Not all tumor cells are equal: huge genetic diversity found incells shed by tumors

The finding underscores how multiple types of treatment may berequired to cure what appears outwardly as a single type of cancer,the researchers say. And it hints that the current cell-line modelsof human cancers, which showed patterns that differed from thetumor cells shed from human patients, need to be improved upon. The new study, published May 7 in PLoS ONE , is the first to look at so-called circulating tumor cells one byone, rather than taking the average of many of the cells. And it’sthe first to show the extent of the genetic differences betweensuch cells. “Within a single blood draw from a single patient, we’reseeing heterogeneous populations of circulating tumor cells,”said senior study author Stefanie Jeffrey, MD, professor of surgeryand chief of surgical oncology research.

For over a century, scientists have known that circulating tumorcells, or CTCs, are shed from tumors and move through thebloodstreams of cancer patients. And over the past five years,there’s been a growing sense among many cancer researchers thatthese cells — accessible by a quick blood draw — could be the keyto tracking tumors non-invasively. But separating CTCs from bloodcells is hard; there can be as few as one or two CTCs in everymilliliter of a person’s blood, mixed among billions of other bloodcells. To make their latest discovery, Jeffrey, along with aninterdisciplinary team of engineers, quantitative biologists,genome scientists and clinicians, relied on a technology theydeveloped in 2008. Called the MagSweeper, it’s a device that letsthem isolate live CTCs with very high purity from patient bloodsamples, based on the presence of a particular protein — EpCAM –that’s on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, theteam first tested whether they could accurately detect theexpression levels of 95 different genes in single cells from sevendifferent cell-line models of breast cancer — a proof of principlesince they already knew the genetics of these tumors. Theseincluded four cell lines generally used by breast cancerresearchers and pharmaceutical scientists worldwide and three celllines specially generated from patients’ primary tumors. “Most researchers look at just a few genes or proteins at atime in CTCs, usually by adding fluorescent antibodies to theirsamples consisting of many cells,” said Jeffrey. “Wewanted to measure the expression of 95 genes at once and didn’twant to pool our cells together, so that we could detectdifferences between individual tumor cells.” So once Jeffrey and her collaborators isolated CTCs using theMagSweeper, they turned to a different kind of technology:real-time PCR microfluidic chips, invented by a Stanfordcollaborator, Stephen Quake, PhD, professor of bioengineering. Theypurified genetic material from each CTC and used thehigh-throughput technology to measure the levels of all 95 genes atonce. DVB-T2 Digital Receiver

The results on the cell-line-derived cells were a success;the genes in the CTCs reflected the known properties of thecell-line models. So the team moved on to testing the 95 genes inCTCs from 50 human breast cancer patients — 30 with cancer thathad spread to other organs, 20 with only primary breast tumors. “In the patients, we ended up with a subset of 31 genes thatwere most dominantly expressed,” said Jeffrey. “And bylooking at levels of those genes, we could see at least twodistinct groups of circulating tumors cells.” Depending onwhich genes they used to divide the CTCs into groups, there were asmany as five groups, she said, each with different combinations ofgenes turned on and off. DVB-S2 Set Top Box

And if they’d chosen genes other than the95 they’d picked, they likely would have seen different patterns ofgrouping. However, because the same individual CTCs tended to grouptogether in multiple different analyses, these cells likelyrepresent different types of spreading cancer cells. The diversity, Jeffrey said, means that tumors may contain multipletypes of cancer cells that may get into the bloodstream, and asingle biopsy from a patient’s tumor doesn’t necessarily reflectall the molecular changes that are driving a cancer forward andhelping it spread. Moreover, different cells may require differenttherapies. One breast cancer patient studied, for example, had someCTCs positive for the marker HER2 and others lacked the marker.When the patient was treated with a drug designed to targetHER2-positive cancers, the CTCs lacking the molecule remained inher bloodstream. ATSC Digital Receiver Manufacturer

When the team went on to compare the diverse genetic profiles ofthe breast cancer patients’ CTCs with the cells they’d studied fromthe cell lines, they were in for another surprise: None of thehuman CTCs had the same gene patterns as any of the cell-linemodels. “These models are what people are using for drug discovery andinitial drug testing,” said Jeffrey, “but our findingsuggests that perhaps they’re not that helpful as models ofspreading cancers.” While the human cell-line cells did showdiversity between each of the seven cell lines, they didn’t fallinto any of the same genetic profiles as the CTCs from human bloodsamples. These results don’t have immediate impacts for cancer patients inthe clinic because more work is needed to discover whetherdifferent types of CTCs respond to different therapies and whetherthat will be clinically useful for guiding treatment decisions. Butthe finding is a step forward in understanding the basic sciencebehind the bits of tumors that circulate in the blood.

It’s thefirst time that scientists have used high-throughput gene analysisto study individual CTCs, and opens the door for future experimentsthat delve even more into the cell diversity. The Stanford team isnow working on different methods of using CTCs for drug testing aswell as studying the relationship between CTC genetic profiles andcancer treatment outcomes. They’ve also expanded their work toinclude primary lung and pancreatic cancers as well as breasttumors. The first authors of the study are former postdoctoral scholarsAshley Powell, PhD, and AmirAli Talasaz, PhD, and researchscientist Haiyu Zhang, PhD. The other corresponding authors areRonald Davis, PhD, professor of biochemistry, and Shanaz Dairkee,PhD, visiting professor.

Other Stanford co-authors include Quake;Marc Coram, PhD, assistant professor of health research and policy;former research scientist Glenn Deng, PhD; Fabian Pease, PhD,emeritus professor of electrical engineering; Michael Mindrinos,PhD, senior research scientist; Melinda Telli, MD, assistantprofessor of medicine; Ranjana Advani, MD, professor of medicine;Robert Carlson, MD, professor of medicine; Joseph Mollick, MD, PhD,clinical instructor of medicine; Shruti Sheth, MD, clinicalinstructor of medicine; Allison Kurian, MD, assistant professor ofmedicine; James Ford, MD, associate professor of medicine and ofgenetics; and Frank Stockdale, MD, PhD, professor emeritus ofmedicine. The team also collaborated with researchers at RutgersUniversity, the Cancer Institute of New Jersey and the SimonsCenter for Systems Biology in New Jersey. The MagSweeper is licensed by Stanford to the sequencing companyIllumina. Jeffrey, Powell, Talasaz, Mindrinos, Pease and Davisreceive royalties for their contributions to the technology;Jeffrey donated her royalties to a nonprofit.

The work was supported by the National Institutes of Health, theCalifornia Breast Cancer Research Grants Program Office of theUniversity of California, the John and Marva Warnock CancerResearch Fund and donations from Andrew and Debra Rachleff andVladimir and Natalie Ermakoff.

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