At A Glance
Name: Subra Suresh
Position: Professor of engineering and head of the department of materials science and engineering, Massachusetts Institute of Technology
Background: MS, Iowa State University — 1979; ScD, MIT — 1981
What does the ability to manipulate cells with lasers have to do with finding better treatments for malaria and cancer? MIT professor of engineering Subra Suresh and his research team, along with colleagues from the Universities of Heidelberg and Ulm in Germany, published a paper in the January 2005 issue of Acta Biomaterialia that answers the above question. Suresh took a few moments last week to discuss his work with Inside Bioassays.
Your training has primarily been in mechanical engineering. How did you come to apply this background to cell biology, and in particular, malaria and cancer biology?
My background has always been mechanical properties of materials. After my PhD, I was a professor of engineering and looking at mechanical properties of metals, ceramics, and polymers. For the last ten years, I’ve been looking at mechanical properties of thin films and pattern structures for micro-electronic applications. So I was going smaller and smaller in size scale. A few years ago, we set up a nanomechanics lab at MIT, and in that lab, we were trying to measure mechanical properties of engineering materials at very small length scales and force levels — on the order of nanometers and picoNewtons. A lot of the things that you do at that scale, with many of these instruments like atomic force microscopes and nanoindenters, can also be used to study living systems. It could be a single DNA molecule, or it could be a single cell, and by functionalizing the tip, we can do the experiment in a fluid environment. That’s why I got interested — all in the context of my background, which is in mechanical properties. Then we thought: Why not really push the limit? If you really want to go to picoNewton force, you can go to an AFM-type system, or you can go to optical tweezers. So we started doing that on healthy red blood cells. That’s a biophysics experiment, but we realized that if we wanted to make connections to biology and medicine, we needed to look at some disease states. We chose red blood cells because there is no nucleus, and it’s a nice, round system, and it’s been studied before with other techniques for a long time. We thought that would be a good system to calibrate with optical tweezers, and then once we are satisfied, we could look at a disease state, where there’s a real connection to biology and medicine.
Then, if you look at red blood cells, there are two diseases that are known to affect the mechanical properties of red blood cells: sickle-cell anemia and malaria.
Sickle-cell anemia seems obvious, but how does malaria affect the structural properties of RBC’s?
The difference between the two is that sickle-cell anemia is a genetic disease, while malaria is infectious. When mosquitoes bite humans, they draw blood, especially the female mosquitoes when they are nurturing eggs in their so-called sexual stage. And when they draw blood, if the person whose blood they are drawing has the gametes for malaria, Plasmodium, those get processed in the gut of the mosquito. When the mosquito feeds again on the human blood, it injects anticoagulants, and it injects the precursor to the malaria parasite. That gets into the human liver, gets processed, and then the liver produces large numbers of merozoites that get into the blood stream. At that point, the merozoites — which are about 1 micron in size, and the red blood cells are about 8 microns — have a tip called the apical prominence, so the merozoite punctures the red blood cell membrane and gets inside. Once it is inside, it’s protected from the white cells in the immune system of the body. The current understanding is that proteins from the surface of the parasite, which is now inside the cell, get transported to the network that lies under the phospholipid bilayer of the red blood cells, and can also create new transmembrane proteins. Essentially the membrane properties can get altered, and in addition, you have a rigid merozoite inside the cell. This is the beginning of the so-called asexual stage of the malaria, and it lasts up to 48 hours. During that time, the merozoite continuously changes its structure, and geometry, and it can undergo nuclear subdivision. A single merozoite, within 48 hours, can sub-divide into up to 20 merozoites. Plus, you have structural changes that take place in the network of the red blood cell, and possibly in the membrane itself. The combination of all of this is that the cell can become stiffer. Up to this point, scientific results have led us to believe that the stiffening was on the order of a factor of two or three, but we showed in our experiments that it could be up to a factor of 10.
To do this, you used optical tweezers, right?
Yes. Optical tweezers have been around for 10 years or so. In fact, using optical tweezers to pull healthy red blood cells is not new, either. There was a group in France that did some work a few years ago, but the problem has been: Typically with optical tweezers, you get forces of a few tens of picoNewtons, at most. Also, in order to interpret the result, you need full 3D simulations of the experiment. The previous groups did only preliminary work, and they could only stretch the cell to 20 or 30 picoNewtons — not nearly enough to deform the red blood cell to match the level of strain it undergoes in the body. So our invention has been several-fold. One, we were able to impose forces on the red blood cell that were several times larger — in excess of 100 picoNewtons — than what has been done to this point. That gives us a new level of flexibility that they could not have before. Also, we were able to stretch the red blood cell in a systematic way over much larger deformation and strain, which nobody has done with optical tweezers before. The third thing is, nobody has used optical tweezers to study malaria before. Those are all the new elements in our work, and the outcome that’s new is that the stiffening effect is significantly more than previously envisioned.
You also reported some data on deformation of pancreatic cancer cells, correct?
Yes. That started from collaboration with a group in Germany at the University of Ulm. The objective there was the following: There is a chemical called SPC — sphingosylphosphocholine — that’s a naturally occurring bioactive lipid in our bodies. For example, SPC is found naturally in HDL cholesterol. It’s been suspected for some time that SPC has an affinity for certain pancreatic cancer cells called PANC-1 cells. Once it gets inside, it can cause molecular deorganization in about one hour. There was some work done last year in Germany that showed SPC affects the keratin molecular network, the intermediate filaments, inside the epithelial cells. In this work, we did mechanical stretching experiments to show that it’s not affected by how you load it, but it’s an intrinsic, chemically induced molecular reorganization. We did force-controlled experiments, the way engineers would do experiments. And we should that irrespective of how you do it, this molecular deorganization takes place, and within 60 minutes of SPC targeting the PANC-1 cell, the keratin molecules get reorganized. They get clustered in the perinuclear region, around the nucleus of the PANC-1 cell. So when you do the mechanical experiments, if you do it before SPC exposure, the force-displacement curve is almost linear. But the moment the SPC [comes in], and the keratin molecules are reorganized around the nucleus, you get a highly nonlinear response. You get significant increases in deformability in the PANC-1 cell, plus you get more hysteresis in the force displacement curve. The consequence of this — and it’s only a hypothesis on our part — is that when SPC targets the cell, it reorganizes the intermediate filaments, a consequence of which is the cell becomes more deformable, and it’s also able to absorb more energy as it absorbs more. We postulate that it can easily move through size-limiting pores in the body, which may play a role in cancer metastasis, so that’s the connection.
How might the results of these experiments, in either the malaria or the cancer cells, lead to better treatments?
Let me discuss malaria. One of the things I did earlier this year is spend some time in Paris, and the Pasteur Institute in Paris is a very well-known place for infectious disease, and they have a large group working on malaria. One of the things they’ve done in recent years is to take the Plasmodium falciparum parasite and clone it, and you can knock off one protein at a time from the cloned parasite. There is a particular protein called RESA — ring-infected erythrocyte surface antigen — that is suspected to be a culprit in the mechanical changes in malaria-infected red blood cells. They’ve been able to make RESA knock-outs, and they’ve been able to do it in a reversible way. By combining their expertise with our technique, we can take a RESA-knockout Plasmodium, put it inside a healthy red blood cell, culture it, and then we can tell them what the contributions are of a single protein on the mechanical characteristics of the infected red blood cell. That means that you can chemically target that particular antigen, so there is potentially a drug connection or vaccine connection there — if it’s important.
The second connection is that there are four Plasmodiums that affect humans. P. falciparum is the most lethal, but the other one that is commonly found in humans is Plasmodium vivax. We can culture the falciparum in the lab, but with vivax you have to draw blood from the patient and immediately test it. Being engineers, we didn’t have access to that. This year though, a group from England went to a region of Thailand with a large population of patients infected with roughly equal numbers of the two types. What they found was striking. They used a different technique in a very crude way, but nevertheless, it was a very interesting result. They found that P. falciparum, when it gets inside the red blood cell, stiffens it. P. vivax, on the other hand, makes it more deformable. The hypothesis — and I have to emphasize that it’s only a crude hypothesis at this point — is that in the case of P. falciparum, the cell becomes very sticky once it gets infected by the parasite. It becomes highly rigid, and less deformable. The combination of these two things is that the infected red blood cells clump together, and they sequester in microvasculature of major organs. On the other hand, in the case of P. vivax, because it becomes more deformable, and also it’s surface area increase, it can easily navigate through size-limiting capillaries and pores. Therefore, it can probably get to the spleen, and the spleen is supposed to do its job — if there’s something wrong in the red blood cell, it’s supposed to remove the inclusion. So in the case of P. vivax, likely, the spleen does its job and circulates the red blood cells back into the bloodstream, where in the case of P. falciparum, the infected red blood cell either doesn’t get to the spleen, or the spleen just takes care of it. This is only a hypothesis, but there may be a connection between how the spleen functions, and the type of strain of the malaria parasite. This is not my work, but work that was done earlier this year. The point I want to make in relation to that is very little is known about how P. vivax changes red blood cell deformability. These preliminary results from Thailand seem to indicate that there may be a good connection between the mechanical deformability and potentially fatality, because P. vivax doesn’t lead to mortality, or severe morbidity. It’s a bit of a stretch at this point, but it’s an area in which a lot more work is needed.
Are there any other disease models that your group has hypothesized this technique might be useful for studying?
I think different types of cancer. There is a broader question of how these cells are able to deform as they move — how they are able to go through size-limiting pores. What we have shown with the optical tweezers is that we can do controlled stretching at picoNewton forces. The other advantage of optical tweezers with respect to cells is that we use two beads at diametrically opposite ends to pull the cell, but there’s no reason you can’t use three or four or five, and attach them to different parts of the cell. So you can explore different types of mechanical deformation, and that really gives great flexibility to develop a wide variety of models, that, to my knowledge, nobody has looked at so far. The other thing is that we can take different types of cells, and knock out the membrane, and take the molecular network, and use the optical tweezers to try and study how molecules deform. That gives a lot of insight, because you could do that as a function of disease state. That gives a connection between molecular changes and mechanical response.
Is using these optical tweezers a relatively tedious task, or can it be implemented in higher-throughput?
No, at this point it’s a scientific tool, not a field tool. Someday maybe, but not yet. You need someone with some physics background or engineering background to do it.
Is it a commercial system, or do you design the instrument?
We designed it; we did not buy it from somebody. We had to put the microscope, and the laser, and the beads together, but all the parts were built commercially.
What’s the next step in this research?
We want to do the knock-out for these particular proteins, and see if there is any effect, because we don’t know yet. If there is any effect, we want to make connections to biochemistry. We are also doing some in vivo experiments now. We have a mouse lab, and we’re collaborating with some microbiologists, and using a different Plasmodium parasite. So we inject the mouse with the parasite, and then we culture it, and do the optical tweezers test as a function of time of culturing of the parasite, in vivo in the mouse, to then see how the trends compare between rodent and human malaria.