Studies by scientists at Massachusetts Institute of Technology (MIT), Beijing Normal University, and the University of California, San Diego (UCSD) have generated new insights into the elastic properties of the basement membrane (BM) that sheaths breast cancer tumors, which could feasibly lead to new strategies for preventing tumor metastasis. Their results, reported in Proceedings of the National Academy of Sciences (PNAS), demonstrated that while this basement membrane is elastic, it also become stiffer as it expands. The team suggested that this property may help basement membranes control how tumors grow, and might also be harnessed to prevent tumor cells from breaking out and spreading to other sites.
“Now we can think of ways to add new materials or drugs to further enhance this stiffening effect, and increase the toughness of the membrane to prevent cancer cells from breaking through,” said Ming Guo, PhD, a lead author of the study and associate professor of mechanical engineering at MIT. The team reports its findings in a paper titled, “Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation.” Co-authors include first author Hui Li, PhD, at Beijing Normal University, Yue Zheng, PhD, and Shengqiang Cai, PhD, at the University of California at Santa Diego, and MIT postdoc Yu Long Han, PhD.
Basement membrane is a thin layer of extracellular matrix that surrounds most animal tissues, and acts as a physical barrier, while still permitting nutrient exchange, the authors explained. BM may be just a fraction of the thickness of a human hair, but it serves as a physical support that holds tissues and organs in place and helps to shape their geometry, while also keeping them separate and distinct.
Although basement membranes play important roles in tissue structural integrity, their physical properties remain largely uncharacterized, and scientists have limited understanding about their mechanical functions. “Given the importance of BMs as a semipermeable barrier maintaining tissue structural integrity, however, their permeability and mechanical properties remain largely unknown, mainly due to the lack of direct measurement methods, especially in situ,” they wrote. “This limits our understanding of the physical role of BMs in various physiological and pathological processes such as tumor development …”.
Most tumors are also sheathed in a protective basement membrane, which acts as a thin, pliable film that holds the cancer cells in place as they grow and divide. Before they can spread to other parts of the body, the cells must first breach this basement membrane. “ … in metastasis, cancer cells must invade through BMs to escape from the primary tumor—a process that causes 90% of cancer-related death,” the team continued. “Indeed, breaks in BMs can be observed in malignant tumors.”
Guo’s group specializes in the study of cell mechanics, with a focus on the behavior of cancer cells and the processes that drive tumors to metastasize. The researchers had been investigating how these cells interact with their surroundings as they migrate through the body. “A critical question we realized hasn’t gotten enough attention is, what about the membrane surrounding tumors?” Guo pointed out. “To get out, cells have to break this layer. What is this layer in terms of material properties? Is it something cells have to work really hard to break? That’s what motivated us to look into the basement membrane.”
Determining the mechanical properties of intact BMs in situ is, however, challenging because of their irregular shape, small thickness, and tight connection to the cells inside, the authors explained. This makes it hard to apply conventional mechanical tests to characterize the mechanical behavior of the BM in situ. Scientists have employed atomic force microscopy (AFM), using a tiny mechanical probe to gently push on the membrane’s surface. The force required to deform the surface can give researchers an idea of a material’s resistance or elasticity. But, as the basement membrane is exceedingly thin and tricky to separate from underlying tissue, Guo says it’s difficult to know from AFM measurements what the resistance of the membrane is, apart from the tissue underneath.
Rather than employ conventional mechanical testing methods, for their newly reported studies, the MIT team and colleagues used a pressure-controlled inflation/deflation approach—similar to blowing up a balloon—to isolate the membrane and measure its elasticity. They first cultured human breast cancer cells, which naturally secrete proteins to form a membrane around groups of cells known as tumor spheroids. They grew several spheroids of various sizes and inserted a glass microneedle into each tumor. They injected the tumors with fluid at controlled pressure, causing the membranes to detach from the cells and inflate like a balloon.
The researchers applied various constant pressures to inflate the membranes until they reached a steady state, or could expand no more, then turned the pressure off. “It’s a very simple experiment that can tell you a few things,” Guo noted. “One is, when you inject pressure to swell this balloon, it gets much bigger than its original size. And as soon as you release the pressure, it gradually shrinks back, which is a classical behavior of an elastic material, similar to a rubber balloon.”
As they inflated each spheroid, the researchers observed that, while a basement membrane’s ability to inflate and deflate showed that it was generally elastic like a balloon, the more specific details of this behavior were more surprising. To blow up a latex balloon typically requires a good amount of effort and pressure to start up. Once it gets going and starts to inflate a bit, the balloon suddenly becomes much easier to blow up. “Typically, once the radius of a balloon increases by about 38%, you don’t need to blow any harder— just maintain pressure and the balloon will expand dramatically,” Guo explained. This phenomenon, known as snap-through instability, is seen in balloons made of materials that are linearly elastic, meaning their inherent elasticity, or stiffness, does not change as they deform or inflate.
The team’s experimental measurements indicated that while the seemingly delicate BM is as tough as plastic wrap, it’s also surprisingly elastic like a party balloon, and able to inflate to twice its original size. However, unlike a typical latex balloon that demonstrates snap-through instability and becomes much easier to blow up after the initial effort, the basement membrane instead becomes stiffer as it inflated, indicating that the material is nonlinearly elastic, and able to change its stiffness as it deforms.
“…we find from our measurements that the elasticity of BM is highly nonlinear with a strong strain-stiffening effect,” the team noted. “This nonlinear stiffening behavior of the BM may have an important role in maintaining tissue mechanical integrity during growth and deformation (e.g., to avoid structural instability), which is a classical behavior leading to drastic expansion or even rupture when inflating most elastomeric balloon.”
And this stiff yet elastic quality may also help basement membranes control how tumors grow. The observation that the membranes appear to stiffen as they expand suggests that they may also restrain a tumor’s growth and potential to spread, or metastasize, at least to a certain extent.
Guo added, “If snap-through instability were to occur, a tumor would become a disaster, it would just explode. In this case, it doesn’t. That indicates to me that the basement membrane provides a control on growth.”
The team plans to measure the membrane’s properties at different stages of cancer development, as well as its behavior around healthy tissues and organs. They are also exploring ways to modify the membrane’s elasticity to see whether making it stiffer will prevent cancer cells from breaking through. “We are actively following up on how to modify the mechanics of these membranes, and apply perturbations on breast cancer models, to see if we can delay their invasion or metastasis,” Guo noted. “This is an analogy to making a stiffer balloon, which we plan to try.”