Studying cells in 3D, the way it should be

When scientists experiment on cells in a flat Petri dish, it’s more been a matter of convenience than anything that recapitulates what that cell experiences in real life. Johns Hopkins professor Denis Wirtz for some time has been growing and studying cells three dimensions, rather than the traditional two dimensions. And pretty much, he’s discovered that a lot of what we think we know about cells is dead wrong.

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Cell in 3D. Image by Anjil Giri/Wirtz Lab

In this recent article by Johns Hopkins writer Dale Keiger, you will discover what Wirtz has discovered through his investigations. Furthermore, you will find out about the man behind these revolutionary ideas that are turning basic cell biology upside-down, as well as challenge a lot of what we thought we understood about diseases like cancer.

Wirtz directs the Johns Hopkins Physical-Sciences Oncology Center and is associate director and co-founder of Johns Hopkins Institute for NanoBioTechnology. He recently launched the Center for Digital Pathology. He is a the Theophilus Halley Smoot professor of chemical and biomolecular engineering.

You can read the entire magazine article “Moving cancer research out of the Petri dish and into the third dimension” online here at the JHU Hub.

Picture this: alumni meet up at AIChE annual

From left, Denis Wirtz, Christopher Hale and Terrence Dobrowsky

From left, Denis Wirtz, Christopher Hale and Terrence Dobrowsky

Two alumni of Johns Hopkins Institute for NanoBioTechnology. Dr. Chris Hale and Dr. Terrence Dobrowsky, recently met up with INBT co-Director Denis Wirtz at the annual meeting of the AIChE, held Nov. 3-8 in San Francisco. Chris and Terrence are currently work at Amgen and Biogen, respectively.

Chris and Terrence were both PhD students in Wirtz’s laboratory in the Department of Chemical and Biomolecular Engineering.

Taking a digital perspective on cancer

Editor’s Note: This story was written by Bryan Kohrs, a junior in Biophysics at Johns Hopkins University  with a strong interest in science writing and science. It first appeared in the 2013 issue of Nano-Bio Magazine.

Denis Wirtz lines up next to many other scientists in the war on cancer. But while others battle with familiar technologies and ideas, Wirtz has armed himself with a new imaging technology, a fresh strategy on how to better combat this dreaded disease and a five year grant from the National Cancer Institute (NCI).

Wirtz, a professor of Chemical and Biomolecular Engineering at Johns Hopkins, is carving out a niche for himself in cancer research by focusing on the look and physical structure of cancerous cells rather than the genes from which the cells originated. He believes that this technology will not only help doctors predict how cancer progresses, but will eventually change the way cancer is treated from a therapeutic standpoint. The technology is the keystone of the new Johns Hopkins Center for Digital Pathology, which Wirtz directs.

To understand how this technology works, consider this analogy: A cell is like a Lego brick. Just as individual Legos can come together to create a building, millions of different cells come together to make a human being. Certain bricks serve different purposes in a building in the same way that distinctive cells carry out certain functions in the body. The properties that make some Legos better suited for one purpose over another are their size and shape. For example, if a piece is flat and wide, it should go on the base. Structural characteristics that make cells unique include overall size, shape, the size and shape of the different cell parts or organelles, the composition of certain organelles, and hundreds of other parameters.

Pei-Hsun Wu of the Wirtz lab examining pancreatic cancer cells. (Photo by Mary Spiro)

Pei-Hsun Wu of the Wirtz lab examining pancreatic cancer cells. (Photo by Mary Spiro)

Wirtz’s technology uses a modified scanning electron microscope and a process called high-throughput cell phenotyping (HTCP) to instantly make hundreds of thousands of highly specific measurements defining each of these structural cellular characteristics of each single cell on a slide. Wirtz has software that uses an algorithm that adds up all of the different measurements and gives a cell a structural “score,” which quantifies the look of the cell with a number. The process will be automated and will take just minutes for a slide of cells to be analyzed and given an overall structural score, which averages the scores of all the cells on the slide.

Anirban Maitra, a professor of Oncology and Pathology at Johns Hopkins School of Medicine who is collaborating with Wirtz on this project, explains the benefits of automating this process, “If you were looking at a cell with the naked eye, you would say it has a large nucleus, medium sized nucleus, or a small nucleus. What automation allows you to do is to spread that crude three-tiered category into hundreds of small denominational events that you could then objectively add up and get a score.”

Over the course of the next five years, Wirtz plans to use HTCPanalysis as a clinically applicable tool that can help doctors treat cancer patients with more personalized therapies.

“Currently, we have a very crude approach to therapy even with the targeted therapies that are being developed. The vast majority of patients in cancer care and oncology get what are called cytotoxic agents, the old agents that were made many years ago,” said Maitra. But by using HTCP to see how cancers that look a certain way respond to certain treatments, doctors will be able to better personalize cancer treatments.

To make the project clinically applicable, Wirtz, with the help of Maitra and Ralph Hruban, also a professor of Oncology and Pathology at Johns Hopkins School of Medicine who is collaborating with the team, will be working to create the first “phenotypic database,” or a cell-feature-focused database. It will combine patient data like age, sex, cancer type, progression, treatment used, genetic sequencing results (analysis of tumor from a genetic standpoint), and so forth in an online, “cloud” database and then also add in the structure score of the patient’s tumor performed from HTCP.

At the moment, Hopkins is the only university with Wirtz’s new technology. The plan is for hospitals across the nation to begin uploading patient information to the database online and sending slides of cancer tumor cells to Hopkins or an alternate research facility using this technology. There, independent researchers will analyze the cells and add the HTCP analysis to the patient information online.

Denis Wirtz, right, working with recent PhD graduate Shyam Khatau. (Photo Will Kirk/Homewood Photography)

Denis Wirtz, right, working with recent PhD graduate Shyam Khatau. (Photo Will Kirk/Homewood Photography)

Doctors can upload all of this data into the cloud and help the database grow initially. Eventually, an oncologist in Chicago,  treating a 70-year-old man with lung cancer, and a HTCP score of X will be able to go online and find that there were two similar patients, a 65-year-old man with lung cancer in Baltimore and a 75-year-old woman with lung cancer in California, both with a score of X as well. The physician would discover that the man in Baltimore was treated with chemotherapy A and died in six months, while the woman in California was treated with chemotherapy B and was cured. Doctors will be able to make more informed treatment decisions.

Classifying the morphological characteristics of cancer is a shift from the traditional genetic approach to categorizing cancer cells. Previously, scientists researched cancer from a genetic standpoint, linking specific genetic mutations to specific cases of cancer. While this has lead to gene-targeted therapies, Wirtz wants to take a different approach to cancer research. He wants to look beyond the genetic origin of cancer and focus on what cancerous cells look like.

“We’ve come to realize that it is the heterogeneity – the diversity of cells that have different characteristics – is also important in evaluating a cancer case. In the end what matters are the cell properties.

That’s what we measure,” Wirtz explained. The rationale for this new approach, Wirtz explained, is that while cells can be identical genetically, they can vary tremendously in structure, just as two identical twins can develop to be very different people, both physically and personally. Cells from one tumor could become metastatic, latch onto a new organ, and start a new tumor that eventually kills the patient, while a genetically identical set of cells could remain localized and die as soon as they detach from the original tumor.

Wirtz’s theory is that the key to cancer treatment prognostication lies not in cancer genetics, but in the physical attributes of cancerous cells. For example, you could say that muscle definition and physical fitness would be strongly correlated with athletics and would therefore be able to be used to predict who out of twenty people would become athletes. Wirtz believes that his technology will allow doctors to do the same thing with cancer.

With this new technology Wirtz hopes to figure out what triggers cancer cells to metastasize. For example, do small, elliptical cells with large nuclei metastasize better than large, rod-like cells with small nuclei? He explained that cells that metastasize have to be super-cells, much like super-heroes are better, faster, and stronger than other humans.

“Millions of cells are shed by tumors every day, but only one or two of them will have what it takes to become metastatic. These  are the decathlon cells. We need to figure out what the physical properties are that give these cells an edge,” Wirtz says.

Maitra poses the question that guides the project in its applications towards therapeutic cancer treatment, “We have a lot of different drugs out there right now. Some work, some don’t. The problem is you only find out if they worked retroactively. You give it to a patient and six months later the metastasis keeps growing and you know if it’s worked or not. But wouldn’t it be nice if we knew going into the treatment that these patients would respond to a particular regimen and these other patients respond to another regimen?”

Maitra believes that conceptually, this project is paradigm shifting. “Wirtz is analyzing cancer in a brand new way. Extending this tool into an open-access cancer database, the project seems to have a bright future for helping doctors treat patients.”

Maitra makes sure to keep the project in perspective while being hopeful about the direction of this project, “It is very preliminary at this point. We have a long way to go before we can actually say this is a clinically applicable technology, but what we are doing right now is working our way up there.”

Center for Digital Pathology

Beauty is in the eye of the microscopist

Scientists have been using microscopes to produce up-close views and gather data about cells and other tiny things for more than 400 years. Many times, however, those images are not just informative, they are beautiful.

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Actin network in mouse fibroblast (Image by Dong-Hwee Kim)

Dong-Hwee Kim, a postdoctoral fellow in the Institute for NanoBioTechnology in the Whiting School of Engineering, frequently uses microscopy in his research on cell mechanics, a field he describes as “one of the fastest growing interdisciplinary fields in biology.”

Several of his images have not only yielded abundant quantitative and qualitative data, but they have netted him awards for scientific imaging.

In 2011 he earned an Image of Distinction Award from the Nikon Small World Competition for his photo of a dis-organized perinuclear actin cap stress fibers in a mouse embryonic fibroblast. That same year, he was awarded an Honorable mention from the American Society for Cell Biology 7th Annual Cell Biology film contest for his movie “Hurricane: Cell Cytoplasm Movements”. And in 2013, Kim took second place in the Biophysical Society’s, The Art of Science Image Contest for his dandelion-like representation of the geodesic actin network in a mouse fibroblast.

Kim says his primary focus in collecting these images has always been purely for scientific purposes. “I am trying to better understand how cells recognize the physical properties of the cell environment and respond to them,” he said.

Engineers have developed theories about cell mechanics and about what they expect to happen at the single cell level. But instead of describing the cell’s response with a computational model or other simulation, Kim was determined to capture actual images of live cells reacting to their surroundings.

Perinuclear actin cap stress fibers in a mouse embryonic fibroblast (Image by Dong-Hween Kim)

Perinuclear actin cap stress fibers in a mouse embryonic fibroblast (Image by Dong-Hwee Kim)

“Direct visualization of cell functions has become one of the most effective ways to support scientific findings, since it is the simplest but most powerful way to con-vince others,” he said.

Using various microscopy techniques, Kim has been able to visualize cell components, such as the nucleus or actin filaments, in very detailed ways. “It is always exciting to become the first one to show something that others haven’t seen yet,” he said.

Denis Wirtz, Kim’s advisor, noticed how beautiful the postdoc’s images were and suggested that he enter some of his work into popular imaging contests. Each contest focuses on a different theme, but the bottom line is that the images must be scientifically relevant as well as visually interesting. For example, the image of the geodesic actin network in the mouse embryonic fibroblasts, which were used for both the Nikon and Biophysical Society image contests, “directly visualized the mechanical and spatial coordinates of filamentous actin cytoskeleton in the cell,” said Kim. But the images also happen to be reminiscent of dandelions and fireworks.

Even if making a pretty picture is not the intent of the image, Kim thinks that having an artistic eye is important in science.

“I think artistic images in science should be based on a combination of aesthetic discrimination and scientific significance, which gives them unparalleled power to support scientific findings and persuade others,” Kim said.

The old saying goes, “a picture is worth 1,000 words.” In this case an expertly executed scientific image “can overcome myriad arguments,” Kim added.

Kim said his favorite imaging tool is the “confocal laser scanning microscopy, which allows high resolution images in cell biology, as well as qualitative and quantitative analysis of images.” He emphasizes that he does not use any software enhancements, such as Photoshop, to beautify his images. However, by attempting to create a beautiful image, he has developed several new imaging protocols.

“In challenging myself to create artistic images, it has sometimes led me to design new scientific methodologies that were not conventionally used in the field, and I think these efforts can contribute to the advancement of science,” Kim said.

This article was written by Mary Spiro, science writer for INBT at Johns Hopkins University, and first appeared in the 2013 issue of Nano-Bio Magazine.

 

Landmark physical characterization of cancer cells completed

An enormous collaborative effort between a multitude of academic and research centers has characterized numerous physical and mechanical properties on one identical human cancer cell line. Their two-year cooperative study, published online in the April 26, 2013 journal Science Reports, reveals the persistent and agile nature of human cancer cells as compared to noncancerous cells. It also represents a major shift in the way scientific research can be accomplished.

Human breast cancer cells like these were used in the study. (Image created by Shyam Khatau/ Wirtz Lab)

Human breast cancer cells like these were used in the study. (Image created by Shyam Khatau/ Wirtz Lab)

The research, which was conducted by 12 federally funded Physical Sciences-Oncology Centers (PS-OC) sponsored by the National Cancer Institute, is a systematic comparison of metastatic human breast-cancer cells to non-metastatic breast cells that reveals dramatic differences between the two cell lines in their mechanics, migration, oxygen response, protein production and ability to stick to surfaces. They have also discovered new insights into how human cells make the transition from nonmalignant to metastatic, a process that is not well understood.

Denis Wirtz, a Johns Hopkins professor of chemical and biomolecular engineering with joint appointments in pathology and oncology who is the corresponding author on the study, remarked that the work adds a tremendous amount of information about the physical nature of cancer cells. “For the first time ever, scientists got together and have created THE phenotypic signature of cancer” Wirtz said. “Yes, it was just one metastatic cell line, and it will require validation with many other cell lines. But we now have an extremely rich signature containing many parameters that are distinct when looking at metastatic and nonmetastatic cells.”

Wirtz, who directs the Johns Hopkins Physical Sciences-Oncology Center, also noted the unique way in which this work was conducted: all centers used the same human cell line for their studies, which makes the quality of the results unparalleled. And, since human and not animal cells were used, the findings are immediately relevant to the development of drugs for the treatment of human disease.

“Cancer cells may nominally be derived from the same patient, but in actuality they will be quite different because cells drift genetically over just a few passages,” Wirtz said.  “This makes any measurement on them from different labs like comparing apples and oranges.” In this study, however, the genetic integrity of the cell lines were safeguarded by limiting the number times the original cell cultures could be regrown before they were discarded.

The nationwide PS-OC brings together researchers from physics, engineering, computer science, cancer biology and chemistry to solve problems in cancer, said Nastaran Zahir Kuhn, PS-OC program manager at the National Cancer Institute.

“The PS-OC program aims to bring physical sciences tools and perspectives into cancer research,” Kuhn said. “The results of this study demonstrate the utility of such an approach, particularly when studies are conducted in a standardized manner from the beginning.”

For the nationwide project, nearly 100 investigators from 20 institutions and laboratories conducted their experiments using the same two cell lines, reagents and protocols to assure that results could be compared. The experimental methods ranged from physical measurements of how the cells push on surrounding cells to measurements of gene and protein expression.

“Roughly 20 techniques were used to study the cell lines, enabling identification of a number of unique relationships between observations,” Kuhn said.

Wirtz added that it would have been logistically impossible for a single institution to employ all of these different techniques and to measure all of these different parameters on just one identical cell line. That means that this work accomplished in just two years what might have otherwise taken ten, he said.

The Johns Hopkins PS-OC made specific contributions to this work. Using particle-tracking microrheology, in which nanospheres are embedded in the cell’s cytoplasm and random cell movement is visually monitored, they measured the mechanical properties of cancerous versus noncancerous cells. They found that highly metastatic breast cancer cells were mechanically softer and more compliant than cells of less metastatic potential.

Using 3D cell culturing techniques, they analyzed the spontaneous migratory potential (that is, migration without the stimulus of any chemical signal) of cancerous versus noncancerous cells. They also analyzed the extracellular matrix molecules that were deposited by the two cell lines and found that cancerous cells deposited more hyaluronic acid (HA). The HA, in turn, affects motility, polarization and differentiation of cells.  Finally, the Hopkins team measured the level of expression of CD44, a cell surface receptor that recognizes HA, and found that metastatic cells express more CD44.

The next steps, Wirtz said, would be to validate these results using other metastatic cell lines.  To read the paper, which is published in an open access journal, follow this link: http://www.nature.com/srep/2013/130422/srep01449/full/srep01449.html

Excerpts from original press release by Princeton science writer Morgan Kelly were used.

 

 

 

 

Cancer data stored in the cloud could improve treatments

These days, storing photos or music remotely in “the cloud”  has become common place. Now Johns Hopkins researchers are applying the concept to the storage of medical data in the hopes of predicting and improving cancer patient treatments and outcomes.

Images courtesy Denis Wirtz/JHU

“The long-range goal is to make these data available through the Internet to physicians who are diagnosing and treating cancer patients around the world,” said Denis Wirtz , associate director of the Johns Hopkins Institute for NanoBioTechnology and professor of chemical and biomolecular engineering. Using a $3.75 million grant over five years from the National Cancer Institute Common Fund Single Cell Analysis Program, Wirtz launched the program in October, with two colleagues from the Johns Hopkins School of Medicine, Anirban Maitra and Ralph Hruban.

Initially the database will focus on information from pancreatic cancer patient cell lines but will expand to other types of cancer, including ovarian.  Data gathered and stored will be at the single cell level, which Wirtz explains, provides better information for predicting how individual patients may respond to certain drugs. Drugs that work well for one patient may do nothing at all, or even be harmful, for another, Wirtz said. Understanding and predicting these outcomes before treatment is a step toward more personalized medicine, he added.

To read more about “cloud pathology,” go to the press release issued by John Hopkins University.

Johns Hopkins Institute for NanoBioTechnology

Johns Hopkins Engineering in Oncology Center

Johns Hopkins Kimmel Cancer Center

 

Breast cancer highlighted at Homewood mini-symposium

A tumor cell breaking free and entering the blood stream. (From animation by Ella McCrea, Nathan Weiss and Martin Rietveld)

Breast cancer will be topic of at least two of the talks planned for a mini-symposium October 10 on the Homewood campus.

UPDATED: Click here for updated list of talk titles.

Students from Johns Hopkins Physical Sciences-Oncology Center (PSOC) and Center of Cancer Nanotechnology Excellence (CCNE) will hold their second mini-symposium of the year on October 10 at 9 a.m. in Hackerman Hall Auditorium. The symposia, scheduled each spring and fall on the Homewood campus, encourage an exchange of ideas between PhD students and postdoctoral fellows associated with these centers. The entire Hopkins community is invited to attend, and no RSVP is required.

Some of the talk titles include, from the department of Chemical and Biomolecular Engineering, “The Pulsing Motion of Breast Cancer Cell is Regulated by Surrounding Epithelial Cells” presented by Meng Horng Lee, a PSOC postdoctoral fellow in the Denis Wirtz lab; “Breast Tumor Extracellular Matrix Promotes Vasculogenesis” presented by Abigail Hielscher, a postdoctoral fellow in the Sharon Gerecht lab; and “Mucin 16 is a Functional Selectin Ligand on Pancreatic Cancer Cells” given by Jack Chen, a pre-doctoral fellow in the lab of Konstantinos Konstantopoulos. Additional speakers include postdoctoral fellow Pei-Hsun Wu, PhD, a from the Wirtz Lab and Koh Meng Aw Yong, a pre-doctoral student affiliated with Princeton University’s Physical Sciences-Oncology Center.

The purpose of these twice a year, student run mini-symposia is to facilitate communication among researchers working in laboratories studying the mechanistic aspects of cancer spread (i.e., those affiliated with the PSOC) and those working on novel means of using nanotechnology for cancer diagnosis or treatment (i.e., those associated with the CCNE). Anjil Giri coordinated the fall mini-symposium, a PSOC pre-doctoral fellow in the Wirtz lab , with Erbil Abaci, a PSOC pre-doctoral fellow with in the Gerecht lab. Visit the INBT website (inbt.jhu.edu) for further details, as additional speakers and talk titles will be announced.