Diagnostics & Imaging Week National Editor

Connect the dots.

In the post-9/11 world, that's considered the best method for identifying some easily-overlooked signals, discerning a pattern in those signals, and using this to make predictions of future disaster, to fight it off.

A comparable disaster in the human body might be avoided in this same way – with the more dots you have for making important connections, the better, according to Cathy Shachaf, PhD, leader of a research team at Stanford University School of Medicine (Stanford, California), developing new methods for imaging cancer cells.

This new method, she says, could provide the ability to simultaneously measure as many as 100 or more distinct features in or on a single cell, providing an improved picture of what's going on in individual cells that may be turning to tumor formation.

An intriguing feature of this proof-of-concept research is the use of nanoparticle "sandwiches," incorporating special dyes, that can be used to produce these multiple "dots,' or points on proteins, which often signal critical cell changes.

Shachaf, an instructor in microbiology and immunology, has for the first time used these nanoparticles to simultaneously image two features within single cells. Although current single-cell flow cytometry technologies can do up to 17 simultaneous visualizations, she says this new method has the potential to do far more.

The group's imaging technology works by enhancing the detection of ultra-specific but very weak patterns, known as Raman signals, that molecules emit in response to light.

Shachaf tells Diagnostics & Imaging Week that the special nanoparticles were first developed by Intel (Santa Clara, California), but that Intel basically abandoned their development as it focused on its core expertise.

Intel "had a biomedical research group working on this for a few years," she says. "But compared to computer chips, this was not as lucrative."

The company had been working in collaboration with the medical school, and engineering the particles is now done in a laboratory run by the study's senior author, Garry Nolan, PhD, associate professor of microbiology and immunology and a member of Stanford's Cancer Center.

The particle sandwiches are composites of dye molecules and atoms of metals, such as silver, gold or copper. These composite organic-inorganic nanoparticles (or COINs) have been produced in 100 different varieties, each with a distinctive signature.

They have reflective properties that amplify a molecule's Raman signals while filtering out its inherent fluorescent response. Raman signals are emitted all the time by various molecules, but they're ordinarily too weak to detect, Shachaf explains.

The special nanoparticles beef up the strength of these signals and they are collected and quantified by a scanner.

Shachaf says that if the technique is ultimately taken to general use – though acknowledging a possible distance between eight to a 10 for ultimate commercialization — it will relatively easy to do the imaging. She characterizes the scanning process involved as a "no-brainer – it's a very easy operational system; you press a button, the instrument scans."

"The difficulty and expense" of the method, she says, are in the construction of the nanoparticles so as to produce the multiple signals and thus offer the major technical hurdles. They are made to give off not just single-wavelength fluorescent echoes but more-complex fingerprints comprising wavelengths slightly different from the single-color beams that are picked up and analyzed by a flow cytometry laser.

These patterns — the Raman signals referred to — occur when energy levels of electrons are just barely modified by weak interactions among the constituent atoms in the molecule being inspected.

The researchers were able to simultaneously monitor changes in two intracellular proteins that play crucial roles in the development of cancer. Two intracellular proteins, Stat1 and Stat6, in particular are considered particularly important in this early development.

Shachaf says the Stanford team was able to simultaneously monitor the changes levels in both proteins in lab-cultured myeloid leukemia cells. The changes in Stat1 and Stat6 closely tracked those demonstrated with existing visualization methods.

This, Shachaf tells D&IW, was the method used for validating the method's proof of principle.

She says that full development of the technique may improve the ability to diagnose cancers — for example, by determining how aggressive tumors' constituent cells are — and eventually to separate living, biopsied cancer cells from one another, based on characteristics indicating stage of progression or degree of resistance to chemotherapeutic drugs.

Shachaf says she hopes to demonstrate simultaneous visualization of nine or 10 COIN-tagged cellular features in the near future and then to take that number to 20 or 30, a new high, before long. "The technology's capacity may ultimately far exceed that number," she adds, reaching as many as 100 features.

The Stanford researchers, she says, are pursuing this goal by collaborating with a working group at the Scripps Research Institute (La Jolla, California) which has developed a raman flow cytometer.

The research describing this imaging technique was published April 15 in the online journal PLoS-ONE.

The study was funded by the Flight Attendant Medical Research Institute (Miami) and the Center for Cancer Nanotechnology Excellence (CCNE), focused on therapy response (CCNE-TR). CCNE-TR brings together scientists and physicians from Stanford, the University of California Los Angeles, Cedars Sinai Medical Center (Los Angeles), the Fred Hutchinson Cancer Center (Seattle), the University of Texas at Austin, Intel, and General Electric Global Research to utilize nanotechnology for cancer therapy management.

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