3D model of lab-made tissue that mimics the lining of the oral cavity

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New research demonstrates that previous models used to examine cancer may not be complex enough to accurately mimic the true cancer environment. Using oral cancer cells in a three-dimensional model of lab-made tissue that mimics the lining of the oral cavity, the researchers found that the tissue surrounding cancer cells can epigenetically mediate, or temporarily trigger, the expression or suppression of a cell adhesion protein associated with the progression of cancer. These new findings support the notion that drugs that are currently being tested to treat many cancers need to be screened using more complex tissue-like systems, rather than by using conventional petri dish cultures that do not fully manifest features of many cancers.

"Research on cancer progression has been drawn largely using models that grow cancer cells in plastic dishes. Our research reveals a major shortcoming in the experimental systems used to study cancer development. When using simplified culture systems in which cells are grown on plastic, cancer cells grow as a two dimensional monolayer and lack the three-dimensional tissue structure seen in human cancer. As a result, complex interactions that occur between the cancer cells and the surrounding tissue layers are not accounted for," said first author Teresa DesRochers, PhD, a graduate of the Sackler School of Graduate Biomedical Sciences at Tufts, currently in the department of biomedical engineering at Tufts University School of Engineering.

The researchers report that the three-dimensional network of cell interactions activates epigenetic mechanisms that control whether genes critical for cancer development will be turned on or off. By imitating the structure of the tumor microenvironment seen in different stages of cancer, the research team was able to observe that cell-to-cell interactions that are inherent in tissue structure are sufficient to turn on the cell adhesion protein, E-cadherin, that can delay cancer development.

Since both invasion and metastasis occur when cells break away from the primary cancer site, an event correlated with loss of E-cadherin, treating cancers to induce re-expression of this protein through epigenetic control may be an important way to control cancer progression.

"Our findings show the reversible nature of E-cadherin when cancer cells are placed in a three-dimensional network of cells that mimics the way cancer develops in our tissues. This confirms that cancer biology needs to move into the "third dimension" where cancer cells can be studied in a network of other cells that can control their behavior. We know now that the plastic dish alone is not good enough," said senior author Jonathan Garlick, DDS, PhD, a professor in the oral and maxillofacial pathology department at Tufts University School of Dental Medicine.

Jonathan Garlick is also a member of the Cell, Molecular & Developmental Biology program faculty at the Sackler School at Tufts and the director of the Center for Integrated Tissue Engineering (CITE) at Tufts University School of Dental Medicine.

This study, published in the January issue of Epigenetics, was performed in collaboration with Laurie Jackson-Grusby, PhD, associate in pathology at Children's Hospital, Boston, and assistant professor at Harvard Medical School. Additional authors of the study are Yulia Shamis, MSc, a PhD student at the Sackler School of Graduate Biomedical Sciences; Addy Alt-Holland, MSc, PhD, an assistant professor at Tufts University School of Dental Medicine; Yasusei Kudo, DDS, PhD, and Takashi Takata, DDS, PhD, both of the department of oral and maxillofacial pathobiology, Graduate School of Biomedical Sciences, Hiroshima University, Japan; and Guangwen Wang, PhD, previously a fellow at Children's Hospital Boston, now a senior scientist at Stemgent.

This research was supported in part by grant #DE017143 from the National Institute of Dental and Craniofacial Research, part of the National Institutes of Health.

DesRochers TM, Shamis Y, Alt-Holland A, Kudo Y, Takata T, Wang G, Jackson-Grusby L, Garlick JA. Epigenetics. 2012 (January); 7 (1): 34-46 "The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma."

Tufts University, Health Sciences Campus

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One of the most important advances in cancer research at Johns Hopkins is viewing cell behavior in three dimensions (3D). Looking at cells in 3D yields more accurate information.

Showing movies in 3-D has produced a box-office bonanza in recent months. Could viewing cell behavior in three dimensions lead to important advances in cancer research? A new study led by Johns Hopkins University engineers indicates it may happen. Looking at cells in 3-D, the team members concluded, yields more accurate information that could help develop drugs to prevent cancer's spread.

The study, a collaboration with researchers at Washington University in St. Louis, appears in the June issue of Nature Cell Biology.

"Finding out how cells move and stick to surfaces is critical to our understanding of cancer and other diseases. But most of what we know about these behaviors has been learned in the 2-D environment of Petri dishes," said Denis Wirtz, director of the Johns Hopkins Engineering in Oncology Center and principal investigator of the study. "Our study demonstrates for the first time that the way cells move inside a three-dimensional environment, such as the human body, is fundamentally different from the behavior we've seen in conventional flat lab dishes. It's both qualitatively and quantitatively different."

One implication of this discovery is that the results produced by a common high-speed method of screening drugs to prevent cell migration on flat substrates are, at best, misleading, said Wirtz, who also is the Theophilus H. Smoot Professor of Chemical and Biomolecular Engineering at Johns Hopkins. This is important because cell movement is related to the spread of cancer, Wirtz said. "Our study identified possible targets to dramatically slow down cell invasion in a three-dimensional matrix."

When cells are grown in two dimensions, Wirtz said, certain proteins help to form long-lived attachments called focal adhesions on surfaces. Under these 2-D conditions, these adhesions can last several seconds to several minutes. The cell also develops a broad, fan-shaped protrusion called a lamella along its leading edges, which helps move it forward. "In 3-D, the shape is completely different," Wirtz said. "It is more spindlelike with two pointed protrusions at opposite ends. Focal adhesions, if they exist at all, are so tiny and so short-lived they cannot be resolved with microscopy."

The study's lead author, Stephanie Fraley, a Johns Hopkins doctoral student in Chemical and Biomolecular Engineering, said that the shape and mode of movement for cells in 2-D are merely an "artifact of their environment," which could produce misleading results when testing the effect of different drugs. "It is much more difficult to do 3-D cell culture than it is to do 2-D cell culture," Fraley said. "Typically, any kind of drug study that you do is conducted in 2D cell cultures before it is carried over into animal models. Sometimes, drug study results don't resemble the outcomes of clinical studies. This may be one of the keys to understanding why things don't always match up."

Fraley's faculty supervisor, Wirtz, suggested that part of the reason for the disconnect could be that even in studies that are called 3-D, the top of the cells are still located above the matrix. "Most of the work has been for cells only partially embedded in a matrix, which we call 2.5-D," he said. "Our paper shows the fundamental difference between 3-D and 2.5-D: Focal adhesions disappear, and the role of focal adhesion proteins in regulating cell motility becomes different."

Wirtz added that "because loss of adhesion and enhanced cell movement are hallmarks of cancer," his team's findings should radically alter the way cells are cultured for drug studies. For example, the team found that in a 3-D environment, cells possessing the protein zyxin would move in a random way, exploring their local environment. But when the gene for zyxin was disabled, the cells traveled in a rapid and persistent, almost one-dimensional pathway far from their place of origin.

Fraley said such cells might even travel back down the same pathways they had already explored. "It turns out that zyxin is misregulated in many cancers," Fraley said. Therefore, she added, an understanding of the function of proteins like zyxin in a 3-D cell culture is critical to understanding how cancer spreads, or metastasizes. "Of course tumor growth is important, but what kills most cancer patients is metastasis," she said.

To study cells in 3-D, the team coated a glass slide with layers of collagen-enriched gel several millimeters thick. Collagen, the most abundant protein in the body, forms a network in the gel of cross-linked fibers similar to the natural extracellular matrix scaffold upon which cells grow in the body. The researchers then mixed cells into the gel before it set. Next, they used an inverted confocal microscope to view from below the cells traveling within the gel matrix. The displacement of tiny beads embedded in the gel was used to show movement of the collagen fibers as the cells extended protrusions in both directions and then pulled inward before releasing one fiber and propelling themselves forward.

Fraley compared the movement of the cells to a person trying to maneuver through an obstacle course crisscrossed with bungee cords. "Cells move by extending one protrusion forward and another backward, contracting inward, and then releasing one of the contacts before releasing the other," she said. Ultimately, the cell moves in the direction of the contact released last.

When a cell moves along on a 2-D surface, the underside of the cell is in constant contact with a surface, where it can form many large and long-lasting focal adhesions. Cells moving in 3-D environments, however, only make brief contacts with the network of collagen fibers surrounding them - "We think the same focal adhesion proteins identified in 2-D situations play a role in 3-D motility, but their role in 3-D is completely different and unknown," Wirtz said. "There is more we need to discover."

Fraley said her future research will be focused specifically on the role of mechanosensory proteins like zyxin on motility, as well as how factors such as gel matrix pore size and stiffness affect cell migration in 3-D.

Notes: Co-investigators on this research from Washington University in St. Louis were Gregory D. Longmore, a professor of medicine, and his postdoctoral fellow Yunfeng Feng, both of whom are affiliated with the university's BRIGHT Institute. Longmore and Wirtz lead one of three core projects that are the focus of the Johns Hopkins Engineering in Oncology Center, a National Cancer Institute-funded Physical Sciences in Oncology Center. Additional Johns Hopkins authors, all from the Department of Chemical and Biomolecular Engineering, were Alfredo Celedon, a recent doctoral recipient; Ranjini Krishnamurthy, a recent bachelor's degree recipient; and Dong-Hwee Kim, a current doctoral student.

Funding for the research was provided by the National Cancer Institute.

Source: Johns Hopkins University

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In cell-based assay labs using functional profiling, they throw away the single cells and work exclusively with three dimensional, floating, tumor spheroids. If you test the same cells as three dimensional spheroids, they are not many-fold resistant in vitro, just as they are in vivo.

Once they have their proper comparison database, they then stratify the database, based on deviations from the median, for each assay system and at each drug concentration. One half standard deviation more "sensitive" than the median is the cut-off for a "sensitive result. One half standard deviation more "resistant" than the median is the cut-off for a "resistant" result.

Upgrading clinical therapy by using drug sensitivity assays measuring "cell death" of three dimensional microclusters of live "fresh" tumor cells improves the conventional situation by allowing more drugs to be considered. The more anti-cancer drug types there are in the selective arsenal, the more likely the system is to prove beneficial.

Because older assay types and peripheral blood assays test on subcultured cells (as opposed to fresh tumor cultures) and test the cells in monolayers (as opposed to three dimensional cell clusters), the cell grown in the lab will not behave the same way as the actual cancer cells do in your body' own environment.

However, all the work in the past twenty years in the cell culture field has been carried out largely on three dimensional clusters of cells (not monolayers). Solid tumor specimens are cultured in concical polypropylene microwells for 96 hours to increase the proportion of tumor cells, relative to normal cells.

Polypropylene is a slippery material which prevents the attachment of fibroblasts and epithelial cells and encourages the tumor cells to remain in the form of three dimensional, floating clusters. Real life 3D analysis makes chemoresponse assays indicative of what will happen in the body.

The "deviations" have to do with the Bayesian method of science, which Dr. Donald Berry, Ph.D., professor and chair of the Department of Biostatistics and Applied Mathematics at MD Anderson, says is more in line with how science works. They are putting the Bayesian approach (which is no stranger to cell culture assays) to test with more than 100 cancer-related phase I and II clinical trials planned or carried out.

In fact, the Bayesian methodology is what gives credit to the accuracy of cell culture assay testing. Bayes theorem has been used to describe the relationship between the accuracy of a predictive test (post-test probability) and the overall incidence of what is being tested (pre-test probability). Bayes' theorem indicates that cell death laboratory assays will be accurate in the prediction of clinical drug sensitivity and resistance in tumors.

Literature Citation:

Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007

Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)

An atlas of fresh human tumors in short term suspension culture, illustrating examples of (1) the appearance of the fresh tumors immediately following scissor mincing and digestion with collagenase/DNAse, (2) appearance of “control” (cultured without drugs) tumor cells, following 96 hours of suspension culture, in anchorage-independent conditions, (3) appearance of cells when cultured with an “effective”/”active” drug, and (4) appearance of cells when cultured with an “ineffective”/”inactive” drug.

[url]http://weisenthal.org/Human_Tumor_Assay_Journal/Atlas.html

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As outlined in the presentation at the American Association for Cancer Research (AACR) Annual Meeting titled, “Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection,” cell function analysis of human tumors provides a novel, real-time view of how such tumors act within their natural microenvironment. This information can, in turn, accelerate the drug development process and improve clinical therapy.

Led by Robert Nagourney, MD, medical director of Rational Therapeutics and the Todd Cancer Institute at Long Beach Memorial Medical Center, the investigators have applied a human tumor micro-spheroid platform that measures both apoptotic and non-apoptotic cell death events and other cellular responses following exposure to a variety of agents.

With its capacity to measure genetic and epigenetic events, the platform provides a functional, real-time adjunct to static genomic and proteomic platforms. By examining small clusters of cancer cells [microspheroids or microclusters] in their native state, they provide a snapshot of the response of tumor cells to drugs, combinations and targeted therapies.

The analysis is unique in that each micro-spheroid examined contains all the complex elements of tumor bio-systems found in the human body and have a major impact on clinical response. Cell function analysis is a conduit that connects novel drugs to clinicians and patients in need. Appropriate use of this platform has the potential to save the pharmaceutical industry millions of dollars, shave years off the drug development cycle and improve clinical therapy.

There are any number of variable that affect drugs, including the rate of excretion of the drugs by the kidneys and liver, protein binding and a myriad of other biological factors. In the body, these cells interact with and are supported by other living cells, both malignant and non-malignant cells. That is why cell-death functional profiling assays study cancer cells in microspheroids-microclusters.

Three-dimensional (3D) tissue culture methods have an invaluable role in tumor biology and provides very important insights into cancer biology. As well as increasing our understanding of homeostasis, cellular differentiation and tissue organization, they provide a well defined environment for cancer research in contrast to the complex host environment of an in vivo model.

Due to their enormous potential, 3D tumor cultures are currently being exploited by many branches of biomedical science with therapeutically orientated studies becoming the major focus of research. Recent advances in 3D culture and tissue engineering techniques have enabled the development of more complex heterologous 3D tumor models.

Source: Nagourney, RA, et al Functional Profiling of Human Tumors In Primary Culture: A Platform for Drug Discovery and Therapy Selection: Proc AACR, 2008

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Highly Specific Prediction of Antineoplastic Drug Resistance With an In Vitro Assay Using Suprapharmacologic Drug Exposures

David H. Kern and Larry M. Weisenthal

Abstract

Bayes' theorem has been used to describe the relationship between the accuracy of a predictive test (posttest probability) and the overall incidence of what is being tested (pretest probability). Bayes' theorem indicates that laboratory assays will be accurate in the prediction of clinical drug resistance in tumors with high overall response rates (e.g., previously untreated breast cancer) only when the assays are extremely (>98%) specific for drug resistance. We developed a highly specific drug-resistance assay in which human tumor colonies were cultured in soft agar and drugs were tested at high concentrations for long exposure times. Coefficients for concentration x time exceeded those reported in contemporaneous studies by about 100-fold. We reviewed 450 correlations between assay results and clinical response over an 8-year period. Results were analyzed by subsets, including different tumor histologies, single agents, and drug combinations. Extreme drug resistance (an assay result ≥ SD below the median) was identified with greater than 99% specificity. Only one of 127 patients with tumors showing extreme drug resistance responded to chemotherapy. This negligible post-test probability of response was independent of pretest (expected) probability of response. Once this population of patients with tumors showing extreme drug resistance had been identified, posttest response probabilities for the remaining cohorts of patients varied according to both assay results and pretest response probabilities, precisely according to predictions based on Bayes' theorem. This finding allowed the construction of a nomogram for determining assay-predicted probability of response.

J Natl Cancer Inst 82:582–588, 1990

[url]http://jnci.oxfordjournals.org/content/82/7/582.abstract?sid=7d0e6015-79b5-4804-ac8e-cdc61b00d3e9