For the study, the researchers created multicellular spheroids using what’s known as the hanging droplet method. They placed droplets of cell culture media in an inverted Petri dish lid, then dispensed about 500 human mammary epithelial cells into each droplet. The dishes were then flipped upside down, allowing the drops to hang from the surface and the cells to form roughly spherical aggregates. Once the cells had come together to form multicellular spheroids, they were embedded in a collagen material that mimics the body’s extracellular matrix.

The design of the experiment enabled the researchers to not only image the movement of cells, but also to measure the forces they exert as they move. The team used cells tagged with fluorescent proteins, making them easy to track using confocal microscopy. The collagen matrix in which the cells were embedded was infused with tiny red tracer particles, which enabled the researchers to see how the matrix changed over time.

The experiment showed that within about five hours of being embedded in the matrix, the cells began to rotate collectively within the culture.

“It’s really striking to see,” said Ian Y. Wong, an associate professor of engineering at Brown and the study’s corresponding author. “These spheroids with hundreds of cells just start spinning around inside the collagen matrix.”

Scientists had seen that kind of rotational motion before. Similar dynamics had been observed in fruit fly egg chambers and in primary cells explanted from mammary tissue. But what came next hadn’t been observed before. After about 12 hours, a few “leader” cells began invading outward from the original sphere, creating small strands of cells that pushed their way through the external collagen. Eventually, these strands elongate the structure of the cell cluster, enabling more cells to move outward.

The tracers embedded in the collagen matrix and a technique called traction force microscopy shed light on why this invasion occurs.

“We saw that the cells were pulling a little harder in places where the original culture was a little bit sharper, where it deviates a little from being spherical,” said Hyuntae Jeong, a postdoctoral researcher in Brown’s School of Engineering and the study’s second author. “One of the consequences of that pulling is that it starts to reshape the collagen that surrounds the cells, aligning the fibers radially and steering the cells outward.”

The researchers further showed that they could alter the invasion pattern by changing the surrounding osmotic pressure — the ability of water to pass between the cell clusters and the external matrix. When “squeezed” under higher osmotic pressure, cells were arrested within the original spheroid and paused their invasion. Increasing pressure during the cell invasion phase caused lines of invading cells to retract back into the original spheroid, underscoring the importance of the physical environment on cell behavior.

The researchers hope the work might be a step toward understanding the complex dynamics of how tissues develop, as well as how cancer cells break free of tumors to spread around the body. There’s much more work to be done, but unpacking how cells interact with each other and their surroundings could be key pieces of the puzzle.

“This is really an argument for understanding the cellular microenvironment,” Wong said. “Cells are getting instructions from each other, but also from their surroundings. And they’re able to reshape their surroundings in important ways. We think it’s worth looking more deeply into these interactions between cells and their surroundings.”

Co-authors of the research were Carles Falcó (University of Oxford), Alex M. Hruska (Brown), W. Duncan Martinson (Francis Crick Institute), Alejandro Marzoratti (Brown), Mauricio Araiza (University of Wisconsin, Madison), Haiqian Yang (MIT), Vera C. Fonseca (Brown), Stephen A. Adam (Northwestern), Christian Franck (Wisconsin), José A. Carrillo (Oxford) and Ming Guo (MIT). The work was primarily supported by Brown University’s Hibbitt Engineering Fellowship, the National Institutes of Health (R01GM140108) and the U.S. Army Research Office (W911NF2310385).