Tissue rigidity might help promote tumor progression, scientists report today in this month's
Tumors are stiffer than normal tissue. To determine if that stiffness contributes to a tumor's malignancy, Valerie Weaver at the University of Pennsylvania in Philadelphia and her colleagues added cells to a three-dimensional gel culture designed to mimic the extracellular matrix, made of synthetic acrylamide or a mix of natural basement membrane and collagen. The researchers increased stiffness of the matrix by boosting either acrylamide cross-linking or collagen levels. When normal mammary epithelial cells were raised in a culture whose stiffness matched a normal extracellular matrix at roughly 170 pascals, they grew and formed normally differentiated structures. With a slight increase of stiffness by up to 1,200 pascals -- still less rigid than a tumor -- the cells showed malignant properties, such as disrupted tissue architecture and significantly increased proliferation.
Prior research has shown that tumors have aberrant levels of integrin, transmembrane extracellular matrix receptors that transduce mechanical signals. Integrins also help regulate cell proliferation through growth-factor dependent extracellular signal-regulated kinase (ERK) activation. When Weaver and colleagues analyzed the integrin activity of soft and rigid cultures using confocal immunofluorescence microscopy, they found that, under stiff conditions, integrins tended to cluster together and form focal adhesions, structures linking integrins to the cytoskeleton. The same was not true under soft conditions, however. Looking at mouse mammary tumors in vivo, the researchers also saw adhesion formation, suggesting tissue rigidity promotes aberrant growth by influencing focal adhesion formation. "We speculate the focal adhesions are recruiting molecules and enhancing signaling that does not normally happen in differentiated structures," Weaver said.
Integrins regulate Rho GTPases, which increase cytoskeletal tension. Using pull-down assays, the researchers found increasing gel stiffness elevated Rho activity. Confocal immunofluorescence microscopy revealed that overexpressing constitutively active V14Rho in normal cells increased the number and size of focal adhesions, disrupted tissue architecture, enhanced growth-factor dependent ERK activation and significantly boosted cell spreading on a soft gel. Drugs inhibiting ERK activation or cell contractility decreased cytoskeletal tension and counteracted V14Rho-triggered growth. It's not clear whether these drugs could affect other vital pathways. This suggests rigidity leads to focal adhesions and aberrant growth by increasing tension in the cell normally generated by Rho.
"For so many years, it was thought all the signals to turn on cell division were soluble growth factors. Then came the concept by Don Ingber that the cell had to be pulled or attached and be under tension for growth. What is really neat here is that Valerie Weaver's paper connects many prior mechanical phenomena regarding cell growth with a molecular mechanism, and how when you don't have the right conditions you might get a cancer cell," Judah Folkman at Harvard Medical School, who did not participate in this study, told
"You would really want to develop ways to measure the effects of changing mechanical rigidity in vivo," Ingber added. "You could have force-sensitive dyes like you now have calcium-sensitive dyes."
"Now we'd like to figure out which integrins are responsible here. They're not all the same," Weaver said. Future experiments could also study different cell types to see what different stiffnesses they require.
These findings could help researchers learn how to "treat cancer in a completely novel way, by going after extracellular matrix and cell rigidity," Weaver added. She and her colleagues plan on testing different transgenic cell lines, to see if certain oncogenes help tumors spread by increasing cell contractility, for instance.