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In Cancer, Softer is Deadlier

11/20/2012
Chris Tachibana

Cancer cells are flexible and deformable—an advantage for squeezing into tissues. Exploiting this biomechanical property could yield new cancer biomarkers and drug targets.


Poke a cancer cell and what will you find? Well, for one thing, it’s softer than its normal neighbors. Cells from nearly all solid tumors—colon to lung to pancreatic—share this trait. Cell deformability might aid invasiveness, allowing cells to detach from a primary tumor and squeeze through tissues to reach the circulatory system and other organs. In reality, metastasis probably requires a range of biomechanical properties (1). Nonetheless, researchers see cancer cell squishability as an opportunity to discover new diagnostic markers and specific anticancer treatments.

Using fluorescence microscopy, Todd Sulchek confirmed that actin filaments become disorganized as cells transform from normal to malignant. Source: PLoS One





Assistant professor Todd Sulchek used atomic force microscopy to study the mechanical properties of various ovarian cell lines. Source: Georgia Institute of Technology

In Sulchek’s lab, an atomic force microscopy is positioned above a petri dish filled with ovarian cancer cells. Source: Georgia Institute of Technology

Lidong Qin, CPRIT Scholar at The Methodist Hospital Research Institute in Houston, discusses research which helps determine the disease stage of prostate cancer and ultimately leads to better treatment. Source: YouTube

Targeting the Cancerous Cytoskeleton

Cancer cells are more flexible than normal cells and in vitro, they have a disarrayed internal structure. In the 1970s, using fluorescence microscopy researchers found that actin filaments—components of the cytoskeleton that function in cell organization, communication, and movement—become disorganized as cells transform from normal to malignant. Researchers don’t know if tumor cells in patients undergo the same architectural changes.

The dramatic rearrangement of actin filaments in cancer cells in vitromight just be a cell culture phenomenon, says Peter Gunning, head of oncology research at the University of New South Wales, but the actin cytoskeleton clearly has fundamental roles in cancer (2). “The actin cytoskeleton is attractive as a drug target because it is involved in cell movement, integration with the environment, and tension sensing, which is involved in cell proliferation,” says Gunning.

However, no current cancer therapies target actin. One challenge is that actin is a major component of muscle. “The trick is to attack the actin cytoskeleton in cancer cells while sparing the major contractile tissues,” says Gunning. Possibilities for cancer-specific targets include actin-associated proteins involved in mechanosensing, which is linked to cell cycle signaling. Although he questions whether tumorigenesis requires complete disorganization of the actin cytoskeleton, Gunning finds cell deformability a relevant and interesting area of cancer research.

Pushing on Cell Softness

To study cell biomechanics—their squeezability—researchers use several methods. These methods include optical tweezers, which use lasers to stretch cells to measure elasticity, and micropipette aspiration, which determines cell stiffness by sucking a cell edge into a glass pipette. Particle-tracking microrheology, which follows the movement of fluorescent microparticles within cells, could be developed to study cell biomechanics in vivo.

In the 1980s, atomic force microscopy (AFM) emerged as a way to measure the biophysical properties of single, living cells. AFM can test a cell’s resistance to compression by poking it, the way a cook tests food for doneness by pushing it with a finger. And in 2007, Jianyu Rao’s and James Gimzewski’s groups at the University of California, Los Angeles used AFM to show metastatic and benign cells from cancer patients had measurably different stiffness.

At the Georgia Institute of Technology, research assistant Wenwei Xu from Todd Sulchek’s group used AFM to rank the malignancy of ovarian cancer cells. The AFM setup in the Sulchek laboratory has a microscopic probe on a cantilever, described by Sulchek as “like a tiny diving board.” The probe tip has a silicon bead that pushes on cells, exerting a force comparable to what cells might experience as they squeeze through tissue.

Using this probe, the researchers found that not only were cells from cancerous ovarian cell lines softer than cells from normal lines, invasive cancer cells were more deformable than less malignant cells (3). By analyzing two ovarian cancer cell lines that originated from the same tumor but have different degrees of invasiveness in mice, Xu and colleagues found that deformability correlated with metastatic potential. The researchers also analyzed gene expression differences in the invasive and noninvasive cell lines.

The findings connected the dots of altered expression of cytoskeletal remodeling proteins, actin filament disorganization seen by fluorescence microscopy, changes in cell stiffness, and increased malignancy. “It all pointed to increased deformation in invasiveness,” says Sulchek. “Doctors have long used palpation to detect and evaluate tumors. This is the same thing on a molecular level.”

Small Volumes, Big Results

Although AFM is not practical for clinical applications, microfluidic devices that run processes and reactions in nanoliter or picoliter volumes might be developed for diagnostics. At The Methodist Hospital Research Institute in Houston and Weill Medical College of Cornell University, Lidong Qin is generated mechanical separation chip (MS-chip) that uses microfluidics to sort cells by flexibility. MS-chips force cells to flow single-file through a maze of posts. Because studies have shown that invasive cancer cells can squeeze through spaces of about 8 microns, Qin place the posts between 7-15 microns apart.

The researchers tested MS-chips using a mixture of same-sized cells from two breast cancer cell lines: one stiff and nonmetastatic, and the other flexible and metastatic (4). The cell lines expressed different fluorescent proteins, one green and one red. Visually following the cells by their fluorescence, the researchers saw that the flexible metastatic cells navigated the tight paths better than the non-metastatic cells. The metastatic cells distributed throughout the chip while the non-metastatic cells clustered at the beginning, impeded by the maze. The team even recorded the flexible cells squeezing through the microscopic barriers while stiffer cells got stuck like Winnie the Pooh wedged in a door.

“The MS-chip separates and enriches cells, making downstream analysis possible,” says Qin, which is an advantage over methods such as AFM. As a proof-of-concept, the researchers used MS-chips to enrich populations of flexible and nonflexible cells from a breast cancer cell line known for heterogeneity in characteristics including biomarker display. Analysis of the collected cells showed that flexibility correlated with higher expression of known tumor-initiating genes and genes related to motility and migration.

Their results showed that MS-chips can separate cells by tumorigenic potential. Qin now plans to use the chips to enrich other types of cancer cells by potential invasiveness. He hopes that finding genes and proteins that correlate with metastatic ability will lead to new biomarkers for more precise cancer diagnoses. Qin says that MS-chips also have applications outside of cancer research. “As cells differentiate, they get stiffer, so we can use this technique to separate cells based on level of differentiation.” Qin’s laboratory is now collaborating with stem cell researchers to collect multipotent cells based on squishiness.

References

1. Wirtz D, Konstantopoulos K, Searson PC. 2011. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature Reviews Cancer 11:512-522.

2. Bonello T, Coombes J, Schevzov G, Gunning P, Stehn J. 2012. Therapeutic targeting of the actin cytoskeleton in cancer. In: Kavallaris M, editor. Cytoskeleton and human disease. Humana Press. p. 181-200.

3. Xu W, Mezencev R, Kim B, Wang L, McDonald J, and Sulchek T. 2012 Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells. PLoS One 7(10): e46609.

4. Zhang W, Kai K, Choi DS, Iwamoto T, Nguyen YH, Wong H, Landis MD, Ueno NT, Chang J, Qin L. 2012. Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells. Proc Natl Acad Sci U S A 109(46):18707-18712.