Chitosan-stabilized nano zero-valent iron (CTS-nZVI) prepared by the liquid-phase reduction method has been shown to achieve a good dispersion effect. However, there has been little analysis on the mechanism affecting its stability and transport in saturated porous media. In this paper, settling experiments were conducted to study the stabilization of CTS-nZVI. The transport of CTS-nZVI in saturated porous media at different influencing factors was studied by sand column experiments. The stability mechanism of CTS-nZVI was analyzed from the point of view of colloidal stability by settling experiments and a zeta potential test. The theoretical model of colloidal filtration was applied for the calculation of transport coefficients on the basis of the column experiments data. Considering attachment–detachment effects, a particle transport model was built using HYDRUS-1D software to analyze the transport and spatial distribution of CTS-nZVI in a sand column. Nano zero-valent iron (nZVI) is a typical in situ remediation material for groundwater pollution ,2. As a Chitosan, as the product of N-deacetylation of chitin, has the characteristics of biodegradability, biocompatibility and non-toxicity. Chitosan-supported nano zero-valent iron (CTS-nZVI) prepared by the liquid-phase reduction method has been shown to achieve a good dispersion effect ,16. Xu e4·7H2O, NaBH4, NaCl, CaCl2 and KCl were purchased from Tianjin Fuchen Chemical Reagent Factory ; glacial acetic acid, NaOH, HCl and HNO3 were purchased from Beijing Chemical Plant . High-purity nitrogen  was purchased from Beijing Shunchi East Ring Dry Ice Business Center . All solutions in this study were prepared with deionized water (DI).All the chemical reagents used in this study were of analytical grade. Chitosan (deacetylation degree ≥ 95%) was purchased from Shanghai Aladdin Biotechnology Co., Ltd. ; FeSOQuartz sand with a particle size of 0.5~1 mm was selected as porous media. The quartz sand was pickled in hydrochloric acid, then cleaned with DI water for many times until the pH of the supernatant was close to DI water and then it was ready for use after air drying.The surface morphology and structure of CTS-nZVI were observed using a scanning electron microscope . The binding energy of CTS-nZVI was recorded by X-ray photoelectron spectroscopy . Functional groups of CTS-nZVI were analyzed by a Fourier transform infrared spectrometer . The settling rate of CTS-nZVI in solution and the concentration of iron were quantified by UV–Vis spectroscopy . The average size and size distribution of CTS-nZVI were measured by the dynamic light scattering technique . Zeta potential of CTS-nZVI was determined using a Brookhaven 90 plus zeta potential analyzer . The pH was measured using a pH-meter .In this study, the settling curves of different pH and ionic strengths were fitted by Origin 2018 software. The standard deviation calculation of the parallel test and the diagram in this paper were plotted using Origin 2018 software. The numerical model of water flow and solute transport in porous media was established by using software version 4.0 of HYDRUS-1D.3 solution, placed in a shaker (Thermostatic oscillator (DSHZ-300A), Jiangsu, China) at a constant temperature of 40 °C and vortexed for 4 h at 200 r/min to obtain 0.5% chitosan solution. An amount of 2.978 g FeSO4·7H2O was dissolved in 100 mL DI water, followed by adding 30 mL of the above 0.5% chitosan solution through a 0.22 μm microporous membrane, and then oxygen was removed with N2 for 30 min and stirred for 10 min to make it fully mixed. At the same time, 100 mL 0.535 mol/L NaBH4 solution was added into the mixed solution, and black iron nanoparticles were formed after 90 min of reaction. The reaction equation is as follows [In this study, CTS-nZVI was prepared by the liquid-phase reduction method. An amount of 1 g chitosan was added into 200 mL of 0.05 mol/L HNO follows :(1)FeSO4The prepared CTS-nZVI was filtered by a vacuum suction filter and cleaned with deoxygenated water for three times to remove the impurities.The CTS-nZVI suspension with a concentration of 150 mg/L was prepared, and the pH range was adjusted to 6~8 using 1% NaOH and 1% HCl solution . After 2) was added to make the corresponding concentration of NaCl (CaCl2) reach 0 mM, 30 mM and 100 mM , and the ionic strength was 0 mM, 30 mM and 100 mM. After treatment with an ultrasonic dissolver for 10 min, the sample was placed in the cuvette, and its absorbance was determined by a spectrophotometer at 508 nm [Water solution of CTS-nZVI with 150 mg/L concentration was prepared first. Then, an appropriate amount of NaCl  are filled at the inlet and outlet of both ends to ensure uniform water distribution. Quartz sand with a particle size of 1–2 mm after pickling was filled in the middle. During the filling process, quartz sand was added slowly, and the glass column was continuously knocked. A peristaltic pump was used to inject 10 pore volumes (PVs) deoxygenated water to establish chemical and hydrochemical equilibration between the sand and water, eventually removing residual colloids. On the other hand, it also plays a role in flushing and removing impurities from the quartz sand .In order to investigate the effects of injection speed, pH, ionic strength and ion species on the transport of CTS-nZVI, the following tests were carried out.The concentration of the CTS-nZVI suspension was 150 mg/L, and 3 PVs suspension was pumped by the peristaltic pump at the injection rates of 9 mL/min and 18 mL/min, and then 5 PVs deoxidized water was injected at the corresponding injection rate. Samples were taken at the outlet of the sand column, and then breakthrough and flushing curves were drawn. In order to reduce the aggregation and precipitation of CTS-nZVI, the suspension was treated by ultrasound about 10 min before and during injection. After the column experiment was completed, the quartz sand in the test column was taken out and soaked in a certain volume of hydrochloric acid to test the concentration of iron in the solution (spectrophotometry) , in ordeThe surface morphologies of chitosan a, bare nAs shown in −1, a C-H stretching vibration absorption peak of the methyl or methylene group near 2868 cm−1, an N-H straining vibration absorption peak of the amino group (-NH2) near 1611 cm−1 and a straining vibration absorption peak of C-CH3 around 1433 cm−1 and 1389 cm−1. There is also a stretching vibration absorption peak of C-OH near 1093 cm−1 [Fourier transform infrared spectroscopy was used to analyze chitosan and CTS-nZVI. As shown in 093 cm−1 .2+ at the binding energies of 709.9 eV and 723.5 eV, and 2p3/2 and 2p1/2 corresponding to Fe3+ at 711.3 eV and 724.9 eV. The characteristic peaks corresponding to Fe0 are found at the binding energies of 709.2 eV and 722.3 eV, indicating that Fe0 was partially oxidized to Fe2+ and Fe3+ during the preparation and characterization.As shown in The settling curves of CTS-nZVI at different pH were obtained by measuring the absorbance with a spectrophotometer, as shown in −1. Through the calculation of the fitting curve, the settling equilibrium time for CTS-nZVI at different pH is determined to be 160–170 min, 110–120 min and 110–120 min. In conclusion, the stability of CTS-nZVI at pH 7 and pH 8 was better than that at pH 6.The settling equilibrium time of CTS-nZVI is defined as the settling rate less than 0.0001 minAs shown in Breakthrough and flushing curves of CTS-nZVI nanoparticles at different pH are provided in It can be seen from The settling curves of CTS-nZVI at different ionic strengths and ionic species are shown in 2+ is lower than that of Na+. The settling rates of CTS-nZVI under different conditions are calculated as shown in 2+ is higher than that of Na+ at the same ionic strength. As the ionic strength increases from 0 to 30 mM, the settling is in equilibrium at the time of 50–60 min. In conclusion, the lower the ionic strength, the better the stability of CTS-nZVI. At the same ionic strength, Ca2+ has a more negative influence on the stability of CTS-nZVI than Na+. According to 2+ can significantly reduce the zeta potential absolute value. The results show that Ca2+ has more influence on the zeta potential of CTS-nZVI than Na+.Ionic strength and ion species effect the zeta potential of CTS-nZVI, which are provided in 2+ has a stronger effect on reducing the mobility of CTS-nZVI than Na+.Breakthrough and flushing curves of CTS-nZVI with different ionic strengths and ionic species are given in +, Ca2+ significantly reduces the stability of CTS-nZVI and increases the deposition of CTS-nZVI on the surface of porous media, leading to an increase in CTS-nZVI mobility. This is consistent with the influence of the ion strength and ion species on the settling experiment, that is, with the increase in ionic strength, the stability of CTS-nZVI gradually decreases, which enhances its deposition on the surface of porous media and reduces its mobility. Compared with Na2+ reduces the stability of CST- nZVI more than Na+.It can be seen from The breakthrough and flushing curves of CTS-nZVI at different injection velocities are illustrated in According to In this study, the stability of CTS-nZVI was analyzed from the perspective of colloidal stability. Brownian motion, the charge of dispersed particles and solvation are three important reasons for the stability of colloidal particles .The charge of colloidal particles is the main factor of colloidal stability. The solid and liquid phases in contact often have opposite sign charges due to ion deposition or dissociation . When th+, Ca2+ has more influence on the stability of CTS-nZVI. This can be explained by the Schulze–Hardy rule [2+ than Na+, the easier it is for CTS-nZVI to agglomerate. Therefore, compared with Na+, Ca2+ reduces the stability of CTS-nZVI.Compared with Nardy rule . The SchThe theoretical model of colloidal filtration depends on two parameters: (1)  factors : particlThe contact efficiency of a single collector is calculated as follows :(2)η0=ηIThe meanings of the parameters are shown in Colloidal filtration theory can be used to predict the maximum transport distance of colloidal particles :(6)Lmax=α and According to +, Ca2+ has a stronger inhibitory effect on the transport of iron nanoparticles. With the increase in the water flow velocity, the maximum transport distance of iron nanoparticles increases significantly.With the increase in pH, the adhesion coefficient α decreases and the maximum transport distance increases, which indicates that the increase in pH is beneficial to the transport of CTS-nZVI in saturated porous media. With the increase in ionic strength, the adhesion coefficient α increases and the maximum transport distance decreases significantly, which indicates that the increase in ionic strength is not conducive to the transport of CTS-nZVI in saturated porous media, and the ionic strength has a great influence on the transport of iron nanoparticles. Compared with NaIn this study, a numerical model of water flow and solute transport in porous media was established by using HYDRUS-1D software. The simulation includes two processes. One is the transport process of the nZVI suspension after being pumped into the saturated sand column from bottom to top, and the other is the transport process of nZVI in the saturated sand column under subsequent flushing by deoxygenated water. The unit of time is min, the unit of mass is μg and the unit of length is cm.H is the water head, cm; θ is the soil volume moisture content; t is the time, min; s is the source and sink term, min−1; α is the angle between the flow direction and the vertical direction . In this study, α = 0°; X is the space position, cm; and K is the unsaturated hydraulic conductivity, cm min−1.The one-dimensional equilibrium flow is described by the Richards equation ,39:(7)∂θThe initial condition of the flow model is the whole saturation state, that is, the initial water content is the saturated water content. The upper and lower boundaries of the flow model are constant flow boundaries.θ is the soil volume moisture content; C is the solute liquid phase concentration, mg·mL−1; S is the solute content in solid media, ug·g−1; the subscripts 1 and 2 represent the two kinetic adsorption sites; D is the dispersion coefficient, cm2·min−1; V is the Darcy velocity, cm·min−1; X is the spatial position, cm; −3; t is the time, min. In this equation, two source and sink terms In this study, a solute transport equation based on the attachment–detachment model was established to simulate the transport of CTS-nZVI in saturated porous media. The expression of the equation is as follows :(8)∂θC∂tThe initial condition of the model is that the solute concentration at each position of the sand column is 0 at the initial time. The lower boundary of the model is defined as concentration flux, and the given concentration boundary and the upper boundary are concentration flux boundaries.This simulation imitates the transport of nZVI nanoparticles in saturated porous media at two different injection rates. The model parameters are shown in From the observation of It can be seen from A Nash–Sutcliffe simulation efficiency coefficient (NSC) evaluation model was used to evaluate the results, and the calculation formula is as follows :(9)NSC=1S1) and the solid concentration of CTS-nZVI nano iron retained in the porous media due to physical straining (S2). It can be seen from In the attachment–detachment model, two source and sink terms (1)The characterization results exhibit that chitosan improves antioxidation and inhibits the aggregation of nZVI particles.(2)2+ has a more negative influence on the stability of CTS-nZVI than Na+. The charge amount of CTS-nZVI changes the repulsive potential energy between particles, which changes the stability of CTS-nZVI.With the higher pH and the lower ionic strength, the stability of nZVI is better. At the same ionic strength, Ca(3)2+ has a more negative influence on the mobility of CTS-nZVI than Na+. As the larger fluid shear force can promote the mobility of nanoparticles, CTS-nZVI exhibits an enhanced transport in saturated porous media with the increase in injection velocity.A series of sand column experiments show that the transport of CTS-nZVI in saturated porous media is related to its stability. When the pH is higher and the ionic strength is lower, the mobility of nZVI is better. At the same ionic strength, Ca(4)According to the colloidal filtration theory, gravity settlement is an important factor affecting the deposition of CTS-nZVI. Transport behaviors of CTS-nZVI are described by the nanoparticles’ spatial distribution in sand columns using an attachment–detachment model in HYDRUS-1D. The simulation results show that straining is an important factor affecting the transport of CTS-nZVI.This study systemically explored the mechanisms of stability and transport of CTS-nZVI in saturated porous media through laboratory experiments, theoretical calculation and simulation modeling. The following conclusions are drawn:Our work reveals the stability mechanism and the transport distance of CTS-nZVI, which will provide an important theoretical and experimental basis for the practical application of in situ injection technology using CTS-nZVI during groundwater remediation.


Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors. Keratinocyte carcinomas (KCs) are the most common cancers in the UK, with over 210,000 tumors reported in 2015 . Mortali3P] in vivo and are dimers of a regulatory and one of four catalytic subunits, namely p110α, p110β, p110δ and p110γ [3P, and subsequent phosphorylation at its residues Thr308 and Ser473. Once activated, Akt regulates a plethora of proteins, including mechanistic  target of rapamycin (mTOR), a master kinase that controls several cellular functions, such as protein synthesis, ribosome biogenesis, cell growth and autophagy [3P thus preventing Akt activation [Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that catalyze the phosphorylation of the 3′ position within the inositol ring of distinct phosphoinositides ,11. Eighnd p110γ . The besnd p110γ , whose mutophagy ,17. The tivation .PTEN and are characterized by hyperplastic changes in the skin [PTEN null mutation which are characterized by epidermal hyperplasia and hyperkeratosis [mTOR which die shortly after birth due to impaired early epidermal differentiation and lack of a protective barrier development [The PI3K/Akt/mTOR signaling pathway is important for epidermal homeostasis ,20,21, athe skin . Additioeratosis , mice wieratosis  and miceelopment .PTEN null mutation developed tumors more rapidly upon chemical treatment as well as spontaneous tumors, mostly squamous papillomas, many of which progressed into invasive cSCC [O-tetradecanoylphorbol-13-acetate [Consistent with its role in epidermal homeostasis, evidence also suggests that the PI3K/Akt/mTOR pathway is important during cSCC development and progression . Activative cSCC . Additioive cSCC , and thaive cSCC ,32. Cons-acetate  and over-acetate .While the importance of the PI3K/Akt/mTOR pathway is supported by several data, much less is known about the specific contribution of each distinct class I PI3K isoform to cSCC. Moreover, whether the use of available isoform-specific inhibitors might represent a potential therapeutic strategy in cSCC remains to be clarified. Recently, an extensive characterization of a panel of patient-derived cSCC cell lines has been reported, validating their use for preclinical testing . Here, wSeveral lines of evidence indicate that the PI3K/Akt/mTOR pathway is critical for keratinocytes ,20,21 anExpression of p110γ and p110δ was also observed . These dHaving confirmed expression of class I PI3K isoforms, we next determined whether these enzymes were involved in regulation of growth and viability of the selected cell lines. First, we observed that treatment with the pan PI3K inhibitor LY294002 reduced the number of N-TERT and Neb1 cells a as wellNext, we tested the effect of these inhibitors on the selected cSCC cell lines. Data showed that treatment with LY294002 (10 µM) reduced number a and viaTaken together, these data confirmed that the PI3K/Akt/mTOR pathway plays a key role in keratinocytes and in cSCC cell growth and viability.To identify the main class I PI3K isoform(s) involved in regulation of keratinocytes and cSCC growth and viability, cells were treated with isoform-specific PI3K inhibitors, namely BYL719 (targeting p110α primarily), GSK2636771 (targeting p110β primarily), AS252424 (targeting p110γ primarily) and CAL101 (targeting p110δ primarily). First, investigation of the effect of these inhibitors on immortalized keratinocytes revealed that BYL719 reduced both cell numbers a,c and vAs a slight reduction in cell numbers was observed upon treatment with the p110β inhibitor GSK2636771 in N-TERT cells a, we nexTreatment with BYL719 appeared to reduce cell numbers and viability less than pan PI3K inhibition a,b; therTaken together, these data demonstrated that BYL719 inhibits growth and viability of keratinocytes more potently than the other inhibitors tested, suggesting that p110α is the main PI3K isoform regulating such cellular functions in these cells.Next, we assessed the effect of the inhibitors on the selected cSCC cell lines. We observed that the p110α inhibitor BYL719 clearly reduced the number a,c and vIn addition, combination of BYL719 and GSK2636771 did not result in any significant increase in the effect of the p110α inhibitor on IC8 and Met1 cell numbers a,c or viTaken together these data indicate that IC8 and Met1 cells retain the same sensitivity to PI3K inhibition as normal keratinocytes and suggest that p110α is the main PI3K regulating growth and viability of these cSCC cell lines.In contrast to IC8 and Met1 cells, BYL719 had only a very small effect on IC1 a and T11Combined results from cell counting and MTT assays indicate that IC1 and T11 cells are more resistant to PI3K inhibition, in particular to p110α inhibition, compared to IC8 and Met1 cells.To determine whether resistance to p110α inhibition was due to a compensatory role of p110β, we next investigated the effect of simultaneous inhibition of p110α and p110β on these cells. Consistent with our previous data, treatment with BYL719 alone reduced IC1 a and T11These data indicate that reduced response to p110α inhibition in IC1 and T11 cells is not due to a compensatory mechanism dependent on p110β, as detected in some breast cancers ,37. TakeIn order to determine whether the limited response to isoform-specific inhibitors observed in IC1 and T11 cells was due to an intrinsic resistance to PI3K inhibition, we next assessed the effect of these compounds on Akt activation in the selected cSCC cell lines. Treatment with BYL719 for 1 h strongly inhibited Akt phosphorylation at both Ser473 and Thr308 residues in Met1 cells without affecting the levels of total Akt, as determined by Western blotting followed by visualization using either Chemidoc™ MP Imaging system  or a filTreatment of Met1  and IC8 Taken together these data demonstrate that p110α is the main isoform regulating Akt activation in IC8 and Met1 cells, consistent with data on cell growth and viability.We next investigated the effect of the isoform-specific inhibitors on T11 and IC1 cells. Data revealed that treatment with BYL719 for 1 h was able to inhibit Akt phosphorylation in T11 cells without affecting total levels of Akt , similarDose response analysis confirmed inhibition of Akt phosphorylation in T11 cells treated with BYL719 but not with GSK2636771 .Inhibition of Akt phosphorylation was also detected in IC1 cells upon 1 h  or 24 h These data indicate that BYL719 inhibits Akt activation in all cSCC cell lines. Together with our previous data, these results indicate that the limited effect of BYL719 on IC1 and T11 cell growth and viability is not due to a generic lack of response to the drug in these cells.Data in the literature indicate that the PI3K/Akt/mTOR pathway could be a valid target in cSCC ,38. The In this study we assessed the sensitivity to pan and isoform-specific PI3K inhibition of four cell lines, part of a recently described panel of patient-derived cSCC cell lines ,35. Impo50s of these inhibitors towards their selective targets are in the low nanomolar range and other PI3Ks might be affected by high concentrations of the compounds [Using isoform-specific inhibitors we further show that the p110α inhibitor BYL719 inhibited growth and viability of the cSCC cell lines IC8 and Met1 more potently than any of the other inhibitors. Similar results were obtained in immortalized keratinocyte cell lines. On the other hand, neither BYL719 nor any of the other selective inhibitors had any major or consistent effect on both cell counting and MTT assays in the cSCC cell lines IC1 and T11, when used at a concentration of 1 µM. These data indicate that, while growth and viability of all investigated cSCC cells is reduced by pan PI3Ks or mTOR inhibition, sensitivity to selective PI3K inhibitors, in particular to BYL719, differs between these cells. It remains to be established whether these inhibitors might affect growth and viability of IC1 and T11 cells if used at concentrations higher than 1 µM. In this respect, it must be noted that treatment with 1 µM of GSK2636771  or AS252ompounds ,52,53, tPIK3CA, detected in many cancers [PIK3CA have been reported in some recurrent, metastatic cSCC [PIK3CA activating mutations have been detected in some cSCC lymph node metastases [Overall, our data indicate that growth and viability of IC1 and T11 cells are more resistant to BYL719 treatment compared to IC8 and Met1 cells. The differential sensitivity to BYL719 was not due to different expression levels of p110α as this was fairly consistent between all cell lines used in this study. Furthermore, data in the literature indicate that activating mutations of  cancers , do not  cancers ,56. Intetic cSCC  and PIK3tastases . These otastases , stronglResistance to p110α inhibition has been documented ,61,62 anWhile BYL719 did not affect their viability and growth, it was able to inhibit Akt activation in IC1 and T11 cells in the experimental conditions used in this study. Whether a rebound Akt activation occurs in these cells upon longer incubation with the inhibitor remains to be established. Nonetheless, these data support the conclusion that monitoring Akt phosphorylation might not be directly indicative of the efficacy of the inhibitor on viability and growth of cSCC cells, as previously observed in breast cancers . It mustFinally, as it was limited to four cSCC cell lines, our study cannot provide a final answer to the question of whether targeting specific PI3K(s) might represent a therapeutic strategy in cSCC, especially considering the many cross talks between this pathway and other signaling cascades. Important information can be obtained from our data nonetheless. Our results have revealed a heterogeneous response of cSCC cell lines to selective PI3K inhibitors (in particular p110α inhibition), possibly suggesting that sensitivity to these drugs might be patient-specific. The observation that BYL719 affects growth and viability of two of the investigated cSCC as well as two immortalized keratinocyte cell lines, however, might already suggest some limitations in the potential use of this inhibitor. Clearly, more studies are now needed to validate whether selective PI3K inhibitors have a therapeutic value in cSCC. Some initial data from this study were presented, as a poster, at the British Society for Investigative Dermatology Annual Meeting Norcroft Conference Centre, University of Bradford, Bradford, UK, 1–3 April 2019 .2. The following inhibitors were used in this study: BYL719 , GSK2636771 , AS252424 , CAL101 , LY294002 , rapamycin , A66 . Cells were incubated with each inhibitor at a concentration of 1 µM (unless otherwise stated) in complete medium supplemented with 10% FBS.cSCC cell lines, previously characterized ,35, and Cells were seeded in 12 well plates (cell counting) or 48 wells (MTT assays) and treated with the indicated inhibitors 24 h after plating. Control cells were treated with vehicle alone (DMSO). After 72 h, cells were manually counted using a Burker chamber and a light microscope at 10x magnification. All experiments were performed in duplicate. Alternatively, cells were incubated with 500 µg/mL Thiazolyl Blue Tetrazolium Bromide  during the last 3 h of incubation. MTT formazan crystals were then dissolved with DMSO and absorbance at 570 and 650 nM was measured using an absorbance microplate reader.v/v) Tween 20 (PBS-T) for 30 min at room temperature, followed by overnight incubation with primary antibodies at + 4 °C. Primary antibodies  were as follow: anti p110α , anti p110β , anti p110γ , anti p110δ , anti pSer473 Akt , anti pThr308 Akt , anti Akt , anti α-Tubulin ; anti Vinculin ; anti GAPDH . After washing with PBS-T, membranes were incubated with peroxidase conjugated secondary antibodies  for 1 h at room temperature, washed with PBS-T and exposed to ECL reagent . Signals were visualized using X-ray Film 18 cm × 24 cm Double Sided  using a film processor or using Chemidoc™ MP Imaging System . For experiments visualized using X-ray films and a film processor, membranes were cut into strips around the molecular weight of each protein of interest and incubated with the corresponding primary antibodies. For experiments visualized using Chemidoc™ Imaging System, the whole membranes were incubated with the specific primary antibody first. After visualization, membranes were re-incubated with anti GAPDH or anti Tubulin as loading control.Cells were seeded in 6 well plates. For analysis of PI3Ks expression levels, cells in normal growing conditions were used. Alternatively, cells were treated with inhibitors for 1 h or 24 h before lysis. For experiments presented in Although limited to four cSCC cell lines, our study has revealed their differential sensitivity to selective PI3K inhibition, confirming the highly heterogeneous nature of cSCC. This highlights the critical need for reliable experimental tools that can allow careful preclinical studies. In this respect, this study has contributed further to the characterization of a panel of cSCC cell lines, which will prove very useful in preclinical testing to identify novel therapeutic strategies.


Renal cell carcinoma (RCC) accounts for over 400,000 new cases and 175,000 deaths annually. Diagnostic RCC biomarkers may prevent overtreatment in patients with early disease. Extracellular vesicles (EVs) are a promising source of RCC biomarkers because EVs carry proteins and messenger RNA (mRNA) among other biomolecules. We aimed to identify biomarkers and assess biological functions of EV cargo from clear cell RCC (ccRCC), papillary RCC (pRCC), and benign kidney cell lines. EVs were enriched from conditioned cell media by size exclusion chromatography. The EV proteome was assessed using Tandem Mass Tag mass spectrometry (TMT-MS) and NanoString nCounter technology was used to profile 770 cancer-related mRNA present in EVs. The heterogeneity of protein and mRNA abundance and identification highlighted the heterogeneity of EV cargo, even between cell lines of a similar pathological group . Overall, 1726 proteins were quantified across all EV samples, including 181 proteins that were detected in all samples. In the targeted profiling of mRNA by NanoString, 461 mRNAs were detected in EVs from at least one cell line, including 159 that were present in EVs from all cell lines. In addition to a shared EV cargo signature, pRCC, ccRCC, and/or benign renal cell lines also showed unique signatures. Using this multi-omics approach, we identified 34 protein candidate pRCC EV biomarkers and 20 protein and 8 mRNA candidate ccRCC EV biomarkers for clinical validation.The online version contains supplementary material available at 10.1007/s12032-021-01554-2. Renal cell carcinoma (RCC) is a lethal disease which accounts for more than 400,000 new cases worldwide and over 175,000 deaths annually . This shExtracellular vesicles (EVs) are nanoparticles 50–1000 nm) that are secreted by all living cells, including cancer cells  and thei00 nm thaVarious species of RNA have been found in EVs, including microRNA (miRNA), long non-coding RNA (lncRNA), and protein-coding mRNA. All of these RNA types have been proposed as diagnostic or prognostic biomarkers for RCC –16. The The EV proteome includes all cytosolic proteins within EVs and the transmembrane or GPI-anchored proteins localized at the bilipid membrane , 22. A sCCM is frequently utilized by EV researchers due to availability and reproducibility , 26. Bec7 EVs/million cells) and Caki1 secreted the least (5.6 × 104 EV/million cells) , pRCC cells (ACHN and Caki2), and immortalized benign epithelial kidney cells (HK2 and RPTEC/TERT1). The number of isolated EVs per mL CCM as measured by nanoFCM varied per cell type  and the non-EV cellular marker calnexin Fig. a. CalnexWhile western blot is a standard in the EV field, detection of positive EV markers is limited by sensitivity and sample input. To robustly characterize the protein cargo of CCM kidney EVs, a previously developed spatial proteomic approach  to exami2 FC > 0.58), all the EV markers were found to be elevated in EVs from HK2, 786-O, and CAKI2 renal cell lines and four of the six markers to be increased in 769-P, ACHN, and CAKI1 EVs. Whereas CD81 and CD63 had increased abundance in all the EV fractions relative to the cell lysate, ALIX, CD9, TSG101, and FLOT1, while robustly detected, did not always display increased abundance in the EV fractions relative to the cell lysates. This reflects the variable abundance of these proteins in the individual renal cell lines and EV fractions, as well as the potential variable distribution of EV sub-populations . U. U22]. Uulations , 31. Whi2 FC > 0.58) between the cell lysate and EV fraction, CANX was found to be the only negative EV marker decreased in all EV samples, following the same expression pattern as shown by western blot, while HSP90B1 was decreased in EVs from five out of the six cell lines.In addition to positive EV markers, the differential abundance of several proteins classically considered to be negative in EVs was also evaluated, including CANX , HSP90B1, and CYC1 . Using a2 FC > 0.58) were considered. Following these criteria, 1279 proteins were annotated that were EV-associated in at least one cell line, 420 proteins in four, and 284 proteins in five cell lines (Table S2). 186 proteins were EV-associated in all six cell line sample sets. Using the bioinformatic tool DAVID [Finally, to specifically assess the enrichment of EV-specific proteins , proteins with high abundance in EV vs cell lysate and/or in EV vs 2 k debris fraction , concordant trends (displaying either increased abundance or decreased abundance) including 76 proteins in ACHN cells/EVs, 59 proteins in CAKI2 cells/EVs, 47 proteins in 786-O cells/EVs, 70 proteins in 769-P cells/EVs, and 39 proteins in CAKI1 cells/EVs were observed (Table S6). These results provide some evidence of EVs reflecting the molecular characteristics of their respective parental cells .Overall, these results confirm that the SEC approach was successful for enriching EVs, and subsequently EV-associated proteins, while reducing the abundance of several proteins commonly categorized as negative markers of EVs.2 FC > 0.58) threshold, the differential abundance of EV-associated proteins between each of these groups was examined. Notably, direct pair-wise comparison of the cell line-derived EVs revealed a high degree of heterogeneity in protein abundance, emphasizing the heterogeneity of EV cargo itself, even within a single histology type (Tables S5 and S7).To better understand the potential biological impact of RCC-derived EVs, the renal cell lines were grouped based on their respective histology: pRCC (ACHN and CAKI2), ccRCC , and a single benign cell line (HK2) . Using aOn comparing pRCC EV cargo (n = 2 cell lines) to benign (n = 1 cell line), 31 proteins were found to be increased and 88 proteins were decreased in abundance in EVs from pRCC cell lines compared to EVs from the HK2 benign cell line (Table S5). Using the bioinformatic tool WebGestalt , 36 for Analysis of the differential abundance of proteins derived from EV released from ccRCC cell lines (n = 3 cell lines) and the benign HK2 cell line revealed that 93 proteins were decreased and 34 were increased in the EVs derived from all three ccRCC cell (Table S5). Cellular processes associated with CCT/TriC protein folding and metabolism were increased in ccRCC EVs relative to HK2-derived EVs, whereas proteins associated with neutrophil degranulation and ERBB2 signaling were decreased in ccRCC EVs .Finally, the differential abundance of proteins in EV derived from pRCC cell lines (n = 2 cell lines) and ccRCC cell lines (n = 3 cell lines) was assessed. Overall, there were six differentially abundant EV proteins between ccRCC and pRCC: five proteins were increased in EVs from all pRCC cell lines assessed relative to all ccRCC EVs , while only one protein, HTRA1, was increased in all ccRCC-derived EV samples relative to pRCC EVs (Table S7). Pathway analysis of the proteins found to be differentially abundant between the pRCC EVs and ccRCC EVs revealed disparate pathways in each of the individual RCC EV comparisons (Tables S10 and S11), albeit with ECM–receptor interaction and focal adhesion being commonly annotated across multiple comparisons.To specifically identify candidate ccRCC or pRCC EV cargo protein biomarkers, the proteins enriched in EV samples were assessed, regardless of relative abundance. To be included in the analysis, a protein must be detected in the EV preparation in all cell lines of the pathological type . 181 proteins were detected in EVs released from all renal cell lines: the benign epithelial kidney cell lines, both pRCC cell lines, and three ccRCC cell lines. While a large number of proteins were enriched in EV from the immortalized benign epithelial cell line, fewer were specifically enriched in RCC EVs. 34 proteins were enriched in pRCC EVs alone, while 20 were uniquely enriched in EV released from ccRCC cells  were run in triplicate using the nCounter PanCancer Progression Panel and data were normalized by total RNA content. Following quality control, 1 replicate of HK2 was excluded due to a technical hybridization error and samples CAKI1 and CAKI2 were excluded due to insufficient RNA input. Of the 770 mRNA transcripts assessed, 461 were detected in one or more samples of CCM EVs, and 159 were present in all five cell line CCM EVs (Table S14). Unsupervised hierarchical clustering of the top 100 most differentially abundant mRNA transcripts indicated that benign renal, ccRCC, and pRCC cell line EVs had distinct mRNA cargos Fig. a. AnalysRELN, CDH2, SERPINE1, STAB1, VCAM1, KCNJ8, SRGNR, EREG, COL6A3, and GDF15 were the 10 most enriched genes in RCC EVs, while HIF1A, KRT19, HOXA7, AGR2, LAD1, EPCAM, LAMA4, MUC1, SCNN1A, and PTRF were the 10 most enriched genes in benign EVs . Ten mRNAs were unique to benign epithelial kidney EVs and absent in EVs released from ccRCC: . Ten mRNLAIR1 mRNA was higher in RNA extracted from the tissue, but measurement of the mRNA transcript directly in EVs was not performed. Qu et al. studied abundance of the long noncoding RNA lncARSR in 786-O and ACHN cells and its sunitinib resistance-promoting effect in xenograft models [GSTA1, CEBPA, PCBD1 in urinary EVs [In this study, a whole proteomic approach and targeted mRNA profiling were leveraged to identify the consensus protein and mRNA signatures of RCC cell lines for the selection of potential biomarkers and to assess biological function of kidney cell line EV cargo. In the literature, only few studies focus on the identity of cargo from EVs derived directly from RCC cells. Jingushi et al. found presence of the LAIR1 protein in RCC tissue as well as in 786-O, Caki1, and Caki2 whole cell lysates and their released EVs . The LAIt models . De Palmnary EVs . Raimondnary EVs . Jingushnary EVs .VHL in ccRCC EVs as compared to benign EVs in concordance with TCGA data reported by Linehan et al. [TCEB1, TCEB2, and RBX1) were significantly more abundant in ccRCC EVs compared with benign EVs, perhaps in response to reduced function of the complex due to loss of VHL. The VHL-Elongin BC-complex stabilizes the HIF1A protein which then degrades. With loss of VHL, HIF1A degradation will be less and this could lead to an increased hypoxia response. The HIF1a-mRNA was significantly higher in both benign and pRCC EVs compared with ccRCC EVs , and COL6A1 was more abundant in ccRCC EVs. CDH2 and VIM, higher expressed in ccA phenotype, were significantly more abundant in ccRCC EVs than in benign EVs.Brannon et al. studied two mRNA signatures, ccA and ccB, in tissue microarrays of ccRCC. The pattern of ccB was associated with a worse prognosis . AlthougThe spatial proteomic approach to robustly characterize the protein cargo of renal cell derived EVs identified over 1900 proteins including well-known EV markers, as well as those considered to be negative markers of EVs. Rather than rely solely on protein identification for classifying a protein as EV cargo, relative protein abundance was utilized as a primary filtering criterion , 40. WhiOf potential interest to the EV field is the identification of a consensus renal EV proteome, which includes proteins that are known to be enriched in EVs, including known EV markers, as well as proteins anticipated to be EV cargo such as those associated with the extracellular matrix, cellular membrane, and endosome/receptor internalization. In addition, proteins were identified that canonically belong to intracellular compartments not usually associated with EVs, including nuclear-associated proteins  and mitochondria-associated proteins , illustrating that the passive and active mechanisms involved in EV protein routing have yet to be fully elucidated. While this report may be the first to annotate a consensus renal EV proteome, it does not offer any insight into whether the proteins identified can be applicable for defining tissue-specific EV features, warranting a more comprehensive analysis of EV protein cargo from a variety of tissue models.Evaluation of the differential abundance of EV cargo proteins revealed cellular pathways commonly annotated and associated with EVs, including ECM–receptor interaction and focal adhesion, albeit with variable abundance when comparing pRCC, ccRCC, and HK2 cell-derived EVs relative to one another. These results support the notion that these protein classes are routed into EVs as a reflection of their respective biological function. Additional studies are needed to delineate the disparate impact these cargo profiles may have on EV internalization by recipient cells and subsequent cellular phenotypes. Proteins were identified that were enriched in RCC EVs relative to HK2 EVs, including TNFAIP6, whose mRNA transcript has been shown to be a potential ccRCC biomarker , and VCAThis study demonstrates the heterogeneity of RNA and protein signatures of EVs, likely due to biological variation as well as limitations of current experimental technology. While this study is a critical first step to identifying tumor-specific EV biomarkers, it has a number of limitations. First, both the proliferation rates and per-cell release of EVs of these cell lines vary. In this study, similar amounts of cells were plated, and the EV counts were normalized by cell count. Secondly, due to limited sample availability in combination with the high sensitivity of the TMT-MS, we used a single pooled sample for cell line EV sample. This allowed mRNA and protein assessment in the same EV populations. To increase the accuracy of the MS-based analysis, relative protein abundance compared to parental whole cell lysates was utilized to determine which proteins were enriched in the EV fractions. This robust approach, with the end goal of reducing the number of potential false-positive hits, serves as a method for filtering possibly protein contaminants that may be present in the final EV analyte, an especially important consideration for high-sensitivity techniques. However, the filtering criterion employed in this study would exclude any proteins that display low abundance in an EV fraction relative to the parental cell as EV-associated and may not fully account for the myriad of mechanisms of protein routing into EVs that may impact select EV protein cargo abundance. Finally, loading exactly equal amounts of EV RNA was limited by current methods for RNA quantitation , 46, 47.This study demonstrates the potential power of EV cargo to be utilized as potential diagnostic and prognostic biomarkers. It also highlights the complicated biology of EV cargo and that further examination is needed to understand how and why RNA and protein is packaged into EVs for expulsion from a cell. Future work remains to be done to characterize the benefits to the cell to secrete EV cargo as well as the potential benefits to cells that internalize EVs.Kidney cell lines were acquired from the American Type Culture Collection (ATCC®). 769-P and 786-O were cultured in RPMI 1640 media (Gibco™). ACHN was cultured in EMEM media (Gibco™). Caki1 and Caki2 were cultured in McCoy’s 5a medium (Gibco™). HK-2 was cultured in DMEM media (Gibco™). These media were all supplemented with 10% (v/v) fetal-bovine serum (FBS) (VWR™), 100 U/mL penicillin, and 0.1 µg/mL streptomycin (Gibco™). RPTEC–hTERT1 (RPTEC) were cultured in DMEM/F12 supplemented with hTERT-immortalized RPTEC growth kit (ATCC) and 0.2% (v/v) Geneticin™ (Gibco™). Cell lines were regularly screened for mycoplasma.2 cell-culture dishes. When confluency reached approximately ~ 60% , the serum-containing media were removed, cultures were washed twice with PBS, and the media were replaced with appropriate media supplemented with 10% (v/v) exosome-depleted FBS, 100 U/mL penicillin, and 0.1 µg/mL streptomycin (Gibco™). For RPTEC/hTERT1, 45 million cells were plated in 500 cm2 cell-culture dishes and confluency reached ~ 60% at day 5. The cultures were washed twice with PBS, and media were replaced with serum- and growth-factor-free DMEM/F12 supplemented with 0.2% (v/v) Geneticin™ (Gibco™). For all cell lines, the media were conditioned for 48 h at 37 °C and 5% CO2. Cells were counted at time of harvest using the Countess™ II Automated Cell Counter (Invitrogen™). CCM was collected in triplicate for each cell line and EVs isolated from each replicate.For 769-P, 786-O, ACHN, Caki1, Caki2, and HK-2, 36 million cells were plated in 500 cmg for 5 min. Precipitated cells were kept, PBS-washed, lysed in 1 × RIPA buffer , and the cell lysates (CL) stored at − 80 °C. The supernatant was kept and centrifuged at 2000×g for 20 min. After this step, the 2 k pellet was kept, PBS-washed, and centrifuged again with same parameters. The 2 k pellets were also resuspended in 1 × RIPA buffer and stored at − 80 °C. The supernatant of the 2000×g was centrifuged at 10,000×g. The remaining supernatant was filtered using a Nalgene™ Rapid-Flow™ bottle top filter with 0.45 µm PES membrane (Thermo Scientific™). All centrifuge steps were performed at 4 °C .After 48 h of incubation, the CCM was harvested and pre-cleaned by differential centrifugation and filtration. The media were centrifuged at 500×CCM was thawed and concentrated in preparation for EV isolation by size-exclusion chromatography followed by additional concentration of the EV sample. All three steps were performed according to the manufacturer’s instructions. Briefly, Centricon® Plus-70 centrifugal filter units  were primed with 50 mL of 0.1 N sodium hydroxide, followed by 65 mL of 0.22 µm-filtered PBS. The retentates of the priming buffers were discarded. Then, 50 mL portions of CCM were concentrated to 500 µL, which was loaded into a SEC column  that had been primed with 13 mL of 0.22 µm-filtered PBS. SEC-output fractions of 500 µL each were collected. The fractions with highest abundance of EVs but with the least protein contaminants are fractions 7 to 10 according to the product manual and our previous findings . For eacThe Flow Nano Analyzer (NanoFCM inc.) was used to assess particle quantity and size-distribution of the EVs sample in accordance with manufacturers’ manual and as we have done previously , 48.EVs were imaged by transmission electron microscopy (TEM) as previously described . BrieflyEV samples were lysed with 5 × RIPA buffer  with added HALT™ Proteinase and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Using the manufacturer’s instructions, the Pierce™ BCA Protein Assay was used to quantify protein content of the EV lysates. Equal amounts of protein (5 µg) were used for western blotting, using our methods as published before , 48. BriFor extraction of RNA from the EV samples, the miRNeasy micro kit (Qiagen) was used according to manufacturer’s protocol, including the optional on-column digestion with RNase-free DNase I (Qiagen). The RNA was eluted in 20 µL RNase-free water and stored in − 80 °C until use.For expression analysis the nCounter PanCancer Progression Panel was used with the low RNA input amplification kit (NanoString Technologies), in accordance with the manufacturer’s instructions . Brieflyhttp://www.genepattern.org) [http://www.nanostring.com). The log2 fold and median normalized data were ranked. Then, the GSEA algorithm generated an enrichment score, which estimated whether certain gene sets were enriched in one or the other. A gene set with nominal p-value (NOM) p-value < 0.05 and False Discovery Rate (FDR) q-value < 0.25 was considered as significantly enriched. The GSEA was repeated with a second run using the 50 Hallmark gene sets from the Molecular Signatures Database [Gene set enrichment analysis (GSEA) was applied to determine the potential functional pathways associated with the differentially abundant mRNA transcripts in EVs from various origins, i.e., RCC subtype or benign. The analyses were run on the Broad Institute Gene Set Enrichment Analysis website (ern.org) , 50. ThiDatabase .g for 10 min at 4 °C prior to buffer exchanged into a compatible digestion buffer  using an Amicon Ultra 0.5 mL centrifuge filter (Millipore) with a 10 K membrane cut-off. Protein lysates were subjected to reduction with 5 mM 1,4-Dithiothreitol (DTT) for 30 min at RT, followed by alkylation with 10 mM iodoacetamide for 45 min at RT in the dark. Urea concentration was reduced < 2 M using 50 mM Tris–HCl, pH 8.0. Samples were subjected to tandem digestion of Lys-C  at a ratio of enzyme-to-substrate 1:50 for 2 h at RT followed by trypsin (Promega) at a ratio of enzyme-to-substrate 1:50 overnight at RT (room temperature). The generated peptides were acidified to a final concentration of 1% formic acid, subjected to clean-up using C-18 SepPak columns (Waters) and then dried. Desalted peptides were labeled with 10-plex TMT (Tandem Mass Tag) reagents (Thermo Fisher Scientific) following manufacturer’s instructions. Equal aliquots of TMT-labeled peptides were pooled and desalted using C-18 Stage Tips, dried down, and resuspended in 3% ACN, 0.1% formic acid prior to ESI-LC–MS/MS analysis. A pooled reference channel composed of a mix of cell lysates from each of the individual cell lines was included to link the two TMT-plexes.Equal parts of CCM from each of the biological replicates used for gene expression analysis were combined for downstream proteomic analyses . For six of the kidney cell lines , the CCM samples were pooled . RPTEC was not used for this experiment. EVs from the CCM were enriched by SEC as described above. Sample preparation for global proteomic characterization were performed as previously described, with some modifications . Briefly5, Max IT—50 ms, charge state include—2–6, dynamic exclusion—45 s, top 20 ions selected for MS2; MS2: resolution—50,000, high-energy collision dissociation activation energy (HCD)—37, isolation width (m/z)—0.7, AGC Target—2.0e5, Max IT—105 ms.For global proteomic analysis, ~ 1 µg of peptide was separated using Easy nLC 1200 UHPLC system (Thermo Fisher Scientific) on an in-house packed 20 cm × 75 µm diameter C18 column (1.9 µm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 μm opening (New Objective)). The column was heated to 50 °C using a column heater (Phoenix-ST). The flow rate was 0.300 μl/min with 0.1% formic acid and 2% acetonitrile in water (A) and 0.1% formic acid, 90% acetonitrile (B). The peptides were separated with a 6–30% B gradient in 84 min and analyzed using the Thermo Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Parameters were as follows: MS1: resolution—60,000, mass range—350 to 1800 m/z, RF Lens—30%, AGC Target—4.0eAll LC–MS/MS files were analyzed by MS-PyCloud, a cloud-based proteomic pipeline developed in Johns Hopkins University to perform database search for spectrum assignments  using MSTo annotate proteins as EV-associated, the following criteria had to be met: (1) proteins with a log2 FC > 0.58 (1.5 FC) between the extracellular vesicle pellet (EV) and the cell lysate (CL) fraction, or (2) proteins with a log2 FC between 0 and 0.58 (1.0–1.5) between the extracellular vesicle pellet (EV) and the cell lysate (CL) fraction and a log2 FC > 0.58 (1.5 FC) between the extracellular vesicle pellet (EV) and the 2 K pellet (2 K). Proteins considered to be significantly increased or decreased between individual EV sample preparations used the criteria of log2 normalized fold change ± 0.58. Proteins categorized as differentially abundant were subjected to overrepresentation enrichment analysis (ORA) using the bioinformatics tool, WebGestalt , 36, andSupplementary file1 (PDF 1017 KB)Supplementary file2 (XLSX 1064 KB)Below is the link to the electronic supplementary material.


Myxofibrosarcoma (MFS) is a highly aggressive malignancy with complex karyotypes and a postoperative recurrence tendency, owing to its strong invasiveness. Although systemic chemotherapy is considered in patients with unresectable MFS, the efficacy of conventional chemotherapy is hitherto unclear. Recently, drug screening analysis using a large number of tumor cell lines has been attempted to discover novel therapeutic candidate drugs for common cancers. However, the number of MFS cell lines is extremely small because of its low incidence—this hinders the conduction of screening studies and slows down the development of therapeutic drugs. To overcome this problem, we established a novel MFS cell line, NCC-MFS5-C1, which was shown to harbor typical MFS genetic abnormalities and thus had useful properties for in vitro studies. We conducted the largest integrated screening analysis of 210 drugs using NCC-MFS5-C1 cells along with four MFS cell lines, which we previously reported. Bortezomib (a proteasome inhibitor) and romidepsin (a histone deacetylase inhibitor) showed stronger antitumor effects than the standard drug, doxorubicin. Therefore, the NCC-MFS5-C1 cell line can potentially contribute to elucidating MFS pathogenesis and developing a novel MFS treatment. TP53 (46%) and RB1 (18%), as well as CDKN2A (16%) and CDKN2B (16%) [TRIO, RICTOR, SKP2, and AMACR are located, is considered to be related to the malignant potential of MFS [Myxofibrosarcoma (MFS) is defined as a fibroblastic malignancy with variably myxoid stroma, pleomorphism, and a distinctive curvilinear vascular pattern . MFS is 2B 16%) . In addil of MFS ,13,14,156% . In aPatient-derived cell lines are fundamental tools for basic research. In particular, cell lines that have not undergone many passages retain the genetic characteristics of the corresponding original tumor and are useful for understanding the molecular biology of tumors ,17,18. IThus, we established an MFS cell line and named it NCC-MFS5-C1 using surgical specimens of MFS. In addition to a detailed characterization of the cell line, we attempted to identify novel candidate drugs by integrating the results of drug screening studies for the following MFS cell lines, as reported in our previous studies: NCC-MFS1-C1 , NCC-MFSThe tumor specimen used in this study were derived from a 60-year-old man with MFS. The patient visited a hospital with a chief complaint of a mass in the right elbow. Needle biopsy indicated that the mass was malignant, and wide resection was recommended. However, the patient left the tumor untreated for 1 year. As a result, the tumor grew larger and was accompanied by bleeding. The patient visited the National Cancer Center Hospital , where a large tumor was detected around the right elbow using MRI a–c. TherThe tumor specimens were formalin-fixed and paraffin-embedded to prepare for 4 μm thick sections. The deparaffinized sections were stained with hematoxylin and eosin (H&E) and validated by expert sarcoma pathologists.2. The cells growing out of the tumor were maintained for 1 year and passaged more than 35 times.Primary cell culture was performed as previously described . The resShort tandem repeats (STRs) were examined at 10 loci using the GenePrint 10 system , as previously described . GenomicMycoplasma contamination was also evaluated using the e-Myco Mycoplasma PCR Detection Kit  according to the instructions. Extracted cellular DNA was used for PCR, and the DNA sequence that was unique to Mycoplasma was amplified. The PCR product was electrophoresed in the agarose gel, and the gel was stained with Midori Green Advance stain . Finally, images were acquired using the Amersham Imager 600 system .http://www.R-project.org, accessed on 28 October 2021) and DNAcopy package . Chromosome locations with copy numbers > 3 or <1 were considered as amplifications and losses, respectively. Genes that showed CNAs were annotated using biomaRt package  and GRCh 37 assembly in Ensembl .Single-nucleotide polymorphism (SNP) array analysis was conducted using the Infinium OmniExpressExome-8 v1.4 BeadChip  according to the manufacturer’s protocol and a previously reported procedure . The gen4) in 24-well culture plates on day 0. We assessed the number of cells based on the absorbance at 450 nm after treatment with Cell Counting Kit-8  (n = 3). The growth curve was obtained on the basis of the absorbance measured every other day until day 4. The population doubling time was acquired on the basis of the growth curve.Cell proliferation was assessed as previously reported . We seed5) were seeded in 96-well round-bottom ultra-low attachment microplates  and incubated for 3 days. Spherical colonies fixed with iPGell  were prepared for paraffin sections, which were stained with H&E.Spheroid formation ability was assessed as previously reported . Cells (4) suspended in a serum-free DMEM/F12 medium in the upper chamber coated with BD Matrigel matrix  (n = 2). DMEM/F12 supplemented with 10% FBS (Gibco), 100 μg/mL penicillin, and 100 μg/mL streptomycin  were added to the lower chamber. The migration of cells from the upper to the lower chamber was measured by electronic sensors every 15 min for 72 h.Invasive potential was assessed using a real-time cell analyzer  along with NCC-MFS1-C1 , NCC-MFS6) suspended in a 100-μL 1:1 mixture of BD Matrigel matrix and D-PBS (-)  and injected subcutaneously into female BALB/c nude mice  (n = 4). After 2 months, the tumors were surgically removed and prepared for H&E staining.Animal experiments were conducted as previously reported . We foll4) were seeded in a 384-well plate and incubated for 1 day (n = 2). On day 2, 10 µM of each drug was added. On day 5, cell proliferation was measured on the basis of the absorbance at 450 nm after treatment with the Cell Counting Kit-8. Cell viability was calculated by comparing with the DMSO control. The acquired data were analyzed using the results of NCC-MFS1-C1 [https://cran.r-project.org, accessed on 28 October 2021).Drug screening tests were performed as previously reported . In this-MFS1-C1 , NCC-MFS-MFS1-C1 , NCC-MFS-MFS1-C1 , and NCC-MFS1-C1 , which w50) value was calculated for drugs that were hit in the preceding screening study and standard treatment drugs, as previously reported [50 values using GraphPad Prism 9.1.1 .The half-maximal inhibitory concentration .We also confirmed that NCC-MFS5-C1 was not contaminated with CDKN2A and CDKN2B in 9p21 and TP53 in 17p13 were identified  in all five MFS cell lines; thus, bortezomib and romidepsin were considered to be promising candidate drugs. Bortezomib is an FDA-approved drug used for the treatment of multiple myeloma [SKP2 overexpression was considered to be related to the malignant potential of MFS, and bortezomib showed its pharmacological effect through the inhibition of SKP2. In our study, bortezomib demonstrated antitumor effects in all five MFS cell lines; nevertheless, the NCC-MFS5-C1 cell lines and the other MFS cell lines that we previously established showed no amplification of the SKP2 gene. Proteasome inhibitors, including bortezomib, may be promising candidate drugs for MFS treatment, albeit further studies are required to investigate the underlying pharmacological mechanisms. Romidepsin is approved for the treatment of cutaneous T-cell lymphoma. Few studies have focused on HDAC inhibition in MFS treatment. Although one study reported that the HDAC inhibitor OBP-801 demonstrated antitumor effects on two MFS cell lines, the underlying pharmacological mechanism remains unelucidated. A recent study revealed frequent amplifications of the HDAC gene family in soft tissue sarcomas [The drug screening test identified multiple promising drugs for the treatment of MFS. Remarkably, bortezomib (a proteasome inhibitor) and romidepsin (an HDAC inhibitor) demonstrated lower IC myeloma . However myeloma . In thissarcomas . This meThis integrated drug screening analysis of five MFS cell lines is the largest analysis that has ever been conducted. However, our study has several limitations. First, MFS is an extremely heterogeneous malignancy. Since the NCC-MFS5-C1 cell line results from the monoclonal proliferation of the original tumor cell, it may not be representative of all MFS, although it was established from a typical MFS case. Second, although this is the largest drug screening analysis conducted for MFS, the number of cell lines used is still insufficient compared to those used for common cancers. For example, over 100 lung cancer cell lines were used in a previous drug screening study . It is eWe successfully established a novel MFS cell line, NCC-MFS5-C1, from a typical case of MFS. NCC-MFS5-C1 cells exhibited multiple CNAs often observed in MFS cells and retained the genetic characteristics of MFS. The in vitro characteristics of NCC-MFS5-C1 cells may also serve as a basis for research on MFS. Drug screening analysis revealed that bortezomib (a proteasome inhibitor) and romidepsin (an HDAC inhibitor) showed a more prominent antitumor effect on MFS than the standard drug, doxorubicin. We believe that this novel MFS cell line contributes to the elucidation of MFS pathogenesis and the development of a novel MFS treatment.


Finally we highlight exciting novel pre-clinical research that has the potential to translate to the clinic successfully. We approach these methods from a pathophysiology based perspective as well and touch upon strategies targeting the interaction between tissue damage induced antigens and T-cells, regimen related endothelial toxicity, T-cell co-stimulatory pathways and other T-cell modulatory approaches, T-cell trafficking, and cytokine pathways. We end this review with a critical discussion of existing data and novel therapies that may be transformative in the field in the near future as a comprehensive picture of GVHD prophylaxis in 2020. While calcineurin inhibitors remain the standard, post-transplant eparinsphamide originally developed to facilitate haploidentical transplantation is becoming an attractive alternative to traditional calcinuerin inhibitor based prophylaxis due to its ability to reduce severe forms of acute and chronic GVHD without compromising other outcomes, even in the HLA-matched setting. In addition T-cell modulation, particularly targeting some important T-cell co-stimulatory pathways have resulted in promising outcomes and may be a part of GVHD prophylaxis in the future. Novel approaches including targeting early events in GVHD pathogenesis such as interactions bvetween tissue damage associated antigens and T-cells, endothelial toxicity, and T-cell trafficking are also promising and discussed in this review. GVHD prophylaxis in 2020 continues to evolve with novel exicitng therapies on the horizon based on a more sophisticated understanding of the immunobiology of GVHD.Graft-vs. host disease (GVHD), both acute and chronic are among the chief non-relapse complications of allogeneic transplantation which still cause substantial morbidity and mortality despite significant advances in supportive care over the last few decades. The prevention of GVHD therefore remains critical to the success of allogeneic transplantation. In this review we briefly discuss the pathophysiology and immunobiology of GVHD and the current standards in the field which remain centered around calcineurin inhibitors. We then discuss important translational advances in GVHD prophylaxis, approaching these various platforms from a mechanistic standpoint based on the pathophysiology of GVHD including  GVHD prophylaxis has come a long way since the initial days of allogeneic transplantation. The improvement in GVHD outcomes has been one of the primary reasons for the reduction in non-relapse mortality over time  that hasIn HLA-matched transplantation, while the backbone of most widely used prophylactic platforms remains calcineurin-inhibitor (CNI)-based, a variety of new drugs have been added to CNI's in an attempt to improve efficacy and reduce toxicity. In this review, we discuss current standards and their evolution over time and highlight some of these translational advances. Further we touch upon novel pre-clinical advances developed on the foundation of a deeper understanding of transplant immunology and promising for translation to the clinic. We begin with a description of the immunobiology of GVHD, to better understand potential targets which have been exploited over the last few decades and currently to develop effective prophylactic therapies for GVHD.One of the first models describing the biology of GVHD was proposed by Antin and Ferrara where they described a sequential cascade initiated by conditioning regimen mediated host tissue injury with the production of inflammatory cytokines (phase 1). This is followed by activation and proliferation of effector T-lymphocytes (phase II) which eventually lead to recruitment and activation of additional mononuclear effectors and amplification of a “cytokine storm” (Phase III) . FurtherBoth neutrophils and monocytes are involved in the initial inflammatory response in the pathogenesis of GVHD. Monocytes are activated by molecules called damage-associated molecular patterns (DAMPs) such as uric acid, ATP, Heparan sulfate, HMGB-1 or IL-33 which can initiate and perpetuate a non-infectious inflammatory response involving the innate immune system. In contrast pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides cause infection-associated inflammation. The DAMP and PAMP mediated inflammatory responses result in activation of the innate immune system (monocytes and neutrophils) which then cause local tissue damage mediated by reactive oxygen species. This eventually culminates in interaction of antigen-presenting cells (APCs) in the innate and adaptive immune and activation of cytokine cascades  leading to the “cytokine storm  which bind corresponding host tissue ligands presenting a potential target for intervention. The T-cell activation process and proliferation process is a crucial target for GVHD prevention.These events lead to a self-potentiating T-lymphocyte activation causing tissue damage via direct cellular cytotoxicity and indirectly via release of soluble mediators  .A number of additional pathways have since been implicated in GVHD pathogenesis including the canonical NOTCH pathway . It has via release of anti-inflammatory cytokines such as IL-10 and TGF-β  are important in immunologic tolerance, partly nd TGF-β . Cytokinnd TGF-β . In addiD deaths . These aChronic GVHD remains the most common late toxicity of allogeneic transplantation with significant morbidity and quality of life implications. cGVHD has its own distinctive immunobiology. Briefly we can conceptualize the pathophysiology of cGVHD in three phases: (1) Inflammation leading to tissue damage (2) chronic inflammation, thymic injury, dysregulated B- and T-cell immunity (3) tissue repair with fibrosis , 12. AltPotential targets for developing novel prophylactic platforms have been identified based on our current and more comprehensive understanding of the biology of GVHD. In this review we discuss both current standards and important translational advances as well as exciting new potential therapies which may be translated to the clinic in the future.The effective prevention of GVHD is critical to the success of allogeneic transplantation. Based on the understanding that aGVHD is primarily mediated by effector T-lymphocytes, prophylactic strategies have focused on T-cell suppression in the recipient. Calcineurin inhibitors (tacrolimus/Tac and cyclosporine/CyA) inhibit the proliferation and activation of T-cells and have been used in combination with either methotrexate (MTX) or mycophenolate mofetil (MMF) as standard prophylaxis in HLA-matched HSCT. In two randomized controlled trials (RCT) in the 1990s, the combination of Tac/MTX was found to be significantly superior to CyA/MTX is the prevention of grade II-IV aGVHD and extensive chronic GVHD in HLA-matched sibling and unrelated donors, although a benefit in overall survival (OS) was not shown , 14. Furin-vivo has been the basis for the development of a number of novel GVHD prophylaxis strategies. These have typically been incorporated into regimens where the backbone comprises CNIs. We summarize some of these approaches below.T-cell depletion or modulation Transplantation across HLA barriers historically has been difficult due to high rates of graft rejection and severe GVHD secondary to strong bidirectional alloreactive responses between donor and recipient. The introduction of post-transplant cyclophosphamide (PTCy) in the context of haploidentical transplantation has been a gamechanger and allowed us to perform such mismatched transplants safely and effectively, typically from related donors.First pioneered at Johns Hopkins, cyclophosphamide is given at doses of 50 mg/kg on days +3 and +4 following the infusion of haploidentical stem-cells. In initial studies reported by the Hopkins group, reduced intensity HSCT with PTCy along with Tac and MMF as GVHD prophylaxis resulted in engraftment in 87% of patients with acceptable rates of grade II-IV (34%) and III-IV aGVHD (6%). Further, rates of chronic severe GVHD were found to be particularly low. Relapse rates were in the 50% range . NumerouThe earlier more simplistic hypotheses postulated that PTCy results in the deletion of alloreactive T-cells with some debate regarding the effect it may have on the T-cell mediated graft-vs. leukemia (GVL) effect. However, more recently it has been shown in mice that Tregs are preserved and that T-effector cell exhaustion may play an important role . A comprThe beneficial effect of PTCy on severe aGVHD and cGVHD has led to its adoption into the HLA-matched setting more recently. In a Phase II RCT (BMT CTN 1203) which compared three different GVHD prophylaxis regimens in RIC MUD HSCT  with standard Tac/MTX prophylaxis, the PTCy arm fared the best with comparable grade II-IV aGVHD (27%) but lower rates of grade III-IV aGVHD (2%) and cGVHD requiring immunosuppression 22%) outcomes. Relapse rates in the PTCy arm were 28%. Overall GVHD and relapse free survival (GRFS) was superior in the PTCy arm meeting the primary end-point of the trial . Again i% outcomeBolstered by this data, PTCy/Tac/MMF is now being compared to standard Tac/MTX prophylaxis in RIC HLA-matched HSCT in a large phase III RCT (BMT CTN 1703). PTCy does have the potential to eliminate the effect of donor mismatch on GVHD outcomes, however, CNI based prophylaxis remains the standard until phase III data is available.in-vivo TCD with the aim to reduce both acute and chronic GVHD with varying success.The polyclonal immunoglobulin product obtained from the sera of rabbits and horses immunized with human thymocytes or T-cell lines is called anti-thymocyte globulin or ATG. ATG has been used as part of transplant conditioning to effect Of the four RCTs that have evaluated ATG in combination with standard CNI/MTX prophylaxis, the first used horse ATG and showed a reduction in aGVHD; however, there were higher rates of infection with resultant no difference in NRM or OS. Importantly, there was a reduction in chronic severe GVHD , 25. TheRabbit ATG is generally considered to deplete T-cells more effectively as well as allow greater expansion of regulatory T-cells (Tregs) . The benIn this context, there is evidence that increased doses or prolonged dosage schedules of ATG may have immunosuppressive toxicity with increased NRM and relapse . IndividIn the realm of matched related donor transplantation, a recent multi-center randomized study from China demonstrated improved acute and chronic GVHD rates without compromising relapse or treatment-related mortality  and meriin-vitro studies appeared to spare regulatory T-lymphocytes. A favorable ratio of Tregs:Teff has been shown to be associated with better GVHD outcomes and hence sirolimus has an immunologic profile that was thought to be potentially beneficial for GVHD prevention. In addition, it has a distinct toxicity profile compared to tacrolimus and is not nephrotoxic. In a large RCT in myeloablative transplants with HLA-matched donors, sirolimus in combination with tacrolimus was compared with the standard Tac/MTX platform. There was no difference in grades II-IV aGVHD and cGVHD, but better grade III-IV aGVHD outcomes with sirolimus/Tac were seen. Non-relapse mortality (NRM) and OS were similar as well  negative selection  through the use of monoclonal antibodies with or without complement  pmplement . The twoex-vivo CD34 selection to PTCy + MMF and conventional Tac/MTX prophylaxis, the results of which are eagerly awaited.An early concern with TCD was that it could affect the powerful GVL effect in HSCT which is also believed to be T-cell mediated. In a RCT in patients transplanted with marrow grafts, TCD was compared to conventional prophylaxis with CyA/MTX; the 3-year disease free survival (DFS) was similar in both groups with lower rates of grade III-IV aGVHD with TCD. Relapse rates were however, higher with TCD, particularly in patients with chronic myelogenous leukemia (CML) . A largein-vivo TCD in the form of anti-thymocyte globulin (ATG), particularly in the haploidentical setting can potentially be helpful in achieving better engraftment rates (The other notable issue with pan-TCD has been a higher incidence of graft failure and slower immune reconstitution (IR) leading to higher rates of infectious complications, particularly viral infections  . The usent rates . Other snt rates  or the unt rates .Given these issues with pan-TCD, more selective methods of T-cell depletion aimed at preventing GVHD while preserving GVL are being explored with the availability of sophisticated clinical grade cell separation techniques.While a number of strategies have been attempted for selective TCD, they did not meet with lasting success. Depletion of CD5+ T-cells and CD8+ T-cells were tried in the 1990s; while rates of GVHD were encouraging, rates of relapse were high leading to the abandonment of these strategies. CD6 depletion is separately discussed in the section on T-cell modulation.More recently Bleakley et al. have looked at CD45RA (naïve) TCD with the understanding that it is primarily the naïve T-cells in an allograft that are alloreactive. Unfortunately, this strategy has not produced encouraging results thus far. Bleakley et al. reported a first in human trial where they performed CD45RA (naïve) TCD via a two-step immunomagnetic bead-based procedure in 35 adult patients. Although 34/35 patients engrafted, rates of aGVHD were high in the 66% range. This was not improved when naïve TCD was combined with a/b TCD (described below) either and this approach is not widely used at this time.While a majority of T-cells express α/β receptors T-cell receptors (TCR), 2–10% of T-cells express γ/δ TCR. These γ/δ T-cells are believed to have important innate immune effects characterized by rapid cytokine release and killing of viral infected and tumor cells . This maDuring T-cell activation, following initial engagement of antigen by the TCR, a number of co-stimulatory and co-inhibitory signals come into play mediated by receptors on T-cells and APCs. This is true in acute GVHD as well. Hence the modulation of these co-stimulatory and co-inhibitory interactions is one of the new frontiers in the prophylaxis of GVHD.The most promising of these has been blockade of the CD28/CTLA-4 axis. CD28 and CTLA-4 are both receptors on the T-cell which bind to B7-1/CD80 and B7-2/CD86 ligands on the APC; however, while CD28 is co-stimulatory, CTLA-4 is co-inhibitory. Abatacept (CTLA4-Ig) is the soluble extra-cellular portion of CTLA-4 complexed with immunoglobulin heavy chain which blocks CD28 and CTLA-4, with more of an effect on CD28 leading ultimately to an inhibitory signal. Murine models from Blazar et al. showed that CD28/CTLA-4 blockade could reduce aGVHD lethality . Kean etSince this approach blocks both stimulatory and inhibitory pathways, the concern for unwanted T-cell activation has been raised; hence more selective approaches to blocking CD28 are also being investigated. As an example, FR104 (CD28-specific pegylated-Fab') with and without sirolimus are being investigated in non-human primate models .ex-vivo expansion of Tregs is possible  comprise a unique subset of T-lymphocytes that can be derived from the thymus or converted from CD4+CD25- cells (inducible or iTregs). Tregs play an important role in immune homeostasis and a favorable balance between Tregs and effector T-cells may be important to prevent GVHD. While possible , there aneficial .Hence, rather than the direct infusion of Tregs, other approaches have been attempted which can upregulate Tregs or enhance their functionality in the post-transplant immune milieu. One such approach involves invariant natural killer T (iNKT) cells.ex-vivo expanded iNKT cells is also an area of investigation.iNKT cells are unique in that they co-express both T and NK cell markers and therefore straddle both the innate and adaptive immune system with a semi-invariant TCR that recognizes glycolipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule CD1d. They modulate the immune system via IL-4 and IL-10. In murine models, iNKT cells reduced GVHD both by a switch to a Th2 cytokine profile and/or IL-4-dependent Treg expansion. These mice were conditioned with a regimen incorporating total lymphoid irradiation plus ATG (TLI-ATG) , 61. ThiIn this contect, REGiMMUNE is a compound in which KRN7000, a synthetic alpha-galactosylceramide derivative and a CD1d ligand, is embedded in a lipid bilayer. REGiMMUNE has been shown to reduce aGVHD mortality by expanding Tregs via iNKT cells in murine models . In a PhAlloreactive CD8+ T-cells bound for the intestines express high levels of integrin β7 (α4β7) that binds to its ligand mucosal addressin cell adhesion molecule 1 (MAdCAM 1) in Peyer's patches and gut-associated lymphoid tissue (GALT) in the intestinal mucosa. Vedolizumab is a humanized moAb which prevents T-cell trafficking to the gut by targeting α4β7integrins on the T-cells. Early proof-of-concept and restrospective analyses have shown promising efficacy with vedolizumab in steroid refractory aGVHD . Given iMaraviroc is an antagonist of CCR5, a chemokine receptor that has been implicated in T-cell trafficking during GVHD pathogenesis. Maraviroc appears to block lymphocyte chemotaxis without actually affecting T-cell function which made it an attractive candidate as a prophylactic agent. However, in a prospective non-randomized study from the BMT CTN, maraviroc in combination with standard Tac/MTX was not superior to standard of care and in this trial the PTCy/Tac/MMF arm fared the best .Interleukin-6 (IL-6), an inflammatory cytokine has been shown to be one of the chief mediators of aGVHD in murine models . TherefoThere are novel therapies which have not yet been successfully translated to clinical practice but hold great promise. These therapies are based on innovative targets based on a more intricate understanding of the pathophysiology of GVHD.As mentioned above in the section on the immunobiology of GVHD, conditioning regimen associated tissue damage exposes antigens which comprise pathogens or Pathogen-Associated Molecular Patterns (PAMPs) and components of damaged cells (Danger-Associated Molecular Patterns or DAMPs) which trigger activation of the innate immune system. Conversely Sialic-acid-binding-immunoglobulin-like lectins (Siglecs) are a particular class of pattern recognition receptors that downregulate innate immune responses . A numbeA role for Siglecs in modulating adaptive T-cell mediated immune responses has also been proposed. Reddy et al. have shown that Siglec-G interacts with CD24c in murine models and this interaction CD24 suppresses TNF-α, IL-1β, and IL-6 via NFkB and therefore is promising in the domain of GVHD prophylaxis .Defibrotide is a polydisperse mixture of predominantly single-stranded polydeoxyribonucleotides which in pre-clinical and human studies has demonstrated profibrinolytic, antithrombotic, anti-inflammatory and angio-protective effects ultimately resulting in stabilization of endothelial cells . DefibroThe canonical NOTCH pathway has been shown to play a critical role in T-cell activation, differentiation, and function in aGVHD pathogenesis . Using hn = 25) and larger studies are needed to further study the effect of MSCs on preventing GVHD. A randomized phase II trial has also shown some beneficial effect on cGVHD and need to be further studied (Attempts have been made to utilize the immunomodulatory properties of MSCs for GVHD prophylaxis based on murine models of HLA-mismatched transplantation with co-transplantation of hematopoietic stem-cells and MSCs . Ning et studied .−/−donor or recipient mice resulted in similar reduction in GVHD (OX40 (CD134) is a co-stimulatory receptor found on T-cells while its ligand OX40L is expressed on dendritic cells, B-cells, and endothelial cells. In 2003 Blazar et al. investigated the OX40 regulation of GVHD in murine models and found that antagonistic anti OX40L moAb or the use of OX40 in GVHD . Further in GVHD  and ther in GVHD  then havvia TNF-alpha and lipopolysaccharides (LPS). In murine models of GVHD, blockade of the ICOS:ICOSL interactions via moAb or ICOS−/− mice resulted in significant decrease in GVHD and GVHD related mortality both mediated by CD4+ and CD8+ cells (Inducible Costimulator (ICOS) is a member of the CD28/CTLA-4 family expressed on activated T-cells while the ICOS ligand (ICOSL), a B7 family member is constitutively expressed on B-cells, macrophages and dendritic cells and upregulated on APCs 8+ cells .ALPN101 is a novel Fc fusion protein of a human ICOSL variant immunoglobulin domain (single domain vIgD™) binding both ICOS and CD28 at higher affinity than wild-type molecules, designed to inhibit both the CD28 and ICOS pathways to dampen co-stimulatory responses during alloreactive T-cell activation. In a murine dose-ranging study, ALPN101 inhibited GVHD responses at all doses and more significantly than the comparator belatacept, an approved CTLA-4-Fc protein CD28 pathway inhibitor .A phase 1/2 dose finding study (BALANCE) is ongoing in patients with steroid sensitive and steroid refractory aGVHD (NCT04227938) investigating this potentially transformative effect and this is another promising target for GVHD prophylaxis.n = 112) with aGVHD rates in the 18% range using bone marrow grafts (CD6 is a co-stimulatory receptor on T-cells that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand on APCs and is involved in T-cell activation and trafficking. Historically CD6 T-cell depletion using a monoclonal antibody (moAb) was evaluated in a single-arm trial  which is constitutively expressed on the vascular endothelium of skin and bone marrow and inducibly expressed on other cells during inflammation. P-selectin is a receptor for PSGL-1, a glycoprotein strongly expressed on all leukocytes . PSGL-1 in-vivo induction of Treg. In preclinical aGVHD models, AAT reduced inflammatory cytokines, altered the ratio of effector and regulatory T-cells and reduced levels of DAMPs  involved in downstream transduction of various cytokine pathways . They arSo far clinical data is limited to a pilot study in myelofibrosis patients where a combination of ATG, ruxolitinib, and PTCy was used as GVHD prophylaxis with acceptable engraftment rates  and a siin vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation.Monoclonal antibodies conjugated with immunotoxins is a method of selective TCD that has been attempted in the past with CD5 as a target among others as described above. T-guard is a immunotoxin combination comprised of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces This was first evaluated in a phase I/II trial in humans in SR aGVHD with a 50% response rate and manageable toxicities albeit with evidence of capillary leak and thrombotic microangiopathy . This maAlthough cGVHD is a distinct clinical and immunologic entity from aGVHD, there are limited interventions that specifically target the prevention of cGVHD. In general we know that patients who have less aGVHD will likely get less cGVHD and so the prevention of aGVHD is important in the prevention of cGVHD. In terms of donor and transplant related interventions, younger same-sex donors and the use of bone marrow product rather than PBSC has been shown to reduce rates of cGVHD .T-cell directed approaches that have been quite successful include ATG and more recently PTCy as outlined above. B-cell directed approaches are an area of interest given our current understanding of the important role that B-cells play in the pathophysiology of cGVHD. Rituximab, a monoclonal antibody targeting CD20 was evaluated in a phase II trial with cGVHD rates in the 48% range cGVHD requiring immunosuppression in the 31% range . This waAnother area of interest is the augmentation of tolerance by the use of low-dose IL-2  to enhance Treg reconstitution creating a favorable immunologic milieu for the prevention of cGVHD. This strategy has had success in the therapy of steroid-refractory cGVHD  and may in-vivo and ex-vivo pan T-cell depletion strategies to more directed immunomodulatory strategies based on a more comprehensive understanding of GVHD immunobiology. Nevertheless, the basic backbone of CNI based prophylaxis has survived the test of time. We have touched upon this current standard in this review and further discussed important translational advances and exciting pre-clinical strategies which may be a part of future prophylactic regimens.GVHD prophylaxis has evolved over the last few decades from direct in vivo T-cell depletion, mechanistic studies have indicated that PTCy has a far more complex impact on the post-transplant immune system including a Treg sparing role which is of great interest in the community , the results of which are eagerly awaited as well. In the last decade the spotlight has shifted to methods of selective ex-vivo TCD with limited success in the clinical setting. a/b TCD which attempts to reduce GVHD without affecting GVL and can be performed without the use of post-transplant immunosuppression is promising and may be an important modality in the future.In the domain of in-vivo or ex-vivo), a new frontier in GVHD prophylaxis is targeting immune checkpoints which regulate T-cell activation. Given the dramatic success of checkpoint inhibitors in the world of solid tumor oncology, there has been tremendous interest and a much better understanding of these checkpoints in recent years. In the case of GVHD of course, researchers have tried to downregulate rather than upregulate T-cell activation following initial antigen engagement by the T-cell receptor complex. Although there are a number of molecules being tested at the bench and detailed in this review, the most promising of these has been blockade of the CD28/CTLA-4 axis with Abatacept (CTLA-Ig) with an eventual inhibitory signal downstream to the T-cell. Results from a RCT with pediatric and adult patients has shown a dramatic reduction in severe acute and chronic GVHD including in mismatched unrelated donors (Separate from direct TCD (d donors . With FDTargeting T-cell trafficking, an early event in GVHD prophylaxis, is being tried with integrin blockers such as vedolizumab. It is logical that inhibiting the very movement of effector T-lymphocytes to target organs should better prevent GVHD rather than trying to arrest the widespread inflammation and cytokine cascades which characterize the final common pathway in GVHD pathogenesis. As a testimony to that, when tocilizumab an IL-6 blocker was evaluated, despite promising phase II single-arm data, was not more effective than standard prophylaxis in a phase III RCT . This haWithin the limitations of this review, we have highlighted some of the exciting pre-clinical science that has the potential to translate into effective prophylactic therapies which target GVHD pathogenesis beyond direct T-cell depletion. Targeting the interaction between DAMPs/ PAMPs on APCs and Siglecs with a downstream inhibitory effect on cytokine cascades as well as investigating a role for Siglecs in modulating adaptive T-cell mediated immunity are areas of interest . EndothePathways critical in T-cell modulation during activation and proliferation such as the Notch pathway has been an area of great interest although not ready for translation at this time . BolsterIn conclusion, while GVHD prophylaxis in 2020 still incorporates the traditional paradigms of CNI based prophylaxis, PTCy is knocking on the door and a number of exciting new translational therapies and pre-clinical advances are on the horizon which promise to challenge the established paradigms.JHA supervised and edited manuscript along with MG. All authors contributed to the article and approved the submitted version.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.