Protocol to detect and quantify tumor hypoxia in mice using photoacoustic imaging.

Photoacoustic imaging (PAI) with co-registered ultrasound (US) is a hybrid non-invasive imaging modality that enables visualization and quantification of tumor hypoxia in live animals. Here, using a breast tumor xenograft model as an example, we present a stepwise protocol describing animal preparation, positioning, instrument setup, and US-PAI image acquisition procedures. This protocol also guides through detailed data analysis, explains functional readouts obtained from PAI, and discusses the potential application of the technology to study the hypoxic tumor microenvironment. For complete details on the use and execution of this protocol, please refer to Dai et al.1.

Sex differences in left-ventricular strain in a murine model of coxsackievirus B3 myocarditis.

Myocarditis is typically caused by viral infections, but most cases are thought to be subclinical. Echocardiography is often used for initial assessment of myocarditis patients but is poor at detecting subtle changes in cardiac dysfunction. Cardiac strain, such as global longitudinal strain (GLS) and global circumferential strain (GCS), represents an increasingly used set of measurements which can detect these subtle changes. Using a murine model of coxsackievirus B3 myocarditis, we characterized functional changes in the heart using echocardiography during myocarditis and by sex. We found that 2D GLS, 4D mode, and 4D strains detected a significant reduction in ejection fraction and GLS during myocarditis compared to baseline and in males compared to females. Furthermore, worse GLS correlated to increased levels of CD45+, CD11b+, and CD3+ immune cells. Our findings closely resemble published reports of GLS in patients with myocarditis indicating the usefulness of this animal model for translational studies of myocarditis.

TNF Signaling Is Required for Castration-Induced Vascular Damage Preceding Prostate Cancer Regression.

The mainstay treatment for locally advanced, recurrent, or metastatic prostate cancer (PrCa) is androgen deprivation therapy (ADT). ADT causes prostate cancers to shrink in volume, or regress, by inducing epithelial tumor cell apoptosis. In normal, non-neoplastic murine prostate, androgen deprivation via castration induces prostate gland regression that is dependent on TNF signaling. In addition to this direct mechanism of action, castration has also been implicated in an indirect mechanism of prostate epithelial cell death, which has been described as vascular regression. The initiating event is endothelial cell apoptosis and/or increased vascular permeability. This subsequently leads to reduced blood flow and perfusion, and then hypoxia, which may enhance epithelial cell apoptosis. Castration-induced vascular regression has been observed in both normal and neoplastic prostates. We used photoacoustic, power Doppler, and contrast-enhanced ultrasound imaging, and CD31 immunohistochemical staining of the microvasculature to assess vascular integrity in the period immediately following castration, enabling us to test the role of TNF signaling in vascular regression. In two mouse models of androgen-responsive prostate cancer, TNF signaling blockade using a soluble TNFR2 ligand trap reversed the functional aspects of vascular regression as well as structural changes in the microvasculature, including reduced vessel wall thickness, cross-sectional area, and vessel perimeter length. These results demonstrate that TNF signaling is required for vascular regression, most likely by inducing endothelial cell apoptosis and increasing vessel permeability. Since TNF is also the critical death receptor ligand for prostate epithelial cells, we propose that TNF is a multi-purpose, comprehensive signal within the prostate cancer microenvironment that mediates prostate cancer regression following androgen deprivation.

Integrating 1H MRS and deuterium labeled glucose for mapping the dynamics of neural metabolism in humans.

In the technique presented here, dubbed 'qMRS', we quantify the change in 1H MRS signal following administration of 2H-labeled glucose. As in recent human DMRS studies, we administer [6,6'-2H2]-glucose orally to healthy subjects. Since 2H is not detectable by 1H MRS, the transfer of the 2H label from glucose to a downstream metabolite leads to a reduction in the corresponding 1H MRS resonance of the metabolite, even if the total concentration of both isoforms remains constant. Moreover, introduction of the deuterium label alters the splitting pattern of the proton resonances, making indirect detection of the deuterated forms- as well as the direct detection of the decrease in unlabeled form- possible even without a 2H coil. Because qMRS requires only standard 1H MRS acquisition methods, it can be performed using commonly implemented single voxel spectroscopy (SVS) and chemical shift imaging (CSI) sequences. In this work, we implement qMRS in semi-LASER based CSI, generating dynamic maps arising from the fitted spectra, and demonstrating the feasibility of using qMRS and qCSI to monitor dynamic metabolism in the human brain using a 7T scanner with no auxiliary hardware.

Photoacoustic and Magnetic Resonance Imaging of Hybrid Manganese Dioxide-Coated Ultra-small NaGdF4 Nanoparticles for Spatiotemporal Modulation of Hypoxia in Head and Neck Cancer.

There is widespread interest in developing agents to modify tumor hypoxia in head and neck squamous cell carcinomas (HNSCC). Here, we report on the synthesis, characterization, and potential utility of ultra-small NaYF4:Nd3+/NaGdF4 nanocrystals coated with manganese dioxide (usNP-MnO2) for spatiotemporal modulation of hypoxia in HNSCC. Using a dual modality imaging approach, we first visualized the release of Mn2+ using T1-weighted magnetic resonance imaging (MRI) and modulation of oxygen saturation (%sO2) using photoacoustic imaging (PAI) in vascular channel phantoms. Combined MRI and PAI performed in patient-derived HNSCC xenografts following local and systemic delivery of the hybrid nanoparticles enabled mapping of intratumoral nanoparticle accumulation (based on T1 contrast enhancement) and improvement in tumor oxygenation (increased %sO2) within the tumor microenvironment. Our results demonstrate the potential of hybrid nanoparticles for the modulation of tumor hypoxia in head and neck cancer. Our findings also highlight the potential of combined MRI-PAI for simultaneous mapping nanoparticle delivery and oxygenation changes in tumors. Such imaging methods could be valuable in the precise selection of patients that are likely to benefit from hypoxia-modifying nanotherapies.

Development and Validation of a Clinically Relevant Workflow for MR-Guided Volumetric Arc Therapy in a Rabbit Model of Head and Neck Cancer.

There is increased interest in the use of magnetic resonance imaging (MRI) for guiding radiation therapy (RT) in the clinical setting. In this regard, preclinical studies can play an important role in understanding the added value of MRI in RT planning. In the present study, we developed and validated a clinically relevant integrated workflow for MRI-guided volumetric arc therapy (VMAT) in a VX2 rabbit neck tumor model of HNSCC. In addition to demonstrating safety and feasibility, we examined the therapeutic impact of MR-guided VMAT using a single high dose to obtain proof-of-concept and compared the response to conventional 2D-RT. Contrast-enhanced MRI (CE-MRI) provided excellent soft tissue contrast for accurate tumor segmentation for VMAT. Notably, MRI-guided RT enabled improved tumor targeting ability and minimal dose to organs at risk (OAR) compared to 2D-RT, which resulted in notable morbidity within a few weeks of RT. Our results highlight the value of integrating MRI into the workflow for VMAT for improved delineation of tumor anatomy and optimal treatment planning. The model combined with the multimodal imaging approach can serve as a valuable platform for the conduct of preclinical RT trials.

1H magnetic resonance spectroscopy of 2H-to-1H exchange quantifies the dynamics of cellular metabolism in vivo.

Quantitative mapping of the in vivo dynamics of cellular metabolism via non-invasive imaging contributes to our understanding of the initiation and progression of diseases associated with dysregulated metabolic processes. Current methods for imaging cellular metabolism are limited by low sensitivities, costs or the use of specialized hardware. Here, we introduce a method that captures the turnover of cellular metabolites by quantifying signal reductions in proton magnetic resonance spectroscopy (MRS) resulting from the replacement of 1H with 2H. The method, which we termed quantitative exchanged-label turnover MRS, only requires deuterium-labelled glucose and standard magnetic resonance imaging scanners, and with a single acquisition provides steady-state information and metabolic rates for several metabolites. We used the method to monitor glutamate, glutamine, γ-aminobutyric acid and lactate in the brains of unaffected and glioma-bearing rats following the administration of 2H2-labelled glucose and 2H3-labelled acetate. Quantitative exchanged-label turnover MRS should broaden the applications of routine 1H MRS.

Photoacoustic Imaging of Tattoo Inks: Phantom and Clinical Evaluation.

Photoacoustic imaging (PAI) is a novel hybrid imaging modality that provides excellent optical contrast with the spatial resolution of ultrasound in vivo. The method is widely being investigated in the clinical setting for diagnostic applications in dermatology. In this report, we illustrate the utility of PAI as a non-invasive tool for imaging tattoos. Ten different samples of commercially available tattoo inks were examined for their optoacoustic properties in vitro. In vivo PAI of an intradermal tattoo on the wrist was performed in a healthy human volunteer. Black/gray, green, violet and blue colored pigments provided higher levels of PA signal compared to white, orange, red and yellow pigments in vitro. PAI provided excellent contrast and enabled accurate delineation of the extent of the tattoo in the dermis. Our results reveal the photoacoustic properties of tattoo inks and demonstrate the potential clinical utility of PAI for intradermal imaging of tattoos. PAI may be useful as a clinical adjunct for objective preoperative evaluation of tattoos and potentially to guide/monitor laser-based tattoo removal procedures.

Performance Characteristics of Photoacoustic Imaging Probes with Varying Frequencies and Light-delivery Schemes.

Photoacoustic imaging (PAI) is an emerging biomedical imaging technique that utilizes a combination of light and ultrasound to detect photoabsorbers embedded within tissues. While the clinical utility of PAI has been widely explored for several applications, limitations in light penetration and detector sensitivity have restricted these studies to mostly superficial sites. Given the importance of PA signal generation and detection on light delivery and ultrasound detector frequency, there is an ongoing effort to optimize these parameters to enhance photoabsorber detection at increased depths. With this in mind, in this study we examined performance benchmarks of a commercially available PAI/ultrasound linear array system when using different imaging frequencies and light delivery schemes. A modified light fiber jacket providing focused light delivery (FLD) at the center of the probe was compared with the built-in fiber optics lining the length of the probe. Studies were performed in vitro to compare performance characteristics such as imaging resolution, maximum imaging depth, and sensitivity to varying hematocrit concentration for each frequency and light delivery method. Monte Carlo simulations of each light delivery method revealed increased light penetration with FLD. In tissue-mimicking phantoms, vascular channels used to simulate blood vessels could be visualized at a depth of 2.4 cm when lowering imaging frequency and utilizing FLD. Imaging at lower frequencies with FLD also enabled enhanced detection of varying hematocrit concentration levels at increased depths, although lateral imaging resolution was reduced. Finally, a proof of concept in vivo probe comparison study in a mouse tumor model provided supportive evidence of our in vitro results. Collectively, our findings show that adjusting imaging frequency and applying FLD can be a straightforward approach for improving PAI performance.

Dynamic photoacoustic imaging of neurovascular coupling in salivary glands.

The purpose of this study was to apply photoacoustic imaging (PAI), a relatively new imaging method, to non-invasively map neurovascular dynamics in salivary glands. Dynamic PAI with co-registered ultrasound (US) was performed in mice to monitor salivary gland hemodynamics in response to exogenous muscarinic receptor stimulation (pilocarpine) and blockade (atropine). Pilocarpine increased salivary gland oxygen saturation (%sO2) within minutes after administration, which was abrogated by atropine. A significant correlation was observed between change in %sO2 measured by PAI and saliva secretion. PAI is a novel imaging method that can be used for functional assessment of neurovascular dynamics in salivary glands.

Sugar alcohol provides imaging contrast in cancer detection.

Clinical imaging is widely used to detect, characterize and stage cancers in addition to monitoring the therapeutic progress. Magnetic resonance imaging (MRI) aided by contrast agents utilizes the differential relaxivity property of water to distinguish between tumorous and normal tissue. Here, we describe an MRI contrast method for the detection of cancer using a sugar alcohol, maltitol, a common low caloric sugar substitute that exploits the chemical exchange saturation transfer (CEST) property of the labile hydroxyl group protons on maltitol (malCEST). In vitro studies pointed toward concentration and pH-dependent CEST effect peaking at 1 ppm downfield to the water resonance. Studies with control rats showed that intravenously injected maltitol does not cross the intact blood-brain barrier (BBB). In glioma carrying rats, administration of maltitol resulted in the elevation of CEST contrast in the tumor region only owing to permeable BBB. These preliminary results show that this method may lead to the development of maltitol and other sugar alcohol derivatives as MRI contrast agents for a variety of preclinical imaging applications.

An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer.

BACKGROUND: Pancreatic cancer is a deadly disease with a very low 5-year patient survival rate of 6-8%. The major challenges of eliminating pancreatic cancer are treatment resistance and stromal barriers to optimal drug access within the tumor. Therefore, effective molecular targeting drugs with high intra-tumor access and retention are urgently needed for managing this devastating disease in the clinic. METHODS: This study has used the following in vitro and in vivo techniques for the investigation of exceptional anticancer drug FL118's efficacy in treatment of resistant pancreatic cancer: cell culture; immunoblotting analysis to test protein expression; DNA sub-G1 flow cytometry analyses to test cell death; MTT assay to test cell viability; pancreatic cancer stem cell assays (fluorescence microscopy tracing; matrigel assay; CD44-positive cell colony formation assay); human luciferase-labeled pancreatic tumor orthotopic animal model in vivo imaging; pancreatic cancer patient-derived xenograft (PDX) animal models; and toxicology studies with immune-competent BALB/cj mice and beagle dogs. RESULTS: Our studies found that FL118 alone preferentially killed cisplatin-resistant cancer cells, while a combination of FL118 with cisplatin synergistically killed resistant pancreatic cancer cells and reduced spheroid formation of treatment-resistant pancreatic cancer stem-like cells. Furthermore, using in vivo-imaging, we found that FL118 in combination with cisplatin strongly inhibited both drug-resistant pancreatic xenograft tumor growth and metastasis. In PDX model, we demonstrated that FL118 alone effectively eliminated PDX tumors, while FL118 in combination with gemcitabine eliminated PDX tumors that showed relative resistance (less sensitivity) to treatment with FL118. These FL118 efficacy results are consistent with our molecular-targeting data showing that FL118 inhibited the expression of multiple antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and ERCC6, a critical regulator of DNA repair, in treatment-resistant pancreatic stem-like cancer cells. Furthermore, FL118 toxicity studies in BALB/cj mice and beagle dogs indicated that FL118 exhibits favorable hematopoietic and biochemical toxicities. CONCLUSION: Together, our studies suggest that FL118 is a promising anticancer drug for further clinical development to effectively treat drug-resistant pancreatic cancer alone or in combination with other pancreatic cancer chemotherapeutic drugs.

Photoacoustic Imaging as an Early Biomarker of Radio Therapeutic Efficacy in Head and Neck Cancer.

The negative impact of tumor hypoxia on radiotherapeutic efficacy is well recognized. However, an easy to use, reliable imaging method for assessment of tumor oxygenation in routine clinical practice remains elusive. Photoacoustic imaging (PAI) is a relatively new imaging technique that utilizes a combination of light and ultrasound (US) to enable functional imaging of tumor hemodynamic characteristics in vivo. Several clinical trials are currently evaluating the utility of PAI in cancer detection for breast, thyroid, and prostate cancer. Here, we evaluated the potential of PAI for rapid, label-free, non-invasive quantification of tumor oxygenation as a biomarker of radiation response in head and neck cancer. Methods: Studies were performed human papilloma virus- positive (HPV+) and -negative (HPV-) patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC). PAI was utilized for longitudinal assessment of tumor hemodynamics (oxygenation saturation and hemoglobin concentration) before, during and after fractionated radiation therapy (fRT). Imaging datasets were correlated with histologic measures of vascularity (CD31), DNA damage (phosphorylated γH2AX) and statistical modeling of tumor growth. Results: A differential response to fRT was observed between HPV+ and HPV- xenografts. Temporal changes in tumor hemodynamics (oxygen saturation and hemoglobin concentration) measured by PAI showed significant association with treatment outcomes. PAI-based changes in oxygen saturation were detected within days after initiation of fRT prior to detectable change in tumor volume, highlighting the potential of PAI to serve as an early biomarker of therapeutic efficacy. Consistent with PAI results, immunohistochemical staining of vascularity (CD31) and DNA damage (phosphorylated γH2AX) revealed distinct patterns of response in HPV+ and HPV- xenografts. Conclusion: Collectively, our observations demonstrate the utility of PAI for temporal mapping of tumor hemodynamics and the value of PAI read-outs as surrogate measures of radiation response in HNSCC.

Visualizing the effects of metformin on tumor growth, vascularity, and metabolism in head and neck cancer.

BACKGROUND: The antidiabetic drug metformin (Met) is believed to inhibit tumor proliferation by altering the metabolism of cancer cells. In this study, we examined the effects of Met on tumor oxygenation, metabolism, and growth in head and neck squamous cell carcinoma (HNSCC) using non-invasive multimodal imaging. MATERIALS AND METHODS: Severe combined immunodeficient (SCID) mice bearing orthotopic FaDu HNSCC xenografts were treated with Met (200 mg/kg, ip) once daily for 5 days. Tumor oxygen saturation (%sO2 ) and hemoglobin concentration (HbT) were measured using photoacoustic imaging (PAI). Fluorescence imaging was employed to measure intratumoral uptake of 2-deoxyglucosone (2-DG) following Met treatment while magnetic resonance imaging (MRI) was utilized to measure tumor volume. Correlative immunostaining of tumor sections for markers of proliferation (Ki67) and vascularity (CD31) was also performed. RESULTS: At 5 days post-Met treatment, PAI revealed a significant increase (P < .05) in %sO2 and HbT levels in treated tumors compared to untreated controls. Fluorescence imaging at this time point revealed a 46% decrease in mean 2-DG uptake compared to controls. No changes in hemodynamic parameters were observed in mouse salivary gland tissue. A significant decrease in Ki-67 staining (P < .001) and MR-based tumor volume was also observed in Met-treated tumors compared to controls with no change in CD31 + vessel count following Met therapy. CONCLUSION: Our results provide, for the first time, direct in vivo evidence of Met-induced changes in tumor microenvironmental parameters in HNSCC xenografts. Our findings highlight the utility of multimodal functional imaging for non-invasive mapping of the effects of Met in HNSCC.

Spatiotemporal Optoacoustic Mapping of Tumor Hemodynamics in a Clinically Relevant Orthotopic Rabbit Model of Head and Neck Cancer.

The purpose of this study was to investigate the usefulness of photoacoustic imaging (PAI) for spatiotemporal mapping of tumor hemodynamics in a rabbit model of head and neck carcinoma. Shope cottontail rabbit papilloma virus associated VX2 carcinomas were established in adult male New Zealand White rabbits (n = 9) by surgical transplantation of tumor tissue in the neck. Noninvasive PAI with co-registered ultrasound (US) was performed to longitudinally monitor tumor growth, oxygen saturation (%sO2), and hemoglobin concentration (HbT). PAI findings were validated with Doppler sonography measures of percent vascularity (PV). Differences in tumor volumes, %sO2, HbT, and PV values over time were analyzed using repeated-measures analysis of variance with multiple comparisons. Two-tailed Spearman correlation analysis was performed to determine the correlation coefficient (r) for comparisons between %sO2, HbT, and tumor volume. US revealed a significant (P < .0001) increase in tumor volume over the 3-week period from 549 ± 260 mm3 on day 7 to 5055 ± 438 mm3 at 21 days postimplantation. Consistent with this aggressive tumor growth, PAI revealed a significant (P < .05) and progressive reduction in %sO2 from day 7 (37.6 ± 7.4%) to day 21 (9.5 ± 2.1%). Corresponding Doppler images also showed a decrease in PV over time. PAI revealed considerable intratumoral spatial heterogeneity with the tumor rim showing two- to three-fold higher %sO2 values compared to the core. Noninvasive PAI based on endogenous contrast provides a label-free method for longitudinal monitoring of temporal changes and spatial heterogeneity in thick head and neck tumors.

Photoacoustic monitoring of tumor and normal tissue response to radiation.

Hypoxia is a recognized characteristic of tumors that influences efficacy of radiotherapy (RT). Photoacoustic imaging (PAI) is a relatively new imaging technique that exploits the optical characteristics of hemoglobin to provide information on tissue oxygenation. In the present study, PAI based measures of tumor oxygen saturation (%sO2) were compared to oxygen-enhanced magnetic resonance imaging (MRI) measurements of longitudinal relaxation rate (R1 = 1/T1) and ex-vivo histology in patient derived xenograft (PDX) models of head and neck cancer. PAI was utilized to assess early changes (24 h) in %sO2 following RT and chemoRT (CRT) and to assess changes in salivary gland hemodynamics following radiation. A significant increase in tumor %sO2 and R1 was observed following oxygen inhalation. Good spatial correlation was observed between PAI, MRI and histology. An early increase in %sO2 after RT and CRT detected by PAI was associated with significant tumor growth inhibition. Twenty four hours after RT, PAI also detected loss of hemodynamic response to gustatory stimulation in murine salivary gland tissue suggestive of radiation-induced vascular damage. Our observations illustrate the utility of PAI in detecting tumor and normal tissue hemodynamic response to radiation in head and neck cancers.

Photoacoustic imaging of salivary glands.

In this work, we utilized photoacoustic imaging (PAI) with co-registered ultrasound (US) to non-invasively assess salivary gland function in vivo. A significant increase in salivary gland oxygen saturation was observed on PAI within minutes after gustatory stimulation of healthy mice reflective of the hyperemic response associated with secretion of saliva. Good correlation was seen between PAI and Doppler sonography. Salivary adenoid cystic carcinomas showed higher oxygen saturation compared to surrounding salivary gland tissue. Our results demonstrate the potential clinical utility of PAI for visualization of salivary gland physiology and pathology.