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To date, the appropriate training required for the reproducible operation of multispectral optoacoustic tomography (MSOT) is poorly discussed. Therefore, the aim of this study was to assess the teachability of MSOT imaging. Five operators (two experienced and three inexperienced) performed repositioning imaging experiments. The inexperienced received the following introductions: personal supervision, video meeting, or printed introduction. The task was to image the exact same position on the calf muscle for seven times on five volunteers in two rounds of investigations. In the first session, operators used ultrasound guidance during measurements while using only photoacoustic data in the second session. The performance comparison was carried out with full‐reference image quality measures to quantitatively assess the difference between repeated scans. The study demonstrates that given a personal supervision and hybrid ultrasound real‐time imaging in MSOT measurements, inexperienced operators are able to achieve the same level as experienced operators in terms of repositioning accuracy. [ABSTRACT FROM AUTHOR]

Imaging plays a critical role in exploring the pathophysiology and enabling the diagnostics and therapy assessment in carotid artery disease. Ultrasonography, computed tomography, magnetic resonance imaging and nuclear medicine techniques have been used to extract of known characteristics of plaque vulnerability, such as inflammation, intraplaque hemorrhage and high lipid content. Despite the plethora of available techniques, there is still a need for new modalities to better characterize the plaque and provide novel biomarkers that might help to detect the vulnerable plaque early enough and before a stroke occurs. Optoacoustics, by providing a multiscale characterization of the morphology and pathophysiology of the plaque could offer such an option. By visualizing endogenous (e.g., hemoglobin, lipids) and exogenous (e.g., injected dyes) chromophores, optoacoustic technologies have shown great capability in imaging lipids, hemoglobin and inflammation in different applications and settings. Herein, we provide an overview of the main optoacoustic systems and scales of detail that enable imaging of carotid plaques in vitro, in small animals and humans. Finally, we discuss the limitations of this novel set of techniques while investigating their potential to enable a deeper understanding of carotid plaque pathophysiology and possibly improve the diagnostics in future patients with carotid artery disease.

Emerging evidence indicates crosstalk between the brain and hematopoietic system following cerebral ischemia. Here, we investigated metabolism and oxygenation in the spleen and spinal cord in a transient middle cerebral artery occlusion (tMCAO) model. Sham-operated and tMCAO mice underwent [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to assess glucose metabolism. Naïve, sham-operated and tMCAO mice underwent multispectral optoacoustic tomography (MSOT) assisted by quantitative model-based reconstruction and unmixing algorithms for accurate mapping of oxygenation patterns in peripheral tissues at 24 h after reperfusion. We found increased [18F]FDG uptake and reduced MSOT oxygen saturation, indicating hypoxia in the thoracic spinal cord of tMCAO mice compared with sham-operated mice but not in the spleen. Reduced spleen size was observed in tMCAO mice compared with sham-operated mice ex vivo. tMCAO led to an increase in the numbers of mature T cells in femoral bone marrow tissues, concomitant with a stark reduction in these cell subsets in the spleen and peripheral blood. The combination of quantitative PET and MSOT thus enabled observation of hypoxia and increased metabolic activity in the spinal cord of tMCAO mice at 24 h after occlusion compared to sham-operated mice.

Imaging skeletal muscle function and metabolism, as reported by local hemodynamics and oxygen kinetics, can elucidate muscle performance, severity of an underlying disease or outcome of a treatment. Herein, we used multispectral optoacoustic tomography (MSOT) to image hemodynamics and oxygen kinetics within muscle during exercise. Four healthy volunteers underwent three different hand-grip exercise challenges (60s isometric, 120s intermittent isometric and 60s isotonic). During isometric contraction, MSOT showed a decrease of HbO2, Hb and total blood volume (TBV), followed by a prominent increase after the end of contraction. Corresponding hemodynamic behaviors were recorded during the intermittent isometric and isotonic exercises. A more detailed analysis of MSOT readouts revealed insights into arteriovenous oxygen differences and muscle oxygen consumption during all exercise schemes. These results demonstrate an excellent capability of visualizing both circulatory function and oxygen metabolism within skeletal muscle under exercise, with great potential implications for muscle research, including relevant disease diagnostics.

Hepatic steatosis is characterized by intrahepatic lipid accumulation and may lead to irreversible liver damage if untreated. Here, we investigate whether multispectral optoacoustic tomography (MSOT) can offer label-free detection of liver lipid content to enable non-invasive characterization of hepatic steatosis by analyzing the spectral region around 930 nm, where lipids characteristically absorb. In a pilot study, we apply MSOT to measure liver and surrounding tissues in five patients with liver steatosis and five healthy volunteers, revealing significantly higher absorptions at 930 nm in the patients, while no significant difference was observed in the subcutaneous adipose tissue of the two groups. We further corroborated the human observations with corresponding MSOT measurements in high fat diet (HFD) - and regular chow diet (CD)-fed mice. This study introduces MSOT as a potential non-invasive and portable technique for detecting/monitoring hepatic steatosis in clinical settings, providing justification for larger studies.

Abstract Pheochromocytomas (PCCs) and paragangliomas (PGLs), together referred to as PPGLs, are rare chromaffin cell-derived tumors. They require timely diagnosis as this is the only way to achieve a cure through surgery and because of the potentially serious cardiovascular complications and sometimes life-threatening comorbidities that can occur if left untreated. The biochemical diagnosis of PPGLs has improved over the last decades, and the knowledge of the underlying genetics has dramatically increased. In addition to conventional anatomical imaging by CT and MRI for PPGL detection, new functional imaging modalities have emerged as very useful for patient surveillance and stratification for therapy. The availability of validated and predictive animal models of cancer is essential for translating molecular, imaging and therapy response findings from the bench to the bedside. This is especially true for rare tumors, such as PPGLs, for which access to large cohorts of patients is limited. There are few animal models of PPGLs that have been instrumental in refining imaging modalities for early tumor detection, as well as in identifying and evaluating novel imaging tracers holding promise for the detection and/or treatment of human PPGLs. The in vivo PPGL models mainly include xenografts/allografts generated by engrafting rat or mouse cell lines, as no representative human cell line is available. In addition, there is a model of endogenous PCCs (i.e., MENX rats) that was characterized in our laboratory. In this review, we will summarize the contribution that various representative models of PPGL have given to the visualization of these tumors in vivo and we present an example of a tracer first evaluated in MENX rats, and then translated to the detection of these tumors in human patients. In addition, we will illustrate briefly the potential of ex vivo biological imaging of intact adrenal glands in MENX rats.

Pheochromocytomas (PCCs) and paragangliomas (PGLs), together referred to as PPGLs, are rare chromaffin cell-derived tumors. They require timely diagnosis as this is the only way to achieve a cure through surgery and because of the potentially serious cardiovascular complications and sometimes life-threatening comorbidities that can occur if left untreated. The biochemical diagnosis of PPGLs has improved over the last decades, and the knowledge of the underlying genetics has dramatically increased. In addition to conventional anatomical imaging by CT and MRI for PPGL detection, new functional imaging modalities have emerged as very useful for patient surveillance and stratification for therapy. The availability of validated and predictive animal models of cancer is essential for translating molecular, imaging and therapy response findings from the bench to the bedside. This is especially true for rare tumors, such as PPGLs, for which access to large cohorts of patients is limited. There are few animal models of PPGLs that have been instrumental in refining imaging modalities for early tumor detection, as well as in identifying and evaluating novel imaging tracers holding promise for the detection and/or treatment of human PPGLs. The in vivo PPGL models mainly include xenografts/allografts generated by engrafting rat or mouse cell lines, as no representative human cell line is available. In addition, there is a model of endogenous PCCs (i.e., MENX rats) that was characterized in our laboratory. In this review, we will summarize the contribution that various representative models of PPGL have given to the visualization of these tumors in vivo and we present an example of a tracer first evaluated in MENX rats, and then translated to the detection of these tumors in human patients. In addition, we will illustrate briefly the potential of ex vivo biological imaging of intact adrenal glands in MENX rats. [ABSTRACT FROM AUTHOR]

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

Photo-or optoacoustic imaging (OAI) allows quantitative imaging of target tissues. Using multi-wavelength illumination with subsequent ultrasound detection, it may visualize a variety of different chromophores at centimeter depth. Despite its non-invasive, label-free advantages, the precision of repeated measurements for clinical applications is still elusive. We present a multilayer analysis of n = 1920 imaging datasets obtained from a prospective clinical trial (NCT03979157) in n = 10 healthy adult volunteers. All datasets were analyzed for 13 single wavelengths (SWL) between 660 nm–1210 nm and five MSOT-parameters (deoxygenated/oxygenated/total hemoglobin, collagen and lipid) by a semi-automated batch mode software. Intraclass correlation coefficients (ICC) were good to excellent for intrarater (SWL: 0.82–0.92; MSOT-parameter: 0.72−0.92) and interrater reproducibility (SWL: 0.79−0.87; MSOT-parameter: 0.78−0.86), with the exception for MSOT-parameter lipid (interrater ICC: 0.56). Results were stable over time, but exercise-related effects as well as inter-and intramuscular variability were observed. The findings of this study provide a framework for further clinical OAI implementation.

Purpose: To establish multi-modal imaging for the assessment of kidney pH, perfusion, and clearance rate using magnetic resonance imaging (MRI) and multispectral optoacoustic tomography (MSOT) in healthy mice. Kidney pH and perfusion values were measured on a pixel-by-pixel basis using the MRI acidoCEST and FAIR-EPI methods. Kidney filtration rate was measured by analyzing the renal clearance rate of IRdye 800 using MSOT. To test the effect of one imaging method on the other, a set of 3 animals were imaged with MSOT followed by MRI, and a second set of 3 animals were imaged with MRI followed by MSOT. In a subsequent study, the reproducibility of pH, perfusion, and renal clearance measurements were tested by imaging 4 animals twice, separated by 4 days. The contrast agents used for acidoCEST based pH measurements influenced the results of MSOT. Specifically, the exponential decay time from the kidney cortex, as measured by MSOT, was significantly altered when MRI was performed prior to MSOT. However, no significant difference in the cortex to pelvis area under the curve (AUC) was noted. When the order of experiments was reversed, no significant differences were noted in the pH or perfusion values. Reproducibility measurements demonstrated similar pH and cortex to pelvis AUC; however, perfusion values were significantly different with the cortex values being higher and the pelvic values being lower in the second imaging time. We demonstrate that using a combination of MRI and MSOT, physiological measurements of pH, blood flow, and clearance rates can be measured in the mouse kidney in the same imaging session. [ABSTRACT FROM AUTHOR]

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

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.

Optoacoustic or photoacoustic imaging (OAI/PAI) is a technology which enables non-invasive visualization of laser-illuminated tissue by the detection of acoustic signals. The combination of “light in” and “sound out” offers unprecedented scalability with a high penetration depth and resolution. The wide range of biomedical applications makes this technology a versatile tool for preclinical and clinical research. Particularly when imaging inflammation, the technology offers advantages over current clinical methods to diagnose, stage, and monitor physiological and pathophysiological processes. This review discusses the clinical perspective of using OAI in the context of imaging inflammation as well as in current and emerging translational applications.

Unlike traditional optical methods, optoacoustic imaging is less sensitive to scattering of ballistic photons, so it is capable of high-resolution interrogation at a greater depth. By integrating video-rate visualization with multiplexing and sensing a range of endogenous and exogenous chromophores, optoacoustic imaging has matured into a versatile noninvasive investigation modality with rapidly expanding use in biomedical research. We review the principal features of the technology and discuss recent advances it has enabled in structural, functional, and molecular neuroimaging in small-animal models. In extending the boundaries of noninvasive observation beyond the reach of customary photonic methods, the latest developments in optoacoustics have substantially advanced neuroimaging inquiry, with promising implications for basic and translational studies. Optoacoustic imaging combines optical contrast and acoustic resolution to surpass the diffraction barrier of high-resolution optical imaging. Excellent penetration with specificity and scalability rendered optoacoustic imaging a promising modality for biomedical interrogation. A wide range of neuroimaging applications enabled considerable advances in basic and translational optoacoustic studies in animal models and in humans. Along with advances and opportunities, numerous limitations have also emerged, which are currently a matter of intense research. Combined with evolving technologies, major developments are expected in noninvasive optoacoustic neuroimaging, facilitating the discovery process. [ABSTRACT FROM AUTHOR]

Imaging plays a critical role in exploring the pathophysiology and enabling the diagnostics and therapy assessment in carotid artery disease. Ultrasonography, computed tomography, magnetic resonance imaging and nuclear medicine techniques have been used to extract of known characteristics of plaque vulnerability, such as inflammation, intraplaque hemorrhage and high lipid content. Despite the plethora of available techniques, there is still a need for new modalities to better characterize the plaque and provide novel biomarkers that might help to detect the vulnerable plaque early enough and before a stroke occurs. Optoacoustics, by providing a multiscale characterization of the morphology and pathophysiology of the plaque could offer such an option. By visualizing endogenous (e.g., hemoglobin, lipids) and exogenous (e.g., injected dyes) chromophores, optoacoustic technologies have shown great capability in imaging lipids, hemoglobin and inflammation in different applications and settings. Herein, we provide an overview of the main optoacoustic systems and scales of detail that enable imaging of carotid plaques in vitro , in small animals and humans. Finally, we discuss the limitations of this novel set of techniques while investigating their potential to enable a deeper understanding of carotid plaque pathophysiology and possibly improve the diagnostics in future patients with carotid artery disease., Competing Interests: VN discloses financial interests to iThera Medical GmbH, Spear UG, i3 Inc and sThesis GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Karlas, Fasoula, Kallmayer, Schäffer, Angelis, Katsouli, Reidl, Duelmer, Al Adem, Hadjileontiadis, Eckstein and Ntziachristos.)

Critical limb ischemia (CLI) remains a challenge for an interdisciplinary therapeutic team due to chronic nonhealing wounds. Against this background, there is a necessity of quality control after revascularization. Beside the isolated evaluation of the macrocirculation by Ankle-Brachial or Toe-Brachial Index measurements, the microcirculation as an additional important factor of wound healing often remains underestimated. The following article gives an overview about the current investigation methods for noninvasive perfusion control of the CLI patient. Therefore, transcutaneous oxygen pressure (tcpO2), the “oxygen-to-see” method which is a combination of white light tissue spectrometry and laser-Doppler flowmetry, fluorescence angiography with indocyanine green, and multispectral optoacoustic tomography will be described. [ABSTRACT FROM AUTHOR]

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3 h timepoint; r = 0.90, P < 0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6 h timepoint; r = 0.70, P = 0.005) and HMGB1 (e.g. 6 h timepoint; r = 0.56, P = 0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration. [ABSTRACT FROM AUTHOR]

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Emerging evidence indicates crosstalk between the brain and hematopoietic system following cerebral ischemia. Here, we investigated metabolism and oxygenation in the spleen and spinal cord in a transient middle cerebral artery occlusion (tMCAO) model. Sham-operated and tMCAO mice underwent [ 18 F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to assess glucose metabolism. Naïve, sham-operated and tMCAO mice underwent multispectral optoacoustic tomography (MSOT) assisted by quantitative model-based reconstruction and unmixing algorithms for accurate mapping of oxygenation patterns in peripheral tissues at 24 h after reperfusion. We found increased [ 18 F]FDG uptake and reduced MSOT oxygen saturation, indicating hypoxia in the thoracic spinal cord of tMCAO mice compared with sham-operated mice but not in the spleen. Reduced spleen size was observed in tMCAO mice compared with sham-operated mice ex vivo . tMCAO led to an increase in the numbers of mature T cells in femoral bone marrow tissues, concomitant with a stark reduction in these cell subsets in the spleen and peripheral blood. The combination of quantitative PET and MSOT thus enabled observation of hypoxia and increased metabolic activity in the spinal cord of tMCAO mice at 24 h after occlusion compared to sham-operated mice., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Imaging skeletal muscle function and metabolism, as reported by local hemodynamics and oxygen kinetics, can elucidate muscle performance, severity of an underlying disease or outcome of a treatment. Herein, we used multispectral optoacoustic tomography (MSOT) to image hemodynamics and oxygen kinetics within muscle during exercise. Four healthy volunteers underwent three different hand-grip exercise challenges (60s isometric, 120s intermittent isometric and 60s isotonic). During isometric contraction, MSOT showed a decrease of HbO 2 , Hb and total blood volume (TBV), followed by a prominent increase after the end of contraction. Corresponding hemodynamic behaviors were recorded during the intermittent isometric and isotonic exercises. A more detailed analysis of MSOT readouts revealed insights into arteriovenous oxygen differences and muscle oxygen consumption during all exercise schemes. These results demonstrate an excellent capability of visualizing both circulatory function and oxygen metabolism within skeletal muscle under exercise, with great potential implications for muscle research, including relevant disease diagnostics., Competing Interests: The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Vasilis Ntziachristos reports a relationship with sThesis GmbH that includes: equity or stocks. Vasilis Ntziachristos reports a relationship with iThera Medical GmbH that includes: equity or stocks. Vasilis Ntziachristos reports a relationship with Spear UG that includes: equity or stocks. Vasilis Ntziachristos reports a relationship with i3 Inc. that includes: equity or stocks. All other authors declare that they have no competing interests., (© 2023 The Authors.)

Hepatic steatosis is characterized by intrahepatic lipid accumulation and may lead to irreversible liver damage if untreated. Here, we investigate whether multispectral optoacoustic tomography (MSOT) can offer label-free detection of liver lipid content to enable non-invasive characterization of hepatic steatosis by analyzing the spectral region around 930 nm, where lipids characteristically absorb. In a pilot study, we apply MSOT to measure liver and surrounding tissues in five patients with liver steatosis and five healthy volunteers, revealing significantly higher absorptions at 930 nm in the patients, while no significant difference was observed in the subcutaneous adipose tissue of the two groups. We further corroborated the human observations with corresponding MSOT measurements in high fat diet (HFD) - and regular chow diet (CD)-fed mice. This study introduces MSOT as a potential non-invasive and portable technique for detecting/monitoring hepatic steatosis in clinical settings, providing justification for larger studies., Competing Interests: V.N. is a founder and equity owner of sThesis GmbH, iThera Medical GmbH, Spear UG, and i3 Inc. All other authors declare that they have no competing interests., (© 2023 The Authors.)

The development of nanotheranostics for precision imaging-guided regulated cell death-mediated synergistic tumor therapy is still challenging. Herein, a novel multifunctional nanotheranostic agent, iRGD-coated maleimide-poly(ethylene glycol)-poly(lactic acid/glycolic acid)-encapsulated hydrophobic gold nanocages (AuNCs) and hydrophilic epigallocatechin gallate (EGCG) (PAuE) is developed for multispectral optoacoustic tomography (MSOT)-guided photothermal therapy (PTT) and chemotherapy. The portions of necroptotic and apoptotic tumor cells were 52.9 and 5.4%, respectively, at 6 h post-incubation after the AuNC-induced mild PTT treatment, whereas they became 14.0 and 46.1% after 24 h, suggesting that the switch of the cell death pathway is a time-dependent process. Mild PTT facilitated the release of EGCG which induces the downregulation of hypoxia-inducible factor-1 (HIF-1α) expression to enhance apoptosis at a later stage, realizing a remarkable tumor growth inhibition in vivo. Moreover, RNA sequence analyses provided insights into the significant changes in genes related to the cross-talk between necroptosis and apoptosis pathways via PAuE upon laser irradiation. In addition, the biodistribution and metabolic pathways of PAuE have been successfully revealed by 3D MSOT. Taken together, this strategy of first combination of EGCG and AuNC-based photothermal agent via triggering necroptosis/apoptosis to downregulate HIF-1α expression in a tumor environment provides a new insight into anti-cancer therapy.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

Optoacoustic or photoacoustic imaging (OAI/PAI) is a technology which enables non-invasive visualization of laser-illuminated tissue by the detection of acoustic signals. The combination of "light in" and "sound out" offers unprecedented scalability with a high penetration depth and resolution. The wide range of biomedical applications makes this technology a versatile tool for preclinical and clinical research. Particularly when imaging inflammation, the technology offers advantages over current clinical methods to diagnose, stage, and monitor physiological and pathophysiological processes. This review discusses the clinical perspective of using OAI in the context of imaging inflammation as well as in current and emerging translational applications. [ABSTRACT FROM AUTHOR]

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques., Competing Interests: Prof. Dr. V. Ntziachristos is a stock owner of iThera Medical GmbH, Munich, Germany. All other authors declare no financial interests., (© 2021 Published by Elsevier GmbH.)

Melanoma is the most aggressive form of skin cancer. Hypoxia is a feature of the tumor microenvironment that reduces efficacy of immuno- and chemotherapies, resulting in poor clinical outcomes. Lactococcus lactis is a facultative anaerobic gram-positive lactic acid bacterium (LAB) that is Generally Recognized as Safe (GRAS). Recently, the use of LAB as a delivery vehicle has emerged as an alternative strategy to deliver therapeutic molecules; therefore, we investigated whether L. lactis can target and localize within melanoma hypoxic niches. To simulate hypoxic conditions in vitro, melanoma cells A2058, A375 and MeWo were cultured in a chamber with a gas mixture of 5% CO2, 94% N2 and 1% O2. Among the cell lines tested, MeWo cells displayed greater survival rates when compared to A2058 and A375 cells. Co-cultures of L. lactis expressing GFP or mCherry and MeWo cells revealed that L. lactis efficiently express the transgenes under hypoxic conditions. Moreover, multispectral optoacoustic tomography (MSOT), and near infrared (NIR) imaging of tumor-bearing BALB/c mice revealed that the intravenous injection of either L. lactis expressing β-galactosidase (β-gal) or infrared fluorescent protein (IRFP713) results in the establishment of the recombinant bacteria within tumor hypoxic niches. Overall, our data suggest that L. lactis represents an alternative strategy to target and deliver therapeutic molecules into the tumor hypoxic microenvironment. [ABSTRACT FROM AUTHOR]

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad-spectrum and high-absorbance near-infrared optoacoustic contrast agent, E4 x12 -Cy7. Our near-infrared tracer is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4 x12 -Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4 x12 -Cy7 and optoacoustically visualized β-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for β-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors., Competing Interests: V.N. is a shareholder in iThera Medical GmbH, Munich, Germany. The remaining authors declare no competing interests., (© 2021 The Authors.)