Students talk cancer nanotech at Homewood March 21

Students affiliated with the Center of Cancer Nanotechnology Excellence (CCNE) and the Physical Sciences-Oncology Center (PS-OC) at Johns Hopkins University have organized a spring mini-symposium for March 21, 10 a.m. in the Hackerman Hall Auditorium at the Johns Hopkins University Homewood campus.

The student-run mini-symposiums aim to bring together researchers from across the campus affiliated with the PS-OC and CCNE. Graduate students training in these centers, both administered by Johns Hopkins Institute for NanoBioTechnology, work in various disciplines from physics to engineering to the basic biological sciences but with an emphasis on understanding cancer metastasis and developing methods for cancer diagnosis or therapy.

The invited speaker for the symposium is postdoctoral researcher Megan Ho of Duke University. Ho earned her PhD in mechanical engineering in the Wang lab in 2008. She is currently focused on developing microfluidic devices to investigate and control the fundamental reactions that form nanocomplexes for gene delivery. (10 a.m.)

Student apeakers, who will talk for 15 minutes, include:

  • Jane Chisholm (Justin Hanes lab/Ophthalmology): Cisplatin nanocomplexes for the local treatment of small cell lung cancer (10:20 a.m.)
  • Yunke Song (Jeff Wang Lab/Mechanical Engineering): Single Quantum Dot-Based Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction (10:35 a.m.)
  • Andrew Wong (Peter Searson Lab/Materials Science and Engineering): Intravisation into an artificial blood vessel (10:50 a.m.)
  • Brian Keeley: (Jeff Wang Lab/Mechanical Engineering): Overcoming detection limitations of DNA methylation in plasma and serum of cancer patients through utilization of nanotechnology. (11:05 a.m.)
  • Sebastian Barretto (Sharon Gerecht Lab/Chemical and Biomolecular Engineering): Development of Hydrogel Microfibers to Study Angiogenesis (11:20 a.m.)

View the symposium flyer here. The mini-symposium is free and open to the entire Johns Hopkins University community. No RSVP is required, although seating is limited.

Johns Hopkins Physical Sciences-Oncology Center

Center of Cancer Nanotechnology Excellence

It’s a small world: Micro/nanotechnology in regenerative medicine and cancer

Sageeta Bhatia

Nanotechnology, regenerative medicine and cancer will be the topic of a special biomedical engineering seminar on March 6 at 3 p.m. in the Darner Conference Room, Ross Building, Room G007 at the Johns Hopkins School of Medicine. Speaker Sangeeta Bhatia, MD, PhD, director, of the Laboratory for Multiscale Regenerative Technologies at Massachusetts Institute of Technology will present “It’s a small world: Micro/Nanotechnology in Regenerative Medicine and Cancer. ” She will discuss the role of micro and nanotechnology for mimicking, monitoring and perturbing the tissue microenvironment.

“I will present our work on reconstructing normal liver microenvironments using microtechnology, biomaterials and induced pluripotent stem cells as well as our work on normalizing diseased cancer microenvironments using both inorganic and organic nano materials,” Bhatia noted in an announcement.  Bhatia is a professor of Health Sciences and Technology and professor of Electrical Engineering and Computer Science at MIT.

The talk is hosted by associate professor of Materials Science and Engineering and affiliated faculty member of the Institute for NanoBioTechnology Hai-Quan Mao. The event is free and open to the Johns Hopkins Community. Refreshments will be served.

 

 

Panel discussion tackles the question: Is undergraduate research for you?

Undergraduates presenting at summer research symposium.

Are you an undergraduate  engineering student who wants to do research but just doesn’t know where to start?

The Johns Hopkins chapter of the Society of Women Engineers  will host a panel discussion Thursday, October 27 at 7 PM in room 132 of Gilman Hall  on the Homewood campus.  The panel discussion is designed to answer your questions about getting started in research at Johns Hopkins University.   Listen to a panel of undergraduate research students in engineering discuss what it’s like to work in an engineering lab.

Undergraduate research experience is extremely important if you want to apply for internships, jobs, scholarships and postgraduate work. Conducting research while you’re an undergraduate also helps put this ideas that you’ve learned in class into action for larger goal. Some undergraduate researchers  even have their work published in peer-reviewed journals.

Johns Hopkins Institute for NanoBiotechnology offers a summer research experience for undergraduates in nano bio.   A criteria for applying to an REU  program is that you have had prior research experience.  Don’t miss your opportunity to learn about this exciting component of your undergraduate academic career.

For more information about the Society of Women Engineers go to http://www.jhu.edu/swe/index.html

For details about  about Johns Hopkins Institute for Nano Biotechnology summer Research Experience for Undergraduates program, go to http://inbt.jhu.edu/education/undergraduate/reu/

Applications for the 2012 summer program will be accepted soon.

Hopkins faculty to present at American Society for NanoMedicine meeting

© Liudmila Gridina | Dreamstime.com

The American Society for NanoMedicine (ASNM) will hold its third annual meeting November 9 -11 at the Universities at Shady Grove Conference Center in Gaithersburg, Md. This year ASNM has worked closely with the Cancer Imaging Program, National Cancer Institute, and National Institutes of Health to create a conference with a special focus on nano-enabeled cancer diagnostics and therapies, and the synergy of the combination of nano-improved imaging modalities and targeted delivery.

The program also focuses on updates on the newest Food and Drug Administration, nanotoxicity, nanoparticle characterization, nanoinformatics, nano-ontology, results of the latest translational research and clinical trials in nanomedicine, and funding initiatives. This year’s keynote speaker is Roger Tsien, 2008 Nobel Prize Laureate. Numerous other speakers and breakout sessions are planned for the three day event. Two speakers affiliated with Johns Hopkins include Justin Hanes and Dmitri Artemov. Hanes is a professor of nanomedicine in the department of ophthalmology at the Johns Hopkins School of Medicine. Artemov is an associate professor of radiology/magnetic resonance imaging research, also at the School of Medicine.

The deadline for the poster abstracts is October 1. The top four posters submitted by young (pre and post doctoral) investigators will be selected to give a short 10-minute (eight slides) oral presentation on November 11.

ASNM describes itself as a “a non-profit, open, democratic and transparent professional society…focus(ing) on cutting-edge research in nanomedicine and moving towards realizing the potential of nanomedicine for diagnosis, treatment, and prevention of disease.” More information about the ASNM can be found on the Society’s official website.

 

 

Agenda set for Oct. 10 mini-symposium on cancer, nanotech

From the spring mini-symposium.

Johns Hopkins Physical Sciences-Oncology Center and Center of Cancer Nanotechnology Excellence will host a mini-symposium on Monday Oct., 10 in the Hackerman Hall Auditorium. Talks on topics related to cancer and nanotechnology begin at 9 a.m.

Speakers include:

  • 9:15 a.m.: The pulsating motion of breast cancer cell is regulated by surrounding epithelial cells. Speaker: Meng Horng Lee
  • 9:40 a.m.: Breast tumor extracellular matrix promotes vasculogenesis. Speaker: Abigail Hielscher
  • 10:00 a.m.: Attachment to growth substrate regulates expression of GDF15, an important molecule in metastatic cancer. Speaker: Koh Meng Aw Yong
  • 10:20 a.m.: Mucin 16 is a functional selectin ligand on pancreatic cancer cells. Speaker: Jack Chen
  • 10:40 a.m.: Particle tracking in vivo. Speaker: Pei-Hsun Wu

These talks are open to the entire Hopkins community. No RSVP is required. Refreshments will be served.

 

 

Nanobio postdocs offer trusted tips on getting grant money

Photo illustration by Mary Spiro.

Three postdoctoral fellows from Johns Hopkins Institute for NanoBioTechnology will offer a one-hour crash course in how to get those research dollars; July 27, 11 a.m. Krieger 205. Free for Hopkins community.

Funding dollars make the research world go ‘round. Few know that better than postdoctoral fellows, who would be out of work without it. As part of Johns Hopkins Institute for NanoBioTechnology’s last professional development seminar of the summer, three INBT affiliated postdoctoral fellows will offer their sage advice on preparing winning research grants.

Topics to be covered on the basic aspects of grant writing include:

  • knowing when to write a grant
  • identifying funding sources
  • planning a timeline
  • how to structure a competitive proposal
  • do’s and dont’s of grant writing and planning
  • basic science writing tips for conveying ideas clearly and succinctly

This seminar will be led by Eric Balzer, postdoctoral fellow with professor Konstantinos Konstantopoulos (ChemBE); Yanique Rattigan, postdoctoral fellow with professor Anirban Maitra (Oncology/Pathology); and Daniele Gilkes, postdoctoral fellow with professor Denis Wirtz (ChemBE).

For additional information on INBT’s professional development seminar series, contact Ashanti Edwards, INBT’s Academic Program Administrator at Ashanti@jhu.edu.

 

 

 

 

‘Just add water’ to activate freeze-dried brain cancer fighting nanoparticles

A fluorescence micrograph showing brain cancer cells producing a green fluorescent protein. DNA encoded to produce the protein was delivered to the cancer cells by new freeze-dried nanoparticles produced by Johns Hopkins biomedical engineers. Image: Stephany Tzeng/JHU

Biomedical engineers and clinicians at Johns Hopkins University have developed freeze-dried nanoparticles made of a shelf-stable polymer that only need the addition of water to activate their cancer-fighting gene therapy capabilities.

Principal investigator Jordan Green, assistant professor in the department of Biomedical Engineering at the Johns Hopkins School of Medicine, led the team that fabricated the polymer-based particles measuring 80 to 150 nanometers in diameter. Each particle, which is about the size of a virus, has the ability to carry a genetic cocktail designed to produce brain cancer cell-destroying molecules. After manufacture, the nanoparticles can be stored for up to 90 days before use. In principle, cancer therapies based on this technology could lead to a convenient commercial product that clinicians simply activate with water before injection into brain cancer tumor sites.

Because this method avoids the common, unpleasant side effects of traditional chemotherapy, “nanoparticle-based gene therapy has the potential to be both safer and more effective than conventional chemical therapies for the treatment of cancer,” Green said. But, he added current gene therapy nanoparticle preparations are just not practical for clinical use.

“A challenge in the field is that most non-viral gene therapy methods have very low efficacy. Another challenge with biodegradable nanoparticles, like the ones used here is that particle preparation typically takes multiple time-sensitive steps.” Green said. “Delay with formulation results in polymer degradation, and there can be variability between batches. Although this is a simple procedure for lab experiments, a clinician who wishes to use these particles during neurosurgery will face factors that would make the results unpredictable.”

In contrast, the nanoparticles developed by the Green lab are a freeze-dried, or “lyophilized,” formulation. “A clinician would simply add water, and it is ready to inject,” Green said. Green thinks this freeze-dried gene-delivery nanoparticle could be easily manufactured on a large scale.

Co-investigator Alfredo Quinones-Hinojosa, a Johns Hopkins Hospital clinician-scientist and associate professor in the departments of Neurosurgery and Oncology at the Johns Hopkins School of Medicine, said he could imagine particles based on this technology being used in conjunction with, and even instead of, brain surgery. “I envision that one day, as we understand the etiology and progression of brain cancer, we will be able to use these nanoparticles even before doing surgery,” Quinones said. “How nice would that be? Imagine avoiding brain surgery all together!”

Currently, patients with glioblastoma, or brain cancer, only have a median survival of about 14 months, Green said. “Methods other than the traditional chemotherapy drugs and radiation—or in combination with them—may improve prognosis,” he said.

Gene therapy approaches could also be personalized, Green said. “Because gene therapy can take advantage of many naturally-existing pathways and can be targeted to the cancer type of choice through nanoparticle design and transcriptional control, several levels of treatment specificity could be provided,” Green said.

The nanoparticles self-assemble from a polymer structural unit, so fabrication is fairly simple, said Green. Finding the right polymer to use, however, proved to be a challenge. Lead author Stephany Tzeng, a PhD student in biomedical engineering in Green’s lab screened an assortment of formulations from a “polymer library” before hitting on a winning combination.

“One challenge with a polymer library approach is that there are many polymers to be synthesized and nanoparticle formulations to be tested. Another challenge is designing the experiments to find out why the lead formulation works so well compared to other similar polymers and to commercially available reagents,” Green said.

Tzeng settled on a particular formulation of poly(beta-amino ester)s specifically attracted to glioblastoma (GB) cells and to brain tumor stem cells (BTSC), the cells responsible for tumor growth and spread. “Poly(beta-amino ester) nanoparticles are generally able to transfect many types of cells, but some are more specific to GBs and BTSCs,” Tzeng said.

The nanoparticles work like a virus, co-opting the cell’s own protein-making machinery, but in this case, to produce a reporter gene (used to delineate a tumor’s location) or new cancer fighting molecule. “It is possible that glioblastoma-derived cells, especially brain tumor stem cells, are more susceptible to our gene delivery approach because they divide much faster,” Tzeng added.

Not only are the particles convenient to use, the team discovered that dividing cells continued to make the new protein for as long as six weeks after application. “The gene expression peaked within a few days, which would correspond to a large initial dose of a therapeutic protein,” said Green. “The fact that gene expression can continue at a low level for a long time following injection could potentially cause a sustained, local delivery of the therapeutic protein without requiring subsequent injection or administration. The cells themselves would act as a ‘factory’ for the drug.”

Once the nanoparticles release their DNA cargo, Tzeng said the polymer quickly degrades in water, usually within days. “From there, we believe the degradation products are processed and excreted with other cellular waste products,” Tzeng said.

Members of the Green Lab are now working on identifying the intracellular mechanism responsible for facilitating cell-specific delivery. “We also plan to build additional levels of targeting into this system to make it even more specific. This includes modifying the nanoparticles with ligands to specifically bind to glioblastoma cells, making the DNA cargo able to be expressed only in GB cells, and using a DNA sequence whose product is only effective in GB cells.”

So far, the team has only successfully transfected brain tumor stem cells using these nanoparticles in a plastic dish. The next step is to test the particle in animal models.

“We hope to begin tests in vivo in the near future by implanting brain tumor stem cells into a mouse and injecting particles. We also hope to begin using functional genes that would kill cancer cells in addition to the fluorescent proteins that serve only as a marker,” Tzeng said.

Other authors who contributed to this work are Hugo Guerrero-Cázares, postdoctoral fellow in Neurosurgery and Oncology, and Joel Sunshine, an M.D.-Ph.D. candidate, and Elliott Martinez, an undergraduate leadership alliance summer student, both from Biomedical Engineering. Funding for this work came from the National Institutes of Health, Howard Hughes Medical Institute, the Robert Wood Johnson Foundation and a pilot-grant from Johns Hopkins Institute for NanoBioTechnology (INBT). Green is an affiliated faculty member of INBT. The research will be published in Issue #23 (August 2011) of the journal Biomaterials and is currently available online.

Freeze-dried gene therapy system avoids virus, complications

Story by Mary Spiro

 

Johns Hopkins Integrated Imaging Center focuses on data

Shyenne Yang positions Drosophila embryos for fluorescence imaging. Photo by Marty Katz/baltimorephotographer.com

Heavy, black curtains and dimmed lights shroud the core of the Johns Hopkins Integrated Imaging Center (IIC). Yet researchers who peer through the advanced microscopes cloaked by these dark draperies view experimental samples more clearly than ever thanks to a combination of the high-tech equipment and the creative expertise offered by the center’s seven-member staff.

When describing Johns Hopkins University’s showpiece microscopy facility, it’s easy to rattle off a laundry list of available equipment and laboratory space able to prepare samples with nearly any contrasting agent found in the literature. The Homewood-based center contains devices that can image a sample in virtually any manner in 2-D, 3-D and even 4-D. IIC’s 3,500 square-foot facility comprising space in Dunning, Jenkins, and Olin Halls, boasts more than $7.5 million worth of state- of-the-art imaging equipment, including a Zeiss laser scanning microscope (LSM) 510 VIS confocal with a Confocor 3 fluorescence correlation spectroscopy (FCS) module—one of only a very few such uniquely configured laser scanning microscopes in the United States.

Director J. Michael McCaffery, a research professor in the Department of Biology at the Krieger School of Arts and Sciences, said the Hopkins community is thrilled to have access to such a versatile microscope with fluorescence correlation spectroscopy that is capable of cross-correlation analysis, with confocal imaging and a fully enclosed environmental system for live imaging. Researchers affiliated with Johns Hopkins Institute for NanoBioTechnology (INBT), the Johns Hopkins Physical Sciences Oncology Center and Center of Cancer Nanotechnology Excellence are also glad to have access to IIC’s menu of facilities.

“Fluorescence correlation spectroscopy allows for high-resolution spatial and temporal analysis of single biomolecules with respect to diffusion, binding, as well as enzymatic reactions in vitro and in vivo,” McCaffery said. In other words, you can see and measure a lot of really tiny stuff with it, something INBT affiliated researchers working at micron/nanometer resolutions are finding incredibly useful.

The center features multiple suites devoted to specific microscopy/imaging functions, as well as facilities for all manner of sample preparation. All these advanced tools help scientists and engineers characterize nanomaterials; and image cells, sub-cellular organelles, and biomolecules/ proteins at very small dimensions. But none of this fancy equipment would be of much use to researchers without the expertise of McCaffery and the IIC staff. McCaffery brings years of experience and a background in cell biology and microbiology. The center’s associate director, William Wilson, an associate research professor in the Department of Materials Science and Engineering at the Whiting School of Engineering, describes himself as a “chemist, turned physicist, who became an electrical engineer, who is now a materials scientist.”

Staff scientist Kenneth J.T. Livi, director of the IIC’s High-Resolution Analytical Electron Microbeam Facility located in Olin Hall, offers his unique perspective on earth and planetary sciences. Researchers can also consult with microscopy specialist/ trained biologist and FACS supervisor Erin Pryce, the FACS manager Yorke Zhang, computer/IT specialist Marcus Sanchez, and research assistants Leah Kim and Adrian Cotarelo, who both are currently earning their bachelor degrees in biology at Johns Hopkins.

From left, IIC director Michael McCaffery, FACS supervisor Erin Pryce, and associate director William Wilson with the BD FACSVantage SE. Photo by Mary Spiro

“Sometimes young researchers haven’t contemplated all the possibilities of how to use and apply an instrument; and don’t realize there are many different ways to utilize familiar tools in order to obtain new, in some cases better, information,” McCaffery said. “Our desire is always to approach a problem from many disparate perspectives to generate convergent data that corroborates each particular assay. Hopefully, results from each individual assay, allows the scientist to arrive at a convergent perspective that yields confidence in the results and conclusions.”

One of the easiest ways to obtain different microscopy data and improve corroboration among assays is simply to change the contrast mechanism.

“The most common contrast mechanisms used to image something are optical contrast (transparent versus opaque), polarization, and fluorescence,” said Wilson. “But there are many different ways you can manipulate how light interacts with the specimen and what you detect out of an objective.”

For example, ultrafast laser sources have made nonlinear optical forms of contrast an exciting new tool. Techniques like two-photon excited fluorescence and second harmonic generation (both available in the IIC) produce excellent spectral and structural information about samples because a smaller effective photon volume is excited. Wilson explained it like this: “Imagine turning your stereo all the way up and hearing the sound distorted. That distortion is created by the higher order acoustic harmonics from your stereo. The same happens with intense laser light resulting in new “colors” being generated from the object irradiated. The cool thing is that the different non-linear processes are often sensitive to different physical proper- ties or structural features, offering complementary information about your sample.”

In some cases, getting more detailed information simply requires looking at the right color range. The two-photon fluorescence and second harmonic signals appear at different wavelengths. If you excite a sample with enough energy to generate third order harmonics, that signal is detected at an even bluer wavelength, Wilson said. “With third harmonic generation, you only get signals from the interface of structures with no interference from anything else. This means you can simultaneously image fluorescence, polar order, and interface dynamics just by popping in a few filters and beam splitters,” he said.

“Over the past ten or so years, physicists and engineers focused on advanced microscopy, have produced better and more advanced laser and optical technologies, generating techniques that many researchers in the biological and biomedical sciences might not know exist,” Wilson said. “There also are a lot of applied physicists who are developing and using these new technologies who don’t know what an interesting sample is. We hope to help bridge this gap, becoming a place where these collaborative synergies can flourish.”

Sample preparation is another area where the center can help researchers. “Cell fractionation, for example, which is the breaking down of whole cells and separating them into their individual components, when combined with biochemical techniques and microscopy, can often allow researchers to pose more precise questions and to better analyze a biological problem,” McCaffery said.

“It is common for someone to come in and want to use a particular instrument or technique they read about in a paper,” McCaffery said. When that happens, McCaffery and Wilson are likely to give researchers “homework.”

“It’s important to remember that the goal is not to make a pretty picture,” Wilson said. “The goal is to answer a question, so sometimes we have to ask them, ‘What is your research question?’” An enviable set of microscopy tools combined with a team that brings years of training and experience from a variety of disciplines sets Johns Hopkins Integrated Imaging Center apart from the microscope on the individual researcher’s lab bench, as well as from facilities nationwide. Wherever possible, McCaffery said, IIC staff tries to be engaged in all of the research that is carried out in the center. “Simply, our involvement leads to better results and better science,” McCaffery added.

Researchers confirm this successful combination.

“The facilities at the IIC have allowed us to obtain critical information about the internal structure of our peptide nanomaterials that would have remained unknown without careful electron and fluorescence microscopy,” said J.D. Tovar, assistant professor of Chemistry. “Equally important, the scientific IIC staff members were vital participants making sure collaborative experiments were done meaningfully and students were trained competently. Our collaboration with Dr. Wilson has given some nice insights and at the same time has posed many more questions for future research.”

Praise like that for the IIC is always nice to hear, staff members say, but they emphasize that the services and tools they provide are just part of the job. “Part of being a scientist is learning not only how to gather information from a wide variety of tools but also understanding how to pose clear questions that lead to the right tools, in a nutshell, how to not waste time. If we can help you do that, then we have achieved our goal,” Wilson said.
This story originally appeared in Johns Hopkins Nano-Bio Magazine.

To read more about IIC’s facilities and services, go here.

Story by Mary Spiro

Photos by Mary Spiro and Marty Katz

 

Gerecht wins NSF CAREER Award for work in blood vessel formation

Sharon Gerecht (Photo:Will Kirk/JHU)

Sharon Gerecht, assistant professor in Chemical and Biomolecular Engineering at Johns Hopkins University, has been awarded the Faculty Early Career Development (CAREER) Award from the National Science Foundation. The $450,000 prize over five years will help Gerecht in her investigation into how hypoxia, or decreased oxygen, affects the development of blood vessels.

Gerecht’s interdisciplinary research brings together her expertise in stem cell and vascular biology with her background in engineering.  Gerecht said she hopes to discover the mechanisms and pathways involved in the formation of vascular networks, as they relate to embryonic development and diseases such as cancer.

Many medical conditions, such as cancer and heart disease, create areas of decreased oxygen or hypoxia in the spaces between cells. But oxygen is required to maintain normal tissue function by blood vessel networks, which bring nutrients to cells. Likewise, the differentiation of stem cells into more complex organs and structures needs a plentiful supply of oxygen from the vasculature to function.

Gerecht’s study will examine how low oxygen levels impact the growth factors responsible for promoting vascular networks. She also will study the growth of vascular networks in engineered hydrogels that mimic the physical attributes of the extracellular matrix, which is the framework upon which cells divide and grow. Finally, her laboratory will focus on discovering how stem cells differentiate to blood vessel cells and assemble into networks under hypoxic conditions.

She will conduct her research through her role as a project director at the Johns Hopkins Engineering in Oncology Center (EOC), a Physical Science-Oncology Center of the National Cancer Institute. Gerecht is also an associated faculty member of the Johns Hopkins Institute for NanoBioTechnology, which administers the EOC.

Gerecht earned her doctoral degree from Technion – Israel Institute of Technology followed by postdoctoral training at Massachusetts Institute of Technology. She joined the faculty of the Whiting School of Engineering at Johns Hopkins in 2007.

The prestigious CAREER award, given to faculty members at the beginning of their academic careers, is one of NSF’s most competitive awards and emphasizes high-quality research and novel education initiatives. It provides funding so that young investigators have the opportunity to focus more intently on furthering their research careers.

Story by Mary Spiro

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