Era of Hope 2011 Investigator Vignette

Title: A Microfluidic Approach for Cancer Cell Surface Protein Analysis

Investigator: Robert Wieder, MD, PhD; UMDNJ-New Jersey Medical School/University Hospital Cancer Center

The focus of this work is to develop a needed mechanism to follow cells for response in a clinical trial. And the reason it’s important is because the novel ways of treating breast cancer will be to address subpopulations of cells that have the capacity to re-grow that are resistant to standard treatments. There is currently no way of following these cells in sequential manner in a clinical trial. Using this device, we will be able to put a needle into a tumor over several steps in a clinical trial and pull out cells and ask the question, are the subpopulation of cells that we are targeting responding to treatment.

If you take a few cells out of a tumor what are you going to do with those cells? You—if you handle them in any way, you’re going to lose them so it’s a technical problem. So what we have is we’re building a device; it’s a concept actually to overcome this technical problem.

It’s a microfluidic device and the way it assays what type of cell it is—is it has immobilized antibodies over patches that are next to each other. And if a cell passes at a certain speed over a patch, if an antibody that’s immobilized on the patch recognizes a surface protein on the cell it binds temporarily and slows down its rate over that patch. As the cell passes over another patch that has an antibody that doesn’t recognize a surface protein the cell grows faster. So as you can imagine if you have eight or ten patches with different antibodies to specific proteins that identify the repertoire of a cell that defines it as a specific cell, you can get a barcode for an individual cell in a population of small cells because—a small number of cells because you’ve only taken out 50 to 100 cells from an aspirate. And so you’re going to be able to tell what percentage of the cells have a specific barcode that identify it as a cell that could potentially metastasize to the lung or have cancer repopulating capacity or have bone marrow metastatic or—capacities and therefore you can see if a percentage of the cells that you take out decreases over time as you’re treating this tumor. It’s going to make clinical trials in the current concept of what is needed in breast cancer doable.

This is a collaboration with an engineer, Rajan Kumar who is our collaborator on this grant and he’s actually building the microfluidic device and he’s providing the technical aspect and I’m providing the medical aspect and the biological aspect, so we work very, very well together.

We’re trying to build a microfluidic device that can separate cells, MDA-MB-231 breast cancer cells that have the capacity to exclusively metastasize to the lungs. The surface protein that we found continues—reliably up-regulated was the interleukin 13 receptor.

So then we built this microfluidic device. It consists of a Plexiglas slide that has a patch that has we figured out the optimum way of immobilizing IL-13 receptor antibody for this patch and this patch has a—a bovine serum albumin as a control and has an inlet so buffer is introduced and flows over the channel. The channel height can be varied by gaskets. The dimensions can be varied. The dimensions will change the sheer force on the cells and so those are very—multiple variables that we are testing.

The cells are introduced a couple of millimeters after the inlet and the cells then flow over the patches of the non-specific and the specific antibody. The whole device is mounted under a microscope which has—is linked to a computer and we can measure the speed of the cells every 15 microseconds.

What is represented here are the rates of flow of cells over the patch. The black lines represent flow over the non-specific BSA patch and the yellow lines represent a flow over the specific patch. So here it represents MCF-7 cells where the flow of cells over these specific patches and the non-specific patches are identical. There is no retardation; 231 cells—the same thing. Let me bring you here to the—the punch line; these cells that express high levels of IL-13 receptor, a fraction of those cells are slow and they’re always slow. None of the cells that pass over the BSA are slow. So after we’ve calculated these essentially 100% specificity is achieved only cells that express the IL-13 receptor are slowed.

Blocking antibodies have demonstrated that this binding is specific where we were able to reverse the inhibition of slow—by incubating the cells with IL-13 receptor antibody beforehand and then they grow faster over the IL-13 patch, so it shows that the binding is specific.

We then reanalyzed the data and looked at only the slowest measurement. We measure the speed of cells over a 200-micron patch what we’ve showed that only cells that go over the IL-13 patch have some of the measurements that are really slow, whereas none of the cells that go over the BSA patch are slow.

So then we look at the slowest measurements. And we compared the slowest measurements over IL-13 and the slowest measurements over BSA. And the curves separated. So what we now have is 100% sensitivity and 100% specificity. And we’ve achieved what we proposed to do in the Army grant to—in—in the first year of the grant that we—we have a device now that can predictably and reliably and specifically separate two populations, one that expresses a protein and one that doesn’t.

A lot more has to be done; multiple patches have to be done. We have to now go ahead and do the animal studies where we’re now going to grow tumors in mice and pull—pull cells out and put them directly on the machine to show that in fact we can identify cells coming right out of a tumor without prior manipulation.

When I saw the data that the curves just separated, it was an epiphany. It was wow; we have something that no one has ever done before and we have now—we will be able to have a device that can be adapted to follow cells in a tumor as it responds to treatment.