Before spreading to other parts of the body, the cells must breach the basement membrane, a material that itself has been tricky for scientists to characterize.
Now MIT engineers have probed the basement membrane of breast cancer tumors and found that the seemingly delicate coating is as tough as plastic wrap, yet surprisingly elastic like a party balloon, able to inflate to twice its original size.
But while a balloon becomes much easier to blow up after some initial effort, the team found that a basement membrane becomes stiffer as it expands.
This stiff yet elastic quality may help basement membranes control how tumors grow. The fact that the membranes appear to stiffen as they expand suggests that they may restrain a tumor’s growth and potential to spread, or metastasize, at least to a certain extent.
The findings, published this week in the Proceedings of the National Academy of Sciences, may open a new route toward preventing tumor metastasis, which is the most common cause of cancer-related deaths.
“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,” says Ming Guo, a lead author of the study and associate professor of mechanical engineering at MIT.
Guo’s co-authors include first author Hui Li of Beijing Normal University, Yue Zheng and Shengqiang Cai of the University of California at Santa Diego, and MIT postdoc Yu Long Han.
Blowing up
The basement membrane envelopes not only cancerous growths but also healthy tissues and organs. The film – a fraction of the thickness of a human hair – 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.
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 says. “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.”
To measure the membrane’s properties, 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.
Instead, the team used a simple technique, 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 a 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 says. “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.”
Elastic snap
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 surprisingly different.
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 percent, you don’t need to blow any harder – just maintain pressure and the balloon will expand dramatically,” Guo says.
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.
But based on their measurements, the researchers found that the basement membrane instead became stiffer, or more resistant as it inflated, indicating that the material is nonlinearly elastic, and able to change its stiffness as it deforms.
“If snap-through instability were to occur, a tumor would become a disaster – it would just explode,” Guo says. “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 says. “This is an analogy to making a stiffer balloon, which we plan to try.”
Cellular phenotypes and molecular functions are fundamentally dependent on signals from outside the cell such as the interactions with the extracellular matrix (ECM). The core matrisome is composed of ~300 unique matrix macromolecules and can be classified into collagens, proteoglycans (such as heparan sulphate proteoglycans, versican and hyaluronan) and glycoproteins (such as laminins, elastin, fibronectin and tenascins)1.
These ECM components are modified post-translationally by an array of secreted remodelling enzymes, such as oxidases and proteases. In addition, the ECM binds soluble factors, such as growth factors and other ECM-associated proteins. Cell surface receptors interact with ECM components and ECM-bound factors to mediate cell adhesion and cell signalling thereby regulating processes as diverse as proliferation, differentiation, migration and apoptosis2. ECM can also demonstrate very different mechanical and topographical properties, which, importantly, can influence cell fate and function via different mechanosignalling routes3.
The ECM has two main forms, which differ in function, composition and location. The interstitial matrix forms porous three-dimensional networks around cells that interconnect cells in the stroma and can connect to the basement membrane, which is the other form of ECM structure. The interstitial matrix guarantees the structural integrity of tissues and organs but also modulates processes such as cell differentiation and migration.
The protein composition of the interstitial matrix mainly includes collagens I, III, V, etc., fibronectin and elastin. Abundance and composition of the interstitial matrix vary between tissue types, between microenvironments within the same tissue and can be remodelled in response to force stress or trauma such as wound repair or tissue regeneration4.
In cancer, remodelling of the interstitial ECM induces a broad range of biophysical and biochemical changes affecting cell signalling, ECM stiffness, cell migration and tumour progression5. In contrast, the basement membrane is a more stable, sheet-like, dense structure that lines the basal surface of, for example, epithelial and endothelial cells, surrounds muscle cells and adipocytes6, and separates tissues into different, well-organised compartments.
The basement membrane consists mainly of collagen IV and laminins, which are interconnected through different network-bridging proteins such as nidogen and heparan sulphate proteoglycans (HSPGs)7. Binding of cells to the basement membrane is essential for establishing epithelial cell polarity and is crucial for many developmental processes and maintenance of tissue homoeostasis8. Remodelling of the basement membrane is required for cancer cells to invade stromal tissue and become a malignant tumour9.
Complex ECM remodelling processes, involving over 700 proteins1, change overall abundance, concentration, structure and organisation of individual ECM components, thereby affecting the three-dimensional spatial topology of the matrix around cells, its biochemical and biophysical properties and consequently its effect on cell fate.
ECM remodelling is an essential and tightly regulated physiological process in development and in restoring tissue homoeostasis during wound repair10. However, it is not surprising that cells dysregulate this process in pathologic conditions such as inflammatory diseases, tissue fibrosis, and cancer11. Recent research highlights the importance of the tumour-mediated systemic aberrations of the ECM for the establishment of metastasis.
In this review, we discuss remodelling mechanisms of extracellular matrices and the implications of these mechanisms during cancer development, and describe recent concepts of ECM remodelling shaping tissues for tumour cells to metastasise. Increasing understanding of these processes opens up the possibilities of therapeutic approaches to target the aberrant ECM and/or the underlying pathologic mechanisms of its remodelling and prevent malignancy.
Changes in ECM composition in cancer
ECM components possess both tumour-suppressing and tumour-promoting properties. For example, depending on its molecular weight hyaluronan (HA) functions as a tumour suppressor or a tumour promoter (reviewed in Bohaumilitzky et al.39). The tumour-resistance of the longest-lived rodent, the naked mole-rat, involves the expression of a unique HA with high molecular mass (HMM-HA) as a major ECM component. HMM-HA signalling through the CD44 receptor activates the expression of key tumour suppressor genes40, leading to a hypersensitive cell-cycle arrest, a common mechanism of tumour suppression41.
In contrast, high levels of HA, and in particular small HA oligosaccharides (LMM-HA), are associated with poor prognosis in several tumours such as colorectal, breast and prostate cancer42–46. Here, dysregulated HA synthetase and HA-degrading hyaluronidase lead to the accumulation of LMM-HA42,47.
LMM-HA directly interacts with cell surface receptors regulating pro-tumourigenic signalling cascades48, including glycolysis, the main source of energy in tumours, and promotes migration49. LMM-HA signalling through CD44 also increases the resistance to cellular stress and thereby may promote tumour development50.
The most common tumourigenic alteration of ECM homoeostasis is an increased deposition of fibrillar collagen13,51,52, which has direct tumour-promoting properties. Additionally, increased deposition of major ECM components including fibronectin, HA and tenascin C into the interstitial matrix results in a fibrotic phenotype, termed desmoplasia, which is similar to the alterations observed during organ fibrosis.
Desmoplasia is a key characteristic of various cancers such as breast cancer and PDAC and is associated with poor prognosis53–55. For example, in a murine breast cancer model, the increased deposition of collagen I results in increased tumour formation and development of metastasis, directly linking ECM remodelling with aggressive tumour progression in vivo56.
Furthermore, collagen V has been associated with altered mitogenic signalling in breast cancer. It can enhance the co-receptor ability of the surface proteoglycan, glypican-1 (GPC1), to modify FGF-2 signalling leading to increased cell proliferation56,57.
Analysing the complex global changes in the ECM may be beneficial for early cancer detection58. Unique ECM signatures that are characteristic of some tumours change during tumour progression and are predictive of clinical outcomes59–61. The combined pathologic alterations of the ECM provide a protein fingerprint by the release of cancer-specific ECM fragments into the circulation that may have diagnostic implications as it was shown for lung, ovarian, breast and colorectal cancer62–64. Moreover, even before tumour development, increased deposition of collagen I and proteoglycans lead to increased breast density, which is the greatest independent risk factor for the development of breast cancer (Box 1)65–67.
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Box 1 breast density and cancer risk
In the breast, mammographic density is indicative of a higher risk for breast cancer development, which is used for preventive screening276,277. High mammographic density is mediated by an increased ECM (e.g., collagen and proteoglycan) deposition and further cross-linking and alignment events which all together also result in stiffer breast tissue. A complex interplay of hormonal, inflammatory and environmental factors may all influence breast density276,277. So far, the detailed underlying mechanisms that trigger these ECM changes and how breast density increases breast cancer risk278 are unclear.
Breast cancer risk is also influenced by the fundamental changes that the mammary gland undergoes during different reproductive states, which are characterised by profound ECM remodelling processes279. Whether or not pregnancy protects or promotes breast cancer is still controversial and largely depends on the age of the woman during her first birth280.
During pregnancy, the breast epithelium and tissue matrix are intensively remodelled to prepare for lactation. After nursing, the lactating glands and surrounding breast stroma are remodelled to its pre-pregnancy-like state in a process called involution.
These remodelling processes are exceptionally rapid and comprehensive. Each state is characterised by a massive change in ECM abundance and composition. Following pregnancy, the breast retains a more developed lobular architecture281, which may also affect the proportion of epithelial cell types in the breast282. Furthermore, even the fully regressed mammary gland shows a higher abundance of ECM components, in particular collagen I, compared to the pre-pregnancy (nulliparous) state283.
During involution, massive ECM proteolysis mediated by various upregulated MMPs leads to the disruption and degradation of the basement membrane, thereby detaching epithelial cells, which triggers cell death of unnecessary secretory mammary epithelium. High MMP activity also leads to the release of bioactive matrix proteins, matrikines, and growth factors and the recruitment of ECM-clearing immune cells284.
Interestingly, ECM remodelling during involution shows high similarities to processes during inflammation and wound healing, which was also shown to provide a bonafide environment for cancer development through pro-inflammatory TGF-β285. Together, these inflammatory conditions and the loss of the barrier function of the basement membrane temporally create a pro-tumourigenic environment that may increase the risk for more aggressive breast cancer280.
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reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547708/
More information: Hui Li el al., “Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation,” PNAS (2021). www.pnas.org/cgi/doi/10.1073/pnas.2022422118
