Sonoporation for Augmenting Chemotherapy of Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic cancer is the third most common cancer diagnosed in the United States, with more than 53,000 new cases in 2017. It is the fourth leading cause of cancer-related death in both men and women. Nonetheless, there has been no significant improvement in survival for pancreatic ductal adenocarcinoma (PDAC) patients over the past 30+ years. For this reason, there is a considerable and urgent clinical need to develop innovative strategies for effective drug delivery and treatment monitoring, resulting in improved outcomes for patients with PDAC.

This chapter describes the development of contrast-enhanced ultrasound image-guided drug delivery (CEUS-IGDD or sonoporation) to be that method and to translate it from the lab to the clinic. The initial clinical focus has been on a Phase I clinical trial for enhancing the effectiveness of standard chemotherapeutics for treatment of inoperable PDAC, which demonstrated a median survival increase from 8.9 months to 17.6 months in ten subjects augmented with sonoporation compared to 63 historical controls (p = 0.011). Recent efforts to optimize this platform and move forward to a larger Phase II clinical trial will be described.

1. Introduction

Pancreatic cancer is the third most common cancer diagnosed in the United States, with more than 53,000 new cases in 2017 [1]. It is the fourth leading cause of cancer-related death in both men and women, with nearly 37,390 deaths in 2018 and 5-year survival rates at around 5% [1, 2]. One of the reasons that the mortality rate nearly parallels the incidence is that cancer is resectable in only 15–20% of patients at the time of diagnosis [1, 3]. Pancreatic ductal adenocarcinoma (PDAC) is notoriously unresponsive to chemotherapy due to a dense desmoplastic stroma and poor blood supply [4, 5], though perfusion is sufficient to observe significant CEUS signal [6–8]. Despite the “curative” intent of treatment for those patients who present with surgically amenable PDAC and undergo resection followed by adjuvant systemic therapy (with or without radiation), their median overall survival is still only 15 months [9]. The 5-year overall survival for these resected patients is 25–30% for those with lymph node-negative disease and only 10% in patients with lymph node-positive disease.

Despite developments in new targeted therapies that have proven effective in other solid tumors, there has been no significant improvement in survival for PDAC patients over the past 30+ years [1, 10]. For this reason, there is a considerable and urgent clinical need to develop innovative strategies for effective drug delivery and treatment monitoring, resulting in improved outcomes for PDAC patients.

Over the last 20 years, the field of ultrasound-directed therapy has been moving toward clinical application to enhance delivery of drugs [6, 11–14] or genetic material [15–23] incardiovascular [24–26], hepatic [6], musculoskeletal [14], and neural [27, 28] disorders. A recent significant therapeutic breakthrough of first-in-human pilot studies, using ultrasound-directed therapy for treatment of PDAC in patients, suggests opportunities for enhancing current therapeutic options. In addition to localized adjunctive chemotherapy, this novel platform represents a multifunctional delivery system capable of targeted delivery to enhance treatment of many disorders. Contrast-enhanced ultrasound (CEUS) image-guided drug delivery (IGDD) has the potential to result in an economic and safe adjunctive to many therapies [8, 12, 29, 30]. CEUS-IGDD is performed by exciting ultrasound contrast agents (UCAs) in the vasculature near cancerous cells. These contrast agents volumetrically oscillate, inducing a physical interaction with their surroundings. To date, many different nonexclusive phenomena that result in an increased cellular porosity have been shown to occur in vitro [30]. This increased cellular porosity translates to highly localized increase in drug or gene uptake [6, 7, 10, 31].

Considering that a major drawback to traditional chemotherapy is the systemic side effects, CEUS-IGDD may provide an effective solution. Use of CEUS-IGDD can increase chemotherapeutic concentrations at the targeted region (i.e., within the primary PDAC) while decreasing systemic concentration. This scenario results in greater treatment efficacy, improving quality of life and, potentially, survival. As the primary tumor is treated more effectively than with chemotherapy alone, there is an increased likelihood of downgrading the tumor, allowing for surgical resection. As the chemotherapy remains systemic, it will still abate metastatic development.

Considered from a viewpoint of translating sonoporation technology into the clinic, key advantages of our approach to pursue ultrasound and microbubble-mediated therapeutic enhancement include the use of existing FDA-/EU-approved UCAs with widely available clinical ultrasound hardware, an emphasis on quantitative data to assess biodistribution in vivo, and quantitative imaging for guidance and monitoring. Initially, our novel technique will be applicable to the ~25% of patients with locally advanced and surgically unresectable PDAC, but we intend to expand the application to ~80% of patients with metastatic disease by sweeping ultrasound through both the pancreas and liver (the primary site for PDAC metastases) by performing IGDD in both organs during chemotherapy. Moreover, this new concept of bedside ultrasound therapy can ultimately be translated to other malignancies, thus providing personalized medicine to a wider range of patients.

2. Materials

To perform CEUS-IGDD, there are three main components: (1) an ultrasound imaging system, (2) an ultrasound contrast agent, and (3) the drug to be delivered. Numerous publications describe a multitude of combinations of these three elements.

1. There are many different ultrasound systems being used in the laboratory setting for IGDD. These can range from top-of-the-line clinical imaging systems to custom-made benchtop units with limited acoustic range but tailored for the desired output.

2. Like the ultrasound system, there are numerous contrast agents being used for drug delivery. These include clinically approved microbubbles as well as agents that can be designed to incorporate such characteristics as shell charge, targeting ligands, and size distribution.

3. Not unexpectedly hundreds of agents are being examined for utility in IGDD. Theoretically, any tissue that can be imaged with ultrasound and has even a modest rate of perfusion is a potential target for CEUS-IGDD. Numerous solid cancers are being examined for small molecule treatment as well as a growing field of genetic disorders, for which DNA therapy is showing promise preclinically.

For the treatment of pancreatic ductile adenocarcinoma, careful consideration was given to each of the three elements. As will be described in this chapter, we began our selection process with a strong focus on the goal of translation from in vitro to in vivo to clinical studies. To accomplish this, we chose to work with the most clinically relevant systems. These include a GE LOGIQ™ E9 (GE Healthcare, Waukesha, WI, USA) clinical scanner, the four major FDA-approved microbubbles (Table 1), and a combination of nab-paclitaxel (Abraxane®, Celgene Corp. Summit, NJ, USA) and gemcitabine (Gemzar®, Eli Lily and Co, Indianapolis, IN, USA). The dosing of both the UCAs and chemotherapeutic is within the FDA limits. Additionally, all acoustic energy use to image via standard CEUS as well as acoustic energy used to cause microbubble cavitation and thereby sonoporation are all within diagnostic limitations. As a result, even during early clinical studies, patients were ensured a minimum standard of care for PDAC.

Table 1.

Commercially available ultrasound contrast agents and critical characteristics

	Definity	SonoVue	Optison	Sonazoid
Shell material	Phospholipid	Phospholipid	Protein	Phospholipid
Concentrations (bubbles/ml)	1.2 × 1010	5.0–8.0 × 108	5.0–8.0 × 108	1.2 × 109
Diameter (μm)	1.1–3.3	2.5	3.0–4.5	2.6
Manufacturer	Lantheus Medical Imaging	Bracco International	GE Healthcare	GE Healthcare/Daiichi

3. Methods

1. Microbubble preparation is the first required step. Careful attention should be paid to manufacturer’s guidelines to avoid microbubble destruction (see Note 1). Phospholipid-shelled UCAs are packaged as a lyophilized powder. These must be reconstituted with the supplied diluent and gently mixed prior to use. The lone protein-shelled microbubble comes in a ready-to-use vial, though as with the other agents they must be adequately mixed.

2. Adjustment of ultrasound probes and parameters should be completed as accurately as possible prior to microbubble/ drug administration. A high degree of acoustic coupling must be achieved for best results (see Note 2). If needed, a dose of microbubble alone can be used to visualize target tissue prior to cavitation and delivery. Whether using a clamp and stand or manually holding probe, proper focal depth and desired acoustic energy (mechanical index, frequency, etc.) must be attained (see Note 3).

3. Injection of microbubble and agent. In most applications of CEUS-IGDD, there is a coadministration of drug with microbubble. This relies on systemic circulation and a matching blood half-life and biodistribution of both agents. In this case, drug is first drawn into the syringe followed by the desired amount of UCA. The syringe must be gently inverted multiple times to ensure adequate mixing. In the case of sonoporation for treatment of PDAC, the study techniques described here use a sequential administration of slow infusion of chemotherapeutic followed by an infusion of UCA. The pharmacokinetics/pharmacodynamics of both agents must be well understood along with the perfusion characteristics of the tumor (see Note 4).

   As stated previously, there can be a progression from the simplest in vitro system, moving to small animal models of pathology and advancing to human studies. In the following section, we describe general techniques that can be used for CEUS-IGDD investigation. This is in no way an exhaustive list but a description of systems the authors have developed proficiency, reliably yielding effective sonoporation for drug delivery.

3.1. In Vitro Studies

There are several setups generally used to evaluate the efficacy of CEUS-IGDD in vitro. Here, a few of the most popular setups are briefly described and some basic advice is provided. All these setups have significant limitations as compared to in vivo studies, and this is touched upon later. Most of these setups can be used with suspension cells or adherent cells:

(a). 6, 12, 24-well setups

This is the easiest way to translate a common in vitro protocol to evaluate the efficacy of CEUS-IGDD. (Option 1) Submerge an ultrasound probe into a well containing cells along with UCAs and drug to be delivered by applying the desired acoustic energy. Due to the limited distance between the plastic and ultrasound probe, there is a high probability that the ultrasound conditions will be different than those expected. Consideration should be given to boundary effects and reverberation as waves bounce off surfaces around and even below the target cell layer. (Option 2) A thin film is placed on the top of the multi-well plate instead of the lid. This is then submerged in a water bath, and acoustic energy applied through the film. By doing this in a water tank, the distance between the ultrasound probe and cells can be maximized resulting in less interference and a larger treatment area. Dedicated setups based on these variations can be designed to minimize acoustic interference.

(b). Closed-cell chamber setups

These closed-cell chambers (e.g., CLINIcell by MABIO International, Paris France, and Petaka G3 by Celartia, Columbus, OH) are the preferred alternative to standard cell culture welled plates. They allow for the removal of air pockets, placing the cells at the optimal location in the ultrasound field, ensuring cell-bubble contact, and minimizing acoustic interference. Furthermore, the ultrasound probe is not in contact with the cell culture media and removes the need for sterilization and risk of cross-contamination.

3.2. Limitations

A plethora of studies have been performed using a variation of in vitro setups to elucidate the optimal ultrasound and microbubbles conditions. Nevertheless, throughout literature, there is extensive conflict on what are the most effective settings are. This primarily stems from the fact that the traditional translational workflow is designed from novel therapeutic “drugs.” In contrast, CEUS-IGDD is a biophysical technique and requires a true recapitulation of the biophysical environment to truly mimic the ultrasound and microbubble behavior. The extensive “cells on a petri dish” experiments miss essential components that significantly affect the behavior of the bubbles and ultrasound, e.g., the dynamic vascular flow, temperature, protein concentration, cellular interactions, oxygen concentration, and more. Furthermore, such in vitro setups also interact with the ultrasound significantly different than traditional tissues inducing standing waves, 0-amplitude nodes at the cell surface, trapping gas pockets, and further complications. Extensive calibration and characterization are needed to fully understand the limitations of every unique setup. Replacing the cell culture plastic with organic tissue, e.g., perfused organoids, is also an option to improve correlation and translatability; nevertheless, this significantly increased the variables, cost, and complications. Last, but not least, replacing a drug with a model drug can result in numerous false positives if the effect is assumed to correlate between the two. Numerous drugs will saturate cells to the extracellular concentration within minutes, meaning CEUS-IGDD would show no benefit if compared to a cell-impermeable dye used as a model drug.

3.3. Preclinical Studies

Evaluating sonoporation in a preclinical state significantly improves its realism and clinical correlation. This can be performed with a single-element ultrasound transducer or multielement probes such as clinical diagnostic probes or custom designs. Depending on the drug regimen, an optimal treatment window needs to be determined. Currently, it is considered optimal to treat when the drug (s) are at their highest concentration in the blood plasma. A representative model should also be used that mimics the correct perfusion, growth, and drug response characteristics.

In our previous work to develop a PDAC model, animals were fully anesthetized with 250 mg/kg tribromoethanol diluted in 2-methyl-2-butanol and 12.5 mg/ml (Sigma-Aldrich) and placed on a heating pad in dorsal recumbency. Hair was removed by shaving, and the abdomen was washed with isobetadine and 70% alcohol. A small incision (0.5 cm) in the abdomen was made below the last rib on the left side, parallel to the linea alba. The pancreas was exteriorized, and cells (1 × 10⁶ MIA PaCa-2^luc) suspended in 20-μl phosphate-buffered saline were injected using a 30-G needle. After placing the pancreas back in the original position, the muscles and the skin were sutured with Ethilon II 5–0 polyamide sutures (Ethicon Inc., Somerville, NJ, USA). Prior to return to their holding cages, the animals were placed under heat lamps for approximately 30 min and monitored for any postoperative complications. All animals were euthanized following institutional guidelines [32].

The treatment process is as follows:

The animal is anesthetized, shaved, and depilated if required. The microbubbles are injected IV via the tail vein as a bolus (one single dose) or an infusion/extended bolus (injection over a few minutes). If an infusion is chosen, catheterization is required. Ultrasound is then applied as soon as possible after microbubble injection due to the inherent instability of the bubbles. The duration of ultrasound varies from a few seconds to 30 min depending on ultrasound conditions and microbubble dose.

3.4. Limitations

Microbubbles are a stabilized gas pocket in the blood; hence, depending on the blood gas saturation, the stability of the microbubbles can be significantly affected. As a result, caution should be exercised if any gas is used during anesthesia, e.g., using O₂ will significantly reduce the stability of most microbubbles. In addition, microbubbles have a limited life span within the vial and at room temperatures. The required stability should be determined prior to the study, and the preclinical model should be carefully considered prior to initiation as using small animals (mice, fish, hamsters) may results in off-site treatment, e.g., kidneys, spleen, liver, indicating increased toxicity and reduced survival. The dose should be scaled appropriately to the blood volume or organ volume of the animal model to accurately mimic human doses. Due to the extensive complications compared to conventional treatment strategies/protocols, it is highly advised to use an ultrasound system capable of visualizing the microbubbles at the target site. If microbubbles cannot be visualized, this may indicate that the microbubble injection failed (the most common issue), or the target site is not perfused, meaning this treatment technique may have limited efficacy in the given model.

3.5. Clinical Translation

To further understand CEUS-IGDD and to implement it in the clinic, three interrelated elements are required: (1) enhanced ultrasound system tools for more sophisticated acoustic control and improved facility for intra- and interlaboratory comparisons and (2) improved mechanistic understanding to focus optimization efforts and mitigate risks associated with clinical translation, building toward (3) a consensus of evidence for clinical efficacy, which will foster expansion of the platform to additional disease treatment scenarios. Our approach is designed to lower the barriers to translation by utilizing clinically approved microbubble UCAs and customizing software on widely available diagnostic ultrasound imaging equipment (while maintaining acoustic outputs in the diagnostic range). To attack these inherently multidisciplinary critical barriers (technological, biological, physical, and medical), our strategy builds on a successful Phase I clinical trial conducted in Norway by team members at Haukeland University Hospital and the University of Bergen in Bergen, Norway [6, 7].

Over a 23-month period (January 2012 to November 2013), ten consecutive patients with inoperable PDAC (ICD-10 C25.0–3) at Haukeland University Hospital were enrolled in this Phase I clinical trial. All had histologically verified, locally advanced (non-resectable Stage III) or metastatic (Stage IV) PDAC. Patients were ambulatory with an Eastern Cooperative Oncology Group (ECOG) performance status 0–1. Patients had to meet the standard criteria at the study hospital for treatment with gemcitabine and no known intolerance to gemcitabine or the UCA SonoVue® (Bracco, Milan, Italy). Historical data from PDAC patients undergoing equal gemcitabine treatment following the same inclusion and exclusion criteria, between 2009 and 2011 at Haukeland University Hospital, were used for comparison of treatment tolerance, safety, and overall survival. The only difference in treatment between the historical control group and our treated group was the addition of ultrasound and microbubbles following chemotherapeutic infusion, i.e., sonoporation.

Gemcitabine was considered the standard of care for the treatment time period of the control patients and throughout this study. It was administered by IV infusion at a dose of 1000 mg/m² over 30 min once weekly for up to 7 weeks (or until toxicity necessitates reducing or withholding a dose), followed by a week of rest from treatment. Subsequent cycles consisted of infusions once weekly for 3 consecutive weeks out of every 4 weeks. Our protocol used the ECOG performance status as a measure of the clinical condition [33]. The ECOG guidelines were used to monitor the effectiveness of the sonoporation treatment, i.e., the longer a patient stayed below an ECOG grade of 3, the more effective the treatment was considered.

A GE LOGIQ 9 ultrasound scanner (GE Healthcare, Waukesha, WI, USA) combined with a 4C curvilinear probe (GE Healthcare) was used for both diagnosis and therapy. The scanner was calibrated in a degassed water bath in order to map the beam profile and optimize the acoustic settings. After the chemotherapeutic dose was delivered, the clinical probe was positioned, aiming directly at the pancreatic tumor and locked in place for 31.5 min. The probe was attached to a ball joint and was positioned near the upper abdomen. Stomach and intestine were avoided in all cases to ensure propagation only through soft tissue to ensure delivery of the aimed ultrasound intensity at the desired area. Once the tumor was located, the probe orientation was fine-tuned in order to locate the largest slice of the tumor and as much vasculature as possible, i.e., the feeding vessels. The probe was then locked in position until the completion of the treatment. The natural breathing motion aided the treatment as the ultrasound slice gently oscillated through the tumor. By visualizing the vasculature and tumor, it could be ensured that the microbubbles were being insonated at the target. These vessels were then used as a reference point for future treatments.

Nine doses of UCA (0.5 ml of SonoVue followed by 5 ml saline) were injected IV every 3.5 min over the 31.5 min to maintain the sonoporation effect throughout the treatment period. To evaluate the efficacy of the combined treatment, the amount of chemotherapy cycles the patient was able to receive was compared. Furthermore, the tumor size was measured over the course of the treatment cycles to monitor and assess tumor growth.

An average of 13.8 ± 5.6 and median 12.5 (range 5–26) treatment cycles of protocol therapy were delivered per patient. In comparison, our historical control group treated with the same chemotherapeutic protocol of gemcitabine alone received an average of 8.3 ± 6.0 and median 7 (range 1–28) treatment cycles (p = 0.008). The survival curve of the combined treatment group compared to the historical control group analyzed with both Gehan-Breslow-Wilcoxon test and Log-rank (Mantel-Cox) test shows that the survival was significantly different with p = 0.0043 and p = 0.011, respectively.

The direct parameters used to evaluate the toxicity of the sonoporation treatment were clinical parameters including vital signs, ECG, and blood chemistry. Overall, all data indicated that gemcitabine in combination with CEUS (i.e., sonoporation) did not induce any unexpected deviation or additional toxicities than chemotherapy alone [7].

3.6. Ongoing Effort

Based on the very encouraging Phase I results reported above, the team decided to incorporate modifications to a state-of-the-art clinical ultrasound system (LOGIQ E10; GE Healthcare, Waukesha, WI, USA) for real-time 3D treatment and monitoring of CEUS-IGDD. The same platform will be continuously optimized based on researcher feedback. Having a system that can be used both experimentally and clinically will dramatically improve research correlation. In the initial stages, we will also validate if there are any microbubble-drug interactions that would benefit CEUS-IGDD/sonoporation. In vitro experiments will be performed on 3D cell culture phantoms to develop two acoustic conditions that improve cell permeability. These results will then be translated into preclinical murine models for further validation. Two murine models will be used to investigate the effect in broader conditions. The results from the preclinical experiments will indicate which microbubble-ultrasound combination has the best efficacy for CEUS-IGDD. This best combination will then be translated into a multicenter Phase II, clinical trial of sonoporation treatment of PDAC.

The LOGIQ E10 will be optimized as the common platform for our upcoming human clinical trial, while the LOGIQ E9 will be used for our preclinical studies. User feedback and access to data are critical for this research-enhanced access acoustic output will be enabled and/or added to the research interface, including:

• RF data capture.

• Display time-gated receive frequency spectra.

• Simulated overlays of pressure and intensity maps.

• Display range-gated detection of stable and inertial cavitation.

Real-time 3D (4D) imaging will also be leveraged to facilitate efficient treatment of tumor volumes. For sonoporation, flexible flash mode controls will be added to the ultrasound system, including fine-scale control of the transmit waveforms (amplitude, bandwidth, frequency, and pulse repetition rate) and spatiotemporal control of 3D ROI for defining treatment zone and reperfusion timing. The system will be configured to only allow acoustic conditions that are approved by national and international guidelines.

Subharmonic imaging (SHI) and subharmonic-aided pressures estimation (SHAPE) have been shown in previous studies performed by our group to be sensitive methods for detecting blood flow as well as interstitial fluid pressures in tumors [34–39]. To monitor tumor vascularity and intra-tumoral pressures, we propose to incorporate SHI and SHAPE tools into the ultrasound system for real-time contrast-specific display and quantification [40–43].

The effect of ultrasound-mediated sonoporation will be evaluated by noninvasive, longitudinal in vivo ultrasound and bioluminescence imaging of tumor volume, vascularization, and oxygenation [44–46]. Bioluminescence imaging allows for rapid detection of metastasis, indicating the tumor development stage, and deep tissue imaging, while our group has extensive experience using photoacoustics (PA) to determine hemoglobin signal, oxygenation levels in detected blood, and oxygenation levels over the entire tumor area [47, 48]. Significant correlation was also observed between PA signals and immunohistochemical marker expression, and these correlations were found to be stronger than UCA-based measurements alone [49]. We will also evaluate the effect of sonoporation on the stroma by histology and B-mode imaging.

Mice will be treated weekly, starting one week after tumor cell implantation for either three weeks or 10 weeks (at Thomas Jefferson University and University of Bergen, respectively). All four UCAs from Table 1 will be tested at low and high acoustic powers (I_SPTA of 200 or 60 mW/cm²). Therapeutic ultrasound will be applied using the modified LOGIQ E10 scanner with a broad bandwidth, curvilinear C1—C6 transducer operating at a 2.1 MHz center frequency. Both sites will use nab-paclitaxel in combination with gemcitabine as the chemotherapy. The dose will be scaled down from the human equivalent dose based on body surface area as per FDA guidelines and injected intraperitoneally to prevent vascular damage in the tail, allowing future intravascular injections [32]. A total of 200 μl of UCA diluted to around 1.2–3.0 × 10⁸ bubbles/ml will be infused via a tail vein over 10 min.

The groups that will be evaluated include two control groups (natural growth and chemotherapy alone) as well as four experimental groups (one for each UCA with ultrasound + chemotherapy). Groups involving ultrasound will be exposed to low and high acoustic powers (as defined above). The expected outcome of this task is to identify which permutations of UCA selection and acoustic power levels for sonoporation have the most significant effect on reducing tumor burden and increasing survival. Specifically, the optimal group should have a greater median survival of at least 5 days and a median tumor volume reduction of 50% compared to animals receiving drug alone. These experiments are currently ongoing.

Final selection of the UCA, from among available clinically approved agents, will be made prior to the start of patient studies, based on the preclinical in vivo studies. While translating results from animals to humans is not trivial, the preclinical experiments will allow us to narrow the acoustic parameter space, although the latter will clearly have to be modified for the planned Phase II, human clinical study. Thomas Jefferson University and University of Bergen see approximately 125 and 80 patients with PDAC per year, respectively. The goal is to enroll 120 patients over 3 years. The patients enrolled in this project will be adults over the age of 21 diagnosed with metastatic or locally advanced and surgically unresectable PDAC, who are scheduled for systemic chemotherapy (i.e., nab-paclitaxel plus gemcitabine) [50]. Written informed consent will be obtained. Subjects who are pregnant or breast-feeding as well as subjects who are clinically unstable, severely ill, or moribund with a life expectancy of less than one month will not be enrolled. Also, subjects with known sensitivities to any UCA products will be excluded. No special classes of subjects will be enrolled.

As part of the standard of care, these patients will receive contrast-enhanced CT and endoscopic ultrasound studies before and after completion of their chemotherapy regimen. Clinical outcomes and CT results will allow responses and local progression to be evaluated based on the Response Evaluation Criteria in Solid Tumors (RECIST) [51], overall survival, CA19–9 and CA125 levels, time to recurrence of pain, and local primary tumor progression (as complete response, partial response, stable disease, or progressive disease). We will obtain SHI and SHAPE data once a month to follow the tumor response. Changes in tumor perfusion and interstitial fluid pressure, in response to treatment, will be observed via SHI and SHAPE, respectively.

Patients will be assigned randomly to one of two groups: standard chemotherapy or standard chemotherapy with sonoporation. In the latter group, the optimal CEUS-IGDD conditions will be applied to a single, primary tumor completely insonified immediately following the infusion of chemotherapy [6, 7]. Treatment will follow the timeline and guidelines of the current standard for chemotherapeutic treatment for PDAC, with sonoporation performed as often as the chemotherapeutic treatment. As in our previous work [6, 7], we will assess treatment success using ECOG guidelines, i.e., the longer a patient maintains an ECOG grade <3, the more effective the treatment is considered. Patient outcomes in the two treatment groups will also be compared using SHI and SHAPE parameters, tumor volume, CT imaging including RECIST criteria, and immunohistochemistry (when available) to assess whether IGDD improves outcomes for PDAC patients.

Following completion of this study, success will be evaluated by local progression-free and overall survival with the main endpoint being an increase in the number of chemotherapy cycles by 75% (from 8 to 14) in the sonoporation group relative to controls. As a secondary endpoint, we hypothesize a median survival of the treatment group being a minimum of 150 days more than in the control arm.