Michal Tomaszewski

Towards Quantitative Evaluation of Tissue Absorption Coefficients Using Light Fluence Correction in Optoacoustic Tomography

Optoacoustic tomography is a fast developing imaging modality, combining the high contrast available from optical excitation of tissue with the high resolution and penetration depth of ultrasound detection. Light is subject to both absorption and scattering when traveling through tissue; adequate knowledge of tissue optical properties and hence the spatial fluence distribution is required to create an optoacoustic image that is directly proportional to chromophore concentrations at all depths. Using data from a commercial multispectral optoacoustic tomography (MSOT) system, we implemented an iterative optimization for fluence correction based on a finite-element implementation of the delta-Eddington approximation to the Radiative Transfer Equation (RTE). We demonstrate a linear relationship between the image intensity and absorption coefficients across multiple wavelengths and depths in phantoms. We also demonstrate improved feature visibility and spectral recovery at depth in phantoms and with in vivo measurements, suggesting our approach could in the future enable quantitative extraction of tissue absorption coefficients in biological tissue.

Oxygen enhanced Optoacoustic Tomography (OE-OT) reveals vascular dynamics in murine models of prostate cancer

Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes, hence non-invasive imaging of oxygen supply and demand in tumours could improve disease staging and therapeutic monitoring. Optoacoustic tomography (OT) is an emerging clinical imaging modality that provides static images of endogenous haemoglobin concentration and oxygenation. Here, we demonstrate oxygen enhanced (OE)-OT, exploiting an oxygen gas challenge to visualise the spatiotemporal heterogeneity of tumour vascular function. We show that tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. LNCaP tumours showed a spatially heterogeneous response within and between tumours, with a substantial but slow response to the gas challenge, aligned with ex vivo analysis, which revealed a generally perfused and viable tumour with marked areas of haemorrhage. PC3 tumours had a lower fraction of responding pixels compared to LNCaP with a high disparity between rim and core response. While the PC3 core showed little or no dynamic response, the rim showed a rapid change, consistent with our ex vivo findings of hypoxic and necrotic core tissue surrounded by a rim of mature and perfused vasculature. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vessel function assessed ex vivo. OE-OT provides a non-invasive approach to reveal the complex dynamics of tumour vessel perfusion, permeability and vasoactivity in real time. Our findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease as well as in patient stratification for chemo- and radio-therapy.

Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects

Optoacoustic tomography (OT) is now widely used in preclinical imaging; however, the precision (repeatability and reproducibility) of OT has yet to be determined. Methods: We used a commercial small-animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, and frame averaging. Variables due to animal handling and physiology, such as anatomic placement and anesthesia conditions, were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and in vivo. Sensitivity to small-molecule dyes was determined in phantoms and in vivo; precision was assessed in vivo using IRDye800CW. Results: OT COV in a stable phantom was less than 2.8% across all wavelengths over 30 d. The factors with the greatest impact on signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4% at 700 nm. Anatomic region-of-interest size showed the highest variation, at 12% and 18% COV in the kidney and spleen, respectively; however, functional SO2 measurements based on a standard operating procedure showed an exceptional reproducibility of less than 4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for in vivo data included respiration rate, degree of user experience, and animal placement. Conclusion: Data acquired with our small-animal OT system were highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.

Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry

BackgroundOptoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures.MethodsMultispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO2MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation.ResultsMCF-7 tumours show SO2MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO2MSOT with a significant rim–core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry.ConclusionsOT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Optoacoustic detection of early therapy-induced tumor cell death using a targeted imaging agent

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU). Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU. Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem. Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893–903. ©2017 AACR.

Measurement of changes in blood oxygenation using Multispectral Optoacoustic Tomography (MSOT) allows assessment of tumor development

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with the spatial resolution and penetration depth similar to ultrasound. We aim to demonstrate that MSOT can be used to monitor the development of tumor vasculature. To establish the relationship between MSOT derived imaging biomarkers and biological changes during tumor development, we performed MSOT on nude mice (n=10) bearing subcutaneous xenograft U87 glioblastoma tumors using a small animal optoacoustic tomography system. The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified between 21% and 100%. The measurements from early (week 4) and late (week 7) stages of tumor development were compared. To further explore the functionality of the blood vessels, we examined the evolution of changes in the abundance of oxy- and deoxyhemoglobin in the tumors in response to a gas challenge. We found that the kinetics of the change in oxygen saturation (SO2) were significantly different between small tumors and the healthy blood vessels in nearby normal tissue (p=0.0054). Furthermore, we showed that there was a significant difference in the kinetics of the gas challenge between small and large tumors (p=0.0015). We also found that the tumor SO2 was significantly correlated (p=0.0057) with the tumor necrotic fraction as assessed by H&E staining in histology. In the future, this approach may be of use in the clinic as a method for tumor staging and assessment of treatment response.

Abstract 4198: Optoacoustic imaging of blood vasculature and study of angiogenesis in orthotopic breast cancer models

The outcomes of anti-angiogenic drugs in breast cancer have been disappointing. There is an urgent clinical need to better understand the existing and emerging anti-angiogenic therapies in order to: select appropriate patients therapy; define ‘windows’ for combination therapy; and reduce healthcare costs of ‘precision medicine’. MultiSpectral Optoacoustic Tomography (MSOT) is emerging as a new imaging modality, cheaper and less toxic than existing functional imaging methods. It is based on the absorption of laser energy in tissues, which produces pressure waves detectable by ultrasound. MSOT can detect binding of O2 to haemoglobin (Hb and HbO2) based on changes in the optical absorption spectrum, making it a very useful tool to image blood vasculature and measure tissue oxygenation. We have used MSOT to study blood vessel formation in a breast cancer xenograft model (MCF7, Estrogen Receptor+, n = 10). MSOT images were acquired at 3 and 6 weeks after innoculation and at 6 weeks, tumours were collected for histopathological study. The endothelial protein CD31 was use to identify blood vessel density. Serum levels of vascular endothelial growth factor (VEGF) were measured at 3 and 6 weeks. Presence/absence of VEGF receptor levels were assessed in MCF7 cell line by WB and IF. The MSOT parameters mean intensity (MI) and maximum intensity (MaI) for total Hb (THb) and O2 saturation (SO2, HbO2/(HbO2+Hb)) did not change significantly during tumour development (p-values: Hb = 0.952, 0.716; HbO2 = 0.102, 0.19; mean/max respectively, SO2 = 0.12), indicating that there is no substantial change in blood vessel density in this tumour model despite a size increase (mean, cm3 3w = 0.139 and 6w = 0.458, p-value = 0.04). The serum levels of the pro-angiogenic cytokine VEGF were significantly decreased (mean, pg/ml 3w = 116.97 and 6w = 86.24). The decrease in VEGF could explain the apparent lack of further blood vessel formation by 6 weeks. Unexpectedly, although the mean intensity for total Hb is lower in the tumour than the reference (cava artery-vein), there is no difference in SO2. Finally, comparing measurements from MSOT to histopathology, HbO2 MI correlates with CD31 staining intensity (CD31si) (Spearman correlation r = 0.57 p-value = 0.041) indicating that both measurements mark presence of blood vessel in the tumour. Microvessel density and CD31si do trend towards correlation with THb MI but this is not statistically significant at present. In conclusion, MSOT is a direct method to image blood vasculature in our tumour model non-invasively and indirectly to determine blood vessel density. The xenograft mouse model from MCF-7 cell line shows good vascularization, oxygenation and stability during tumour growth. In the next steps, we will investigate in this xenograft model the utility of MSOT biomarkers to monitor response to anti-angiogenic therapies, hence establishing the potential of the technique as a companion diagnostic. Citation Format: Isabel Quiros-Gonzalez, Michal Tomaszewski, James Joseph, Sarah E. Bohndiek. Optoacoustic imaging of blood vasculature and study of angiogenesis in orthotopic breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4198.

Quantitative imaging of tumor vasculature using multispectral optoacoustic tomography (MSOT)

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are often more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with spatial resolution and penetration depth similar to ultrasound. We hypothesized that MSOT could reveal both tumor vascular density and function based on modulation of blood oxygenation. We performed MSOT on nude mice (n=8) bearing subcutaneous xenograft PC3 tumors using an inVision 256 (iThera Medical). The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified from 21% to 100% and back. After imaging, Hoechst 33348 was injected to indicate vascular perfusion and permeability. Tumors were then extracted for histopathological analysis and fluorescence microscopy. The acquired data was analyzed to extract a bulk measurement of blood oxygenation (SO2MSOT) from the whole tumor using different approaches. The tumors were also automatically segmented into 5 regions to investigate the effect of depth on SO2MSOT. Baseline SO2MSOT values at 21% and 100% oxygen breathing showed no relationship with ex vivo measures of vascular density or function, while the change in SO2MSOT showed a strong negative correlation to Hoechst intensity (r=- 0.92, p=0.0016). Tumor voxels responding to oxygen challenge were spatially heterogeneous. We observed a significant drop in SO2 MSOT value with tumor depth following a switch of respiratory gas from air to oxygen (0.323±0.017 vs. 0.11±0.05, p=0.009 between 0 and 1.5mm depth), but no such effect for air breathing (0.265±0.013 vs. 0.19±0.04, p=0.14 between 0 and 1.5mm depth). Our results indicate that in subcutaneous prostate tumors, baseline SO2MSOT levels do not correlate to tumor vascular density or function while the magnitude of the response to oxygen challenge provides insight into these parameters. Future work will include validation using in vivo imaging and protocol optimization for clinical application.

Evaluation of MultiSpectral Optoacoustic Tomography (MSOT) performance in phantoms and in vivo

MultiSpectral optoacoustic tomography (MSOT) is an emerging modality that combines the high contrast of optical imaging with the spatial resolution and penetration depth of ultrasound, to provide detailed images of hemoglobin concentration and oxygenation. To facilitate accurate determination of changes in the vascularity and oxygenation of a biological tissue over time, a tumor in response to cancer therapy for example, an extensive study of stability and reproducibility of a small animal MSOT system has been performed. Investigations were first made with a stable phantom imaged repeatedly over time scales of hours, days and months to evaluate the reproducibility of the system over time. We found that the small animal MSOT system exhibited excellent reproducibility with a coefficient of variation (COV) in the measured MSOT signals of less than 8% over the course of 30 days and within 1.5% over a single day. Experiments performed in vivo demonstrated the potential for measurement of oxyhemoglobin over time in a realistic experimental setting. The effect of breathing medical air or oxygen under conditions of fixed respiration rate and body temperature within normal organs, including the spleen and kidneys, were investigated. The COV for oxyhemoglobin signals retrieved from spectral unmixing was assessed within both biological (different mouse) and imaging (different scan) replicates. As expected, biological replicates produced a large COV (up to 40% within the spleen) compared to imaging replicates within a single mouse (up to 10% within the spleen). Furthermore, no significant difference was found between data acquired by different operators. The data presented here suggest that MSOT is highly reproducible for both phantom and in vivo imaging, hence could reliably detect changes in oxygenation occurring in living subjects.