Cells studied in 3-D may reveal novel cancer targets

Stephanie Fraley

Stephanie Fraley, a doctoral student in chemical and biomolecular engineering, was lead author of the study. Photo by Will Kirk/HomewoodPhoto.jhu.edu

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.

“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.”

collagen fibers

Reflection confocal micrograph of collagen fibers of a 3D matrix with cancer cells embedded. Image by Stephanie Fraley/Wirtz Lab

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–contacts too small to see and too short-lived to even measure, the researchers observed.

“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.

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

Related links:

Johns Hopkins Engineering in Oncology Center

Department of Chemical and Biomolecular Engineering

Watch a related video on YouTube

Story by Mary Spiro

Drazer wins NSF Career Award

German Drazer

German Drazer (Photo: Will Kirk)

German Drazer, assistant professor in the Department of Chemical and Biomolecular Engineering and affiliated faculty member of Johns Hopkins Institute for NanoBioTechnology was recently named a recipient of the National Science Foundation Faculty Early Career Development (CAREER) awards, given in recognition of a young scientist’s commitment to research and education. Drazer was given the award for “Deterministic and Stochastic Transport of Suspended Particles in Periodic Systems: Fundamentals and Applications in Separation Science.” The grant will support his investigations into the transport phenomena that arise in the motion of suspended particles in spatially periodic systems, and the translation of these phenomena into new principles for the manipulation of suspended particles in fluidic devices.

Read more about the work in the Drazer Lab here.

INBT researchers use LEGO to study what happens inside lab-on-a-chip devices

Animator, scientist partner to illustrate cover of Advanced Materials

AM_3_U1resizeThe cover of the January 19, 2010 issue of the journal Advanced Materials features a photo illustration executed by Martin Rietveld, web director and animator at Johns Hopkins Institute for NanoBioTechnology. Rietveld’s work illustrates an article about chemomechanical actuators—grippers that open and close like a hand in response to chemical reactions. The paper is based on the research of lead author, doctoral student Jatinder Randhawa in the laboratory of David Gracias, associate professor of chemical and biomolecular engineering and faculty affiliate of the Institute for Nanobiotechnology. Randhawa conceptualized the illustration of his research for the journal cover.

Says Gracias, “Chemomechanical actuation is intellectually appealing since it is widely observed in nature, but chemomechanical actuation is relatively unexplored in human engineering where the dominant strategy to actuate structures is based on electromechanical actuation (i.e. with electrical signals). Here, microstructures open and close reversibly in response to chemical surface oxidation and reduction without the need for any wires or batteries.”

Related links:

Chemomechanical Actuators: Reversible Actuation of Microstructures by Surface-Chemical Modification of Thin-Film Bilayers. Jatinder S. Randhawa, Michael D. Keung, Pawan Tyagi, David H. Gracias.

Johns Hopkins Institute for NanoBioTechnology Animation Studio

David Gracias INBT Faculty Profile

Chemical and biomolecular engineer Denis Wirtz named Smoot professor

Denis Wirtz. Photo by Will Kirk/JHU

Denis Wirtz. Photo by Will Kirk/JHU

Denis Wirtz, Johns Hopkins University professor of chemical and biomolecular engineering and director of the Engineering in Oncology Center, has been named the Theophilus Halley Smoot Professor in the Whiting School of Engineering. University president Ronald J. Daniels and the Board of Trustees determined the recipient.

Wirtz is the founding associate director of the Johns Hopkins Institute for NanoBioTechnology. He was recently named a 2009 fellow of the American Academy for the Advancement of Science in the Engineering Section for his contributions to cell micromechanics, cell adhesion, and for the development and application of particle tracking methods that probe the micromechanical properties of living cells.

He is on the Editorial Boards of Biophysical Journal, Cell Adhesion and Migration and J. Nanomedicine. In 2005, he was named a fellow of the American Institute for Medical and Biological Engineering. Wirtz won the National Science Foundation Career Award in 1996 and the Whitaker Foundation Biomedical Engineering Foundation Award in 1997.

Wirtz came to Johns Hopkins faculty in 1994 and completing a postdoctoral fellowship in Physics and Biophysics at ESPCI (ParisTech). Wirtz earned his PhD in Chemical Engineering from Stanford University in 1993.

An announcement from the Whiting School’s dean Nick Jones stated that, “Throughout his time at Johns Hopkins, Denis has distinguished himself as an outstanding scholar and teacher. Additionally, Denis’ role as a catalyst for interdisciplinary research and collaboration at the university has proven extremely effective, both in terms of the research he conducts and the support he has attracted over the years. I am confident that his current research into the physical basis for cell adhesion and de-adhesion will prove critical to our understanding of the metastasis of cancer and enable important breakthroughs in the diagnosis and treatment of cancer in the years to come.”

The Smoot Professorship was established in 1981 through the estate of Theophilus H. Smoot, who joined Johns Hopkins as a research assistant in the Department of Mechanical Engineering in 1942 and later a research associate in the department in 1946. Upon the passing of Mr. Smoot in 1976 and his widow, Helen A. Smoot in 1980, the Theophilus Halley Smoot Fund for Engineering Science was created.  The first Smoot Professorship was awarded in 1981 to Stanley Corrsin, a professor and former chair in the department of mechanical engineering. Robert E. Green, Jr., professor in the department of materials science, held the professorship from 1988 through 2007.

Presentation of the Smoot professorship will occur in the spring.

Wirtz Lab

Named Professorships of The Johns Hopkins University

Johns Hopkins Institute for NanoBioTechnology

Johns Hopkins Engineering in Oncology Center

Story by Mary Spiro and from materials provided by the Whiting School of Engineering.