3aBA1 – Ultrasound-Mediated Drug Targeting to Tumors: Revision of Paradigms Through Intravital Imaging – Natalya Rapoport

3aBA1 – Ultrasound-Mediated Drug Targeting to Tumors: Revision of Paradigms Through Intravital Imaging – Natalya Rapoport

Ultrasound-Mediated Drug Targeting to Tumors: Revision of Paradigms Through Intravital Imaging


Natalya Rapoport – natasha.rapoport@utah.edu

Department of Bioengineering
University of Utah
36 S. Wasatch Dr., Room 3100
Salt Lake City, Utah 84112

Popular version of paper 3aBA1, “Ultrasound-mediated drug targeting to tumors: Revision of paradigms through intravital imaging”

Presented Wednesday morning, May 25, 2016, 8:15 AM in Salon H

171st ASA Meeting, Salt Lake City


More than a century ago, Nobel Prize laureate Paul Ehrlich formulated the idea of a “magic bullet”. This is a virtual drug that hits its target while bypassing healthy tissues. No field of medicine could benefit more from the development of a “magic bullet” than cancer chemotherapy, which is complicated by severe side effects. For decades, the prospects of developing “magic bullets” remained elusive. During the last decade, progress in nanomedicine has enabled tumor-targeted delivery of anticancer drugs via their encapsulation in tiny carriers called nanoparticles. Nanoparticle tumor targeting is based on the “Achilles’ heels” of cancerous tumors – their poorly organized and leaky microvasculature. Due to their size, nanoparticles are not capable to penetrate through a tight healthy tissue vasculature. In contrast, nanoparticles penetrate through a leaky tumor microvasculature thus providing for localized accumulation in tumor tissue.  After tumor accumulation of drug-loaded nanoparticles, a drug should be released from the carrier to allow penetration into a site of action (usually located in a cell cytoplasm or nucleus). A local release of an encapsulated drug may be triggered by tumor-directed ultrasound; application of ultrasound has additional benefits: ultrasound enhances nanoparticle penetration through blood vessel walls (extravasation) as well as drug uptake (internalization) by tumor cells.

For decades, ultrasound has been used only as an imaging modality; the development of microbubbles as ultrasound contrast agents in early 2000s has revolutionized imaging. Recently, microbubbles have attracted attention as drug carriers and enhancers of drug and gene delivery. Microbubbles could have been ideal carriers for the ultrasound-mediated delivery of anticancer drugs.  Unfortunately, their micron-scale size does not allow effective extravasation from the tumor microvasculature into tumor tissue. In Dr. Rapoport’s lab, this problem has been solved by the development of nanoscale microbubble precursors, namely drug-loaded nanodroplets that converted into microbubbles under the action of ultrasound[1-6]. Nanodroplets comprised a liquid core formed by a perfluorocarbon compound and a two-layered drug-containing polymeric shell (Figure 1.Schematic representation of a drug-loaded nanodroplet). An aqueous dispersion of nanodroplets is called nanoemulsion.

A suggested mechanism of therapeutic action of drug-loaded perfluorocarbon nanoemulsions is discussed below [3, 5, 6]. A nanoscale size of droplets (ca. 250 nm) provides for their extravasation into a tumor tissue while bypassing normal tissues, which is a basis of tumor targeting. Upon nanodroplet tumor accumulation, tumor-directed ultrasound triggers nanodroplet conversion into microbubbles, which in turn triggers release of a nanodroplet-encapsulated drug.  This is because in the process of the droplet-to-bubble conversion, particle volume increases about a hundred-fold, with a related decrease of a shell thickness. Microbubbles oscillate in the ultrasound field, resulting in a drug “ripping” off a thin microbubble shell (Figure 2. Schematic representation of the mechanism of drug release from perfluorocarbon nanodroplets triggered by ultrasound-induced droplet-to-bubble conversion; PFC – perfluorocarbon). In addition, oscillating microbubbles enhance internalization of released drug by tumor cells.

This tumor treatment modality has been tested in mice bearing breast, ovarian, or pancreatic cancerous tumors and has been proved very effective. Dramatic tumor regression and sometimes complete resolution was observed when optimal nanodroplet composition and ultrasound parameters were applied

Rapoport 3A


Rapoport 3B


Rapoport 3C


(Figure 3. A – Photographs of a mouse bearing a subcutaneously grown breast cancer tumor xenograft treated by four systemic injections of the nanodroplet-encapsulated anticancer drug paclitaxel (PTX) at a dose of 40 mg/kg as PTX. B – Photographs of a mouse bearing two ovarian carcinoma tumors (a) – immediately before and (b) – three weeks after the end of treatment; mouse was treated by four systemic injections of the nanodroplet-encapsulated PTX at a dose of 20 mg/kg as PTX; only the right tumor was sonicated. C – Photographs (a, c) and fluorescence images (b, d) of a mouse bearing fluorescent pancreatic tumor taken before (a, b) and three weeks after the one-time treatment with PTX-loaded nanodroplets at a dose of 40 mg/kg as PTX (c,d). The tumor was completely resolved and never recurred) [3, 4, 6].

In the current presentation, the proposed mechanism of a therapeutic action of drug-loaded, ultrasound-activated perfluorocarbon nanoemulsions has been tested using intravital laser fluorescence microscopy performed in collaboration with Dr. Brian O’Neill (then with Houston Methodist Research Institute, Houston, Texas) [2]. Fluorescently labeled nanocarrier particles (or a fluorescently labeled drug) were systemically injected though the tail vein to anesthetized live mice bearing subcutaneously grown pancreatic tumors. Nanocarrier and drug arrival and extravasation in the region of interest (i.e. normal or tumor tissue) were quantitatively monitored. Various drug nanocarriers in the following size hierarchy were tested: individual polymeric molecules; tiny micelles formed by a self-assembly of these molecules; nanodroplets formed from micelles. The results obtained confirmed the mechanism discussed above.

  • As expected, dramatic differences in the extravasation rates of nanoparticles were observed.
  • The extravsation of individual polymer molecules was extremely fast even in the normal (thigh muscle) tissue; In contrast, the extravasation of nanodroplets into the normal tissue was very slow. (Figure 4. A – Bright field image of the adipose and thigh muscle tissue. B,C – extravasation of individual molecules (B – 0 min; C – 10 min after injection); vasculature lost fluorescence while tissue fluorescence increased. D,E – extravasation of nanodroplets; blood vessel fluorescence was retained for an hour of observation (D – 30 min; E – 60 min after injection).
  • Nanodroplet extravasation into the tumor tissue was substantially faster than that into the normal tissue thus providing for effective nanodroplet tumor targeting.
  • Tumor-directed ultrasound significantly enhanced extravasation and tumor accumulation of both, micelles and nanodroplets (Figure 5. Effect of ultrasound on the extravasation of Fluorescence of blood vessels dropped while that of the tumor tissue increased after ultrasound). Also, pay attention to a very irregular tumor microvasculature, to be compared with that of a normal tissue shown in Figure 4.
  • The ultrasound effect on nanodroplets was 3-fold stronger than that on micelles thus making nanodroplets a better drug carriers for ultrasound-mediated drug delivery.
  • On a negative side, some premature drug release into the circulation that preceded tumor accumulation was observed. This proposes directions for a further improvement of nanoemulsion formulations.
Rapoport 1



Rapoport 2



Rapoport 5


2aBAa7 – Ultrasonic “Soft Touch” for Breast Cancer Diagnosis – Mahdi Bayat

2aBAa7 – Ultrasonic “Soft Touch” for Breast Cancer Diagnosis – Mahdi Bayat

Ultrasonic “Soft Touch” for Breast Cancer Diagnosis


Mahdi Bayat – bayat.mahdi@mayo.edu

Alireza Nabavizadeh- nabavizadehrafsanjani.alireza@mayo.edu

Viksit Kumar- kumar.viksit@mayo.edu

Adriana Gregory- gregory.adriana@mayo.edu

Azra Aliza- alizad.azra@mayo.edu

Mostafa Fatemi- Fatemi.mostafa@mayo.edu


Mayo Clinic College of Medicine
200 First St SW
Rochester, MN 55905


Michael Insana- mfi@illinois.edu

University of Illinois at Urbana-Champaign
Department of Bioengineering
1270 DCL, MC-278
1304 Springfield Avenue
Urbana, IL 61801


Popular version of paper 2aBAa7, “Differentiation of breast lesions based on viscoelasticity response at sub-Hertz frequencies”

Presented Tuesday Morning, May 24, 2016, 9:30 AM, Snowbird/Brighton room

171st ASA Meeting, Salt Lake City



Breast cancer remains the first cause of death among American women under the age of 60. Although modern imaging technologies, such as enhanced mammography (tomosynthesis), MRI and ultrasound, can visualize a suspicious mass in breast, it often remains unclear whether the detected mass is cancerous or non-cancerous until a biopsy is performed.

Despite high sensitivity for detecting lesions, no imaging modality alone has yet been able to determine the type of all abnormalities with high confidence. For this reason most patients with suspicious masses, even those with very small likelihood of a cancer, opt in to undergo a costly and painful biopsy.

It is long believed that cancerous tumors grow in the form of stiff masses that, if found to be superficial enough, can be identified by palpation. The feeling of hardness under palpation is directly related to the tissue’s tendency to deform upon compression.  Elastography, which has emerged as a branch of ultrasound, aims at capturing tissue stiffness by relating the amount of tissue deformation under a compression to its stiffness. While this technique has shown promising results in identifying some types of breast lesions, the diversity of breast cancer types leaves doubt whether stiffness alone is the best discriminator for diagnostic purposes.

Studies have shown that tissues subjected to a sudden external force do not deform instantly, rather they deform gradually over a period of time. Tissue deformation rate reveals another important aspect of its mechanical property known as viscoelasticity. This is the main material feature that, for example, makes a piece of memory foam to feel differently from a block of rubber under the touch. Similar material feature can be used to explore mechanical properties of different types of tissue. In breast masses, studies have shown that biological pathways leading to different breast masses are quite different. While in benign lesions an increase in a protein-based component can potentially increase its viscosity, hence a slower deformation rate compared to normal tissue, the opposite trend occurs in malignant tumors.

In this study, we report on using an ultrasound technique that enables capturing the deformation rate in breast tissue. We studied 43 breast masses in 42 patients and observed that a factor based on the deformation rate was significantly different in benign and malignant lesions (Fig. 1).

The results of this study promise a new imaging biomarker for diagnosis of the breast masses. If such technique proves to be of high accuracy in a large pool of patients, then this technology can be integrated into breast examination procedures to improve the accuracy of diagnosis, reduce unnecessary biopsies, and help detecting cancerous tumors early on


Figure 1 Error bar chart for benign and malignant

Figure1- Distribution of relative deformation rates for malignant and benign breast lesions. A significantly different relative deformation rates can be observed in the two groups, thus allowing differentiation of such lesions.


4pBA1 – Ultrasound Helps Detect Cancer Biomarkers – Tatiana Khokhlova

4pBA1 – Ultrasound Helps Detect Cancer Biomarkers – Tatiana Khokhlova

The clinical evaluation of solid tumors typically includes needle biopsies, which can provide diagnostic (benign vs. cancer) and molecular information (targetable mutations, drug resistance, etc). This procedure has several diagnostic limitations, most notably, the potential to miss the mutations only millimeters away. In response to these limitations, the concept of “liquid biopsy” has emerged in recent years: the detection of nucleic acid cancer biomarkers, such as tumor-derived microRNAs (miRNAs) and circulating tumor DNA (ctDNA). These biomarkers have shown high diagnostic value and could guide the selection of appropriate targeted therapies. However, the abundance of these biomarker classes in the circulation is often too low to be detectable even with the most sensitive techniques because of their low levels of release from the tumor.

How can we make tumor cells release these biomarkers into the blood? The most straightforward way would be to puncture the cell membrane so that its content is released. One technology that allows for just that is high intensity focused ultrasound (HIFU). HIFU uses powerful, controlled ultrasound waves that are focused inside human body to ablate the targeted tissue at the focus without affecting the surrounding organs. Alternatively, if HIFU waves are sent in short, infrequent but powerful bursts, they cause mechanical disruption of tissue at the focus without any thermal effects. The disruption is achieved by small gas bubbles in tissue that appear, grow and collapse in response to the ultrasound wave– a phenomenon known as cavitation. Depending on the pulsing protocol employed, the outcome can range from small holes in cell membranes and capillaries to complete liquefaction of a small region of tumor. Using this technology, we seek to release biomarkers from tumor cells into the circulation in the effort to detect them using a blood test and avoiding biopsy.
Figure caption: Experimental setup and the basic concept of “ultrasound-aided liquid biopsy”. Pulsed high intensity focused ultrasound (HIFU) waves create, grow and collapse bubbles in tissue, which leads to puncturing of cell membranes and capillary walls. Cancer-derived microRNAs are thus released from the cells into the circulation and can be detected in a blood sample.
To test this approach, we applied pulsed HIFU exposures to prostate cancer tumors implanted under the skin of laboratory rats, as illustrated in the image above. For image guidance and targeting we used conventional ultrasound imaging. Blood samples were collected immediately before and at periodic intervals after HIFU treatment and were tested for the presence of microRNAs that are associated with rat prostate cancer. The levels of these miRNAs were elevated up to 12-fold within minutes after the ultrasound procedure, and then declined over the course of several hours. The effects on tissue were evaluated in the resected tumors, and we found only micron-sized areas of hemorrhage scattered through otherwise intact tissue, suggesting damage to small capillaries. These data provided the proof of principle for the approach that we termed “ultrasound-aided liquid biopsy”. We are now working on identifying other classes of clinically valuable biomarkers, most notably tumor-derived DNA, that could be amplified using this methodology.


Tatiana Khokhlova – tdk7@uw.edu
George Schade – schade@uw.edu
Yak-Nam Wang – ynwang@uw.edu
Joo Ha Hwang – jooha@uw.edu
University of Washington
1013 NE 40th St
Seattle, WA 98105

John Chevillet – jchevill@systemsbiology.org
Institute for Systems Biology
401 Terry Ave N
Seattle, WA 98109

Maria Giraldez – mgiralde@med.umich.edu
Muneesh Tewari – mtewari@med.umich.edu
University of Michigan
109 Zina Pitcher Place 4029
Ann Arbor, MI 48109

Popular version of paper 4pBA1
Presented Thursday afternoon, October 30, 2014, at 1:30 pm
168th ASA Meeting, Indianapolis