The tumour stroma -or tumour microenvironment- is an important constituent of solid cancers and it is thought to be one of the main obstacles to quantitative translation of drug activity between the preclinical and clinical phases of drug development. The tumour-stroma relationship has been described as being both pro- and antitumour in multiple studies. However, the causality of this complex biological relationship between the tumour and stroma has not yet been explored in a quantitative manner in complex tumour morphologies.To understand how these stromal and microenvironmental factors contribute to tumour physiology and how oxygen distributes within them, we have developed a lattice-based multiscalar cellular automaton model. This model uses principles of cytokine and oxygen diffusion as well as cell motility and plasticity to describe tumour-stroma landscapes. Furthermore, to calibrate the model, we propose an innovative modelling platform to extract model parameters from multiple in-vitro assays. This platform provides a novel way to extract meta-data that can be used to complement in-vivo studies and can be further applied in other contexts.Here we show the necessity of the tumour-stroma opposing relationship for the model simulations to successfully describe the in-vivo stromal patterns of the human lung cancer cell lines Calu3 and Calu6, as models of clinical and preclinical tumour-stromal topologies. This is especially relevant to drugs that target the tumour microenvironment, such as antiangiogenics, compounds targeting the hedgehog pathway or immune checkpoint inhibitors, and is potentially a key platform to understand the mechanistic drivers for these drugs.The tumour-stroma automaton model presented here enables the interpretation of complex in-vitro data and uses it to parametrise a model for in-vivo tumour-stromal relationships.The online version of this article (doi:10.1186/s12911-017-0461-1) contains supplementary material, which is available to authorized users. Tumours function as complex, interactive, multi-cellular aberrant organs. Within the tumour microenvironment, malignant cells exist amongst extracellular matrix, immune cells, vasculature, lymphatic vessels, fibroblasts, and other stromal cells. This tumour microenvironment can exert pro- and/or anti-tumorigenic actions, depending on context, while malignant cells create a permissive and supportive tumour matrix by secreting stroma-modulating growth factors, including PDGF, EGFR, VEGF, and TGF-β . These fStromal composition, architecture, and quantity varies between patient tumour types, while tumour heterogeneity is a hallmark of patient tumours. It is now well-recognised that complex tumour-stromal interactions underlie tumour growth, progression, and invasion. Recently, it has been demonstrated that the tumour microenvironment can affect tumour pathophysiology , therapeDespite the important role the tumour microenvironment, few pre-clinical tumour models include an extensive desmoplastic stroma or three dimensional interaction . Here isThe TSM is a predictive multiscale lattice-based cellular automaton model described by eight parameters Figs.  and 2b wτ) in the time domain.The model is built on a fixed 2D regular lattice in which each square element (or voxel) represents groups in the range of thousands of cells of one kind. These cells, based on their position in space, behave metabolically different. Further, the model considers delays inherent to the biological processes, such as cell cycle progression, which are orchestrated by an internal clock defined by the variable tau    and (13)).the tumour mass is composed of tumour tissue  and stroma ;all processes contain pure time delays, i.e. the cells have to spend a minimum time in the cell cycle before dividing; we avoid hereby the non-realistic instantaneous cell division;hypoxia and necrosis appear sequentially following increasing exposure to oxygen deprivation;hypoxia is reversible, providing oxygen levels are recovered;stromal tissue has a purely synergistic biochemical relationship with tumour tissue, supplying oxygen and nutrients; andthe high stiffness of the stroma may limit tumour progression.The model equations were written assuming that,These simple assumptions are enough to demonstrate the morphological and biomechanical roles of the stroma in different tumour phenotypes.xso{xso, yso} ⊂ V  ∈ ℝ2 as the map of oxygen computed for the first single point (xs ∈ V). Now let us define the boundary conditions as (1) Diritchlet boundary at the stromal cell, where the concentration of oxygen should match the oxygen concentration in blood giving the constant PO2(xs) = PO2blood and (2) Neumann bounds at the stroma cell, where there is a maximum (∇PO2(xs) = 0), and oxygen decreases gradually until infinity \documentFor the tumour cells, the oxygen uptake is directly proportional to the division rate. The proliferation constant is represented by a time constant Now substituting the oxygen levels on  with theSubsequently, if we take the continuous deterministic fraction of dividing cells just defined in Eq.  in discrpression  substitupression  gives thpression .In turn, stromal cells may be recruited with probability  and moveThe equations and a graphical display are summarised below and in Fig. selection of time step update time,randomly select each possible event and compare the probability of each event update the state,repeat 3–4 until all possible events have been checked,repeat from 2 until simulation time is reached.The TSM was run stochastically at a time step of mentclass2pt{minimThe PEM transforms both 2Dimensional cell culture and 3Dimensional tissue slice culture biomarker expressions into the in-vivo parameter values of the TSM using a number of mathematical strategies graphically summarised in Fig. Tumour parameters see Fig. , fit andwhere the linear matrix product in Eq.  was solvThe results for both parameters for the cell line MCF7 are (20–40 h ) and somWe now concentrate on the spatial distribution of hypoxia biomarkers to estimate oxygen diffusion parameters. IHC was carried out on paraffin embedded tissue slice sections as has been previously described in Davies et al. . To calcThis method utilises the absolute curve shift between the curves at 24 and 48 h for 20% expression of HIF1α and 10% of necrosis as the standards to calculate the delays. The parameters can be calculated withwhere The heterogeneity module uses imaging data to output a measure of stromal heterogeneity, which we define based on the fractal dimension of the 2D image. We estimate fractal dimensions via a counting-box algorithm, which counts boxes of decreasing sizes needed to cover the region of interest of our image. The total count of boxes , where the heterogeneity is a deviation from the “integer” dimension, thuswhere Finally, the stromal parameters are described directly by the total quantification and by the heterogeneity factor, aswhere Now accounting for the available data we obtain,−1 (Str/T)−1 and 0.0031 h−1 (Str/T)−1. This calculation has been summarised in Fig. for both parameters respectively which results in values of 0.064 h2 atmospheres were used, then 5 and 21% O2 with a total of 12 measurable points. Since the chemiluminescent exposure may have been slightly different, the sample results have been linearly normalised for the reference average of 21% O2.To find the oxygen concentration at which HIF1α is expressed, the MCF7 cell line was cultivated in-vitro at different oxygen concentrations  and then Western blots  were runThe results show a fair exponential distribution (as expected) with formulafor which an arbitrary expression of 20% of HIF1α would need around 0.064 mmol/L of oxygen in tissue or around 5% of atmospheric oxygen in culture see Fig. . The estIn this module, we attempt to calculate oxygen diffusion parameters as described in Eq.  by meansmentclass2pt{minimwhereby ussed in .The complete parameter set has been estimated for the cell line MCF7 only, which results are presented in Table Once the algorithms have been presented and the parameters estimated, it follows to analyse the potential of their tumour-stromal model in the different phenotypes.Using the parameters obtained from the in-vitro platforms for MCF7 summarised in Table However, the values reported for MCF7 obtained from the fitting of other in-vivo models  resulted in Ref.  - is dueble Fig. . The actble Fig.  but willn=10) and n=8) respectively , which negatively correlates to the amount of necrosis in these images, see Additional file The simulations produced by the original parameter set  will be an interesting exercise which will provide with other insights of the TSM. Especially interesting will be the usage of a cell resolution lattice and a sufficiently short time step to properly capture diffusion of macromolecules and changes in cell polarity. These computations require high specification hardware and computation time.The PEM was a useful platform to generate in-vivo-relevant parameter values from in-vitro data; nevertheless its usage relies upon the availability of the in-vitro assays mentioned above. To circumvent this, we proposed a feasible parameter extrapolation method which resulted valid and may be useful to other scientists. The results generated by the PEM-calibrated TSM for the two cell lines Calu3 and Calu6 demonstrate that the tumour-stroma mutually-positive relationship assumed here does not represent both the stromal patterns and growth rates simultaneously. This means that while the stroma constrains growth by limiting space to grow, these effects do not explain the slower growth rate of Calu3 Fig. . ExperimOur in-vitro-calibrated mathematical models describe aspects of in-vivo tumour biology extrinsic to the tumour cell, which may provide a valuable addition to existing predictive models, such as cancer pharmacokinetic-pharmacodynamic-efficacy models developed using cell line-derived xenograft models. This adds another dimension to simplistic tumour models which may allow us to better predict the effect of drugs in morphologically-complex clinically-relevant pathophysiologies , 9.Additional file 1:Supplementary material: This file contains additional information on the experiments conducted and the raw results as well as algorithmic detail of the scripts that quantify the distribution of IHC stain within a tissue culture, and the calculation of the oxygen concentrations at either interface of the tissue slice. (PDF 927 kb)Additional file 2:mainTSM_Code: Main script for the Tumour-Stroma Model (TSM). This script is a stand-alone script that simulates the model given certain parameter values and initial conditions. (CPP 9.34 kb)Additional file 3:PEM_Code: Compressed folder containing the codes for the Parameter Estimation Model algorithms and example figures. These scrips are stand-alone and contain all the necessary information to replicate the data in the manuscript. (7Z 8793 kb)


The discovery of EphA1, a novel cancer-associated receptor tyrosine kinase (RTK), in 1987 paved the way to identification of the 16 members of the Eph RTK family. Many of these were initially identified in a variety of human cancers. Eph RTKs mainly function, during development, to regulate cell positioning and migration. However, Eph receptors and their ephrin ligands are also widely and aberrantly expressed in a number of diseases, in particular, in many human malignancies, where their level of expression often correlates with increased aggressiveness [Eph family of RTKs have been shown to have a complex role in tumour formation, progression and metastasis. One aspect of this complexity is the potential for more than one of a number of Eph RTKs to be expressed in a particular tumour whilst exerting the same functional effect. A further aspect is that an Eph RTK can have either tumour promoting or inhibitory effects depending on the tumour type and the stage of the disease. The higher level of expression of the Eph receptors, compared with normal tissues, together with their functional role in cancer has led to an increased interest in developing therapeutic strategies targeting Eph proteins in many cancers .Therapies targeting Eph receptors may operate through a number of mechanisms. Where Eph proteins function as tumour promoters, therapies that target these proteins by blocking their function or their downstream signalling mechanisms may be effective. Despite most Eph proteins being functional tyrosine kinases it has been demonstrated that oncogenic functions are largely kinase independent and hence kinase inhibitors are generally ineffective or even contraindicated -5. AnothEphA2 expression has been demonstrated in many cancers; including colon, skin, kidney, breast, glioblastoma and lung. Functionally, EphA2 expression has been shown to influence tumour angiogenesis, migration, proliferation and adhesion. Many strategies have been used for targeting EphA2, in both pre-clinical and clinical setting including the use of monoclonal antibodies, immunoconjugates, small molecule antagonists, immunotherapy, RNAi-mediated EphA2 silencing, EphA2-derived peptides and targeting EphA2 using nanoparticles. Combination of EphA2-directed therapies with other anticancer therapies has also been evaluated , 7.High expression of EphA3 has also been observed in many types of solid cancers and in leukaemias . A monocEphB4 expression has been detected in both embryonic vasculature and in tumour angiogenesis and progression; it plays an important role in cell signalling, morphology, adhesion, migration and invasion. This includes EphB4 signalling which leads to activation of downstream signals including VEGF expression. EphB4 also has a tumour suppressor effect in some cancers including colon and breast. The differing roles of EphB4 in tumour progression have led to the development of many strategies in therapeutic targeting of this protein. One of these approaches is the use of a therapeutic antibody which results in EphB4 degradation, thereby inhibiting tumour angiogenesis and tumour growth. Another approach has been to competitively block EphB4-ephrinB2 signalling using a soluble, monomeric-human serum albumin (EphB4-HSA) fusion protein as a decoy receptor. In pre-clinical models, this approach has been successfully exploited to inhibit tumour growth in colon, lung, breast, glioma, melanoma and prostate cancer [In summary, we and others have developed agents which target Eph proteins as novel therapeutics which have been tested successfully in pre-clinical and early phase clinical studies. Because the Eph RTKs are expressed at low levels in normal adult tissues, several therapies targeting members of the Eph RTK family have been shown to have a highly favourable toxicity profile making them relatively tumour- specific agents. As we move into an era of individually tailored therapies based on tumour phenotype, the development of anti-Eph therapies will provide an important, low toxicity addition to the therapeutic arsenal.


These same advantages have also been successfully exploited in the design of responsive 19F probes. Part of the recent growth of this technique can be attributed to novel designs of 19F probes with improved imaging parameters due to the incorporation of paramagnetic metal ions. In this review, we provide a description of the theories and strategies that have been employed successfully to improve the sensitivity of 19F probes with paramagnetic metal ions. The Bloch-Wangsness-Redfield theory accurately predicts how molecular parameters such as internuclear distance, geometry, rotational correlation times, as well as the nature, oxidation state, and spin state of the metal ion affect the sensitivity of the fluorine-based probes. The principles governing the design of responsive 19F probes are subsequently described in a “how to” guide format. Examples of such probes and their advantages and disadvantages are highlighted through a synopsis of the literature.Fluorine magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) of chemical and physiological processes is becoming more widespread. The strength of this technique comes from the negligible background signal in  These attributes make it comparable to the 1H nuclei  and very broad signals that are easily removed with appropriate pulse sequences  and T2-based 1H MRI contrast agents, which are primarily gadolinium- and iron oxide nanoparticle-based, generate contrast by modulating the relaxation rates of the naturally-occurring water molecules. The significant background from the endogenous water can render certain imaging more difficult. In addition, 19F nuclei have a large chemical shift range, >300 ppm, that readily allows for the design of fluorine probes featuring two distinct resonances that can be independently imaged if they are sufficiently separated. As will be discussed later, this property is advantageous in the design of ratiometric responsive 19F MRI probes. Ratiometric probes can independently map the distribution of the probe and target analyte in vivo and enable tracking of different cell types or monitoring of multiple markers simultaneously.The fluorine nuclei  . This issue is best addressed via the incorporation of appropriate paramagnetic metal ions that shorten the relaxation rates of the 19F nuclei.Despite the advantages of 19F MRS/MRI signal intensity is simply to increase the local concentration of 19F nuclei. This is most often accomplished by increasing the number of 19F nuclei on the probe. Complications of this approach are attributed to the hydrophobicity of fluorine which decreases the solubility of a probe in water and affects biodistribution and clearance. Nonetheless, many perfluorocarbons have successfully been used as 19F MRS/MRI oximetry sensors and 19F cell tracking agents provided they are injected as stable emulsions formulated to optimize clearance  and perfluorooctanoic acid (PFOA) are both known to affect the function of the pancreas, thyroid, and liver  ligand, for instance, adopts a pseudo C4 symmetry in which the acetate arms are arranged in a propeller-like fashion above the plane of the coordinating nitrogen atoms. Four possible isomers exist for this system based on the combination of two macrocyclic ring configurations (square prismatic or square antiprismatic) and the two possible arrangements of the acetate arms −  ester)), the lanthanide complex of the tetra trifluoroethyl-substituted DOTMP ) analog  to 70 ppm (TmIII) range. As discussed above, the presence of these different isomers limits the efficacy of these complexes to function as 19F MR probes. The metal complexes of the tetra substituted DOTA with aryl-CF3 groups , the percentage of which varies according to the lanthanide  as one isomer. In extreme cases, a single isomer of a DOTA analog fluorinated on a single arm can be obtained, as for compounds 8–10 tetrakisacetamide))  or iron(II). The metal should be selected with care as different paramagnetic metal ions have different effects on the relaxation of nearby nuclei.Increasing the number of T1 and T2 of a 19F nuclei is described by the Bloch-Wangsness-Redfield theory. A 19F nuclei adjacent to a paramagnetic center in a magnetic field can relax via five different mechanisms: (1) chemical shift anisotropy, (2) inter-nuclear dipole-dipole interaction, (3) electron-nucleus contact interaction, (4) electron-nucleus dipole-dipole interaction, and (5) Curie relaxation  and transverse (R2 = 1/T2) relaxation rates of 19F nuclei in paramagnetic metal complexes are described by Equations (1) and (2), respectively , the Bohr magneton (μB), and the electron angular momentum, J(J+1) according to Equation (3). The electron angular frequency (ωe) is a function of the magnetic field strength (B0) according to Equation (4) in which ℏ is the reduced Planck's constant. The τR+e term is dependent on the rotational correlation time (τR) and the electron spin longitudinal relaxation time (T1e) according to Equation (5).In these equations, R1 and R2 to the effective magnetic moment (μeff) of the paramagnetic metal, the 19F–metal distance (d), the rotational correlation time (τR), the applied magnetic field strength (B0), and the temperature (T). Optimizing the sensitivity of 19F MRS/MRI probes requires understanding each of these relationships.In Equations (1) and (2), the first term represents the dipolar relaxation characterized by the stochastic electron magnetization of the electron-nucleus dipole-dipole interaction. The second term is the Curie relaxation arising from the interaction between the fluorine nuclear spin and the magnetic dipole induced by the applied magnetic field. Together, they relate eff) of the paramagnetic metal affects both the electron-nucleus dipole-dipole interaction and, to a greater extent, the Curie relaxation mechanism. The effective magnetic moment of transition metal complexes is a function of both their oxidation state and their ligand fields. Each lanthanide ion has a characteristic μeff value  and DOTA ligand scaffold tetra substituted with aryl-CF3 groups (11)  ]2−  exemplify that the diverse electronic and structural parameters of transition metal complexes can also be exploited to optimize 19F paramagnetic relaxation enhancement (PRE).Recently, our lab reported the relaxation properties of the Fet et al. , 2017. TT1 but also on T2. The line-broadening characteristic of the shorter T2 of the fluorines of certain paramagnetic complexes significantly affect the sensitivity of the probe. An extreme example being the GdIII complex of DOTAm-F12 that has such a short T2 that the complex could not be observed by MRI. Note that for such complexes with rapid transverse relaxation, the use ultrashort TE (UTE) and zero TE (ZTE) acquisition pulse sequences can sometimes increase the SNR in 19F-MRI  is more than an order of magnitude lower than that of small diamagnetic probes.Not all paramagnetic metals affect 2–5, 7, and 10–12 . As mentioned above, transition metals such as FeII (μeff ~ 5) and NiII (μeff ~ 3)  can also generate high 19F MRI SNR ratios of 20–30 despite their lower magnetic moments.Similar trends have been observed with other lanthanide complexes, such as ty Table . Do note19F–M distances, the effect of the paramagnetic metal ion on the relaxation rates of the fluorine nuclei can be notable. The fluorinated complex of Jiang  with respect to environmental fluctuations in pH, temperature, and the concentration of either O2 or bovine serum albumin (BSA) .Even at greater R1 and the R2 of the fluorine nuclei have a steep dependence on the distance d separating the 19F nuclei from the metal ion. The 19F–M distance is a key parameter in determining the sensitivity of metal-based fluorine probes. The effect of the paramagnetic metal ion on R2 extends to longer distances than that of R1. Both too long and too short a distance can be detrimental. Too long a 19F–M distance and the effect of the paramagnetic metal ion on the relaxation times of the fluorine nuclei is severely diminished. Too short a distance, and the substantial shortening of T2 decreases the sensitivity of the fluorine probe. A higher T2/T1 ratio and thus a more sensitive fluorine probe is obtained if the 19F nuclei are positioned between 4.5 and 7.5 Å from the metal ion  given above, both the 19F–M ion distance can have an impact on the relaxation rates of fluorine nuclei and the sensitivity of the agent. One such example are the lanthanide complexes containing a single aryl-CF3 moiety positioned either on an acetate-based DOTA  exists in solution as a mixture of multiple isomers with the primary species representing only 50% of the fluorine signal intensity by NMR. On the other hand, the principal isomer of the phosphinate analog (7) accounts for 87% of the total 19F signal intensity  of DOPA scaffold are 5–12% smaller than those of DOTA scaffold with an exception of TbIII and DyIII being 14–124% greater than those of DOTA complexes. Global fitting of the relaxation rates collected at multiple field strengths to the Bloch-Wangsness-Redfield theory determined that the differences in R1 and R2 are due to a 0.3 Å shorter 19F–Ln distance in the DOPA complex than in the DOTA one , ErIII (0.79), Tm (0.75), and Dy (0.74) that yielded the highest T2/T1 ratio. As is apparent from this example, the best metal ion for one fluorinated ligand is not necessarily the same for another one.Within this range, even minor changes in T2/T1 ratio and the sensitivity of the probe has been observed with other complexes. The YbIII -DO3A )-based complex with a fluorinated aryl phosphonate of Angelovski,  may result in decomplexation of the fluorinated moiety under biological conditions.Supramolecular self-assembly enables more facile synthesis of paramagnetic fluorine probes via the formation of ternary complexes. Given the hardness of lanthanide ions, fluorinated carboxylate ligands are particularly well-suited for this approach -3-(2-(((E)-octadec-9-en-1-yl)amino)-2-oxoethyl)-11-oxo-3,6,9,12-tetraazatriacont-21-enoic acid) complexes , and the diffusion coefficient of the perfluorocarbon affect the R1 and R2 of the 19F enabled further improvement of the assembly  compared to non-metallated perfluorocarbon. Additionally, a shorter linker between the Gd and the perfluorocarbon core of the DTPA complexes (14 and 15) vs. the Gd-DOTA chelate (16) further increased the fluorine relaxation rates  had a greater impact on the relaxation rates of the fluorines than Gd-DOTA-DSPE -2,3-bispropoxy)(hydroxy)phosphoryl)oxy)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid)and Gd-DOTA-C6-DSPE-2,3-bispropoxy)(hydroxy)phosphoryl)oxy)-ethyl)amino)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid), they can be used to track cells in vivo 24 h post injection in mice. Do note, though, that it is not possible to distinguish between live and dead cells labeled with fluorine probes 24 h post injection, especially if the cells are engrafted subcutaneously.These previous examples positioned the paramagnetic metal ions on the surface of the nanoparticles and, thus, a significant distance away from the R1 and R2 of fluorine nuclei depend on the strength of the magnetic field  increased the image SNR ~ 13-fold compared to the diamagnetic YIII analog   as well as by the field strength of the instrument (B0) and the temperature of the sample. Both R1 and R2 increase as the magnetic field strength (B0) increases. This increase is far more significant if the rotational correlation time, τR, is also optimized. Macromolecules, which tumble more slowly (longer τR), have higher R2. As can be seen in Figure R2 increases drastically for 19F nuclei with τR > 1,000 ps, especially at high magnetic field (>7 T). The resulting extreme line broadening explains why fluorines embedded in bones and teeth in the body are not visible by MRI. The effect of τR on R1 is more complicated  and polymer (23) , whereas the R2 decreased from 440 to 367 s−1, which is unusual for a polymer . Since variations in each of these three variables can readily be observed both by MRS and by MRI, paramagnetic fluorine complexes offer unique mechanisms for the development of responsive MR probes that often yield much greater response than their purely organic analogs  and is a function primarily of the nature of the paramagnetic metal ion and the distance d separating it from the fluorine nuclei. The second is the lanthanide induced shift (LIS) effect. It is just as readily be observed with 19F than with 1H nuclei and is governed by the McConnell-Robertson theory. Unlike PRE, LIS is affected not only by the nature of the paramagnetic lanthanide ion and the distance d separating it from the fluorine nuclei, but also by the second order crystal field coefficient of the complex, 19F offers valuable opportunity to design ratiometric responsive MR probes that can distinguish between changes in signal intensity due to the presence of an analyte vs. that due to a change in the concentration of the probe.Advantageously, a paramagnetic metal ion that alters 19F basis of paramagnetic probes as compared to their organic counterparts. It is the substantial shorter T1 and higher T2/T1 ratio of paramagnetic fluorinated complexes that enables more scans to be acquired in the same amount of time thereby yielding higher SNR both by 19F MRS and MRI. Analysis of the Bloch-Wangsness-Redfield Equations (1–5) indicate that at constant applied magnetic field (the same scanner) and for the same metal ion, T1 and T2 are a function of primarily one parameter: the distance d separating the 19F nuclei from the metal ion. This parameter can be altered significantly by binding to or reacting with a substrate. Herein lies a substantial advantage of paramagnetic metal ions in fluorine probes: they readily enable the design of responsive and ratiometric molecular probes.The PRE effect described in the previous section of this review is the basis for the improved sensitivity on a per T1 and T2 on the distance separating the fluorine nuclei from the lanthanide ion has already been applied to the design of responsive paramagnetic fluorine probes. As depicted in Figure The dependence of III. As described in the previous section, GdIII has a notable effect on the T1 of nearby nuclei, and, importantly, a substantially more pronounced one on T2. Indeed, among the lanthanides, GdIII typically offers the lowest T2/T1 ratio    or decrease (turn off) the 19F–Gd distance  and Cu-POSS (36) contain either a redox sensitive ferrocene or a Cu complex with a bis(2-pyridylmethyl)amino ligand (PMEA) along with trifluoromethyl groups appended to a cubic polyhedral oligomeric silsesquioxane (POSS) for water solubility  mediated reduction of paramagnetic CuII in 36 to diamagnetic CuI modestly increased T1 (from 1180 ms to 1460 ms) and T2 (from 420 to 540 ms) causing a 3-fold increase in signal intensity. Thus, their work demonstrates the ability to apply PRE to the design of 19F redox sensitive responsive probes. Que's laboratory utilized this strategy and the PRE of CuII to detect cellular hypoxia by 19F MR  to CuI  and subsequent ligand dissociation increases T2 of the 19F and restores their signal intensity. A second generation version of this probe (38) was designed to be more sensitive  or long (TE = 100 ms for CuI) echo pulse sequences  than EuII amino)ethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid)  to diamagnetic CoIII (d6) shifts the 19F signal by 3.6 ppm and increases the T1 and T2 of the 19F nuclei 60- and 121-fold, respectively  reaction time and the low sensitivity of the probe compared to in vivo levels of H2O2 still limit the application of this probe to in vivo imaging.This approach also enables direct detection of reactive oxygen species. Oxidation of Co-NODA-CFT2 in the design of responsive fluorine probes. The first, as mentioned above, is that such probes are not ratiometric. A lack of signal can be attributed both to a lack of target and to a lack of probe. Similarly, signal intensity is not a direct measure of the concentration of the target as it is equally affected by the concentration of the probe. The second disadvantage is that upon release, the organic fluorine moiety regains its very long T1. Such probes are thus plagued by the same sensitivity issue as the purely organic fluorinated probes  is traditionally defined as the difference in chemical shift, Δδ, between a paramagnetic complex and its diamagnetic analog (Δδ = δpara – δdia). In terms of responsive paramagnetic fluorine probes, the induced shift, 19FLIS, is defined as the difference between the chemical shift of a 19F nuclei in the presence and absence of the probe's analyte (Equation 6).One of the first applications of lanthanides in NMR spectroscopy was as induced shift reagents , TmIII and HoIII are the best choices in the design of 19FLIS probes for MRI.Where 19F nuclei from the metal ion, d    to high spin (S = 2) in hexadentate triazacyclonone ligands with fluorinated 2-picolyl substituents , which bodes well for future in vivo applications.The chemical shift of fluorine nuclei can also be modulated by altering the spin state of the metal ion. A recent example exploits the temperature induced transition of Fe51 . In this case, the lanthanide ion functions only to increase the sensitivity of the probe. HoIII shortens the T1 and T2 of the fluorine, thereby significantly increasing the number of scans that can be recorded in a given amount of time, and thus improving the SNR of the probe.The standard approaches that are used in the design of diamagnetic fluorine probes can also be applied to their paramagnetic analogs. An example is the α-chymotrypsin esterase probe 1 Figure . Hydroly19F MR imaging, a significant advantage of 19F probes is their ability to also function in other modalities. Fluorinated polyaminocarboxylate-based complexes, for instance, can also be effective chemical exchange saturation transfer (CEST) MR contrast agents. Such dual-modalities are particularly well-suited for the design of ratiometric agents. False negatives are more readily avoided if one of the two modalities is responsive to the targeted marker while the other one is not. This enables the distribution of the contrast agent to be mapped independently of that of the marker. Two examples of dual 19F-CEST agents have been reported. A fluorinated Eu-DOTAm-Gly complex with two trans trifluoromethyl groups (2, Figure 19F and CEST contrast in vitro despite multiple isomers in solution (Cakic et al., ex vivo by 19F MRI, possibly due to interactions with tissues that substantially reduced T2. This first dual-modality 19F contrast agent is not responsive.Although this review has focused on 12, Figure 19F MR probe and a paraCEST contrast agent. Importantly, whereas the 19F signal intensity is independent of pH, the CEST signal is modulated by pH. The % saturation transfer of the complexes increases 5-fold between pH 4 and 6.2. The complex accurately determines the pH independently of the concentration of the contrast agent between pH 6.9 and 7.4, a range that is relevant to cancer diagnosis. Advantageously, the similar sensitivity of the agent in both modalities facilitates ratiometric determination of pH.Fe-DOTAm-F12 which was developed in our lab, has 12 equivalent fluorine nuclei and one isomer in solution (R1 of the fluorine nuclei and increased chemical shift spectral window due to the paramagnetic induced shift.The development of paramagnetic fluorine probes over the last decade mirrors, in many ways, that of paraCEST agents, the paramagnetic derivatives of CEST contrast agents. The introduction of paramagnetic metal ions in the design of fluorine probes, most notably lanthanides, iron, and cobalt, offer several advantages. Those include increased sensitivity of the fluorine probes due to decreased T1 and T2 of the fluorine nuclei change substantially upon binding to or reacting with a desired analyte. This causes either the appearance, disappearance or notable chemical shift of the 19F peaks. Moreover, the absence of background signal in 19F MRI is an added advantage of this class of probes over the more common Gd-based and iron oxide nanoparticle-based contrast agents which have to contend with the signal of endogenous water. The recent work published on paramagnetic fluorine probes, backed by accurate theoretical models, enable scientists to design molecules with maximum sensitivity by optimizing the nature, specially the oxidation and spin state, of the paramagnetic ion and its distance from the fluorine nuclei all while maximizing the number of chemically equivalent 19F nuclei. Those same parameters are used in the design and optimization of responsive probes. Notably, the large spectral windows of paramagnetic fluorine probes enable the design of ratiometric responsive probes which can independently report on both the distribution of a probe and that of its targeted analyte. Paramagnetic fluorine probes are also uniquely suited to multicolor, or multifrequency, imaging, which enables the tracking of different types of cells.Advantageously, both the effects on relaxation rates and chemical shift offer new approaches to the development of responsive fluorine probes that are not available with their diamagnetic counterparts, and these strategies lead to a significantly greater response. Indeed, paramagnetic fluorinated complexes can be readily designed such that the chemical shifts and/or the relaxation times R1 induced by the paramagnetic ion is minimal at high magnetic fields. These probes are better suited for use with scanners of low and medium magnetic field strengths, below 6 T. The structure and conformation of the probe in solution, parameters that can be more difficult to predict, have a significant impact on the properties of the probe. Complexes existing as a single isomer in solution with all fluorine nuclei chemically equivalent are more sensitive and easier to image, but many lanthanide complexes exist in solution as interconverting isomers. The potential toxicity of the complex should also be kept in mind, especially given the high concentrations of probes required for in vivo imaging. Although the toxicity and pharmacological properties of this new class of probes has not yet been evaluated, conclusions drawn from Gd-based contrast agents are likely to extend to paramagnetic fluorine probes. Kinetically labile lanthanide complexes, for instance, are expected to present higher toxicity. Nonetheless, we foresee that these first generation of paramagnetic fluorine probes will open avenues for many more applications for in vivo imaging. In particular, the ability of these probes to track different cells and biomarkers, and their response to different stimuli, render them particularly promising for molecular imaging.Paramagnetic fluorine probes have drawbacks too that should be kept in mind. The increase in sensitivity due to lower VP, KP, and KS: searched and analyzed the literature; VP, KP, and KS: co-wrote the manuscript.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.


Tumours are not merely masses of abnormally proliferating cancer cells. Today, we have a clearer view of cancer complexity in which the participation of cancer and host cells leads to a tremendous heterogeneity of neoplastic diseases concerning the genetics, epigenetics, proteomics and biochemistry of the tumour . Such inCorrespondingly, drug treatment is progressively shifting from the use of chemicals producing toxics effects in general processes of cell division, with the goal of killing the tumour cell, to compounds targeting specific cell behaviours with the goal of disarming the malignancy of the tumour cell  2,3]. T,3. T2,3]Metastasis is the primary target for cancer chemotherapy, independently of the kind of drug being used. The main reason is that the primary tumour can often, if not always, be aggressed by surgery and/or radiotherapy whereas metastases, spread in several tissues and organs, are believed to be better reached with drugs that follow the pharmacological rules of distribution in the body.The generation of these novel drugs, whether selective and specific monoclonal antibodies or small organic molecules, requires a deep knowledge of the nature of the tumour cell and particularly of tumour metastasis. Similarly, to the primary lesions, tumour metastases are characterised by the interactions with healthy cells and extracellular matrix leading to a complex microenvironment in dynamic evolution but extremely important for the metastatic growth ,8.The twin research that joins the biochemistry and molecular biology of cancer metastasis with the study of novel targets and novel approaches to combat their growth is even more mandatory in a scientific era in which the improvements of human health are transforming cancers into chronic diseases and therefore significantly prolonging the life-time expectancy.


Approximately 10% of pleomorphic adenomas occur in the minor salivary glands with the palate being the most common site. Pleomorphic adenomas account for the majority of palatal tumours; however, minor salivary gland tumours have a higher risk of malignancy compared to tumours of the major salivary glands, so appropriate diagnostic evaluation should be prompt. We present a case of a 52-year-old man with a longstanding history of a soft palate pleomorphic adenoma which required excision under general anaesthetic via a mandibular swing approach. As well as the surgical approach to access this tumour; this case is unique as it is the largest soft palate pleomorphic adenoma reported in the literature. We discuss the appropriate preoperative investigations and airway considerations for this patient, as well as the factors to consider when planning operative management of palatal tumours. Pleomorphic adenomas (PAs) are the commonest tumour of both the major and minor salivary glands, with approximately 10% of PAs occurring in the minor salivary glands. The palate is the most common site of minor salivary gland PA, but other sites may also be affected, including the lips, cheek, floor of mouth, and nasal cavity [To the best of our knowledge, this case is the largest pleomorphic adenoma of the soft palate which has been excised via a mandibular swing procedure. This case required careful preoperative planning with regards to surgical approach and airway management.A 53-year-old man presented to our department complaining of longstanding progressive dysphagia and a “hot potato” voice. He had only been managing a soft or liquid diet, and he slept with two pillows due to dyspnoea when lying flat and on exertion. He also complained of significant weight loss.He reported that he had a lump in his mouth for thirty years and had previously presented to ENT services both in England and abroad, but he did not attend follow-up.Clinical examination revealed a large left-sided oropharyngeal mass originating from the soft palate and extending inferiorly. The overlying mucosa was normal in appearance.Magnetic resonance imaging (MRI) demonstrated a large tumour which originated from the soft palate and extended to the larynx, measuring 9.96 cm in craniocaudal dimensions . ComputeRadiological assessment was consistent with either a myoepithelioma, a pleomorphic adenoma, or a low-grade mucoepidermoid tumour. Fine-needle aspiration cytology (FNAC) was consistent with an epithelial neoplasm, possibly of salivary gland origin . The mulDue to the impossibility of oropharyngeal or nasopharyngeal intubation, he underwent a local anaesthetic tracheostomy with transnasal humidified rapid-insufflation ventilatory exchange (THRIVE). Access to the tumour was gained via a lip-split mandibulotomy and mandibular swing procedure. The whole encapsulated mass was excised from the soft palate and left parapharyngeal space via a submucosal dissection Figures  and 4. TVideo fluoroscopy demonstrated significant dysphagia due to reduction in pharyngeal bulk and failure of approximation of tongue base. Having been fed via a nasogastric tube for 6 weeks, he is currently tolerating a premashed diet with supraglottic swallowing manoeuvres. He is undergoing further swallow rehabilitation and remains under clinical surveillance.Histopathological examination of the excised mass demonstrated a benign encapsulated neoplasm composed of epithelium in a variety of architectural patterns consistent with a pleomorphic adenoma Figures  and 6.Pleomorphic adenomas (PAs) are the commonest tumour of both major and minor salivary glands with about 80–90% of PA occurring in the parotid gland . PA makeAt 9 × 7.5 × 3.9 cm, the PA in our case represents the largest soft palate PA reported in the literature. The previous largest soft palate PA was 7 × 6 cm [PAs demonstrate a wide variety of histological features and growth patterns. Three main histologic subgroups of pleomorphic adenoma have been identified: myxoid, cellular, and mixed (classic) type , almost Due to the higher risk of malignancy with IMSGTs, appropriate preoperative diagnostic investigations are appropriate. von Stempel et al.  suggest Intraoral ultrasound is also suggested as an adjunct , particuFine-needle aspiration cytology aims to differentiate benign from malignant disease, allowing more accurate counselling of the patient and aiding with surgical planning. FNAC has an accuracy of 82–97% for the preoperative diagnosis of pleomorphic adenoma –16. WhenAnaesthetic considerations when managing IMSGT of the palate are important, particularly given the size of the tumour we report. Due to the extent of the tumour, neither endotracheal nor fibreoptic nasal intubation was an option; therefore, the decision to proceed to tracheostomy was made. In conjunction with our anaesthetic colleagues, transnasal humidified rapid-insufflation ventilatory exchange (THRIVE) was used during the tracheostomy. THRIVE consists of high-flow humidified nasal oxygen therapy, delivered at up to 70 L/min, using OptiFlow . It has been shown to maintain oxygen saturations and extend apnoea time in patients with difficult airways . THRIVE The best approach for management of palatal pleomorphic adenomas is a wide excision with negative margins , especiaSize of the tumourSuspicion of malignancyProximity and the projection of the tumour to the oropharyngeal wall or the neckVascularityRelation of the tumour to the neck neurovascular bundleThere are many surgical approaches possible for tumours of the soft palate, including intraoral, transcervical, and mandibulotomy and mandibular swing. The surgical approach taken depends on the size and location of the tumour, and as this tumour extended from the soft palate to the parapharyngeal space, a transmandibular approach was deemed necessary to allow adequate exposure in order to ensure complete tumour excision and control of any feeding vessels in order to prevent haemorrhage. Five considerations have been suggested by Papadogeorgakis et al.  when decOur tumour was mainly oropharyngeal, with extension to the infratemporal fossa. The inferior component was also suprahyoid. Therefore, it was felt that a lip-split mandibulotomy would give good access and delivery of the tumour. We were prepared, however, to extend the neck incision should a combined cervical approach have been necessary.Our approach gave excellent tumour exposure. From the outset, we were able to fully mobilise and separate the tumour laterally and ensure no major feeding vessels. This approach allowed adequate excision and reduced the chance of capsule rupture and recurrence. The disadvantages of a mandibulotomy include malunion, malocclusion, injury to the mental nerve, and the cosmetic and functional consequences of the lip-split.Soft palate pleomorphic adenomas of this size are rare. Appropriate diagnostic evaluation with FNAC and radiological investigations should be prompt due to the higher risk of malignancy in minor salivary glands compared to tumours of the major salivary glands.Excisional biopsy is necessary to provide an accurate histopathological diagnosis, and careful preoperative planning should be undertaken in order assess the best surgical approach to ensure complete excision.A comprehensive airway strategy should be planned before excision of any palatal lesion, including the use of adjuncts, such as THRIVE, if deemed appropriate.


The nude mouse animal model was established by subcutaneously injecting 5 × 107/ml HepG2 cells into the shoulder flank of each mouse. Biodistribution studies were performed at 0.5, 1, 2 and 4 h after intravenous administration of 0.37 MBq of 99mTc-3PRGD2. Immunohistochemistry was performed to evaluate the expression level of integrin αvβ3 in the HCC tissues. Dynamic imaging was performed using list-mode after the administration of 55.5 MBq of 99mTc-3PRGD2, to reconstruct the multiphase images and acquire the best initial scan time. At 8, 12, 16, 20 and 24 days after inoculation with HepG2 cells, 55.5 MBq of 99mTc-3PRGD2 and 37 MBq of 18F-FDG were injected successively into the nude mouse model, subsequently, simultaneous SPECT/PET imaging was performed to calculate the tumour volume and tumour uptake of 99mTc-3PRGD2 and 18F-FDG.99mTc-3PRGD2 at the different time points was higher than that of all the other organs tested in the experiment, except for the kidney. Integrin αvβ3 expressed highly in early stage HCC and declined for further necrosis of the tumour tissue. Subcutaneous tumours were visualized clearly with excellent contrast under 99mTc-3PRGD2 SPECT/CT imaging, and the multiphase imaging comparison showed the tumours were prominent at 0.5 h, suggesting that the best initial scan time is 0.5 h post-injection. The comparison of the imaging results of the two methods showed that 99mTc-3PRGD2 integrin receptor imaging was more sensitive than 18F-FDG metabolic imaging for the detection of early stage HCC, meanwhile the tumour uptake of 99mTc-3PRGD2 was consistently higher than that of 18F-FDG. However, as tumour necrosis further increased in HCC tissues, the uptake of 18F-FDG was higher than that of 99mTc-3PRGD2.The biodistribution study first validated that the tumour uptake of 99mTc-3PRGD2 is a valuable tumour molecular probe for the detection of early stage HCC compared with 18F-FDG, meriting further investigation of 99mTc-3PRGD2 as a novel SPECT tracer for tumour imaging.Our study demonstrated that  Hepatocellular carcinoma (HCC) is a primary cancer of the liver with a significant impact worldwide due to its high incidence and mortality. Early detection and diagnosis of HCC can bring great benefit for the prognosis of patients. Currently, the definitive diagnosis of HCC depends primarily on conventional imaging modalities, such as ultrasonography, magnetic resonance imaging or computed tomography . Howeverd-glucose (18F-FDG) PET/CT is the most widely employed tumour-functional imaging modality worldwide, however, the positive detection rate for HCC is still not ideal, particularly in terms of the low specificity and sensitivity for the detection of early stage HCC 2 (99mTc-3PRGD2) for the detection of HCC and specifically to compare the diagnostic performance of 99mTc-3PRGD2 integrin receptor imaging and 18F-FDG metabolic imaging in HCC tumour-bearing mice.Integrin αvβ3, a member of the integrin receptor family, is highly expressed in both tumour cells and activated endothelial cells and plays a pivotal role in tumour growth, invasion and metastasis –11. The 2 lyophilized kit was obtained from the Medical Isotope Research Center of Peking University. The synthesis of the labelling precursor and subsequent 99mTc-labelling were performed according to previously described methods [18F-FDG was supplied by the Atom High-tech Company of China . Doses were prepared by dissolving the radiotracer in saline.The HYNIC-3PRGD methods . The via2 in air. The cells were split or harvested when they reached 90–100% confluency to maintain exponential growth. Each mouse was implanted subcutaneously with 5 × 107/ml HepG2 cells into the left shoulder flank. The tumour volume was measured every other day with vernier callipers and calculated using the following formula: (length × width2)/2.This study was approved by our hospital’s Ethics Committee for medical research. A total of 36 female athymic nu/nu mice were purchased from the Medical Animal Center, FuZhou General Hospital of Nanjing Military Command . The human HCC HepG2 cell line was purchased from the Institute of Cytobiology, Chinese Academy of Science. The HepG2 cells were cultured in DMEM supplemented with 10% foetal bovine serum  and 1% penicillin and streptomycin solution (Gibco) at 37 °C in a humidified atmosphere of 5% CO99mTc-3PRGD2 in 0.1 ml. At 0.5, 1, 2 and 4 h after administration, the mice were sacrificed by humane euthanasia, and the important organs  were excised, dried, weighed and counted using a γ-counter. The percentage of the injected dose per gram (%ID/g) and the target/non-target (T/NT) ratios were calculated to evaluate the biological distribution characteristics of 99mTc-3PRGD2 in the nude mice.A total of 16 HepG2 hepatoma-bearing mice, with a maximum tumour diameter of 1.0 cm, were randomly divided into 4 groups, with 4 animals in each group. Each mouse was injected intravenously with 0.37 MBq of In total, 12 mice were chosen randomly for HepG2 cell inoculation at different time points. When the subcutaneous tumours grew to various sizes (0.06–1.0 g), the mice were sacrificed by humane euthanasia, and the tumours were excised, fixed in 10% formalin, embedded in paraffin, cut into slices (6-μm-thick) and dried. After dewaxing in xylene and hydrating in gradient ethyl alcohol (from 100 to 80%), the sections were incubated with the mouse anti-integrin αvβ3 monoclonal antibody , followed by sequential incubation with reagent A (reaction enhancer) and B (enzyme-conjugated goat anti-mouse/rabbit IgG polymer) of an immunohistochemical EliVision plus kit, then washed with phosphate buffer solution (PBS) and stained with fresh diaminobenzidine (DAB) solution. Finally, the sections were visualized under an Olympus optical microscope . The appearance of brown colouring in the cytoplasm or cytomembrane of the tumour tissues was regarded as positive integrin αvβ3 expression.99mTc-3PRGD2 in 0.1 ml saline via the tail vein. At 20 min after administration, the mice were quickly transferred into a shielded chamber connected to an isoflurane anaesthesia unit. After the induction of anaesthesia, the mice were immediately placed supine on the scanning bed. The 99mTc-3PRGD2 imaging commenced 30 min after the tracer injection. First, rectangular scans in the regions of interest (ROIs) from both the SPECT and CT were selected by X-ray. CT scanning was performed using the “normal” acquisition settings at 55 keV and 615 μA, and then SPECT data were acquired from 30 min to 3 h and 4 to 4.5 h post-injection (pi) in list-mode using the following settings: 84 projections per frame over 30 min and a 20% energy window centred on 140 keV.All imaging studies were performed using microSPECT/PET/CT equipment , which enables simultaneous submillimeter imaging of single-photon and positron-emitting radiolabeled molecules. Four HepG2 hepatoma-bearing mice were injected with 55.5 MBq of 99mTc-3PRGD2 was expressed as the percentage of the injected dose (%ID) and the percentage of the injected dose per unit volume (%ID/cm3). The background ROI (0.01 cm3) was drawn over the muscle of the contralateral forelimb, and then the T/NT ratio of tumour to muscle was calculated for the semi-quantitative analysis.CT reconstruction was performed using a cone-beam filtered back-projection algorithm . SPECT image reconstruction of the list-mode raw data was performed using a pixel-based ordered-subsets expectation maximization algorithm (POSEM), with the initial scan time set as 0.5, 1, 2 and 4 h pi (a 30-min scanning time). The new SPECT data and CT data were fused together using PMOD software . To achieve a higher signal-to-noise ratio, Gaussian smooth 3D  was used and the image colour gradation was adjusted. For each SPECT/CT fusion image, the ROI covering the entire tumour was generated automatically by setting a maximum pixel value threshold of 30% or more, and then the tumour volume and radioactivity counts were calculated using PMOD. The tumour uptake of 99mTc-3PRGD2 and 37 MBq of 18F-FDG successively. A 45-min simultaneous SPECT/PET acquisition was obtained, beginning at 45 min after the tracer injection. Throughout the scanning, the body temperature of the mice was monitored using a constant heating and feedback device. To achieve signal separation of the two tracers, the energy peak was set to 140 keV for 99mTc-3PRGD2 and 511 keV for 18F-FDG. All photopeak windows were set to a width of 20%. The SPECT and PET images were reconstructed using POSEM. The tumour volume and tumour uptakes of 99mTc-3PRGD2 and 18F-FDG (%ID and %ID/cm3) were calculated using PMOD software.The HepG2 hepatoma-bearing mice were reared at 26–27 °C. Four mice were chosen for simultaneous SPECT/PET imaging at 8, 12, 16, 20 and 24 days after inoculation with HepG2 cells, and they were fasted but allowed to drink water for 12 h before imaging. The selected mice were injected intravenously with 55.5 MBq of t test for the comparisons between groups and regression analyses for the correlation analysis with the SPSS 18.0 statistical software package. The level of significance was set at P < 0.05.The quantitative data are expressed as 99mTc-3PRGD2 at the different time points was 4.301 ± 0.313 %ID/g (0.5 h), 6.902 ± 0.717 %ID/g (1 h), 5.045 ± 0.193 %ID/g (2 h) and 2.099 ± 0.388 % ID/g (4 h), with a statistically significant difference between each group . 99mTc-3PRGD2 was primarily excreted through the urinary system as evidenced by the higher renal uptake . Moderate accumulation was also found in the intestine and liver, illustrating that 99mTc-3PRGD2 was partially metabolized by the liver or intestine. Other organs with relatively low radioactivity were the spleen, lung, stomach, blood and muscle. Figure T/NT ratios of tumour to muscle and tumour to liver, indicating that the highest T/NT ratios were obtained at 2 h pi .As shown in Fig. HCC cells have large and atypical nuclei and mitosis in haematoxylin-eosin (HE)-stained slices. Immunohistochemistry showed that integrin αvβ3 positive expression, as visualized by brown staining, was observed predominantly in the cytoplasm and cytomembrane, as well as the vascular epithelial cells Fig. . The exp99mTc-3PRGD2 was observed at all time points examined, whereas abdomen uptake was moderate and radioactivity accumulation in the lung, mediastinum and muscle was relatively low. Furthermore, radioactivity distribution in all body tissue types decreased over time. These results were consistent with that of the biodistribution study.The subcutaneous tumours were clearly visualized under multiphase SPECT/CT imaging, with excellent contrast at 0.5, 1, 2 and 4 h pi Fig. . The acc3 (0.5 h), 3.61 ± 0.87 %ID/cm3 (1 h), 3.02 ± 0.75 %ID/cm3 (2 h) and 2.63 ± 0.71 %ID/cm3 (4 h), suggesting that the highest tumour uptake occurred when the initial scan time was 0.5 h pi. The T/NT ratios of tumour to muscle were 3.39 ± 1.33 (0.5 h), 3.17 ± 1.33 (1 h), 3.66 ± 0.92 (2 h) and 2.86 ± 0.49 (4 h). Although the highest T/NT ratio was found when the initial scan time was 2 h pi, there were no statistically significant differences among the four groups (P > 0.05). Both the direct visualization and quantitative analysis indicated that the best initial scan time was 0.5 h pi.The tumour uptake was 4.61 ± 1.17 %ID/cm99mTc-3PRGD2 integrin receptor imaging and 18F-FDG metabolic imaging during the growth of tumour with the microSPECT/PET/CT system , there were no statistically significant differences among the different groups, which were divided according to the imaging day . A regression analysis was used to assess the relationship between the tumour volume and tumour uptake of 99mTc-3PRGD2 and 18F-FDG . The relationship between the tumour volume and %ID of 18F-FDG was also modelled as quadratic polynomial, with an R2 value of 0.96. The conversion formula was y = −6.61x2 + 7.68x−0.18, illustrating that the tumour uptake of 18F-FDG was consistently less than that of 99mTc-3PRGD2 from day 8 to day 16. When all the tumour volume was greater than 0.30 cm3 from day 20 to day 24, the uptake of 18F-FDG, in turn, was higher than that of 99mTc-3PRGD2 during this time period.The tumour uptake of 99mTc-3PRGD2 stands out for its superior αvβ3-targeting ability, biodistribution and pharmacokinetics [99mTc-3PRGD2 for the diagnosis of HCC, especially no studies have compared the efficacy of 99mTc-3PRGD2 integrin receptor imaging with 18F-FDG metabolic imaging. Integrin αvβ3 express lowly in normal liver tissues including hepatocytes, stellate cells, Kupffer cells, and other nonparenchymal cells [99mTc-3PRGD2 as the radiotracer.Among the various forms of radioactive medications, kinetics –15. It hkinetics –19. Howeal cells . Studiesal cells . In this99mTc-3PRGD2 in the non-invasive monitoring of HCC tumours, 18F-FDG was chosen as the positive control. The imaging results indicated that 99mTc-3PRGD2 SPECT/CT has an advantage over 18F-FDG PET/CT for HCC imaging in two aspects. First, 99mTc-3PRGD2 SPECT/CT could distinctly and sensitively detect the HCC tissues at 8 days after HepG2 cell inoculation, a time point at which the smallest tumour volume was only 0.041 cm3 but the uptake of 99mTc-3PRGD2 reached 10.67 %ID/cm3 at this point. In contrast, the tumours were unclear under 18F-FDG images so the imaging could not be used to accurately detect early stage HCC. Furthermore, the tumour uptake of 99mTc-3PRGD2 was significantly higher than that of 18F-FDG from day 8 to day 16. This finding suggests that 99mTc-3PRGD2 has the potential to be used as a SPECT tracer for early detection of HCC over 18F-FDG. Second, it is well known that 18F-FDG PET/CT imaging is affected by many factors, such as glucose levels, insulin, and temperature. Muscle or brown adipose tissue can also competitively uptake 18F-FDG [99mTc-3PRGD2 integrin receptor imaging shows less interference from these factors, resulting in the acquisition of clearer images. Moreover, 99mTc-3PRGD2 can be more widely applied to improve the screening rates for early HCC detection because it is more cost-effective, feasible and practical as a SPECT tracer.To further estimate the usefulness of  18F-FDG , 23. In 99mTc-3PRGD2 and 18F-FDG comes from two completely different biological processes they are targeting. In this study, we found that αvβ3 expression in the xenografted HepG2 tumours was high in the early stage and showed an increasing trend until the tumours became necrotic. Given that 99mTc-3PRGD2 is a target-specific radiotracer whose biodistribution is determined by its receptor, the high expression level of αvβ3 in early HCC contributes to the high tumour uptake of 99mTc-3PRGD2; therefore, 99mTc-3PRGD2 imaging is able to sensitively detect early HCC while 18F-FDG imaging is used to evaluate glucose metabolism, the main mechanism of 18F-FDG uptake in malignant tumours largely depends on the presence of facilitated glucose transporters, especially type 1 (Glut 1) and a rate-limiting glycolytic enzyme, especially hexokinase (HK) type II. However, HCCs have different glucose-regulating mechanisms and enzyme expression patterns, and the low expression of Glut 1 and the existence of dephosphorylase are the main reasons for the low 18F-FDG uptake [18F-FDG in HCC is also related to the degree of differentiation and lesion size, with well-differentiated HCC exhibiting a high rate of gluconeogenesis compared with that in normal liver tissues, resulting in similar uptake of 18F-FDG [18F-FDG PET/CT imaging.We analysed the possible reasons for the different imaging results with G uptake –26. The  18F-FDG . In part99mTc-3PRGD2 SPECT/CT was more sensitive than 18F-FDG PET/CT for the detection of early HCC, it is worth noting that HCC uptake of 18F-FDG was higher than that of 99mTc-3PRGD2 in advanced stage HCC, indicating that the diagnostic efficiency of 18F-FDG PET/CT might be better than 99mTc-3PRGD2 SPECT/CT for imaging advanced HCC. On one hand, the tumour uptake of 99mTc-3PRGD2 may have decreased due to the concomitant maturity of blood vessels, tumour necrosis, and larger interstitial space that accompanied the rapid growth of the tumour [18F-FDG increased in advanced stage HCC [99mTc-3PRGD2 SPECT/CT and 18F-FDG PET/CT imaging could aide in the detection of HCC by compensating for the deficiency of 18F-FDG in the detection of early stage HCC and 99mTc-3PRGD2 in the diagnosis of advanced HCC. Moreover, multimodality imaging using multiple tracers can also provide complementary information on tumour-associated functions with excellent sensitivity and high selectivity; thus, multimodality imaging might improve the tumour diagnosis, differential diagnosis and therapeutic monitoring of HCC compared with those of a single radiological modality alone.Except e tumour . On the tage HCC . Therefo99mTc-3PRGD2 can be applied to guide anti-angiogenic therapy by visualizing and quantifying the expression level of αvβ3 [99mTc-3PRGD2 imaging has the potential to select suitable patients who could benefit from sorafenib chemotherapy and supply an objective basis for its curative effect.In the complex pathogenesis of hepatic carcinoma, angiogenesis is one of the crucial events, relying on the migration and invasion of vascular endothelial cells, which is regulated by various cell adhesion receptors including the integrin family . Among t of αvβ3 , 31. Thu99mTc-3PRGD2 can assess activation of hepatic stellate cells and diagnose liver fibrosis [T/NT ratio and influence the detection of HCC. Finally, the relation between the pathologic grade and tumour uptake of 99mTc-3PRGD2 was not assessed in the present study; therefore, we cannot verify whether various pathologic types of liver cancer impact 99mTc-3PRGD2 SPECT/CT imaging.It should be noted that our results are preliminary and obtained using a very limited number of animals; therefore, insufficient data may have caused errors in our analysis. Further studies with a substantial number of animals or a comparable translational human clinical trial are still needed. Moreover, most HCCs arise in the setting of chronic hepatitis induced by HCV or HBV infection. However, current animal models cannot mimic the inflammatory microenvironment that leads to the pathogenesis of HCC and the pathogenic sequence of human HCC that starts with fibrosis and cirrhosis prior to the development of HCC. Several studies have reported that fibrosis , 33. The99mTc-3PRGD2 is more sensitive for the detection of early stage HCC than 18F-FDG, but in advanced HCC, the diagnostic performance of 18F-FDG might be better than that of 99mTc-3PRGD2 because the tumour uptake of 99mTc-3PRGD2 significantly declined due to tumour necrosis. Combined 99mTc-3PRGD2 SPECT/CT and 18F-FDG PET/CT imaging would be helpful for the imaging diagnosis of HCC. We believe that 99mTc-3PRGD2 is worth further investing as a promising radiotracer for the diagnosis of HCC and guidance of the anti-angiogenic therapy.In conclusion,


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Oxygen-dependent fluctuation of labile Fe(ii) was visualized by a new N-oxide-based fluorescent probe for Fe(ii) ion. 2+. In this study, a universal fluorogenic switching system is established, which is responsive to Fe2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe2+ to generate various fluorescent probes of Fe2+–CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe In particular, several tumor cells can adapt to the harsh conditions of hypoxia, resulting from immature angiogenesis in solid tumors, by developing sophisticated protective machinery against hypoxia through activation of HIF-1.21Iron, the most abundant transition metal in our bodies, is involved in a number of biologically important processes such as respiration, oxygen transport, and energy production in collaboration with oxygen, based on its electrochemical properties with interconvertible multiple oxidation states.23 and PhenGreen-SK25 have been widely used as fluorescent probes for Fe ions in living cells, and have facilitated development of the important concept of “labile iron species”. However, their turn-off response as well as poor metal selectivity limits their utility. The majority of other examples of fluorescent probes for Fe ions show response to Fe3+ and/or a turn-off readout,29 both of which characteristics are not suitable for measurement of the delicate alteration of labile iron consisting of Fe2+ in living cells. Recently, several redox state-selective fluorescent probes for Fe2+ with a turn-on readout have been developed.30 RhoNox-1, reported by our group, is the first example of detection of labile iron in live cells with a turn-on response, where N-oxide acts as a selective fluorogenic switch to Fe2+.31 Subsequently, Chang et al. reported IP-1 on the basis of a chelation-assisted oxidative C–O bond cleavage, which was successfully applied to detection of biologically stimulated accumulation of labile iron.32 Wang et al. reported that acetylhydroxylamine-modified naphthalimide, the N–O bond of which is cleaved by Fe2+ as in the case of N-oxide, could work as a fluorogenic probe for Fe2+ to visualize Zn-induced accumulation of Fe2+ as well as ischemia-mediated Fe2+ accumulation.33 Very recently, Wells and Renslo et al. reported a unique puromycin analogue, the activity of which is stimulated through O–O bond cleavage of trioxolane selectively by Fe2+. Using this analogue, the authors reported that cancer cells have higher labile Fe2+ level than non-cancerous cells.34 Among these successful strategies, our N-oxide chemistry-based approach offers the advantage of high selectivity for Fe2+. The previous examples of N-oxide based probes have a rhodamine scaffold,35 which limits their utility to only a single color region .To study the dynamics of intracellular iron in living cells, calcein2+ with emission at the blue (CoNox-1), green (FluNox-1 and FluNox-2), and deep-red (SiRhoNox-1) regions is reported, verifying the versatility of the N-oxide chemistry. The N-oxide chemistry-based approach, involving deoxygenation of dialkylarylamine N-oxide by Fe2+, worked well as a universal fluorogenic switch for all the dyes tested and the deep-red dye, SiRhoNox-1, showed a remarkable response among the probes. Moreover, application of SiRhoNox-1 to image the fluctuation of redox balance of Fe2+/Fe3+ in tumor cells under hypoxia revealed that redox equilibrium shifts towards Fe2+ along with a decrease in oxygen tension. This is the first example of use of a redox state-selective fluorescent probe for Fe2+ for elucidation of hypoxia-mediated alteration in the intracellular redox balance of labile iron.Herein, development of a series of fluorescent probes of Fe2+ in living cells by exploiting N-oxide chemistry (λex/λem = 555/575 nm).31 After careful consideration of the principle of fluorescence switching enabled by the N-oxide chemistry, it was found that isolation of a nitrogen atom from the π-conjugation system of xanthene chromophore by N–O bond formation at one of the dialkylarylamines is the critical factor for achieving the dark turn-off state of the probes  in their π-conjugation system. To prove this concept, coumarin-6H (λex/λem = 403/495 nm), morpholinorhodol (510/535 nm), and Si-rhodamine B (645/660 nm) were chosen as parent fluorophores because they satisfied the following structural and photophysical requirements: (1) more than one dialkylarylamine(s) involved in their π-conjugation system, (2) high quantum yields under aqueous conditions, and (3) excitation/emission wavelength at blue, green, and deep-red regions. CoNox-1, FluNox-1, and SiRhoNox-1 were synthesized from coumarin-6H, morpholinorhodol, and Si-rhodamine B in modest yields, 63%, 57%, and 55%, respectively, by treating the corresponding dyes with m-CPBA. Diethylamino rhodol N-oxide was also synthesized as a rhodol-based probe, but its fluorescence response was not sufficient because of bright background signal as well as a low response rate (see ESIvide infra). These results indicate that the N-oxidation reaction works universally for oxygenation of dialkylarylamines within fluorophores.Previously, RhoNox-1 and HMRhoNox-M were reported to be useful fluorescent probes for detecting labile Fehemistry ; howevere probes  and an eε = 29 500 M–1 cm–1) with a shoulder at 335 nm, while coumarin-6H had an intense absorption at 402 nm with ε = 43 500 M–1 cm–1  of 15 000 M–1 cm–1 and 1250 M–1 cm–1, while the corresponding parent dyes, morpholinorhodol and Si-rhodamine B have strong absorption bands at 510 nm with 48 500 M–1 cm–1 and 645 nm with 12 500 M–1 cm–1, respectively  of 0.04 (CoNox-1), 0.08 (FluNox-1), and 0.08 (SiRhoNox-1), than the parent fluorophores; 0.92 (coumarin-6H), 0.15 (morpholinorhodol), and 0.34 (Si-rhodamine B) (Φ × ε) as shown in their fluorescence spectra  > RhoNox-1 (kobs = 7.0 × 10–4 s–1) > FluNox-2 (kobs = n.d.). It is hypothesized that the fast response and high off/on fluorescence contrast of SiRhoNox-1 is attributed to its closed spirolactone form at neutral pH. In accordance with its extremely low extinction coefficient (1250 M–1 cm–1 at 575 nm) at neutral pH  than RhoNox-1, which exists predominantly in quinoid form (kobs = 3.6 × 10–4 s–1) in neutral buffer. These factors synergistically improved the reaction kinetics and off/on contrast of SiRhoNox-1 for Fe2+.The N-oxide compounds showed considerably lower quantum yields (amine B) . As sugge Fig. S2. Among t2+ for 1 h , which acts as a Fe2+ ion chelator  at various time points  using SiRhoNox-1 as an indicator of labile Fe2+ (2 compared with 20% O2 (2) at all time points  as an inhibitor of NADPH-dependent reductases41 such as NADPH-cytochrome c reductase, the enzymatic activity of which is strongly activated under hypoxic conditions. In the presence of Bpy, the signal enhancement was completely suppressed for cells incubated under 1% and 5% O2 ,51 a direct importer of Fe2+ ion, no significant increase was observed in total iron contents of the cells incubated under hypoxia (1% O2), as quantified by atomic absorption spectroscopy . Western blot analysis of HIF-1α showed that the protein expression level of HIF-1α increased and reached a maximum at 4 h under 1% O2; no significant protein expression of HIF-1α was observed in the cells incubated under 5% O2  did not affect intracelluar ferritin level , FluNox-1 (rhodol), and SiRhoNox-1 (Si-rhodamine B) in addition to the previously described probe, RhoNox-1 (Rhodamine B). All the probes presented here exhibited a selective turn-on response against Fe2+ in aqueous buffer, with SiRhoNox-1 characterized by a distinctly high off/on contrast as well as preferred reaction kinetics. All the probes were applicable to live-cell imaging, and SiRhoNox-1 also showed extremely high signal/background ratio and good response rate within cells. Using SiRhoNox-1, it was observed that the redox balance of labile Fe species is altered under hypoxic conditions and that the up-regulation of labile Fe2+ is clearly dependent on O2 level but independent of total cellular amount of iron (iron uptake), ferritin degradation, HIF-1α-mediated signal transduction, and hypoxia-activated enzymes. Furthermore, it was demonstrated that these phenomena occurred in the central hypoxic core of the 3D spheroid tumor models by means of SiRhoNox-1. This is the first fluorescent imaging tool capable of capturing a slight equilibrium shift of cellular redox balance to labile Fe2+ under hypoxia. Successful development of this series of Fe2+-selective fluorescent probes with various color emissions on the basis of the N-oxide chemistry would encourage and enable the progress of biological study in iron-related physiological and pathological events.Using N-oxide chemistry a color series of Fe


P<0.001) and in tumor tissues (P<0.001) from patients with CRC. Interestingly, the increased PSG9 levels correlated with poor survival (P=0.009) and microvessel density (MVD) (P=0.034). The overexpression of PSG9 strongly promoted the proliferation and migration of HCT-116 and HT-29 cells. However, PSG9 depletion inhibited the proliferation of SW-480 cells. Using a human umbilical vein endothelial cell tube-forming assay, we found that PSG9 promoted angiogenesis. The overexpression of PSG9 also increased the growth of tumor xenografts in nude mice. Co-immunoprecipitation experiments revealed that PSG9 was bound to SMAD4. The PSG9/SMAD4 complex recruited cytoplasmic SMAD2/3 to form a complex, which enhanced SMAD4 nuclear retention. The PSG9 and SMAD4 complex activated the expression of multiple angiogenesis-related genes . Together, our findings illustrate the innovative mechanism by which PSG9 drives the progression of CRC and tumor angiogenesis. This occurs via nuclear translocation of PSG9/SMAD4, which activates angiogenic cytokines. Therefore, our study may provide evidence for novel treatment strategies by targeting PSG9 in antiangiogenic cancer therapy.PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family and has been shown to contribute to the progression of colorectal cancer (CRC) and cancer-related angiogenesis. Here, we aim to investigate abnormal PSG9 levels in patients with CRC and to emphasize the role of PSG9 in driving tumorigenesis. Serum from 140 patients with CRC and 125 healthy controls as well as 74 paired tumors and adjacent normal tissue were used to determine PSG9 levels. We discovered that PSG9 was significantly increased in serum ( PSG genes  and exhibit sequence similarity to the carcinoembryonic antigen (CEA) cell adhesion molecule (CEACAM) family [PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family. PSGs are encoded by ten ) family . CEA has) family . HoweverPrevious studies have recognized PSGs as secreted proteins that are the most abundant trophoblastic proteins in maternal blood during human pregnancy . Human PCRC ranks as the fifth most common cancer in males and as the fourth most common cancer in females in China . In compThe TGF-β signaling pathway can induce a proangiogenic environment, as it stimulates tumor angiogenesis . TGF-β1 In the present study, we first revealed the overexpression of PSG9 in patients with CRC. Then, we further investigated potential crosstalk between PSG9 and TGF-β/SMAD4 signaling, which is an intensively studied oncogenic pathway. We also explored whether PSG9 is a key component in the regulation of TGF-β signaling-induced tumor angiogenesis via enhancement of nuclear retention of SMAD4, and finally, we determined whether the expression of proangiogenic molecules is affected by the PSG9/SMAD4 interaction.We searched the Oncomine microarray database to analyze the expression of PSGs in CRC patients. Four  of 11 PSG members showed differential mRNA expression levels in tissues from CRC patients . The folTo analyze PSG9 expression in CRC tissues, a tissue microarray was stained with a PSG9 antibody. Finally, 74 subjects were enrolled for subsequent analysis. Negative, weak and moderate PSG9 staining was regarded as low expression, while strong PSG9 staining was regarded as high expression Figure . In cancThe MVD in 74 CRC tissues was analyzed by staining for CD31. The microvessels stained by the CD31 antibody were distributed within the mesenchymal tissues surrounding the carcinoma nests Figure . TherefoFurthermore, 9 (20.93%) patients out of 43 with low PSG9 expression had died. However, 16 (51.61%) out of 31 subjects with high PSG9 expression had died. A Kaplan-Meier analysis revealed that the overall survival rates in the high PSG9 expression group were significantly shorter than those in the low expression group Figure . Kaplan-We proposed that PSG9 might be one of the drivers that promotes the progression of CRC during carcinogenesis. We thus determined whether the change in PSG9 expression effects the proliferation of CRC cells. We selected HT-29 and HCT-116 cells to upregulate PSG9, which have relatively low PSG9 levels, and SW-480 cells to downregulate PSG9, which have high PSG9 levels, respectively . InitialNext, we asked whether PSG9 overexpression promotes the formation of tumor xenografts. We injected HT-29 and HCT-116 cells that were stably transfected with pcDNA3.1 and pcDNA3.1-PSG9 into BALB/c nude mice . As expein vitro. HUVECs were cultured with endothelial cell media (ECM) or conditioned media (CM) from HCT-116-pcDNA3.1 cell culture and HCT-116-pcPSG9 cell culture for 24 hours. Besides, we produced recombinant human PSG9 proteins . It was found that PSG9 and VEGFA were distributed in the cell membrane, cytoplasm and nucleus of HUVEC cells . Throughproteins . For thiproteins . As a reproteins . Similarproteins . Thus, w3.4 . Following this, we compared the levels of SMAD4 in HT-29 cells stably transected with PSG9 and HT-29 cells stably transfected with control vector. We found that the nuclear localization of SMAD4 was increased, but no changes were detected in the levels of SMAD4 in the cytoplasm Figure . IF staiThe location of SMAD4 in the cytoplasmic and nuclear fractions of TGF-β- treated HCT-116-pcDNA3.1 and HCT-116-pcPSG9 cells was examined in the presence of the TGF-β-receptor I (TβRI) inhibitor SB431542, which blocked SMAD3 and SMAD4 nuclear translocation. By Western blot of HCT-116-pcDNA3.1 and HCT-116-pcPSG9, we found that the PSG9 level in the nucleus was higher than that in the cytoplasm Figure . TGF-β tIt is well known that SMAD4/TGF-β signaling activates many angiogenesis- related genes by binding to their promoter regions. Next, an angiogenesis membrane-based antibody array was used to screen differential proteins whose expression is affected by PSG9. Antibody arrays allowed us to detect 58.18% (32/55) of angiogenic proteins in HCT-116 cells Figure . The proIGFBP-3, PDGF-AA, GM-CSF, and VEGFA genes , that might represent the genes that are most likely activated by PSG9 and further determined their mRNA levels in HCT-116-pcDNA3.1 and HCT-116-pcPSG9 cells. Stable transfection of cells with PSG9 increased the mRNA levels of the s Figure . A chroms Figure . We founA Figure . CollectWe report here for the first time that through an enhancement of angiogenesis, PSG9 is a potential driver of CRC. High PSG9 levels were found to be related to increased tumor growth. Importantly, elevated PSG9 levels in CRC patients were significantly associated with poor outcomes after surgery.Angiogenesis induction is pivotal to tumor growth and metastases . Our finet al. demonstrated that SMAD4 exerted a tumor-promoting role in hepatocellular carcinoma (HCC) [Previously, a large amount of data supported an early tumor-suppressive role for TGF-β. However, it was also shown that TGF-β signaling could promote tumor progression and metastasis . Howeverma (HCC) . Our resma (HCC) . Here, wTGF-β is sufficient to upregulate the expression of multiple cytokines, which in turn attracts neighboring endothelial cells and promotes angiogenesis , 27. OurIn summary, our findings indicate that the interaction of PSG9 with the transcription factor SMAD4 promotes its functions and is therefore critical for TGF-β-activated expression of angiogenesis-associated genes, cell growth, and cancer metastasis. Our findings that the overexpression of PSG9 promotes cancer cell growth and vasculogenesis provide insights into mechanisms and strategies for therapeutic intervention in advanced CRC.Serum samples were collected from 140 patients with CRC prior to surgery and from 125 healthy volunteers at the Tianjin Medical University Cancer Hospital from May 2014 to December 2014. All donors provided informed consent, and the study was conducted under the approval of the Institutional Ethics Committee. The concentration of PSG9 proteins in the serum samples was measured by an ELISA kit  according to the manufacturer's instructions.The HCol-Ade180Sur-07 tumor tissue microarray (TMA)  consisted of 90 paired colorectal adenocarcinoma tissues and matched normal mucosa; this TMA included 74 completed cases and 16 censored cases. Patients underwent surgery from January 2009 to October 2009, and the follow-up information was available from February 2009 to May 2014. The TMA slide was de-paraffinized and rehydrated. The slide was pre-treated with EDTA antigen retrieval solution (pH=8.0) and rinsed in water. The sections were then blocked in 2% goat serum and were incubated with the primary antibody overnight at 4°C. The PSG9 staining intensity and percentage of stained cells were analyzed as described previously . The intThe hotspot method was utilized to assess the microvessel density (MVD) on the CD31-stained tumor tissue sections as previously descried , 38. MVDSW620, HCT-116, and HT-29 cells were obtained from the American Type Culture Collection , while SW-480 and RKO cells were obtained from the Culture Collection of the Chinese Academy of Sciences . Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell Research Laboratories  and were cultured in Endothelial Cell Medium (ECM). Full-length PSG9 DNA was cloned into the pcDNA3.1-myc-his (−) plasmid  to generate a PSG9 expression plasmid. The PSG9-shRNA and scrambled shRNA control plasmids were purchased from GeneChem . SMAD2, SMAD3, and SMAD4 plasmids were purchased from Vigene Biosciences .E. coli Trans BL21 (DE3) cells were transformed with the pET-21α vector containing the PSG9 gene to obtain PSG9 protein.Stable cell lines with PSG9 overexpression and control vectors of HT-29 (HT-29-pcPSG9 and HT-29-pcDNA3.1) and HCT-116 (HCT-116-pcPSG9 and HCT-116-pcDNA3.1) cells were generated by transfection and were selected and maintained with neomycin . Stable SW-480 cells with PSG9 downregulation (SW-480-shPSG9) and controls (SW-480-shCon) were generated by stable transfection with PSG9 shRNA (shPSG9) and control shRNA (shCon); these were selected and maintained with puromycin (1 μg/ml). Total RNA was extracted using TRIzol and was reverse transcribed using a TransScipt II cDNA synthesis SuperMix kit  for standard real-time PCR (RT-PCR) analysis. Real-time PCR was performed using a SYBR green detection system in an ABI 7500 qPCR machine ; 18S was used as an internal reference gene. The gene-specific primers are summarized in A CCK8 assay  and a Click-iT®EdU (5′-ethynyl-2′-deoxyuridine) Flow Cytometry Assay Kit  were used to determine cell proliferation abilities. For the colony formation assays, cells were seeded into 6-well plates and grown for 21 days. The migration assay was performed in Transwell chambers containing polycarbonate filters .5 cells/well). The purified PSG9 protein, VEGFA and PSG9 antibody (5 μg/ml) were incorporated into the ECM and cultured with HUVECs. After 24 hours, images of capillary-like networks were recorded using a phase-contrast microscope. Images were analyzed with the Angiogenesis Analyzer plugin for Image J , which is available on the NIH website .Initially, Matrigel (BD Biosciences) was polymerized (200 μl/well of a 48-well culture plate) for 30 minutes at 37°C. Serum-starved HUVECs were resuspended in conditioned medium and reseeded in Matrigel-coated wells (107 cells per mouse) subcutaneously in the back adjacent to the left lower limb. Tumor dimensions were measured every 3-6 days. Mice were sacrificed at 38 days (~5 weeks) after injection, and the tumors were surgically isolated. IHC staining for CD31 in xenograft tumor tissues was performed according to the method described above.Animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of Beijing Chaoyang Hospital, Capital Medical University (Beijing China). Twenty male BALB/c nude mice (~6 weeks old) were purchased from Vital River . Each mouse was inoculated  and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked in 5% milk and then incubated with various antibodies followed by incubation with the appropriate secondary antibodies. The antibodies used in the present study are summarized in Cells seeded on chamber slides were fixed in ice-cold formaldehyde, blocked with 5% bovine serum albumin and incubated with anti-PSG9 (1:100) and anti-SMAD4 antibodies (1:50) overnight at 4°C. Antibody-bound cells were detected by Cy3- or FITC-conjugated secondary antibody . DAPI waCell lysates were incubated with 2 μg of antibody and placed on a rotator overnight at 4°C. After extensive washing in RIPA lysis buffer, the immunoprecipitated complexes were resuspended in reducing buffer and boiled for 10 minutes. After centrifugation to pellet the Protein A/G Agarose beads (Santa Cruz), the supernatants were subjected to SDS-PAGE and immunoblotting.A chromatin immunoprecipitation (ChIP) assay kit was provided by Beyotime  and was used according to the manufacturer's instructions. The primers for ChIP were described in The expression of angiogenesis-related proteins in cell lysates was determined using a Human Angiogenesis Array Kit , which consists of 55 antibodies against angiogenesis-related proteins spotted onto a nitrocellulose membrane. The array was performed according to the manufacturer's instructions using 300 μg of cell lysates. The data from developed X-ray film were digitalized with a transmission-mode scanner and were quantified using Image J analysis software as described previously . The ave2 test or Mann-Whitney U test. The significance of the in vitro and in vivo data was determined by Student's t test. Overall survival (OS) rates were assessed by the Kaplan-Meier test, and the log-rank test was used to plot survival curves. P<0.05 was considered to be significant.The significance of the data from patient specimens was determined by the χ


Swift and adequate fluid loading is a cornerstone of septic shock therapy. Yet, careful assessment of volume responsiveness and volume amount during the resuscitation process is a prerequisite. Both overzealous initial fluid administration and late fluid overload are harmful and may be associated with increased mortality.Static  pressure readings are erroneous for monitoring fluid resuscitation and should be abandoned. Dynamic measurements  better predict fluid responsiveness than static filling pressures but the conditions necessary for these parameters to correctly evaluate preload dependency are frequently not met. The passive leg raising maneuver as a means to alter biventricular preload in combination with real-time measurement of cardiac output changes is an easy-to-use, fast, relatively unbiased, and accurate bedside test to guide fluid management and to avoid fluid overload during early septic shock treatment. Moreover, PLR may also be particularly useful to assist various treatments that trigger fluid removal during the “de-resuscitation” phase of septic shock.The passive leg raising maneuver in combination with real-time measurement of cardiac output changes is an easy-to-use, fast, relatively unbiased, and accurate bedside test to guide fluid management during septic shock. The recently published Surviving Sepsis Campaign guidelines strongly recommend ample volume resuscitation during the first day of septic shock treatment . Such agJournal of Intensive Care, Krige et al. prospectively investigated the use of a novel generation Vigileo FloTrac™ system during a PLR maneuver in medico-surgical patients with vasopressor-dependent circulatory shock [At the beginning of this millennium, several authors proposed to use physiologic heart-lung interactions during positive pressure ventilation as more reliable predictors of fluid responsiveness in septic shock . Variatiry shock . The stuThrough the years, awareness has risen that after initial aggressive fluid resuscitation the focus should shift towards obtaining a net and even negative fluid balance . Late liIn conclusion, PLR testing in combination with more sophisticated real-time hemodynamic monitoring actually seems to be the most appropriate method to guide a bedside “do not harm” volume resuscitation strategy in septic shock. Future studies should assess the PLR maneuver using calibrated intermittent  or continuous  monitoring and evaluate whether this test could become pivotal in managing fluid removal during the “de-resuscitation” phase of septic shock.