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CARS visualizes lipids in cancer metastasis

Apr 2009
Caren B. Les,

Researchers have provided mechanistic evidence linking a high-fat diet to increased cancer metastasis by measuring the precise impact of fats on cancer cells using coherent anti-Stokes Raman scattering (CARS) microscopy and a laser-based tool called intravital flow cytometry.

Ji-Xin Cheng, who led the investigative team at Purdue University said that, for more than 30 years, doctors have observed a strong correlation between lipid-rich tumor biopsies from cancer patients and aggressive clinical cancer behaviors, including early death.

Cheng, an assistant professor of biomedical engineering and of chemistry, said it has been widely observed that diet and obesity account for 30 percent of preventable causes of cancers, but that the relationship between diet and cancer incidence and mortality remains controversial. For example, he said, omega-3 fish oil and olive oil are widely believed to prevent cancer, but there is no scientific evidence either to support or dispute these claims. Clinical studies have yielded conflicting results, he added.

A contributing factor to the controversy is the lack of mechanistic understanding of how fat affects cancer development, he added. Doctors cannot classify lipid-rich tumors as a distinctive type of carcinoma, nor do they know how to treat patients with lipid-rich tumors differently from those with other types of tumors, Cheng said, adding that their study demonstrated that lipid-rich tumors are consequences of excess lipids in tumor environments.

The experiment

The researchers conducted an experiment on mice, into which they implanted lung cancer tumors. The animals were separated into two groups, one fed a high-fat diet and the other, a lean diet. The CARS technique documented how the increased dietary fat caused the cancer cells to undergo changes that enabled them to metastasize, and the flow cytometry method counted the number of cancer cells in the bloodstream of the mice in both groups.

Researchers have found that excessive dietary fat has caused a significant increase in cancer metastasis in laboratory animals. The two round objects in the image are lipid-rich cancer cells attached to collagen fibrils. They were simultaneously visualized using CARS and sum frequency generation imaging. Cancer cells attach to collagen before spreading to organs in the body. Courtesy of Thuc Le and Ji-Xin Cheng.

Study results showed that, although the increase in lipids had no impact on the original tumors implanted in the mice, the rate of metastasis rose 300 percent in the mice fed a high-fat diet. The researchers also examined the animals’ lungs and counted the number of cancer cells that had migrated there as a result of metastasis. Those findings supported the other results showing increased metastasis in the animals fed a high-fat diet.

The CARS technique

The CARS technique documented how increasing lipids from fat intake induced changes to the cancer cell membranes. Unlike fat cells, cancer cells have limited capacity to store lipids in the cytoplasm. Excess lipids were channeled into the cancer cell membranes. Those changes, including processes called membrane phase separation and membrane rounding, enhance cancer metastasis. Without excess lipids, cancer cells stick together and form tight junctions in tumors, but increasing lipids causes them to take on a rounded shape and separate from each other, Cheng said. This change enables them to separate from each other and spread throughout the body via the bloodstream.

Cheng said that CARS is a lipid-sensitive imaging technique and that the Raman-based method is sensitive to the vibrational signatures of chemical bonds. Unlike Raman, however, CARS employs multiple photons to address the molecular vibrations and produces a signal in which the emitted waves are coherent with one another. As a result, CARS is orders of magnitude stronger than spontaneous Raman emission, Cheng explained. Because CARS signals arise from vibrations of intrinsic chemical bonds of an object, they require no labeling for visualization. CARS also is highly sensitive to lipid-rich structures because of the abundance of C-H bonds, he added.

A typical CARS microscope with picosecond pulse excitation can perform simultaneous CARS, sum frequency generation and two-photon excitation fluorescence imaging.

Intravital flow cytometry

The intravital flow cytometry technique works by shining a laser through the skin and into blood vessels, where dyed cancer cells are visible. Cheng said that, in the study, the technique detected circulating tumor cells expressing both green and red fluorescent proteins. The cancer cell line that they used was genetically engineered to carry genes from jellyfish, which express variants of fluorescent protein. Circulating tumor cells fluoresce because of the fluorescent proteins, whereas other cells do not. The number of circulating tumor cells is enumerated by counting fluorescent objects in the bloodstream, Cheng said.

The technique does not require blood to be drawn from humans and animals, an advantage for the long-term monitoring of cancer metastasis. A primary challenge, however, is the ability to label circulating tumor cells so that they can be distinguished from other blood cells. Cheng said that a lot of dye is needed because an average human has approximately 5 l of blood, and that long-term toxicity of dyes to humans has yet to be evaluated.

Label-free detection

Cheng noted that their discovery of lipid-rich circulating tumor cells should inspire the development of combined CARS intra-vital flow cytometry for label-free detection of circulating tumor cells. CARS can detect lipids without labeling and thus can discriminate lipid-rich circulating tumor cells from lipid-poor blood cells.

He said that recent advances in optics may help to overcome challenges in intravital CARS flow cytometry. Adaptive optics should enable the technique to be performed on blood vessels farther from the skin and should allow visualization of blood components with better clarity by removing optical distortion. A big challenge for tissue imaging is the scattering of light by components of tissues, including cells, which deteriorate image quality. Adaptive optics minimizes such scatterings through optics modulation.

Other improvements could include CARS signal generation from a single picosecond synchronously pumped optical parametric oscillator for increased penetration depth and video rate CARS microscopy for faster laser scanning speed.

Currently, CARS application is limited to the lab bench on small animals. Bedside application is dependent on the successful development of CARS endoscopy, Cheng said. Other developments might include the application of CARS to elucidate not only how obesity increases metastasis but also how it might play a direct role in initiating the development of cancers.

Dietary implications

Based on findings of the study, it appears that if a patient already has cancer, eating a high-fat diet greatly increases the risk of its spreading, according to Cheng. However, he added that doctors should advise their patients with many caveats, especially since their study was performed on lab animals and may not hold true in humans. He added that the study did not dwell on obesity as a cause of cancer but rather on obesity or a high-fat diet as accelerators of cancer spread.

The researchers also documented that linoleic acid, which is found in polyunsaturated fats, caused increased membrane phase separation, whereas oleic acid, found in monounsaturated fats, did not. These findings support earlier evidence from other research that consuming high amounts of polyunsaturated fat may increase the risk of cancer spreading.

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