The researchers studied two lines of metastatic tumour cells: MDA-MB-231 breast-cancer cells, which prefer rigid substrates, and SKOV-3 ovarian-cancer cells, which prefer compliant substrates. They cultured both cell lines on soft (2.83 kPa, similar to fatty tissue) and hard (34.88 kPa, similar to bone) gel substrates, as well as on a collagen-coated glass control, and assessed three important malignant characteristics: the cells' ability to grow, to survive chemotherapy and to migrate (Phys. Biol. 12 026001).

"We recently discovered that ovarian cancer cells, which preferentially metastasize to a soft environment, become more aggressive when grown on soft gels – the exact opposite of breast-cancer cells, which often metastasize to stiffer environments like bone," explained lead-author Michelle Dawson. "We performed this study to understand this disparate behaviour and elucidate the pathways responsible for the different mechanical preferences of ovarian and breast cancer cells."

Dawson and colleagues first quantified cell proliferation on the three substrates. They found that significantly less breast-cancer cells and more ovarian-cancer cells proliferated on soft substrates, compared with hard substrates and glass. Analysing cell viability after treatment with the anti-cancer drug doxorubicin revealed that breast-cancer cells on glass showed 20% higher viability over those on soft substrates. Ovarian-cancer cells were most resistant to doxorubicin on soft substrates.

To quantify the impact of rigidity on cell migration, the researchers labelled cells with a dye to track cell motion. Breast-cancer cells displayed higher velocities on hard substrates than on soft substrates, with even larger gains in speed seen on glass. Ovarian-cancer cells exhibited lower velocities, but were still most motile on soft substrates. The authors note that this phenomenon was mitigated in less metastatic cell lines, suggesting that it may be a property of metastatic cells.

Microarray analysis

To isolate the molecules responsible for these opposing responses, the researchers used microarray analysis to assess the expression of genes associated with the actomyosin cytoskeleton (the protein responsible for regulating cytoskeletal tension). They found that breast-cancer cells expressed higher levels of genes associated with actomyosin contraction, such as myosin light chain, myosin heavy chain, myosin light chain kinase (MLCK) and RhoA. Ovarian-cancer cells, meanwhile, expressed higher levels of genes associated with actin filament stabilization.

The researchers hypothesized that these different gene expressions could be responsible for the cell lines' different responses to matrix rigidity, and investigated whether chemically blocking these molecules could mitigate this behaviour. To do this, they treated the cells to inhibit either Rho kinase (ROCK, which is activated by RhoA to increase cytoskeletal tension), MLCK or non-muscle myosin II activity. Inhibiting ROCK or MLCK produced rigidity-independent behaviour in both cell lines. Inhibition of non-muscle myosin in breast-cancer cells increased spreading on soft substrates and decreased spread on hard substrates.

They also tested whether increasing contractility in ovarian-cancer cells – to mimic the more contractile breast-cancer cells – could recover function on hard substrates. They found that this treatment increased the spread area and cell motility on hard substrates, but that, just like breast cancer, the ovarian-cancer cells with increased contractility now collapsed on soft substrates.

Traction force cytometry

To investigate how the cells interact with their underlying substrates, the team performed traction-force cytometry. Both breast- and ovarian-cancer cells exerted two to three times more force on their preferred substrates (hard and soft, respectively). ROCK inhibition eliminated this force increase in both cell lines, but had no effect on forces exerted on less preferred substrates.

Inhibiting MLCK increased force exertion by breast-cancer cells on both substrates, whereas in ovarian-cancer cells, it decreased force on the preferred soft substrate but did not affect cells on a hard substrate. Increasing contractility in ovarian-cancer cells with LPA increased force exertion on hard substrates but decreased force on soft substrates.

To investigate these disparities, the researchers analysed the spatial polarization (the force distribution relative to the cell centre) of the exerted forces. They found that while the total amount of force exerted by each cell did not correlate with cell motility, the polarization of these forces did.

"These studies can tell us a lot about bad ways to target metastasis, as well as new potential targets," explained first-author Daniel McGrail. "For instance, researchers have previously shown that inhibiting ROCK does indeed block breast-cancer bone metastasis; however, it may also unintentionally increase lung metastasis by reducing cell contractility enough to allow for the cells to survive in this softer tissue."

Dawson added that the team hopes to apply its cell-behaviour findings to patient biopsies, to enable a personalized approach to cancer treatment. "However, this study required the generation of more than 1000 individual substrates by hand – something far from scalable to the clinic," she said. "To address this, we are developing a high-content method to multiplex several of the experiments and automate the downstream analysis to enable large-scale translation."

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