Linking cell extrusion to cancer
Image: Epithelium, with DNA in turquoise and actin in red [Rosenblatt Lab]
Professor Jody Rosenblatt discovered epithelial cell extrusion, a mechanism that rocked the world of cell biology, by accident.
While studying wound healing in chick and mouse embryos during postdoctoral research at University College London, she stumbled across a process she just couldn't explain.
As well as the wounds she had made herself in the epithelial tissue lining the surfaces of the embryos, she spotted much smaller, single-celled wounds in the background that she hadn't made.
As she says: “I was always getting distracted by things that I wasn't supposed to be working on, and in this case, thought, hmmmm, what is that all about?”
The young Rosenblatt and her supervisor, Martin Raff, initially assumed these cells were apoptotic but further scrutiny using fluorescence confocal microscopy revealed quite a different story.
Although destined for apoptosis, the cells weren't actually committing suicide, but instead were being extruded by the contraction of neighbouring cells.
Results accompanied by stunning imagery were published in Current Biology, with the researchers concluding that the dying cell signals to its neighbours to extrude it, thereby preserving the epithelial barrier layer.
Signalling activates the formation of an actin and myosin ring within the dying cell and its neighbours, with the neighbouring cells then contracting and squeezing the cell out of the epithelial layer.
“At the beginning of this study, I didn't know anything about apoptosis or epithelia, so this really was a baptism of fire,” says Rosenblatt. “But I started to look at many different types of epithelia in tissues, and when we realised that most of the cells were extruding while still alive, that was an epiphany.”
Early images of cell extrusion, with cell membranes in labelled in green and DNA labelled in blue. [Rosenblatt Lab]
Rosenblatt's discovery sparked years of research into the signalling processes and mechanisms behind cell extrusion, a topic that her research group remains very much entrenched in today.
On completing her postdoc, she returned to her home turf, Salt Lake City, Utah, setting up her laboratory at the Huntsman Cancer Institute and Department of Oncological Sciences at the University of Utah, later becoming Professor.
During her time at Utah, Rosenblatt and her colleagues used an array of light microscopies including spinning disc confocal and light sheet microscopy to study cell extrusion in zebrafish.
“We used all the microscopes that were available to us but a real breakthrough was getting into zebrafish instead of drosophila,” she says. “These have been just amazing as, for example, you can just see everything, which has been huge for studying what cancer cells do.”
Helium microscopy of view of zebrafish skin. [Rosenblatt Lab]
Studies revealed that cells destined to extrude produce a lipid, Sphingosine 1-Phosphate, which binds to a receptor in neighbouring epithelial cells to extrude them.
But moreover, these years of research confirmed that overcrowding causes epithelial cells to die. When epithelial cells become too crowded, they activate a stretch-activated channel called Piezo1 to trigger extrusion of cells that then go on to die.
Research has clarified that cell extrusion is critical for regulating cell numbers, with aggressive metastatic cancers and asthma resulting from defective extrusion signalling.
For example, when extrusion is disrupted, the cells that would normally be extruded build up and also escape underneath the epithelium, eventually driving tumour formation and metastasis.
On the other hand, the constriction of an asthma attack can cause crowding to such an extent that it leads to too much cell extrusion airway epithelia. This destroys the barrier to the outside world, so inflammation ensues.
As Rosenblatt points out, she and colleagues have now clearly shown that all of these processes are controlled by mechanical forces and are keen to find out exactly how.
“We want to figure out how crowding translates into the signalling mechanism that is governed by the sphingosine 1-phosphate lipid,” she says. “So understanding how mechanical forces are translated into a chemical signalling readout is going to be really critical.”
Confocal projections of bronchioles from mouse ex vivo lung slices stained for epithelia with E-cadherin (green), actin (red), and DNA (blue) showing from left to right, 1) no constriction, 2) constriction with methacholine, 3) constriction with methacholine where gadolinium is added to block extrusions. [Rosenblatt Lab]
It is this desire to better understand the mechanobiology behind epithelial cell turnover, that has brought Rosenblatt back to the UK to set-up her laboratory at Randall Centre for Cell and Molecular Biophysics and the Comprehensive Cancer Centre at King's College London.
“This field of research as well as cell tissue biology is a lot stronger in Europe than in the US and I felt like our work could go a lot further over here,” she says. “The Randall Centre is just perfect for all of this as we have a lot of researchers working on muscle biology, asthma, as well as mechanobiology, so [our new set-up] is pretty ideal.”
Indeed, the Randall Centre encompasses molecular, cellular, and muscle biophysics with a translational focus on allergy, asthma and cancer. Cell imaging features heavily across all disciplines.
Myriad methods including structured illumination and 4Pi microscopy are used to boost resolution in fluorescence lifetime and single molecule imaging with researchers visualising biological processes in the context of diseased states.
Right now, Rosenblatt's group consists of herself, four post-doctoral researchers and a PhD student, and their research will take three key directions.
They will continue to investigate the fundamental mechanisms of cell extrusion, study invasion in metastasis and more deeply explore the role of cell extrusion in asthma and other inflammatory diseases.
“We're really going full steam-ahead with cancer as well as asthma,” she says.
Professor Jody Rosenblatt and fellow researchers. [Rosenblatt Lab]
Rosenblatt's past research has shown that gadolinium – the contrast agent used in MRI – can block cell extrusion by inhibiting the Piezo1 channel.
As she points out: “If we can block [this activated channel], then maybe we can not only block inflammation, but also block the entire inflammatory cycle and prevent further asthma attacks.”
The researcher is hopeful that human pilot trials will ensue.
As she adds: “One next line of work is to really understand how extrusion could promote modelling of the smooth muscle that promotes this ridiculous airway constriction, which is the most life-threatening aspect of the disease,” she adds.
In a similar vein, Rosenblatt also hopes to tackle metastatic disease, such as pancreatic cancer, which she describes as a 'death sentence' at the moment.
“We have this unprecedented view of the different stages in metastasis in zebrafish and now want to answer some big questions, such as what are the different types of [cancer] cells and can we detect them early on just by screening blood,” she says. “We could then see if patients have a propensity to get pancreatic cancer and provide preventative treatment earlier.”
Setting up the lab
Rosenblatt has spent the past months getting the necessary licences to work with zebrafish and mice, and has submitted several significant papers, which are, as she says, a culmination of ten years of research. She is also hoping that successful grant applications will be in place this Autumn.
One thing that was attractive to Rosenblatt about returning to the UK was funding schemes such as project grants, that have been more open to exploring new ideas.
As she says: “These not only allow small labs to function in lean times but help young scientists pioneer new directions.”
“This is so important, as we never know what approach will lead to a breakthrough,” she adds. “I think this is what has kept the UK’s research lean and mean, and why there are more Nobel Laureates per capita here than other countries.”
The researcher is also looking forward to accessing a wealth of instrumentation whilst at King's College London. Her laboratory will be home to a new spinning disc confocal microscope, which will be crucial for capturing live movies of zebrafish.
“Having a system where we can carry out high throughput screening is going to be so important but also following zebrafish cells for a long time and seeing how they seed to secondary metastasis sites is going to be critical,” she says.
Rosenblatt's group will also use a EVOS fluorescence microscope, from Thermo Fisher Scientific, to study thick lung tissue sections ex vivo while two-photon microscopy may be used to investigate smooth muscle in asthma studies. And the researcher is also excited about using high-resolution fluorescence microscopy at King's College London's new Microscopy Innovation Centre as well as the many light microscopies at its Nikon Imaging Centre.
“A lattice light sheet is also being built here, so all of this was a big selling point that drew me to King's,” she says. “The researchers in my lab are not afraid of trying any kind of microscope; just seeing what works the best has always been a good approach for us.”