High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology

Abstract

Objectives

We be under the necessity developed a relevant preclinical model associated by a specific imaging protocol dedicated to onco-pharmacology studies in mice.

Materials and Methods

We optimized both the animal model and an ultrasound imaging action to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to means to an end by photoacoustic imaging the intratumoral hypoxia, that is a crucial parameter responsible with regard to resistance to therapies. Finally, we compared ultrasound given conditions to x-ray micro computed tomography and volumetric measurements to validate the relevance of this come near on the NCI-H460 human orthotopic lung tumefaction.

Results

This study demonstrates the aptitude of ultrasound imaging to detect and mentor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This come enabled us to characterize key biological parameters of the like kind as oxygenation, perfusion status and vascularization of tumors.

Conclusion

Such some experimental approach has never been reported before and it would provide a nonradiative tool conducive to assessment of anticancer therapeutic efficacy in mice. Considering the distraction of ultrasound propagation through the lung parenchyma, this strategetics requires the implantation of tumors strictly located in the exterior posterior part of the lung.

Citation: Raes F, Sobilo J, Le Mée M, Rétif S, Natkunarajah S, Lerondel S, et al. (2016) High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives as being Onco-Pharmacology. PLoS ONE 11(4): e0153532. doi:10.1371/diary.pone.0153532

Editor: Bernhard Ryffel, French National Centre instead of Scientific Research, FRANCE

Received: February 2, 2016; Accepted: March 30, 2016; Published: April 12, 2016

Copyright: © 2016 Raes et al. This is ~y open access article distributed under the provisions of the Creative Commons Attribution License, what one. permits unrestricted use, distribution, and propagation in any medium, provided the creative author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The authors esteem no support or funding to recite.

Competing interests: The authors have declared that in ~ degree competing interests exist.

1. Introduction

Because lung cancer smooth remains the leading cause of cancer-related death, there is a need to bring out more accurate and predictive preclinical protocols and proper cancer models. Orthotopic lung cancer models take the advantage of being more foretelling regarding clinical relevance, including the knack of primary tumors to develop impulsive metastasis but also more predictive regarding the therapeutic response. The implementation and examination of such orthotopic models allows us to improve our intellectual powers of the biology of cancer to expound preclinical in vivo results in humans, especially on account of the potential therapeutic response of anticancer agents. Studies vexation into account more representative parameters from clinical situations, particularly hypoxia, are of great interest to boost radically new measure for new anticancer treatments [1–3].

One grave parameter in oncology is tumor body assessment before but also during treatments [4]. In a clinical setting, the pulmonic tumor measurements are predominantly performed with X-ray computed tomography (CT) imaging [5]. For pulmonary preclinical oncology, imaging objectives are to improve the niceness for determining volumes, without irradiation effects or interferences with the anti-tumefaction response.

Thanks to technological developments because both X-ray sources and detectors, CT dedicated to molecular animal imaging provides a sub-millimetric solution making this tool efficient for the characterization of lung tumor volumes. However, the beamy brightness dose delivered to tumors remains a restriction, especially when a study requires repeated exams [6].

Bioluminescence imaging (BLI) brought with regard to a revolution in preclinical oncology research but this method provides quantitative knowledge about tumor proliferation without any potential sizing. Moreover, since BLI is at the disposal of upon metabolism, it is not trusty when tumors become hypoxic [7].

In clinical practices, lung ultrasound (US) has been gaining in popular regard among clinicians and has become one essential tool in critically ill negotiation [8,9]. However regarding human pulmonic oncology, there is no possible employment of US except for invasive endoscopy of cancer nodules and lymph nodes [10,11]. The principal point limitation of endoscopy and ultrasound is the finding out of these nodules if proximity with the probe is not close enough. This entrance limitation is due to the non-attendance of US propagation through the lung parenchyma for the cause that of air.

On the contrary, preclinical boisterous resolution US and photoacoustic imaging (PAI) are giving ground of hope modalities to investigate lung tumor passage and hypoxia respectively but considering the specified constraints of US, the implantation of tumors in the outward posterior lung region is required.

The vast cell NCI-H460 orthotopic lung carcinoma model that we chose to improve, is based forward a study by Gagnadoux et al. [12], chief to the growth of a unfrequented intrapulmonary nodule located near the later after diaphragmatic surface.

Here we recommend refining of the like kind an onco-pharmacology protocol in a translational come near while overcoming physical US limitations allowing lung swelling exploration. In this longitudinal study we assessed orthotopic lung swelling volumes in mice by in vivo 3D US and also hypoxic tumor status by PAI. Furthermore, we compared our facts to different imaging methods with the mark to validate this new approach.

2. Materials and Methods

2.1 Ethics Statement

All procedures up~ the body animals were performed in accordance through European ethical guidelines (European directives 2010/63/EU) and were approved ~ dint of. the Regional Committee for Animal Care and Ethics in Animal Experiments (C2EA-03 Comité d’éthique en expérimentation animale Campus CNRS d’Orléans).

2.2 Cell Culture

The NCI-H460-luc2 human lung cancer cell line was obtained from Perkin Elmer (France). This cancer elementary corpuscle line was maintained according to the supplier’s instructions.

2.3 Animals

Pathogen-liberated 6 to 8 week-old pistil-bearing nude Balb/c mice were purchased from Charles River Laboratories (France). Mice were acclimated with a view to 7 days in the laboratory ahead of experimentation and were maintained in sterilized filter-stopped cages inside a controlled ventilated rack (USA) with access to food and irrigate ad libitum. They were examined quotidian for clinical signs, distress, decreased pertaining to physics activity and weighed 3 times a week.

2.4 Subcutaneous and Intra-Bronchial Cell Xenograft

Human lung cancer xenografts from NCI-H460-luc2 cells were established in Balb/c unclothed mice. We first performed subcutaneous implantation in regular government to get reference data from measurement techniques on standard conditions, so that 10 mice were anaesthetized ~ means of inhalation of 1.5% isoflurane with air (Isoflo®, AXIENCE S.A.S, France) and inoculated ~ means of different tumor burdens (either 1×105 to 2.5×106 swelling cells in 100 μL PBS) in the dorsal flank. For orthotopic implantation, 24 mice were inoculated (1.25×105 or 2.5×105 tumefaction cells in 25 μL PBS) using a 1.9F×50cm harsh-end silicon catheter inserted into the bronchus via a laryngoscope (S1A Fig). This discriminating procedure to get superficial cell displacement into a posterior part of a look black lobe via a main bronchus requires interventional imaging. The affirmation of the radio-opaque catheter is checked ~ dint of. planar radiography (Faxitron MX20, Faxitron X-ray corp, USA) (S1B Fig) then categorical deposition of 99mTc-labelled tumor cells is controlled by Single Photon Emission Computed Tomography (Nano Spect CT, Mediso, Hungary), (S1C Fig).

2.5 Computed Tomography

Mice were anesthetized ~ the agency of 1.5% isoflurane and placed adhering a bed in prone position. For tumor measurements, computed tomography was performed using a microscopic animal imager (eXplore CT 120, Trifoil Imaging, USA) by an external respiratory gating device (Biovet, USA). Some mice admitted an intravenous injection of the vascular contrariety agent eXIA160™ (Binitio Biomedical, Canada). Tumor volumes were obtained through manually delineating margins of tumors from sagittal sections of CT images using Microview separation + 2.3 software.

2.6 Bioluminescence Imaging

BLI was performed once a week until the end of the study (Day 28) using each IVIS-Lumina II (Perkin Elmer, France) generating a pseudo-colored statue representing light intensity and superimposed more than a greyscale reference image. Each mouse was IP injected with 100mg/kg luciferin potassium wit (Promega, France). Mice anesthetized by 1.5% isoflurane were placed attached a thermostatically controlled heating pad (37°C) for the time of imaging. Acquisition binning and duration were arrange depending on tumor activity. Signal intenseness was quantified as the total looseness (photons/seconds) within ROIs drawn manually right and left the tumor area using Living Image 4.0 software (Perkin Elmer, France). The summary of signals from the prone and inattentive positions was considered for each search.

2.7 Ultrasound and Photoacoustic Imaging

Mice anesthetized by 1.5% isoflurane were placed adhering a thermostatically controlled heating pad in bending forward position with the paws taped from beginning to end the ECG electrodes attached to the catalogue. Respiratory gating was derived from ECG. A uncolored aqueous warmed ultrasonic gel (Supragel®, LCH, France) destitute of any air bubbles was applied betwixt the skin and the transducer. Tumors were imaged by the VisualSonics Vevo®LAZR System (FUJIFILM VisualSonics Inc, Canada). 3D scans of US likeness being recorded digitally. The tumor territory in coronal planes was measured ~ means of manually delineating margins using Vevo®LAB 1.7.2 software. The software therefore calculated the corresponding volume of every one coronal slice. For hypoxia assessments, tumors were investigated through PAI with OxyHemo-Mode so that medium values of SO2 were determined and answering. hypoxic volumes documented. Tumor perfusion standing and VEGFR2 expression were assessed by contrast enhanced ultrasound (CEUS) imaging following IV injection (tail vein) of Vevo MicroMarker™ and Target-Ready MicroMarker™ coupled by either anti-VEGFR2 or isotype regulate antibodies (eBioscience, USA). Imaging protocols were performed with the destruction-replenishment sequences. Data were processed with the VevoCQ™ software. A elucidation parameter that should be respected by reason of accurate 3D US acquisition is the positioning of the transducer (S2A Fig). The US piece of timber has to be directed towards the lung tumefaction with the best angle possible, in the same manner that the entire tumor can be detected during the acquisition. Despite the carriage of artifacts and shadows due to the ribs, at what time changing the angle of the transducer it is practicable to select the most efficient positioning during the transducer that allows detection. The minimal bigness of tumors that can be detected is 1mm in diameter, however the bigger the tumor greatness, the fewer artifacts from the ribs drive firmly together tumor detection. Transducers with central common occurrence at 21MHz and 40MHz, were used as antidote to B-Mode imaging of large and stolid tumors respectively. PAI was performed with the 21MHz transducer.

2.8 Sacrifice and Organ Removal

Mice in a state of being liable to anesthesia were sacrificed by cervical disjointing and tumors were collected from harvested land animal for immediate ex vivo assessments. In grade to assess the accuracy of in vivo US measurements, tumors were collected at the period of the study (Day 28) and were imaged ex vivo with 3D US. The dedicated plate filled with ultrasonic gel allowed us to standard tumor volumes accurately, avoiding any passage of collected tissues due to the motion of the US transducer during the 3D acquisition (S2B Fig).

2.9 Volumetric Measurements

Tumors were carefully dissected soon afterward immersed in suitable graduated cylinders filled by water. Volumes were assessed by measuring the load down of water removed from the cylinder to constrain the concave meniscus adjusted to the upper brim of the baseline graduation mark (S2C Fig). These measurements were performed in triplicate.

2.10 Statistical Analysis

Statistical dissection was performed using GraphPad Prism software rendering 5.0 (GraphPad, USA). Correlation graphs and R squared coefficients were obtained ~ means of nonlinear regression.

3. Results

3.1 Validation of Techniques on the side of Tumor Volume Measurements on Subcutaneous Models

To assess the real accuracy of each modality to find out tumor volumes, the subcutaneous tumor shape allows us to avoid disrupting contributions of the like kind as respiratory movements and well-known problems associated through US chest exams. Comparison of results was achieved from subcutaneous tumors with sizes ranging from 15 to 700mm3. Correlation decomposition between US measurements achieved in vivo Vs ex vivo (R2 = 0.94), Vs CT (R2 = 0.93), or swelling weight (R2 = 0.98) and volumetric assessments (R2 = 0.96) clearly validated the faculty of US and CT protocols to perform accurate determination of tumor volumes (Fig 1).

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Fig 1. Correlation separation between different methods for tumor mass assessment.

Nonlinear regression of data points collected from orthotopic subcutaneous tumors (n = 10 animals). The correlation coefficient R squared is provided in the lower right hand of harvested land graph.

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3.2 Control of the Tumor Cell Implantation

For subcutaneous tumors, altogether the 10 xenografted animals were enrolled with regard to assessment of the tumor sizes at the time determined by the various measurement methods. For orthotopic lung tumors, seven days afterward engraftment the tumor growth was confirmed ~ means of BLI (S1D Fig), either in the left or up~ lung. Based on BLI intensity, 12 mice through tumor activity ranging from 7.45×105 to 8.55×107 Photons/sec were selected with regard to the study.

3.3 Assessment of Tumor Volumes in Lung through In Vivo US Imaging

As in human lung US, we observed artifacts from the pleural fill due its echogenicity. As pointed thoroughly by the white arrows (Fig 2A), the pleural cover with ~s appears bright, as it is an increased ultrasound reflection at the interface between pleura and healthy lung. We noticed standing still regularly spaced hyperechogenic lines, named A lines, that are repetition artifacts of the pleural note. The break in the pleural and A lines allowed us to prove to be identical the lung tumor with certainty, fair at early stages (Fig 2B). A indicative artifact observed below the lung swelling, such as a bright shadow corresponding to the meeting point between tumefaction and healthy pulmonary parenchyma, enabled us to identify tumor margins. This posterior enhancement is explained ~ means of the difference in ultrasound velocity betwixt tumor and lung parenchyma.

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Fig 2. In vivo identification and monitoring of orthotopic lung tumors.

(A) & (B) Detection of lung tumors ~ the agency of High Resolution Ultrasound Imaging. (A) 2D B-Mode acquisition attached a healthy lung in mouse. Vertical white arrows point out the pleural row. Vertical yellow arrows correspond to A lines, representing reverberations of the pleural draw. (B) On 2D B-Mode Ultrasound imaging of a lung long-suffering an orthotopic NCI-H460luc tumor (2.8mmx2.4mm), perpendicular red arrows point out the margins of the tumor, highlighted by the typical bright image artifact. We also remark white and fulvid arrows indicating the pleural line and A lines respectively. (C) From 2D to 3D Ultrasound B-Mode imaging of a lung swelling in mouse. The red area corresponds to the lung swelling in the thoracic cavity of the catch mice. The red grid corresponds to the tumor volume obtained by tracing margins steady each 2D B-mode slices from the 3D acquisition. (D) Assessing swelling burden with BLI (left) and US (becoming), data are presented as mean ±SEM and statistically analyzed. A pair-way repeated-measure analysis of disagreement followed by Bonferroni post-tests was used toward the data of over time career. Differences were considered significant at p< 0.05. Left: Signal activity from in vivo longitudinal monitoring of tumefaction proliferation by BLI following the evidence of 1.5×105 or 2.5×105 swelling cells (Photons/sec). Right: In vivo tumor volumes measured by US imaging using a transducer mounted forward a 3D motor, comparing the swelling growth between 2 different tumor burdens (mm3). Results stand in the place of mean±SEM (n = 5 animals per groups). (***p<0.001; ****p<0.0001).

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Positioning the transducer through an appropriate angle made it practicable to record successive 2D images by a stepper motor for 3D acquisitions. Processed volumes were displayed being of the kind which a red grid (Fig 2C).

When compared to US swelling volumes in the two different groups of lung tumors, BLI exhibited extremely different patterns of evolution (Fig 2D). In the at the outset group (1.25x105cells), BLI increased progressively until day 21 then remained unchanged it being the case that US determined volumes increased during the not notched study. In the second group (2.5x105cells), unremitting increase in volumes determined by US was to a great extent different from the BLI pattern exhibiting pronounced return. from day 14 to day 21, afterwards stagnation. In vivo US follow up of lung tumors allowed us to determined length tumor volumes precisely and highlighted the signifying differences between growth curves from the two initial tumor burdens.

3.4 Comparison of Techniques toward Tumor Volume Measurements in Lungs

At the cessation of the study (Day 28), non-show difference enhanced CT was performed with the goal to compare data from US and CT. The 3D reconstituted volumes of health-giving lungs and tumors were processed (Fig 3). Isosurfaces of hale lungs were obtained with an automated segmentation it being the case that tumor volumes required manual delineation up~ 2D slices.

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Fig 3. Assessment of tumefaction volumes with micro Computed Tomography.

3D interpretation of CT scan anterior and ensuing reconstructions, after processing the tumor outline on 2D slices from coronal, brow-band or sagittal plane. Healthy lung parenchyma is blue whereas tumor is highlighted in golden.

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The correlation decomposition of data obtained by each means on orthotopic lung tumors is reported in Fig 4. We compared tumors with different sizes, data obtained from in vivo and ex vivo US, CT, volumetric measurements and weights. CT scrutinize provides an accurate estimation of tumefaction volumes compared to ex vivo US (R2 = 0.87), tumefaction weight (R2 = 0.98) and volumetric measurements (R2 = 0.80).

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Fig 4. Correlation analytics between different modalities for tumor contortion assessment.

Nonlinear regression of data points collected from orthotopic lung tumors (n = 12 animals). These graphs present a resemblance the correlation between 3D US imaging in vivo and ex vivo, in vivo 3D CT scans, ex vivo volumetric measurements and tumefaction weight.

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For in vivo measurements, results clearly show gross correlation of volumes processed through in vivo US, CT and swelling weight (R2 = 0.75). These results cook not reflect an overestimation of swelling volumes measured by US or CT for the re~on that compared to the tumor weight. Such a correlation is furthermore observed with ex vivo US and volumetric measurements (R2 = 0.72 and R2 = 0.65 particularly).

3.5 Contrast Enhanced Ultrasound, Power Doppler and Photoacoustic Imaging

Since it was likely to perform in vivo US B-Mode imaging, the sufficiency to measure different US and PAI parameters was investigated. Interestingly we noticed that micro US imaging enabled the visualization of swelling perfusion status after the injection of MicroMarker™ contrariety agent (Fig 5A). We noticed a well enhanced indication through contrasted enhancement demonstrating an effective perfusion status. CEUS imaging was in greater numbers efficient and sensitive in highlighting vascularization parameters than Power Doppler. Thanks to Power Doppler (~t one injection of contrast agent required), we were able to utter the larger vessels. Due to the minikin size of other vessels and contribution of parameters such as slow madcap flow motion, heartbeats and breathing, the visualization of narrow-minded blood flows is challenging, even through ECG and breathing gating (data not shown).

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Fig 5. Contrast enhanced ultrasound imaging and Photoacoustic imaging of hypoxia up~ orthotopic lung tumors in mice.

(A) B-degree image of a lung tumor with corresponding contrast image before IV injecting of Vevo MicroMarker™. Maximum Intensity Projection ~wards injection of MicroMarker™. (B) B-quality image of a hypoxic lung tumor with corresponding OxyHemo photoacoustic images. With the OxyHemo-Mode, red areas show well oxygenated parts of the tumefaction whereas blue and dark areas designate the presence of hypoxia. Regarding the 3D volumes, the red grid corresponds to the hypoxic country of tumor and green grid corresponds to the not toothed tumor. (C) B-mode image of a well oxygenated lung swelling with corresponding OxyHemo photoacoustic images showing absence of any hypoxic core.

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Regarding PAI, we obtained accusation on the oxygenation status of tumors and it was possible to reveal the presence of hypoxia interior tumors noninvasively (Fig 5B). We noticed oxygenated areas in the outer boundary of the tumor as compared to the hypoxic heart. 3D acquisitions allowed quantification of hypoxia and volumes since both regions (Vtumor = 29.4mm3, SO2tumor = 11.4%; Vhypoxia = 10.2mm3, SO2hypoxia = 0.4%, every part of numbers given as single values despite the tumor shown here) (Fig 5B). It is of influence to observe that some mice through comparable total tumor volumes exhibited a well oxygenated tumor (Vtumor = 47.3mm3, SO2tumor = 51.7%, everything numbers given as single values since the tumor shown here) (Fig 5C).

Targeted CEUS allowed us to assess the relative pronoun VEGFR2 expression in lung tumors. The VevoCQ software calculated the discriminating Targeted Enhancement (dTE) values for harvested land region of interest, the same resolution was completed for both the VEGFR2 and isotype govern antibody conjugated contrast agents. From parametric images the spatial dispensing of the bound contrast agent be possible to clearly be seen (Fig 6A and 6C). Comparison of dTE betwixt microbubbles labeled with VEGFR2 and isotype ascendency antibodies was significant (Fig 6D). Moreover, hypoxic areas determined ~ dint of. PAI matched the spatial distribution of VEGFR2 (Fig 6E).

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Fig 6. Molecular imaging of VEGFR2 ~ dint of. Targeted Contrast Enhanced Ultrasound Imaging.

(A) & (C) Parametric images of the spatial partition of contrast agent bubbles. (A) Isotype command conjugated microbubbles. (C) VEGFR2 conjugated microbubbles (Target Ready Vevo MicroMarker™). (B) Corresponding B-Mode similitude of the tumor. (D) Differential Targeted enhancement of VEGFR2 and Isotype sway conjugated microbubbles (dTE corresponds to the divergence between the echo power from both targeted and free bubbles, and the be sounded back power from free bubbles only). Statistical separation was performed with the Student’s unpaired t touchstone (n = 4 animals per group). (****p<0.001). (E) Corresponding PA picture highlighting hypoxic areas where VEGFR2 is mainly expressed.

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3.6 Limitations of Imaging Modalities

CT images performed following vascular stand out in opposition agent injection allowed clear delineation of the tumefaction from surrounding tissues (more specifically the liver) (Fig 7A). On the obstinate, non-contrast enhanced CT performed forward this orthotopic lung cancer model was greater amount of difficult to analyze especially for copious tumors, resulting in less accurate settlement of tumor volume (Fig 7B).

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Fig 7. CT scans and bioluminescence signals of mice direction orthotopic lung tumors.

(A) 2D CT look into of a mouse bearing a lung tumefaction, after IV injection with 100μL of a mingle with PBS and eXIA 160, a iodinated vascular opposition agent. Delineation of the tumor is perceptible on both planes with a fulvous dashed line. (B) CT scan obtained exclusively of contrast agent injection. Delineation of the tumor is visible on both planes with a yellow dashed line. (C) Longitudinal BLI study on a mouse bearing a lung swelling between day 7 and day 35 (Photons/sec/cm2/steradian), hind implantation of 1.25×105 tumor cells.

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Fig 7C corresponds to the development of BLI signals within the same animal over time. These results prove that BLI signals from the lung tumefaction were disrupted during the tumor germination since an increase in BLI was observed up to daytime 21 then regression was observed when in fact the tumor volume was still increasing (Fig 2D).

4. Discussion

The use of preclinical tumor models allows us to follow biological parameters or tumor growth and experiment the efficacy of therapeutic agents. The experiments described hither have proven their utility in providing researchers a recently made known competitive technique to assess orthotopic lung swelling growth by US imaging in vivo, allowing non-invasive and non-emission of rays based investigations.

Each of the imaging modalities used for the period of this study has advantages and disadvantages. CT is applicable to image the respiratory system resulting from to the low density within the lung extension [13]. Regarding lung cancer detection, CT has been proven to have ~ing useful in detecting lung tumors located in the summit and hilar area but less able for tumors located near the diaphragmatic superficies [14], because the lack of show difference with the underlying tissue makes it arduous to measure larger tumor volumes and requires capacious knowledge of both CT imaging and ~ing agents with animal anatomy. The appliance of CT vascular contrast agents, like as eXIA™, was necessary to play accurate measurements of tumor volume on the same level if tumors were small and enlarging around the periphery of the lungs. Unfortunately, repeated conversion to an act of contrast agent is not recommended in onco-pharmacological studies to be paid to potential effects on tumor growing or therapies. The main limitation of CT imaging is the ir~ dose delivered to the tumor. Indeed in this radiosensitive tumefaction model, the impact of repeated imaging examinations (1 through week versus 1 per 2 weeks, by a conventional chest CT scan delivering 38.9±3.9 Gy) attached tumor growth is significant. The sway of doses on tumor proliferation and stanch risk of potentiation of an anti-tumor effect may affect the relevance of facts about efficacy assessment of anticancer agents [6,15,16].

Although BLI is a quantitative technique exploited routinely to assess tumefaction proliferation, it is strictly dependent concerning metabolic conditions, namely the presence of ATP and O2. As shown in Fig 7C, the in the first stages tumor burden in these mice was stolid (1.25×105 cells), nevertheless leading to the arrangement of a hypoxic core and to the grow less of the bioluminescent signal. These swelling hypoxic conditions are a critical question to be considered to ensure the relevance of obtained data [7,17,18]. Because both BLI and US are non-invasive, measurements were performed from end to end in the same animal. Results confirmed that quantification of BLI signals from the lung is fastidious, and obtained data should be used by caution regarding the tumor growth. Nevertheless BLI quiet remains a great resource in the at daybreak stage of studies to control baconian method of lung tumors and for allocation of animals into homogenous groups.

Among other useful imaging modalities, US is noninvasive, non-radiating, does not necessarily require contrast agents and thus offers the favorable opportunity of being fast, cheap and with minimal impact on the investigated process. Moreover, this method is not subject to burst due to the appearance of hypoxia. Given that the image of tumors is not ellipsoid 3D acquisitions were unavoidable to accurately measure volumes. Combined through high resolution US imaging, PAI is a substantial-time noninvasive and quantitative imaging modality ~ the sake of the study of tumor hypoxia and heterogeneity. SO2 mapping can be performed thanks to the existing differences in optical absorbing. between oxygenated and deoxygenated hemoglobin [19]. PAI, combining lofty optical absorption contrast with ultrasound domineering resolution, provides both anatomical and functional premises. In oncology, numerous parameters have to be assessed on tumors, therefore this multimodality is in a great degree relevant to monitor therapeutic response or illness burden. However, due to the restraint of US propagation through the lung parenchyma, the version to clinical investigations cannot be considered.

Thanks to ultrasound molecular imaging, it was possible to assess the expression and spatial distribution of VEGFR2-targeted microbubbles similar to compared with microbubbles labeled with isotype superintend antibodies. Differences in acoustical echoes between free bubbles and those linked to the molecular target are the key feature to facilitate discrimination [20]. This opens up interesting perspectives to characterize changes at a corpuscular level [21] and for investigations steady targeted therapies [22], more especially anti-angiogenic strategies based attached anti-VEGFR treatments [23].

In close, we described for the first time a Photoacoustic and Ultrasound imaging strategetics to investigate orthotopic lung tumor growth and assess key biomarkers such being of the cl~s who hypoxia or VEGFR2 expression in vivo. Hypoxia is a major parameter involved in lung tumor opposition towards radio and chemotherapies. For personalized healing art, hypoxia can be assessed by PET tracers labelled with 18Fluorine [24,25] and chemotherapies dedicated to hypoxic tumors be in actual possession of already been included in phase III clinical trials [26,27]. This means/high throughput validated imaging resource would subsist of great interest to longitudinally come both tumor growth and hypoxic rank in animals when testing the competency of new anticancer therapies. This avoids ~ one risk of disruption of tumor advance compared to other imaging methods in the same state as BLI and CT.

Considering its cleverness to provide high resolution molecular imaging, it is practicable to imagine an additional potent application of PAI for intra-tumoral micro-biodistribution of therapeutic agents such as monoclonal antibodies [28].

Supporting Information

S1 Fig. Orthotopic lung tumor implantation and assessment of the engraftment.

(A) The diagram represents the route of inoculation. The catheter is inserted in the depth bronchus through the trachea, so that the tumor grows in the lower lobes of the lungs, close-fisted the posterior diaphragm surface. (B) The in vivo restrain of the catheter positioning by planar X-sight is performed in order to refrain from the implantation in the wrong place. (C1) Control of the accuracy of lonely dwelling deposition into the deep bronchus ~ dint of. SPECT/CT imaging of 99Tcm-labeled cells. (C2) Sagittal explore demonstrating the suitable location, at the rear part of the lung. (D) Tumor growth is confirmed by BLI on ~light 7.

doi:10.1371/journal.pone.0153532.s001

(TIF)

S2 Fig. In vivo and ex vivo bulk methods to determine the lung tumor volumes.

(1) Transducer positioning allowing despite the conduction of ultrasound through the tumor parenchyma (delineated yellow area). (2) Set up during the term of ex vivo 3D US acquisitions. An excavated layer is filled with ultrasound conductive gel around the tumor tissue in order to help any movement, and the transducer is positioned above. (3) Volumetric determination by immersing the tumor in a graduated cylinder filled with water. The water is removed from the graduated cylinder to adjust the concave meniscus at the upper brim of the baseline graduation mark and in that case weighed.

doi:10.1371/journal.pone.0153532.s002

(TIF)

Acknowledgments

We distinctly thank Dieter Fuchs, Philippe Trochet and Jithin Jose from FUJIFILM VisualSonics on account of assistance in ultrasound imaging analysis and clever advice for photoacoustics.

Author Contributions

Conceived and designed the experiments: FR SL ALP. Performed the experiments: FR. Analyzed the premises: FR JS SN SL ALP. Contributed reagents/materials/algebra tools: FR JS MLM SR SN SL ALP. Wrote the drafts: FR JS MLM SR SN SL ALP.

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Those inclination embody medical history, symptoms or dissection for the underlying reason behind ED, verge of life and basic health, and different factors.

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