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.

 

 

 

 

Recent publications from the Johns Hopkins Physical Sciences-Oncology Center

Johns Hopkins Physical Sciences-Oncology Center has had a productive quarter publishing from February to May 2013. Here are some of the most recent publications in support or the center’s core research projects, including a huge collaborative work drawing on the knowledge and research findings of the entire PS-OC network.

Screen Shot 2013-05-15 at 4.27.37 PMThat paper, A physical sciences network characterization of non-tumorigenic and metastatic cells, was the work of 95 authors from all 12 of the National Cancer Institute’s PS-OC  program centers. JHU’s PS-OC director Denis Wirtz, the Theophilus H. Smoot Professor in the Johns Hopkins Department of Chemical and Ciomolecular Engineering, is the corresponding author on this massive effort. We will be discussing the findings of that paper in a future post here on the PS-OC website. Until then, here is a link to that network paper and 13 other recent publications from the Johns Hopkins PS-OC.

  • A physical sciences network characterization of non-tumorigenic and metastatic cells.Physical Sciences – Oncology Centers Network, Agus DB, Alexander JF, Arap W,Ashili S, Aslan JE, Austin RH, Backman V, Bethel KJ, Bonneau R, Chen WC,Chen-Tanyolac C, Choi NC, Curley SA, Dallas M, Damania D, Davies PC, Decuzzi P,Dickinson L, Estevez-Salmeron L, Estrella V, Ferrari M, Fischbach C, Foo J,Fraley SI, Frantz C, Fuhrmann A, Gascard P, Gatenby RA, Geng Y, Gerecht S,Gillies RJ, Godin B, Grady WM, Greenfield A, Hemphill C, Hempstead BL, HielscherA, Hillis WD, Holland EC, Ibrahim-Hashim A, Jacks T, Johnson RH, Joo A, Katz JE,Kelbauskas L, Kesselman C, King MR, Konstantopoulos K, Kraning-Rush CM, Kuhn P,Kung K, Kwee B, Lakins JN, Lambert G, Liao D, Licht JD, Liphardt JT, Liu L, LloydMC, Lyubimova A, Mallick P, Marko J, McCarty OJ, Meldrum DR, Michor F,Mumenthaler SM, Nandakumar V, O’Halloran TV, Oh S, Pasqualini R, Paszek MJ,Philips KG, Poultney CS, Rana K, Reinhart-King CA, Ros R, Semenza GL, Senechal P,Shuler ML, Srinivasan S, Staunton JR, Stypula Y, Subramanian H, Tlsty TD, TormoenGW, Tseng Y, van Oudenaarden A, Verbridge SS, Wan JC, Weaver VM, Widom J, Will C, Wirtz D, Wojtkowiak J, Wu PH.  Sci Rep. 2013 Apr 25;3:1449. doi:10.1038/srep01449. PubMed PMID: 23618955; PubMed Central PMCID: PMC3636513. http://www.ncbi.nlm.nih.gov/pubmed/23618955
  • Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis. Gilkes DM, Bajpai S, Wong CC, Chaturvedi P, Hubbi ME, Wirtz D, Semenza GL.Mol Cancer Res. 2013 May 7. [Epub ahead of print] PubMed PMID: 23378577. http://www.ncbi.nlm.nih.gov/pubmed/23378577
  • Predicting how cells spread and migrate: Focal adhesion size does matter. Kim DH, Wirtz D. Cell Adh Migr. 2013 Apr 29;7(3). [Epub ahead of print] PubMed PMID: 23628962. http://www.ncbi.nlm.nih.gov/pubmed/23628962
  • Hypoxia-inducible Factor 1 (HIF-1) Promotes Extracellular Matrix Remodeling under Hypoxic Conditions by Inducing P4HA1, P4HA2, and PLOD2 Expression in Fibroblasts. Gilkes DM, Bajpai S, Chaturvedi P, Wirtz D, Semenza GL. J Biol   Chem. 2013 Apr 12;288(15):10819-29. doi: 10.1074/jbc.M112.442939. Epub 2013 Feb 19. PubMed PMID: 23423382; PubMed Central PMCID: PMC3624462. http://www.ncbi.nlm.nih.gov/pubmed/23423382
  • Perivascular cells in blood vessel regeneration. Wanjare M, Kusuma S, Gerecht S. Biotechnol J. 2013 Apr;8(4):434-47. doi: 10.1002/biot.201200199. PubMed PMID: 23554249. http://www.ncbi.nlm.nih.gov/pubmed/23554249
  • Focal adhesion size uniquely predicts cell migration. Kim DH, Wirtz D. FASEB J. 2013 Apr;27(4):1351-61. doi: 10.1096/fj.12-220160. Epub 2012 Dec 19. PubMed PMID: 23254340; PubMed Central PMCID: PMC3606534. http://www.ncbi.nlm.nih.gov/pubmed/23254340
  • Notch4-dependent Antagonism of Canonical TGFβ1  Signaling Defines Unique Temporal Fluctuations of SMAD3 Activity in Sheared Proximal Tubular Epithelial Cells. Grabias BM, Konstantopoulos K. Am J Physiol Renal Physiol. 2013 Apr 10. [Epub ahead of print] PubMed PMID: 23576639. http://www.ncbi.nlm.nih.gov/pubmed/23576639
  • Integration and regression of implanted engineered human vascular networks during deep wound healing. Hanjaya-Putra D, Shen YI, Wilson A, Fox-Talbot K, Khetan S, Burdick JA, Steenbergen C, Gerecht S. Stem Cells Transl Med. 2013 Apr;2(4):297-306. doi: 10.5966/sctm.2012-0111. Epub 2013 Mar 13. PubMed PMID: 23486832. http://www.ncbi.nlm.nih.gov/pubmed/23486832
  • Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis. Gilkes DM, Chaturvedi P, Bajpai S, Wong CC, Wei H, Pitcairn S, Hubbi ME, Wirtz D, Semenza GL. Cancer Res. 2013 Mar 28. [Epub ahead of print] PubMed PMID: 23539444. http://www.ncbi.nlm.nih.gov/pubmed/23539444
  • Simultaneously defining cell phenotypes, cell cycle, and chromatin modifications at single-cell resolution.Chambliss AB, Wu PH, Chen WC, Sun SX, Wirtz D.FASEB J. 2013 Mar 28. [Epub ahead of print] PubMed PMID: 23538711.http://www.ncbi.nlm.nih.gov/pubmed/23538711
  • Interstitial friction greatly impacts membrane mechanics. Wirtz D. Biophys J.2013 Mar 19;104(6):1217-8. doi: 10.1016/j.bpj.2013.02.003. Epub 2013 Mar 19.PubMed PMID: 23528079; PubMed Central PMCID: PMC3602747.http://www.ncbi.nlm.nih.gov/pubmed/23528079
  • Functional interplay between the cell cycle and cell phenotypes. Chen WC, Wu PH, Phillip JM, Khatau SB, Choi JM, Dallas MR, Konstantopoulos K,Sun SX, Lee JS, Hodzic D, Wirtz D.Integr Biol (Camb). 2013 Mar;5(3):523-34. doi:10.1039/c2ib20246h. PubMed PMID: 23319145 http://www.ncbi.nlm.nih.gov/pubmed/23319145
  • High-throughput secretomic analysis of single cells to assess functional cellular heterogeneity. Lu Y, Chen JJ, Mu L, Xue Q, Wu Y, Wu PH, Li J, Vortmeyer AO, Miller-Jensen K, Wirtz D, Fan R. Anal Chem. 2013 Feb 19;85(4):2548-56. doi:10.1021/ac400082e. Epub 2013 Feb 1. PubMed PMID: 23339603; PubMed Central PMCID:  PMC3589817.http://www.ncbi.nlm.nih.gov/pubmed/23339603\

 

Self-assembling drug molecules could fight cancer

A popular method of targeted drug delivery for anti-cancer drugs involves doping another material with the desired pharmaceutical to obtain better targeting efficiency to tumor sites. The problem with this method, researchers have discovered, is that the quantity of drug payload per delivery unit can vary widely and that the materials used for delivery can have toxic side effects.

But what if you could turn the drug molecule itself into a nanoscale delivery system, cutting out the middleman completely?

TEM image of nanotubes formed by self-assembly of an anticancer drug amphiphile. These nanotubes possess a fixed drug loading of 38% (w/w). Image from Cui Lab.

TEM image of nanotubes formed by self-assembly of an anticancer drug amphiphile. These nanotubes possess a fixed drug loading of 38% (w/w). Image from Cui Lab.

Using the process of molecular self-assembly, that is what Honggang Cui, an assistant professor in the Department of Chemical and Biomolecular Engineering at Johns Hopkins University, is attempting to do. His efforts have netted him the prestigious Faculty Early Career Development (CAREER) Award from the National Science Foundation. Cui, an affiliated faculty member of the Johns Hopkins Institute for NanoBioTechnology, will receive the $500,000 award over five years.

Cui explained that a current method of delivering anti-cancer drugs is to enclose them in a nanoscale carrier made of natural or synthetic materials, but this method presents several challenges. “The amount of drug loaded per carrier is very much limited and varies from batch to batch. Even in the same batch, there is a drug loading variation from carrier to carrier. Additionally, the carrier material itself may have toxic side effects,” he said.

Cui’s research seeks to eliminate the need for the carrier by coaxing the drug molecules themselves to form their own carrier through the process of self-assembly. His team is developing new molecular engineering strategies to assemble anti-cancer drugs into supramolecular nanostructures.

“Such supramolecules could carry as much as 100 percent of the drug, would possess a fixed amount of drug per nanostructure and would minimize the potential toxicity of the carrier,” Cui said.

To learn more about research in the Cui lab go to http://www.jhu.edu/cui/

 

Molecular culprit linked to breast cancer spread

Johns Hopkins researchers have uncovered a protein “partner” commonly used by breast cancer cells to unlock genes needed for spreading the disease around the body. A report on the discovery, published Nov. 5 on the website of the Proceedings of the National Academy of Sciences, details how some tumors get the tools they need to metastasize.

“We’ve identified a protein that wasn’t known before to be involved in breast cancer progression,” says Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine at the Johns Hopkins University School of Medicine and director of the Vascular Program at the university’s Institute for Cell Engineering. “The protein JMJD2C is the key that opens up a whole suite of genes needed for tumors to grow and metastasize, so it represents a potential target for cancer drug development.” Semenza also is associate director of the Johns Hopkins Physical Sciences-Oncology Center.

Semenza and his colleagues made their finding when they traced the activity of HIF-1, a protein known to switch on hundreds of genes involved in development, red blood cell production, and metabolism in normal cells. Previous studies had shown that HIF-1 could also be hijacked to switch on genes needed to make breast tumors more malignant.

Would-be tumor cells face a host of challenges as they make the transition from working with their host to working against it, such as the need to evade the immune system and to produce more cancer cells, explains Weibo Luo, Ph.D., an instructor in the Institute for Cell Engineering and Department of Biological Chemistry who led the project. All of these efforts require switching on the right genes for the job.

To learn more about how HIF-1 works, the researchers tested a range of human proteins to see whether they would interact with HIF-1. They then sifted through the 200 resulting hits, looking for proteins involved in chemical changes to sections of DNA that determine whether or not the genes they contain are available for use. “In order for HIF-1 to switch genes on, they have to be available, but many of the genes HIF-1 activates are normally locked down in mature cells,” explains Luo. “So we thought HIF-1 must have a partner that can do the unlocking.”

That partner turned out to be JMJD2C, Luo says. Delving deeper, the researchers found that HIF-1 switches on the JMJD2C gene, stimulating production of the protein. HIF-1’s presence also enables JMJD2C to bind to DNA at other HIF-1 target genes, and then loosen those DNA sections, enabling more HIF-1 to bind to the same sites and activate the target genes.

To test the implications of their discovery, the research team injected mice with breast cancer cells in which the JMJD2C protein was not produced. Tumors with depleted JMJD2C were much less likely to grow and metastasize to the lungs, confirming the protein’s role in breast cancer progression, says Luo.

“Active HIF proteins have been found in many types of tumors, so the implications of this finding go beyond breast cancer,” says Luo. “JMJD2C is both an important piece of the puzzle of how tumors metastasize, and a potential target for anti-cancer therapy.”

Other authors of the research report are Ryan Chang, Jun Zhong, Ph.D., and Akhilesh Pandey, M.D., Ph.D., all of the Johns Hopkins University School of Medicine.

This work was supported by grants from the National Heart, Lung, and Blood Institute (contracts N01-HV28180 and HHS-N268201000032C), and by funds from the Johns Hopkins Institute for Cell Engineering.

On the Web:

Johns Hopkins Physical Sciences-Oncology Center: http://psoc.inbt.jhu.edu/

Link to article: http://www.pnas.org/content/early/2012/10/31/1217394109.abstract

Semenza lab: http://www.hopkinsmedicine.org/institute_cell_engineering/experts/gregg_semenza.html

Q&A with Semenza: http://www.hopkinsmedicine.org/institute_cell_engineering/experts/meet_scientists/gregg_semenza.html

Original press release by Shawna WilliamsCatherine Kolf and Vanessa McMains

 

 

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

 

Konstantopoulos named BMES fellow

Konstantinos Konstantopoulos (Photo by Mary Spiro)

Konstantinos Konstantopoulos, professor and chair of the Department of Chemical and Biomolecular Engineering at Johns Hopkins University’s Whiting School of Engineering has been named a Fellow of the Biomedical Engineering Society (BMES). Konstantopoulos was one of only nine fellows elected to the Society’s Class of 2012.

BMES states that Konstantopoulos received this honor in recognition of his “seminal bioengineering research contributions involving the discovery and characterization of novel selectin ligands expressed by metastatic tumor cells.”  Formal installation of fellows will take place at the BMES annual meeting  October 24-27 in Atlanta.

Konstantopoulos is an affiliated faculty member of Johns Hopkins Institute for NanoBioTechnology. He is also a project leader with the Johns Hopkins Physical Sciences-Oncology Center. Together with Martin Pomper, a School of Medicine professor of radiology and co-principal investigator of the Johns Hopkins Center of Cancer Nanotechnology Excellence, Konstantopoulos is researching mechanochemical effects on metastasis.

Specifically, his work investigates the effects of fluid mechanical forces at different oxygen tension microenvironments on tumor cell signaling, adhesion and migration. Fluid flow in and around tumor tissue modulates the mechanical microenvironment, including the forces acting on the cell surface and the tethering force on cell-substrate connections. Cells in the interior of a tumor mass experience a lower oxygen tension microenvironment and lower fluid velocities than those at the edges in proximity with a functional blood vessel, and are prompted to produce different biochemical signals. These differential responses affect tumor cell fate that is, whether a cell will live or die, and whether it will be able to detach and migrate to secondary sites in the body.

According to the BMES website, members who demonstrate exceptional achievements and experience in the field of biomedical engineering, as well as a record of membership and participation in the Society, have the opportunity to become fellows. Fellows are selected and conferred  by the BMES board of directors through a highly selective process. Nominations for each of these categories may be made by Society members and the board of directors.

Learn more about research in the Konstantopoulos Lab here.

 

 

Coated nanoparticles move easily into brain tissue

Real-time imaging of nanoparticles green) coated with polyethylene-glycol (PEG), a hydrophilic, non-toxic polymer, penetrate within normal rodent brain. Without the PEG coating, negatively charged, hydrophobic particles (red) of a similar size do not penetrate. Image by Elizabeth Nance, Kurt Sailor, Graeme Woodworth.

Johns Hopkins researchers report they are one step closer to having a drug-delivery system flexible enough to overcome some key challenges posed by brain cancer and perhaps other maladies affecting that organ. In a report published online Aug. 29 in Science Translational Medicine, the Johns Hopkins team says its bioengineers have designed nanoparticles that can safely and predictably infiltrate deep into the brain when tested in rodent and human tissue.

“We are pleased to have found a way to prevent drug-embedded particles from sticking to their surroundings so that they can spread once they are in the brain,” said Justin Hanes, Lewis J. Ort Professor of Ophthalmology and project leader in the Johns Hopkins Center of Cancer Nanotechnology Excellence.

Standard protocols following the removal of brain tumors include chemotherapy directly applied to the surgical site to kill any cancer cells left behind. This method, however, is only partially effective because it is hard to administer a dose of chemotherapy high enough to sufficiently penetrate the tissue to be effective and low enough to be safe for the patient and healthy tissue. Furthermore, previous versions of drug-loaded nanoparticles typically adhere to the surgical site and do not penetrate into the tissue.

These newly engineered nanoparticles overcome this challenge. Elizabeth Nance, a graduate student in chemical and biomolecular engineering, and Johns Hopkins neurosurgeon Graeme Woodworth, suspected that drug penetration might be improved if drug-delivery nanoparticles interacted minimally with their surroundings. Nance achieved this by coating nano-scale beads with a dense layer of PEG or poly(ethylene glycol). The team then injected the coated beads, which had been marked with a fluorescent tag,  into slices of rodent and human brain tissue. They found that a dense coating of PEG allowed larger beads to penetrate the tissue, even those beads that were nearly twice the size previously thought to be the maximum possible for penetration within the brain. They then tested these beads in live rodent brains and found the same results.

Elizabeth Nance. Photo by Ming Yang.

The results were similar when biodegradable nanoparticles carrying the chemotherapy drug paclitaxel and coated with PEG were used. “It’s really exciting that we now have particles that can carry five times more drug, release it for three times as long and penetrate farther into the brain than before,” said Nance. “The next step is to see if we can slow tumor growth or recurrence in rodents.”

Woodworth added that the team “also wants to optimize the particles and pair them with drugs to treat other brain diseases, like multiple sclerosis, stroke, traumatic brain injury, Alzheimer’s and Parkinson’s.” Another goal for the team is to be able to administer their nanoparticles intravenously, which is research they have already begun.

Additional authors on the paper include Kurt Sailor, Ting-Yu Shih, Qingguo Xu, Ganesh Swaminathan, Dennis Xiang, and Charles Eberhart, all from The Johns Hopkins University.

Story adapted from an original press release by Cathy Kolf.

 

Additional news coverage of this research can be found at the following links:

Nanotechnology/Bio & Medicine

Death and Taxes Mag

New Scientist Health

Nanotech Web

Portugese news release (in Portugese)

German Public Radio (in German)

Light-activated synthetic protein illuminates disease destruction

Illustration of collagen’s rope-like structure. Click to watch video. (INBT Animation Studios)

Johns Hopkins researchers have created a synthetic protein that, when activated by ultraviolet light, can guide doctors to places within the body where cancer, arthritis and other serious medical disorders can be detected. The synthetic protein does not zero in directly on the diseased cells. Instead, it binds to nearby collagen that has been degraded by disease or injury.

“These disease cells are like burglars who break into a house and do lots of damage but who are not there when the police arrive,” said S. Michael Yu, a faculty member in the Whiting School of Engineering’s Department of Materials Science and Engineering. “Instead of looking for the burglars, our synthetic protein is reacting to evidence left at the scene of the crime,” said Yu, who was principal investigator in the study.

The technique could lead to a new type of diagnostic imaging technology and may someday serve as a way to move medications to parts of the body where signs of disease have been found. In a study published in the Aug. 27-31 Online Early Edition of Proceedings of the National Academy of Sciences, the researchers reported success in using the synthetic protein in mouse models to locate prostate and pancreatic cancers, as well as to detect abnormal bone growth activity associated with Marfan syndrome.

Collagen, the body’s most abundant protein, provides structure and creates a sturdy framework upon which cells build nerves, bone and skin. Some buildup and degradation of collagen is normal, but disease cells such as cancer can send out enzymes that break down collagen at an accelerated pace. It is this excessive damage, caused by disease, that the new synthetic protein can detect, the researchers said.

A key collaborator was Martin Pomper, a School of Medicine professor of radiology and co-principal investigator of the Johns Hopkins Center of Cancer Nanotechnology Excellence. Pomper and Yu met as fellow affiliates of the Johns Hopkins Institute for NanoBioTechnology. “A major unmet medical need is for a better non-invasive characterization of disrupted collagen, which occurs in a wide variety of disorders,” Pomper said. “Michael has found what could be a very elegant and practical solution, which we are converting into a suite of imaging and potential agents for diagnosis and treatment.”

The synthetic proteins used in the study are called collagen mimetic peptides or CMPs. These tiny bits of protein are attracted to and physically bind to degraded strands of collagen, particularly those damaged by disease. Fluorescent tags are placed on each CMP so that it will show up when doctors scan tissue with fluorescent imaging equipment. The glowing areas indicate the location of damaged collagen that is likely to be associated with disease.

In developing the technique, the researchers faced a challenge because CMPs tend to bind with one another and form their own structures, similar to DNA, in a way that would cause them to ignore the disease-linked collagen targeted by the researchers.

To remedy this, the study’s lead author, Yang Li, synthesized CMPs that possess a chemical “cage” to keep the proteins from binding with one another. Just prior to entering the bloodstream to search for damaged collagen, a powerful ultraviolet light is used to “unlock” the cage and allow the CMPs to initiate their disease-tracking mission. Li is a doctoral student from the Department of Chemistry in the Krieger School of Arts and Sciences at Johns Hopkins. Yu, who holds a joint appointment in that department, is his doctoral adviser.

Yu’s team tested Li’s fluorescently tagged and caged peptides by injecting them into lab mice that possessed both prostate and pancreatic human cancer cells. Through a series of fluorescent images taken over four days, researchers tracked single strands of the synthetic protein spreading throughout the tumor sites via blood vessels and binding to collagen that had been damaged by cancer.

Similar in vivo tests showed that the CMP can target bones and cartilage that contain large amounts of degraded collagen. Therefore, the new protein could be used for diagnosis and treatment related to bone and cartilage damage.

Although the process is not well understood, the breakdown and rebuilding of collagen is thought to play a role in the excessive bone growth found in patients with Marfan syndrome. Yu’s team tested their CMPs on a mouse model for this disease and saw increased CMP binding in the ribs and spines of the Marfan mice, as compared to the control mice.

Funding for the research was provided by the National Science Foundation, the National Institutes of Health and the Department of Defense. The synthetic protein process used in this research is protected by patents obtained through the Johns Hopkins Technology Transfer Office.

Along with Yu, Li and Pomper, co-authors of this study were instructor Catherine A. Foss and medical resident Collin M. Torok from the Department of Radiology and Radiological Science at the Johns Hopkins School of Medicine; Harry C. Dietz, a professor, and Jefferson J. Doyle, a doctoral student, both of the Howard Hughes Medical Institute and Institute of Genetic Medicine at the School of Medicine; and Daniel D. Summerfield a former master’s student in the Department of Materials Science and Engineering.

Adapted from an original press release by Phil Sneiderman.

 

Killing prostate cancer cells with out harming the healthy cells

Experimenting with human prostate cancer cells and mice, cancer imaging experts at Johns Hopkins say they have developed a method for finding and killing malignant cells while sparing healthy ones.

The method, called “theranostic” imaging, targets and tracks potent drug therapies directly and only to cancer cells. It relies on binding an originally inactive form of drug chemotherapy, with an enzyme, to specific proteins on tumor cell surfaces and detecting the drug’s absorption into the tumor. The binding of the highly specific drug-protein complex, or nanoplex, to the cell surface allows it to get inside the cancerous cell, where the enzyme slowly activates the tumor-killing drug.

Researchers say their findings, published in the journal American Chemical Society Nano online Aug. 6, are believed to be the first to show that chemotherapies can be precisely controlled at the molecular level to maximize their effectiveness against tumors, while also minimizing their side effects.

Senior study investigator Zaver Bhujwalla, Ph.D., a professor at the Johns Hopkins University School of Medicine and its Kimmel Cancer Center, notes that a persistent problem with current chemotherapy is that it attacks all kinds of cells and tissues, not just cancerous ones.

In the theranostic imaging experiments, overseen by Bhujwalla and study co-investigator Martin Pomper, M.D., Ph.D., investigators directed drugs only to cancer cells, specifically those with prostate-specific membrane antigen, or PSMA cell surface proteins. Both Pomper and Bhujwalla are affiliated faculty members of Johns Hopkins Institute for NanoBioTechnology.

“Our results show a non-invasive imaging approach to following and delivering targeted therapy to any cancer that expresses PSMA,” says Bhujwalla, who also serves as director of the Johns Hopkins In Vivo Cellular and Molecular Imaging Center (ICMIC), where the theranostic imaging studies were developed.

Bhujwalla says the new technique potentially will work against any cancer in which tumors elevate production of certain cell surface proteins. Examples would include breast cancers with HER-2/neu and CXCR4 proteins, and some liver, lung and kidney cancers also known to express particular proteins. She notes that PSMA is expressed in the vessels of most solid tumors, suggesting that the nanoplex reported in the latest study could be used in general to image and treat a variety of cancers.

In their latest series of experiments, primarily in mice injected with human prostate tumor cells, Bhujwalla and the Johns Hopkins team tested their ability to track with imaging devices the delivery of anti-cancer drugs directly to tumors. Some of the tumors comprised cells with PSMA, while other so-called control tumors had none. Included in the drug nanoplex were small strands of RNA, cell construction acids that can be used instead to block and turn down production of a well-known enzyme, choline kinase, whose levels usually rise with tumor growth. All nanoplex components were imaged inside the tumor, in addition to dropping choline kinase production, which decreased by 80 percent within 48 hours of nanoplex absorption into cells with ample PSMA. When researchers used antibodies to block the action of PSMA, down went the level of nanoplex uptake and drug activation in cancerous cells as measured by dimming of the image.

Different concentrations of the drug nanoplex, tagged with radioactive and fluorescent molecules, were mixed in the lab with prostate cancer tissue cells, some of which had extra PSMA and others which had none. Only those cells with extra PSMA showed nanoplex uptake, as measured by image intensity, which later decreased when PSMA-blocking chemicals were added (back to levels seen in cells with almost no PSMA).

Additional experiments involving injections of three different concentrations of the drug nanoplex showed no damage to other vital mouse organs, such as the kidney and liver, nor any uptick in the mouse immune system response.

“Our theranostic imaging approach shows how the best methods of detection and treatment can be combined to form highly specialized, more potent and safer forms of chemotherapy,” says Pomper, a professor at Johns Hopkins who also serves as an associate director at ICMIC.

He says that an important goal for theranostic imaging is to move it beyond standard chemotherapy that attacks one target molecule at a time.

“With theranostic imaging, we can attack multiple tumor targets, making it harder for the tumor to evade drug treatment,” says Pomper, who is already working with colleagues at Johns Hopkins to identify other molecular targets.

The most recent studies were performed at Johns Hopkins over two years, starting in 2010, with funding support from the National Cancer Institute, part of the National Institutes of Health. The corresponding grant numbers are P50-CA103175, RO1-CA138515 and RO1-CA134675.

In addition to Bhujwalla and Pomper, other Johns Hopkins researchers from the Russell H. Morgan Department of Radiology involved in this imaging study were lead investigators Zhihang Chen, Ph.D., and Mary-France Penet, Ph.D. Additional study co-investigators were Sridhar Nimmagadda, Ph.D.; Li Cong, Ph.D.; Sangeeta Banerjee, Ph.D.; Paul Winnard Jr., Ph.D.; Dmitri Artemov, Ph.D.; and Kristine Glunde, Ph.D.

Press release by David March

Therapeutic nanolex containing multi-modal imaging reporters targeted to prostate specific membrane antigen (PSMA), which is expressed on the cell surface of castrate resistant PCa. (Image by Chen et al. from ACS Nano 2012).

 

Tumor cells change when put into a ‘tight spot’

Konstantinos Konstantopoulos addresses audience at 2012 NanoBio Symposium. Photo by Mary Spiro/JHU

“Cell migration represents a key aspect of cancer metastasis,” said Konstantinos Konstantopoulos, professor and chair of the Department of Chemical and Biomolecular Engineering at Johns Hopkins University. Konstantopoulos was among the invited faculty speakers for the 2012 NanoBio Symposium.

Cancer metastasis, the migration of cancer cells from a primary tumor to other parts of the body, represents an important topic among professors affiliated with Johns Hopkins Institute for NanoBioTechnology. Surprisingly, 90 percent of cancer deaths are caused from this spread, not from the primary tumor alone. Konstantopoulos and his lab group are working to understand the metastatic process better so that effective preventions and treatments can be established. His students have studied metastatic breast cancer cells in the lab by tracking their migration patterns. The group has fabricated a microfluidic-based cell migration chamber that contains channels of varying widths. Cells are seeded at one opening of the channels, and fetal bovine serum is used as a chemoattractant at the other opening of the channels to induce the cells to move across. These channels can be as big as 50 µm wide, where cells can spread out to the fullest extent, or as small as 3 µm wide, where cells have to narrowly squeeze themselves to fit through.

A current dogma in the field of cell migration is that actin polymerization and actomyosin contractility give cells the flexibility they need to protrude and contract across a matrix in order to migrate. When Konstantopoulos’s students observed cells in the wide, 50 µm-wide channels, they saw actin distributed over the entirety of the cells, as expected. They also observed that when the cells were treated with drugs that inhibited actin polymerization and actomyosin contractility, they did not migrate across the channels, also as expected.

However, when the students observed cells in the narrow, 3 µm-wide channels, they were surprised to see actin only at the leading and trailing edges of the cells. Additionally, the inhibition of actin polymerization and actomyosin contractility did not affect the cells’ ability to migrate.

“Actin polymerization and actomyosin contractility are critical for 2D cell migration but dispensable for migration through narrow channels,” concluded Konstantopoulos. The data challenged what many had previously believed about cell migration by showing that cells in confined spaces did not need these actin components to migrate.

These findings are indeed important in the context of cancer metastasis, where cells must migrate through a heterogeneous environment of both wide and narrow areas. Konstantopoulos’s data gives a better mechanistic understanding of the different methods cancer cells use to migrate in diverse surroundings.

Future studies in the Konstantopoulos lab will focus on how tumor cells decide which migratory paths to take. INBT-sponsored graduate student Colin Paul has developed an additional microfluidic device that contains channels with bifurcations. He hopes to determine what factors guide a cell in one direction versus another. The Konstantopoulos lab hopes to continue to understand exactly how tumor cells migrate so that new therapies can eventually be developed to stop metastasis.

Story by Allison Chambliss, a Ph.D. student in the Department of Chemical and Biomolecular Engineering with interests in cellular biophysics and epigenetics.

Watch a video related to this research here.

Konstantopoulos reported these findings in October 2012 The Journal of the Federation of American Societies for Experimental Biology.  Read the article online here.