Ideally, monoclonal antibodies provide selective treatment by targeting the tumour, without affecting normal tissues. Therefore, antibody imaging is of interest, preferably in early stages of drug development. However, the imaging signal consists of specific, as well as non-specific, uptake. The aim of this study was to assess specific, target-mediated uptake in normal tissues, with immuno-PET in a phase I dose escalation study, using the anti-CD44 antibody RG7356 as example.89Zirconium-labelled RG7356  was administered after a variable dose of unlabelled RG7356 (0 to 675 mg). Tracer uptake in normal tissues  was used to calculate the area under the time antibody concentration curve (AUC) and expressed as tissue-to-blood AUC ratios.Data from thirteen patients with CD44-expressing solid tumours included in an imaging sub-study of a phase I dose escalation clinical trial using the anti-CD44 antibody RG7356 was analysed. n = 7), tumour uptake of the antibody was observed.Within the dose range of 1 to 450 mg, tissue-to-blood AUC ratios decreased from 10.6 to 0.75 ± 0.16 for the spleen, 7.5 to 0.86 ± 0.18 for the liver, 3.6 to 0.48 ± 0.13 for the bone marrow, 0.69 to 0.26 ± 0.1 for the lung and 1.29 to 0.56 ± 0.14 for the kidney, indicating dose-dependent uptake. In all patients receiving ≥ 450 mg  contains supplementary material, which is available to authorized users. Treatment of cancer has improved as a result of immunotherapy with monoclonal antibodies (mAbs). Ideally, mAbs selectively target tumour cells, resulting in limited toxicity compared to classical chemotherapy.However, lack of mAb selectivity may result in significant toxicity and/or suboptimal tumour targeting, leading to therapy failure. Therefore, it is important to confirm tumour selectivity of a novel candidate mAb to minimize toxicity and maximize efficacy, preferably in early stages of drug development. Currently, toxicity is assessed by dose escalation in traditional phase I trials, using dose-limiting toxicity and a maximum tolerated dose to establish the therapeutic dose for the next stages of drug development . For the ideal mAb, selective tumour targeting is expected, with limited target antigen-mediated specific uptake in normal tissues.89Zr-labelled mAbs provides a non-invasive tool for in vivo visualization and quantification of mAbs [Recently, there is increasing interest in the use of imaging techniques to measure the mAb biodistribution in vivo without requiring blood or tissue samples . After i of mAbs –4. Quant89Zr-labelled RG7356 confirmed tumour targeting of CD44+ tumours in xenograft-bearing mice. Since RG7356 is not cross-reactive with murine CD44, studies in mice do not provide any information regarding accessible binding sites in physiologically normal organs. Assessment of biodistribution in cynomolgus monkeys showed uptake in normal CD44+ tissues, for example, spleen and bone marrow [89Zr-labelled RG7356 was performed to assess biodistribution and tumour uptake of RG7356 in a subgroup (13 of 65 patients) of this phase I trial. The previous publication on the main trial [89Zr-RG7356 was localized to the spleen, bone marrow and liver, while tumour accumulation of 89Zr-RG7356 was observed with co-administration of unlabelled antibody (≥ 199 mg), suggesting that the modest efficacy was not related to poor drug delivery to the tumour per se [89Zr-labelled RG7356 by performing quantitative analysis of the imaging data.Investigational RG7356 is an anti-CD44 recombinant humanized mAb, which targets the constant region of the extracellular domain of CD44 and provides antibody-dependent cellular phagocytosis of the malignant cells by macrophages . A precle marrow . PET imain trial  includedr per se . In the 89Zr-RG7356 and to evaluate whether uptake of 89Zr-RG7356 in normal tissues was dose-dependent as indication of target-mediated specific uptake. With this example, we aim to provide a general approach for application of immuno-PET to evaluate new drug-target-combinations in first-in-human studies.The aim of this study was to quantify biodistribution and tumour uptake of ClinicalTrials.gov Identifier NCT01358903) [Patients with advanced CD44-expressing solid tumours were included in a multicentre phase I clinical study with RG7356 1358903) . As part358903 . 89Zr  according to Good Manufacturing Practice (GMP) standards, as previously described [89Zr-labelled RG7356 (1 mg) within 2 h after administration of a variable dose (range 0–675 mg) of unlabelled RG7356. Pre-loading with unlabelled mAb was preferred from a logistical perspective (infusion of the unlabelled mAb in a phase I oncology unit), assuming no difference in uptake between pre-loading and co-infusion.For PET imaging, RG7356  was labelled with escribed –13. InfoWhole body PET and low dose CT (ldCT) scans were acquired on a Gemini TF-64 PET/CT scanner  and scheduled at 1, 24 and 96 h post injection (p.i.). Images were reconstructed as described previously . After c89Zr-RG7356 was defined as focal uptake exceeding local background. PET scans were considered positive if at least one tumour lesion showed 89Zr uptake at 96 h p.i..Visual assessment of biodistribution and tumour uptake was performed by a nuclear medicine physician. Tumour uptake of mean in Bq mL− 1). For the lung and brain, VOIs were semi-automatically defined on the ldCT and projected on the PET images. The thresholds for lung VOIs were based on Hounsfield units , therefore excluding tissue with higher Hounsfield units . VOIs of the liver, spleen and kidney were manually delineated on the PET images themselves, using the ldCT as reference. Fixed sized VOIs with volumes of 8.6 and 2.9 mL were placed on lumbar vertebrae and aortic arch (on ldCT) to estimate ACmean in the bone marrow and blood pool, respectively. Tumour lesions were manually delineated, and peak activity concentrations (ACpeak) were derived per tumour VOI [Volumes of interest (VOIs) of the liver, spleen, kidney, lung, bone marrow, blood pool and brain were delineated to derive mean activity concentrations (ACmour VOI . AssuminCmAb = (AC/ID) × DmAb, where CmAb is the mAb concentration in mg/mL and DmAb is the total (= labelled + unlabelled) antibody dose administered in milligrams.For all VOIs, standardized uptake values (SUVs) were calculated by dividing AC (decay corrected to time of injection) by injected dose (ID in Bq) corrected for body weight. Additionally, all radioactivity concentrations were converted to antibody concentrations by the following formula: − 1 was calculated. For each tissue, the tissue-to-blood AUC ratio was obtained by dividing the tissue AUC by the blood pool AUC. Data, averaged over the number of patients per dose cohort, are presented as mean ± SD.All measured time points were combined to derive a time antibody concentration curve. Subsequently, using trapezoidal integration, the AUC in mg h mLn = 1), 100 mg (n = 3), 200 mg (n = 2), 450 mg (n = 5) and 675 mg (n = 2). These dose cohorts were determined based on safety as assessed in the main phase I trial. Subsequently, the imaging dose was increased when imaging and tumour characteristics were not satisfactory . Patient details are provided in Table Thirteen patients with advanced CD44-expressing solid tumours were included. Five cohorts with different doses of antibody were evaluated: 1 mg , decreasing with increasing antibody doses , relative high tissue-to-blood AUC ratios were observed. The spleen showed the highest tissue-to-blood AUC ratio for the 1 mg dose cohort (10.6), followed by the liver (7.5), bone marrow (3.6), kidney (1.3), lung (0.7) and brain (0.08). For liver, spleen and kidney, a constant tissue-to blood AUC ratio was reached for doses ≥ 450 mg and for the lung at doses ≥ 200 mg. For the 675 mg dose cohort, the liver showed the highest tissue-to-blood AUC ratio of 0.85 ± 0.08, followed by the spleen (0.72 ± 0.09), kidney (0.59 ± 0.08), bone marrow (0.40 ± 0.04), lung (0.27 ± 0.03) and brain (0.07 ± 0.02).89Zr-RG7356 at 96 h p.i. per tumour lesion is shown in Table n = 7), tumour uptake was observed. An example image of visible tumour uptake is shown in Fig. 89Zr-labelled-RG7356 was observed in the lung, indicating diffuse tumour localisation. Quantification was not feasible due to the diffuse localisation. Quantification of mAb uptake in focal tumour lesions resulted in an average SUVpeak of 3.7 ± 1.7 for the 450 mg cohort and 6.5 ± 1.3 for the 675 mg cohort. Average tumour AUC was 1.5 ± 0.5 mg h/mL for the 450 mg cohort and 3.2 ± 0.5 mg h/mL for the 675 mg cohort. Average tumour to blood AUC ratio was 0.46 ± 0.15 for the 450 mg cohort and 0.65 ± 0.07 for the 675 mg cohort. For the 675 mg cohort, the average tumour blood volume fraction was 67%.Assessment of tumour uptake of 89Zr-labelled anti-CD44 antibody RG7356 in normal tissues to identify specific, target-mediated uptake on immuno-PET in a dose escalation phase 1 study.In this study, we assessed dose-dependent and dose-independent uptake of the 89Zr-RG7356 was observed, indicating target antigen-mediated specific uptake in these tissues. A very similar pattern of dose-dependent uptake in the spleen, liver and bone marrow has been reported previously in the preclinical study with 89Zr-RG7356 in cynomolgus monkeys, indicating that such preclinical immuno-PET studies can be predictive with respect to normal tissue uptake in human [Both dose-dependent and dose-independent uptake were observed, reflecting specific as well as non-specific uptake of RG7356. For tissues without antigen expression, a linear increase in antibody concentrations can be expected for increasing antibody doses, driven by perfusion and blood volume of the tissue. However, our results suggest a mechanism that extracts antibody from the blood pool to tissues, in addition to the non-specific uptake mechanisms  , 17. Alt89Zr-labelled antibodies, with minimal release of 89Zr, has been demonstrated in many in vitro and in vivo preclinical as well as clinical studies [89Zr in normal tissues, except for the observation that free 89Zr, arising after internalisation and intracellular catabolism of the conjugate, may accumulate in bone tissue (not bone marrow) [89Zr accumulation in the bone  to a single patient [We observed tumour uptake in all patients receiving ≥ 450 mg, with an extremely high tumour blood volume fraction estimated for the 675 mg cohort. Although this might suggest target-mediated specific tumour uptake, another study design would have been more informative on this point . This re patient . AlthougNo focal tumour uptake was visualized in the lowest dose cohorts 1–200 mg). This observation cannot be explained by the level of CD44 expression or the percentage of CD44-positive tumour cells  approaches.89Zr-labelled mAb can be used as a general method during phase I dose escalation studies to evaluate the therapeutic potential of an antibody. Evaluation of dose-dependent and dose-independent normal tissue uptake with immuno-PET reflects specific and non-specific uptake. Antibody quantification obtained by molecular imaging provides an additional, non-invasive method to study in vivo mAb biodistribution, besides traditional PK obtained by blood sampling. Especially for a candidate mAb with a potential future as ADC, the resulting information  may justify the patient burden and cost for additional scans in a limited number of patients in a phase I setting.This exploratory imaging study demonstrates how immuno-PET with a For a mAb which continues in further stages of drug development, in vivo measurements of antibody concentrations in tissue and tumour can be of value for PK/PD modelling/dose optimization and response prediction to guide individualized treatment.This study demonstrates how immuno-PET in early clinical drug development provides a non-invasive technique to quantify dose-dependent uptake in normal tissues, indicative of specific, target-mediated uptake.Additional file 1: Table S1.Specific scoring criteria for CD44 expression in patient samples. (DOCX 14 kb)Additional file 2: Supplementary Data.Information on the quality control of the radiolabelled tracer. (DOCX 16 kb)Additional file 3: Figure S1.Example of immunohistochemistry staining for CD44 for (a) patient 9, rectum carcinoma, biopsy of a liver metastasis CD44 score: 2+; (b) patient 8, squamous cell carcinoma of the head and neck, biopsy of a neck metastasis, CD44 score: 3+. (TIFF 4368 kb)Additional file 4: Figure S2.Example graph of blood pool (a), normal tissue (b) and tumour (c) for patient 8. The mediastinal tumour (patient 8) corresponds with Fig. Additional file 5: Figure S3.89Zr-RG7356 at 96 h p.i.. From left to right: low dose CT, attenuation-corrected PET and fused image. (a) Tumour lesion: left side of the skull . (b) Tumour lesion: sacrum . (TIFF 6335 kb)Additional examples of focal tumour uptake of


We present an in-silico model of avascular poroelastic tumour growth coupled with a multiscale biphasic description of the tumour–host environment. The model is specified to in-vitro data, facilitating biophysically realistic simulations of tumour spheroid growth into a dense collagen hydrogel. We use the model to first confirm that passive mechanical remodelling of collagen fibres at the tumour boundary is driven by solid stress, and not fluid pressure. The model is then used to demonstrate the influence of collagen microstructure on peritumoural permeability and interstitial fluid flow. Our model suggests that at the tumour periphery, remodelling causes the peritumoural stroma to become more permeable in the circumferential than radial direction, and the interstitial fluid velocity is found to be dependent on initial collagen alignment. Finally we show that solid stresses are negatively correlated with peritumoural permeability, and positively correlated with interstitial fluid velocity. These results point to a heterogeneous, microstructure-dependent force environment at the tumour–peritumoural stroma interface. The importance of mechanics in cancerous growth, invasion and metastasis is well-established . I). I2]). Given the potentially correlated nature of these observations, it is instructive to build a reductionist mathematical model with which to isolate and test each component separately. In order to make such a model biologically relevant, it should be parametrised by measurable biophysical properties at multiple scales. This motivates the development of a multiscale, multiphase model that can be coupled with a model of tumour growth. A brief overview of pertinent models is given here; see e.g. , 9] for for9] foPioneering work by  investigTo our knowledge there exists no multiple length scale description of biphasic tumour growth. The model we present here uses the biphasic formulation proposed by  to extenHere we conduct simulations of in-vitro tumour spheroid growth into a collagen hydrogel to test that i) tumour growth causes passive microstructural remodelling; ii) collagen microstructure influences macroscale fluid flow; and iii) solid stress is correlated with tissue permeability and interstitial fluid flow. The paper is structured as follows: ‘Material and methods’ provides a description of multiscale tumour modelling and its numerical implementation, including a link to our open-source software; ‘Results’ presents the set of hypothesis tests using the proposed model; and ‘Discussion’ critiques the results and methods.T, embedded in a shell of peritumoural stroma, ΩP, with a boundary interface between the tumour and peritumoural stroma, ΓI, and an external boundary, ΓE. For simplification, spherical symmetry is assumed and only one eighth of the total domain is analysed; symmetry surfaces are collectively denoted by ΓS. The tumour is defined at the macroscopic scale only, while the peritumoural stroma (PTS) is treated as a multiscale material. As with our previous work V is the u′ is the directional derivative of the microscale displacement, Here z, and k is the fibre permeability, defined as k = (rT ⋅ c ⋅ r)−1, where r is the fibre direction cosine matrix (i.e. the mapping between the fibre and fluid frames) and c is the diagonalised fibre drag coefficient matrix. The diagonal components are set equal to expressions previously derived for parallel , , , to nameIt is worth noting the limitations of the current model, particularly with a view to its application to in-vivo tumour growth and patient-specific modelling . First, S1 FileHere we provide the finite element form of the coupled macro-micro conservation equations.(PDF)Click here for additional data file.S1 TableThis table lists the material parameters used in all simulations, unless stated otherwise in the text.(PDF)Click here for additional data file.


A solitary fibrous tumour is a rare, mainly benign spindle cell mesenchymal tumour most commonly originating from the pleura. An intrapericardial location of a solitary fibrous tumour is extremely unusual. We present a case of an asymptomatic patient with a slow-growing massive benign cardiac solitary fibrous tumour.A 37-year-old asymptomatic female patient was referred to our hospital with an enlarged cardiac silhouette found on her screening chest X-ray. The echocardiographic examination revealed pericardial effusion and an inhomogeneous mobile mass located in the pericardial sac around the left ventricle. Cardiac magnetic resonance (MRI) examination showed an intrapericardial, semilunar-shaped mass attached to the pulmonary trunk with an intermediate signal intensity on proton density-weighted images and high signal intensity on T2-weighted spectral fat saturation inversion recovery images. First-pass perfusion and early and late gadolinium-enhanced images showed a vascularized mass with septated, patchy, inhomogeneous late enhancement. Coronary computed tomography angiography revealed no invasion of the coronaries. Based on the retrospectively analysed screening chest X-rays, the mass had started to form at least 7 years earlier. Complete resection of the tumour with partial resection of the pulmonary trunk was performed. Histological evaluation of the septated, cystic mass revealed tumour cells forming an irregular patternless pattern; immunohistochemically, the cells tested positive for vimentin, CD34, CD99 and STAT6 but negative for keratin (AE1-AE3), CD31 and S100. Thus, the diagnosis of an intrapericardial solitary fibrous tumour was established. There has been no recurrence for 3 years based on the regular MRI follow-up.Intrapericardial SFTs, showing slow growth dynamics, can present with massive extent even in completely asymptomatic patients. MRI is exceedingly useful for characterizing intrapericardial masses, allowing precise surgical planning, and is reliable for long-term follow up.The online version of this article (10.1186/s12885-017-3574-0) contains supplementary material, which is available to authorized users. A solitary fibrous tumour (SFT) is a rare primary tumour most commonly originating from mesenchymal tissue of the pleura. Complete surgical resection is the main treatment if possible. Histological appearance shows spindle-shaped cells and collagen fibres. Immunohistochemical features are vimentin, CD34, CD99 positivity and S-100 protein negativity , 3. We hA 37-year-old asymptomatic female patient was referred to our hospital with an enlarged cardiac silhouette found on her screening chest X-ray Fig. , panel dAdditional file 1: Transthoracic 2D echocardiography movie in the parasternal long-axis. (MP4 1.76 MB)Additional file 2: Cine movie MRI images in the short-axis planes. (MP4 440 kb)A solitary fibrous tumour (SFT) is a rare spindle cell mesenchymal neoplasm that most commonly originates from the pleura, but for which extrapleural anatomic locations have also been reported  , 5. PrimAs a first-line imaging modality, chest X-ray typically shows marked cardiomegaly, and echocardiography could verify an intrapericardial mass and pericardial effusion. MRI can provide additional information about the morphology, location and extent of intrapericardial masses, and it can help to further characterize the tumour tissue. In general, MRI characteristics of SFT vary because of the altering degree of vascularity, cystic degeneration, haemorrhage and necrosis. SFTs usually show intermediate signal intensity on T1-weighted images and variable signal intensity on T2-weighted images [SFT shows characteristic CD34 expression in 95% of the cases, and CD99 can also be positive . Most ofOur case confirms the conjecture that intrapericardial SFTs are typically slow-growing masses because, according to the consecutive X-ray images, the intrapericardial mass started to form at least 7 years ago.Although the majority of SFTs of the thorax are benign and are cured by complete resection, 10–20% are locally aggressive or malignant . MalignaAlthough intrapericardial SFT is an extremely rare condition, the slow-growth, considerable size of the tumour, and its typical MRI appearance can raise the suspicion of SFT. The “patternless pattern” histopathological finding, vimentin, CD34 and CD99 positivity and specific STAT6 immunostaining can be valuable indicators of this rare mesenchymal tumour. Owing to the high recurrence rate of extrapleural SFTs, long-term follow-up is crucial, and magnetic resonance imaging is a reliable method for the early detection of local recurrence.Additional file 3:The patient’s clinical history organized as a timeline. (PDF 287 kb)


The aim of this pilot study was to determine Clara cell protein (CC16) concentration in bronchoalveolar lavages (BAL) fluid from full-term and preterm  neonates requiring respiratory support, having symptoms of neonatal respiratory distress syndrome, and at risk of bronchopulmonary dysplasia (BPD). We hypothesized that CC16 may be predictive of BPD diagnosis regardless of gestational age. BAL fluid CC16 was measured by ELISA at birth and at day 7 of life. Both groups that developed BPD showed significantly decreased BAL fluid CC16 levels compared to those infants that did not develop the disease. CC16 positively correlated with diagnosis of BPD and negatively with the severity of the disease. These results suggest that BAL fluid CC16 levels may have a diagnostic value at day 7 for BPD in both term and preterm infants. This study demonstrates the potential utility of BAL fluid CC16 levels as a biomarker for BPD in term infants. Bronchopulmonary dysplasia (BPD) also called chronic lung disease (CLD) of infancy is a neonatal-pediatric disease associated with lung injury in preterm infants or anomalous lung development in full-term neonates –3 and isClara cell secretory protein of 10–16 kDa (CC16), also known as CC10, Club cell protein, CCSP, or uteroglobin , 10, a m Isidro Espinoza de los Reyes approved the study (#212250-19071) and signed informed consent was obtained. The study was conducted according to the Declaration of Helsinki (1964) ethic principles for human participants.The Institutional responsible Ethical Committee and Research Committee of the National Institute of Perinatologyn = 5) or not (n = 5). Clinical data was collected from medical records such as gestational age, birth weight, gender, Apgar score, prenatal steroids, and surfactant treatment. One infant had prophylactic surfactant, one neonate received rescue surfactant, and, finally, one infant had one more dose of rescue surfactant. Exclusion criteria were infants with chromosomal abnormalities, congenital lung and heart diseases, and malformations. The clinical characteristics of the study population are depicted in Ten newborns were included in the study: term (>37 weeks' gestation) and preterm (28–36 weeks' gestation) neonates who required endotracheal intubation for mechanical ventilation for more than 7 days and diagnosis of failure of ventilation treatment (3), with signs and symptoms of neonatal respiratory distress syndrome (RDS), in whom bronchopulmonary dysplasia (BPD) was diagnosed subsequently (Only term neonates presented the following characteristics: septic shock (two neonates) and transitory tachypnea of the newborn (three neonates) with two of the latter newborns also developing transitory pulmonary hypertension. Therefore, neonatal mechanical ventilation was secondary to the initial diagnosis.BPD diagnosis and severity were defined according to the National Institute of Child Health and Human Development (NICHD) and National Heart, Lung, and Blood Institute (NHLBI) Workshops –8: GradeBronchoalveolar lavages (BAL) fluid samples were collected only when indicated clinically , at day 1 (birth) and day 7 (1 week). A nonbronchoscopic method was used to obtain tracheal aspirate. Briefly, after tracheal intubation, a fixed volume of saline solution (0.5 mL) was applied and immediately aspirated (the recovered volume was 0.3 mL). The fluid was kept at all times at 4°C, under ice, and transported to the laboratory within 30 min after its collection. Samples were immediately centrifuged for 10 min at 3500 rpm/4°C and the supernatant was frozen at −70°C. Total protein concentration in BAL samples was determined by Bradford assay . CC16 protein concentration was assayed in BAL fluid in duplicate by a commercial CC16 ELISA kit  with an intra-assay coefficients of variation (CV) of 3.4% and interassay CV of 4.7%.U, two-tailed test , or number of individuals as appropriate. Statistical differences were assessed by Mann–Whitney led test . Correla2O in six infants. BPD was diagnosed in five infants from which three were term and two were preterm neonates. One neonate presented Grade 1 BPD (one preterm neonate), three neonates Grade 2 BPD (two term and one preterm neonates), and one neonate Grade 3 BPD (one term neonate). The clinical characteristics are depicted in In this pilot study, the objective was to determine Clara cell protein (CC16) concentration in BAL fluid from term and preterm infants with respiratory failure in whom bronchopulmonary dysplasia (BPD) was diagnosed subsequently. All neonates required ventilatory support for more than 7 days. The peak inspiratory pressure exceeded 17 cm Hp = 0.0079∗∗ in day 1 and 0.29 ng/mL ± 0.06 versus 1.07 ng/mL ± 0.29, p = 0.0317∗ in day 7; p = 0.0159∗ and p = 0.5476 without or with BPD, resp.; BAL fluid CC-10 levels were found significantly diminished in neonates that developed BPD compared to those that did not develop the disease  and severity of the disease . Furthermore, CC16 levels at day 1 also correlated with maximal weight loss at day 7  . IUGR, prophylactic, and rescue surfactant did not correlate significantly with any variable.There was a significant correlation between both CC16 determinations in BAL with BPD diagnosis , and mechanical ventilation . FuIt has been shown in a mouse model that CC16 administration increases epithelial proliferation, protects against oxidative stress , and is We acknowledge that the results and analysis are limited by the very small sample size; however, we developed this pilot study with the intention to incorporate a wider study in our own center and other centers based on the obtained results. This exploratory work will contribute to the development of a subsequent study by the information obtained of BAL CC16 concentration in BPD.BAL fluid CC16 levels were found to be decreased in both term and preterm ventilated neonates who subsequently developed BPD. CC16 levels at day 7 may be useful to diagnose the disease. This study demonstrates the potential utility of BAL fluid CC16 levels as a biomarker for BPD in term infants.


Sleep apnea (SA), a condition associated with increased cardiovascular risk, has been traditionally associated with obesity and aging. However, in patients with fluid-retaining states, such as congestive heart failure and end-stage renal disease, both prevalence and severity of SA are increased. Recently, fluid shift has been recognized to play an important role in the pathophysiology of SA, since the fluid retained in the legs during the day shifts rostrally while recumbent, leading to edema of upper airways. Such simple physics, observed even in healthy individuals, has great impact in patients with fluid overload. Correction of the excess fluid volume has risen as a potential target therapy to improve SA, by attenuation of nocturnal fluid shift. Such strategy has gained special attention, since the standard treatment for SA, the positive airway pressure, has low compliance rates among its users and has failed to reduce cardiovascular outcomes. This review focuses on the pathophysiology of edema and fluid shift, and summarizes the most relevant findings of studies that investigated the impact of treating volume overload on SA. We aim to expand horizons in the treatment of SA by calling attention to a potentially reversible condition, which is commonly underestimated in clinical practice. Sleep apnea (SA) is a condition characterized by repeated episodes of complete or partial airflow cessation during sleep, typically referred as apnea and hypopnea. Individuals with SA usually present witnessed episodes of snoring, choking, and are more likely to suffer from daytime sleepiness , depressThe apnea–hypopnea index (AHI), defined as the total number of episodes of apnea and hypopnea per hour of sleep, is routinely used to diagnose SA and to classify it as mild (AHI between 5 and 15), moderate (15–30), or severe (>30) . The preEven though aging and obesity are clearly the most relevant associated risk factors, the prevalence of SA is much higher among patients with edematous states, such as end-stage renal disease (ESRD)  and congDespite its high prevalence in edematous patients, SA is often overlooked because of its oligosymptomatic nature , 17. EveAlthough prevention of fluid accumulation is a plausible alternative strategy to alleviate SA in edematous patients, current guidelines do not include treatment of edema as part of the therapeutic effort against this condition. In this review, we discuss the impact of edema on the pathogenesis of SA in patients with CHF, ESRD and nephrotic syndrome, as well as the corresponding implications for innovative therapeutic strategies.Edema is defined as an abnormal buildup of fluid anywhere in the body. When utilized with no qualifier, the term “edema” usually refers to the accumulation of plasma transudate in the interstitial space, as in CHF, nephrotic syndrome, and hepatic cirrhosis –26.For fluid to accumulate at the interstitial space, a positive sodium balance must establish. Since the kidneys are ultimately responsible for maintaining sodium balance, it follows that edema formation always demands some degree of renal sodium retention. Nevertheless, impaired sodium excretion is insufficient to ensure fluid accumulation. For instance, in primary hyperaldosteronism, excess sodium reabsorption by the distal nephron translates into hypertension, rather than edema formation. To reach the interstitial space, fluid retained by the kidneys must be driven by an imbalance of Starling forces at the complex interface between the intravascular and interstitial compartments .Under normal conditions, small amounts of fluid do reach the interstitial compartment due to a slight predominance of hydrostatic over oncotic forces. Actually, the normal interstitium contains about 10 L of fluid, an amount kept within narrow limits by three mechanisms : the actAn important consequence of these physical characteristics of the normal interstitial matrix is that fluid cannot move freely across the interstitium following gravity and, therefore, will not accumulate in the lower limbs while standing, or in the cervical region after several hours in the recumbent position.Although the classical view centered on Starling forces still predominates, recent evidence suggests that this theory should be revised taking into account the segmentation of the capillary wall and the adsorption of ions by interstitial macromolecules , 29. An When sodium excretion is hindered by intrinsic renal disease, a positive sodium balance establishes, leading to expansion of the extracellular (EC) and intravascular volumes. If Starling equilibrium is not disrupted, sodium retention will cause hypertension, according to Guyton’s theory , but notIn patients on chronic dialysis, maintenance of fluid balance is entirely dependent on an artificial procedure. Therefore, development of edema in this context usually results from insufficient fluid removal  or poor In a number of situations, effective arterial blood volume (EABV), hence renal perfusion, cannot be maintained despite normal or even increased total blood volume. In this context, the kidneys  react to the reduction of EABV by retaining sodium and water, which nevertheless escape the intravascular space because of a disequilibrium of Starling forces, promoting further sodium retention. In this manner, the retained fluid tends to accumulate at the interstitial compartment, instead of recomposing the EABV .This mechanism of edema formation resulting from chronic reduction of the EABV is known as underfilling. Unlike what happens with overflow, here renal dysfunction is not the primary cause of sodium retention. Rather, the kidneys act as expected, responding to hypoperfusion by reabsorbing as much sodium as possible. Underfilling is central to the pathogenesis of edema in CHF, hepatic cirrhosis and some cases of nephrotic syndrome.Under normal conditions, the heart easily meets the needs of all tissues, keeping cardiac output at physiological levels. In CHF, the weakened myocardium can no longer maintain adequate perfusion of the peripheral territories, including the renal circulation. The consequent fall of EABV stimulates the kidneys to retain sodium. On the other hand, the malfunctioning pump leads to venous damming of blood. Retrograde transmission of the resulting venous hypertension to the capillaries promotes the passage of fluid to the interstitial space. This process is continuously fueled by the renal retention of sodium, which nevertheless fails to restore the EABV. Therefore, the basic mechanism of edema formation in CHF is underfillling .About one-third of patients with nephrotic syndrome exhibit clear signs of hypovolemia despite massive EC fluid expansion. In these patients, edema formation is believed to result from hypoalbuminemia, hence decreased systemic oncotic pressure, leading to an imbalance of starling forces, fluid displacement to the interstitium, EABV reduction, and incessant renal sodium retention. This sequence is fully compatible with the concept of underfilling , 34. HowIf fluid escape into the interstitium persists, the initially slow accumulation of edema raises gradually the local hydraulic pressure, until it becomes positive. When this happens, the normally tight architecture of the interstitium is disrupted, leading to an abrupt increase of compliance, enabling the interstitium to accommodate increasing amounts of fluid with a small rise of hydraulic pressure. The shift of fluid throughout the interstitium is no longer restricted, being now governed by gravity: during daytime, edema accumulates in the lower limbs; at night, interstitial fluid tends to be redistributed rostrally, reaching the cervical region. These movements largely explain the occurrence of airway obstruction and SA in edematous states.Sleep apnea can be classified as obstructive sleep apnea (OSA), associated with airway obstruction and, therefore, respiratory effort, or central sleep apnea (CSA), in which the main pathogenic factor is respiratory center instability. The most common sleep disorder is OSA. CSA is far less common although equally as dangerous as OSA.Both SA modalities are more prevalent in patients with CHF, compared to the general population , 35–37, In CHF, fluids displaced from the lower body during the night can accumulate at cervical and head areas, thus promoting upper airway obstruction and OSA. It has been postulated that systemic fluid retention, with consequent venous engorgement and mucosal fluid accumulation, can increase tongue volume, facilitating airway obstruction . Of noteIn men with CHF and OSA, Kasai and colleagues showed that application of lower body positive pressure (LBPP) in the awake state, thus forcing rostral fluid shift, was accompanied by a significant increase in neck circumference and an increase in upper airway resistance in proportion to the volume of fluid displaced from the legs . InteresIn CHF patients, fluid retention and fluid shift from the legs can also lead to pulmonary congestion. In this case, however, SA is unrelated to obstruction. Rather, it seems to result from a central respiratory mechanism, thus conforming to the CSA type.2 to increase, now leading to hyperventilation and generating a Cheynes–Stokes-like pattern  and/or pulmonary congestion and CSA, through the mechanisms discussed earlier. Increased leg fluid retention, and consequently nocturnal overnight rostral fluid shift, can also be favored by excessive sodium intake .The presentation of SA in patients with ESRD is quite distinct from that in the general population. First, the typical history of loud snoring and witnessed apnea during sleep is seldom obtained. Second, the association with age, gender, and body mass index is less clearcut . Third, Sleep apnea can exert a high impact on CKD mortality , given iUremia has been implicated as a possible cause of SA , 58. ThiRostral fluid shift may exert a similar influence in CKD as in CHF. In a study of 26 patients on conventional hemodialysis, Elias and coworkers  showed tThe importance of fluid retention in the pathogenesis of SA is not restricted to CKD and ESRD. In patients with nephrotic syndrome, even with normal renal function, the treatment of hypervolemia, with contraction of EC volume and disappearance of lower limb edema, was shown to alleviate SA .per se contributed to the presence of SA in patients on hemodialysis that can be partly reversible through fluid removal by ultrafiltration , the SA severity tends to increase during interdialytic periods, reaching a maximum immediately before each session , 16, 51.The importance of fluid retention is highlighted by the behavior of SA in patients with nephrotic syndrome, even when renal function is normal. In patients with steroid-responsive nephropathy, Tang et al.  showed tReducing leg swelling by wearing compression stockings during the day attenuated SA in patients with venous insufficiency , 76 and Table In the search for alternatives to CPAP, it is imperative to understand the pathogenic role of fluid overload and overnight fluid shift. The association of CPAP with strategies aimed at limiting edema and rostral fluid shift may reduce the need for high airway pressure, thus improving tolerance. Further work is required in order to assess cardiovascular outcomes of treating SA by interference on fluid overload/redistribution. Additionally, CPAP alone has failed to improve mortality among patients with OSA. Nevertheless, it is unclear if adding a fluid restriction strategy would change such outcomes.Concept and design: BS and RE; data interpretation: BS, TK, FC, RZ, and RE; manuscript writing: BS, TK, RZ, and RE; final approval of manuscript: BS, TK, FC, RZ, and RE.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This model combines automata approaches with image processing algorithms to simulate the cellular response of tumours exposed to ionizing radiation, modelling the alteration of oxygen permeabilization in blood vessels against repeated doses, and introducing mitotic catastrophe (as opposed to arbitrary delayed cell-death) as a means of modelling radiation-induced cell death. Published data describing cell death in vitro as well as tumour oxygenation in vivo are used to inform parameters. Our model is validated by comparing simulations to in vivo data obtained from the radiation treatment of mice transplanted with human prostate tumours. We then predict the efficacy of untested hypofractionation protocols, hypothesizing that tumour control can be optimized by adjusting daily radiation dosage as a function of the degree of hypoxia in the tumour environment. Further biological refinement of this tool will permit the rapid development of more sophisticated strategies for radiotherapy.The concept of hypofractionation is gaining momentum in radiation oncology centres, enabled by recent advances in radiotherapy apparatus. The gain of efficacy of this innovative treatment must be defined. We present a computer model based on translational murine data for  To limit radiation toxicity, conventional radiation treatment delivers a daily 2 Gy fraction, which provides enough time for the healthy tissue to repair damage while the tumour, which receives a higher dose, is unable to complete repair1. Though successful for most individuals, conventional radiotherapy may be limited in some hypersensitive patients, who suffer from toxicity in surrounding normal tissues, or in some patients whose tumours develop radiation resistance1. Some of these issues have been somewhat ameliorated in the past ten years, with technical breakthroughs in tumour imaging and medical physics leading to better targeting of the tumour using the latest generation of image-guided-, intensity-modulated radiotherapy or proton-therapy3. Better tumour targeting and reduced normal tissue irradiation open the door to more radical protocols where the dose per fraction may be significantly increased, and all fractions given over a shorter period, typically one or two weeks. In addition, new protocols may benefit from recent discoveries in molecular and cellular radiobiology which are often neglected in conventional radiotherapy. Common radio-oncology studies have already shed light on important radiation-induced processes such as mitotic death and cell cycle inhibition1. In addition, cancer cell signalling pathways, micro-environmental changes modifying endothelial cell behaviour, tissue oxygenation, and immune responses are all important factors that should be considered in the tumour response during radiotherapy4.The ability of ionizing radiation (IR) to control tumour growth through the induction of DNA damage is the primary basis of radiation therapy strategies. Unrepaired DNA damage can lead to mitotic cell death or proliferation blockade of malignant cells1. However, clinical trials are costly and increasingly more difficult to launch. Furthermore, the research scope in each trial remains limited, and cannot cover every new potential optimization or tumour type.However, moving to new radiation protocols is complicated, as there is no optimal statistical characterization of the outcome and physicians will be reluctant to risk to have worse outcomes than those from conventional approaches. How can we quickly build confidence in new approaches, optimize selection of protocols, and compete with 70 years of knowledge in conventional therapy? Until now, medical strategies to optimize radiotherapy have been essentially based on clinical trials using assay/error escalation of doses and computational dosimetry searching for an optimized balance between high tumour control probability (TCP) and low normal tissue complication probability (NTCP)in silico the response of tumour cells and their micro-environment to ionizing radiation. As a first step for proof-of-principle, we develop a model which models a well described in-vivo situation in mouse, where orthotopic transplants of human prostate tumours are treated with radiation5.This article describes a method to bridge the gap between basic knowledge in radiation biology and the establishment of optimized radiotherapy protocols by introducing cellular automata models that model in silico clinical trials. In this model, individual tumour cells are independent automata evolving in an oxygenated environment perfused by blood vessels. The integration of our biological knowledge into this model allowed us to better apprehend the complex spatiotemporal relationship between oxygen and tumour cell death as the blood vessels accumulate damage after each fraction delivered to the tumour. After validating the model parameters for the simple vascular microenvironment of orthotopic tumours in nude mice, we predict TCP curves for such in vivo conditions under various hypotheses related to changes in blood vessel properties following exposure to fractionated doses of radiation. Our predictions and biological assumptions illustrate clearly the importance of hypoxia and blood vessel damage during radiotherapy. These results give a glimpse of what is currently lacking from empirical and one-size-fits-all radiotherapy protocols. Our model opens the prospect of more elaborate models which could eventually predict the response of individual human tumours in real clinical situations.With increasing computer power, non-deterministic stochastic models of cell behaviour are now able to model in a reasonable amount of time the tumour response to various insults, allowing computational biologists to conduct et al.5. Briefly, PC3 tumours were transplanted in mice as described. Tumours were exposed to 2 Gy daily for 2 weeks to measure the biological impact of a conventional radiotherapy protocol using small animal radiotherapy and macroscopic and microscopic imaging tools . All experiments were performed in accordance with relevant guidelines and regulations as described in a previously published article5.All biological data were extracted from previously published manuscripts, especially Potiron In order to study the response of tumours to radiotherapy and the impact of the tumour microenvironment (vascularization and oxygen levels) on the radio-sensitivity of tumours cells, a 2D and a 3D cellular automaton model was implemented. We describe the 2D model here, and demonstrate its equivalence to a 3D model using an extrapolation factor, while considerably economizing computation time.6, the tumour microenvironment is simulated as a matrix of pixels with integer values that represent either a cell type, an open space or a blood vessel. For each time iteration, the status of all pixels is updated based on biological rules, allowing us to take advantages of an image processing algorithm to model complex spatial events such as oxygen (O2) diffusion from blood vessels or cell division. The rules and states of the model are summarized in Supplementary Fig. In silico experiments consists of a 2D array of 600 × 600 pixels for the tumour and peritumoral tissue, with an extra dimension corresponding to the number of simulated days . Figure 9. We will also show subsequently that such simplification is accurate enough for modelling O2 diffusion from blood vessels to tissue. Oxygen maps are computed using simple diffusion law and O2 maps are updated for each time iteration step as illustrated in Fig. As we have shown previously for normal breast tissuein vivo data to benchmark our simulations. A well-defined human prostate tumour cell line, PC3, has been grown as xenograft in mice12 or orthotopically in our lab5. Tumour growth curves from these various studies may thus be compiled into one dataset by allowing the time reference for each individual experiment to be shifted so that tumour volume would match the same time points. The resulting data points are shown in Fig. 14. We first test the model by assuming that only the outer cell layer is dividing. Note that to compare simulated 2D growth curves to in vivo 3D volume, we had to scale the cell number N at each time point so that the simulated disk represents the cross-section of a full tumour. The spherical tumour volume can then be approximated as Ve in the following manner:r is the radius of the simulated disk, N the cell number for one time point in the 2D simulated tumour and sc the average area of one tumour cell derived experimentally from microscopic sections15 (i.e. 15 × 15 µm2).Figure in vivo data . This parameter is therefore not modified further in this work and only the number of dividing layers is investigated by parameter sweep. Figure in vivo data12. Therefore, n = 3 is used thereafter for all subsequent simulations involving radiation. However tumour growth has been shown to follow a sigmoidal pattern in both avascular and vascular tumour18: the model we used here may not hold for larger tumour sizes, where a plateau of growth will be reached. Our model in its actual state would then have to be considered only for tumour reaching sizes up to the inflection point of the sigmoid.The imaging process is performed in multiple steps. First, non-dividing cells are identified by applying a binary erosion on the tumour mask with n iterations, resulting in a smaller mask of cells which are too deep inside the tumour to divide (i.e. the resulting mask is more than n inside layers deep). Similarly, we define the area where new cells can occupy space (the growth area) by applying a binary dilation with n iterations on the tumour mask. Pixels of type 3 and 4  are excluded from the growth area which restricts the amount of space available. Next, the outermost layer of tumour cells is duplicated first by randomly filling the growth area with the same number of cells present in the first layer. We repeat this process iteratively for the second and third layers until the nc (i.e. pixel) of the tumour or healthy tissue (noted O2(c)).Our tumour model incorporates the O2 diffusion through blood vessels assuming that the oxygen consumption of tumour cells is steady (i.e. the tumour is in homeostasis leading to no net change of oxygen from cell consumption). O2 maps, as illustrated in Fig. O2(c) is computed by applying a spatial Gaussian filter to a Boolean mask defined by all pixels of type 4 (blood vessels).As illustrated in Fig. First, the sigma value for the Gaussian filter is derived by fitting the O2 profile measured radially away from one single blood vessel using the diffusion equation:21 after converting pressure to percentage of the atmospheric pressure at sea level, which is 760 mm Hg (i.e. 21% in ambient air).The fitting gives a range of plausible values for D (sigma squared of the Gaussian and diffusion coefficient from the vessel) and s  with a 95% confidence interval. We get a fit value of sigma of 2.7 with a confidence interval of 1.6 to 3.7, assuming a pixel size of 15 µm. The data for the model are derived from measurements made of the partial pressure of O2 in mm Hg in anesthetized and un-anesthetized mice and rats5; other studies report average O2 concentrations of around 3%22. We search in the space of simulated oxygenation levels, generated from the O2 maps, which are dependent on the range of plausible values of D (sigma) and on the blood vessel density; the value of D allows the two oxygen conditions (hypoxia and average O2 concentration) to be filled. Figure 22. By fixing the blood vessel density (type 4 objects) crossing the 2D tumour perpendicularly at 3.8%, only one value from the plausible range of D (coefficient of diffusion D = 5.76 pixels corresponding to a sigma of 2.4) fulfilled both O2 conditions, and this was used in all simulations.Next, we estimate the density of blood vessels from tumours sections using immunohistochemistry, which shows that 3.8% of the space is occupied by blood vessels . This process is illustrated in Fig. If a vessel has been “hit”, then we assume the amount of O2 passing through the endothelial wall is increased by a fixed multiplicative “leak factor” Lf, we start with a value of 1 (i.e. no effect) and increase it incrementally until we reproduce in vivo measurements of the fraction of hypoxic cells in the tumour following the protracted dose experiment we conducted in the past5. Lf values between 1.3 and 1.7 lead to reasonable fit and we used Lf equal to 1.5 for the rest of this work  at 36 hours27 given by:Secondly, radiation-induced cell death has been the focus of radiation oncology for many decades and is well understood and characterized. For instance, the survival probability of tumour cells after irradiation is traditionally calculated by fitting clonogenic survival data using the linear quadratic (LQ) modelIn vitro cells are typically cultured under 21% O2. However, this scenario differs from natural conditions in vivo. For most tissues, O2 concentrations vary from 2 to 5% and may fall to less than 1% in tumours22. O2 levels modulate clonogenic survival. For this reason, the O2 enhancement ratio (OER) was introduced in radiotherapy and refers to the enhancement of therapeutic or detrimental effect of ionizing radiation in presence of O228. Another way to characterize OER is by the hypoxia reduction factor (HRF) which is the ratio of the doses for a specific iso-effect under a given oxygenation condition compared against the condition of 1 Gy at 21% O229. In order to predict cell death for a given cell c in the tumour, we apply the LQ fit of the clonogenic survival at a dose corrected for the O2 level 15 as shown in Supplementary Fig. 30 as described in ref. m is the maximum HRF and K is the oxygen partial pressure at which the HRF is half the maximum value. The fitting with initial condition at 2.7 for m and 0.002 for K leads to the values m = 2.804 and K = 0.001076, as described in Supplementary Table fracD is then:Cell survival for each dose fraction This model leads to accurate prediction of cell death for the various calibrated doses and O2 levels as shown in Fig. Tumour cells have blood vessels intercalated with an O2 leakiness parameter;Radiation can make blood vessels leakier for O2;Conversely, high radiation doses can disable a vessel’s capacity to perfuse O2;in vitro clonogenic survival modulated by O2 concentrations.Tumour cell death is derived from Tumour cells can grow as long as there is free space around them.All tumour cells are growth-arrested when exposed to ionizing radiation and the duration of the arrest is proportional to the dose received.Tumour cells behaviour does not change with repetitive exposure (no adaptation).To summarize material and methods, our model has so far incorporated the following assumptions:in vitro clonogenic survival data to the actual death of the tumour in vivo. The clonogenic survival assay in mammalian cells was first developed by Puck and Marcus in 195632 and has been the basis of the fractionation protocol for standard radiotherapy for the last 50 years. However, the data derived from such measurements is insufficient to inform our computer model. First, we cannot differentiate the different types of cell death. For example, apoptosis and mitotic death – where a cell actually disappears from the tumour– cannot be modelled in the same way as senescence – where the cell remains inside the tumour but can no longer divide. For now, we will ignore senescence. In addition, we do not know how long it will take for a cell with a lethal damage (i.e. type 5) to be cleared from the tumour. Death and cell clearing is not instantaneous after exposure and tumours undergoing radiotherapy do not shrink at the rate one would expect from measured in vitro cell death. Recent clonogenic survival data on lung cancer cells have in fact shown that it can take as long as 14 days post-exposure before a cell actually disappears33. Similarly, we have shown using time lapse imaging of normal human breast cells in vitro34 that cell death is asynchronous following exposure to the same dose of x-rays with large variations between cells.There is one last aspect of the model that deserves more attention regarding the translation of 35 and it circumvents the need to introduce another parameter required for the cell death delay model that we and others have previously used33. It also neglects senescence and apoptosis as other mechanisms for radiation-induced tumour cell death.In our model, we introduce the concept of “mitotic death” as the main mechanism of radiation-induced tumour cell death. This concept implies that a cell which has been marked with a lethal damage (type 5 – cyan colour) will be removed from the simulation only when it undergoes division upon availability of space. Supplementary Fig. 5 and changing this value will have important effect on simulations  curves are generated by simulating 100 “The resulting simulated TCPs have the well-known sigmoid shape which can be fitted using the sigmoid equation Fig. :10\documDt for one particular protocol leading to a TCP of 50% (0.5 on the sigmoid), i.e a 50% probability of achieving tumour control using a particular protocol) is computed by solving the previous equation with the fitted parameters such that TCP(Dt) = 50. The lower and upper 95% confidence interval of the TCPs are also computed by measuring the observation bounds, taking into account the uncertainty in the fitting, and the random variations in the observations. The error on the evaluation of the TCP of 50% is then directly derived from the confidence bounds on the TCP of this dose.The total dose Fitting of TCPs obtained for dose fractionation mimicking clinically relevant protocols are shown in Fig. First, as expected, TCP50 decreases with the dose fraction used. Secondly, results show that neglecting the low oxygenation of the tumour environment lead to under estimation of the dose required to fully control the tumour . When blood vessel death is included, hypoxia does not drop severely anymore with 8 Gy fractions -based formula with an overall time factor and a tumour repopulation time delay during fractionated radiotherapy courses. This formalism is used almost universally for calculating isoeffect doses of different fractionation schemes and is therefore of fundamental relevance in daily clinical practice36. However, the applicability of this model to high dose per fraction is controversial38. This radiobiological debate has become increasingly relevant due to the widespread adoption of stereotactic body radiation therapy (SBRT), where irradiation is delivered in a few fractions or even in a single fraction of very high dose39. Last decade, several models were proposed for SBRT therapeutic schemes, such as the modified Linear-Quadratic (MQL) model40, the Linear-Quadratic-Linear (LQL) model41, the generalized Linear-Quadratic (gLQ) model42 or the universal cell survival (UCS) curve43. In addition to those simple mathematical formalisms, complex stochastic models that include more realistic biological processes have been developed to take into account phenomena such as cell cycle phase, vascularization or hypoxia. In their reviews, Marcu et al.44 and Enderling et al.46 presented models that simulate tumour growth and/or radiotherapy incorporating tumour oxygenation (i.e. vascularized tumours). These reviews concluded on the need for better description of biological processes to improve computer models in the field of radiation oncology. Emphasis on tumour oxygenation, tumour heterogeneity and cancer stem cells were noted as important factors in this modelling effort.Different modelling approaches have been proposed in the literature for radiotherapy. In 1989 Fowler 47 where oxic rate is modelled with partial derivative equations, we use a simpler and faster formalism simulating diffusion processes via Gaussian image filtering. Other simpler oxygen models include the Krogh model assuming cylindrical diffusion48 or even spherical diffusion kernels9. In addition, we introduce mitotic death as the main mechanism for radiation-induced death. And lastly, all the parameters of our model are kept to a minimum and have a physical or biological meaning that can be obtained experimentally  modelling and for its influence on alpha/beta modulations. For now, we have followed the approach described in Carlson 23 further complicating the very high dose response. For this reason, more work is necessary to better understand the biology behind cell-cell interactions between tumour and blood vessel as function of dose and time.With these caveats in mind, one can infer important rules that may improve cancer treatment. For instance, our simulations showed that weekends increase the number of tumour cells re-entering cycle which give them an opportunity to escape death. We would therefore recommend increasing the dose on Friday to further reduce proliferation during weekends. Simulation also shows that the core of the tumour is more easily controlled by repeated radiation fractions, whereas cells on the outer layers of the tumours are more likely to survive treatment. Therefore, a large dose for the first exposure may help to control the outer layer better. The inclusion of our data regarding biological response of blood vessels following repetitive irradiation, allows assessment of the impact of blood vessel maturation and increased blood vessel perfusion for various radiotherapy protocols in our model. The results suggest that the best strategy is to use 8 Gy fractions to maximize tumour oxygenation via radiation-induced perfusion while having minimum increased hypoxia due to blood vessel death. However, the impact of endothelial apoptosis on the tumour response was not fully characterized. For instance, in addition to blood vessel loss for doses larger than 8 Gy, it is also known that endothelial apoptosis can induce radio sensitization for doses above 15 Gyin vitro data, where the cellular heterogeneity in cell-cycle arrest for tumours is linked to DNA repair, so that damaged cells which spend less time repairing in G2 have a higher death rate during mitosis35. This would involve the gathering of detailed in vitro measurements by tracking individual cells for many generations as we previously did for non-malignant human breast cells34. We have started an ambitious program between our laboratories to monitor mitotic catastrophe, growth arrest and proliferation as a function of dose, O2 concentrations and time interval between repetitive exposures. The automata will be constantly refined as this information is generated. By looking at various tumour cell lines and characterizing the integrity of their cell cycle checkpoint via genomic analysis, we will also be able to better model the variability in tumour response and hopefully better understand what makes a tumour resist or adapt to radiotherapy. Finally, one aspect of this model that will also require further development is to include the response of the surrounding healthy tissue. This will be critical to validate any fractionation protocol, since a protocol needs to maximize tumour control while minimizing toxicity. Thus, tumour specificity as well as patient’s radiation sensitivity will be an essential aspect of future work.In terms of model refinements, it would be interesting to modify the automata model to link DNA repair kinetic and cell cycle arrest to mitotic catastrophe. One could envision a model, given the right 32.Once further developed, we believe these new modelling methods will provide a powerful tool for precision medicine in radiotherapy taking us into a new paradigm beyond the linear-quadratic model for clonogenic survival in radiation oncology, introduced some sixty years agoSupplementary Tables and FiguresSupplementary Software


The aim of this guideline is to provide clinicians with up-to-date, evidence-based information regarding management of distension media in operative hysteroscopy, with particular reference to prevention and management of complications that may arise from fluid overload.http://www.rcog.org.uk/womens-health/clinical-guidance/development-rcog-green-top-guidelines-policies-andprocesses). The classification of evidence levels and grade of recommendations are given in Appendix This guideline was developed using the methodology described by the RCOG for developing RCOG Green-top Guidelines , MEDLINE (through PubMed), EMBASE (through Embase.com) were searched for potentially eligible records. We searched the databases using a combination of MeSH  and relevant index terms. We used MeSH or index terms for the following key word: “operative hysteroscopy”, “TCRE”, “TCRF”, “hysteroscopy” AND “glycine”, “resectoscope”, “hysteroscopy” AND “fluid overload”, “hysteroscopy” AND “distension media”, “hysteroscopy” AND “management”. The search was limited to humans and papers in the English language. Relevant guidelines were also searched using the same criteria in the National Guidelines Clearinghouse, the National Electronic Library for Health, the Organising Medical Networked Information (OMNI) and the Canadian Medical Association (CMA) Infobase. The literature search, study selection and data extraction were carried out by two authors (SU and DM) independently, and a third author (JC) made the final decision in case of disagreement. Three authors  graded the level of evidence.Hysteroscopy enables visualisation of the uterine cavity and allows the diagnosis and surgical treatment of intrauterine pathology. To achieve this, the uterine cavity needs to be distended by a medium which could either be fluid or carbon dioxide . Carbon Operative hysteroscopic procedures are usually carried out using resectoscopes which are larger diameter, continuous flow operating hysteroscopes. They incorporate a working element that moves an electrically activated wire loop. These devices were initially developed to use monopolar current, which require non-electrolyte distending media such as glycine and sorbitol. Such solutions are however hypotonic so that excessive absorption can cause a number of complications including hyponatremia, a variable degree of hypo-osmolality, and certain solution-specific problems that are described below. Isotonic electrolyte-containing solutions cannot be used with monopolar energy because this leads to activation of ions that disperse the electric current and reduce the power density. Hence the heat generated in tissues is insufficient to destroy or have a tissue effect .Resectoscopes have now been developed to use bipolar electrical current with the advantage that they are compatible with electrolyte-containing distension solutions such as physiological normal saline and Ringer’s lactate. Use of these solutions reduces the risk of hyponatremia, but excessive absorption can, as with monopolar current, lead to expansion of the extracellular fluid volume with the potential to generate fluid overload, pulmonary oedema, hypertension and cardiac failure.Operative hysteroscopy can also be performed using small diameter, continuous flow hysteroscopes which incorporate a small, usually 5Fr or 7Fr, diameter working channel down which mechanical or electrosurgical instruments can be passed. Tissue removal systems refer to operative hysteroscopes that have been designed to simultaneously cut and aspirate tissue from within the uterine cavity. These systems usually incorporate their own fluid monitoring equipment but fluid overload can still occur. Smaller diameter operative hysteroscopes are less likely to cause fluid overload due to smaller diameter inflow channels and the generally less invasive nature of procedures that can be undertaken with such technology. However fluid overload may still occur and vigilance when using any operative hysteroscopic technology is mandatory.Data evaluating fluid deficit during hysteroscopic surgery are lacking preventing a standard definition of fluid overload. A decrease in serum sodium of 10 mmol/L corresponds to an absorbed volume of approximately 1000 mL when using 1.5 % glycine  and it iWith the advent of bipolar electrosurgical systems using isotonic solutions, fluid deficits >1000 mL will be tolerated by healthy women but a safe, upper limit is not still well defined and will depend upon an individual’s size, age and medical fitness.In the absence of evidence to define an upper safe threshold for isotonic media the BSGE/ESGE Guideline Development Group recommends a limit of 2500 ml. This is in line with other national guidelines .These thresholds apply to otherwise healthy fit women. However, in the elderly or those women with co-morbid conditions such as cardiovascular disease and renal impairment, lower thresholds should apply and it is suggested that upper fluid deficit levels of 750 ml for hypotonic solutions and 1500 ml for isotonic solutions .The incidence of fluid overload during hysteroscopic surgery is generally low . SeveralThe complications of distension media during hysteroscopic surgery depend primarily on the type of medium used and the complexity of the operation. The main complications that occur are related to fluid absorption during the procedure leading to fluid overload with or without electrolyte imbalance.Intrauterine pressure – the higher the pressure, the greater the degree of absorption into the body; systemic absorption of fluid increases considerably when intrauterine pressure exceeds mean arterial pressure A fluid deficit of more than 1000 ml should be used threshold to define fluid overload when using hypotonic solutions in healthy women of reproductive age. [GPP]In the absence of a consensus, a fluid deficit of 2500 ml should be used threshold to define fluid overload when using isotonic solutions in healthy women of reproductive age. [D]The incidence of fluid overload will vary according to case mix and type of hysteroscopic surgery. In general when using large diameter resectoscopes the incidence of fluid overloads should be less than 5 %. [GPP]The clinical course and outcome of all women with fluid overload should be audited. This should include unrecognised fluid overload in women presenting post-operatively as well as all women where fluid overload was identified during surgery. [GPP]Surgeons should understand the factors that can lead to complication related to distension media. [GPP]Surgeons should understand the factors that influence the severity and nature of complications arising from excessive systemic fluid absorption. [GPP]Surgeons should be aware of the potential complications when using different distension media during hysteroscopic surgery. [D]Surgeons should be cognisant of symptoms associated with distension media complications to allow timely recognition. [D]Fluid absorption of over 1000 ml of hypotonic solution can cause clinical hyponatraemia. [C]Mild symptoms can develop even with absorption of over 500 ml of a hypotonic solution. [D]Larger volumes of isotonic solution need to be absorbed to cause symptomatic fluid overload but there are no data to define a safe threshold. [A]Isotonic medium is considered safer than hypotonic media as fluid absorption does not cause hyponatraemia. [GPP]Fluid deficit should still be closely monitored when using either hypotonic or isotonic distension media. [C]A maximum fluid deficit of 1000 ml should be set when using a hypotonic solution in a healthy woman and surgery immediately stopped on reaching this limit. [GPP]A maximum fluid deficit of 2500 ml should be set when using an isotonic solution in a health woman and surgery immediately stopped on reaching this limit. [GPP]Lower thresholds for fluid deficit should be considered in the elderly and women with cardiovascular, renal or other co-morbidities. Suggested upper limits are 750 ml for hypotonic solutions and 1500 ml for isotonic solutions although these limits may need to be reduced depending upon the clinical condition of the woman during surgery. [D]Most serious complications associated with fluid media are related to excessive absorption during surgery and rarely gas or air embolism during the procedure. [D]Isotonic, electrolyte-containing distension media such as normal saline should be used with mechanical instrumentation and bipolar electrosurgery because it is less likely to cause hyponatraemia where there is fluid overload. [D]Hypotonic, electrolyte-free distension media such as glycine and sorbitol should only be used with monopolar electrosurgical instruments. [GPP]Carbon dioxide gaseous media should not be used for operative hysteroscopy. [B]Preoperative administration of GnRH agonists should be considered in premenopausal women before hysteroscopic resection of fibroids. [B]Intracervical injection of dilute Vasopressin can be considered before dilatation of the cervix. [B]The intrauterine pressure needed for distension should be maintained as low as possible to allow adequate visualisation and kept below the mean arterial pressure. [D]Delivery of the distension medium can be safely and effectively achieved using simple gravity, pressure bags or automated delivery systems. [D]Automated pressure delivery systems facilitate the creation of a constant intrauterine pressure and accurate fluid deficit surveillance which is advantageous with prolonged cases such as endometrial resection or hysteroscopic myomectomy. [GPP]Mechanisms should be in place to monitor fluid deficit during operative hysteroscopic surgery. [GPP]Closed systems should be used as they allow more accurate measurement of the fluid output. [GPP]Drapes that contain a fluid reservoir should be used as they allow measurement of the fluid output. [D]Automated fluid measurement systems are more accurate than manual measurement but they can still overestimate fluid deficit. Their use should be considered for prolonged complex hysteroscopic procedures where fluid absorption is anticipated. [GPP]Measurement of the fluid deficit should be done at a minimum of 10 min intervals during hysteroscopic surgery. [B]Where feasible, the use of local anaesthesia with sedation should be considered for performing operative hysteroscopic procedures rather than general anaesthesia because fluid overload may be minimised.


Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM  is evaluated as target for FGS in combination with the novel Artemis imaging system.The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein.All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent.This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery. Curative intended oncologic surgery aims to completely resect all malignant cells. Discriminating tumour from healthy tissue during surgery is therefore of paramount importance . As a covβ3 integrin, or they are abundantly present in only a limited percentage of tumour (types), like folate receptor, EGFR and HER2.The first NIR imaging systems for clinical application proved the feasibility of the concept but were in fact more proto-types than standard clinical equipment. The next generation NIR imaging systems, as used in this study, are more versatile, smaller, cheaper, and more sensitive and should meet uniform standards warranting an exponential increase for clinical applications. Therefore, presently the biggest challenge for clinical introduction of FGS is the development of specific tumour targeting NIR fluorescent agents that comply with these second generation imagers. Various established membrane-bound tumour markers are under evaluation as targets for (NIR) fluorescence imaging in pre-clinical settings, such as EGFR, HER2/Neu, VEGF(R), folate receptor alpha, uPAR and various integrins –17. AlthIn this study we evaluate Epithelial Cell Adhesion Molecule (EpCAM) as target for FGS. EpCAM is a transmembrane glycoprotein involved in cell-cell interactions and cell-stroma adhesion . EpCAM eIn the current study we conjugated the same monoclonal antibody 323/A3 to the clinically relevant NIR fluorescent dye IRDye800CW and evaluated this conjugate in tumour models of clinically relevant orthotopic colorectal, breast and head-and-neck cancer models. To simulate clinical conditions we evaluated the performance of the probe with the recently introduced commercially available Artemis imaging system  in compa2. Cells were cultured for not more than 10 passages and regularly checked for Mycoplasma infection by PCR.For colorectal, breast and head-and-neck cancer, we selected two cell lines with different tumour characteristics. Luciferase transfected cells were used to follow orthotopic tumour growth by bioluminescence imaging (BLI). MCF-7 and OSC-19 cells were transfected with Luciferase 2 and green fluorescent protein as described previously . All cel6 per tube in ice cold phosphate-buffered saline (PBS), they were incubated with 0.4 μg/ml 323/A3 anti-EpCAM antibody or isotype control MOPC21 for 30 min on ice. Then cells were washed three times in ice cold PBS and incubated with a goat anti-mouse IgG1-AF488 secondary antibody . The cells were washed three times in ice cold PBS and resuspended in 400 μL PBS containing propidium iodide to exclude dead cells from the analysis. Flow cytometry was performed using the LSRII (BD Biosciences). The experiments were performed in duplicate and EpCAM expression was estimated as the geometric mean of fluorescence intensity measured in 10,000 viable cells. For quantitative determination of EpCAM numbers per cell type the Qifikit (Dako) was used.EpCAM expression of HT29(−/+)luc2, COLO320, OSC-19-luc2-cGFP, FaDu-luc2, MCF-7-luc2-cGFP and MDA-MB-231 cells was evaluated by flow cytometry. Cells were cultured until 90% confluence and detached with trypsin. Viability of the cells was evaluated using trypan blue. After adjusting the number of cells to 0.5 × 101k isotype control monoclonal antibody MOPC21  were used [α = 2 × 109 M−1) for EpCAM and is directed against the EGF-like domain I epitope on the extracellular domain of the EpCAM molecule, whereas MOPC21 has an unknown specificity after testing on human and rodent tissues [EpCAM specific monoclonal chimeric antibody 323/A3 and the IgG tissues –35. Both2. At 4, 24, 48 and 96 h 20 μl of sample, diluted in 40 μL PBS was evaluated using HPLC in PBS at a flow rate of 0,5 ml/min for 60 min, detected at 2 channels, 280 and 780 nm.The stability of 323/A3-800CW in human serum was evaluated using HPLC . Serum and sodium azide dilution were filtrated through a 0.22 μm filter in a 15 ml tube. A 24-wells plate  was prepared with 0.02% sodium azide and serum/probe in a ratio of 1:1 and PBS as control and incubated at 37 °C under 5% COA cell binding assay was performed to confirm the EpCAM specificity of 323/A3-800CW. HT29-luc2 , COLO320 , OSC-19-luc2-cGFP , FaDu-luc2 , MCF-7-luc2-cGFP  and MDA-MB-231 cells  cells were seeded in a black 96-well plate . At ±90% confluence the cells were washed twice with PBS. 323/A3-800CW and MOPC21-800CW were added in a concentration range of 0–8 μg/ml and incubated for 1 h at 37 °C. After incubation, the cells were washed twice with culture medium without supplements. Bound antibody was imaged with an Odyssey scanner (LI-COR), scanning at the 800 nm channel. To correct the fluorescence signal for the number of tumour cells per well a cell nucleus staining was performed: The cells were fixed/permeabilized with acetone/methanol for 10 min, washed with PBS, and incubated with TO-PRO-3 (Invitrogen) at 1:1000 for 5 min at room temperature. After washing twice with PBS, the plate was imaged with the Odyssey scanner at the 700 nm channel to detect TO-PRO-3 fluorescence. The ratio of the 800 and 700 nm fluorescence was plotted. The experiments were performed in triplicate.ad libitum. Their general health state was monitored by weight measurements throughout the experiments. Tumour growth was monitored longitudinally by visual inspection of the tumours, caliper measurements and/or by bioluminescence imaging. Bioluminescence imaging was performed by intraperitoneal injecting of 150 mg/kg of D-luciferin solution  in PBS, in a total volume of 50 μL. After 10 min, mice were imaged with the IVIS Spectrum imaging system . Imaging procedures were performed under isoflurane gas anaesthesiaColon cancer models5 HT29 cells in 40 μL RPMI1640 medium. Tumour growth was followed by calliper measurements and after 10 days, when the tumours reached a volume of approximately 75 mm3, imaging experiments started. Orthotopic HT29-luc2 tumours were induced as described previously [To induce colon tumours, mice were subcutaneously injected at four sites with 5 × 10eviously .5 MCF-7-luc2-cGFP cells were inoculated in two contralateral mammary fat pads. Oestrogen pellets  were implanted subcutaneously. Tumour growth was followed by visual inspection and bioluminescence measurements as described above.To induce orthotopic breast tumours, 2.5 × 10Breast carcinoma model4 OSC-19-luc2-cGFP cells. When tumours were visible and bioluminescence signal ranged between 5 × 109 and 1 × 1010 relative light units (RLU) imaging experiments started.Orthotopic tongue tumours were induced in the tip of the tongue through a submucosal injection of 4 × 10Peritonitis carcinomatosa5 MCF-luc2-cGFP cells. Tumour growth was followed twice a week by bioluminescence as described above. Imaging experiments initiated when multiple tumour nodules were formed of various sizes.Multiple small MCF-luc2-cGFP tumours were induced in the peritoneum by intraperitoneal injection of 2.5 × 10Nude Balb/c female mice , aged 4–6 weeks, were housed in individually ventilated cages and provided with food and sterilized water Real-time NIR fluorescence imaging and operative resection of the tumours was performed using the next generation Artemis imaging system . An earlier iteration of this system was extensively validated . This syNext to the Artemis imaging system, the Pearl Impulse small animal imaging system (LI-COR) was used as a preclinical reference to visualize tumours and calculate the tumour-to-background ratios (TBRs). Data from the Artemis and Pearl imaging systems were analysed using imageJ  and the Pearl Cam Software, respectively.2, 1 nmol (≈150 μg) of 323/A3-800CW (n = 3), 1 nmol (≈150 μg) MOPC-800CW (n = 3) or 1 nmol (≈1.1 μg) of 800CW carboxylate was injected intravenously. NIR fluorescence signals were measured at 0, 4, 24, 48, 72 and 96 h after injection using the PEARL small animal imaging system and the intraoperative Artemis imaging system after which TBRs were calculated.Subcutaneous HT29 colon tumours were used to confirm in vivo EpCAM specificity of 323/A3-800CW and to measure fluorescence over time. When the subcutaneous HT29 colon tumours were 36 ± 6 mmn = 3) or 1 nmol MOPC21-800CW (n = 3) was intravenously injected in each group. Fluorescence imaging of mice bearing orthotopic tumours was performed 72 h after administration for optimal TBR as determined in the subcutaneous HT29 colon carcinoma model. TBRs of orthotopic tumours were measured and tumours were resected under NIR fluorescence guidance using the Artemis imaging system. Ex vivo, fluorescent measurements of resected tissue were performed on a back table. Tumours were sliced and fluorescence measurements were performed on the sections to evaluate the distribution of the probe. Resected tumours from the head-and-neck cancer and breast cancer models were assessed by BLI imaging and GFP fluorescence imaging .After in vivo confirmation of the EpCAM specificity and establishment of the optimal time frame for imaging with the subcutaneous model, the clinically more relevant orthotopic MCF-7-luc2-cGFP breast, OSC-19-luc2-cGFP tongue and HT29-luc2 colon tumours were evaluated. Hence, 1 nmol of 323/A3-800CW (n = 3), as described above and fluorescence imaging using the Pearl and Artemis imaging system was performed. A midline abdominal incision was made and the abdominal skin was removed. Fluorescence imaging of the mice with both imaging systems was performed followed by resection of the peritoneum and again fluorescence images were taken. Fluorescence imaging of the abdominal area was performed to search for residual intraperitoneal tumour nodules. Presence of tumour nodules and tumour specific NIR fluorescence was confirmed by bioluminescence imaging and GFP fluorescence imaging, as described before. Receiver Operator Curve (ROC) analysis was performed for the detection of micrometastases in the peritoneum. The overlay of the BLI and GFP signals was used as the ground truth for tumour metastases and the ascending TBRs as positive cut off criteria. The area under the curve (AUC), including the sensitivity and specificity rates at the optimal TBR cut off were computed. The signals of NIR fluorescence and the overlay with BLI and GFP was confirmed against pathology in the primary tumor but not in the micrometastases.The MCF-luc2-cGFP peritonitis carcinomatosa tumour model was used to determine the minimal tumour sizes that could be detected by intra-operative fluorescence imaging using the EpCAM specific antibody 323/A3-800CW in combination with 2 imaging systems. Because an enhanced permeability and retention (EPR) effect in these micrometases is not expected as indicated recently by Hall et al.  no MOPC2Multiple regions of interest were drawn in the tumour and in adjacent normal tissue and divided by each other to calculate TBRs. TBRs of subcutaneous colon and orthotopic breast tumours were calculated with skin overlying the tumour and adjacent normal tissue. Tongue tumours were imaged through an epithelial cell layer covering the tumour and normal tissue. For colon tumours the peritoneum was opened.Three of the six nude Balb/C mice with bilateral orthotopic MCF-7-luc2-cGFP breast tumours were pre-injected with unconjugated 323/A3 antibody . After 48 h, all six mice were intravenously injected with 1 nmol 323/A3-800CW. Then, 72 h after injection of 323/A3-800CW fluorescence imaging of all mice was performed using a Pearl imaging system. Mice were sacrificed and tumours were collected. Quantification of fluorescence was done as described before . In brieex vivo fluorescence measurements, tumours were snap frozen in isopentane and kept at −80 °C. Tissues were sectioned at 10 μm and fluorescence imaging was performed using the Odyssey imager. The presence of OSC-19-luc2-cGFP and MCF-7-luc2-cGFP tumour cells was confirmed by fluorescence microscopy (Nikon Eclipse e800). All histologic sections were stained with standard haematoxylin-eosin stain (HE) after acetone fixation. To confirm the presence of HT29 and HT29-luc2 cells, sections were stained with an anti-human wide-spectrum cytokeratin antibody . Primary antibodies or controls were incubated for 60 min at room temperature. All slides were three times washed with PBS and incubated with Envision anti-rabbit (DAKO) for 30 min at room temperature. Subsequently, the slides were washed with PBS and staining was visualized by using 3,3-diaminobenzidine. Sections were counterstained with haematoxylin, dehydrated and mounted with pertex. Frozen OSC-19-luc2-cGFP and MCF-7-luc2-cGFP tumours were stained with an anti-cGFP staining . Sections stained with anti-cGFP were fixated with 4% formalin for 10 min. After washing with PBS, cells were treated with 0,1% saponin/PBS for 10 min and incubated with the anti-GFP antibody, diluted in 0,1% saponin/PBS for 60 min at room temperature. Adjacent sections were fixated with aceton for 10 min followed by three washes with PBS and stained for cytokeratin as described before.After t-test was used to calculate the overall difference between the EpCAM specific and control groups. The two-way repeated measurement ANOVA was corrected using the Bonferroni correction. A P-value equal or lower than 0.05 was considered significant.For statistical analysis, SPSS statistical software package  was used. TBRs were calculated by dividing the fluorescent signal of the tumour by fluorescent signal of surrounding healthy tissue. TBRs are reported in mean and standard deviation. A two-way repeated measurement ANOVA was used to assess the relation between TBRs in the dose groups and time points. Furthermore a paired Student’s The colon adenocarcinoma cell line HT29 showed intermediate high expression of EpCAM while COLO320 hardly had any EpCAM expression , see Fig. p < 0.01) difference was observed between 323/A3-800CW and MOPC21-800CW signals on MCF-7, HT29, OSC-19 and FaDu cells, but not on the low EpCAM expressing COLO320 and MB-MDA-231 cells  and the control antibody MOPC21) were labelled with IRDye 800CW Fig.  in compalls Fig. . The flu were lablls Fig. .Fig. 2Cop < 0.01, n = 3 in both groups), see Fig. p < 0.01) between EpCAM specific (323/A3-800CW) and non-specific control (MOPC21-800CW) could be observed from 24 h post injection (TBR 3.2 ± 0.1 and 1.7 ± 0.2 respectively). MOPC21-800CW and IRDye 800CW showed a significantly lower TBR at all time points (p < 0.01).In vivo specificity of 323/A3-800CW was shown by competition with unconjugated antibody. Pre-injection of 1 mg unconjugated 323/A3, resulted in a decrease of the percentage of injected dose per gram of tumour from 3.2 ± 0.9% ID/g to 1.0 ± 0.1% ID/g (p < 0.01) in TBRs were observed between the orthotopic colon (HT29) mice injected with 323/A3-800CW (TBR 13.5 ± 3.6) and MOPC21-800CW (TBR 1.8 ± 0.4) . Examples of in vivo and ex vivo images that were acquired with the Artemis imaging system after injection of 323/A3-800CW or MOPC21-800CW are shown for the orthotopic colon  Fig. . For the.4) Fig. . Signifilon Fig.  and b, blon Fig.  and headlon Fig. . Camera p < 0.0001) for detecting micro metastases with a sensitivity of 93% and specificity of 92% at the curve’s optimal TBR cut off value  323/A3-800CW, using both the Pearl and Artemis imaging system  of 323/A3-800CW to visualize tumours. This is the equivalent of 0.5 mg/kg for humans when converted using the body-surface-area method . Using t outcome , 45.Although the anti-EpCAM/800CW conjugate performed well in various tumour types, this probe could still be optimized. The use of antibodies with relatively high affinity, like 323/A3, might culminate in heterogeneous tumour staining, concentrated around the tumour vascularization rather than homogeneous throughout the tumour. The use of an antibody with low or intermediate EpCAM affinity for EpCAM might promote a more homogenous distribution and avoid adverse effects at the cost of a lower total tumour uptake , 46. An FGS is a promising technique to ensure intra-operative fluorescence feedback of tumour margins. Clinical success is only achieved by using a dedicated camera system and tumour-specific agents. This study aimed to achieve complete preclinical validation of an EpCAM targeting fluorescence agent in combination with a next-generation Artemis imaging system. We showed the ability to visualize, primary tumours and millimetre sized tumour nodules and metastases that were otherwise invisible for the human eye. As a novel EpCAM specific optical agent can be used in a wide variety of tumours, together with the knowledge from previous clinical trials and the results from this study, this paves the way for a fast and cost effective clinical translation.