Vanadium has a good therapeutic potential, as several biological effects, but few side effects, have been demonstrated. Evidence suggests that vanadium compounds could represent a new class of non-platinum, metal antitumor agents. In the present study, we aimed to characterize the antiproliferative activities of fluorescent vanadyl complexes with acetylacetonate derivates bearing asymmetric substitutions on the β-dicarbonyl moiety on different cell lines. The effects of fluorescent vanadyl complexes on proliferation and cell cycle modulation in different cell lines were detected by ATP content using the CellTiter-Glo Luminescent Assay and flow cytometry, respectively. Western blotting was performed to assess the modulation of mitogen-activated protein kinases (MAPKs) and relevant proteins. Confocal microscopy revealed that complexes were mainly localized in the cytoplasm, with a diffuse distribution, as in podocyte or a more aggregate conformation, as in the other cell lines. The effects of complexes on cell cycle were studied by cytofluorimetry and Western blot analysis, suggesting that the inhibition of proliferation could be correlated with a block in the G2/M phase of cell cycle and an increase in cdc2 phosphorylation. Complexes modulated mitogen-activated protein kinases (MAPKs) activation in a cell-dependent manner, but MAPK modulation can only partly explain the antiproliferative activity of these complexes. All together our results demonstrate that antiproliferative effects mediated by these compounds are cell type-dependent and involve the cdc2 and MAPKs pathway. Cell cyn tumors , and by n tumors ,13,14,15n tumors ,17. Vanadium could also influence the invasive and metastatic potential of tumor cells, regulating cell–substrate adhesion, cell–cell contact, and actin cytoskeletal changes . In 2013, Sgarbossa et al. synthetized and fully characterized six vanadyl complexes with acetylacetonate derivates bearing asymmetric substitutions on the β-dicarbonyl moiety . All comThe aim of this work was to investigate the antiproliferative activity of complex c and d; we chose these two complexes because the oxidovanadium (IV) was functionalized with fluorescent ligands, which allowed us to study the intracellular localization of complexes. Moreover, to investigate the antiproliferative mechanism of these complexes, we studied cell cycle modulation and MAPKs activation. To perform these studies, we utilized four different cell lines. HCT 116 and HT-29 are two colon cancer cell lines, hTERT-HME1 is a non-transformed epithelial cell line of human breast origin immortalized with hTERT, while the last one is an immortalized human podocytes cell line. We chose two tumoral and two non-tumoral cell lines with the aim of demonstrating the possibly different behavior among them. Moreover, we chose as non-tumoral two cell lines of breast and renal origin, respectively, as non-tumoral colon cancer cell lines are not available for experimental use. We studied the antiproliferative activity of these vanadyl compounds in colorectal adenocarcinoma because intestinal cells are sensitive to metal compounds. One of the most effective drugs utilized in colorectal cancer therapy is oxaliplatin, a diaminocyclohexane platinum compound routinely utilized in the treatment of patient with this pathology ,21. OtheIn hTERT-HME1 and HCT 116, complex d was respectively detected as a few small green and a few slightly larger granules in the cytoplasm of almost all the cells analyzed. Complex c was present in both cell lines in small quantity and in a very low percentage of cells as small aggregates. Both ligands were present in very low quantity in a few cells as very small granules, either in hTERT-HME1 or HCT 116 .Both complexes spread out in podocyte cytoplasm as a diffuse reticulum in cells treated with complex c and with a prevalent plasma-membrane localization for complex d ; ligandsFinally, in HT-29, both complexes and ligands were present in cytoplasm but with different distributions. In fact, complex c was present as granules or aggregates primarily localized around the nuclear membrane, while complex d showed a more diffuse localization, with some granules and probably also an intranuclear localization. Ligand c spread out in the cytoplasm of all cells, while ligand d was mainly present as cytoplasmic granules .50 obtained from proliferation assay, and their activity was compared with that of VOSO4 and ligands c and d . Treatment with complex d for 24 h induced a block in S-G2/M phase of the cell cycle in hTERT-HME1 and in podocytes, but in the last cell lines there was no statistical significance. The proportion of cells in the G1 phase decreased in all the cell lines analyzed but HT-29 a,b. In hTERT-HME1, podocytes, and HCT 116, the S and G2/M populations increased relative to control, but the difference was statistically significant only for the G2/M phase in HCT 116. In all cell lines analyzed, complex c induced less variation in cell cycle phase distribution than complex d. To better understand the mechanisms of cell cycle variation induced by vanadyl complexes, the modulation of phosphorylation of two proteins involved in cell cycle progression, cdc2 and p53, was investigated a,b. 4, but not with ligands, increased cdc2 phosphorylation in a significant manner (p < 0.001). Additionally, in HCT 116, both complexes induced a significant increase in cdc2 phosphorylation (p < 0.05), which was lower than that induced by VOSO4 (p < 0.001); the degree of phosphorylation of this protein by the ligands was not statistically significant. In contrast, in HT-29, both complexes and ligands induced a statistically significant phosphorylation of cdc2, even though the increase was greater for complexes ; the treatment with VOSO4 did not modify cdc2 phosphorylation in a statistically significant manner. None of the treatments modulated p53 phosphorylation in podocytes, HCT 116, and HT-29. The absence of p53 phosphorylation in hTERT-HME1  and significantly decreased p42/44 activation (p < 0.05)  but not by complex d; the effect observed treating cells with complex c was similar if cells were treated with ligand c (p < 0.01). Complex d and VOSO4 induced p42/44 phosphorylation . Both ligands inhibited p42/44 activation, but only ligand d in significant manner (p < 0.05). Complexes, ligands, and VOSO4 all increased p38 activation  a,b. Neittivation .p < 0.05) but not in HCT 116, in which we observed a significant decrease in JNK phosphorylation (p < 0.05). VOSO4 was able to turn on p38 and p42/44 in HCT1 116, but not in HT-29; in HCT 116, complex d had an opposite effect, as it reduced p38 and p42/44 phosphorylation and complex c did not modulate the activation of these proteins. In HT-29, both complexes and ligands were able to induce p38 phosphorylation, and they did not modulate p42/44 activation. VOSO4 did not modify p38 and p42/44 phosphorylation in HT-29  and SB202190 (100 nM), respectively (p < 0.001) (The pre-treatment with AZD6244 (p42/44 inhibitor) or with SB202190 (p38 inhibitor) reduced the antiproliferative effect of complex c only in podocytes; the IC< 0.001) .50 of complex d moved from 4.42 ± 1.07 to 6.81 ± 1.14 (p < 0.05) and 7.99 ± 1.12 (p < 0.01) in presence of AZD6244 and SB202190 respectively. Neither AZD6244 nor SB202190 modified the antiproliferative effect of either of the complexes in hTERT-HME1 or in HT-29. The pre-treatment of cells with the JNK inhibitor, SP 600125, did not influence proliferation of any of the studied cell lines treated with both complexes -dependent MAPKs modulation; in HT-29 and podocytes, the antiproliferative activity of complexes seemed to be correlated with the activation of JNK and p38. The p38 involvement in antiproliferative effects of V(IV) has been demonstrated in human bronchial epithelial cells, in which p38 could induce the transcription of NF-kB, involved in both cell cycle progression and apoptosis . The inc50.Vanadium salt can also activate p42/44 . In HCT 4; ligands could also influence the interaction with other targets, such as COX-2. Additionally, the different vanadyl effect on cdc2 activation in HCT 116 and HT-29 could be correlated with the different status of PI3K in the two cell lines: vanadium upregulates cdc2 [Although HCT 116 and HT-29 are both adenocarcinoma colorectal cell lines, they show different biochemical and morphological characteristics. Many studies have been performed to analyze the differences present in different colorectal cancer cell lines, evidencing that they can be used as preclinical models, as they showed a molecular heterogeneity similar to that observed in patients . For exaThe mechanism of action of vanadyl compounds is not clear; we observed that the effect is not dependent upon p53, as also demonstrated by Clark et al. . ROS genAltogether, these results underline the role of the two complexes analyzed in modulating cell cycle and cell proliferation in a cell-specific manner. The two complexes have a different distribution inside the cell and a different aggregation state. The presence of the methoxy group on the complex surface changes the uptake interaction/behavior because of its higher polarity than the naphthalene backbone, which changes the complex interaction with the cellular membrane and with the water inside the cell, and consequently changes the intracellular distribution. This can be responsible for the observed differences. Furthermore, since the reported redox potential for V(IV)/V(III) couple of similar complexes  has been4), 4’,6-diamidino-2-phenylindole (DAPI), Mowiol® 4-88, RNase, propidium iodide, EDTA, protease inhibitor cocktails, and monoclonal anti-β-actin antibody were purchased from Sigma-Aldrich . VOSO4 was freshly dissolved in medium. DMEM, DMEM-F12, RPMI 1640, and fetal bovine serum (FBS) were purchased from Aurogene . EGF, insulin, hydrocortisone, penicillin/streptomycin antibiotic mixture, amphotericin B, and glutamine were purchased from Sigma-Aldrich .The synthesis of the ligands and complexes was performed according to the protocol described by Sgarbossa and co-workers . ComplexLigand c was purchased from Sigma-Aldrich. Ligand d was synthesized according to a previously reported procedure . SpectroHCT 116, HT-29, and hTERT-HME1 cell lines were purchased from American Type Culture Collection ; immortalized human podocytes were kindly provided by Prof. Gianluca Miglio . Human podocytes were obtained by infection of cultures of renal cells with a hybrid Adeno5/SV40 virus and characterized as previously reported . HCT 11650 calculated from the antiproliferative assay  1X and then treated with complex c and d and their respective ligands. Complexes were used at the ICve assay , and lig6 cells were grown in a 10 cm plate for 24 h and then incubated with complex c, d, and the respective ligands or VOSO4 for 24 h. Complexes were used at the IC50 calculated from the antiproliferative assay . After doublet exclusion, extended analysis of the DNA content and calculations of the percentage of cells in each phase of the cell cycle were performed using Flowing Software 2.5.1 (released 4.11.2013).Cell cycles were analyzed by flow cytometry. The 1.5 × 10ve assay , and lig reached . The 1 ×4 for different times. Complexes were used at the IC50 calculated from the antiproliferative assay  according to manufacturer’s instructions. Cell lysates were then resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes . The primary antibodies used for immunoblotting were anti-p42/44 MAP Kinase, anti-phospho-p42/44 MAP kinase (Thr202/Tyr204), anti-phospho-p38 (Tyr 1068); anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185) and anti-SAPK/JNK, anti-phospho-cdc2 (Tyr15); anti-phospho-p53 (Ser15); all from Cell Signaling Technology . The primary antibodies were diluted at 1:1000 in PBS containing 0.1% Tween-20 (PBS-T) and incubated overnight at 4 °C, except for anti-phospho-p42/44, which was diluted at 1:2000. The secondary antibody utilized for all the primary antibodies reported above was horseradish peroxidase-conjugated goat anti-rabbit IgG  or horseradish peroxidase-conjugated horse anti-mouse IgG , according to the manufacturer’s instruction. Both the secondary antibodies were diluted at 1:2000 in PBS-T and incubated for 1 h at room temperature. To confirm the homogeneity of the protein loaded, the membranes were stripped and incubated with monoclonal anti-β-actin antibody (1:5000), 30 min at room temperature, and subsequently with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000) , 1 h at room temperature. Protein bands were visualized using Western Lightning Plus-ECL  and quantified on the films by densitometry using the ImageJ software [Cells were grown in 6-well plates for 24 h and then starved and treated with complexes, ligands, or VOSOve assay , and lig reached . Cells wsoftware .4 in serum-free medium was added to cells with a multichannel pipette. Vehicle and medium-only containing wells were added as controls. Plates were incubated at 37 °C in 5% CO2 for 6 days, after which cell viability was assessed by ATP content using the CellTiter-Glo Luminescent Assay . All luminescence measurements (indicated as relative light units) were recorded on a Victor X4 multimode plate reader .Cells were seeded in 100 µL complete medium at appropriate density (1500–2000 cells/well) in 96-well plastic culture plates in triplicate. The following day, cells were pre-incubated for 30 min with the irreversible MAPK inhibitors SB202190 and AZD6244, respectively, for p38 and p42/44MAPKs, or with the reversible inhibitor SP600125 for JNK; all the inhibitors were used at 100 nM. After serial dilutions (0.206–50 µM), 100 µL of each compound, ligand, and VOSO50 for each compound calculated using GraphPad Prism 6. Dose–response curves were analyzed using a log (inhibitor) vs. normalized response–variable slope model. Where indicated, the results are given as the mean ± S.E.M. Statistical analyses were performed by one-way ANOVA using Dunnett’s post hoc test (GraphPad). Differences of means were considered significant at a significance level of 0.05 .Experiments were performed at least three times. Graphs were constructed, data analyzed, and IC


In ovarian cancer patients the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK kinase signaling pathways are frequently dysregulated, making them potential targets of therapeutic inhibitors. In this study, we used four human ovarian cancer cell lines grown in two- and three-dimensional models to investigate the potential efficacy of combining two inhibitors, which target these pathways, against ovarian cancer. The inhibitor combination was found to have cell line- and model-dependent synergistic antiproliferative effect.Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors. Ovarian cancer is a deadly disease that has a five-year survival rate below 50% . AlthougMutations within the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK kinase signaling pathways are characteristic of the low-grade serous (LGSOC), mucinous (MOC), endometrioid (ENOC) and clear cell (CCOC) ovarian cancer subtypes ,5,6,7. BPIK3CA gene occurs in 6–12% [PIK3CA ovarian cancer cells in vitro [The PI3K/AKT/mTOR signaling pathway is thought to be aberrant in approximately 70% of ovarian cancers . Inactivin 6–12% ,18 of cain 6–12% ,20 and rin 6–12% . Severalin 6–12% ,23,24,25in vitro ,23. Addiin vitro ,22,25,26in vitro ,28,29.BEZ235 is a dual PI3K and mTOR inhibitor that has shown considerable efficacy against a wide range of different cancer cell lines including ovarian, breast and bladder cancers ,30,31. IIncreased activation of the RAS/RAF/MEK/ERK signaling pathway occurs in approximately 40% of ovarian cancers, most commonly in low-grade ovarian cancers ,36. MutaSCH772984, which was developed to treat BRAF and MEK inhibitor resistant melanoma, is a highly specific inhibitor of ERK . It has The clinical relevance of the responsiveness of ovarian cancer cells to anti-cancer drugs may be influenced by the unique features and microenvironment niche of those cells . In the Complexity can be added to in vitro cancer models to provide a better representation of tumors and their response to treatments. Examples of these include simple forced suspension and hanging drop models, which restore the microenvironment features outlined above, and more complex co-culture systems, with the inclusion of stromal cells, which influence the development and progression of cancers, and ECM constituents, which can impact gene expression, cell growth and survival and reduce the penetration of anticancer drugs into tumors ,50. ThesThe anti-tumor effect of kinase inhibitors targeted at the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways is likely to be anti-proliferative rather than through the promotion of cell death (cytostatic vs. cytotoxic). This means that the research outcomes may be much more promising in 2D monolayers, where proliferation is more pronounced, than in 3D models. Additionally, the gene expression profiles of these pathways are different in 3D models and are thought to be more representative of in vivo models than 2D monolayer models . While, This study aims to provide a comparison of the antiproliferative effect of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathway inhibitors in 2D monolayer and 3D ovarian cancer models. The PI3K/mTOR inhibitor BEZ235/Dactolisib and the ERK inhibitor SCH772984 were used, both alone and in combination. Additionally, the impact of culture model on findings of synergistic interactions of these inhibitors was investigated. We hypothesise that the synergistic effects of the PI3K and ERK inhibitors on ovarian cancer cells is cell line- and cell model-dependent.v/v) foetal bovine serum (FBS) (GIBCOv/v) KnockoutThe four human ovarian adenocarcinoma cell lines, OV-90, OVCAR5, OVCAR8, and SKOV3 were kindly donated by Dr Kenny Chitcholtan  and had been authenticated using STR testing by CellBank . These were maintained in Dulbecco’s Modified Eagle Medium nutrient mix F12 (DMEM-F12)  and cells were sub-cultured when near confluent. Cells were subcultured by discarding the working media and rinsing with phosphate-buffer solution (PBS) before incubation with 1× trpsin- ethylenediaminetetraacetic acid (trypsin-EDTA)  until the cells became detached (after approximately 10 min). Cells suspended in trypsin-EDTA were collected in 15 or 50 mL tubes, flasks were rinsed with PBS and that PBS was added to the tubes before centrifugation at 0.2 rcf for 5 min. The supernatant was then discarded, and the cell pellet was resuspended in working media and a portion  was added to culture flasks and/or counted and added to well plates for subsequent experiments.Experiments involving the generation of 2D monolayer and 3D aggregate cultures were carried out in 24 well tissue culture plates . Cells were collected when near confluent by trypsinisation as outlined above. The approximate number of viable cells in the cell suspension was determined on a haemocytometer using trypan blue exclusion (Gibcov/v) (due to a higher concentration of 50 µM treatment being used). The stock solutions were diluted with working media to a 2× treatment concentration. Five hundred Forty eight hours after establishment, monolayers and 3D cell aggregates were treated with BEZ235 and SCH772984 , alone or in combination, for 72 h, or for 24 h for the apoptosis assays. To make up stock inhibitor solutions, BEZ235 and SCH772984 were dissolved in dimethylsulfoxide (DMSO)  to a concentration of 10 mM. In each experiment all treatments had the same DMSO concentration which was equal to the vehicle control. In general, the DMSO concentration was kept at or below 0.1% (v/v) except in the combination experiments for SKOV3 monolayers (0.2%) and OVCAR8 0.56%), OVCAR5 (0.52%) and SKOV3 (0.47%) aggregates  cell aggregates were collected in 1.5 mL microtubes and the media discarded by pelleting the aggregates and discarding the supernatant and rinsing once with PBS. Aggregates were then trypsinized at 37 Prior to counting of the cells, 50 Synergistic interactions between BEZ235 and SCH772984 were determined using the methodologies described by Chou . CompuSyA simple hanging drop technique was adapted from that of Foty . FollowiImages of individual hanging drop spheroids were captured using a Nikon Eclipse Ts2 inverted microscope (4×/0.10 NA objective lens). The equivalent diameter and sphericity of each spheroid was then determined using AnaSP computer software (Piccinini ). To detTo confirm and compare the 3D morphology of OV-90 and OVCAR8 aggregates, the aggregates were first established on polyHEMA-coated plates for 7 days. Aggregates were then stained for one hour at 37 For sectioning the hanging drop spheroids and polyHEMA aggregates were rinsed with PBS and stained for one hour with PI . Significance was determined by one-way or two-way ANOVA with Dunnett’s and Tukey’s tests for multiple comparisons. Normality was determined by Shapiro-Wilk test with percentage and ratio data log transformed. The statistical test used to determine significance is indicated in the figure legend. A minimum of three biological replicates were carried out for each experiment.To investigate the effect of the dual PI3K/mTOR inhibitor BEZ235 on cell viability, OV-90, OVCAR8, OVCAR5 and SKOV3 cells were cultured for 48 h as both monolayers and cell aggregates and then exposed to the inhibitor for 72 h. BEZ235 significantly reduced cell viability relative to the controls in all four cell lines and was more effective against monolayers than aggregates . Signifirized in . OverallUsing the same approach as with BEZ235, monolayer and aggregate cultures of the four cell lines were next exposed to the ERK inhibitor SCH772984. Overall the cell lines were less sensitive to SCH772984, with significant reductions in cell viability relative to the control at concentrations of 0.1 nd above . The inhrised in .To investigate the effect of the combination of BEZ235 and SCH772984 on proliferation the four cell lines were cultured in monolayers and aggregates for 48 h then exposed for 72 h to the inhibitor combination at the concentrations and ratios as determined for each cell line and culture method and described in To investigate the effect of BEZ235 and SCH772984, alone and in combination, on apoptosis, OV-90, OVCAR8, OVCAR5 and SKOV3 were established as monolayers and aggregates for 48 h before 24 h of exposure to BEZ235 and SCH772984, either alone at their respective 1× ECAcross all of the cell lines, in both the monolayer and aggregate models, the percentage of cells in apoptosis was found to be low and treatment with the inhibitors, alone and in combination, did not increase apoptosis in most of the cell lines. A statistically significant increase in apoptosis was seen in OVCAR5 aggregates, which were treated with BEZ235 alone and with the 1/2× and 1× ECCell lines which are cultured in forced suspension models form cell aggregates which vary in their size and shape. OV-90 cell culture in forced suspension on polyHEMA coated plates formed small aggregates that averaged 50 ± 9.1 ctively, c. OV-90 ctively, d. For thTo investigate the cell density and viability of the aggregates, OV-90 and OVCAR8 cells were cultured on polyHEMA coated plates for 7 days to form aggregates. Aggregates were then stained with Hoechst and PI and imaged by fluorescent confocal microscopy. Hoechst and PI staining showed that OV-90 aggregates appeared as cell clusters often containing only a small number of cells. There were few PI positive cells and these were typically an individual cell or cells within the smaller clusters a. OVCAR8To investigate cell density and viability of the spheroids OV-90 and OVCAR8 spheroids were produced by culturing in hanging drop for 7 days. Aggregates were stained with PI prior to fixing and embedding. Eight µm thick sections were taken from the center of the spheroid and stained with Hoechst and images were obtained by fluorescent microscopy. OV-90 cell formed large spheroids that contained several hollow regions and abundant PI staining b. In conTo compare the anti-proliferative effect of BEZ235 and SCH772984 in the forced suspension aggregates and hanging drop spheroids, OV-90 and OVCAR8 aggregates and spheroids were established for 7 days on polyHEMA coated plates or in hanging drops, before treatment with the inhibitors. The establishment time in these experiments was increased to 7 days in order to allow the spheroids to form dense structures. This was also the case for the suspension aggregates in order to allow a direct comparison between the two models.p = 0.0248).In response to BEZ235 proliferation decreased in a dose-dependent manner in OV-90 aggregates, with statistically significant reductions relative to the control at concentrations of 0.5 nd above A. A simipheroids C suggestp = 0.0265).Proliferation was reduced in a dose dependent manner in OV-90 suspension aggregates and spheroids in response to SCH772984, with statistically significant reductions relative to the control at concentrations of 0.5 pheroids B. No sigCH772984 D. HoweveThe Compusyn software was used to predict EChibitors . The EC5gregates . In cont to 7 d) . We wereTo investigate the effects of the inhibitor combination on OV-90 aggregates and spheroids BEZ235 and SCH772984 were combined at the EC0 values . These cpheroids . Treatmepheroids . CI valuTo investigate the effect of BEZ235 and SCH772984, alone and in combination, on the morphology of OV-90 spheroids the equivalent diameter and sphericity was tracked by imaging 7 day old spheroids prior to exposure to the inhibitors and 72 h later. AnaSP software  was usedThe equivalent diameter tended to decrease in the spheroids which were treated with SCH772984 and the inhibitor combination. This decrease was statistically significant for the spheroids treated with SCH772984 alone and the 1 and 2× ECdiameter A. A statbination B.This study presents the novel combination of a PI3K/mTOR inhibitor and an ERK inhibitor as a potential treatment for ovarian cancer and directly compares the effect of these inhibitors in both monolayer and three-dimensional in vitro ovarian cancer models. The combination of BEZ235 and SCH772984 used in this present study showed a synergistic antiproliferative effect against ovarian cancer cell lines, which was cell line dependent. The ovarian cancer cell lines were also found to be less sensitive to BEZ235 and SCH772984, alone and in combination, in the 3D culture models in comparison to 2D monolayer models. Additionally, the response of the cell lines to the inhibitors was influenced by the type of 3D culture model that was used. The inhibitors had a synergistic effect reducing proliferation of OV-90 cells in 2D and in both 3D models. Conversely, the OVCAR8 cells cultured as hanging drop spheroids showed no sensitivity to SCH772984, whereas they were sensitive when grown as suspension aggregates. These findings provide a direct comparison of the anti-cancer effect of kinase inhibitors targeted at proliferation and survival signaling pathways in 2D and 3D culture models. Additionally, the potential efficacy of the novel combination of BEZ235 and SCH772984 against ovarian cancer is established, as well as SCH772984 alone, which, to our knowledge, had yet to be investigated against ovarian cancer 2D and 3D in vitro models. These findings provide support for further investigation of these inhibitors against ovarian cancers.The combination of a PI3K/mTOR inhibitor (PF502) and MEK inhibitor (PD901) have previously been shown to have a synergistic antiproliferative effect against ovarian cancer cell lines cultured as monolayers . In the v/v) in the inhibitor combination experiments for SKOV3 monolayers (0.2 %) and OVCAR8 (0.56%), OVCAR5 (0.52% and SKOV3 (0.47%) aggregates. Therefore, a potential cytotoxic or cytopathic effect of DMSO cannot be ruled out in these experiments. Although, ECAll of the cell lines showed a reduction in proliferation in response to BEZ235 in both monolayer and aggregate models. However, a 10-fold higher concentration was required to gain a statistically significant decrease in proliferation for the cells grown in the 3D aggregate models and this effect was reflected in the higher ECin vitro . Interesin vitro , was effOverall, treatment with BEZ235 and SCH772984, alone and in combination, was not found to increase apoptosis in any of the cell lines in monolayers and was only found to increase in OVCAR5 aggregates. These results suggest that targeting of the PI3K/mTOR and RAS/RAF pathways results in decreased proliferation, but does not significantly increase cell death. The effects of the inhibitors may, therefore, be predominantly cytostatic rather than cytotoxic against these cell lines. However, it is possible that higher concentrations or longer treatment times may be required to induce apoptosis. Overall rates of apoptosis in cells treated with the vehicle control were low, which is consistent with previous research . Ovarianter 96 h . Others feration . In contell type . The actell type ,69,70,71The presence of activating mutations within the targeted pathways generally predict sensitivity to PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathway inhibitors ,36. Therhibition ,73. Althhibition .The OVCAThe forced suspension aggregate models produced cell aggregates with a large variation in both size and morphology. While this heterogeneous suspension of cell aggregates may provide a representative model of a patients ascitic fluid, which can contain aggregates of various sizes and morphology, these models do not allow for an investigation of the effect of the inhibitors on large dense aggregates. The size of 3D aggregates is an important factor in inhibitor sensitivity with increased size resulting in increased resistance . Spheroinolayers . Spheroixic core . In the xic core . This reA comparison of OV-90 and OVCAR8 polyHEMA aggregates and hanging drop spheroids revealed that these cell lines formed 3D structures which varied in size and morphology. This difference was most obvious in the OV-90 cell line, which formed small loose and irregularly shaped cell clusters in the forced suspension model but will form large spheroids in hanging drop, which are consistently bigger than 700 findings ,77. Whilfindings ,77, whicfindings . Furtherfindings ,80. The findings . This mafindings .This present study investigated the novel combination of PI3K/mTOR and ERK inhibitors against ovarian cancer and provides evidence of the potential efficacy of the combination of BEZ235 and SCH772984 against ovarian cancer with a robust synergistic interaction seen in OV-90 2D monolayers, 3D aggregates and 3D spheroids as well as OVCAR8 monolayers and OVCAR5 aggregates. Additionally, the ERK inhibitor SCH772984 shows cell line-dependent antiproliferative effects, suggesting that this inhibitor has potential as an ovarian cancer treatment. Additionally, this study provides a direct comparison of the effect of these targeted anti-proliferative inhibitors in monolayer and three-dimensional, which was yet to be investigated in ovarian cancer models. It was found that the type of model used was found to play an important role in the sensitivity of cell lines to these inhibitors with 3D aggregate models being less sensitive than their 2D monolayer counterparts. Furthermore, OVCAR8 cells grown in hanging drops differed in their response to the individual inhibitors in comparison to those grown as forced suspension aggregates. Further work is required to establish potential biomarkers to ensure these combination treatment strategies are given to patients who will benefit from it.


Correction to: J Exp Clin Cancer Res 40, 322 (2021)https://doi.org/10.1186/s13046-021-02125-zFollowing publication of the original article , the autAdditional file 2: Fig. S1 a, Expression levels of different class I HDAC genes in different pediatric small-round-blue-cell tumors, carcinomas and normal tissues by box plot presentation using a comparative study of the amc onco-genomics software tool (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). The number of samples in each cohort is given in brackets. b, Differential expression levels of class I HDAC genes in primary EwS at different tumor sites by box plot presentation using the GSE63157 study set and the amc onco-genomics software tool. The number of samples in each cohort is given in brackets, ND: not determined. p-value < 0.05. c, Retroviral gene transfer of EWS-FLI1 cDNA into MSC lines L87 and V54.2 [4] results in HDAC3 and HDAC8 induction as measured via qRT-PCR, while no change of HDAC1 and HDAC2 expression was observed. Induction of EWS-FLI1-dependent EZH2 expression served as control.Figure S2 a, Tube formation assay with the EwS cell lines CHLA-10 and SK-N-MC after incubation with 3μM MS275 or 4nM FK228 over-night compared to WT control. Both HDACi clearly enhanced endothelial differentiation potential . b, Analysis of neurogenic differentiation potential of the EwS cell lines CHLA-10, EW7 and SK-N-MC treated for six days with 0.5μM MS-275 or 0.2nM FK228. The neurogenic differentiation marker GFAP  and GAP43 (growth associated protein 43) were significantly upregulated after incubation with both HDACi as demonstrated by qRT-PCR.Figure S3 a, Cell cycle analysis of CRISPR/Cas9 HDAC1 or HDAC2 knock outs compared to their controls (Cas9) in three different EwS cell lines are shown. Distributional analysis of cell cycle phases of HDAC1 or HDAC2 knock outs compared to their control were performed by propidium iodine staining and flow cytometry measurement, respectively. b, To analyze apoptosis in HDAC1 and HDAC2 CRISPR/Cas9 knock outs, DNA double strand breaks were measured with anti-phospho-histone H2AX-FITC conjugated mAbs and counterstained with DAPI. Left, the frequency of γ-H2AX positive foci per cell was summarized in bar graphs. Right, fluorescence images show a representative experiment with HDAC1 (top) and HDAC2 (bottom) in two different EwS cell lines each, compared to their controls.Figure S4 a, Western blot analysis of class I HDAC protein levels and their compensation in CRISPR/Cas9 HDAC1 and HDAC2 knock outs compared to their controls (Cas9). Protein levels were detected by antibodies against HDAC1, HDAC2, HDAC3 and HDAC8. β-actin or GAPDH antibodies were used as loading control. b, Heat map of 229 genes differentially expressed in three different EwS lines CHLA-10, EW7 and SK-N-MC after CRISPR/Cas9 HDAC1 knock out, are shown. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162786). c, Circos plots of downregulated genes (left column) and heatmaps of pathways and ontology terms the downregulated genes are enriched for (right column). The plots are based on gene lists for three EwS cell lines , containing the 300 strongest downregulated genes after HDAC1 or HDAC2 knock out, respectively. The lists of downregulated genes for HDAC knock out effects in the top row is based on averaged expression data from HDAC1 and HDAC2 knock outs. The circos plots show overlaps in the gene sets, where each gene is a spot on the inner arc. Purple lines indicate genes shared by the gene lists, and blue lines indicate functional overlaps in the lists. A blue line connects two different genes belonging to the same enriched ontology term. The strongest enriched ontology terms are depicted in the heatmaps. The cells are colored by p-value. Grey cells indicate that a term is not significantly enriched in a gene list. Hence, the heatmap shows common and unique enrichments for the three cell lines.Figure S5 a, HDAC3 or HDAC8 expression after transient shRNA transfection measured by qRT-PCR in EwS cell lines CHLA-10, SK-N-MC or EW7, respectively. Induction of three different shRNAs was done with Doxycycline for 72 hours. b, Proliferation of EwS cells after transfection with HDAC3 (top) or HDAC8  specific shRNA. Further proliferation of SK-N-MC HDAC1 knock out cells with transient HDAC3 or HDAC8 knock down . Control cells were transfected with irrelevant shRNA. Proliferation and cell impendence was measured by the xCELLigence assay every 4 hours. Data are shown as mean ± SEM . c, Analysis of the invasive potential of EwS cell line CHLA-10 after transient shRNA transfection with HDAC3 (top) or SK-N-MC HDAC1 knock out with HDAC8 (bottom) specific shRNA 48 hours after seeding. d, Evaluation of tumorigenicity of CRISPR/Cas9 knock outs of HDAC1 and their controls (Cas9) in EwS cell line CHLA-10. Immune deficient Rag2-/-γC-/-mice were injected s.c. with 4x106 EwS cells. Mice with an average tumor size >10 mm in diameter were considered positive and sacrificed. Kaplan-Meier plots of individual experiments with six mice per group are shown. Log-rank test was used to test for differences in survival.Figure S6 a, Proliferation of SK-N-MC and CHLA-10 after treatment with Doxorubicin and/or HDACi (MS-275 or FK228) was analyzed with the xCELLigence system. Cell impedance was measured every 4 hours. Data are shown as mean ± SEM .Figure S7 a, Proliferation of EwS CRISPR/Cas9 HDAC 1 knock outs and their controls (Cas9) in CHLA-10 or SK-N-MC cells after treatment with Vincristine (top 2 panels) or combined treatment of SK-N-MC with MS-275 and Vincristine (bottom panel). Proliferation and cell impendence were measured by the xCELLigence assay every 4 hours. Data are shown as mean ± SEM . b, Heatmaps of pathways and ontology terms that are enriched among up- and downregulated genes. The plots are based on gene lists for two EwS cell lines , containing the 300 strongest differentially expressed genes after FK228, Vincristine or combined treatment, compared to solvent controls, respectively. The strongest enriched ontology terms are depicted in the heatmaps. The cells are colored by p-value. Grey cells indicate that a term is not significantly enriched in a gene list. Hence, the heatmap shows common and unique enrichments for the two cell lines. c, Spheroid growth was monitored in Greiner bio-one CELLSTAR® Cell-Repellent Surface 96-well round bottom plates. Left, CHLA-10 or EW7 cells were plated in Matrigel-containing medium and cells were treated for 48 hours with the inhibitors as indicated. Results were compared to solvent controls. Right, primary EwS tumor cells derived from PDX mice. Cell viability was measured with CellTiter Glo® Luminescent assay (quadruplets/group).Figure S8 a, Western blot analysis of apoptosis susceptibility after FK228 or MS-275 and/or A-395 treatment, respectively. Protein levels measured by antibodies against, PARP, CASP3, and GAPDH as loading control. CHLA-10, EW7 or SK-N-MC cells were treated for 48 hours with inhibitors. b, Left, heat map of 824 genes, 3-fold differentially expressed in different EwS tumor samples (CHLA-10 and SK-N-MC) at the end of treatment, are shown. Right, zoomed in heat map with 132 genes contains only those genes with a p-value < 0.05. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162788). Cells were treated for 27 hours with solvent control or EEDi (A-395), HDACi (FK228) or with both inhibitors. c, Volcano plot of differentially expressed genes of EwS cells at the end of treatment . The plot shows fold changes of log2 expression values (log FC) and p-values obtained from differential expression analysis comparing tumors treated with A-395 + FK228 to solvent controls. Depicted in red are genes obtaining p-value < 0.05 and absolute log FC > 1; in blue, genes with p-value < 0.05 and absolute log FC ≤ 1; in green, genes with p-value ≥ 0.05 and absolute log FC > 1; and in black, genes with p-value ≥ 0.05 and absolute log FC ≤ 1. Positive log FCs indicate higher expression of the gene in the treated cell lines. D, GSEA enrichment plots of up- and downregulated gene sets after combined A-395 and FK228 treatment. NES: normalized enrichment score. GSEA: http://www.broadinstitute.org/gsea/index.jspAdditional file 3:Figure S9. Whole Western blots with molecular weight markers in selected figures.The supplementary material file captions were updated after initial publication to accurately describe the contents. The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected.


The anti-tumor properties of mesenchymal stem cell (MSCs) expressing TNF-related apoptosis inducing ligand (TRAIL) or MSC-TRAIL have been well documented by several reports. However, some tumors are resistant to TRAIL due to the existence of cancer stem cells (CSCs). Chemo-sensitization of tumors and their CSCs has been reported to enhance TRAIL-mediated inhibition. In this study, we examined the effect of pre-treatment using first-line chemotherapies on MSC-TRAIL-induced inhibition in non-small cell lung cancers (NSCLCs)–derived CSCs. We found that these chemotherapies were able to induce a chemo-sensitization effect to the CSC, thus improving the MSC-TRAIL-induced inhibition. We also noticed that the effect of chemo-sensitization was cell type specific and selecting chemotherapies for the right NSCLC subtypes might help in inducing a more meaningful combinatory effect. As such, this study has proven that chemo-sensitization of the CSCs was able to enhance the MSC-TRAIL-induced inhibition in NSCLC cell lines. Pre-clinical studies have demonstrated the efficacy of mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or MSC-TRAIL against several tumors. However, due to the existence of cancer stem cells (CSCs), some tumors, including non-small cell lung cancer (NSCLC), exhibit TRAIL resistance. This study was designed to evaluate the capacity of using first-line chemotherapies including cisplatin, 5-fluorouracil (5-FU) and vinorelbine to act as a chemo-sensitizer on CD133+ (prominin-1 positive) CSCs derived from NSCLC cell lines  for the purpose of MSC-TRAIL-induced inhibition. We showed that MSC-TRAIL was resistant to all three chemotherapies compared to the NSCLC cell lines, suggesting that the chemotherapies had little effect on MSC-TRAIL viability. Pre-treatment using either cisplatin or 5-FU, but not with vinorelbine, was able to increase the efficacy of MSC-TRAIL to kill the TRAIL-resistant A549-derived CSCs. The study also demonstrated that both 5-FU and vinorelbine were an effective chemo-sensitizer, used to increase the anti-tumor effect of MSC-TRAIL against H460- and H2170-derived CSCs. Furthermore, pre-treatment using cisplatin was noted to enhance the effect of MSC-TRAIL in H460-derived CSCs; however, this effect was not detected in the H2170-derived CSCs. These findings suggest that a pre-treatment using certain chemotherapies in NSCLC could enhance the anti-tumor effect of MSC-TRAIL to target the CSCs, and therefore the combination of chemotherapies and MSC-TRAIL may serve as a novel approach for the treatment of NSCLC. Lung cancer is an uncontrolled growth of malignant cells that can occur in any part of the lungs . These mTumor necrosis factor-related apoptosis-inducing ligand (TRAIL)  is one of several members of the TNF gene superfamily that induces apoptosis. TRAIL activates the extrinsic apoptosis pathway through binding to its two specific agonistic receptors, known as DR4 and DR5, and three antagonistic decoy receptors, known as DcR1, DcR2 and osteoprotegerin (OPG) . The antSeveral limitations, including its short half-life and poor bioavailability, have hindered the translation of TRAIL into the clinic ,18. OwinMesenchymal stem cells or MSCs are adult multipotent stem cells that can be derived from different sources, including adipose tissue, the umbilical cord and bone marrow . The abiThe synergistic anti-tumor activity of the combination of chemotherapies and MSC-TRAIL seen in several tumor models suggests that this combination could be an effective anti-cancer therapy ,38,39. T® PCS-500-011©) were purchased from the American Type Culture Collection . The cells were cultured in specific growth medium containing knockout Dulbecco’s modified Eagle medium (DMEM-KO), 1% penicillin/streptomycin, 2 mM of l X glutamine (200 mM stock), 10% fetal bovine serum (FBS), 5 ng/mL fibroblast growth factor (FGF) basic and 5 ng/mL recombinant epidermal growth factor (rhEGF).Human adipose-derived mesenchymal stem cells  was purchased from the American Type Culture Collection , whereas the other two cell lines (both constitutively expressing luciferase), human adenocarcinoma (A549) (cat no: JCRB1414) and large cell lung cancer (H460) (cat no: JCRB1407), were purchased from Cell Bank Australia . The A549 cell line was cultured in Roswell Park Memorial Institute-1640 (RPMI)-1640 complete medium containing 1% penicillin/streptomycin, 1X non-essential amino-acid solution and 10% heat-inactivated FBS. For the H460 cell line, a complete medium was prepared by adding 15% heat-inactivated FBS to RPMI-1640 medium containing 1% penicillin/streptomycin and 0.08 µg/mL insulin (4 mg/mL stock). H2170 complete medium was prepared by adding 10% FBS and 1% penicillin/streptomycin to RPMI-1640. The cells were maintained in 75 cm2 flasks  and harvested using 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) when the cells reached 80% confluence. All cells were grown at 37 °C in a humidified atmosphere of 5% CO2. All culture reagents were obtained from Gibco .Three types of human non-small cell lung cancer cell lines  were used in this study. The lung squamous cell carcinoma cell line (H2170)  encoding the membrane-bound TRAIL tagged with a red fluorescence protein (mCherry) using lentivirus. The MSC-TRAIL was successfully characterised based on TRAIL expression and multipotent characteristics , as shown in our previous report .The CD133+ population from all of the NSCLC cell lines was isolated and successfully characterised based on clonogenicity, sphere formation and aldehyde dehydrogenase expression . In brie5 cells in 2 mL of a 6-well plate) using formulas as below:The cumulative population doublings (CPD) and population doubling time (PDT) in all the MSCs (MSC-WT (wild-type), MSC-EV (empty vector) and MSC-TRAIL) and NSCLC cell lines  were analysed by comparing the number of cells harvested at every 72 h to the initial number of cells seeded (2.5 × 10H—No. of cells harvested5 cells)I—No. of cells seeded  used for the treatment of NSCLC were determined in the NSCLC cell lines  and MSCs (MSC-WT (wild-type), MSC-EV (empty vector) and MSC-TRAIL) using a proliferation/MTS assay. Cells were seeded in a 96-well plate (5.0 × 103 in 50 µL complete medium) and grown overnight prior to the addition of chemotherapeutic drugs. To produce treatment stocks consisting of 10 mM of cisplatin and vinorelbine tartrate and 0.25 M of 5-FU, different volumes of solvents were added into each of the drugs with different weights calculated, according to their molecular weight. Different concentrations of drugs  and 40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM and 0.6 mM for 5-FU) were added into each of the wells containing the tumor cells in triplicate. After 48 h of treatment, 10 µL of MTS was added into each well and incubated for another 3 h. The samples were then subjected to absorbance reading at 490 nm using a plate reader . Cell proliferation was calculated according to the following formula: cell proliferation (%) = [absorbance (cells with treatment)/absorbance (cells without treatment)] × 100. The IC50 values of the chemotherapeutic drugs for each of the cell lines were calculated using a linear regression formula (y = mx + c) from a scatter plot, wherein the x-axis represents the concentration of drugs (in log10), the y-axis expresses the percentage of cell viability, “m” is the gradient, and “c” is the y-intercept value. The IC50 values of the drugs were then calculated by determining the anti-log of the derived drug’s concentration value (in log10) from 50% of cell viability.The IC50 value, for 24 h. Briefly, the NSCLC cell lines were seeded in a 24-well plate (4.0 × 105 cells) in 500 µL of medium containing the chemotherapeutic drugs according to their IC50 values. The next day, sensitized cells were harvested and re-seeded again (1.0 × 104 cells in 50 µL complete medium) with either MSC-TRAIL (1:1 ratio) or rhTRAIL  in a 96-well plate for another 24 h. The NSCLC cells without any treatment  were used as a control for the experiment. The analysis of cell viability/proliferation assay was performed the next day using either the luciferase assay for both A549 and H460 (both cells express luciferase constitutively), or MTS for the H2170 cell line (which does not express luciferase). For the luciferase assay, 600 µg/mL of D-luciferin was added into each well containing the cells and subjected to a bioluminescence reading. For the proliferation assay, 10 µL of MTS  was added into each of the wells. The samples were then incubated for 4 h and subjected to absorbance reading at 490 nm using a plate reader .The effect of sensitization in NSCLC-derived CD133+ CSCs using chemotherapies targeting MSC-TRAIL was assessed by treating the sorted (CD133+ and CD133−) and unsorted NSCLC cell lines  with the chemotherapeutic drugs  first, based on the calculated ICt-test with p < 0.05 considered statistically significant. Analyses were performed using Excel 2010, version 14.0 .Data are presented as means ± standard deviation (SD) of three independent experiments. Comparisons between two groups were performed using the two-tailed The proliferation rate between MSCs  versus NSCLC cell lines  was evaluated by analysing the cumulative population doublings (CPD) for each cell type A. As indTo compare the chemo-sensitivity of NSCLC cell lines  versus MSC variants , cells were treated with serially diluted concentration of either cisplatin, 5-FU or vinorelbine for 48 h. At the end of the 2 days of treatment, MTS was added into each well and the plates were subjected to an absorbance reading at 490 nm. A distinct separation in terms of cell viability between MSC-TRAIL and NSCLC cell lines was detected when the cells were treated with cisplatin at concentrations lower than 40 µM, indicating that treatments higher than 40 µM were highly toxic to all cells, including the MSCs A. Signif50 values of cisplatin, 5-FU and vinorelbine in the NSCLC cells lines were derived from the chemo-sensitivity assays , compared to both A549  and H2170 , respectively. The IC50 values of all three chemotherapies in the MSC variants and NSCLC cell lines are simplified in The ICy assays , before ll lines . The sen50 values) for 24 h. The sensitized CSCs were subsequently reseeded and then exposed to MSC-TRAIL or rhTRAIL for another 24 h. The CD133− cells from the A549 cell line were highly sensitive to both cisplatin and 5-FU, shown by a lower cell viability (45.0% ± 3.7% for cisplatin and 36.2% ± 3.2% for 5-FU) compared to the CD133+ (71.6% ± 6.0% for cisplatin and 60.0% ± 3.1% for 5-FU) populations  compared to the CD133+  populations A–C. The Treatment using either one of the three chemotherapies did not induce changes in the percentage of cell viability between the CD133+ and CD133− cells in the H2170 cell line. There were no significant changes in cell viability in the CD133+ CSCs between MSC-TRAIL used as a single treatment and in combination with cisplatin, as depicted in Several studies have reported the anti-tumor activity of MSC-TRAIL against lung cancer, specifically in pre-clinical models of NSCLC ,49,50. H50 values in the MSC variants compared to the NSCLC cell lines  as no significant changes in cell viability were detected between the single rhTRAIL treatment and in combination with chemotherapy. This observation might be due to the short half-life of TRAIL, at about 32 min, leading to the ineffective TRAIL concentration during the 24 h of rhTRAIL treatment in regard to the TRAIL-resistant A549 cell line [Although our previous study showed that the A549-derived CD133+ CSCs were highly resistant to MSC-TRAIL ; pre-treell line  may contpoptosis . Furtherell line .Chemo-sensitization of H460-derived CD133+ CSCs using either cisplatin, 5-FU or vinorelbine prior to MSC-TRAIL treatment was able to significantly enhance the killing effect of MSC-TRAIL against the CSCs . Pre-treThe efficacy of rhTRAIL in targeting H2170-derived CD133+ cells was improved through cisplatin chemo-sensitization A. HoweveWe have demonstrated that a pre-treatment with first-line chemotherapies was able to enhance the sensitivity of the NSCLC-derived CD133+ cells to the effects of MSC-TRAIL. However, the exact mechanism leading to this observation remains to be elucidated. One particular reason might be due to the ability of these chemotherapies to regulate the expression of cellular proteins, which may subsequently enhance the transcription of TRAIL receptors, such as DR4 and DR5, leading to the greater sensitivity of tumor cells and CSCs to TRAIL. For example, a pre-treatment using cisplatin has been reported to down-regulate FADD-like IL-1β-converting enzyme (c-FLIP) activity, which is an anti- apoptotic protein, leading to an increase in DR5 receptor expression in glioma-derived CSCs . On the This study has demonstrated that the sensitizing of NSCLC cell lines and their CSCs, specifically the TRAIL-resistant A549 cell line, using first-line chemotherapies could enhance the effect of MSC-TRAIL against NSCLC. However, different chemotherapies may have different chemo-sensitizing effects on the NSCLC subtype. Therefore, selecting chemotherapies for the right NSCLC subtypes is crucial for a meaningful combination effect. Despite the promising outcomes from this in vitro study, we are uncertain if these observations could be replicated in vivo. Therefore, we intend to conduct further studies on the lung cancer mouse model in order to verify the efficacy of this strategy. The favorable results from this study suggest an alternative approach of using MSC-TRAIL as a complement for the treatment of NSCLC patients. However, to achieve optimal results, the chemotherapies should have no effect on the MSCs, and this can be assured by choosing chemotherapies that specifically target the tumor without harming MSC-TRAIL.


ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation.Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in   •Healthy donor iPSCs differentiate into radial germ layers on micropatterns•Differentiation can be quantified using high-content image analysis•iPSC lines show donor-specific variation in germ layer patterning•ITGB1 is associated with expanded endoderm formationA deleterious nsSNV in  ITGB1.In this study, Vickers and colleagues combine micropatterning and high-content image analysis of healthy donor iPSC lines to identify genetic variants that influence cellular differentiation. iPSC lines displayed donor-specific variation in germ layer patterning. Outlier cell lines were identified, including one line that displayed expanded endoderm differentiation and was associated with a deleterious nsSNV in  Howevermedicine . Studiesmedicine .The Human Induced Pluripotent Stem Cell Initiative (HipSci) was established to create a large, high-quality reference panel of iPSCs with accompanying genetic, proteomic, and phenotypic data. Genetically diverse, large-scale collections of iPSCs such as HipSci have enabled the identification of genetic factors that influence gene expression and cellular phenotypes in both pluripotent and differentiated cells . Using tin vitro model that reflects in vivo genetics to map normal genomic variation to more complex cell behaviors. For this, we employed a recently described micropatterning-based differentiation platform  identify each micropatterned colony, (2) select colonies quality controlled on colony area and roundness, (3) determine the outer border and geometrical center of each colony, and (4) segment individual nuclei based on the expression intensity in the DAPI channel and measure the fluorescence intensity of the proteins of interest localized within each nucleus B. ColoniThe image analysis pipeline calculated the distance of each nucleus from the colony centroid using the equation in To evaluate whether the pipeline could provide quantitative comparisons of the differentiation capacities of different iPSC lines, we first investigated the level of variation in differentiation of a single iPSC line, uoxz_4, from a healthy donor. This line lacks any nsSNVs in genes related to cell adhesion or germ line differentiation. The mean protein expression of each germ layer marker on each micropatterned substrate was normalized to the highest value per well and plotted as a function of distance from the colony center D. The baUsing our differentiation and analysis platform, we compared a panel of iPSC lines from three additional healthy donors, lacking any nsSNVs in genes related to cell adhesion or germ line differentiation. This enabled us to characterize the reproducibility of germ layer differentiation phenotypes within and between cell lines from different donors. Following differentiation with 50 ng/mL BMP4 and 100 ng/mL NODAL for 48 h, we evaluated spatial patterning of SOX2, BRA, and SOX17 A–2C.FiguWe observed variation in the quantity and spatial patterning of the germ layers between iPSC lines from different donors. In qanu_1, the peak of SOX2 expression was detected up to 300 μm from the colony center A. In conin vitro cellular behaviors, specifically the differentiation of germ layer fates.We previously identified 103 rare, destabilizing, and deleterious nsSNVs in a subset of healthy donor cell lines from the HipSci resource . These gFor our analysis, we searched for further genes with nsSNVs. Exome sequencing datasets for over 700 lines available through HipSci identified rare and deleterious nsSNVs in 124 genes related to germ layer differentiation. nsSNVs were classified as rare if present in fewer than five of the HipSci cell lines. nsSNVs were classified as deleterious to protein function based on the computational model Condel. This identified 270 nsSNVs that were present in 229 lines from 176 donors, details of which can be found in TBXT). The third category comprised two lines from one donor that were phenotypic outliers on fibronectin and had deleterious nsSNVs in both germ layer specification- and cell adhesion-associated genes  and SOX17 expression (r = −0.48) (r = 0.50)  A and S4B= −0.48) A and 3C. = 0.50) C. In mos = 0.50) D–S4F.ITGA6   .We tested cell lines from donors pamv, yuze, and ffdc to understand whether the identified polymorphisms could influence their differentiation propensity. The lines pamv_1 and pamv_3 displayed high overall SOX17 expression  C, with tFGFR1, and presented high expression of BRA   B and 4D.y center E.We next sought to investigate extrinsic  or intrinsic  drivers of the outlier phenotype observed in the ffdc iPSC lines. Previous studies have shown that mesendodermal fates are restricted to the colony edge due to expression of the secreted BMP inhibitor Noggin (NOG) in the colony center . HoweverTBXT, which encodes BRA, and ITGB1. To investigate whether these polymorphisms contribute to the outlier SOX17 phenotype, we first searched for further iPSC lines that harbored nsSNVs in TBXT or ITGB1. We identified oikd_5, which harbors a deleterious nsSNV in TBXT and had not been included in the initial panel of HipSci cell lines for phenotypic characterization   . P5D2 try (P5D2) . We founy (P5D2) C. When cy (P5D2) C. The diiPSC lines have been shown to display variable propensity to differentiate, including preference toward certain lineages, loss of differentiation capacity, and teratoma formation . Recent in vivo development than two-dimensional (2D) micropatterns  with approval from the UK National Health Service (NHS) Health Research Authority .iPSC lines were obtained from the HipSci cell bank at the Wellcome Trust Sanger Institute, Cambridge  in Essential 8 (E8) medium (Thermo Fisher) supplemented with 1% penicillin-streptomycin (Sigma). For routine maintenance, cultures underwent daily medium changes and were passaged every 4–5 days at approximately a 1:6 split ratio. Cell cultures were routinely tested for mycoplasma and all were negative for contamination. Details of the cell lines used are listed in http://amigo.geneontology.org/amigo/landing) and Gorilla (http://cbl-gorilla.cs.technion.ac.il/).Gene Ontology analysis was performed using the web-services AmiGO 2  <0.005 in both the 1000 Genomes Project  and ExACAn adapted version of a previously described protocol was used to fabricate UV lithography micropatterned 96-well plates . BrieflyITGB1 antibody  and collected as a single-cell suspension in seeding medium (SM) consisting of 74% DMEM, 20% Knockout Serum Replacement (KOSR), 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 1% Glutamax, and 2% B27 minus retinoic acid, supplemented with 20 ng/mL basic fibroblast growth factor (bFGF)  and 10 μM ROCKi (Rho-associated protein kinase [ROCK] inhibitor [Sigma-Aldrich]). Cells were incubated with an anti-dy P5D2;  for 5 miWhen cells had reached confluency , germ layer differentiation was induced using N2B27 medium consisting of 93% DMEM, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 1% Glutamax, 2% B27 minus retinoic acid, 1% N2 supplement, and supplemented with 50 ng/mL BMP4 (R&D), 100 ng/mL NODAL (R&D), and 10 ng/mL bFGF (Thermo Fisher). Cells were incubated for 48 h at 37.5°C prior to fixation.Cells were fixed using 4% paraformaldehyde , permeabilized with 100% methanol  then blocked using 5% donkey serum . Primary antibodies diluted in 5% donkey serum were applied to wells overnight at 4°C. Following three washes with PBS, wells were incubated with secondary antibodies and DAPI  for 1 h at RT. Finally, wells were washed three times with PBS. All antibodies used are listed in Images were acquired using the Operetta CLS (PerkinElmer) microscope with a 20× 1.0 NA water objective and the Leica TCS SP8 confocal microscope with a 10× objective. An automated high-content image analysis pipeline was built in house using Harmony 4.5 software (PerkinElmer) to identify each micropatterned colony, select colonies quality controlled on area and roundness, determine the geometrical center of each colony, identify individual nuclei using the expression intensity in the DAPI channel, and measure the fluorescence intensity of each protein marker in each nucleus see . Single-Statistical analysis of mean protein expression (percentage of positive cells) across all cell lines was performed using the Kruskal-Wallis test with Dunn's multiple comparison post hoc test. Outlier cell lines based on mean protein expression (percentage positive) were identified by comparing the mean expression in one cell line with the mean of all cell lines pooled together using a two-tailed Student's t test, with p values <0.001 considered significant. Cell lines with outlier radial trends of protein expression were identified using the Kolmogorov-Smirnov test by comparing the radial protein expression in each cell line with the radial protein expression profile of the control iPSC line uoxz_4, with p values <0.01 considered significant. Spearman's rank correlation coefficients were calculated to analyze the relationship between expression of germ layer proteins. The Kruskal-Wallis test was performed in Prism. The Student's t test and Kolmogorov-Smirnov test were performed in R 3.4.3. Data are presented as the mean and standard deviation (SD).A.V., M.T., D.D., F.W., conceptualization; A.V., A.L., M.T., M.P., V.S., F.F., data curation; A.V., M.T., A.L., M.P., V.S., F.F., formal analysis; A.V., M.T., F.W., funding acquisition; A.V., investigation; A.V. and M.T., methodology; F.W., project administration; D.D. and F.W., supervision; A.V., M.T., A.L., M.P., and V.S., visualization; A.V. and F.W., writing – original draft; A.V., M.T., A.L., M.P., D.D., and F.W., writing – review & editing. All authors contributed to the article and approved the submitted version.F.M.W. is currently on secondment as Executive Chair, UK Medical Research Council.D.D. is an employee of King's College London and an employee of bit.bio. D.D. declares no other affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.


FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B’s predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples. Adaptor proteins are usually non-enzymes and are essential components that inherit important roles in various cell signalling pathways. In order to mediate protein–protein or protein–membrane interactions, they contain modular domains and/or linear peptide motifs. They are involved in regulating diverse aspects of cell surface receptor functions, including downstream signal transduction and transport or recycling of receptors. β-arrestin and its involvement in G-protein-coupled receptor signalling is a notable example of such an adaptor protein and its importance in the cell .Another adaptor protein that is a Shisa-like protein is FAM159B. These Shisa-like proteins possess an N-terminal domain with six conserved cysteines and are thus related to the Shisa family of single transmembrane proteins that contain eight conserved cysteines. Vertebrates possess two forms of FAM159: FAM159A and FAM159B. FAM159B is thought to be a transmembrane adaptor that regulates other transmembrane proteins and receptors [To identify genes with elevated expression in the human pancreas, Danielsson et al.  performe2+ entry and Na+ currents of β-cells. Furthermore, after knockdown of FAM159B with a specific siRNA, a significant reduction of the exocytotic response was observed. In contrast to the findings of Danielsson et al. [Using a simultaneous pancreatic single-cell, patch-clamp and RNA sequencing (patch–seq) approach to identify genes associated with islet cell function and dysfunction, Camunas-Soler et al.  found thn et al. . WhereasBeyond these limited data regarding the presence of FAM159B in adult islet cells and endocrine cells of the gut, little is known of FAM159B expression in other normal human tissues. Notably, no information is available related to the possible presence of FAM159B in human tumours and cancer cell lines.Therefore, the aim of the present study was to evaluate the expression of FAM159B in a large series of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples using immunohistochemistry to obtain a broad expression profile of this protein in humans and elucidate its possible functions. For this purpose, the commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778  was initially characterised for its specificity using Western blot analyses and immuncytochemistry in the neuroendocrine tumour cell line BON-1, which endogenously expresses FAM159B. Our subsequent immunohistochemical investigations revealed high expression of FAM159B, particularly in pancreas islets, specific neuroendocrine cells of the intestinal tract and small-cell lung cancer (SCLC). Based on these findings, the range of neuroendocrine neoplasms studied was broadened and, ultimately, a diverse panel of bronchopulmonary and gastroenteropancreatic neoplasms (BP-NEN and GEP-NEN) of different malignancy and origin were examined for FAM159B expression. Staining results were then correlated with clinical data, such as tumour stage and grade, glucagon or insulin secretion and overall survival of the patients, and with the expression data of different neuroendocrine tumour markers.r = 16,000–18,000  value. Overall, moderate-to-strong FAM159B expression (average IRS > 5) was seen in pituitary adenomas, medullary and anaplastic thyroid carcinomas, parathyroid adenomas, adenocarcinomas of the lung, SCLCs, ovarian carcinomas and lymphomas. Low expression of FAM159B (3 ≤ IRS ≤ 5) occurred in papillary thyroid carcinomas, squamous cell carcinomas of the lung, gastric adenocarcinomas, colon carcinomas, pancreatic adenocarcinomas, breast carcinomas, cervical cancer and melanomas. No or very weak expression (IRS < 3) was found in glioblastomas, follicular thyroid carcinomas, gastrointestinal stromal tumours, hepatocellular and cholangiocellular carcinomas, renal clear cell cancer, urinary bladder cancer, pheochromocytomas, endometrial cancer, prostate and testicular cancer and sarcomas .With respect to FAM159B expression in various tumour cell lines, a very strong immunosignal was obtained in the neuroendocrine tumour cell line BON-1, SCLC cell line OH-1 and prostate cancer cell lines PC-3, DU145 and LNCaP. A moderate-to-strong FAM159B expression was observed in neuroblastoma cell lines SK-N-SH and SH-SY5Y, SCLC cell lines NCI-h69 and NCI-h82, hepatocellular carcinoma cell lines SK-HEP-1 and HuH-7, colon carcinoma cell lines SW480, HT-29 and LoVo, renal cell carcinoma cell line A498, urinary bladder carcinoma cell line RT-112, breast cancer cell lines MCF-7 and BT-474, ovarian carcinoma cell line A2780 and cervical cancer cell lines SW756, SiHa, CaSki and HeLa. A very weak expression was observed in the lung adenocarcinoma cell line A549, hepatocellular carcinoma cell lines HepG2 and Hep3B, ovarian carcinoma cell line SK-OV-3, urinary bladder carcinoma cell line T24, cervical cancer cell line ME-180 and the epidermoid carcinoma cell line A431. Neuroblastoma cell line SiMa and the breast cancer cell line MDA-MB-231 were devoid of FAM159B immunosignal.The evaluated neuroendocrine neoplasms originated from 144 (51.8%) male and 125 (45.0%) female patients; for 9 (3.2%) patients, the sex was unknown. The overall mean age of the patients at diagnosis was 58.5 years . Forty-seven (16.9%) of the corresponding primary tumours were classified as T1, 46 (16.6%) as T2, 57 (20.5%) as T3 and 24 (8.6%) as T4. In 104 (37.4%) cases, the extent of the primary tumour was unknown. One hundred and twenty-three (44.2%) patients already had lymph node metastases at diagnosis, 83 (29.9%) patients showed none, and the lymph node status was unknown for 72 (25.9%) patients. One hundred and ten (39.5%) patients had distant metastases, 89 (32.0%) had none, and no data were available for 79 (24.4%) patients. Of the 172 patients with GEP-NEN, 17 patients (9.9%) had UICC stage I disease, 12 (7.0%) had stage II disease, 23 (13.4%) had stage III disease, 98 (57.0%) had stage IV disease, and records of 22 (12.7%) patients had no information regarding UICC stage. The stage of the disease was not determined for the 97 bronchopulmonary tumour patients and the 9 patients with unknown tumour origin. Regarding histological grading, 99 (35.6%) patients had grade 1, 94 (33.8%) patients had grade 2 and 76 (27.3%) patients had grade 3 tumours. Tumour grading was unknown for 9 (3.2%) patients.The median follow-up time was 59.7 months overall. One hundred and fifty-eight (56.8%) patients were alive at the end of the follow-up period, 87 (31.3%) had died due to tumour-related causes and 33 (11.9%) had no data available. The median survival time of patients who died during follow-up was 27.7 months, differing significantly by sex .Representative stainings of different BP-NEN and GEP-NEN are shown in Again, both cytoplasmic and membranous expression was observed, and substantial variation in immunostaining across individual patients and sometimes even between different samples originating from the same patient was noticed. These findings are illustrated by the lengths of the boxes and whiskers in U test: p = 0.035). In contrast, a significant positive correlation was observed between FAM159B IRS values and tumour grade  and levels of the proliferation marker Ki-67 . Nonetheless, Kaplan–Meier analysis revealed that FAM159B expression does not influence patient overall survival. No difference between FAM159B-positive or -negative tumours was observed when using IRS values of 3 (threshold of positivity) or 6  as the cut-off value. When analysing interrelationships with several typical markers of, and receptors for, neuroendocrine tumours determined on the same set of samples as part of previous studies [Analysing the collective data of all BP-NEN and GEP-NEN revealed a significant difference between the FAM159B IRS scores for patients with or without lymph node metastases at diagnosis. Patients without lymph node metastases showed higher FAM159B IRS values  and Ki-67 index  persisted. Again, Kaplan–Meier analysis could not demonstrate any statistically significant differences between patients with FAM159B-positive or -negative tumours. However, a positive correlation between IRS values of FAM159B and those of SST4 and CXCR4 was noted, as well as a trending positive association with PD-L1 . As previously observed, FAM159B IRS was significantly positively correlated with tumour grade  and Ki-67 levels . Similarly, Kaplan–Meier analysis showed no influence of FAM159B expression on overall survival of patients. When evaluating the associations between FAM159B and typical neuroendocrine tumour markers, positive associations were observed between the IRS values of FAM15B and those of the dopamine receptor D2, SST1, SST2, SST3, SST5, CXCR4 and PD-L1.Analyses of only GEP-NEN revealed significantly higher FAM159B IRS values in patients free of lymph node metastases vs. those with metastases  but not of glucagon . Furthermore, a positive correlation was observed between the IRS values of FAM159B and neuron-specific enolase (NSE)  but not chromogranin A .Finally, because double-labelling experiments have shown the presence of FAM159B in insulin-producing β-cells and in some glucagon-producing α-cells, pancreatic neuroendocrine tumours were additionally evaluated for insulin or glucagon expression. Here, a significant relationship was found between the IRS values of FAM159B and those of insulin  of prepared cell lysates. As no signal was detected when analysing the WGA bead fraction, FAM159B seems to lack noticeable glycosylation within BON-1 cells. The specificity of the antibody for FAM159B was further demonstrated by transfection of BON-1 cells with a specific siRNA and by use of pre-adsorption controls with the immunising peptide. We also confirmed the adaptor protein’s previously predicted size of approximately 17 kDa both in BON-1 cells and in mouse pancreas.We applied immunocytochemistry to characterise FAM159B subcellular localisation. Endogenously expressed FAM159B in BON-1 cells is found within the cytosol, most likely within distinct vesicles or associated with other proteins. The FAM159B immunosignal was significantly diminished after BON-1 cells were transfected with a specific siRNA. The reduction in signal intensity represents successful transfection and thus decreased protein synthesis. Because the cells were incubated for 24 h after the start of transfection, FAM159B seems to have a lifespan of approximately 24 h, as the signal intensity would not be reduced if the lifespan was much longer. No signal would have been detected at this time point if FAM159B’s lifespan was much shorter than 24 h.In formalin-fixed, paraffin-embedded human tissue samples, the polyclonal anti-FAM159B antibody yielded a highly efficient and selective immunohistochemical staining of distinct cell populations. Through positive staining of pancreatic islets and endocrine cells of the gastrointestinal tract, the only existing immunohistochemical data of FAM159B expression could be confirmed ,4. As an2+ and Na+ ion currents [In the present study, for the first time, FAM159B expression has been evaluated and visualised in various normal human tissues using immunohistochemistry and double-labelling experiments. A strong expression of FAM159B was found in particular in neuronal structures such as pyramidal cells of the cortex, the trigeminal ganglia, dorsal root and enteric ganglia and neuroendocrine tissues and cells, including pancreatic islets and neuroendocrine cells of the bronchopulmonary and gastrointestinal tracts, but also in syncytiotrophoblasts of the placenta. Because all these tissues and cells are involved in the release of various neurotransmitters and hormones, FAM159B may directly or indirectly play a role in their secretory function. In addition to neuronal and neuroendocrine cells, FAM159B was found to be expressed in various epithelia, such as bronchial, gastrointestinal, bile duct and gallbladder epithelia and in epithelia of the distal tubules of the kidney. At these sites, FAM159B may be involved in ion transport processes. The presence of FAM159B in neuroendocrine cells of the intestinal tract and in pancreatic islets has been shown previously ,3,4 and In the present study, a wide range of different human tumour entities and cancer cell lines was evaluated for possible expression of FAM159B. To date, no respective data are available from the literature. Our immunohistochemical studies revealed strong expression of FAM159B in tumours of endocrine or neuroendocrine origin, such as pituitary adenomas, BP-NEN and GEP-NEN, medullary and anaplastic thyroid carcinomas and parathyroid adenomas. This finding may be due to the fact that moderate-to-strong expression of FAM159B is normally observed in the corresponding non-neoplastic endocrine and neuroendocrine cells. However, intense FAM159B expression was not confined to tumours of endocrine or neuroendocrine origin. A moderate-to-strong presence of the protein was also found, e.g., in lung adenocarcinomas, ovarian carcinomas and lymphomas. Additionally, in many other tumour entities , individual tumours showed high FAM159B expression. In contrast, pheochromocytomas, neuroendocrine tumours originating from the adrenal medulla, did not noticeably express the adaptor protein. Fittingly, cancer cell lines with or without a neuronal or neuroendocrine background showed similarly strong immunosignals. Additionally, in BP-NEN and GEP-NEN, distinct differences in FAM159B expression were observed depending on the origin of the primary tumour. Together, these findings suggest functions of FAM159B that go beyond direct or indirect participation in neurotransmission and/or hormone secretion. Correlations with different markers for neuroendocrine tumours revealed a positive relationship between FAM159B and NSE (but not with chromogranin A), as well as with various receptors and membrane proteins commonly expressed in these tumours , associated either with good prognosis, such as the D2 dopamine receptor and the different somatostatin receptors or with a negative prognosis, such as the chemokine receptor CXCR4 and PD-L1 ,9. ThereThe rabbit polyclonal anti-FAM159B antibody (HPA011778) was purchased from Atlas Antibodies . The sequence of the peptide used for the immunisations of the rabbits was as follows: TKPQRLDTGLKLQHLEASSTQEGKSNGKTKALNSNAASNATN ETYYEADDIIQEKTMDATQIHIA. The respective peptide PrEST Antigen FAM159B (APrEST71583) was also obtained from Atlas Antibodies .BON-1 cells , a neuroendocrine tumour cell line endogenously expressing FAM159B, were seeded into 60 mm Petri dishes and grown to 80% confluence. Cells were lysed in detergent buffer , 5 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 100 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 5 mg/mL aprotinin, 1 mg/mL pepstatin A). For detection of FAM159B in mouse pancreas, 50 mg of tissue was weighed out, 200 µL of the detergent buffer was added and the pancreas was sonicated for 10 s. Afterwards, the samples were gently inverted for 1 h at 4 °C before centrifugation for 30 min at 14,800 g at 4 °C. All samples were then treated with wheat germ lectin–agarose (WGA) beads. The supernatant of the samples, as well as the bead fractions, were subjected to 10% SDS-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride membranes. The blots were then incubated with rabbit polyclonal anti-FAM159B antibody HPA011778 at a dilution of 1:500 overnight at 4 °C, followed by incubation with a peroxidase-conjugated secondary anti-rabbit antibody  and enhanced chemiluminescence detection .When indicated, the expression of endogenous FAM159B in BON-1 cells was silenced using a chemically synthesised, single-stranded RNA oligonucleotide , Waltham, MA, USA) according to the manufacturer’s instructions. An empty vector was used as a negative control .For adsorption controls, HPA011778 was pre-incubated for 2 h at room temperature with 10 µg/mL of the peptide PrEST antigen  used for rabbit immunisations.BON-1 cells were grown overnight on glass coverslips until 80% confluence was achieved. After washing of the cells with phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 20 min at room temperature. After thorough washing with PBS, cells were incubated with anti-FAM159B HPA011778 overnight at 4 °C, followed by incubation with an Alexa-Fluor-488-conjugated secondary antibody  for 2 h at room temperature. Samples were then mounted  and examined using a Zeiss LSM 510 META laser scanning confocal microscope .If required, endogenous FAM159B expression in BON-1 cells was silenced using a chemically synthesised, single-stranded RNA oligonucleotide , Waltham, MA, USA) according to the manufacturer’s instructions. An empty vector was used as a negative control .When indicated, HPA011778 was pre-adsorbed for 2 h at room temperature with 10 µg/mL of the peptide PrEST antigen  used for rabbit immunisations.n = 2–5 each site) were also evaluated. These specimens were obtained from the Department of Pathology of the Ernst Moritz Arndt University  and the Laboratory of Pathology and Cytology Bad Berka .For the initial assessment of FAM159B expression in various human tumour specimens, 259 formalin-fixed and paraffin-embedded tumour samples  were obtFor the subsequent evaluation of FAM159B expression in bronchopulmonary and gastroenteropancreatic neuroendocrine neoplasms (BP-NEN and GEP-NEN), 764 tumour samples from 278 patients , with 416 primary tumour samples from 206 patients and 313 metastasis samples from 116 patients, were included in the investigation. For 35 samples from 45 patients, it was not known whether they originated from the primary tumour or the metastasis. Samples from primary tumours and metastases were available for several patients. Of the tumours, 97 (34.9%) originated from the lung , 27 atypical carcinoids (AC), 38 small-cell lung cancer (SCLC) and 8 large-cell neuroendocrine carcinomas (LCNEC)), 18 (6.5%) from the stomach, 16 (5.7%) from the duodenum/jejunum, 58 (20.9%) from the ileum, 5 (1.8%) from the appendix, 11 (4.0%) from the colon, 15 (5.4%) from the rectum and 49 (17.6%) from the pancreas. The localisation of 9 (3.2%) primary tumours was unknown.The samples were made available by the Institute of Pathology and Cytology Bad Berka  and had been surgically removed between 1998 and 2016 at the Department of General and Visceral Surgery, Zentralklinik Bad Berka . The clinical data were gathered from the patient records.All procedures performed in this study involving human participants were in accordance with both the ethical standards of the institutional or national research committee and the 1964 Helsinki declaration and its later amendments. Permission for this retrospective analysis was obtained from the local ethics committee (Ethikkommission der Landesärztekammer Thüringen). All data were recorded and analysed anonymously.2 culture flasks to 80% confluence. In preparation for the cytoblocks, the cells were washed once with PBS and transferred into 10% buffered formalin for 2 h. After centrifugation for 10 min at 3500 × g, the supernatant was removed, and 1 mL of pooled human plasma was added to the cell samples. The samples were briefly vortexed, 100 µL of fibrinogen was added to each sample and the samples were vortexed again. The resulting clots were immersed in 10% buffered formalin for 24 h and subsequently embedded in paraffin blocks.Neuroblastoma cell lines , a neuroendocrine tumour cell line (BON-1), SCLC cell lines , a lung adenocarcinoma cell line (A549), hepatocellular carcinoma cell lines , colon carcinoma cell lines , a renal cell carcinoma cell line (A498), urinary bladder carcinoma cell lines (T24 and RT-112), breast cancer cell lines , ovarian carcinoma cell lines (SK-OV-3 and A2780), cervical cancer cell lines , prostate cancer cell lines  and an epidermoid carcinoma cell line (A431)  were grown in 75 cmTM mounting medium . For immunohistochemical controls, HPA011778 was adsorbed for 2 h at room temperature with 10 µg/mL of the peptide PrEST antigen  used for rabbit immunisations.From the paraffin blocks, 4 µm sections were prepared and floated onto positively charged slides. Immunostaining was performed using an indirect peroxidase labelling method as described previously . BrieflyFor dual immunofluorescence experiments, samples were incubated overnight at 4 °C with anti-FAM159B HPA011778, together with either a mouse monoclonal anti-insulin antibody  or a mouse monoclonal anti-glucagon antibody . Next, samples were incubated with Cy3-conjugated anti-rabbit and Alexa-Fluor-488-conjugated anti-mouse antibodies. After mounting  the samples were examined using a Zeiss LSM 510 META laser scanning confocal microscope .FAM159B staining of the tumour slides was scored using the semi-quantitative immunoreactivity score (IRS) according to Remmele and Stegner . The perP values ≤ 0.05 were considered statistically significant.SPSS 25.0.0.0  was used for the statistical analyses. Because the data were not normally distributed, a Kolmogorov-Smirnov test, Kruskal–Wallis test, Mann–Whitney test, chi-square test, Kendall’s τ-b test or Spearman’s rank correlation was performed. For survival analysis, the Kaplan–Meier method with a log-rank test was used. We thoroughly characterised the polyclonal rabbit anti-human FAM159B antibody HPA011778 and demonstrated its suitability for both Western blot and immunocytochemistry analyses in basic research, as well as for visualising FAM159B expression in formalin-fixed, paraffin-embedded tissue samples. By applying this antibody, we could detect FAM159B expression in various non-neoplastic and neoplastic tissues and cancer cell lines. Our results also implicate FAM159B in the direct or indirect regulation of diverse membrane proteins, including receptors, ion channels or others such as PD-L1, that may be typically (but not exclusively) expressed in neuronal and (neuro)endocrine tissues. In this context, FAM159B may also regulate secretory functions, among other actions.