A representative image of cancer cells. Image: NCI/Unsplash
- When one cancerous cell arises in a population of otherwise normal cells, they work together to remove the cancerous cell from the tissue.
- This removal is well-documented, but scientists still wish to know the chemical and physical signals that tell normal cells what they have to do.
- A team in TIFR Hyderabad conducted a series of tests in which it unearthed novel, even counterintuitive findings to answer this and other questions.
- The study, which independent scientists have commended, adds “an important piece to the puzzle of how” our cells’ microenvironment affects cancers.
Hyderabad: Scientists from the Tata Institute of Fundamental Research, Hyderabad, have revealed new insights into an important defence mechanism the body uses to respond to early-stage cancer.
The findings are important because they shed light on fundamental concepts that could guide future work on helping the body respond better in these early stages, before the cancer gets worse.
“This paper is a textbook example of basic research that could be translated into novel ideas and new technologies,” Tuli Dey, a cancer biologist at the Savitribai Phule Pune University, said. “This should encourage our funding bodies to fund basic research.”
A common metaphor for cancer is that of a battle: cancerous cells arising due to genetic mutations rapidly defeat normal cells, leading to the spread of cancer. Biologists’ name for this process, in which cells compete with each other for space and resources, is straightforward: cell-cell competition.
Scientists hold cell-cell competition to be a fundamental defence mechanism that protects the body against cancer when the cancer is just taking root. When one cancerous cell arises in a population of otherwise normal cells, they work together to remove the cancerous cell from the tissue. This process is called cell extrusion.
While cell extrusion is well-documented, scientists are still looking for answers to several questions. For example, how do the normal cells throw out the cancerous cell? And which chemical and physical signals tell normal cells what they have to do?
The team of researchers at TIFR Hyderabad, led by Tamal Das, a principal investigator, answered some of these questions in their study published on January 11, 2022.
Das was interested in conducting the study given his long-standing interest in how cells behave collectively, in a group. Shilpa P. Pothapragada, a PhD student with Das and a member of the study, was drawn in by the interdisciplinary field of mechanobiology – the study of how physical and mechanical forces influence cellular processes.
‘Tissue’ is the name for the material from which our bodies are made. It consists of cells and their products.
The most common tissue in any organism is epithelial tissue. It covers the body’s surfaces and cavities, among other things. Examples include skin and the lining of the gut.
Since around 80% of all cancers are epithelial in origin, Das told The Wire Science that “cancer can be, in some ways, called a disease of the epithelial tissue”.
For researchers interested in studying the collective behaviours of cells, epithelial tissues are a great place to start. The tissue’s cells are tightly packed, which means they perform many of their functions together, as a group.
Now, these cells are surrounded by the extracellular matrix (ECM). This is a mesh of proteins, minerals and other macromolecules that physically and chemically support the cells in a tissue.
Scientists already know that the ECM changes in many ways in the presence of a cancer. One of them is that it becomes stiffer. This happens because two components of the ECM – a protein called collagen and a compound called hyaluronic acid – start to form bonds with each other. And scientists know that a stiffer ECM is positively correlated with oncogenesis, the genesis of cancer.
The TIFR team’s question was this: if cancer is often a disease of the epithelial tissue, why are cancers of the epithelial tissue so rare?
That is, around 80% of all cancers are epithelial in origin, according to Das. This means 80% of the cancers that do happen, happen in epithelial tissues. But because the mutations that lead to cancer are ubiquitous, the total number of epithelial cancer cases should be a lot higher. But this isn’t the case.
For answers, the team investigated the epithelial defence against cancer. The idea is that epithelial tissue, among all tissues, has a built-in defence that pushes back newly formed cancerous cells from invading the tissue. One unit in this defence is cell extrusion.
The team members grew epithelial cells in Petri dishes in the lab. Here, they mixed two populations of cells. One population consisted of normal cells and the other consisted of cells genetically modified to become cancerous.
They let the mixed population grow as a single layer – much like how epithelial cells grow in the body – on top of a hydrogel, an extremely porous material made mostly of water. (The hydrogel was a proxy for the ECM.)
The researchers found that when the cells were growing on less stiff hydrogel, the normal cells would extrude the cancerous cells more efficiently than when the cells were growing atop more stiff hydrogel.
Second, cancerous cells that weren’t extruded on the stiffer substrates continued to grow and occupy more and more space in the layer – mimicking the way a cancer progresses in its early stages.
For Das, this was one of the most surprising findings of the study.
He said that “since stiffer substrates exert a lot of forces on the cells, I was under the impression that cell extrusion would work better on stiffer substrates. However, what we found was the complete opposite.”
Gautam I. Menon, a mechanobiologist and a professor at Ashoka University, Haryana, commended the study for highlighting “one route to understanding why conditions such as obesity and ageing, which lead to tissue stiffening, correlate with an increased risk of cancer.”
He also said this study reaffirms his belief that “the role of [physical] forces cannot be neglected in a host of biological processes.”
The molecular mechanism
Having found that the stiffness of the ECM could affect how well normal cells can evict cancerous cells from the tissue, Das and his team set about trying to find out how exactly this happened – at the level of molecules.
Imagine every cell is a building. The cell’s components, like ribosomes, mitochondria, endoplasmic reticulum, etc., are arranged in the rooms on each floor. The walls of this building are the cell’s cytoskeleton: it’s made mostly of proteins and helps the cell maintain its shape and size.
There are three kinds of cytoskeleton in cells. One of them is called microfilaments. They are made up mainly of a protein called actin. This actin is bound together using many other proteins, one of which is filamin.
The TIFR researchers found that filamin changes its position inside cells in two ways.
First, when a cancerous cell and a normal cell come in contact with each other, the researchers found that the filamin in the normal cell moved closer to the cell-cell interface, suggesting it had a part to play in cell extrusion.
Second, the filamin’s position changed for the worse when the ECM became stiffer. That is, for cells grown on relatively stiffer hydrogel, the filamin was found to localise around the cell’s nucleus, instead of moving towards the cell-cell interface.
Based on these findings, the team had an idea: when the ECM stiffens, filamin accumulates around the nucleus and reduces the amount of filamin at the cell-cell interface. And this causes cell extrusion to fail.
To test their hypothesis, Pothapragada & co. genetically modified cells to produce more filamin, and grew them as a single layer on stiffer hydrogel. They found that the cells’ ability to produce more filamin allowed them to push out the cancer cells.
This confirmed their prediction that the lack of filamin at the cell-cell interface was responsible for the failure of the cell extrusion process.
Next, they asked: how does the filamin sense when the ECM is stiffer?
Put another way, how do changing physical forces outside the cell induce the filamin to move inside the cell?
Biologists call this mechanotransduction: it’s the process by which physical signals outside the cell are converted to chemical signals inside.
The team found that a protein that binds with filamin, called Cdc42, helped move the filamin to the cell-cell interface, while another protein, called RefilinB, recruited the filamin to get around the nucleus.
When they inhibited Cdc42 activity by treating cells with a chemical inhibitor, they found that even on less stiff substrates, the filamin could no longer collect at the cell-cell interface.
Similarly, when they inhibited RefilinB activity by genetically modifying the protein, the filamin couldn’t localise around the nucleus.
Intervening in the fight
At this point, the researchers knew that a stiffer ECM caused cell extrusion to fail, and how. Next they tried to find out if they could somehow help the cell extrude cancerous cells even when the ECM was stiffer.
For this, the researchers used cells that had been modified to inhibit RefilinB activity. So even when the ECM got stiffer, their filamin couldn’t localise around the nucleus. This would mean there would be more filamin available at the cell-cell interface.
To the team’s delight, the normal cells could effectively extrude cancerous cells even on the stiffer surface.
Nagaraj Balasubramanian, an associate professor at the Indian Institute of Science Education and Research, Pune, where he studies how cells stick to the ECM, appreciated the study for adding “an important piece to the puzzle of how the ECM microenvironment affects cancers.”
“The techniques used in the paper are time-consuming and challenging,” Dey, the cancer biologist, said.
Pothapragada said the team was almost ready to communicate the first draft of the paper when in March 2020 the Indian government imposed a nationwide lockdown. So they had to completely halt their work for sometime, after which the team had to work in shifts.
When they finally submitted the final draft of their paper to a journal that accepted it, its editors allowed the team extra time to answer their queries. This was needed, Pothapragada said, “because the second wave hit us soon after.”
The next steps
Das told The Wire Science the team has been parallely studying the kind of forces acting on cancerous cells when they’re being extruded; he said a compressive force seems to stand out. That is, the cancerous cells seem to be squeezed. This isn’t surprising – but the team needs to figure out what’s doing the squeezing.
The team is collaborating with Max Bi, a theoretical physicist at the Northeastern University College of Science, Boston, to uncover the source of these forces. Bi is modelling a system based on results from Das’s lab and making predictions, and Das & co. are testing them.
The researchers are also trying to replicate their findings in a more physiologically relevant model system: organoids. An organoid is a miniaturised version of an organ, like the heart or a lung. Organoids are a bridge between in vitro cell culture models and in vivo animal models. For Das’s team, the challenge is to grow organoids as well as to figure out a way to control the stiffness of their ECM.
Balasubramanian told The Wire Science that he is also interested in knowing whether epithelial cells’ response to changing ECM stiffness has any effects on slow- v. fast-spreading cancers.
Sayantan Datta (they/them) are a queer-trans science writer, communicator and journalist. They currently work with the feminist multimedia science collective TheLifeofScience.com, and tweet at @queersprings.