer cells from blood ever since they discovered that tumors shed malignant cells that migrate throughout the vasculature—a process known as metastasis. “If you pluck out these cells, you have a direct indicator of what the cancer looks like,” says Karp. “Then you can screen drugs to get those that will have the greatest impact.” Doctors might also be able to detect such cells during the earliest stages of metastatic cancer, when it’s more readily treatable.
CANCER-CELL CAPTURE DEVICE: Jellyfish’s long, sticky tentacles grab prey and other food particles from water. Researchers have copied this design by coating the channels of a microfluidic chip with long, tentacle-like strands of DNA that bind a protein on the surface of leukemia cells. The device can process 10 times more blood than existing chips in the same amount of time.
See full infographic: JPG SANDCASTLE WORM: PHEBE LI FOR THE SCIENTIST. DIAGRAM: KIMBERLY BATTISTA
The problem is, CTCs make up a tiny fraction of cells in the bloodstream of a person with cancer, meaning an effective diagnostic must process relatively large volumes of blood. However, an existing test, which uses magnetic particles to isolate CTCs, processes just 7.5 milliliters of blood, only a fraction of one percent of the 5 liters of blood in an adult human. Dialysis-like microfluidic devices promise to handle larger volumes and improve efficiency, but the best current prototypes still feature extremely narrow microchannels to ensure CTCs pass within reach of CTC-binding antibodies along the perimeter. “Channel height is extremely low in a lot of the proposed devices, meaning you can barely flow any blood through,” says Karp. (See “Capturing Cancer Cells on the Move,” The Scientist, April 2014.)
Karp wanted to change that. “We asked ourselves, ‘What creatures can capture things at a distance?’” he recalls. One of his graduate students suggested jellyfish, whose long, sticky tentacles grab prey and other food particles from water. Within a year, Karp and his colleagues had designed a microfluidic chip on which 800-micron-wide microchannels are lined with long, tentacle-like strands of DNA that bind a protein on the surface of leukemia cells as they pass through the channels. (See illustration below.) In 2012, Karp showed that the jellyfish-inspired device could process 10 times more blood than existing chips in the same amount of time and trap an average of 50 percent of circulating leukemia cells.1 Karp estimates that a device the size of the standard microscope slide could collect hundreds or thousands of tumor cells in minutes. Encouraged by such results, Karp’s team is now improving the platform, designing chips that can catch any CTC of interest.
The jellyfish is far from the only intriguing organism to have served as a blueprint for scientists in the field of bioinspired medicine. Researchers have taken cues from the adhesive chemistry perfected by mussels and marine worms to create tissue glues that stick in wet and turbulent conditions; from red blood cell membranes to help drug-carrying nanoparticles avoid immune attack; and from the slippery slides that help carnivorous pitcher plants catch prey to produce novel antibacterial surfaces. (See “Bioinspired Antibacterial Surfaces.”) Nature, it seems, provides a compendium of biomedical solutions.
“Nature has used the power of evolution by natural selection to develop the most efficient ways to solve all kinds of problems,” says Donald Ingber, founding director of the Wyss Institute for Biologically Inspired Engineering in Boston. “We’ve uncovered so much about how nature works, builds, controls, and manufactures from the nanoscale up. Now we’re starting to leverage those biological principles.”
Sticking points
Looking to nature is not a new concept, and bioinspiration is just one of several approaches bioengineers employ to devise new medical treatments and devices. But in the last few years, the approach has come to the fore with several promising new products, even if most of them remain a few years away from human trials. “Almost every research institute now has a center for biomimicry or biologically inspired engineering,” says Ingber. “It’s just reaching that tipping point where it’s going to begin to have an impact.”
TISSUE GLUE: The sandcastle worm (Phragmatopoma californica) builds reef-like shelters by gluing together grains of sand with two separate secretions: one containing negatively charged polyphosphate proteins and the other positively charged polyamine proteins. Researchers mimicked this idea with synthetic polyelectrolytes to create an injectible fluid that can patch fetal membrane ruptures in an in vitro model.
See full infographic: JPGSANDCASTLE WORM: PHEBE LI FOR THE SCIENTIST. DIAGRAM: KIMBERLY BATTISTA
Medical adhesion is one area where bioinspiration promises to make an impression. Stitches and staples are still the standard for suturing wounds and closing up surgical incisions, but these technologies can damage tissue, leave gaps for bacteria to infiltrate, and increase the risk of inflammation. For years, surgeons have been in need of new medical adhesives that can bond tissue strongly inside the body without provoking inflammation.
Heeding the call, bioengineers have again turned to the sea. Phillip Messersmith of the University of California, Berkeley, for example, is focused on the protein-filled secretions marine mussels use to fasten themselves to wave-battered rocks. The proteins in these liquid secretions are rich in an amino acid called dihydroxyphenylalanine (DOPA), which features reactive catechol chains. These catechol chains bond tightly with each other in a mussel’s own secretions but also bond with metal atoms present on the surface of rocks. Using this strategy as a blueprint, Messersmith and colleagues chemically synthesized a variant of DOPA to crosslink biocompatible polymers.
Their glue has successfully fastened transplanted insulin-producing islet cells to the outer surface of the liver and nearby tissues in mice.2 The technique could potentially provide an alternative to standard methods of islet transplantation in which islets are infused into the liver vasculature, where they trigger an inflammatory response that quickly kills off about half of the transplanted cells—and impairs the surviving cells’ ability to produce therapeutic insulin. The researchers are also testing the bioinspired adhesive’s ability to repair ruptured fetal membranes, which can lead to premature birth and other serious complications. (See “Mimicking Mussels,” The Scientist, April 2013.)
Gene Test Finds Which Breast Cancer Patients Can Skip Chemo
9/28/2015 Marilynn Marchione, AP Chief Medical Writer
In this Sept. 5, 2013 file photo, chemotherapy is administered to a cancer patient via intravenous drip in Durham, N.C. In a study sponsored by the National Cancer Institute and results published online Monday, Sept. 28, 2015, by the New England Journal of Medicine, a gene-activity test that was used to gauge early-stage breast cancer patient’s risk accurately identified a group of women whose cancers are so likely to respond to hormone-blocking drugs that adding chemo would do little if any good while exposing them to side effects and other health risks. (Gerry Broome, Associated Press)Many women with early-stage breast cancer can skip chemotherapy without hurting their odds of beating the disease – good news from a major study that shows the value of a gene-activity test to gauge each patient’s risk.
The test accurately identified a group of women whose cancers are so likely to respond to hormone-blocking drugs that adding chemo would do little if any good while exposing them to side effects and other health risks. In the study, women who skipped chemo based on the test had less than a 1 percent chance of cancer recurring far away, such as the liver or lungs, within the next five years.
“You can’t do better than that,” said the study leader, Dr. Joseph Sparano of Montefiore Medical Center in New York.
An independent expert, Dr. Clifford Hudis of New York’s Memorial Sloan Kettering Cancer Center, agreed.
“There is really no chance that chemotherapy could make that number better,” he said. Using the gene test “lets us focus our chemotherapy more on the higher risk patients who do benefit” and spare others the ordeal.
The study was sponsored by the National Cancer Institute. Results were published online Monday by the New England Journal of Medicine and discussed at the European Cancer Congress in Vienna.
The study involved the most common type of breast cancer – early stage, without spread to lymph nodes; hormone-positive, meaning the tumor’s growth is fueled by estrogen or progesterone; and not the type that the drug Herceptin targets. Each year, more than 100,000 women in the United States alone are diagnosed with this.
The usual treatment is surgery followed by years of a hormone-blocking drug. But many women also are urged to have chemo, to help kill any stray cancer cells that may have spread beyond the breast and could seed a new cancer later. Doctors know that most of these women don’t need chemo but there are no great ways to tell who can safely skip it.
A California company, Genomic Health Inc., has sold a test called Oncotype DX since 2004 to help gauge this risk. The test measures the activity of genes that control cell growth, and others that indicate a likely response to hormone therapy treatment.
Past studies have looked at how women classified as low, intermediate or high risk by the test have fared. The new study is the first to assign women treatments based on their scores and track recurrence rates.
Of the 10,253 women in the study, 16 percent were classified as low risk, 67 percent as intermediate and 17 percent as high risk for recurrence by the test. The high-risk group was given chemotherapy and hormone-blocking drugs. Women in the middle group were randomly assigned to get hormone therapy alone or to add chemo. Results on these groups are not yet ready – the study is continuing.
But independent monitors recommended the results on the low-risk group be released, because it was clear that adding chemo would not improve their fate.
After five years, about 99 percent had not relapsed, and 98 percent were alive. About 94 percent were free of any invasive cancer, including new cancers at other sites or in the opposite breast.
“These patients who had low risk scores by Oncotype did extraordinarily well at five years,” said Dr. Hope Rugo, a breast cancer specialist at the University of California, San Francisco, with no role in the study. “There is no chance that for these patients, that chemotherapy would have any benefit.”
Dr. Karen Beckerman, a New York City obstetrician diagnosed with breast cancer in 2011, said she was advised to have chemo but feared complications. A doctor suggested the gene test and she scored very low for recurrence risk.
“I was convinced that there was no indication for chemotherapy. I was thrilled not to have to have it,” and has been fine since then, she said.
Mary Lou Smith, a breast cancer survivor and advocate who helped design the trial for ECOG, the Eastern Cooperative Oncology Group, which ran it, said she thought women “would be thrilled” to skip chemo.
“Patients love the idea of a test” to help reduce uncertainty about treatment, she said. “I’ve had chemotherapy. It’s not pretty.”
The test costs $4,175, which Medicare and many insurers cover. Others besides Oncotype DX also are on the market, and Hudis said he hopes the new study will encourage more, to compete on price and accuracy.
“The future is bright” for gene tests to more precisely guide treatment, he said.
Source: Associated Press
Back-to-the-Future with Tumor Cell-Based Avatars
Researchers Looking for Alternatives to Individual Avatars Have Found Reason to Be Hopeful in Tumor-Cell Based Predictive Models
Formidable barriers, including time and expense required to breed and maintain mice engrafted with human tumor tissue, impede the widespread use of mouse avatars.
- Mice grafted with human tumors, known as patient-derived xenograft (PDX) mice, have migrated from cancer research labs to the clinic. But as limitations to modeling patient individual tumors in mice emerge, some investigators are turning to cell-based models and applying new methodologies to support and grow cells in culture.
Conceived by Heinz-Herbert Fiebig and colleagues at the University of Freiburg in the early 1980s, it was hoped that PDX mice would more accurately reflect an individual patient’s tumor in a model system and predict tumor responses to drug therapies. Dr. Fiebig is the founder and CEO of Oncotest, a company that specializes in preclinical pharmacological contract research.
Since their introduction, commercial labs, including Oncotest, the Jackson Laboratory, and Discovery Group plc Horizon (Horizon), have provided access to a wide range of PDX mice made from donated tumor tissue. The tissue, cryopreserved for future use after biopsy, serves as the basis for offering drug-testing services to researchers and pharmaceutical companies. Oncotest, for example, says it provides drug-testing services to 16 of the 20 largest pharmaceutical companies, using a library of more than 350 PDX mouse models.
And beyond PDX mouse model libraries for pharma companies, companies now offer individualized avatar mice directly to patients developed using their own tumors. Champions Oncology provides mouse avatars directly to patients, at a cost of $10,000 to $12,000. Proponents of these mouse models say they can facilitate the identification of a personalized therapeutic regimen, may prove more useful than genomic analysis, and eliminate the cost and toxicity associated with nontargeted chemotherapeutics.
But formidable barriers impede the widespread use of mouse avatars, scientists say, including the time and expense required to breed and maintain mice engrafted with human tumor tissue. Development of an individualized avatar takes anywhere from three to six months, more time than some critically ill patients can survive and, in about 30% of cases, Champions points out it hasn’t been able to grow the patient’s tumor in mice.
In a study published in Cancer in April 2014, Justin Stebbing, M.D., Ph.D., and colleagues at Imperial College, London, reported that they worked with Champions to develop avatars with the company’s TumorGraft system for 22 patients with advanced sarcoma. But nine patients died before the results were ready. “Within a couple of months after their surgery or biopsy, they get chemotherapy and they pass away,” says Champions CEO Ronnie Morris. “We build the avatar, but the patient can’t use it.”
In this study, the scientists said that of implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although several patients died before completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of the 16 (81%) remaining individuals. No patients died during the TumorGraft-predicted therapy.
On the other hand the authors noted that a primary advantage of Champions’ TumorGraft is “that it allows discrimination between the different standard-of-care therapies that may be available, as well as other potential treatments not normally indicated for that tumor.
“Our increased understanding of tumor heterogeneity, even within a single subtype, means that knowing how patients with the same tumor previously responded to a particular drug is no guarantee that the current patient will respond similarly. TumorGraft overcomes this problem by helping guide oncologists to those treatments that are most likely to provide a positive clinical outcome.”
- Search for Alternatives
Given the obstacles to using individual avatars to guide patient therapy, researchers in several laboratories are currently looking for alternatives, turning in some cases to tumor-cell based predictive models in a back to the future approach utilizing up-to-date pharmacogenomics and novel cell culture technologies to improve the longstanding odds against success culture of tumor cells from biopsied material.
The team of Jeffrey Engelman, M.D., Ph.D., director of thoracic oncology and molecular therapeutics at Massachusetts General Hospital Cancer Center, has successfully established cell culture models from biopsy samples of lung cancer patients for functional pharmacologic studies. Dr. Engelman noted that while “Genetics has been extremely useful to guiding treatment, in many cases tumor genetics are ambiguous or do not reveal a mutation that informs a therapeutic strategy. These functional pharmacologic studies can identify effective therapeutic choices even when the genetics fail to do so.”
Dr. Engelman and colleagues described in Science a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome drug resistance. Their cell culture models were derived from patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected to genetic analyses and a pharmacological screen.
With the system they could identify multiple effective drug combinations, they said. These included the combination of ALK and mitogen-activated protein kinases (MAPK) inhibitors active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation. A combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant-resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, the authors said their strategy could help direct therapeutic choices for individual patients.
- Several Approaches
Noting the historical difficulty of coaxing tumor cells obtained from tumor biopsies to grow in culture, Dr. Engelman told GEN that his team typically tries three or four different approaches to optimize the growth of cells from a single biopsy, including 3D culture, organoids, and feeder layers to support the best cancer cell growth. “We want to get the biopsy to the high-throughput screening phase as quickly as possible and get the results to inform patient therapy as quickly as possible,” he said.
While the application described in their publication involved lung cancer, he notes that his lab is trying the approach on breast cancer, colorectal tumors, and melanoma. “What’s interesting for us is that there are cancers for which no work has ever been done before,” he noted.
To date, the investigators are “not applying the cell culture technology to the clinic, but are inching closer to doing so,” Dr. Engleman said. “We are confident in the results we get from the screen and believe the data is quite valuable, but we want to make sure there is clinical outcome with therapeutics prior to having a patient enroll in a clinical trial or embark on a specific therapy.”
Dr. Engelman also believes that the technology can be commercialized, but that he is “focused on making it work.” These initial studies demonstrated success in developing NSCLC models NSCLC models in 50% of collected specimens. However, the team believes that success rates could be further improved by using biopsies acquired for specifically for cell line generation.
The authors noted that with their pharmacologic platform, they discovered several previously undescribed combinations in EGFR mutant and ALK-positive lung cancers that were validated in follow-up studies and in vivo. They speculate that a similar approach could be explored in the future as a diagnostic test to identify therapeutic strategies for individual patients (under the auspices of an IRB-approved protocol).
In their study, they screened the cells after they became fully established cell lines, often requiring two to six months, a time frame that would make this approach less than ideal as a routine diagnostic test. But they say, their results of the program provides the groundwork for performing screens on viable cells obtained within weeks of a biopsy using newer technologies that would permit screening of the cancer cells while still in the presence of the stroma present in the biopsy.
In a proof of concept study in Nature Methods, investigators working at MGH, Harvard Medical School, the Karolinska Institute, and other institutions showed that circulating tumor cells (CTCs) can be captured in viable form and used to establish cell cultures, potentially bypassing the need for a biopsy as a source of tumor cells to culture.
The investigators captured the CTCs using microchip technology (the Cluster-Chip) developed to capture CTC clusters independently of tumor-specific markers from unprocessed blood. The device isolates the CTC clusters through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and, the investigators said, even two-cell clusters can be efficiently captured.
Maheswaran et al., in Cancer Research, used the device to show that the culture of CTCs in the blood of patients with breast cancer enabled them to study patterns of drug susceptibility linked to the genetic context that is unique to an individual tumor.
The investigators established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets.
The authors noted that with optimization of CTC culture conditions, this strategy could help identify the best therapies for individual cancer patients over the course of their disease.
These and other investigators believe, that cell-based methods, once optimized, could bypass the need for whole animal cancer avatars, providing another resource to help inform the choice of therapies likely to be effective in a given patient.
Linking Phenotypes and Modes of Action Through High-Content Screen Fingerprints
The Use of High-Content Screening as a Powerful Technique for Monitoring Phenotypic Responses
Felix Reisen, Amelie Sauty de Chalon, Martin Pfeifer, Xian Zhang, Daniela Gabriel, Paul Selzer
Fig. 2. Phenotypes of snuclei are colored purple, the cytoplasm redix tool compounds targeting different cellular compartments. In all figures nuclei are colored purple, the cytoplasm red.
- In today’s drug discovery campaigns we observe a clear trend toward more complex assay environments. While target-based high-throughput screening (HTS) still plays an important role, phenotypic screening techniques are gaining importance. Phenotypic screening assays are believed to be more closely linked to a given disease state than target-based approaches where the molecular hypothesis might not be relevant for disease pathogenesis.
One approach to phenotypic drug discovery is high-content screening (HCS), an HTS technique based on automated microscopy. HCS allows for highly multiplexed assay readouts that can be used to simultaneously assay several modes of action or toxicity. Additionally, HCS enables screening in a controlled and disease-relevant environment by even using patient-derived cell cultures.
While there are many advantages to phenotypic screening, additional knowledge about the targets being modulated to bring about the desired phenotype can be highly beneficial, for example, in lead optimization, by helping interpretation of structure activity relationships. In addition, knowledge of the target can also help to identify related targets that may bring about challenges in designing selective lead molecules.
Various techniques have been developed to support target identification for compounds active in phenotypic assays. These include approaches such as affinity chromatography, biochemical fractionation, radioactive ligand binding assays, drug affinity responsive target stability. Alternative approaches are based on in vivo chemical genomic assays developed in yeast Saccharomyces cerevisiae or in silico approaches using historic knowledge about compound target associations. In silico methods predict possible targets for a compound by comparing the similarity of the compound’s profile (using chemical similarity, gene expression profile, or HCS experiments) to those of previously characterized compounds with known target.
For the rest of the story, click here.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. GEN presents here one article “Linking Phenotypes and Modes of Action Through High-Content Screen Fingerprints.” Authors of the paper are Felix Reisen, Amelie Sauty de Chalon, Martin Pfeifer, Xian Zhang, Daniela Gabriel, and Paul Selzer.
Immuno-Oncology Landscape Expands
New Techniques Enable Closer Look into Genetic & Cellular Alterations in Tumor Microenvironment
- For years, researchers and physicians have suspected, and have worked to demonstrate, how the immune system affects susceptibility to, defense against, and progression of certain cancers. It is now understood that the immune system has the ability to influence the fate of developing cancers by not only functioning as a tumor promoter that facilitates cellular transformation, promotes tumor growth, and sculpts tumor cell immunogenicity, but also as an extrinsic tumor suppressor that either destroys developing tumors or restrains their expansion.
In the last few decades, drugs, biologicals, and vaccines targeting certain attributes of the immune system, known as immunotherapeutics, have become available, and emerging clinical data suggest that cancer immunotherapy is likely to become a key part of the clinical management of cancer for years to come.
Although immunotherapies represent a major step forward in cancer care, providing in some cases unprecedented response rates, there is still much work to do to discover new druggable targets, find biomarkers to predict response, as well as gain deeper understanding of why some cancer types are incredibly responsive to immunotherapeutic treatments while others are not.
- How Immunotherapies Work
Figure 1. Inhibitory costimulatory checkpoints are a natural immune mechanism for self-tolerance and minimization of collateral tissue damage. Inhibitory checkpoint receptors such as PD-1, LAG-3, TIM-3, and CTLA-4 are expressed by T cells, and their ligands are expressed by macrophage and dendritic cells. Tumor cells can express multiple inhibitory ligands to repress T-cell function and thereby evade clearance by the immune system.
- A deeper understanding of cancer as a disease requires the acknowledgement of its inherent heterogeneity. As with the cancer cells within a tumor, the immunological microenvironments in which they grow are similarly heterogeneous. Emerging and well-established scientific tools and techniques for the analysis of cancer cells, immune cells and their microenvironment can be combined to yield new insights into the nature of tumorigenesis, immune system recruitment, and treatment optimization.In general, immunotherapies direct an individual’s immune system to fight cancer by either stimulating it to attack cancer cells or by introducing manufactured immune system components to augment immune function. Immunotherapy treatments work in different ways. Some boost the body’s immune system in a very general way. Others help train the immune system to attack cancer cells specifically.
- On an immuno-oncological level, the genetic and cellular alterations that define a cancer cell provide the immune system with the means to be recruited to the tumor and generate T-cell responses to recognize and eradicate those cells. Elimination of cancer by T cells is only one step in the cancer immunity cycle. T-cell activation is controlled by both stimulatory and inhibitory checkpoints. Tumors use the expression of inhibitory ligands as a mechanism of suppressing cytotoxic T-cell response and inducing an immunosuppressive environment.
- Identification of specific cancer T-cell inhibitory signals, such as PD-L1, has prompted the development of a new class of cancer immunotherapy that specifically hinders immune effector inhibition, reinvigorating and potentially expanding preexisting anticancer immune responses (Figure 1).
- The presence of environment-altering immunosuppressive innate myeloid lineages in the tumor microenvironment may further explain the limited activity observed with previous immune-based therapies and why these therapies may be more effective in combination with agents that target other steps of the cycle.
- Understanding the Tumor and Its Microenvironment
In addition, the presence and quantity of various immune cell types in the tumor microenvironment may have prognostic value. Many scientists believe that a deepening appreciation of oncology genomics and the quantity and type of antigens expressed by the tumor cells, when coupled with an analysis of the patient’s immune system, will greatly progress the field and unlock the next generation of immunotherapies.
Flow cytometry and immunohistochemistry are established tools for the labeling and analysis of immunological and oncology cellular components. New techniques are likewise becoming more widely used that enable simultaneous detection of proteins and nucleic acids at single-cell resolution.
New Cellular Analysis Tools
- eBioscience, a business unit of Affymetrix, has recently expanded commercialization of two such novel assays that provide exciting new technologies in the armament of cellular analysis techniques for immuno-oncology research. The first is PrimeFlow™ RNA Assay, which is the only commercially available assay for the simultaneous detection of RNA and protein expression within millions of cells at single-cell resolution using a standard flow cytometer. The assay is compatible with cell surface and intracellular antibody staining, using traditional fluorochromes for multiparameter cellular analysis.
- With this technology an immune-oncology researcher could explore gene expression heterogeneity among different rare tumor-infiltrating immune cell subsets with single-cell resolution and without laborious cell sorts, as well as compare kinetics of both RNA and protein in the same cell.
http://www.genengnews.com/Media/images/Article/thumb_eBioscience_Fig21361229223.jpg
Figure 2. The PrimeFlow RNA Assay workflow contains several steps: antibody staining, fixation and permeabilization including intracellular staining if desired, followed by target hybridization with a target-specific probe set containing 20 to 40 oligonucleotide pairs. Next, branched DNA signal amplification is achieved through a series of sequential hybridization steps consisting of pre-amplifiers, amplifiers, and labeled probes, followed by detection by flow cytometric analysis. This results in excellent specificity, low background, and a high signal-to-noise ratio. For simplicity, two RNA targets are shown in the schematic above (red and green), and only 3 of the 20 to 40 oligonucleotide target probe pairs per target RNA are shown.
http://www.genengnews.com/gen-articles/immuno-oncology-landscape-expands/5577/
- S. Shalapour et al. recently published a study in the journal Nature (April 29, 2015) applying these techniques to mouse models of castrate-resistant prostate cancer demonstrating that the presence of a very specific and rare (0.04–3% of total) B cell population in the tumor microenvironment correlates to a immunotherapeutic response allowing a CTL-dependent eradication of oxaliplatin-treated tumors.
- ViewRNA® In Situ Hybridization (ISH) Cell and Tissue Assays comprise the second new technique from eBioscience. Similar to the PrimeFlow RNA assay, but compatible with microscopy, these assays enable the visualization of single-copy RNA transcripts within adherent and suspended single cells or single cells in tissue sections, and in the case of ViewRNA ISH Tissue Assays, the spatial separation of tumor subclones by phenotypic RNA expression. Similarly, this technique can be used to visualize and quantitate cellular and molecular attributes of tumor-infiltrating immune cells to elucidate biomarkers of resistance and response. Leveraging these novel cell analysis approaches, immuno-oncology researchers can analyze cellular diversity in the tumor microenvironment as well as the diversity of immune cell responses at a single-cell level.
- Breakthrough responses to new immunotherapies are stimulating a renewed interest in basic immune biology. With our quest to develop strategies to harness the human immune response against cancer to achieve durable responses and/or complete eradication of cancer in patients safely, we must explore multiple approaches simultaneously. Which immune checkpoints can be manipulated? Are there dual therapies that can be applied to improve responses? Are there biomarkers inherent to the immune system in general, the specific tumor and the tumor microenvironment that can be used to stratify responders?
- Multiple approaches to cancer therapy exist, and few are as complicated as immune-based therapy. That being said, few therapies in recent history have demonstrated such extraordinary and durable responses for the patients who do respond. As such, many believe that this will be an intensifying area of research and clinical focus for years to come.
New Research for Prostate Cancer Therapies
Dr. Glenn Bubley has been treating patients with prostate cancer for more than 25 years.
“When a patient’s diagnosis is latter-stage prostate cancer, the standard treatment is androgen deprivation therapy [ADT],” says Bubley, Director of the Genitourinary Cancer Program in the Cancer Center at Beth Israel 
Deaconess Medical Center. “ADT works by lowering testosterone production and thereby depriving prostate tumors of the ‘fuel’ that helps them grow.”
But, he adds, although this hormone therapy is almost always effective, all tumors eventually grow resistant to ADT — and cancer recurs. Over the past two years, Bubley has been part of a BIDMC scientific team that has been testing a targeted treatment alternative for late-stage prostate cancer using a unique type of study known as a “Co-Clinical Trial.”
This new approach to clinical research — in which specially-created mouse models with genetic mutations are matched with tumor tissue from human cancer patients in order to test new therapies — was developed by BIDMC Cancer Center Director Pier Paolo Pandolfi, MD, PhD.
“Targeted therapies are designed to attack cancers by pinpointing the genes and genetic mutations that underlie diseases,” says Pandolfi (right). “The problem is that cancer cells are genetically complex, sometimes containing hundreds of genetic mutations. We needed to develop a way to cut down on all this ‘genetic noise’ to get at the root of the disease. The Co-Clinical Trial enables us to streamline and expedite the process in order to more quickly test a variety of new cancer drugs.”
Here’s how it works: In the Co-Clinical Trial, human participants are matched with animal models that have been genetically engineered to carry different combinations of just a few major human prostate cancer genes.
“When the animals develop tumors — just as the human patients did — they will receive the same therapies as the patients receive,” says Bubley (right). But, he adds, because each animal has only a few mutations, the researchers will be able to quickly assess which treatments are effective and which are not — and will be able to go back and adjust treatment accordingly for the human patients.
A particular advantage to this approach, say Bubley and Pandolfi, will be the ability to test combinations of different drugs to treat prostate cancer and overcome ADT resistance.
“Going forward, we think that combinations of targeted and conventional therapies may prove to be effective, particularly for drug-resistant disease,” says Bubley. “And the only realistic way to be able to quickly test numerous different drug combinations will be through the Co-Clinical Trial process.”
Nanocarriers May Carry New Hope for Brain Cancer Therapy
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