IMPaCT Investigator Highlights (Text Version) - Dr. Evan T. Keller

Title: Novel Application of Aptamer Selection (Pheno-SELEX) to Target the Invasive Phenotype Successfully Creates Anti-Metastatic Aptamers

Investigator: Evan T. Keller, DVM, PhD. University of Michigan.

Our current award that we’re concentrating on is trying to understand a way to prevent the spread of cancer.

So one of the big challenges to cancer is that when it spreads there’s a lot of different molecules that can cause the spread. And so we don’t know what those molecules might be and it could be very hard to target those molecules without knowing what they are. So we want to develop a method to be able to target those molecules without knowing them but will inhibit the spread of cancer.

So one of the ways to we thought we could approach this is to use something called aptamers. Aptamers are short little pieces of RNA or DNA, which can fold into different forms and the overall goal is to bind them to very invasive cancer cells which is the kind that would spread, and then compare how they bind to those cells versus the cells that don’t invade. And so we want to pick the ones that will invade the most. So our overall goal ultimately is to be able to have development of aptamers which will inhibit metastases.

So the method we’re using is somewhat like evolution in a test tube where we have hundreds and hundreds and actually more like trillions of different molecules that have different shapes and then we see which of those molecules will stick to the cancer cell. And then we can identify those molecules that stick to the cancer cell the best, and from those we can amplify those so that we get more and more of those and we keep doing that over and over so we can get the molecules that will stick the best to the cancer cells and prevent the spread.

So now we’re left after this process is what we call evolution of aptamers that we can now take these and now test them. First of all, the first thing you want to do is test them. Do they bind to the invasive cells? So here you could see that when we do the cancer cells there’s little red dots. These little red dots show that those aptamers are binding the invasive cells whereas the control aptamer doesn’t. Next, the most important step really is, are they functional? Just because they bind doesn’t mean t hey’re going to inhibit invasion. So we next take these cells and then we put in the aptamers and test the aptamer versus what we call a scrambled or controlled aptamer if it combined to the cells.

So this is how much invasion that occurs in the cancer cell by itself. When we put in the control we don’t see any change in invasion. But when we put in the aptamer that we developed we see that we get a big block of invasion. Additionally we looked at another prostate cancer cell line to see if it was general for prostate cancer. And again we could see inhibition of invasion when we used that particular aptamer again. Then we looked to see is this a general effect for invasion itself. And to do that we used a different cancer cell line totally. We used an osteo-sarcoma which is a bone cancer cell line and again we found inhibition of invasion in that particular cell line when the aptamer is present. So overall this shows that in the test tube that this aptamer is able to decrease invasion of cancer cells. That’s great but that invasion doesn’t always translate into being able to inhibit the spread of cancer to a distant site.

So next we went to ask can this aptamer inhibit invasion in vivo in a mouse model? So to do that type of experiment we take mice and we inject cancer cells into the heart and when we do that type of thing, the cancer will spread throughout the body. So some mice will receive the aptamer and some of them will receive a control aptamer. And then we monitor them over a period of time. And so what we find is that there’s more metastases in the control but when we have the aptamer present we had a marked inhib ition of the spread of cancer, so decreased metastases. There were less mice with tumors when the aptamer was present and there were less total number of metastases, and this is represented in this figure here. When we had the control we can see multiple metastases or spread of tumors. When we give the aptamer we see a decrease in tumor take.

So this really brings home that we can that we are able to inhibit the spread of cancer from the prostate in this model. Now again we wanted to ask--is this general to prostate cancer or does it go onto other cancers? And this time we used an osteo-sarcoma cell line. Osteo-sarcoma is bone cancer that can be injected into the leg; that’s where it would normally develop in the person and it spreads to the lungs. And again when they had just control aptamer all the animals developed metastases. When they had the aptamer that we created we found a marked inhibition of the number of animals that had metastases down to only 20 percent. And again we look at the actual metastatic burden in the lung and we found that the animals that received control again had a greater tumor in the lung versus the animals that had the test aptamer. Eventually it comes back up and interestingly what you need to remember is aptamer administration stopped at this time point here, so it seemed like it was going there was inhibition a nd then it had a chance to escape from that.

And finally we look at survival. We found that those animals that had the aptamer drug had longer survival compared to those that just had the control. So overall these results have shown that we were able to create an aptamer that can actually inhibit invasion and that can also result in inhibition of metastases in an in vivo model.

Now we have a library of different aptamers that we want to test, so we’re going to look for the ones that seem to be most effective and then be able to develop further from there. These can be used potentially for imaging since they bind to the cancer cells specifically and also we’ll be able to use these to pull out what molecule it binds to on the cancer cells so we can get a better feel for what mechanisms may be occurring that allow the cancer to spread.