Theranostic Applications of Microbubble Sonography: An Overview - HD

Introduction

Hello, I'm Arthur Fleischer, the Vanderbilt professor of Radiology at Vanderbilt University Medical Center, Nashville, Tennessee.

It is a pleasure to give a short presentation on the theranostic applications of Microbubble Sonography.

Understanding Theranostics and Microbubbles

The word theranostic implies that we have diagnostic applications as well as therapeutic applications of this new microbubble, applications using sonography.

Bubbles are, of course around in our environment.

These are macro bubbles as opposed to microbubbles.

The word theranostic implies that it has both diagnostic and therapeutic applications.

And the way microbubbles can be used this way is, to label them, to have them to go to tumor micro microvessels using Vasogenic endothelial growth factor as a target inflammation, we can target pectin and thrombosis, alpha v beta three.

We can also use these micro bubbles to assess tumor response.

And a very large area of interest is enhancing drug delivery, using microbubbles.

And it is very interesting to see that over the 40 years that I've been in ultrasound, that ultrasound started as a therapeutic agent primarily.

And the future applications of ultrasound for therapy using, microbubbles to deliver drug and enhance, treatment is becoming a very important application of ultrasound.

Structure and Function of Microbubbles

The microbubbles that are used, uh, have a lipid shell.

There are three to five microns, which is about a third to a half the size of a red blood cell.

Inside the microbubble is a gas.

We use perfluorocarbon, uh, for example, in definitive, uh, as I said, the diameter can vary depending on the microbubble oscillating.

And, these oscillating microbubbles allow us to image using a harmonic so we can pick up the microbubble.

Over the surrounding, uh, soft tissue echoes these microbubbles circulate through the body, uh, for several passes through the lungs and eventually are ventilated out, uh, of the body.

This is a picture of a microbubble oscillating, as you can see here.

And if we turn up the mechanical index, the mi, we can actually explode the microbubble.

And, this is a beautiful example of that.

So the microbubbles that are used for definitive, one of the contrast agents are shown here with a five micron, uh, scale.

Now, basically what we try to do with the microbubbles is we pass it through the capillaries.

The capillaries have about the size of a red blood cell, about seven microns, and we only need a few of these microbubbles to pass through to get a very strong signal when we image with harmonics.

Perfusion Imaging with Microbubbles

Now, if we want to calculate the perfusion of a tissue, which is basically defined as blood flow in ML per second over volume, we need to know the vascular area and the mean blood velocity.

We can term the mean blood velocity is basically related to the slice thickness and the replenishment rate.

The replenishment rate we can determine by breaking the microbubbles and watching the fusion.

Now, we can image using continuous imaging where the micro bubbles are, uh, susceptive to the, the beam and some of them actually rupture.

We can turn up the mi, the mechanical index, break the micro microbubbles, and then watch them as they replenish the area of interest.

Or we can use interval delay imaging or pulsing, which is what we're used to in mr, for example, and watch the microbubbles replenish.

Now, when we qu quantitate the amount of profusion, this is a typical time intensity curve with the intensity as DB on the vertical axis time on the horizontal axis, once we establish a steady state, we can break the microbubbles and we can determine the replenishment rate, which is beta, and the overall vascularity, which is alpha and alpha times beta is basically related to perfusion.

Applications in Anti-Angiogenic Therapy

Now, anti-angiogenic therapy is a major application for lots of, uh, cancers.

If we can block the production of new blood vessels, we can, control the metastatic properties of a tumor.

And this diagram shows the VE GF receptor, um, projecting down from the endothelial cell.

And when there is connection of the anti VEGF, the VGF is not produced and the tumor, uh, does not metastasize.

So this is basically the focus of anti-angiogenesis therapy.

Dr. Ach and I, uh, have looked at some of these patients with hepatocellular carcinomas that are, um, refractory to chemotherapy and being treated with an anti angiogenesis, uh, drug.

And here is the initial scan on a patient with a metastatic tumor in the, in the, uh, I'm sorry, primary tumor in the liver, as you can see here, and you can see the relative vascularity.

Now at 15 days post-treatment, the volume of the tumor has not changed significantly.

But as I'll show you with the profusion studies, um, there's a significant change.

So here is the baseline scan of the tumor.

We break the bubbles and we watch the reperfusion and we quantitate that.

Now at day 15, the same lesion is shown here.

Perhaps you can't see it well.

But here, here's the same lesion.

And when we graph these profusions, we can see that there's a significant difference between baseline and day 15 treatment in that there's much less flow on day 15, even though the volume of the tumor has not changed.

And this would be considered a good responder to anti-angiogenesis therapy.

On the other hand, this is a patient that has a hepatocellular carcinoma here.

This is the baseline image, and as we can see at day 15, that there is actually more flow in the tumor.

Perhaps you can make that out here.

And when we look at the time intensity curves in this patient, we can see that in fact, there's more flow after 15 days of treatment, this being baseline.

And so this would be considered a poor responder Lifestyle in France uses ultrasound and microbubbles to determine a tumor response.

A 40% or more change is considered a, a positive response.

This has also been shown to be much more accurate than the resist R-E-S-I-S-T criteria used in CT scanning and McCarville in her pediatric patients have shown that contrast can be helpful in assessing whether or not a tumor is refractory to treatment.

Targeted Microbubbles and Ligand Binding

So this is a diagram of a typical microbubble with a ligand extending from the shell of the microbubble.

And there is potential for putting, uh, drugs in the center of this because the center is basically a gas and, uh, drugs can be put in the center of this microbubble, theoretically brought to the area of interest.

And then a high mechanical index applied, uh, thus delivering the drug exactly where it needs to go.

The, the, uh, ligand is put on the microbubble using a strep avidan, if you wanna call it glue.

And, uh, this is shown here in the VEGF model.

And this is another diagram of what the micro labeled microbubble looks like.

So basically we can follow, um, the labeled microbubbles.

This is at baseline.

We have the microbubbles coming in and circulating.

And the ones where there is a VEGF receptor stay around.

And we can see this, it's at a follow up scan, we can determine the number of labeled microbubbles.

And this is a optic, uh, image of some of the microbubbles shown us red dots in some of the tumor vessels.

So basically, if we're looking, uh, for the labeling of microbubbles, we image after about two and a half minutes, we apply a high mechanical index, destroy the microbubbles, and the ones that are, uh, freely floating are gone.

And the ones that are bound, uh, remain.

And this is a picture of, of A1C one centimeter, uh, tumor in a mouse hindin.

These are millimeter gradus, and the green dots are actually the microbubbles that are coming into this, uh, tumor.

And, um, this is another picture of that with, uh, microbubbles coming into a, uh, exteriorized tumor.

And this is with the veg G receptor model.

These are courtesy of Dr. Ach, and he compared the high VEGF tumor to the low VEGF tumor.

As you can see, uh, visually, there's less green in the image to the right, uh, using staining of VEGF uh, material.

Now, this has been, um, recently reported by the group at Stanford Jurgen Wilman using a kinase domain receptor.

This is all, uh, in patients with breast and or ovarian cancer.

This, I believe is one of the first papers describing this in ovarian and breast cancer.

There has been work with labeled microbubbles in prostate cancer.

Um, of 48 patients.

93% of breast cancer patients showed an increased signal.

85% of ovarian cancer patients showed an increased signal.

And 93, um, were, uh, shown to be strong in breast cancer patients and none in 67% of benign lesions.

And in ovarian cancer is 77% and none in 78%.

So I believe this is one of the first images, uh, that, uh, he lent to me showing at the top a carcinoma where the aerials are showed lots of echoes, uh, dots.

And this is a, um, malignant lesion versus the image at the bottom, which was benign.

A, a cys adeno rova that did not have these labeled microbubbles.

Um, this is also in, uh, patients shown here with, um, more of the signals seen in the carcinoma than the two benign lesions on either side.

Challenges in Drug Delivery and Tumor Vasculature

So when we think about what causes, uh, drugs not to be delivered and uh, to their target, part of this is related to binding of the microbubble, uh, destruction by enzymes and inability to go through the tumor interstitial.

This beautiful diagram shows that tumors have a very abnormal blood supply.

The vessels of tumors are leaky.

They, um, are high acidity, and if these tumor vessels could be obliterated, the tumors themselves could be much more susceptible and much more radio sensitive and chemo sensitive to therapeutic agents.

This is a picture of a microbubble, uh, showing the, um, lipid covering.

And this is the diagram showing how some of these ligands on the outer aspect of the microbubble can be arranged using a peg um, receptor.

Advanced Therapeutic Applications

Oxygen Delivery to Tumors

So there has been some work which, uh, tries to put oxygen into a microbubble and make it, uh, go to the tumor.

And then ultrasound is applied in the tumor, thereby releasing the oxygen and making the tumor less hypoxic.

We know that hypoxic tumors are typically very difficult to treat, and this is a potential application for a microbubble delivering oxygen.

One of the problems is getting the oxygen past the lungs.

And so there has been some work, some intra arterial injection of oxygenated microbubbles.

Gene Delivery via Sonoporation

This is a diagram showing what's called sono porion, where there is incorporation of genes inside of a microbubble.

The microbubble gets to its target, the ultrasound is applied, the microbubble, uh, is broken, and the gene is the area of interest.

Blood-Brain Barrier Disruption

This is some work from my associate Charles Caskey, showing how the microbubbles oscillate, uh, under an ultrasound feel, and then basically break.

And when they break, they push the fluid inside the vascular space past the gap junction, and he actually photographs some of these changes in microbubbles.

Um, this is a very small, uh, pilot study where Charles put, uh, microbubbles, uh, and doxorubicin into, uh, a mouse that had a brain tumor.

These are optic images on week three and week four, and we could show that ultrasound helped disrupt the blood-brain barrier, which is a very big application of microbubbles, not only in cancers, but perhaps in Alzheimer's and other diseases.

Um, this is a group of Mr images of these mice showing that the microbubbles can be, um, uh, periodically used to break the blood break through the blood-brain barrier.

Um, as we can see on day zero, there's a, a blush from the microbubbles in the right hemisphere of this mouse.

There has been work using microbubbles to get the antibody to amyloid past the blood-brain barrier in, in mice with Alzheimer's disease.

And they showed a 75% improvement in memory using the ultrasound.

And this again, isn't in a mice model.

There has also been work using the microbubbles in an MR focused ultrasound field for opening up the blood brainin barrier and establishing better, uh, flow of stem cells, stem cell treatment across the blood-brain barrier.

And, um, this is very exciting work.

Nanoparticle and Drug-Encapsulated Microbubbles

There's also been some work using a nanoparticle microbubble in a breast cancer xenograft.

Um, they used encapsulated nanoparticle microbubbles and used ultrasound to get the microbubble to the tumor and get the treatment, which was in the nanoparticle into the tumor.

And these tumors exhibited complete remission using this treatment.

There is a group at University of Alabama Birmingham, which has tested the use of microbubbles that encapsulate doxorubicin and the doxorubicin microbubble gets to the tumor.

The ultrasound is applied and basically breaks the microbubble and therefore delivers the doxorubicin.

Clinical Trials in Pancreatic Cancer

There are some clinical trials that have been described in pancreatic cancer patients, and one from Norway showed a doubling of the mean survival in in pancreatic cancer patients.

Um, this, uh, admittedly is a small series, but it shows tremendous uh, potential.

There is also a group in China using FU high intensity focused ultrasound with microbubbles enhancing the drug therapy of pancreatic cancer.

High-Intensity Focused Ultrasound (HIFU) Applications

Fu uh, can use microbubbles and FU itself has been used in uterine fibroid ablation, reducing pain associated with bone metastases.

It's also recently been shown to be very effective in decreasing the tremors associated with some types of Parkinsonism.

There's liver, breast and pancreatic tumors that have been tested with fu.

And finally, prostate can be ablated with fu.

This is a ongoing trial, just, uh, started in University of Virginia in 15 patients with breast cancer where FU is added to, um, Keytruda, which is an immunotherapy agent and has thought that the haifu would unmask the breast cancer cells and leading to better treatment.

Post-HIFU Assessment

The final slide shows some work done by Ed Grant and his associates at University of Southern California using contrast after FU of the prostate.

You can see the tumor, uh, blood flow in the original tumor at the top with, um, the follow up showing complete ablation of the prostate.

Conclusion

Um, using contrast I'd like to give, uh, thanks to Charles Caskey, my colleague here at Vanderbilt Imaging Center, um, Andre Leach, who's at Jefferson, Jurgen Woolman, who's at Stanford, and Ed Grant at Southern California.

I hope that you've, uh, obtained a overview of the theranostic applications of ultrasound, and it's only going to grow in its clinical applications in the next few years.

Thank you.