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Photoacoustic imaging (PAI) provides information on haemoglobin levels and blood oxygenation (sO2). To facilitate assessment of the variability in sO2 and haemoglobin in tumours, for example in response to therapies, the baseline variability of these parameters was evaluated in subcutaneous head and neck tumours in mice, using a PAI system (MSOTinVision-256TF). Tumours of anaesthetized animals (midazolam-fentanyl-medetomidine) were imaged for 75 min, in varying positions, and repeatedly over 6 days. An increasing linear trend for average tumoural haemoglobin and blood sO2 was observed, when imaging over 75 min. There were no significant differences in these temporal trends, when repositioning tumours. A negative correlation was found between the percent decrease in blood sO2 over 6 days and tumour growth rate. This paper shows the potential of PAI to provide baseline data for assessing the significance of intra- and inter-tumoural variations that may eventually have value for predicting and/or monitoring cancer treatment response. Keywords: Photoacoustic imaging, Hypoxia, Head and neck subcutaneous tumours, Pimonidazole, Blood sO2, Hemoglobin

IntroductionAcoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor’s microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy.MethodsIn this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7–10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy.ResultsResults show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

Optoacoustic imaging (OAI) can detect haemoglobin and assess its oxygenation. However, the lack of a haemoglobin signal need not indicate a lack of perfusion. This study uses a novel method to assist the co-registration of optoacoustic images with dynamic contrast enhanced ultrasound (DCE-US) images to demonstrate, in preclinical tumour models, the value of combining haemoglobin imaging with a perfusion imaging method, showing that a lack of a haemoglobin signal does not necessarily indicate an absence of perfusion. DCE-US was chosen for this particular experiment because US is extremely sensitive to microbubble contrast agents and because microbubbles, like red blood cells but unlike currently available optical contrast agents, do not extravasate. Significant spatial correlations were revealed between the DCE-US properties and tumour blood-oxygen saturation and haemoglobin, as estimated using OAI. It is speculated that DCE-US properties could be applied as surrogate biomarkers for hypoxia when planning clinical radiotherapy or chemotherapy.

Abstract Targeted therapies specific to the BRAF-MEK-ERK signaling pathway have shown great promise in the treatment of malignant melanoma in the last few years, with these drugs now commonly used in clinic. Melanoma cells treated using these agents are known to exhibit increased levels of melanin pigment and tyrosinase activity. In this study we assessed the potential of non-invasive imaging approaches (photoacoustic imaging (PAI) and magnetic resonance imaging (MRI)) to detect melanin induction in SKMEL28 human melanoma cells, following inhibition of Hsp90 and BRAF signaling using 17-AAG and vemurafenib, respectively. We confirmed, using western blot and spectrophotometry, that Hsp90 or BRAF inhibitor-induced melanoma cell differentiation resulted in an upregulation of tyrosinase and melanin expression levels, in comparison to control cells. This post-treatment increase in cellular pigmentation induced a significant increase in PAI signals that are spectrally identifiable and shortening of the MRI relaxation times T 1 and $${{\boldsymbol{T}}}_{{\bf{2}}}^{{\boldsymbol{\ast }}}$$ T 2 ∗ . This proof-of-concept study demonstrates the potential of MRI and PAI for detecting the downstream cellular changes induced by Hsp90 and BRAF-MEK-targeted therapies in melanoma cells with potential significance for in vivo imaging.

We demonstrate a versatile phase-change sub-micron contrast agent providing three modes of contrast enhancement: 1) photoacoustic imaging contrast, 2) ultrasound contrast with optical activation, and 3) ultrasound contrast with acoustic activation. This agent, which we name ‘Cy-droplet’, has the following novel features. It comprises a highly volatile perfluorocarbon for easy versatile activation, and a near-infrared optically absorbing dye chosen to absorb light at a wavelength with good tissue penetration. It is manufactured via a ‘microbubble condensation’ method. The phase-transition of Cy-droplets can be optically triggered by pulsed-laser illumination, inducing photoacoustic signal and forming stable gas bubbles that are visible with echo-ultrasound in situ. Alternatively, Cy-droplets can be converted to microbubble contrast agents upon acoustic activation with clinical ultrasound. Potentially all modes offer extravascular contrast enhancement because of the sub-micron initial size. Such versatility of acoustic and optical ‘triggerability’ can potentially improve multi-modality imaging, molecularly targeted imaging and controlled drug release.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy.Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40–2.00 ml PS101/kg body weight (n = 8–15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20.Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Gold nanorods (GNRs) are increasingly being investigated for cancer theranostics as they possess features which lend themselves in equal measures as contrast agents and catalysts for photothermal therapy. Their optical absorption spectral peak wavelength is determined by their size and shape. Photothermal therapy using GNRs is typically established using near infrared light as this allows sufficient penetration into the tumour matrix. Continuous wave (CW) lasers are the most commonly applied source of near infrared irradiation on GNRs for tumour photothermal therapy. It is perceived that large tumours may require fractionated or prolonged irradiation. However the true efficacy of repeated or protracted CW irradiation on tumour sites using the original sample of GNRs remains unclear. In this study spectroscopy and transmission electron microscopy are used to demonstrate that GNRs reshape both in vitro and in vivo after CW irradiation, which reduces their absorption efficiency. These changes were sustained throughout and beyond the initial period of irradiation, resulting from a spectral blue-shift and a considerable diminution in the absorption peak of GNRs. Solid subcutaneous tumours in immunodeficient BALB/c mice were subjected to GNRs and analysed with electron microscopy pre- and post-CW laser irradiation. This phenomenon of thermally induced GNR reshaping can occur at relatively low bulk temperatures, well below the bulk melting point of gold. Photoacoustic monitoring of GNR reshaping is also evaluated as a potential clinical aid to determine GNR absorption and reshaping during photothermal therapy. Aggregation of particles was coincidentally observed following CW irradiation, which would further diminish the subsequent optical absorption capacity of irradiated GNRs. It is thus established that sequential or prolonged applications of CW laser will not confer any additional photothermal effect on tumours due to significant attenuations in the peak optical absorption properties of GNRs following primary laser irradiation.

Imaging has become an essential tool in modern radiotherapy (RT), being used to plan dose delivery prior to treatment and verify target position before and during treatment. Ultrasound (US) imaging is cost-effective in providing excellent contrast at high resolution for depicting soft tissue targets apart from those shielded by the lungs or cranium. As a result, it is increasingly used in RT setup verification for the measurement of inter-fraction motion, the subject of Part I of this review (Fontanarosa et al 2015 Phys. Med. Biol. 60 R77–114). The combination of rapid imaging and zero ionising radiation dose makes US highly suitable for estimating intra-fraction motion. The current paper (Part II of the review) covers this topic. The basic technology for US motion estimation, and its current clinical application to the prostate, is described here, along with recent developments in robust motion-estimation algorithms, and three dimensional (3D) imaging. Together, these are likely to drive an increase in the number of future clinical studies and the range of cancer sites in which US motion management is applied. Also reviewed are selections of existing and proposed novel applications of US imaging to RT. These are driven by exciting developments in structural, functional and molecular US imaging and analytical techniques such as backscatter tissue analysis, elastography, photoacoustography, contrast-specific imaging, dynamic contrast analysis, microvascular and super-resolution imaging, and targeted microbubbles. Such techniques show promise for predicting and measuring the outcome of RT, quantifying normal tissue toxicity, improving tumour definition and defining a biological target volume that describes radiation sensitive regions of the tumour. US offers easy, low cost and efficient integration of these techniques into the RT workflow. US contrast technology also has potential to be used actively to assist RT by manipulating the tumour cell environment and by improving the delivery of radiosensitising agents. Finally, US imaging offers various ways to measure dose in 3D. If technical problems can be overcome, these hold potential for wide-dissemination of cost-effective pre-treatment dose verification and in vivo dose monitoring methods. It is concluded that US imaging could eventually contribute to all aspects of the RT workflow. [ABSTRACT FROM AUTHOR]

In modern radiotherapy, verification of the treatment to ensure the target receives the prescribed dose and normal tissues are optimally spared has become essential. Several forms of image guidance are available for this purpose. The most commonly used forms of image guidance are based on kilovolt or megavolt x-ray imaging. Image guidance can also be performed with non-harmful ultrasound (US) waves. This increasingly used technique has the potential to offer both anatomical and functional information.This review presents an overview of the historical and current use of two-dimensional and three-dimensional US imaging for treatment verification in radiotherapy. The US technology and the implementation in the radiotherapy workflow are described. The use of US guidance in the treatment planning process is discussed. The role of US technology in inter-fraction motion monitoring and management is explained, and clinical studies of applications in areas such as the pelvis, abdomen and breast are reviewed. A companion review paper (O’Shea et al 2015 Phys. Med. Biol. submitted) will extensively discuss the use of US imaging for intra-fraction motion quantification and novel applications of US technology to RT. [ABSTRACT FROM AUTHOR]