Bracco (Switzerland)

Immune checkpoint inhibitors (ICIs) have emerged as a highly effective treatment option for patients with metastatic melanoma. As not all patients respond to ICI immunotherapy, imaging biomarkers are required to accurately monitor early response to therapy. Therefore, the aim of this study was to evaluate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring the effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine melanoma model. Murine melanoma allografts (B16-F10) were implanted subcutaneously in n = 10 therapy and n = 10 control female C57BL/6 mice. CEUS with VEGFR2-targeted microbubbles was performed on day 7 and 12. The therapy group received 3 intraperitoneal injections on days 7, 9, 11 of combined anti-PD-L1/anti-CTLA-4 immunotherapy, the control group received a placebo. CEUS assessed tumour perfusion during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity at 8 minutes (SI8min) and 10 minutes (SI10min)). For pathophysiological validation immunohistochemistry was performed. At follow-up, the CEUS perfusion parameter WiAUC demonstrated a significantly higher decrease in the therapy than in the control group (p = 0.021). At follow-up, the signal enhancement in the late phase was significantly lower in the therapy than in the control group (SI8min p = 0.003; SI10min p = 0.002). Immunohistochemistry revealed significantly more apoptotic tumour cells (p = 0.001), more tumour infiltrating lymphocytes (p = 0.049), lower tumour cell proliferation (p = 0.001), lower microvascular density (p = 0.003) and lower VEGFR2 expression (p = 0.003) in the therapy than in the control group. CEUS with VEGFR2-targeted microbubbles allowed for monitoring early treatment effects of a combined anti-PD-L1/anti-CTLA-4 immunotherapy on melanoma allografts with significantly lower tumour perfusion and significantly lower binding of VEGFR2-targeted microbubbles in the therapy than in the control group.

Abstract Microbubbles (MBs) exhibit cavitation upon exposure to ultrasound (US), which creates opportunities to adopt them in new therapeutic approaches. The present study reports an efficient, translatable approach to precisely control the spaciotemporal delivery of therapeutic microRNAs (AmiR‐21 and miR‐100) and TK‐p53‐NTR triple therapeutic gene, co‐loaded in PLGA‐PEG‐PEI polymer nanoparticles (NPs) to tumor models of triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC) using US‐mediated targeted destruction of BR38 MBs. PLGA‐PEG‐PEI conjugated triblock co‐polymer NPs are synthesized and characterized for their physicochemical properties, and optimized for co‐loading of miRNAs and TK‐p53‐NTR. Quantitative in vivo imaging and ex vivo tissue analysis of 4T1 (TNBC) subcutaneous tumors in BALB/c mice reveal 19 ± 0.5% (p < 0.01) increase in delivery of miRNAs, and 48 ± 1.79% (p < 0.001) increase in delivery of TK‐p53‐NTR upon US treatment, which results to 48 ± 6.98% (p < 0.01) reduction in tumor growth as compared to contralateral tumors without US. This significantly increases the survival rate of animals as compared to pDNA control group. Similar treatment effects are observed in both TNBC and HCC tumor models. This novel combined therapeutic approach, entailing both miRNAs and suicide gene therapy has strong potential for future applications in cancer therapy.

The aim of this study was to evaluate the added value of ultrasound molecular imaging of the vascular growth factor receptor 2 (VEGFR2) expression, using the clinical grade contrast agent BR55, for the early evaluation of antiangiogenic treatment efficacy in a chemo-induced rat mammary tumor model. In this preclinical study, chemo-induced rat mammary tumors were obtained after a single injection of N-nitroso-N-methylurea intraperitoneally in 46 prepubescent (age 38 ± 2 days) female rats. All experiments were performed under the authorization of the Direction Générale de la Santé, Geneva, Switzerland. Once tumor reached 0.8 cm in the largest cross-section, animals were enrolled in a sunitinib- or vehicle-treated group. Ultrasound molecular imaging was performed using BR55, a clinical grade targeted contrast agent against VEGFR2, before therapy and up to 72 hours. Anatomical changes of tumor over time, that is, area of the tumor largest cross-section and tumor volume, were measured in B-mode. Signal from microbubbles was detected in a nonlinear contrast mode (power modulation) using the iU22 diagnostic ultrasound system (Phillips, United States) equipped with a L12-5 linear transducer (transmit frequency 5 MHz). Peak enhancement and wash-in area under the curve were extracted from the time intensity curves generated by a dedicated quantification software for contrast ultrasound, so-called VueBox (Bracco Suisse SA, Switzerland). The signal of bound BR55 microbubbles in the tumor was quantified 10 minutes after injection. Altogether, these parameters were used to monitor tumoral response to treatment at the anatomical, functional, and molecular levels. At each time point, a cohort of tumors was harvested for the assessment of CD31 and VEGFR2 expression by immunohistochemistry staining. Under sunitinib therapy, assessment of the expression of VEGFR2 by ultrasound molecular imaging with BR55 reveals a significant difference as early as 12 hours after first dosing (-25%), whereas tumor size significant change occurs only after 24 hours. At the end of the therapeutic protocol, 72 hours after the onset of treatment, molecular changes are more marked with a 80% decrease compared with only ~40% for the anatomic parameters. Ultrasound molecular imaging observations suggesting a decrease in VEGFR2 expression in treated tumors were corroborated by semiquantitative grading of VEGFR2, showing a decrease expression over time. Functional parameters measured in the perfusion phase also show a decrease along treatment, significant for 24 hours and of 48% of peak enhancement at the end of protocol. Anatomical, functional, and molecular evaluations are feasible in a single examination using BR55 ultrasound targeted contrast agent. Ultrasound molecular imaging of VEGFR2 can depict an early response to antiangiogenic treatment in a rat mammary tumor model. This imaging modality has a potential for early assessment of each patient's response, which could be useful to take decisions on therapeutic protocol, providing as such an imaging tool for personalized medicine.

To evaluate the feasibility and time saving of fusing CT and MR enterography with ultrasound for ultrasound molecular imaging (USMI) of inflammation in an acute small bowel inflammation of swine. Nine swine with ileitis were scanned with either CT (n = 3) or MR (n = 6) enterography. Imaging times to load CT/MR images onto a clinical ultrasound machine, fuse them to ultrasound with an anatomical landmark-based approach, and identify ileitis were compared to the imaging times without anatomical road mapping. Inflammation was then assessed by USMI using dual selectin-targeted (MBSelectin) and control (MBControl) contrast agents in diseased and healthy control bowel segments, followed by ex vivo histology. Cross-sectional image fusion with ultrasound was feasible with an alignment error of 13.9 ± 9.7 mm. Anatomical road mapping significantly reduced (P < 0.001) scanning times by 40%. Localising ileitis was achieved within 1.0 min. Subsequently performed USMI demonstrated significantly (P < 0.001) higher imaging signal using MBSelectin compared to MBControl and histology confirmed a significantly higher inflammation score (P = 0.006) and P- and E-selectin expression (P ≤ 0.02) in inflamed vs. healthy bowel. Fusion of CT and MR enterography data sets with ultrasound in real time is feasible and allows rapid anatomical localisation of ileitis for subsequent quantification of inflammation using USMI. • Real-time fusion of CT/MRI with ultrasound to localise ileitis is feasible. • Anatomical road mapping using CT/MRI significantly decreases the scanning time for USMI. • USMI allows quantification of inflammation in swine, verified with ex vivo histology.

Aim of the present study was to investigate the sensitivity of high resolution ultrasound (HRU), standard contrast-enhanced ultrasound (CEUS) and CEUS using a novel vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agent for the detection of hepatic metastases in a mouse model of colorectal cancer using clinical standard technology. The human colon cancer cell line HT29, transfected with luciferase cDNA for in vivo bioluminescence monitoring, was injected intrasplenically into CB17.SCID mice. Mice were monitored weekly by bioluminescence and after 2 and 4.5 weeks by HRU and CEUS. Contrast media (untargeted BR1, targeted BR55) was applied and digital cine loops from the arterial phase (15 - 45 sec), portal venous phase (50 - 120 s) and late phases (3 - 5 min, 1hour) of the whole liver were analyzed. Data were correlated with postmortem histopathology. Without contrast enhancement, lesions > 4 mm were reliably detected. After use of untargeted CEUS, lesions > 2 mm were reliably detected and enhanced rim vascularization and late-phase wash-out was shown. With BR55, lesions > 0.8 mm were reliably detected with excellent documentation of vascularization. A persistent contrast enhancement was seen > 30 min after injection. Contrast-enhancement patterns with BR55 significantly correlated with CD31 (R2 = 0.74) and VEGFR2-immunohistochemistry (R2 = 0.66). Detection of metastases by HRU and CEUS was earlier and more accurate than monitoring via bioluminescence. In vivo monitoring of hepatic micrometastases can thus be performed without prior modification of cancer cells using standard technology.

The role of ultrasound contrast agents (UCA) initially designed for diagnosis has evolved towards a therapeutic use. Ultrasound (US) for triggered drug delivery has many advantages. In particular, it enables a high spatial control of drug release, thus potentially allowing activation of drug delivery only in the targeted region, and not in surrounding healthy tissue. Moreover, UCA imaging can also be used firstly to precisely locate the target region to, and then used to monitor the drug delivery process by tracking the location of release occurrence. All these features make UCA and ultrasound attractive means to mediate drug delivery. The three main potential clinical indications for drug/gene US delivery are (i) the cardiovascular system, (ii) the central nervous system for small molecule delivery, and (iii) tumor therapy using cytotoxic drugs. Although promising results have been achieved in preclinical studies in various animal models, still very few examples of clinical use have been reported. In this chapter will be addressed the aspects pertaining to UCA formulation (chemical composition, mode of preparation, analytical methods…) and the requirement for a potential translation into the clinic following approval by regulatory authorities.

With contrast-enhanced ultrasound (CEUS) now established as a valuable imaging modality for many applications, a more specific demand has recently emerged for quantifying perfusion and using measured parameters as objective indicators for various disease states. However, CEUS perfusion quantification remains challenging and is not well integrated in daily clinical practice. The development of VueBox™ alleviates existing limitations and enables quantification in a standardized way. VueBox™ operates as an off-line software application, after dynamic contrast-enhanced ultrasound (DCE-US) is performed. It enables linearization of DICOM clips, assessment of perfusion using patented curve-fitting models, and generation of parametric images by synthesizing perfusion information at the pixel level using color coding. VueBox™ is compatible with most of the available ultrasound platforms (nonlinear contrast-enabled), has the ability to process both bolus and disruption-replenishment kinetics loops, allows analysis results and their context to be saved, and generates analysis reports automatically. Specific features have been added to VueBox™, such as fully automatic in-plane motion compensation and an easy-to-use clip editor. Processing time has been reduced as a result of parallel programming optimized for multi-core processors. A long list of perfusion parameters is available for each of the two administration modes to address all possible demands currently reported in the literature for diagnosis or treatment monitoring. In conclusion, VueBox™ is a valid and robust quantification tool to be used for standardizing perfusion quantification and to improve the reproducibility of results across centers.

To implement a retrospective intrinsic landmark-based (ILB) gating protocol for contrast-enhanced ultrasound (CEUS) and to compare its efficiency to non-gated, manually gated and extrinsically gated CEUS. CEUS of the liver was performed in healthy mice (n = 5) and in NEMO knockout mice with dysplastic livers (n = 5). In healthy animals, first-pass kinetics of non-specific microbubbles was recorded. Knockout mice were analysed regarding retention of VEGFR2-specific microbubbles. For retrospective gating, a landmark which showed respiratory movement was encircled as a region of interest (ROI). During inspiration, the signal intensity within the ROI altered, which served as gating signal. To evaluate the accuracy, non-gated, extrinsically gated and ILB-gated time-intensity curves were created. For each curve, descriptive parameters were calculated and compared to the gold standard (manual frame-by-frame gating). No significant differences in the variation of ILB- and extrinsically gated time-intensity curves from the gold standard were observed. Non-gated data showed significantly higher variations. Also the variation of molecular ultrasound data was significantly lower for ILB-gated compared to non-gated data. ILB gating is a robust and easy method to improve data accuracy in functional and molecular ultrasound liver imaging. This technique can presumably be translated to contrast-enhanced ultrasound examinations in humans. • Quantitative analysis of the uptake of contrast agents during ultrasound is complex. • Intrinsic landmark-based gating (ILB) offers a simple implementable method for motion correction. • Results using ILB-gating are comparable to extrinsic gating using external biomonitoring devices. • Functional and molecular imaging of mobile organs will benefit from ILB gating.