Studying Cells in 3-D Could Reveal New Cancer Targets

[gpawelski]

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

[gpawelski]

Cell-based chemoresponse assays test fresh "live" cells in their three dimensional (3D), floating clusters (in their natural state), not passaged cells (cell-lines). Established cell-line is not reflective of the behavior of "fresh" tumor cells in primary culture in the lab, much less in the patient. 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 (3D), floating clusters. Real life 3D analysis makes chemoresponse assays indicative of what will happen in the body.

One of the problems with genetic tests is in evaluating the data which exists to validate the predictive accuracy of them. Generally, a large number of archival specimens are batch processed together, within a very narrow time frame, by the same research team, so all the technical variables are minimized, which makes it much easier to get good results than in a "real world" setting, where specimens are tested over a period of weeks, months, years, by different people, with different laboratory reagents, as occurs in the "real world."

Evaluating "real world" data, requires specimens that are tested as they are logged into the lab in question, in "real time." No one is publishing "real world" studies, except private laboratories performing cell-based chemoresponse assays, which can only do "real world" studies, because their studies require fresh, viable specimen, which must be accessioned and tested in "real time," under "real world" conditions.

The "cell-death" assays are not growing anything. They are testing a drug or combinations of drugs with cells that are in their natural state (live or fresh). Three dimensional tumor cell clusters. Clusters maintain natural cell-cell interactions. This makes the assays indicative of what will happen in the body. The protocol takes "fresh" patient tumor cells and floats them in newer 3D cell suspensions.

As the researchers at Johns Hopkins and Washington University have found out, our body is 3D, not 2D in form, undoubtedly, this novel step better replicates that of the human body. Traditionally, in-vitro (in lab) cell-lines have been studied in 2 dimensions (2D) which has inherent limitations in applicability to real life 3D in-vivo (in body) states. Recently, other researchers have pointed to the limitations of 2D cell line study and chemotherapy to more correctly reflect the human body.

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)

[gpawelski]

Semin Cancer Biol. 2005 Oct;15(5):365-77.

Three-dimensional tissue culture models in cancer biology.

Kim JB.

Ludwig Institute for Cancer Research, First Floor - Breast Cancer Laboratory, Department of Surgery, Royal Free and University College London Medical School, Charles Bell House, 67-73 Riding House Street, London W1W 7EJ, UK. jongbkim2001@yahoo.co.uk

Abstract

Three-dimensional (3D) tissue culture models have an invaluable role in tumour biology today providing some 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 tumour 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 tumour models.

PMID: 15975824 PubMed

[gpawelski]

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

[gpawelski]

University of Pittsburgh researchers have devised a three-dimensional system in laboratory culture that mimics the growth patterns of colon cancer stem cells in patients. Their findings were presented at the American Association for Cancer Research special conference on Colorectal Cancer: Biology to Therapy, held Oct. 27-30, 2010.

The assay, which uses green fluorescent "reporter" proteins to watch the process of stem cell differentiation, is designed to understand how these cancer stem cells behave, and to identify and test therapies that could halt production of the endless generations of new cancer stem cells that continually revive a tumor.

"Colon cancer stem cells are thought to be the root of therapy resistance, metastases and recurrence in colon cancer, so our approach is to find a way to remove the ability of these stem cells to self-renew," said the study's lead investigator, Julie Chandler, a graduate student in pathology.

"While many labs have investigated notch inhibitors and others have investigated cancer stem cells, our unique approach combines both in a three-dimensional culture that mimics what happens in patients," she said. Animal models, which are immunodeficient and use human xenografs, may not provide accurate information about colon cancer stem cell behavior, Chandler added.

Colon cancer stem cells have the ability to repopulate a tumor after treatment, using stem cells that are resistant to treatment. Such treatment forces a response in these cells, which are genetically unstable, forcing the cells to adapt and pass on resistance to daughter stem cells.

In the same way that adult intestinal stem cells self-renew, colon stem cells give rise to different kinds of cells, including daughter stem cells and fully differentiated cells, such as the goblet epithelial cells that line the colon. Researchers would like to force cancer stem cells to differentiate and behave like goblet cells because these cells do not self renew. Chandler said the notch pathway that controls differentiation in stem cells is inactivated in goblet cells. One way to possibly do that is to use agents that shut down the notch pathway, such as gamma secretase inhibitors, she said. Cancer treatment may then be able to destroy tumors that are now populated by fully differentiated goblet cells.

In their new assay, Chandler used a three-dimensional culture matrix in which she could watch a single cancer stem cell divide and produce progeny, which is called an "independent organoid."

To see the kind of cells a colon cancer stem cell produces, they labeled a protein that is specific only to goblet cells. To date, the researchers have found that some colon cancer stem cells produce many differentiated cells, such as goblets and others, while others produce more primitive, self-renewing cells.

In this way, the researchers can test the ability of notch pathway inhibitors to force progeny cancer stem cells to differentiate into harmless goblet cells.

"Green goblet cells are no longer capable of promoting cancer growth," Chandler said. "It may be that a certain notch inhibitor or similar drug is all that is needed to prevent cancer recurrence and metastasis that so often follows an initial response to treatment. This new tool will help us determine if that is so."

Source: American Association for Cancer Research