Our model couples Poiseuille and Darcy descriptions of vascular and interstitial flow, respectively, and incorporates spatially heterogeneous blood vessel lumen and interstitial permeabilities to generate accurate predictions of tumour fluid dynamics. Our platform enables highly-controlled experiments to be performed which provide insight into how tumour vascular heterogeneity contributes to tumour fluid transport. We detail the application of our framework to an orthotopic murine glioma (GL261) and a human colorectal carcinoma (LS147T), and perform sensitivity analysis to gain an understanding of the key biological mechanisms which determine tumour fluid transport. Finally we mimic vascular normalization by modifying parameters, such as vascular and interstitial permeabilities, and show that incorporating realistic vasculatures is key to modelling the contrasting fluid dynamic response between tumour samples. Contrary to literature, we show that reducing tumour interstitial fluid pressure is not essential to increase interstitial perfusion and that therapies should seek to develop an interstitial fluid pressure gradient. We also hypothesise that stabilising vessel diameters and permeabilities are not key responses following vascular normalization and that therapy may alter interstitial hydraulic conductivity. Consequently, we suggest that normalizing the interstitial microenvironment may provide a more effective means to increase interstitial perfusion within tumours.Cancers exhibit spatially heterogeneous, unique vascular architectures across individual samples, cell-lines and patients. This inherently disorganised collection of leaky blood vessels contribute significantly to suboptimal treatment efficacy. Preclinical tools are urgently required which incorporate the inherent variability and heterogeneity of tumours to optimise and engineer anti-cancer therapies. In this study, we present a novel computational framework which incorporates whole, realistic tumours extracted  The structure of tumours varies widely, with dense and chaotically-formed networks of blood vessels that differ between each individual tumour and even between different regions of the same tumour. This atypical environment can inhibit the delivery of anti-cancer therapies. Computational tools are urgently required which facilitate a deeper understanding of the relationship between blood vessel architectures and therapeutic response. We have developed a computational framework which integrates the complex tumour vascular architecture to predict fluid transport across all lengths scales in whole tumours. We apply our model to two tumour cell-lines and show that differences in their inherent vascular structures influence flow through cancerous tissue. We also use our platform to predict the fluid dynamic response following vascular normalization therapy in realistic, static tumour networks and show that the response is dependent on tumour vascular architecture. We hypothesise that therapy may alter the permeability of interstitial tissue to fluid transport and show that lowering interstitial fluid pressure is not a necessary therapeutic outcome to increase tumour perfusion. In silico modelling is one such tool which can meet this need by testing novel therapeutic strategies at a much faster rate and cheaper cost than preclinical experimentation [Architectural heterogeneities in cancerous tissue limit the delivery of anti-cancer drugs by inhibiting their ability to circumnavigate the entire tumour to all cancerous cells . In solientation .For a systemically-administered agent to effectively target diseased tissue, it must travel from the site of delivery to the site of disease, whilst minimally interacting with normal tissues and not degrading . This isThe irregular microenivronment is typically characterised by hypoxia, acidosis and elevated interstitial fluid pressure (IFP) –14, whicμm in vivo [in silico methods provides scope to provide such detail.Heterogeneities in the underlying morphology of tumours, such as vessel diameters and lengths, and inter-branch distance, exist across individual tumours and tumour cell-lines . These v in vivo . HoweverMathematical models have been used to investigate the tumour microenvironment and have provided detailed insights which may otherwise be unavailable experimentally. Seminal models have indicated that a leaky tumour vasculature induces elevated IFP, reduced fluid penetration into the interstitium , 27, andin vivo experimentation [in silico modelling to predict tumour vascular blood flow [Subsequent studies have sought to incorporate the spatially heterogeneous effects of tumour vasculature using computer-generated synthetic networks which retain key features of tumour vascular architecture –34, or bentation , 36. Morood flow  and introod flow . Howeverex vivo optical imaging of cleared tissue specimens have enabled large samples (up to 2 cm3 with > 105 blood vessels) to be imaged in three-dimensions, at resolutions down to a few microns [REAlistic Numerical Image-based Modelling of biologicALTissue substratEs) which combines optical imaging of cleared tissue with mathematical modelling and in vivo imaging, within a unified framework, to generate quantitative, testable predictions regarding tumour transport [in silico predictions of blood flow, vascular exchange and interstitial transport. REANIMATE enables new hypotheses to be generated and tested in a manner that would be challenging or impossible in a conventional experimental setting. We have previously used REANIMATE to explore the impact of vascular network topology on fluid transport and vascular disrupting therapy (Oxi4503) to two colorectal cell-lines (LS147T and SW1222) [Recent advances in  microns . We haveransport . The pla SW1222) .in vivo ASL-MRI data [We develop here a computational model to efficiently simulate both intra- and extravascular fluid transport across large, discrete microvascular networks . Our modMRI data . FollowiMRI data , the vasMRI data .In this study, we detail the generalised model which allows for spatially heterogeneous hydraulic conductances and conductivities. A comprehensive description is provided of its application to whole tumour vascular networks. We then apply our model to an orthotopic murine glioma (GL261) and a human colorectal carcinoma xenograft (LS147T) and reproduce physiological conditions observed in literature. We perform sensitivity analysis to the model parameters associated with transvascular fluid delivery, such as vascular hydraulic conductance and interstitial hydraulic conductivity, to explore the impact on the tumour IFP and interstitial fluid velocity (IFV) profiles.Finally, we use our computational framework to explore the biomechanics underpinning vascular normalization. Vascular normalization is a method that applies anti-angiogenic therapy to restore tumour vascular structure and function to physiological levels , 43–45. Preclinical and clinical evidence indicates that anti-VEGF  therapy creates a transient window of vessel normalization which improves tumour oxygenation and the delivery of therapeutic agents , 49. Howin silico model in combination with realistic, static vasculatures to replicate fluid dynamics changes observed experimentally during vascular normalization [Here, we recruit our lization –53 by moin vivo arterial spin-labelling MRI (ASL-MRI) was performed on a subset of GL261 and LS147T tumours, from which a mean tumour perfusion of 130 ± 50 and 19 ± 8 ml/min/100g was measured [Orthotopic murine gliomas and human colorectal carcinoma xenograft from the GL261 and LS147T cell-lines (n = 6 for each), respectively, were grown subcutaneously in 8–10 week old, female mice. Following 10 to 14 days of growth, measured , respectmeasured . Full demeasured .Whole-tumour blood vessel networks were segmented from the OPT data for both tumour types. A combination of three-dimensional Gaussian and Frangi filters were applied to the data to enhance vessel-like structures allowing for the segmentation of the blood vessels from the background see . Skeletoin silico development and testing. Vessel diameters ranged from 17.9 ± 9.3 and 8.9 ± 2.8 μm, with branching lengths of 68.7 ± 48.3 and 88.8 ± 49.4 μm, respectively , thereby permitting predictions of whole tumour fluid dynamics which incorporate the inherent architectural heterogeneities occurring at the micron-scale.Our computational framework is compartmentalised into two models. The first predicts blood flow through the tumour vasculature and the second predicts interstitial fluid flow throughout the cancerous tissue through use of non-singular Green’s functions. Our method enables application to whole, large vascular networks .The segmented tumour networks consist of a series of vessel segments connected by nodal junctions or, in the case of boundary nodes, one-segment nodes which form a boundary to the microvascular network see . We defiKik represents network conductanceS is the set of all segments,lj, dj and μj denote the length, diameter and effective blood viscosity of segment j, respectively.Following the notation of Fry et al. , the matin vivo blood viscosity laws, which prescribe the effective blood viscosity as a function of vessel diameter and haematocrit, to compute μj and consequently incorporate non-Newtonian effects in each individual microvessel [We apply empirical rovessel . Networkrovessel .pk0 is the target pressure at node k, τj is the wall shear stress in segment j, τj0 is the corresponding target shear stress, kp and kτ are weighting factors associated with the pressure and shear deviations from the target values, λi is the Lagrange multiplier associated with node i and wk is the vessel length associated with node k. Setting dL/dpi = 0 and combing with , u, within the porous interstitial domain:κ is the hydraulic conductivity of the interstitial tissue. Here we assume that interstitial pressure tends towards a constant value, p∞, in the far-field region,Darcy’s law has been effectively used to describe the passage of fluids –33, 65 oJv and Lp are the fluid flux across and the hydraulic conductance of the vessel wall, respectively, S is the surface area of the vasculature, σ is the oncotic reflection coefficient and, Δp and ΔΠ are the fluid and oncotic pressure gradients between the vasculature and surrounding tissue.Tumours are leaky due to large pores along a vessel’s lumen, and so the vasculature exhibits a strong fluid and oncotic interaction. Following the approach of Baxter and Jain  and subsxj and ϕj are the spatial coordinates and strength, respectively, of point source j, and δ(x − xj) is the delta function. The term ϕj(x)δ(x − xj) represents a point source of fluid flux from the vasculature to the surrounding interstitial domain.The tumour vascular architecture is used to spatially parameterise the locations of a discrete set of sources of flux into the interstitial domain. Assuming these sources both supply or drain the interstitium, conservation of mass yieldsG, for the adjoint problem for Applying the substitution r0. We assume continuity of G and ∇G at the interface r = r0, which enables the following radially symmetric Green’s function to be derived:For a given source, we distribute the delta function over a sphere of finite radius, Ns sources across the vasculature with a maximum spacing of δ, and assign a source radii, ri0, equal to the corresponding blood vessel radius  and Πv,lated by ) and Πb,i and assuming flux at the interface is continuous, we definerv,i is the vessel radius and li is the length of vessel i.Integrating over the volume of vessel Ns equations to be solved for the source fluxes pi,b, using Starling’s law, , subsequently, tissue IFP and IFV fields can be computed using  an an16) anlues see , the resg’s law, , in the g’s law, . Here, fed using  and 14)Ns equatiIn effect, our computational framework enables a detailed, quantitative assessment of blood and interstitial flow for tissues, both healthy and pathological, where their entire vascular networks are characterised, by encapsulating the flow of fluid between the vascular and interstitial domains. Similar to a Green’s function model for O2 transport , our modThe computational framework was coded using C++  and run in vivo, which necessitates a pragmatic approach to boundary condition assignment. Under the assumption that vessels along the tumour surface are connected to peritumoural vessels [in vivo measurements of mean perfusion from ASL-MRI  defining the surface of the tumour; 2) computing the IFV vectors across the tumour surface; and 3) approximating the total tissue perfusion.Application of our boundary assignment method requires us to accurately compute mean perfusion across the tumours for a comparison against equivalent experimental data gathered boundary’ function applied to all nodes defined during vascular segmentation  To approximate tumour perfusion requires us to define a set of normal vectors to the tumour surface to compute pressure gradients. We identify the centre of the tumour and duplicate the hull, which is then expanded to form a 10 μm gap between the two surfaces (see 1) The hull of a tumour is calculated using the Matlab  ‘tion see . The Mataces see . IFP is aces see . Any infp∞, was set to 0 mmHg. Due to a lack of experimental data, the vascular conductance and interstitial conductivity, Lp and κ, respectively, were given uniform values based on literature . Asectively .in silico experiments to establish the tumour parameters, such as vascular and interstitial permeability, which change tumour IFP and perfusion following vascular normalization therapy, and show how vascular architectural heterogeneities affect treatment response.In the following section we apply our computational framework to a GL261 orthotopic murine glioma and a LS147T human colorectal carcinoma xenograft to form baseline flow solutions see Figs  and 4. NOur vascular flow simulations are in good agreement with those in computational , 76 and in vivo ASL-MRI measurements of 110 ± 70 and 19 ± 8 ml/min/100g for GL261 and LS147T, respectively. This implies that imposing physiologically realistic pressure boundary conditions generates physiologically realistic perfusion, and consequently accurate drug delivery solutions [Tissue perfusion  was further validated by olutions .in vivo . Consid[in vivo  and the ions see .ρ = 0.41, p < 0.001, where ρ and p are the Pearson’s correlation coefficient and its corresponding p–value between tumour radius and vascular flow, respectively).Examining the tumour radial IFP profiles, the LS147T network exhibited similar configurations as observed both experimentally in LS147T , in otheIn comparison, the GL261 network also exhibited a traditional, yet steeper, IFP profile with a wider variance throughout the tumour see . The IFVρ = −0.584, p < 0.001—including in silico predictions from [High IFP has been associated with low vascular density in A-07-GFP tumours . Similarons from ). This mons from  directlyHere, we perform sensitivity analysis to understand the impact of parameter variance on predictions of tumour fluid dynamics. In doing so, not only do we understand the sensitivity to the model, but gain insight into the biological mechanisms at play.source parameters, which include the vascular blood pressure, pb,i and the source radius, ri0, for segment i, and the maximal spacing between each source along a given vessel, δ  in which IFP displayed an increased gradient compared to greater values of κ along with decreasing IFV (see κ ≥ 8.33 × 10−9 cm2 / mmHg s displayed similar IFP profiles with mean IFP decreasing with increasing κ (26.2 ± 1.5 to 22.5 ± 1.6 mmHg).Interstitial hydraulic conductivity, ther see . For κ ≤ IFV see . In compIn the next section we apply our computational model to predict changes in tumour fluid dynamics following vascular normalization therapy.in silico modelling.Vascular normalization is a therapeutic strategy used to restore tumour vasculature to a structural and functional state exhibited by healthy blood vessels , 51, 79.Lp and σ), as a result of increase pericyte coverage, ranges linearly from typical tumour values in the core to physiological levels at the periphery. Here, vessel diameters were decreased by a factor of 1.99 [Lp and σ to 0.44 × 10−7 cm mmHg−1 s−1 and 0.91 [We next use our computational framework to investigate the effectiveness of vascular normalization in both GL261 and LS147T tumours where normalization therapy is administered intraperitoneally , 82. We  of 1.99 , and setand 0.91 , respectSimilarly to experimental , 52 and Our therapeutic predictions of vascular normalization are inconsistent with the magnitude of observed effects, such as reduction in IFP, compared to experimentation , 53. Thiκ  IFP and (right) IFV for (blue) pre- and (red) post-normalization. Error bars represent standard deviation.(TIF)Click here for additional data file.S2 FigLp, σ, vessel diameters and κ, and (red) κ. Error bars represent standard deviation.Plots show (left) IFP and (right) IFV for normalization of (blue) (TIF)Click here for additional data file.S3 FigError bars represent standard deviation.(TIF)Click here for additional data file.


In this study, we designed a backpack that can reduce the moment arm of backpack load by placing the center of gravity of the backpack close to the axis of the spine. In order to investigate the effect of sagittal spinal alignment compared with the general backpack, we conducted the study using radiological images. The participants in this study were 18 adults . The subjects participated in the experiment without carrying the backpack, wearing the normal backpack, and wearing a backpack designed to reduce the load moment arm by placing the center of gravity close to the body. Spinal alignment parameters were measured and analyzed using 3D radiography measurement software based on radiographic images taken under three conditions. The overall angle of lumbar lordosis, upper arc, lower arc, difference between pelvic incidence and lumbar lordosis, lower cervical lordosis, and sagittal vertical axis were measured. In the case of wearing the backpack rather than without the backpack, there was a significant difference in the overall angle of lumbar lordosis, lower arc, lower cervical spine angle, difference between pelvic incidence and lumbar lordosis, and sagittal vertical axis. In the case of wearing the backpack with reduced moment arm, the overall angle and lower arc of lumbar lordosis were significantly increased compared to those with the normal backpack. The difference between pelvic incidence and lumbar lordosis was significantly decreased. The results showed that a normal backpack caused imbalance of sagittal spinal alignment, and the backpack reducing the load moment arm by placing the backpack’s center of gravity close to the vertebral joint played a positive role in reducing the change of lumbar alignment compared with the normal backpack. Due to the recent increase of sedentary life, the normal alignment of the spine is damaged, and this abnormal posture causes pain and musculoskeletal problems . RecentlIn general, lumbar lordosis is defined as the angle between L1–S1, and thoracic kyphosis is defined as the angle between T4–T12 . It is aThe alignment of the spine is greatly affected by backpacks, which are an important means of carrying. In a previous study, hyperlordotic spinal posture increased significantly after walking with wearing a backpack . This hyThus, discussions about the alignment of the lumbar and cervical spine with wearing backpacks are ongoing; however, the conclusions vary. The above study used an anatomic maker of the skin rather than an accurate measurement method such as radiation; however, previous studies have reported that using anatomic markers without images like radiation was quite limited in analyzing spinal alignment . TherefoIt is recommended that the weight of the backpack not exceed 10–15% of user’s weight . ExcessiIn this study, the backpack was designed to reduce the moment arm of the backpack load by keeping the backpack load position close to the axis of the spine. In order to investigate how it affects spinal alignment compared with general backpacks, we conducted a study using radiological images.Eighteen adults  over 19 years old participated in this study. Subjects who had severe communication disorders such as cognitive impairment or aphasia, congenital malformations or diseases of the spine, pelvis, feet, orthopedic or neurosurgery diseases of the spine and legs, severe limitations in the range of joint movement, and who were judged to be inappropriate to participate in this study by the researcher were excluded from this study. This study was conducted with the approval of the Bioethics Committee of Gimcheon University (GU-201709-HRa-06-02). All subjects were fully informed about the experiment and agreed to participate in the study.The subjects participated in the experiment without carrying the backpack, wearing the normal backpack, and wearing a backpack designed to reduce the load moment arm by placing the center of gravity close to the body. Typical backpacks are 30 cm width, 15 cm depth, and 45 cm height, with the design having a strap starting from the top of the backpack connecting to the lower part on the side . The bacIn a previous study on the weight of a backpack, the weight of the backpack was mainly studied with weights of 5%, 10%, and 15% of the weight of the subject. Usually over 10% has been shown to affect the subject’s spine . TherefoRadiographic images were acquired in all 3 conditions: without carrying the backpack, with the normal backpack, and with the backpack with reduced moment arms. Digital X-ray radiography  was used to acquire the lateral projection radiograph of the whole spine of the subject. As a test method, the centerline of the X-ray was positioned between thoracic spine no. 7–10 according to the subject’s height, and the two images of X-ray irradiation in both up/down directions were stitched to express the whole image of the spine. To minimize the distortion of the image caused by the diverging phenomenon of the X-ray, the focus to detector distance (FID) was set to 229 cm, and the external auricular meatus and femur head in the X-ray field size were used as reference points to include the whole spine of the subject. In this study, both feet of the subject stood at shoulder width so that the weight of the bag was evenly distributed throughout the spine, and the subject was instructed to keep straightening their knee for weighting baring. In order to minimize the overlap of the shoulder and humorous parts with the thoracic spine column in the image, the position of the subject’s hand was placed on the support located at the height of the shoulder, forming an angle of about 30 degrees between the humorous long axis and the coronal plane.Spinal alignment indices were measured and analyzed using 3D radiography measurement software  based on radiographic images taken under 3 conditions: without carrying the backpack, with the normal backpack, and with the backpack with reduced moment arms. An expert with more than two years of radiologist experience marked the apex of the lumbar lordosis and the inflection point on the sagittal image . The upper arc of lumbar lordosis is the angle formed by the horizontal line in the apex of lumbar lordosis and parallel to the upper edge of the lower spine at the inflection point. The lower arc is the angle formed by the horizontal line in the apex of lumbar lordosis and parallel to the upper edge of the first sacrum. The overall lumbar lordosis was presented by adding the lower and upper arcs. Pelvic incidence is the angle between the vertical line at the midpoint of the sacral plate and the line connecting the same point to the center of the bicoxofemoral axis . The difStatistical analysis of all collected data was performed using the SPSS version 20.0 statistics program for Windows . The general characteristics of the subjects were presented as mean and standard deviation of all variables measured using descriptive statistics. Repeated measure ANOVA was used to compare each spinal alignment under the three conditions. In order to test the statistical significance, the significance level α was set to .05 for all analyses.The subjects of the study were 18 adults including 10 females and 8 males. The subjects were 22.8 ± 1.8 years old, 165.9 ± 8.2 cm in height and 66.7 ± 12.2 kg in weight. The subjects’ weights ranged from 48 kg to 85 kg. The weight of the backpack was adjusted to 10% of the weight by increasing or decreasing the number of acrylic plates.Radiographic images were analyzed to compare the lower cervical spine alignment under three conditions: the absence of a backpack and with backpacks of two different designs. The lower cervical spine angle was significantly decreased with the backpack compared to without the backpack. There was no significant difference in cervical spine alignment by backpack design .Lumbar lordosis was reduced with a normal backpack compared with no backpack. However, the overall lumbar lordosis was significantly increased when wearing the backpack with reduced moment arm compared with when wearing the normal backpack. The same pattern appeared in the lower lumbar spine, but the change of the total lumbar spine was affected by the lower lumbar spine. There was no significant difference in the upper lumbar spine .The difference between pelvic incidence and lumbar lordosis was significantly increased with a normal backpack compared with no backpack. However, when wearing a backpack with reduced moment arms, there was no significant difference compared to the state without the backpack .In the state of wearing the backpack, the shortest distance between the sagittal vertical axis, i.e., the centerline of the 7th cervical vertebra, and the posterior upper corner of the sacral spine increased significantly compared to the state of no backpack. However, there was no significant difference in sagittal vertical axis by backpack design .A backpack is important equipment that can contain educational materials, sports equipment, and personal items . A typicPrevious studies reported that increasing thoracic kyphosis, lumbar lordosis and scoliosis did not contribute significantly to the increase of lower back pain or dorsal pain. However, there is a limitation in the previous studies in that the alignment and clinical features of the spine were confirmed using anatomic markers on the surface of the back rather than the radiological method on sagittal and frontal curvature ,19. TherThe sagittal balance of the spine is very important for spinal health. It has been reported in previous studies that patients with spinal sagittal imbalance should restore lumbar lordosis in order to correct sagittal imbalance . In prevIn a previous study, wearing a backpack reduced lumbar lordosisLBL by flexing lumbar to move the back load into the base of supportBOS ,10. SomeIn this study, pelvic incidence was measured together, which is closely related to lumbar lordosis and does not change after growth. Characteristics of pelvic incidence do not change after growth, which is useful for determining the state of lumbar lordosis. In adult spinal deformity, the recovery of lumbar lordosis is important, and pelvic incidence is the criterion for determining how much to restore lumbar lordosis. Patients with large pelvic incidence should have more lumbar lordosis correction because of greater lumbar lordosis. Lumbar lordosis is known to be suitable for ± 9 degrees of pelvic incidence. Health-related disability was severe when the difference between pelvic incidence and lumbar lordosis was above 11 degrees . In thisSagittal balance is determined by the C7 plumb line . The norIn previous studies, wearing a backpack tended to increase the head-on-neck extension (HNL), corresponding to the upper cervical spine angle, which reduces the LBL to move the load behind the back into the BOS. At the same time, it is reported that tilting the torso forward increased HNL to keep the gaze straight forward . Upper aBackpacks are the most common means of carrying in everyday life, so it is important to study not only standing posture but also walking posture . TherefoThe results in this study showed that wearing a backpack caused an imbalance of sagittal spinal alignment, similar to the results of a previous study. In particular, it reduced lumbar lordosis, which is important for sagittal alignment and affected pelvic and cervical spine alignment. The backpack’s load caused stress in the back of the spine. The backpack designed in this study reduced the moment arm of the vertebral joint and separation. It was also shown to reduce lumbar alignment changes compared to conventional backpacks. Therefore, not only the weight of the backpack but also the center of gravity is a very important factor regarding spinal load. Further studies related to the design of backpacks are necessary for spine health.


Background: A common practice in the management of critically ill patients is fluid resuscitation. An excessive administration of fluids can lead to an imbalance in fluid homeostasis and cause fluid overload (FO). In pediatric critical care patients, FO can lead to a multitude of adverse effects and increased risk of morbidity.Objectives: To review the literature highlighting impact of FO on a multitude of outcomes in critically-ill children, causative vs. associative relationship of FO with critical illness and current pediatric fluid management guidelines.Data Sources: A literature search was conducted using PubMed/Medline and Embase databases from the earliest available date until June 2017.Data Extraction: Two authors independently reviewed the titles and abstracts of all articles which were assessed for inclusion. The manuscripts of studies deemed relevant to the objectives of this review were then retrieved and associated reference lists hand-searched.Data Synthesis: Articles were segregated into various categories namely pathophysiology and sequelae of fluid overload, assessment techniques, epidemiology and fluid management. Each author reviewed the selected articles in categories assigned to them. All authors participated in the final review process.Conclusions: Recent evidence has purported a relationship between mortality and FO, which can be validated by prospective RCTs . The current literature demonstrates that “clinically significant” degree of FO could be below 10%. The lack of a standardized method to assess FB  and a universal definition of FO are issues that need to be addressed. To date, the impact of early goal directed therapy and utility of hemodynamic parameters in predicting fluid responsiveness remains underexplored in pediatric resuscitation. In a critically ill patient, fluid balance is imperative in management and maintaining homeostasis. More often than not, patients are resuscitated with fluids to maintain adequate intravascular volume. A common issue seen in pediatric intensive care units is fluid imbalances and hemodynamic instability. Aggressive fluid administration can lead to fluid overload (FO), a condition in which there is a positive fluid balance in the patient. This phenomenon has been associated with a multitude of unfavorable effects and can further complicate the patient's condition. While FO itself is no indicator of mortality, the adverse effects of FO on an already at risk population puts them at an increased risk of morbidity and mortality. Research on this commonly practiced standard of care has shown that inundation is not always the best method for hemodynamic optimization. Current literature shows that there is a lack of a standard definition of fluid overload and a standard guideline by which to direct fluid therapy. The purpose of this study was to review the literature highlighting the impact of FO on a multitude of outcomes in critically-ill children, analyzing the causative vs. associative relationship of FO with critical illness, and addressing the current pediatric fluid management guidelines.The literature search was conducted using PubMed/Medline, and Embase databases for studies on fluid overload in pediatric critical care patients <18 years, from the earliest available date until June 2017. A total of 477 articles were obtained; after duplicate removal and review, 48 articles were selected for data extraction  in maintaining an intact vascular biology. EGL, a web of membrane-bound glycoproteins and proteoglycans on the luminal surface of endothelium, binds to plasma proteins and effectively excludes proteins from the sub-endothelial cleft, thereby resulting in a local oncotic gradient between plasma and cleft which opposes trans-capillary efflux . The lawOf note, some data regarding glycocalyx and natriuretic peptides is partly theoretical, and partly derived from adult studies and there is no pediatric-specific data in this context –10.Despite the burgeoning evidence regarding association between FO and adverse patient outcomes, there is no consensus on the optimal method for calculating FO. Lombel et al. highlighted that depending upon the definition used, there was a significant variability in the number of patients identified with >10% FO .Most pediatric studies utilize the FB method introduced by Goldstein et al. . The forMore practical WB(weight based) methods can be alternatively used to estimate degree of FO. The formulas are:orTable 2). Nonetheless, reports have shown correlation between the two and a similar predictive potential of mortality ], where TBW = 0.6 × weight (kg) . HoweverRe-analysis of FACTT  showed that presence of covert AKI was independently associated with a higher risk of death. Incidence of AKI was greater in the conservative arm before adjustment but in liberal arm post-adjustment . Secondapost-hoc analysis of FACTT, Grams et al. showed FO as a causal intermediate in the association between diuretic administration and decreased mortality in AKI patients, suggesting that the mortality benefit associated with furosemide was due to a decrease in FO  and increased RRT-free days (p = 0.0017) (n = 610) in the PICARD study also had a significantly higher %FO at CRRT initiation (p = 0.01) and cessation (p = 0.004)  in a critically ill patient, FO being one of them. RRT provides control in fluid management and allows for the ability to achieve a negative fluid balance . Associa 0.0017) . Non- su= 0.004) . A subse= 0.004) .Table 4)  .In neonates with congenital heart disease undergoing cardiac surgery, fluid management is of particular importance. These patients are administered large amounts of fluid pre, intra, and post-operatively to prevent hypotension. The mainstay of management in these patients is diuretic therapy, with furosemide being the most widely administered drug . FO in tp = 0.05), higher risk of death and were more likely to require prolonged MV (p < 0.03) and oxygen support than neonates without AKI even post-adjustment (Association between FO and BPD (bronchopulmonary dysplasia) has been an active area of research. Studies document an association between FB and weight loss in the first few weeks of life and subsequent development of BPD in preterm neonates –66. In tjustment . These sV p < 0.0 and oxygjustment . In the justment . Literatjustment .p < 0.001), VFDs (ventilator-free days) and ICU-free days were significantly lower in the conservative arm  but similar to adults in the liberal arm, and promulgated the use of a Bayesian pediatric trial mirroring FACTT. No association with mortality was noted  fluid resuscitation within first 6 h in the treatment of septic patients  has been an area of intense research since its inception in the landmark Rivers' study wherein a mortality benefit was sought using protocolized  is the preferred resuscitating fluid but development of significant hyperchloremic acidosis necessitates a switch to hypotonic fluids such as 1/2NS or more physiological choices with lower chloride but high sodium concentrations such as LR (lactated Ringer's), Hartmann's solution, or plasmalyte . In pediIn cases of pediatric sepsis, different studies have shown varying and conflicting data on which fluids provide adequate resuscitation without increasing mortality. The SSCG recommend crystalloids as the initial resuscitation fluid of choice, with addition of albumin only if substantial amounts of crystalloids are required . HoweverIn a recent large matched cohort study on pediatric sepsis, LR resuscitation was not associated with reduced mortality, AKI, or dialysis even when matched by fluid volume and proportionate LR utilization . MortaliEarly FO has garnered much attention as it seems to be a more decisive factor than late FO in predicting survival because the peak of illness is typically in first few days post-admission and the CFB curve generally flattens out after this period , 75, 76.The lack of a specific recommendation on optimal timing of RRT institution still plagues clinicians. CRRT remains the modality of choice in pediatric critical care. The American College of Critical Care Medicine recommends that a threshold of 10% should be construed as the time for intervention . An expeThe prediction of FR (fluid responsiveness) in critically ill children remains largely unembraced to date despite its potential role in obviating FO as only 40–60 % of patients typically respond to fluids by increasing the cardiac output by more than 10–15% . The hemLiterature on fluid resuscitation in pediatric patients is vast but there is no general consensus on the type and dose of resuscitation fluid. Though there is an association between fluid overload and the risk of BPD in a neonate, there is no consensus or guideline on whether fluid restriction or the management of FO will reduce BPD incidence. This is just one example showing the current lack of knowledge on fluid management in children. Further studies and well-designed RCTs are necessary to provide physicians with optimal guidelines. The limited predictability of fluid responsiveness in children along with limited knowledge on the utility of early goal directed therapy further complicates the management of FO. There is a need for more studies to better clarify the effects of FO and create a standardized set of guidelines to assist physicians with management and decrease adverse outcomes.Managing fluid overload in a critically ill pediatric patient is no simple task and poses a multitude of complications. To begin with, the lack of a proper consensus on a method for calculating FO due to the significant number of uncertainties in estimating intake and output makes management a challenge. While FO itself is not a direct marker of mortality, the adverse effects of FO lead to patients being vulnerable to an increased risk of morbidity and mortality. FO puts patients at a risk of being underdiagnosed with AKI and delays treatment, raises odds for mortality associated with CCRT complications, can lead to increased hospital and ICU stay, and prolonged ventilator support in the critically ill population. Given the current lack of knowledge on fluid management in children, further studies and RCTs are necessary to provide guidelines to physicians.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. RR, SKS, NW and SBB conceptualized the study. RR, SKS, NW and SBB wrote the review. MV and VK made significant contributions in writing, editing and revising the study.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.


There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear.In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFNγ for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry.We discovered that IFNγ increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes.Here we clarify for the first time an additional mechanism of anti-tumour effect—as exerted by anti-PD-1 antibody decreasing Treg— we anticipate that our findings will lead to the development of new methods for cancer treatment. In the microenvironment of cancer, the role of innate immunity is inhibited in a process known as immune tolerance. One of the mechanisms of immune tolerance is the immune checkpoint mechanism, whereby T cells are suppressed to prevent excessive immune responses. Several types of immune checkpoint molecules are known, namely the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3), in addition to programmed cell death 1 (PD-1) and its ligand 1 (PD-L1) . PD-1 isNormal cells are believed to express PD-L1 in an inflammatory environment, suppress T cells, and prevent excessive tissue damage from long-term persistence and spread of inflammation . HoweverImmune checkpoint molecules also play an important role in Treg that are involved in suppressing the function of cytotoxic T cells. Treg expresses CTLA-4, which is an immunity checkpoint molecule on the cell surface that suppresses the activity of antigen-presenting cell (APC), resulting in the suppression of T cell activation . The antThis study used osteosarcoma as a tumour model to elucidate its relationship to the anti-tumour effect of anti-PD-1 antibody and Treg. Osteosarcoma is reported as a tumour that is susceptible to immunotherapy  with greIn this study, we first evaluated the mechanism of PD-L1 expression in osteosarcoma cells in vitro to establish osteosarcoma as a tumour model. The anti-tumour effect was also confirmed in vivo from the changes in tumour volume and overall survival time of anti-PD-1 antibody administration using a subcutaneously implanted mouse model of osteosarcoma. Furthermore, changes in the tumour microenvironments were evaluated in detail, and the relationship between anti-PD-1 antibody and Treg was examined. From these experiments, we report that anti-PD-1 antibody suppresses tumour-infiltrating Treg and exerts an antitumour effect.Murrin osteosarcoma cell line , human osteosarcoma cell lines HOS  and SaOS-2  were cultured in α-MEM containing 5% fetal bovine serum (FBS). 143B human osteosarcoma cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% FBS. All cell cultures were maintained in 5% CO2 at 37 °C. For all cell lines, frozen aliquots of 10^6 cells were stored at a passage below 5 and a fresh aliquot used after 5 consecutive passages. All of cell lines were additionally tested by Riken cell bank and found to be negative for mycoplasma.Mice were housed and maintained at the Committee for Animal Experiments of Shinshu University. Mice were housed with an inverse 12 h day-night cycle with lights on at 9 am in a temperature-controlled room. All mice were allowed free access to water and a maintenance diet. Based on the national regulations and guidelines, all experimental procedures were reviewed by the Committee for Animal Experiments and finally approved by the president of Shinshu University. The animal protocol was approved by the Committee for Animal Experiments of Shinshu University .Male C3H/HeSlc mice ) were bought by Japan SLC . The established protocol  is used n = 3). Cells were detached with trypsin, and surface markers were detected by flow cytometry.Recombinant IFNγ  species-matched to human osteosarcoma cell lines  and murine osteosarcoma cell line (LM8) was administered and incubated for 24 h . Mouse anti-PD-1 antibody  was intraperitoneally administered three times per week at 20 mg/kg of body weight per dose for a total of five doses. 200 μl of PBS was intraperitoneally administered to the control group. Mice were followed up until reaching the humane endpoint or until natural death. Macroscopic tumour size was calculated using the established method [Murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of 4-week old mice (1 × 10d method  three tin = 3). To obtain a sufficient amount of tumour at 2 weeks after the initial administration of 4H2, the initial administration was withheld until day 7. 4H2 was intraperitoneally administered three times per week at 20 mg/kg of body weight per dose for a total of five doses. 200 μl of PBS was intraperitoneally administered to the control group. All mice were euthanized at day 21, and the tumour and spleen were excised.Subcutaneously transplanted mouse models of osteosarcoma were prepared (n = 5). In the short-term administration group, 4H2 was intraperitoneally administered twice a week at 20 mg/kg per dose for a total of four doses. In the long-term administration group, administration of anti-PD-1 antibody was continued twice a week until the endpoint. For the control group, PBS was administered twice a week until the endpoint. Mice were followed up until reaching our euthanasia criteria or until natural death. Changes in tumour size, body weight, and μCT were observed over time.Subcutaneously transplanted mouse models of osteosarcoma were prepared , cells from the spleen and tumour were resuspended in red blood cell lysis buffer  and incubated at room temperature for 1 min to remove erythrocytes. Cells were filtered again through a 40 μm cell strainer. Finally, the cells were re-pelleted and re-suspended in RPMI 1640 medium.Mice were euthanized by inhaled isoflurane overdose. The tumour of the dorsal subcutaneous and spleen were excised in a clean environment. The excised spleen was physically triturated and suspended in RPMI 1640 medium . Cells from tumours were isolated using a previously reported  method wP values less than 0.05 was considered statistically significant.The mice were separated to experimental and control group at random. Statistical analysis was performed with SPSS software ver. 25  using the unpaired two-tailed t-test or Bonferroni’s multiple comparison test. Kaplan-Meier curves were created by the same software and log rank analysis was used for comparison between groups. p = 0.0022, 143B; 1.83, p = 0.011, SaOS-2; 2.14, p = 0.027, and LM8; 1.3, p = 0.0017. Figure p value: iso vs 0 = 0.0019, iso vs 100 = 0.0056, 0 vs 100 = 0.031. 143B; 0 group 2.70, 100 group 5.65, p value: iso vs 0 = 0.010, iso vs 100 < 0.0001, 0 vs 100 = 0.0010. SaOS-2; 0 group 1.54, 100 group 3.85, p value: iso vs 0 = 0.25, iso vs 100 = 0.025, 0 vs 100 = 0.039. LM8; 0 group 0.99, 100 group 13.19, p value: iso vs 0 = 0.93, iso vs 100 = 0.00034, 0 vs 100 = 0.00034. Figure Osteosarcoma cell lines were used to verify the mechanism of PD-L1 expression in osteosarcoma. Analysis of human  and mouse (LM8) osteosarcoma cell lines by flow cytometry revealed that IFNGR1 was expressed in all cell lines  was administered to subcutaneously implanted models of murine osteosarcoma cell line LM8 and evaluated in vivo. First, anti-PD-1 antibody was administered three times per week for a total of five times and compared to the control (Fig.  was admiTo investigate changes in the tumour microenvironment following the administration of anti-PD-1 antibody, all mice were euthanized 2 weeks after the initial administration of anti-PD-1 antibody, spleen and tumour were collected, and cells were isolated Fig. a. Expresp = 0.73, Fig. p = 0.15, Fig. 5 /g, 4H2; 3.6 ± 0.8 × 105 /g. p = 0.047, Fig. p = 0.14, Fig. p = 0.13, Fig. p = 0.013Fig. p = 0.046, Fig. In the spleen, the administration of anti-PD-1 antibody showed no changes in the ratio of T cell to B cell (Control; 0.53 ± 0.16, 4H2; 0.54 ± 0.19. We found that the long-term administration of anti-PD-1 antibody suppressed tumour volume and prolonged overall survival. Even in the group administered with 4H2 for 2 weeks, the speed of tumour growth increased at approximately 1 week after final administration (Fig. 3, short-term 412.8 ± 302.8 mm3, long-term 132.3 ± 41.2 mm3, p value: control vs short-term = 0.72, control vs long-term = 0.093, short-term vs long-term = 0.074. Day 17; control 904.8 ± 549.8 mm3, short-term 392.9 ± 260.9 mm3, long-term 132.8 ± 16.3 mm3. p value: control vs short-term = 0.097, control vs long-term = 0.014, short-term vs long-term = 0.057. Day 21; control 1844.1 ± 1041.5 mm3, short-term 649.7 ± 384.5 mm3, long-term 198.3 ± 124.0 mm3. p value: control vs short-term = 0.043, control vs long-term = 0.0080, short-term vs long-term = 0.037. Day 24; short-term 726.5 ± 430.8 mm3, long-term 260.1 ± 92.3 mm3. p = 0.045. Day 28; short-term 1200.4 ± 952.8 mm3, long-term 586.1 ± 253.2 mm3. p = 0.20. Day 31; long-term 694.4 ± 231.2 mm3. Day 35; long-term 800.5 ± 202.9 mm3. Figure p = 0.0002. Figure p value: control vs short-term = 0.0044, control-term vs long-term = 0.00024, short-term vs long-term = 0.013. Figure For the long-term administration group, 4H2 was administered twice a week from day 0 until death or having reached our euthanasia criteria. The results were compared with a control group in addition to a short-term administration group that received only four administrations in total (Fig. In this study, we selected osteosarcoma as a tumour model due to its susceptibility to immunotherapy. First, the pattern of expression for PD-L1 in osteosarcoma cells was confirmed. Next, we verified the effectiveness of anti-PD-1 antibody on osteosarcoma in animal experiments and clarified the mechanism of action of anti-PD-1 antibody by evaluating the tumour microenvironment. The above results demonstrate that osteosarcoma is suitable as a model for evaluating the action of anti-PD-1 antibody on tumour.Two types of PD-L1 expression in the tumour are known: an innate mechanism induced by genetic mutation and an adaptive mechanism induced by the stimulation of inflammatory cytokine, the latter of which is primarily caused by IFNγ  or by a Furthermore, we confirmed the inhibition of tumour growth by anti-PD-1 antibody and the prolonged overall survival for subcutaneously implanted mouse models of osteosarcoma in vivo. These results demonstrate that osteosarcoma is a suitable model for evaluating the effect of anti-PD-1 antibody on tumours.Many studies have been conducted on changes in the tumour microenvironment during the use of immune checkpoint inhibitors in various carcinomas –37. It hIt is widely known that Treg suppresses immunity against tumours in the tumour microenvironment . Treg isIn this study, we showed that the survival time of subcutaneously transplanted mouse models of osteosarcoma was prolonged by the administration of anti-PD-1 antibody. Furthermore, the survival time is likely to extend further with a long-term administration of anti-PD-1 antibody. As expected, long-term administration of anti-PD-1 antibody was found to prolong survival time compared to short-term administration in this study.We revealed for the first time that anti-PD-1 antibody suppresses the infiltration of Treg into tumour and exerts an antitumour effect. Although osteosarcoma was used as a tumour model in this study, future studies should investigate these results further in the context of other tumours. If the mechanism of action in tumour suppression of anti-PD-1 antibody is established in many types of cancer, it will enable us to provide an explain regarding the higher response rate of anti-PD-1 antibody compared to anti-PD-L1 antibody in clinical practice . In termWe believe that this study not only affects the use of anti-PD-1 antibody in clinical practice, but also serves to facilitate the development of new treatments for tumours with a focus on the relationship between anti-PD-1 antibody and Treg.It is limitation of this study that the most results are from mouse transplantation model of osteosarcoma. Because it is difficult to be proven this model behaves the same as the human disease, further studies like using humanized mice or collecting the data from clinical specimen of osteosarcoma used anti-PD-1 antibody are needed.The suppression of Treg by the administration of anti-PD-1 antibody is a new finding and may offer new insights into the mechanism of action of the anti-PD-1 antibody. The results of this study could be a useful asset in designing a combination therapy of anti-PD-1 antibody with other drugs. Moreover, because an antitumour effect can be anticipated through the suppression of Treg, clinical importance can also be placed in the possible therapeutic effects for tumours with no expression of PD-L1.Additional file 1: Table S1. List of antibodies.


Social networks such as Twitter offer the clinical research community a novel opportunity for engaging potential study participants based on user activity data. However, the availability of public social media data has led to new ethical challenges about respecting user privacy and the appropriateness of monitoring social media for clinical trial recruitment. Researchers have voiced the need for involving users’ perspectives in the development of ethical norms and regulations.This study examined the attitudes and level of concern among Twitter users and nonusers about using Twitter for monitoring social media users and their conversations to recruit potential clinical trial participants.We used two online methods for recruiting study participants: the open survey was (1) advertised on Twitter between May 23 and June 8, 2017, and (2) deployed on TurkPrime, a crowdsourcing data acquisition platform, between May 23 and June 8, 2017. Eligible participants were adults, 18 years of age or older, who lived in the United States. People with and without Twitter accounts were included in the study.P<.005). We found associations between respondents’ Twitter literacy and their concerns about the ability for researchers to monitor their Twitter activity for clinical trial recruitment (P=.001) and whether they consider Twitter monitoring for clinical trial recruitment as eavesdropping (P<.001) and an invasion of privacy (P=.003). As Twitter literacy increased, so did people’s concerns about researchers monitoring Twitter activity. Our data support the previously suggested use of the nonexceptionalist methodology for assessing social media in research, insofar as social media-based recruitment does not need to be considered exceptional and, for most, it is considered preferable to traditional in-person interventions at physical clinics. The expressed attitudes were highly contextual, depending on factors such as the type of disease or health topic , the entity or person monitoring users on Twitter, and the monitored information.While nearly half the respondents—on Twitter  and on TurkPrime —indicated agreement that social media monitoring constitutes a form of eavesdropping that invades their privacy, over one-third disagreed and nearly 1 in 5 had no opinion. A chi-square test revealed a positive relationship between respondents’ general privacy concern and their average concern about Internet research (The data and findings from this study contribute to the critical dialogue with the public about the use of social media in clinical research. The findings suggest that most users do not think that monitoring Twitter for clinical trial recruitment constitutes inappropriate surveillance or a violation of privacy. However, researchers should remain mindful that some participants might find social media monitoring problematic when connected with certain conditions or health topics. Further research should isolate factors that influence the level of concern among social media users across platforms and populations and inform the development of more clear and consistent guidelines. Previous research suggested that Twitter provides a “rich and promising avenue for exploring how patients conceptualize and communicate about their specific health issues”  diagnosis may be increased by taking part in the study. The study team, by echoing the information about an individual’s diagnosis, may amplify this information, so it’s more likely to come to the attention of the public or an employer.We used this feedback as guidance and motivation for designing the following research study to ascertain people's attitudes and level of concern about the use of social media monitoring on Twitter for targeted clinical trial recruitment.Scientists have pointed out a lack of the inclusion of public views to inform future practices in social media research and social media-enabled recruitment ,21. Furtnonexceptionalist methodology  to contribute to moving science forward.Finally, we used a set of vignettes to assess the association between the level of concern and different variables, such as the disease or health topic of the clinical trial and the entity that monitors social media user activity on Twitter.Eligible participants were adults, 18 years of age or older, who lived in the United States. People with and without Twitter accounts were included in the study.We used two online methods for recruiting study participants, who made up our convenience sample: the open survey was (1) advertised on Twitter between May 23 and June 8, 2017, and (2) deployed on TurkPrime, a crowdsourcing data acquisition platform, between May 23 and June 8, 2017 . Accessiage targeting to adults aged 18 or older, (2) location targeting to the United States, (3) language targeting to users who understand English, and (4) keyword and hashtag targeting for words and hashtagged words that Twitter users have tweeted or searched for on Twitter related to four main categories. The four categories to which targeted keywords or hashtags were related were (1) social media and social media surveillance, (2) research participant recruitment and clinical trial enrolment, (3) ethics and Internet privacy, and (4) clinical research and clinical trials. Each ad included a brief description , an image related to survey taking, a request for volunteers needed, a request for providing feedback, and a link to the questionnaire. Twitter ads were posted by the principal investigator's Twitter handle . Our recruitment target was 500 participants. The daily maximum ad budget was set at US $49 with a total budget of US $980 for the entirety of the project. Respondents on Twitter had the opportunity to enter a raffle to win one of 10 US $100 gift cards upon completion of the survey. Duplicate and fraudulent responses were identified and removed as described by Teitcher et al  data, including Access Key ID and Secret Access Key, are encrypted with AES-256 encryption, the standard adopted by the National Institute of Standards and Technology.Study data were collected and managed using Research Electronic Data Capture (REDCap), an electronic data capture tool, hosted at the University of Southern California. REDCap is a secure, Web-based application designed to support data capture for research studies, providing (1) an intuitive interface for validated data entry, (2) audit trails for tracking data manipulation and export procedures, (3) automated export procedures for seamless data downloads to common statistical packages, and (4) procedures for importing data from external sources .The paid ads posted on Twitter included a link to the survey hosted on REDCap. Respondents filled out a multipage survey online on either a mobile device or desktop. On TurkPrime, each respondent was provided with a unique link to a separate survey hosted on REDCap. The datasets used for analysis were generated directly from REDCap using the platform’s reporting tools. Please see the Twitter and TurkPrime recruitment sections for further details.A total of 603 participants completed the survey and passed the attention-check questions in this study: 94 (15.6%) on Twitter and 509 (84.4%) on TurkPrime. Among the initial 704 respondents on Twitter alone, we used Excel filters to identify and remove 70 respondents (9.9%) who did not show correct completion of the attention-check questions and 540 respondents (76.7%) who gave fraudulent responses with unique characteristics. Regarding the fraudulent responses, all of them (1) showed the same age ; (2) were submitted about 5-10 minutes apart from each other over a period of 5 days; (3) used email addresses with a consistent pattern, namely, a random English word followed by three to six random letters ; and (4) were confirmed to be fraudulent when respondents were asked to verify the information provided through the survey about their first name, age, and highest degree or level of school they had completed. For each filtered entry, we manually reviewed the email address to identify fraudulent emails . Finally, we manually sent a message to each email address and asked the users to verify the information they provided in their survey responses. Among the initial 738 responses on TurkPrime, we removed 229 responses (31.0%) that did not show correct completion of the attention-check questions.Very or somewhat concerned and a low level of concern as responses that indicated Not too or not at all concerned. Finally, we also analyzed the responses using descriptive and inferential statistical techniques  to determine, generally, where respondents had strong concerns regarding the use of Twitter monitoring in clinical trial recruitment and where respondents had a weaker understanding of Twitter’s functions and usage policies. In particular, we looked to see if the concern regarding the use of Twitter monitoring in clinical trial recruitment was correlated with greater or less knowledge of Twitter, the type of monitoring used, or the method of outreach to the potential recruit. We report the results in aggregated form with all individually identifying information removed.We did not use any methods to adjust the sample, such as weighting of items or propensity scores. We analyzed the data on two levels: (1) at the respondent level to test control variables  and (2) at the vignette level to test independent variables . Survey responses were first analyzed through descriptive statistical methods to assess the distribution of participants across our dependent and independent variables, such as the degree of privacy concern and demographic factors. Next, data regarding the different levels of concern for each vignette were further analyzed using pivot tables to identify any relationships between the levels of general privacy concern and the participants’ attitudes regarding the vignettes. We defined a high level of concern as responses that indicated The study was reviewed and approved by the IRB at the University of Southern California (HS-17-00348).Overall, the sample of 603 participants showed the following distribution see : 324 werWe further assessed Twitter usage among the 603 survey participants see . Of the We attempted to assess the level of Twitter literacy and knowledge among study participants see . OverallWe sought to learn more about general privacy concerns associated with the use of the Internet see . Of the sponsored tweets) to try to increase the likelihood that a clinical trial recruitment message gets seen by as many individuals as possible. Finally, 259 out of 603 respondents (43.0%) expressed some level of concern about Twitter keeping track of whether they clicked on a Twitter recruitment message related to a health study, for example, “Seeking participants for a #Cancer study.”We also assessed respondents’ concerns about Internet research activities, which pertain to the use of their Twitter data for research purposes see . We founHypothesis 1 states that social media monitoring on Twitter for clinical trial recruitment is perceived as eavesdropping and an invasion of privacy.active listening may be perceived by participants as eavesdropping on their conversations about their health  who expressed some level of general concern about their privacy while using the Internet; we combined We also examined the responses of those participants  who expressed little or no general concern about their overall privacy while using the Internet; this allowed us to assess whether those with little general privacy concern might still have elevated privacy concern about Twitter monitoring. Those with lower general Internet privacy concern indicated lower concern in response to the questions about eavesdropping, privacy, and confidentiality see . SimilarHypothesis 2 states that the expectation of Internet privacy relates to the level of concern about Internet research and Twitter monitoring for clinical trial recruitment.Very concerned with Somewhat concerned responses—and compared them to the entire population reported in We wanted to gauge whether the presence of general Internet privacy concern is related to increased concern about Internet research see . Therefo216=143.0, P<.005.We then stratified responses based on Twitter use to assess whether active users of the social media platform expressed different levels of privacy concern regarding the use of Twitter for research purposes  who expressed little or no general privacy concern, we found that this population generally had lower levels of Internet research privacy concern see . For exaoses see . RespondFinally, we stratified the responses to the Twitter-monitoring vignettes see  based onWe also isolated responses for those respondents  who expressed little or no general concern about their overall privacy while using the Internet; this allowed us to assess whether those with little general privacy concern might still have elevated privacy concern about the types of Twitter monitoring described in the vignettes. As reported in P<.001), as reported in Finally, we performed chi-square tests to explore whether there was a relationship between general Internet privacy concern and levels of concern expressed with each vignette. The tests revealed a statistically significant relationship in all cases  between respondents’ Twitter literacy and their concerns about the ability of researchers to monitor their Twitter activity, generally, for the purpose of clinical trial recruitment (see P=.004) between respondents’ Twitter literacy and their concerns about researchers monitoring particular information types on Twitter  for the purpose of clinical trial recruitment. Overall, there was a significant association (P=.03) between respondents’ Twitter literacy and their overall concerns with researchers monitoring Twitter activity.There was a significant association (ment see . This reP<.001) and an invasion of privacy (P=.003). There was no significant association, however, between Twitter literacy and whether respondents felt that Twitter monitoring jeopardized confidentiality (P=.43).Related to the CIRB’s concerns, we also found a significant association between respondents’ Twitter literacy and whether they considered Twitter monitoring for clinical trial recruitment as eavesdropping  is doing the Twitter monitoring was the most common factor that impacted concern across all scenarios, regardless of whether the overall Internet privacy concern was low or high; the exception was with the HIV/AIDS scenario, where respondents who expressed overall concern noted that The nature of the disease/medical condition being monitoredfor was the main contributing factor. For the obesity and HPV scenarios, a noticeably larger portion of the respondents who expressed some concern also noted that the Use of Twitter as a method in which the researchers contacted you was also a contributing factor.We further stratified responses for each vignette’s subquestions, isolating responses for those who expressed some concern—indicated All relevant data that support the findings of this study are available in the data repository figshare:Responses from Twitter users: Monitoring Twitter for clinical trial recruitment .Responses from TurkPrime workers: Monitoring Twitter for clinical trial recruitment .Public social networks such as Twitter provide access to user information, including personal and sensitive data, without necessarily requiring an individual's knowledge or consent. While previous studies explored the unique ethical challenges of social media as a health research tool and research data source ,20,39,40The goal of this study was to contribute data that reflect public views of Twitter users and nonusers and to inform the scientific discourse about the use of Twitter user data for clinical trial recruitment. We discuss our findings in relation to our hypotheses see  and contactive listening may be perceived by participants as eavesdropping on their conversations about their health, an invasion of their privacy, and a potential breach of confidentiality, we found that the majority of respondents did not share this view. While the CIRB’s concerns have some basis, with 4 in 10 respondents feeling Twitter monitoring is eavesdropping and an invasion of privacy, the concern was not widespread, even among those expressing higher levels of general online privacy concern. This suggests that while clinical researchers should be mindful that some Twitter users will be wary of being monitored for the purpose of clinical trial recruitment, these concerns should not prevent the recruitment strategy from being pursued. Tactics such as Privacy by Design  to be addressed privately and not through a public reply.Participant in response to the HIV/AIDS vignetteI think the public reply instead of a dm [direct message] could be embarrassing.Participant in response to the obesity vignetteThe nature of this can be very embarrassing and a public reply could be damaging.Participant in response to the HPV vignetteThis may be due to the stigma  associatThis study was limited to two populations: Twitter users and TurkPrime workers. The range of ages, education levels, and socioeconomic statuses of these populations could be more limited than those found in the general public. A total of 22% of US adults use Twitter, nearly equally among white, black, and Hispanic adults across all ages but with the highest usage among those 18-29 years of age . TurkPriAdditionally, this was an exploratory study prompted by the feedback from a national research organization  and the sample size of this study was limited. More robust studies with a larger sample could yield additional insights. Finally, we acknowledge that while we chose seven hypotheses for this initial study, there are certainly other issues and variables that deserve further attention related to the subject in future studies.nonexceptionalist methodology for assessing social media in research, insofar as social media-based recruitment in itself does not need to be considered exceptional from the participant’s perspective and, for most, it is considered preferable to traditional in-person interventions at physical clinics. Notwithstanding these findings, researchers should also remain mindful that some participants might find social media monitoring problematic when connected with certain conditions. The expressed attitudes were highly contextual, depending on factors such as the type of disease or health topic and the entity or person who monitored users on Twitter. Further research should isolate factors that influence the level of concern among social media users across platforms and inform the development of more clear and consistent guidelines.The data we presented here contribute to the critical dialogue with the public about the use of social media in clinical research. Public social networks such as Twitter offer the clinical research community a novel opportunity for identifying and engaging potential study participants based on user activity data. However, the availability of public social media data has led to new ethical challenges about respecting user privacy and the appropriateness of monitoring social media for clinical trial recruitment. The results of this study suggest that most users do not think monitoring Twitter for the purpose of clinical trial recruitment constitutes inappropriate surveillance or a violation of privacy. Our data further support the previously suggested use of the


This article has been corrected: The authors made a mistake in the grant number in Funding. The authors declare that this correction does not change the results or conclusions of this paper. The authors sincerely apologize for this error. The correct grant number of Guangdong Provincial Key Research and Development Program is marked in bold font below.5769-5785 . https://doi.org/10.18632/aging.102165Original article: Oncotarget. 2019; 11:5769–5785. 2019B110233002), Major Project of Collaborative Innovation of Guangzhou Science and Technology Program (grant number 201704020175) and Natural Science Foundation of Guangdong Province (grant number 2018A030313592).This study was supported in part by National Natural Science Foundation of China (grant number 81872006), Guangdong Provincial Key Research and Development Program (grant number


The purpose of this study was to assess the feasibility, efficacy and toxicity of fiducial marker implantation and tracking in CyberKnife® stereotactic radiation therapy (SBRT) applied to extracranial locations.This is a retrospective, single-centre, observational study to collect the data of all patients treated by stereotactic radiation therapy with fiducial marker tracking at extracranial locations, conducted between June 2014 and November 2017. Information regarding the implantation procedure, the types of toxicity related to marker implantation and the number of markers implanted/tracked during treatment were collected. Complication rates were evaluated using the CTCAE v4 [Common Terminology Criteria for Adverse Events] scale. The technical success rate was based on the ability to optimally track the tumor throughout all treatment fractions.Out of 2505 patients treated by stereotactic radiation therapy, 25% received treatment with fiducial marker tracking. The total number of implantation procedures was 616 and 1543 fiducial markers were implanted. The implantation-related complication rate was 3%, with 16 Grade 1 events and 4 Grade 2 events. The number of treated patients and the number of implanted markers has gradually increased since the technique was first implemented. The median treatment time was 27 min (range 10–76). 1295 fiducials were effectively tracked throughout all treatment fractions, corresponding to a technical success rate of 84%. The difference between the number of fiducials implanted and those tracked during treatment decreased significantly as the site’s experience increased.Fiducial marker implantation and tracking is feasible, well-tolerated, and technically effective technique in SBRT for extracranial tumors. Extracranial radiation therapy under stereotactic conditions  is defined as a high-precision treatment method that delivers a high dose of radiation to a target volume with a steep dose gradient, ensuring that surrounding critical organs are spared. Organ movement and deformation between and during treatment fractions must be characterized, verified, and taken into account during treatment planning. This challenge has led to the development of several methods to monitor the position of the target and normal tissue avoidance structures. Image-guided radiotherapy (RT) is the use of various imaging modalities before or during radiotherapy treatments to align and verify the anatomical agreement between simulation anatomy, treatment of RT and the patient at the time of treatment. The image guided in SBRT (IG-SBRT) is necessary to ensure accurate target coverage despite the necessary reductions in target volume margins expected to reduce the dose to the organs at risk.Imaging techniques may be divided into radiation-based and non-radiation-based systems. Non-radiation-based systems include electromagnetic tracking, ultrasound and MRI systems integrated into treatment machine. Radiation-based systems include static as well as real time tracking, using either kV, MV, or hybrid methods. Registration of tumor targets and normal tissue avoidance structures to the treatment delivery machine is possible via motion control devices. There are three categories of motion control techniques: the gating, the damping and the tracking. The gating consist to follow the respiratory cycle using a surrogate in order to trigger the beam only in a specific time. The damping device such as abdominal compression or breath hold maneuvers can restrain the motion. Tracking device make possible to move the radiation beam to follow the moving target.The CyberKnife® allows for frameless SBRT of extracranial locations with real-time target and motion tracking using Kv imaging methods. Two target tracking methods are currently available with this device: the fiducial tracking that requires the use of fiducial markers which are radio-opaque markers implanted around or within the tumor, and the Xsight® Lung Tracking System that is fiducial-free. Both tumor tracking methods can be combined with the Synchrony® Respiratory Tracking System, which synchronizes the beam delivery with the motion of the target due to respiration. Trackable fiducials, whose position can be measured rapidly, accurately, and objectively by a fiducial tracking system contribute for accurate patient positioning and targeting because they can provide the accuracy of fiducial-based IGRT while remaining as fast, easy, and objective as skin mark alignment. However, SBRT with fiducial tracking do not require and is not defined by one device. Each fiducial marker must remain in a fixed position in relation to the tumor and to the other markers. This ensures tracking of target translation movements  and rotational movements . Because the geometrical relationship between the fiducial marker and the target volume represents the greatest uncertainty in the CyberKnife® tracking system, optimal placement of the markers is essential . Sub-optThe purpose of this study was to assess the feasibility, efficacy and toxicity of fiducial marker implantation and tracking in CyberKnife® stereotactic radiation therapy applied to extracranial locations.The success rate is defined by the number of fiducial markers implanted in relation to the number of markers actually tracked throughout the treatment. Thus, for each patient, the number of fiducials that the CyberKnife® were not able to track has been recorded. This study was approved by our Institutional Clinical Research Committee.This is a retrospective, single-centre, observational study to collect the data of all patients treated by stereotactic radiation therapy with fiducial marker tracking at extracranial locations, conducted between June 2014 and November 2017 at the Hartmann Radiotherapy Institute. Patients requiring fiducial implantation were selected at the SBRT dedicated meetings. Information about the physician who performed the procedure, the implantation path, the success rates/rates of implantation-related complications, and the number of markers implanted/tracked during treatments were collected for all patients in whom fiducials were implanted. Eight different radiologists performed the implantation procedures. The implantation procedure and the type of fiducials implanted evolved over time, according to the experience acquired by the radiologist. The exclusion criteria were the same as for a biopsy .All procedures were image-guided and performed on patients under short-duration local or general anaesthesia. Images were taken before, at regular intervals during, and following the procedure to check for fiducial positioning and the absence of immediate complications.The fiducials were implanted close to the tumor, observing as closely as possible the fiducial implantation recommendations . Each pap < 0.0001, indicating a learning curve by the radiologists. The main radiologist had significantly fewer missing fiducials than the other radiologists, p < 0.0001. On average this radiologist had 0.23 fewer fiducials missing and this difference persisted across all the measured dates  have received IG-SBRT for extracranial locations with fiducial marker tracking. Thus, 1543 fiducial markers were implanted and 2757 sessions with fiducial tracking were performed. The median patient age at the time of treatment was 71 years (range [33–94]). The median time intervals between fiducial implantation, the simulation scan and the first treatment session were, respectively, 11 days (range [5–41]) and 15 days (range [2–36]). The median total prescribed dose was 36 Gy , the median fractional dose was 10 Gy  and the median number of fraction was 5 . The median duration for one treatment was 29 mn . The rate of implantation-related complications was measured at 3%, with 16 Grade 1 events, 4 Grade 2 and a single grade 3 event. The number of patients treated and the number of implanted markers increased since the technique was first implemented, with close to 350 patients treated with fiducial tracking over the year 2017. 468 procedures, i.e. 76% of all procedures, were performed by the same radiologist. 1295 fiducials were actually tracked throughout all treatment fractions, representing 84% of the implanted fiducials. Moreover, there is a significant decrease in the number of missing fiducials over time, All results are summarised in Tables Out of 450 patients treated by lung SBRT for primary or secondary tumors, 89 received fiducial implantations. 15 patients had previously received lung SBRT without fiducial implantation. The fiducial implantation procedure consisted in implanting a single ‘Concerto’ or ‘Gold Anchor’-type seed through guided needle puncture. Fiducial implantation in the lungs was generally performed percutaneously via the trans-thoracic route, or through endovascular access if the patient had severe respiratory co-morbidities .One hundred two fiducials were implanted and 89 patients were treated. The procedure was performed by the same radiologist in 69 patients, i.e. in 78% of the procedures. The trans-thoracic implantation route was used in 90%, and the endo-vascular route was used in 10% of the implanted patients. All seeds were correctly positioned in the close vicinity of the tumor. In two of the 89 patients, a second seed had to be implanted within the same operative step due to immediate migration of the first seed. The median number of implanted and tracked fiducials per patient was, respectively, 1, mean 1.1, SD 0.32, range .The implantation procedures were complicated by eight Grade 1 events: seven cases of pneumothorax occurring immediately after the puncture, not requiring a drain, and one episode of minimal haemoptysis. The implantation procedures were complicated by two Grade 2 events: two cases of pneumothorax occurring immediately after the puncture and requiring a drain. No cases of lung bleeding were reported. No events of Grade 3 or higher were reported. No patients required surgery or intensive care hospitalisation. No cases of secondary migration were observed on the tracking scan. The median dose was 60 Gy (range [36–60]) and the median number of fraction was 3 (range [3–6]). All implanted fiducials were used to reliably determine the lesion location, ensuring effective tracking and continuous radiation of the lesion throughout the treatment. The technical success rate was, therefore, 100%.in the years 2014 and 2015, and later using joined Fleximark-type seeds. The fiducials were implanted transperineally in the prostate in the transverse and sagittal planes under endorectal ultrasound guidance.All patients undergoing SBRT treatment in the prostate underwent fiducial implantation. The procedure consisted in placing non-joined seeds One hundred fifty patients received SBRT treatment with fiducial implantation and tracking for prostatic tumor. The median dose was 36 Gy (range [36–36.25]) and the median number of fraction was 6 . In total, 544 seeds were implanted, i.e. a median number of 4 seeds per patient, mean 3.6, SD 0.84, range [2–5]. 88 procedures were performed by the same radiologist (58%). The tolerance rate was excellent. The implantations were complicated by seven Grade 1 events: 4 transient haematuria events and 3 perineal pain events. In addition, one fiducial had migrated out of the prostate and into the perineum immediately after implantation.Four hundred seventy fiducials were tracked, i.e. 86% of the implanted fiducials. The total median number of fiducials tracked per patient was 3, mean 3.1, SD 0.57, range [2–4]. For 14 patients, two of the four implanted fiducials could not be selected at the planning stage, and therefore during tracking, as they were placed too close together or in a co-linear pattern. For all other patients (136 patients), at least three fiducials were tracked throughout the treatment. All fiducials selected at the planning stage were actually tracked during the treatment sessions. No fiducial migration was observed between the tracking scan and the last treatment fraction. The technical success rate was measured at 91%.Fiducial implantation was performed under arterial CBCT guidance, percutaneously and/or endovascularly, depending on tumor location and patient co-morbidities. The endovascular route was preferred, particularly for centrally located tumors. The advantage of the endovascular route is that is provides a better view of the tumor volume shown by the contrast medium injection. The radiologist selected the level of scan sections and the puncture site. Under radiological guidance, the radiologist performed 2 needle punctures, dropping one to two joined seeds via the needle. The markers were placed perpendicularly according to a cube-shaped pattern centred around the tumor. The edges of the cube had to be in two different spatial planes., 461 fiducials were tracked, i.e. 74% of the implanted markers, with a median number of 3 tracked fiducials per patient . The technical success rate was, therefore, 75%.One hundred fifty-one patients received SBRT treatment with fiducial implantation and tracking for primary or secondary tumoral lesions of the liver. The median dose was 45 Gy (range [40–45]) and the median number of fraction was 3 (range [3–5]). Six hundred seventeen fiducials were implanted, with a median number of 4 implanted fiducials per patient . One hundred ten procedures (73%) were performed by the same radiologist. The tolerance rate of the implantation procedure was excellent. Fiducial implantation was complicated by two Grade 2 events, i.e. a sub-capsular haematoma in 2 patients. In addition, three cases of secondary migration inside the liver were noted: one occurring during the implantation procedure and two secondary migrations observed on the dosimetry scan. FinallyOutcomes about fiducial markers implanted and tracked into lymph nodes, pancreas, bones and adrenal glands are summarized in Tables Implantation of fiducial markers for SBRT treatment of extracranial targets is a feasible, well-tolerated and effective technique, as summarised in the Table To our knowledge, the studied cohort of 616 patients who received SBRT treatment with fiducial tracking is the largest reported in the literature. Such numbers provide grounds for a reliable and comprehensive evaluation of fiducial implantation and tracking practices, since all extracranial locations were analysed. The uniqueness of this study is that it objectively evaluates the number of fiducials actually tracked for each patient during treatment, and therefore provides data not only on the toxicity profile of the implantation procedure, but also on the efficacy of fiducial tracking and the technical success of SBRT treatment. Thus, we propose that four fiducials markers need to be implant in order to track three markers concerning prostate and liver target. We also suggest that joined markers be used whenever technically possible to reduce the number of needles and therefore puncture-related complications.Because fiducial implantation is an invasive technique, it must not only ensure optimal tumor tracking, but also be acceptable for patients and involve minimal complications. It has been shown that fiducial tracking allow for high precision tumor localization and real time target tracking without the need to restrain the patient’s breathing. In this study, implantation and tracking of fiducial marker is safe because the reported rate of grade 2 or more related toxicities is 1% with six grade 2 and one grade 3 events. The size of the dataset is relevant to have a representative sample of toxicities in this setting. The safety seems good enough to justify using this invasive technique regarding the other options. However, fiducial placement has some drawbacks. It is an invasive procedure and not applicable for all patients and thus limits the application range of fiducial tracking SBRT. Otherwise, fiducial markers alter the image quality of the planning CT and make sometimes the tumor delineation more difficult.Many studies define the technical success of an implantation procedure as the fact of placing at least one fiducial in the vicinity of the target , 5; otheThis is due to the fact that the CyberKnife® fiducial tracking method has a weakness:It considers the geometrical relationship among fiducials as rigid, and the geometrical relationship between the fiducials and the target as invariant. Thus, it does not take into consideration the possibility of anatomical deformations which can change the geometrical relationships among the fiducials themselves, or between the fiducials and the target.This study also demonstrates the learning curve of the team translating in an improved efficacy of fiducial marker tracking as experience is gained. There has been a decrease in the difference between the number of implanted fiducials and the number actually tracked during treatment in the 2 years since this technique was implemented in our department. There is a significant decrease in the number of missing fiducials over time. This learning curve can be explained by several reasons. First, increasing the number of implantation procedure make it technically more efficient. Second, regular team meetings provide feedback and solutions to radiologists to improve placement. Continuous interactions between the members of the treatment team – radiographers, medical physicists, radiation oncologists, radiologists – help to identify the difficulties due to fiducial implantation and tracking, and to resolve them depending on treatment constraints Third, the implantation equipment has improved and adapted to the demand over time . Thus, radiologists gather technical expertise and achieve to be more efficient according recommendations over time as the proportion of tracked vs. implanted fiducials becomes greater.The toxicities related to the implantation are not statistically different according to the radiologist (p = 0.15). However, the rates of toxicity decrease overtime with 9, 7 and then 5 events over the three and half years. The event rates are too low to show a statistically significant difference. This study demonstrates the learning curve of the radiologist translating in an improved efficacy of fiducial marker tracking and probably in toxicities related to implantation as experience is gained. Thus, we suggest a minimum of 100 implantations procedure per year to ensure a high quality of fiducial tracking SBRT program. We recommend multi-disciplinary collaboration for implementation and application of fiducial tracking SBRT. Each patient’s proposed course of SBRT treatment should be discussed and reviewed by the multi-disciplinary stereotactic team. We think useful to develop processes for initial and ongoing training, to write departmental protocols, to structure follow up for assessment of the toxicity and efficacy of the procedure in order to evaluate and improve continuously clinical practices.A medical-economic evaluation covering the management of implantation complications, the number of fiducials to be implanted, and the number of postponed treatment sessions should, logically, demonstrate that ensuring a lower level of toxicity and higher efficacy in fiducial implantation and tracking will also help reduce per-treatment costs as the number of patients under treatment gradually increases .This study has certain limits. Firstly, it is a retrospective and single-centre study, which means the level of data evidence reported is low. However, the goal of the study was to report a preliminary experience based on a large patient population, while remaining strict on data collection quality and toxicity ratings, in order to provide reliable and comprehensive results. In addition, no long-term treatment results are reported in this cohort. This type of results, however, regarding SBRT treatment for different tumor locations is already well known, and the purpose of this work was to focus on the feasibility and toxicity of fiducial implantation and the efficacy of fiducial tracking during treatment. Lastly, successfully tracked fiducials do not guarantee good localization. Indeed, the fiducials were implanted close to the tumor, and he distance between the fiducials and the target was not recorded. It would be interesting to collect data related to this parameter in order to report on implantation quality and target tracking reliability.The rate grade 2 or more implantation related complications was very low (2%) in this report. Trans-thoracic fiducial implantation is responsible for a pneumothorax rate that varies across the different studies from 10 to 23% –11. In aTrans-perineal fiducial implantation was reliable and very well tolerated in our study, with complication rates close to those reported in the literature . One advFor intra-prostatic markers, the initial use of non-joined seeds was responsible for a decrease in the number of tracked fiducials during treatment, as the fiducials were placed too closely (less than 2 cm) and only one of the two implanted fiducials could actually be detected and tracked. Later on, by implanting joined seeds, we were able to correct this problem and increase the number of fiducials tracked during treatment sessions.The cube-shaped implantation pattern is a technique that uses limited cutaneous and adrenal entry ports: only one puncture was required for inserting a set of 2 markers. Thus, the very low Grade 2 complication rate (1%), lower than those reported elsewhere , can be Optimal fiducial marker implantation is essential for a successful SBRT treatment when fiducial tracking is required. Fiducial marker implantation and tracking is a feasible, well-tolerated and effective technique in SBRT treatment for extracranial tumors. Two and a half years after installing the CyberKnife®, over 3000 patients have received SBRT treatment, 616 using fiducial marker implantation and tracking. This new line of activity has allowed us to develop cooperation ties between the radiology and radiotherapy teams, and to discuss fiducial implantation upstream in order to select the best treatment strategies.