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Optical resolution photoacoustic microscopy for the study of craniosynostosis in mouse models (#423)
G. Tserevelakis1, K. Makris2, S. Amarioti1, G. Mavrothalassitis2, G. Zacharakis1
1 FORTH-IESL, Heraklion, Vassilika Vouton, Greece
Craniosynostosis is a serious pathological condition occurring in 1 every 2000 births, characterized by a premature fusion of infants’ cranial sutures, changing, in this manner, the growth pattern of the skull. In this direction, studies aiming to a comprehensive genetic etiology understanding and novel therapeutic interventions design  require the development of high resolution imaging tools being able to provide valuable diagnostic information. This paper demonstrates the detailed investigation of craniosynostosis in mouse skulls, by means of optical resolution photoacoustic microscopy.
The system  is built around a purposefully modified inverted optical microscope serving as a platform for the photoacoustic microscopy setup. A variable repetition rate diode pumped ns laser is employed for efficient signal excitation (energy per pulse: 29.4 μJ, pulse width: ~10 ns, selected repetition rate: 5 kHz) emitting infrared radiation at 1064 nm and 532nm after frequency doubling. The dyed skulls (Alizarin Red S, for bone staining and Alcian Blue for cartilage staining) are raster scanned over the focused beam via XY motorized stages and the generated acoustic waves are detected in transmission mode by a 20 MHz spherically focused ultrasonic transducer.
The generated three-dimensional reconstructions permitted the determination of skulls’ geometrical features such as the local radii of curvature, the μm precision measurement of several anatomical distances, as well as, the depth variability of cranial sutures in different specimens. Statistically significant differences between groups of healthy, treated and untreated animals were found as regards to the investigated parameters.
Optical resolution photoacoustic microscopy was proven to be a powerful diagnostic tool for the study of craniosynostosis in mouse models. Features such as the increased spatial resolution and the superior imaging contrast, render the proposed approach highly competitive when compared to state of the art techniques such as X-ray microtomography.
This work was supported by the Grants “Skin-DOCTor” implemented under the "ARISTEIA" Action of the "OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING", co-funded by the European Social Fund (ESF) and National Resources and from the EU Marie Curie Initial Training Network “OILTEBIA”.
Keywords: photoacoustic, microscopy, diagnosis, craniosynostosis, imaging
Whole Body In vivo Photoacoustic Imaging for the monitoring of ICG Biodistribution and Pharmacokinetics (#551)
J. Lavaud1, 2, 3, M. Yin4, A. Forbrich4, P. Trochet4, A. Needles4, J. Jose4, J. - L. Coll1, 2, 3, A. Heinmiller4, V. Josserand1, 2, 3
1 Optimal small animal imaging platform, Grenoble, France
Non-invasive whole-body photoacoustic imaging (PAI) combined with anatomical pure Ultrasounds (US) imaging offers great opportunities in translational oncology research for the non-invasive exploration of physiopathological information gived by endogenous contrast (oxygenated and deoxygenated hemoglobin, melanin, fat...) as well as exogenous compounds biodistribution and pharmacokinetics.
Here we present a new preclinical whole-body PAI setup that enable to non-invasively monitor a contrast agent biodistribution (Indocyanine Green; ICG) within complete anatomical and functional profile.
Mouse whole-body PAI was performed using a VevoLAZR-X system (FUJIFILM VisualSonics Inc.), equipped with a 15 MHz linear array transducer paired with 34 mm optical fibers displayed on epi- and trans-illumination sides of a motorized glass stage.
Performance of the trans and epi- illumination setup was evaluated on a tissue-mimicking phantom where five tubing filed-up with Methylene-blue were distributed at various depths. The signal from each tube was measured and compared to the conventional illumination setup measurements.
Simultaneous 3D whole-body US and multi-wavelengths PAI of a living mouse were performed before, 30 min, 1h, 24h and 48h after bolus i.v. Injection of ICG .
From pure US acquisition the Volume Of Interest for each organ was drawn and used for PAI analyses.
The addition of trans illumination to existing epi-illumination improved the total illumination scheme and thus tended to normalize PA signals in depth.
In vivo on mice, the extended 3D motor allowed for full coverage of an adult mouse’s body and 3D rendered US images provided clear delineation of main organs.
From multi-wavelengths PAI, oxy and deoxy hemoglobin contents were separated from the ICG signal thus simultaneously providing functional (hemoglobin content and tissue oxygenation mapping) and molecular information (ICG biodistribution and pharmacokinetics in the main organs). ICG was shown to be first quickly uptaken by the liver and then eliminated by stomach and guts as broadly previously described.
We illustrated that anatomical, functional and molecular whole-body PAI can be achieved with the VevoLAZR-X imaging system through novel illumination and an extended cross sectional 3D imaging setup. The use of epi- and trans-illumination provided more uniform light distribution, thereby increasing signal at depth throughout the whole animal. By using multispectral PAI protocol, functional and molecular information could be obtained in a complete anatomical profile. This provides a setup optimized for further investigations into whole-body drug delivery and pharmacokinetics.
Keywords: Ultrasound, Photoacoustic, Whole-body, Biodistribution
Analyzing the necessary detection bandwidth for optoacoustic esophagus imaging (#297)
H. He1, 2, A. Buehler1, 2, D. Bozhko1, 2, X. Jian3, Y. Cui3, V. Ntziachristos1, 2
1 HelmholtzZentrum München, Institute for Biological and Medical Imaging, Neuherberg, Germany
Optoacoustic (photoacoustic) endoscopy has shown potential to reveal complementary contrast to optical endoscopy methods, indicating clinical relevance. However operational parameters for accurate optoacoustic endoscopy must be specified for optimal performance. In this study, we interrogated the frequency bandwidth that is best suited for human esophageal imaging. This investigation relates to the selection of a transducer for developing an optoacoustic endoscope for human studies.
We simulated the frequency response of esophagus wall and then validated the simulation results with experimental measurements of pig esophagus. Phantoms and fresh pig esophagus samples were measured using two detectors with central frequencies of 15 or 50 MHz, and the imaging performance of both detectors was compared. We analyzed the frequency bandwidth of optoacoustic signals in relation to morphological layer structures of the esophagus and found the 50MHz detector to differentiate layer structures better than the 15-MHz detector. Furthermore, we identify the necessary detection bandwidth for visualizing esophagus morphology and selecting ultrasound transducers for future optoacoustic endoscopy of the esophagus.
This study examined what optoacoustic detection bandwidth is necessary to reconstruct the layered structure of the esophageal wall. Simulations suggested that a wide frequency band is necessary, and this was confirmed in imaging experiments with ex vivo pig esophagus. The 50-MHz detector was better than the 15-MHz detector at resolving the LP and SM layers, although the 15-MHz detector was able to image deeper. Optoacoustic signals ranging from 5 to 20 MHz corresponded mainly to gross structure in the MP layer, signals from 20-50 MHz corresponded to finer structure, especially in the LP and SM layers, and signals from 50-80 MHz corresponded to even finer features of the LP and SM layers. Small vessel structures could not be adequately imaged even when detecting the full bandwidth of the 50-MHz detector (5-80 MHz). These results suggest that ultrawide bandwidth detectors ranging from a few MHz to 100 MHz can provide reasonable resolution of esophageal layers.
The present study makes clear that ultrawide-bandwidth transducers are necessary for optoacoustic endoscopy capable of resolving the layers of the esophageal wall. It further suggests the need to strike a reasonable balance between resolution and imaging depth in order to permit accurate early staging of esophageal cancer. The present work may help guide the design of optoacoustic endoscopes that are more accurate than ultrasound and provide more clinically useful information than confocal or OCT.
1. M. Vieth, C. Ell, L. Gossner, A. May, and M. Stolte, "Histological Analysis of Endoscopic Resection Specimens From 326 Patients with Barrett’s Esophagus and Early Neoplasia," Endoscopy, vol. 36, pp. 776-781, 24.08.2004 2004.
2. B. J. Reid, P. L. Blount, and P. S. Rabinovitch, "Biomarkers in Barrett's esophagus," Gastrointest Endosc Clin N Am, vol. 13, pp. 369-397, 4 2003.
3. J. Mannath and K. Ragunath, "Role of endoscopy in early oesophageal cancer," Nat Rev Gastroenterol Hepatol, Nov 03 2016.
4. J. M. Yang, C. Favazza, R. Chen, J. Yao, X. Cai, K. Maslov, et al., "Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo," Nat Med, vol. 18, pp. 1297-1302, Aug 2012.
5. H. He, A. Buehler, and V. Ntziachristos, "Optoacoustic endoscopy with curved scanning," Opt Lett, vol. 40, pp. 4667-70, Oct 15 2015.
6. H. He, G. Wissmeyer, S. V. Ovsepian, A. Buehler, and V. Ntziachristos, "Hybrid optical and acoustic resolution optoacoustic endoscopy," Opt Lett, vol. 41, pp. 2708-10, Jun 15 2016.
7. V. Ntziachristos, "Going deeper than microscopy: the optical imaging frontier in biology," Nat Methods, vol. 7, pp. 603-14, Aug 2010.
8. A. Taruttis and V. Ntziachristos, "Advances in real-time multispectral optoacoustic imaging and its applications," Nature Photonics, vol. 9, pp. 219-227, 2015.
9. D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, et al., "Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo," Nature Photonics, vol. 3, pp. 412-417, 2009.
10. A. Taruttis, G. M. van Dam, and V. Ntziachristos, "Mesoscopic and Macroscopic Optoacoustic Imaging of Cancer," Cancer Res, vol. 75, pp. 1548-1559, Apr 15 2015.
This work was supported by the European Union project FAMOS (FP7 ICT, Contract 317744). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 732720 (ESOTRAC). H. He acknowledges support from China Scholarship Council (CSC) Scholarship. We thank Dr. Chapin Rodriguez for his attentive reading and improvements of the manuscript. We thank Dr. habil. Krzysztof Flisikowski for providing the pig esophagus samples.
Schematic of the optoacoustic endoscopy system and simulated sensitivity fields of both transducers
(a) Schematic illustration of the endoscopy set-up. (b) Photograph of the distal end of the two endoscopy probes with central frequencies of 15 or 50 MHz. (c,d) Simulated sensitivity fields of the (c) 15 MHz detector and (d) 50 MHz detector, which indicate the near field length. HRJ: Hybrid Rotary Joint.
Optoacoustic imaging of ex vivo pig esophagus using 15 and 50 MHz transducers
(a) Photograph of esophageal tissue;(b) Unstained histological image;(c,d) Optoacoustic images of the same longitudinal slice similar to that in panel (b) generated using data from the (c) 15-MHz detector or (d) 50-MHz detector. (e,f) Optoacoustic images of the same tissue cross-section generated using data from the (e) 15 MHz detector or (f) 50 MHz detector.
Keywords: optoacoustic imaging, esophagus imaging, ultrawide bandwidth
Photoacoustic endoscopy with speckle illumination (#97)
A. M. Caravaca Aguirre1, E. Bossy1
1 Universite Grenoble Alpes (UGA), Laboratoire Interdisciplinaire de la Physique (LIPhy), Grenoble, France
Optical scattering limits the penetration depth and resolution of current optical-resolution photoacoustic techniques. To overcome this problem at expenses of increasing the invasiveness, endoscopic approaches are an appealing solution. Conventional approaches generally involve mechanically raster scanning a focused spot over the sample and acquiring an acoustic signal for each spot. Here, we demonstrate that a full-field illumination approach with multiple known speckle patterns generated by a multimode fiber (MMF) can also provide diffraction-limited optical-resolution photoacoustic images.
A digital micromirror device (DMD) modulates the light coupled into a MMF provides a set of speckle patterns at the distal tip of the fiber where an absorbing sample is placed. A fiber-based optical hydrophone attached next to the MMF records the photoacoustic waves produced by the sample. The experiment consists of two steps; first, we project N patterns onto the DMD and record each speckle pattern at the output of the fiber with a CMOS camera (calibration). Afterwards, the sample is illuminated by the MMF and a photoacoustic signal is recorded for each calibrated speckle (measurement). The intensity fluctuations from shot to shot codes for the position at which it is measured. Computational methods based on correlation, pseudo-inverse and compress sensing are investigated.
As a proof of principle, we experimentally image micro-structured test samples using a 100 μm diameter MMF. Fig. 1a shows the image taken with a CMOS camera of the distal tip of the MMF with a Selba sample in place composed of four circles with 10μm diameter. Fig. 1b-d compares the performance of the reconstruction of the three different reconstruction methods. The first method is based on cross-correlation between the photoacoustic signal under multiple speckle illumination with the calibrated known speckle patterns, following approaches from ghost imaging. This method fails to reconstruct the image due to the noise and lack of orthogonality of the speckle basis. The second method is based on computing the pseudo-inverse of the reference matrix obtained from the calibration step leading to a faithful image of the sample resolving the four circles. A third method based on compressed sensing exploits the sparsity of the sample achieving reconstructed images with a better SNR.
We experimentally demonstrate the feasibility of an optical resolution photoacoustic endoscope based on a multimode and a fiber hydrophone. The DMD allows for fast calibration approaches to reach calibration and measurement times of a few seconds, as compared with current approaches limited to hours. Additionally, speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, that can demand less computation time in case of compressed sensing approaches.
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (project COHERENCE, grant agreement 681514). The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n. PCOFUND-GA-2013-609102, through the PRESTIGE program coordinated by Campus France.
Experimental photoacoustic image reconstruction of four circles imaged with the endoscope
Figure 1: Experimental photoacoustic image reconstruction of four circles imaged with the endoscope. (a) CMOS camera image of the distal tip of the fiber with the sample in place. Photoacoustic image reconstructed using (b) Ghost-imaging approach, (c) Pseudo-inverse approach and (d) compressed sensing. Scale bar is 20 um.
Keywords: Endoscopy, computational optics, Photoacoustics, Multimode fibers
Added Value of Quantitative Multiparametric Breast Ultrasound (#479)
P. Kapetas1, R. Woitek1, 2, P. Clauser1, K. Pinker1, 3, M. Bernathova1, T. H. Helbich1, P. A. Baltzer1
1 Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna, Wien, Austria
Breast ultrasound (US) shows high accuracy and sensitivity but varying specificity and a high interobserver variability (1). Recently, functional US modalities (e.g. Doppler, Contrast-Enhanced US, elastography) have been developed, offering quantitative indices that may reduce interobserver variability (2-4). The added value of combining several, quantitative US modalities has not yet been explored in detail (5). Therefore, this study aims to evaluate quantitative multiparametric breast US for the differentiation of benign and malignant lesions and to identify potential US imaging biomarkers.
124 patients, each with one biopsy-proven, sonographically evident breast lesion were included in this prospective, IRB-approved study. Each lesion was examined with B-mode US, elastography (Acoustic Radiation Force Impulse -ARFI), Doppler US and Contrast Enhanced US (CEUS). Quantitative indices were recorded for each modality: lesion maximum, intermediate and minimum Shear Wave Velocity (SWVmax, SWVmid and SWVmin) for ARFI, Pulsatility (PI) and Resistive Index (RI) for Doppler US and 11 different indices for CEUS. Diagnostic accuracy of measurements was compared using Receiver Operating Characteristics (ROC) analysis. Multivariate logistic regression was used to determine independent predictors of malignancy.
65 lesions were malignant and 59 benign. SWVmax and RI showed the highest diagnostic performance as measured by the area under the ROC curve (0.871 and 0.805 respectively). At a cut-off value of 3.1 m/s, SWVmax showed sensitivity of 85.94% and specificity of 85.19%. An RI cut-off of 0.68 revealed sensitivity of 77.78% and specificity of 80.56%. Multivariate logistic regression showed that SWVmax, RI and mean Transit Time (local) (mTTl) were independent predictors of malignancy.
Quantitative indices acquired by elastography (SWVmax) and Doppler US (RI) can accurately differentiate benign from malignant breast lesions and may potentially be used as sonographic imaging biomarkers.
1. Scaperrotta G. et al., Eur Radiol 2008 Nov;18(11):2381-9
2. Kapetas P. et al., Acta Radiol 2017 Feb;58(2):140-147
3. Özdemir A. et al., J Ultrasound Med 2001 Oct;20(10):1091-101
4. Saracco A. et al., Acta Radiol 2012 May 1;53(4):382-8
5. Cho N. et al., Radiology 2012 Jan;262(1):80-90
Keywords: Breast cancer, Ultrasound, Doppler ultrasonography, Elastography, Contrast agents
Photoacoustic bone imaging – proof of principle for non-invasive quantitative and localized in vivo imaging of intraosseous signals (#124)
J. Humbert1, O. M. Will2, T. Peñate Medina2, O. Peñate Medina2, C. - C. Glüer2, O. Jansen1, M. Both1
1 University Hospital Schleswig-Holstein, Campus Kiel, Department of Radiology and Neuroradiology, Kiel, Schleswig-Holstein, Germany
Topographic bone ultrasound (US) is limited to the bone surface, where US waves are mostly reflected causing posterior shadowing. The aim of this study was to investigate whether photoacoustic imaging (PAI) can overcome the constraints of conventional US and non-invasively image intraosseous proceedings in a mouse model. PAI has only been tested on excised human bone samples [1, 2], and in superficial rat osteosarcoma lesions . For proof of principle, indocyanine green (ICG) containing liposomes were injected into the tibiae of 8 BALB/c mice and visualized and quantified by in vivo PAI.
Eight 2-6 week old BALB/c mice were anesthetized with 75/0.5 mg/kg body weight ketamin/medetomidin. Transverse three-dimensional photoacoustic tomography of the first 10 mm of the proximal tibia was performed on the Vevo LAZR (FUJIFILM VisualSonics Inc., Toronto, CA) before and after intratibial injection of 10 µl of liposomes containing 0.33 µg/µl ICG at multiple wavelengths (680 nm, 700-950 nm in 25 nm steps, 970 nm). Spectral unmixing for ICG liposomes was performed and a VOI fitted around the bone. The percentage of ICG signal in the tibia before and after the injection was compared by Mann-Whitney test. Additionally, a spectral scan (wavelengths 680-970 nm) showing the transverse plane of the proximal tibial corpus was executed for verification of the signal origin.
Before the intratibial injection was performed, the unmixed photoacoustic signal was very low in the tibia (Fig. 1A). Background signals were probably caused by the overlap in the photoacoustic spectra of ICG (about 800 nm) with oxygenated and deoxygenated blood (peak at 850 and 750 nm). The photoacoustic signal detected after the intratibial injection of ICG liposomes was located in the tibial head, collum and inside the medullary cavity in the proximal tibial body (Fig. 1B-D). The depth of the signal differed between mice depending on the penetration depth of the contrast agent into the tibial shaft. After intratibial injection, there was a significant increase in percentage of PA signal in the VOI fitted around the tibia (average PA signal 3.06±3.88% before injection; 61.36±22.94% after injection; p=0.0003; Fig. 1A). The spectral scan after the injection showed that the photoacoustic signal inside the tibia exhibited the expected peak at the absorption maximum of liposomal ICG.
This study provides the proof of principle that photoacoustic imaging is feasible for the non-invasive in vivo imaging of contrast agents inside mouse bone, supplying both quantitative and localisation information. This system is especially interesting for the use in pre-clinical studies on bone metastasis, where for example breast cancer or leukemia cells pre-labeled or transfected with a photoacoustic contrast agents could be longitudinally visualized. Further experiments are needed to evaluate the sensitivity, as well as the feasibility in other bones or animals.
1) Lashkari B, Yang L, Mandelis A. The application of backscattered ultrasound and photoacoustic signals for assessment of bone collagen and mineral contents. Quant Imaging Med Surg 2015;5(1):46–56.
2) Steinberg I, Turko N, Levi O, et al. Quantitative study of optical and mechanical bone status using multispectral photoacoustics. J Biophotonics 2016;9(9):924–33.
3) Hu J, Yu M, Ye F, et al. In vivo photoacoustic imaging of osteosarcoma in a rat model. J Biomed Opt 2011;16(2):20503.
PA signal in vivo (A-C) and ex vivo (D) in mouse bone after intratibial injection of ICG liposomes.
A: Average increase in percentage of PA signal in the proximal 10 mm of tibiae after intratibial injectionof ICG liposomes (p=0.0003, n=7/8 mice respectively); B-D: Exemplary PA images unmixed for ICG liposomes (green) after intratibial injection of ICG liposomes: B: transverse unmixed in vivo image of proximal tibia; C: in vivo 3D images of tibiae of different mice; D: excised mouse tibia.
Keywords: photoacoustic imaging, bone imaging, liposomes, contrast agent
Motion-corrected cardiac metabolic imaging using a simultaneous Positron Emission Tomography – Ultrafast Ultrasound Imaging system (#430)
B. Berthon1, J. Porée1, J. Sourdon2, 3, T. Viel2, 3, M. Pérez-Liva2, 3, A. Garofalakis2, 3, M. Tanter1, B. Tavitian2, 3, J. Provost1
1 Institut Langevin, ESPCI Paris, PSL Research University, CNRS 7587, INSERM U979, Paris, France
Although a promising approach for the characterization of cardiac viability, cardiac metabolic imaging with 18FDG has seldom been used in part due to the challenges of accounting for heart motion during the PET reconstruction process, which is a difficult task that requires long ECG-gated acquisition times, complex 4D reconstruction algorithms, or the use of a Positron Emission Tomography (PET) -Magnetic Resonance Imaging device. Herein, we demonstrate that a PET – Ultrafast Ultrasound Imaging (UUI) system drastically enhances metabolic cardiac imaging using UUI-based motion correction.
30-min, 8-frame ECG-gated, simultaneous PET-UUI was performed in rats (n = 5) using a small-animal PET-CT (Nano-PET-CT, Mediso, Hungary) and a 12-MHz ultrasound probe connected to a commercially-available UUI system (Aixplorer, Supersonic Imagine, France). The ultrasound probe was positioned using a 6-degree-of-freedom micropositioner (H811, Physik Instrumente, Germany) of which the positioning coordinates were co-registered to the PET-CT device coordinates during a single prior calibration procedure. UUI was performed using 20 compounded tilted plane waves per frame at 750 frames/s. 2-D motion fields were mapped using a regularized vector Doppler approach and used to recast the images in the material coordinates, onto which the PET data were projected to correct for motion.
Figure 1 shows the large improvement in terms of image quality due to the motion-correction process by comparing (a) a motion-corrected image with (b) a single-frame image. The resolution was similar in the single-frame image and in the motion-corrected one, while the contrast increased from 10.2 to 15.1 dB. In comparison, images obtained by directly compounding all 8 frames without motion compensation showed a contrast of 11.2 dB and a strongly degraded resolution. These results indicate that the motion correction process successfully summed the contributions from specific material points, which enables an improvement in contrast without degrading resolution.
We have shown that simultaneous PET-UUI acquisition using an ultrasound imaging probe inside the PET gantry drastically enhances cardiac metabolic imaging without any change to the PET acquisition process. Such an improvement could be leveraged to better characterize cardiac viability by imaging and also to reduce image acquisition times.
This project was funded in part by Plan Cancer (ASC16026HSA-C16026HS) and by LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24) within the French program “Investments for the Future” under reference ANR-10-IDEX-0001-02 PSL. The project also received the support of the Inserm Technology Research Accelerator in Biomedical Ultrasound. In vivo imaging was performed at the Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant - PIV), supported by France Life Imaging (grant ANR-11-INBS-0006) and Infrastructures Biologie-Santé (IBISA).
Keywords: Ultrafast Ultrasound Imaging, Positron Emission Tomography, cardiac metabolic imaging, motion correction
Optoacoustic imaging of non-melanocytic skin cancer: relationship to vasculature, inflammation and prognosis (#306)
I. Quiros Gonzales1, L. Ansel-Bollepalli1, S. J. Aitken2, S. E. Bohndiek1
1 University of Cambridge, CRUK Cambridge Institute & Cavendish Laboratory, Cambridge, United Kingdom
Optoacoustic tomography (OT) has been shown to be useful in melanoma detection, however, little is known about its application in non-melanocytic cancer. Squamous cell carcinoma (SCC) leads to changes in the skin including soreness and bleeding that could be assessed by OT. In this study, we characterized the OT profile of an inflammation-based SCC murine model (Topical 2-step carcinogenesis model, single application of 7,12-dimethylbenz[a]-anthracene + weekly application of 12-O-tetra decanoylphorbol-13-acetate) and compared to inflammatory, prognosis and vascular markers.
Swiss mice (n=4; total tumour number n=14) were exposed to the carcinogenesis model. OT (700-950nm) was performed over the whole skin surface exposed to the carcinogen and healthy skin. Oxy- and deoxy-haemoglobin (Hb) spectral unmixing was performed and total Hb (THb) and oxygen saturation (SO2) were calculated. Regions of interest (ROIs) were drawn as (Fig. 1A): Sk: Immediate skin border; DSk: Area 1 mm deep below Sk; TUM: Tumour tissue protruding from the skin surface; and SkUT: Skin under the tumour, up to 1 mm deep (F1A). After OT imaging, serum, tumours and healthy skin samples were collected. Serum concentrations of VEGF, NO and abundance of white cells were measured. IHC was performed for vascular (CD31, aSMA) and hypoxia (CA-IX) and proliferation (Ki67) markers.
Tumour tissue presents higher THb and SO2 than skin (2414±162 vs. 1207±301 for THb; p=0.02 and 0.40±0.02 vs. 0.22±0.03 for SO2; p=0.002) (F1B), these changes can be detected in tumour tissue but more extensively in SkUT (SO2 0.26±0.03 vs. 0.50±0.04, p=0.0002). These changes cannot be explained by alterations in blood vessel density (MVD) or maturity (CD31/ASMA) (F1C). Tumour tissue presents high CA-IX stained areas compared to healthy skin (p=0.03) (F1D). This CA-IX (SkUT) staining inversely correlates with THb and SO2 (pTHb=0.001 and pSO2=0.01) (F1E), pointing the potential application of OT as an indirect method to measure hypoxia and blood flow in SCC. No association was found with inflammation features, although the correlation with NO may suggest endothelial regulation (F1F). To monitor progression, we studied the levels of Ki-67 (F1G); they correlate significantly with SO2 in the superficial and deeper areas of the skin (Fig. 1H, p=0.001 and p=0.003).
OT is able to detect changes in THb in the tumour area and surrounding skin between healthy and malignant, correlating with markers of hypoxia and aggressiveness. Further work is needed to establish the clinical implications of these findings.
Figure 1. SCC tumours in mouse. A) Macroscopic, optoacoustic and H&E (inset). B) MSOT parameters, total haemoglobin (THb) and oxygen saturation (SO2). C) Vascular density (MVD) and maturity (CD31/ASMA colocalization). D) CA-IX staining, epithelia (yellow) and stroma (red). E) MSOT correlation with CA-IX in SkUT. F) NO correlation with tumour THb. G) and H) Ki-67 levels and correlation with MSOT.
Keywords: Skin cancer, Optoacoustic imaging, hypoxia, progression