Platinum chemoresistance results in high-grade serous ovarian cancer (HGSOC) disease recurrence. Recent treatment advances using checkpoint inhibitor immunotherapy has not benefited platinum-resistant HGSOC. In ovarian cancer, DNA methyltransferase inhibitors (DNMTi) block methylation and allow expression of silenced genes, primarily affecting immune reactivation pathways. We aimed to determine the epigenome and transcriptome response to sequential treatment with DNMTi and carboplatin in HGSOC.In vitro studies with azacitidine or carboplatin alone and in sequential combination. Response was determined by cell growth, death and apoptosis. Genome-wide DNA methylation levels and transcript expression were compared between untreated and azacitidine and carboplatin sequential treatment.Sequential azacitidine and carboplatin significantly slowed cell growth in 50% of cell lines compared to carboplatin alone. The combination resulted in significantly higher cell death in 25% of cell lines, and significantly higher cell apoptosis in 37.5% of cell lines, than carboplatin alone. Pathway analysis of upregulated transcripts showed that the majority of changes were in immune-related pathways, including those regulating response to checkpoint inhibitors.Sequential azacitidine and carboplatin treatment slows cell growth, and demethylate and upregulate pathways involved in immune response, suggesting that this combination may be used to increase HGSOC response to immune checkpoint inhibitors in platinum-resistant patients who have exhausted all currently-approved avenues of treatment.The online version contains supplementary material available at 10.1186/s12885-022-09197-w. Standard treatment for high-grade serous ovarian cancer (HGSOC) is cytoreductive surgery followed by a combination of platinum (carboplatin or cisplatin) and taxane (paclitaxel) chemotherapies. Although initial recurrences are often platinum-sensitive, development of resistance to platinum-based chemotherapy usually occurs with subsequent recurrences .When chemoresistance occurs, the disease-free period between recurrences progressively gets shorter. The most recent definition of platinum-resistant disease is disease recurrence within 6 months of completion of first-line platinum-based chemotherapy . The aveRecent advances in the treatment of many solid tumours using checkpoint inhibitor (CPI) immunotherapy targeting the CTLA4 and PD1–1/PDL-1 axes held great promise for HGSOC, which is often characterised by increased tumour-infiltrating lymphocytes (TILs), a biomarker of response to CPIs. Unfortunately, the results of both monotherapy and combination CPI immunotherapy has not significantly increased progression-free or overall survival for HGSOC , 15. TheCurrent evidence indicates that when low-doses of DNMTi are given, methylation is blocked but the dose is not cytotoxic –19. In oTherefore, the aim of the study was to investigate if sequential DNMTi and platinum-based chemotherapy on HGSOC can increase expression of transcripts associated with immune responses, whilst occurring in parallel to decreased proliferation and/or increased cell death, therefore priming platinum-resistant HGSOC for increased response to CPI immunotherapy.BRCA status of each cell line have been described  [Eight ovarian cancer cell lines were used in this study; KURAMOCHI, OVSAHO, COV362, OVCAR4, COV318, TYKNU, OVKATE, and OAW28. These cell lines were confirmed by genomic and proteomic profiling to be representative of HGSOC . The BRCildtype) , 21.2. Azacitidine (Sigma-Aldrich) and carboplatin (Sigma-Aldrich) were resuspended at 1 mM and 27 mM (10 mg/mL) respectively. All cell lines were treated with 0.5 μM azacytidine alone, 20 μM carboplatin alone and sequential combination of 0.5 μM azacitidine and 20 μM carboplatin. For azacitidine treatment, cells were treated for 72 h with 0.5 μM azacitidine, with media and azacitidine replaced every 24 h. For carboplatin treatment, cells were treated with 20 μM carboplatin for 48 h. For sequential combination azacitidine and carboplatin treatment, cell lines were first treated with 0.5 μM azacitidine for 72 h followed by 20 μM carboplatin for 48 h.The cell lines were cultured in high glucose DMEM (10% FBS) (Gibco). All cells were incubated at 37 °C with 5% COThese doses of carboplatin (20 μM) and azacitidine (0.5 μM) used were lower than maximum plasma concentrations of each drug when used as chemotherapy agents , 23. A mIncuCyte® Cytotox Green was used to stain dead cells after treatment and apoptotic cells were stained with IncuCyte® Annexin Red reagents. The quantification of each population of cells was performed using IncuCyte® ZOOM (Essen BioScience). The eight cells lines were seeded in 96-well plates in triplicate wells at 20% confluency; 8000 cells/well for each cell line, 10,000 cells/well for TYKNU; and the doubling time for the cell lines ranged from 40 to 96 h. They were incubated for 24 h before treatment was added (dosage and timing described above) with Cytotox Green  and Annexin Red  per manufacturer’s protocol.2 ± SEM. Cell confluence was used as a surrogate marker of cell growth in the live-cell assay.The wells were imaged using IncuCyte® Zoom live imaging system at 10x objective using the green and red channels . Phase contrast and dual-colour fluorescence images  were collected for all experiments with spectral unmixing set as 8% of red removed from green) every 6 h for 7 days. The average live (cell confluence), dead (green fluorescence) and apoptotic (red fluorescence) cell count was determined from the replicated wells and data was expressed mean (green or red) confluence per mmThe normalised cell death and apoptosis was obtained by dividing the green or red fluorescence area over the phase confluence area  cells / confluence of total cells; normalised cell apoptosis = confluence of apoptotic (red) cells / confluence of total cells).The processing definitions for analysing the confluences of phase and green and red fluorescence are described in Additional File Cell pellets were collected after 5 days of no treatment (untreated) or 5 days of sequential combination treatment (72 h with azacytidine and 48 h with carboplatin) from one independent experiment.Cell pellets for DNA extraction were resuspended in 200 μL 1x phosphate buffered saline (Invitrogen). DNA was then extracted using the Zymo Quick-DNA Miniprep Kit (Zymo Research) per manufacturer’s protocol. DNA was quantified using the Qubit™ DNA Broad Range Assay Kit (Invitrogen) and purity assessed by 260/280 ratios (> 1.8) using the Epoch Microplate Spectrophotometer (BioTek). DNA integrity was analysed using the TapeStation and D1000 ScreenTape Assay. Cell pellets with a minimum of 250 ng genomic DNA were used for methylation array.Total RNA was extracted by TRIzol (Invitrogen) extraction method according to standard laboratory protocol. RNA was quantified using the Qubit™ RNA High Sensitivity Assay Kit (Invitrogen) and purity assessed by 260/280 ratios (> 1.8) as above. RNA integrity was analysed using the TapeStation and RNA ScreenTape Assay (Agilent Technologies). Cell pellets with a minimum of 1 μg total RNA and RNA Integrity Number (RIN) ≥8 were used for RNA sequencing.In total, four HGSOC cell lines, COV362, OVCAR4, TYKNU, and OAW28 fulfilled the minimum requirements for methylation array and RNA sequencing to be performed. Additionally, these 4 cell lines show the most significant changes in cell confluence and relative cell death and apoptosis compared to standard-of-care platinum treatment . Genomic DNA extracted from cell lines as above were bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research). Genome-wide DNA methylation profiles were obtained using the Infinium MethylationEPIC Beadchip (Illumina), which interrogates over 850,000 methylation sites across the genome.P-value> 0.01, non-CpG probes, and SNP-related probes were removed from the analysis. Probes on the X and Y chromosomes were excluded from the analysis. Normalisation and background correction were applied to the methylation data using PBC within the ChAMP package.Data preprocessing and analyses were performed in the statistical programming environment R v3.6.2 with the package Chip Analysis Methylation Pipeline for Illumina HumanMethylation450 and EPIC (ChAMP) v3.11 . Probes P values for multiple hypothesis testing, false discovery rate (FDR) was calculated using Benjamini-Hochberg correction, and methylation sites in CpG sites/regions with FDR-adjusted P-value< 0.275 were considered statistically significant.Differentially methylated probes (DMPs) were identified using the package within ChAMP, which calculated the difference in β-value between the untreated and azacitidine and carboplatin-treated groups. The β-value is the ratio of intensities between methylated and unmethylated alleles, with β being between 0 (unmethylated) and 1 (fully methylated) . The DMPhttp://great.stanford.edu/) was used to analyse the functional significance of differentially methylated regulatory regions [The Genomic Regions Enrichment of Annotations Tool (GREAT) ( regions .Whole transcriptome sequencing (RNA-Seq) was performed by AGRF. Total RNA was extracted from cell lines and quality of the total RNA evaluated as above. Briefly, the ribosomal RNA (rRNA) was removed from the extracted total RNA by Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina), and strand-specific RNA-seq libraries were built by TruSeq Stranded Total RNA (Illumina) according to the manufacturer’s protocol. Quality control of the libraries was performed by Agilent 2100 Bioanalyzer (Agilent Technologies). RNA sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina), and 100 bp paired-end reads were generated, with a sequencing depth of 30-60 M reads. This is sufficient for characterisation of gene expression levels.The RNA-Seq reads were first trimmed with Cutadapt to remove adapter sequences, primers, poly-A tails and low quality reads. The reads were aligned to a reference genome GRCh37/hg19 using the sequence alignment tool HISAT2. Counts for mapped reads for genomic features such as genes, exons, promoters, gene bodies, genomic bins, and chromosomal locations were created using featureCounts.Differential expression analysis between the untreated and treatment groups was performed using the DESeq2 package v3.12. The estimation of transcript abundances is based on normalised base counts from all samples, dividing by size factors. The biological processes altered by gene expression changes between the groups were assessed using Ingenuity Pathway Analysis (IPA). The RNA sequencing data is available for download at the GEO repository (GSE169617).Cellular confluence was measured to determine if ovarian cancer cell growth is affected by combined azacitdine and carboplatin. The sequential combination of azacitidine and carboplatin significantly slowed the growth of all cell lines Fig. . Cells tBRCA mutations  Fig. A. This wons Fig. B. In BRCnly Fig. C.Fig. 2Pp < 0.05). For KURAMOCHI, COV362, OVCAR4, and TYKNU, confluence was significantly lower when treated with combined azacitidine and low-dose carboplatin compared to a single treatment of higher dose carboplatin . For TYKNU, combination azacitidine and carboplatin showed significantly higher cell death compared to the higher dose of carboplatin (30 μM)  or higher-dose 30 μM) carboplatin only Fig. A. This wons Fig. B and C. 0 μM carbBRCA-wildtype cell lines . One cell line, OVCAR4 had significantly higher cell apoptosis after combined azacitidine and carboplatin compared to higher-dose of 30 μM carboplatin. Two cell lines, COV318 and OAW28 had significantly higher cell apoptosis when treated with combined azacitidine and carboplatin compared to treatment with low-dose carboplatin (20 μM)  Fig. A. This wnes Fig. B. In BRCtin Fig. C. All ceP > 0.01 in more than one sample and probes that are not represented by a minimum of three beads on the array.To investigate the potential for sequential azacitidine and carboplatin to induce biological pathways related to CPI sensitivity, global demethylation was assessed in untreated cells compared to cells treated with sequential azacitidine and carboplatin. The reference genome used in all analyses was GRCh37/hg19. Probe filtering resulted in 711,405 probes after removing those that failed with a detection P < 0.05 after multiple comparison correction FDR = 0.275 was considered significant. In total, there were 86,979 DMPs found to be significantly different between the untreated and sequential azacitidine and carboplatin groups  ≥ 0.1).Differential methylation was assessed in CpG sites (DMPs); p = 3.55e-37), the main mechanism of programmed cell death after treatment with carboplatin . 36 annotated genes were upregulated after combination treatment compared to the untreated group . Analysis by IPA was performed to investigate the pathways significantly upregulated by the combination azacitidine and carboplatin treatment. One of the most significantly upregulated pathways after combination treatment was the apoptosis signaling pathway (p = 1.03e-02), which is triggered to respond to carboplatin-induced DNA damage , TNFR1 signaling, (p = 2.5e-03), death receptor signaling (p = 8.72e-03)), which are involved in response to chemotherapy. Also of note was the enrichment of the antigen presentation pathway, which is essential in presenting neoantigens or foreign peptide antigens to trigger T cell immune response [Three of the most highly enriched were pathways related to apoptotic programmed cell death . The network constructed from the ten upregulated genes indicated that the majority of changes were in pathways relating to immune responses, including cytotoxic T lymphocyte-mediated apoptosis (p = 1.44e-02), antigen presentation pathway (p = 1.65e-02), CTLA4 signaling in cytotoxic T lymphocytes (p = 3.79e-02), and PD-1 and PD-L1 cancer immunotherapy (p = 4.42e-02) , which is involved in many of these immune-related pathways.An integrated methylome-transcriptome analysis was performed to examine the association between CpG methylation and gene expression in the tumour cells after combination treatment. The DMPs obtained from the TSS1500 and TSS200 regions were selected for subsequent analysis as promoters and their surrounding regions are primarily responsible for gene expression regulation. We focused on promoter CpG island-containing genes that were simultaneously hypomethylated and upregulated after combined azacitidine and carboplatin treatment, and ten genes were identified  class I after sequential azacitidine and carboplatin treatment of HGSOC cell lines.The integrated methylome-transcriptome analysis also showed hypomethylation and upregulation of β2m after combined azacitidine and carboplatin. β2m is a component of MHC class I heterodimers responsible for antigen presentation, which shuttles peptide fragments to the cell surface for recognition by CD8+ T lymphocytes , 37. In + and CD8+ PD-1-positive tumour-infiltrating T-lymphocytes in the tumour microenvironment compared to the untreated tumours, suggesting an enhanced response to PD-1 inhibition [+ and CD8+ T-cells [Recent advances in the treatment of solid tumours using CPI immunotherapy has only resulted in modest improvement to overall response rates in HGSOC. Two clinical trials of CPI immunotherapy in patients with platinum-resistant recurrent ovarian cancer showed objective response rate of only 9.6% (12/25) for anti-PD-L1 and 9% (11/97) for anti-PD-1 , 40. Prehibition . A recen T-cells , indicat T-cells . Our hypThe results from this study are consistent with our previous study in melanoma, in which the sequential combination of a DNMTi and platinum chemotherapy induced greater cell death and slowed cell proliferation . SeveralThere are limitations to our cell line model-based study, which lacks tumour heterogeneity and microenvironment. It would be beneficial to replicate this study in an in vitro 3D cultures from primary tumours, ascites, or metastases, especially from platinum-resistant patients, to model the ovarian cancer tumour microenvironment . AdditioThe results of this study indicate that sequential azacitidine and carboplatin can slow tumour cell growth, and sequential combined low doses of azacitidine and carboplatin can slightly increase HGSOC cell death compared to clinically-relevant dose of carboplatin, which is an important outcome especially in platinum-resistant disease. The combination of sequential azacitidine and carboplatin treatment demethylates and upregulates pathways that are involved in immune response and immune checkpoint blockade. Taken together, the results suggest that the addition of azacitidine to standard-of-care carboplatin can be used in increase HGSOC response to immune checkpoint inhibitors.Additional file 1: Supplementary Table 1: Suggested processing definitions using the Incucyte® Zoom.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Zataria multiflora (ZMSLN) enhanced the anticancer efficacy of the essential oil against breast cancer (MDA-MB-468) and melanoma (A-375) cells.The cancer burden is rising rapidly worldwide, and it annually causes about 8.8 million deaths worldwide. Due to chemical drugs’ side effects and the emergence of resistance, the development of new green drugs has received much attention. We aimed to investigate whether solid-lipid nanoparticles containing essential oil of ZMSLN was prepared by the high-pressure homogenizer method; particle size 176 ± 8 nm, polydispersity index 0.22 ± 0.1, entrapment efficiency 67 ± 5%. The essential oil showed a dose-dependent antiproliferative effect on MDA-MB-468 and A-375 cells at all examined concentrations . Interestingly, after treating both cells with 75 μg/mL of ZMSLN, their viabilities were reduced to under 13%.The finding showed that ZMSLN had a distinct antiproliferative efficacy; it could thus be considered a green anticancer candidate for further in vivo and in vivo studies. Cancers have many financial, emotional, and physical burdens in all societies and comprise approximately 17% of global deaths . CardiovZataria multiflora Bioss, the Lamiaceae family, is one of the most important medicinal plants in Pakistan, Afghanistan, and southern Iran; it is applied as an antiseptic and antitussive in respiratory tract disorders [Z. multiflora essential oil (ZMEO) also possesses many biological activities, including anticancer and antioxidant effects [Bone marrow depression, extreme fatigue, alopecia, loss of self-esteem or immunity, and a remarkable decline in white blood cell count are common side effects of synthetic or semisynthetic anticancer drugs , 8. Thusisorders . The Z.  effects , 12. EOs effects , 14. Sol effects , 16. We MDA-MB-468 (ATCC HTB-132 and A-375 (ATCC CRL-1619) as breast cancer and melanoma cell lines were purchased from Pasteur Institute of Iran. Stearic acid, tween 80, span 60, phosphate-buffered saline (PBS) tablets, penicillin-streptomycin, trypsin, dimethyl sulfoxide (DMSO), absolute ethanol (99.8%), and MTT -2, 5diphenyltetrazolium bromide) were bought from Merck Co. (Germany). Shellmax (China) and Gibco (USA) supplied cell culture medium (RPMI 1640) and fetal bovine serum (FBS). ZMEO was bought from Zardband Pharmaceutical Co. (Iran).ZMSLN was prepared according to our described high-pressure Homogenizer method . ZMEO . The formulation was poured into a quartz cell and situated in the hole of the device for analysis.To determine the shape of ZMSLN and free SLNs, it was first diluted two times with distilled water; one drop was then impregnated on a copper grid (200-mesh carbon-coated). Next, it was stained with phosphotungstic acid (2%) solution, dried at room temperature, and subjected to a TEM device, Philips CM30 TEM microscope (Netherlands).n = 3). ZMEO content in the supernatant was then determined by the UV-Visible method; absorbance at λ max was placed in the regression equation.ZMEO was first diluted in ethanol in a 150–350 μg/mL concentration, and absorbance was then screened (200–400 nm) to determine the maximum absorption wavelength (λ max). The standard calibration curve (with regression equation) for ZMEO was then plotted  in 96-well culture plates overnight. Wells content was then replaced with 75 μg.mL−1 RPMI complete medium (containing 10% FBS and penciling/streptomycin 1%). The cells were treated with various concentrations of ZMEO and ZMSLN , as well as free SLNs; treated plates were incubated for 24 h (37 °C and 5% of CO2 concentration). Wells’ content was then discarded and washed with PBS to the removed milky color of ZMSLN. After that, 100 μL of MTT reagent (0.5 mg/mL dissolved in RPMI) was treated for each well and incubated for 4 h at 37 °C and 5% of CO2 concentration. Finally, 100 μL/well DMSO was added to solubilization the precipitate formazan crystals to the obtained purple color. The absorbance (A) of wells was measured at 570 nm by the ELISA Plate Reader. Cell viabilities at different concentrations were calculated by eq. The in vitro cytotoxicity of ZMEO and ZMSLN on the breast cancer (MDA-MB-468) and melanoma (A-375) cancer cells was estimated by MTT assay according to our previous study with slight modification . ZMEO wa50s of the samples. One-way ANOVA  with the least significant difference test (L.S.D) at the 5% level was used to compare anticancer activities.All the tests were carried out in triplicate; results were expressed as mean ± SD. CalcuSyn software  was applied for calculating the ICDLS characterized the ZMSLN with a particle size of 176 ± 8 nm and a polydispersity index of 0.22 ± 0.1  at all concentrations. Moreover, a significant difference (P < 0.001) is observed amongst the control group and treated cells with free SLNs; viability was reduced to 79%.As shown in Fig. P < 0.001). Besides, the viability of the cell after treatment with free SLNs was significantly (P < 0.001) reduced (to ~82%) in comparison with the control group.From Fig. 50 values of ZMEO against MDA-MB-468 and A-375 cells were obtained at 380 and 59 μg.mL-1. Because even the lowest concentration of the ZMSLN reduced cell viability of both cell lines below ~ %13, IC50 of ZMSLN was thus not calculating precisely. However, it could be concluded that ZMSLN had a distinct antiproliferative effect.IC2 non-expression (triple-negative) is an invasive and poorly differentiated cell line. It is one of the commonly used cell lines in the literature [Breast and skin cancer are the two most frequent types of cancers worldwide , 20. Theterature , 24. Skiterature . Malignaterature . Amongstterature , 28.50 values were obtained as 76, 70, and 104 μg.mL−1 [50 380 μM was more potent than thymol with IC50 497 μM against human non-small cell lung carcinoma after a 24 h incubation period [Elbe et al.; thymol was more potent than carvacrol [Natural products, especially EOs, have been widely used for their therapeutic properties as chemopreventive/chemotherapeutic agents in vitro and in vivo studies –31. For  μg.mL−1 . Further μg.mL−1 . For ins μg.mL−1 –36. For  μg.mL−1 . The cytn period . RecentlCroton argyrophyllus EO with the particle size of 201.4 ± 2.3 nm and entrapment efficacy 89.63 ± 1.02% was reported; an improvement in antioxidant properties of EO in the treatment of neurodegenerative diseases was observed [Eugenia caryophyllata EO were presented a unique nanoparticulate system with a vast antimicrobial activity. The particle size was between 397 ± 10 nm and 786 ± 11, with entrapment efficacy at approximately 70% [et al. designed and prepared a SLN from ZMEO using hot homogenization and precipitation methods; the prepared SLNs had a mean size of 650 nm [et al. applied the ultrasonic Homogenizer method to produce several SLNs with different sizes [SLNs contain a hydrophobic solid matrix core with one layer of a phospholipid coating . The higobserved . In anotobserved , 44. SLNtely 70% . Anotherf 650 nm . Ehsanfant sizes .et al. prepared ZMSLN with a particle size of 255 ± 3 nm to improve and control antifungal efficiency in some fungal pathogens [Cinnamomum zeylanicum EO against Leishmania tropica and Leishmania major was increased when size was reduced to the nanoscale range, 52 ± 4 nm [50 8.94 μM) compared with free variabilin (IC50 87.74 μM) against the PC-3 cell line [Furthermore, several studies showed that formulating EO in nanocarriers leads to higher activity. For example, Nasseri athogens . In our athogens . In addi2 ± 4 nm . Also, c2 ± 4 nm . Besides−1. This is due to the maximum capacity of this nanoformulation at exanimated concentrations; with decreasing concentrations, lower efficacies become visible. In our previous report, the anticancer properties of chitosan nanoparticles containing Citrus sinensis and Citrus limon essential oils at exanimated concentrations were also not dose-dependent [−1), further research to investigate toxic effects on normal cells and other cancer cell lines in vitro and animal models are suggested.In the current study, the anticancer activity of ZMEO and ZMSLN was evaluated; results represented that ZMEO at the nanoscale size (176 nm) had a substantially more anticarcinogenic effect compared with bulk form against MDA-MB-468 and A-375 cancer cell lines. It may relate to the higher efficiency of cellular uptake and intracellular absorption of ZMSLN than the bulk form of ZMEO . Moreoveependent . HoweverZ. multiflora EO (ZMSLN) with a particle size of 176 ± 8 nm, polydispersity index 0.22 ± 0.1, and entrapment efficiency 67 ± 5% was first prepared. The comparative study showed that ZMSLN had a higher anticancer activity on tested cancer lines (MDA-MB-468 and A-375) than the EO treatment group. Furthermore, after treating both cell lines with the lowest examined concentration of ZMSLN, 75 μg/mL, their viabilities were decreased under %13. ZMSLN could thus be introduced as a green potent antiproliferative candidate. However, more studies should be evaluated to reveal the exact mechanism of anticancer activity of ZMSLN.Solid-lipid nanoparticle-containing


The cell lines used in this study  were recSNCA expression. The determination that the cells are HEK-293 rather than SH-SY5Y in origin does not impact the validity of the system or results reported in the article, including the validation of the reported assay and cell lines for identifying compounds that modulate SNCA expression. Nevertheless, readers should consider potential implications of the HEK-293 cellular context when using this system in future studies.The intention of this study was to validate cell line assays for high-throughput screening to identify compounds which lower A reviewer raised that HEK-293 cells are not expected to differentiate in response to retinoic acid as is reported in the S4 Fig legend. This figure showed that autophagy blockade increased α-synuclein levels. The authors stand by the validity of this result which has since been confirmed in other studies (e.g. [The underlying data to support all graphs reported in the article are available upon request from DRS.Daniel.Scoles@hsc.utah.edu.DPH is deceased, and so as of the time of this notice, DRS will serve as this article’s corresponding author: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.


However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.Ewing’s sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1 In at least one out of three patients, CHM1319-specific TCR-transgenic T cell homing to affected bone marrow could be monitored. Tumor regression was observed without causing symptoms of graft versus host disease (GvHD) [Ewing’s sarcoma (EwS) represents a highly malignant pediatric solid tumor entity. EwS carries a low somatic mutational burden and a low rate of tumor-infiltrating T cells, implying a low immunogenicity. Upregulation of the endochondral bone protein Chondromodulin-I CHM1) in EwS is driven by the fusion oncogene EWS-FLI1. In this context, CHM1 has been identified as a promising target antigen for immunotherapy in EwS patients  in EwS i,3,4. Our in vivo . In the e (GvHD) .+ T cell stimulation [+ T cells upon TCR activation which, when binding to CD83 on APC, results in increased antigen-specific CD8+ T cell numbers [CD83 is a transmembrane glycoprotein of the immunoglobulin superfamily and is predominantly expressed on several activated immune cells . Two isomulation . Further numbers .319-specific TCR-transgenic T cells in vitro.In this analysis, we hypothesized that an immunogenic signature can be induced using different cell mediators in EwS cell lines. In particular, we hypothesized an immunogenic effect of cell mediators used in the differentiation of dendritic cells out of CD14+ myeloid cells. We hypothesized that mCD83 up-regulation in EwS is feasible, causing significantly improved susceptibility of HLA-A*02:01 positive EwS cell lines when treated with CHM1+ EwS line A673 and HLA-A*02:01- EwS cell line SBSR-AKS were cultured in medium containing X-Vivo 15 and 1% human AB serum. The protocol applied to the ‘supplemented’ EwS cell lines is generally used to differentiate CD14+ monocytes into DCs [5 tumor cells was supplemented with 1000 U/mL IL-4  and 800 U/mL GM-CSF . Medium was renewed at the same concentrations on day three. On day six, in accordance to DC maturation protocols, the medium was again renewed and supplemented with IL-6 (1000 U/mL), IL-1β (10 ng/mL), TNF (10 ng/mL) and PGE2 (1 µg/mL) . Further analyses commenced at day 7 . The antibodies ICAM-1, PD-L1, PD-L2, CD83, MHC class I and II and respective isotypes were purchased from Miltenyi Biotec. Respective APC-, APC-Vio770- or PE-conjugated IgG isotypes served as staining controls. Control EwS cell lines cultivated in RPMI 1640 medium  constituted respective control groups.Surface expression of CD83 (APC-Vio770), MHC class I and II (APC), ICAM-1 (PE), PD-L1 (PE) and PD-L2 (APC-Vio770) were determined by flow cytometry analysis using MACSQuant2), respectively, or (2) three out of four DC maturation cytokines, respectively. The reason why we chose this approach was to juxtapose the possible immunogenic effect in respective contrast to the effect of the three remaining cell mediators in order to screen for the strongest impact of each single mediator. To determine the point of time of CD83 surface upregulation, we performed FACS analyses at day four, day six, and day seven of the maturation protocol  either only one of the DC maturation cytokines . For analysis of the flow cytometry data, gates were set to identify living cells as well as single cells only. Furthermore, quadrants were set according to the respective APC-, APC-Vio770- and PE isotype-labelled control samples.319/HLA-A*02:01-specific TCR transgene was introduced into T cells using the retroviral vector pMP-71 [+ T cells were activated with 3 µg/mL OKT3 according to manufacturer’s recommendation and 100 U/mL IL-2 (Novartis) was added for T cell proliferation. Two days later, spin infection with virus supernatant from the packaging cell line RD114 was performed. On the next day, T cells were re-infected with the same approach. After verifying successful transduction via FACS staining, T cell subsets were purified with anti-phycoerythrin (PE) magnetic beads (Miltenyi Biotec) coupled to CHM1319/HLA-A*02:01-specific PE labeled multimers as previously published [The CHM1r pMP-71 ,13. Puriublished . Upon ge319/HLA-A*02:01-specific recognition and killing of A673 EwS cells cultured in cytokine supplemented medium compared to the control group cultured in standard control medium, was assessed using interferon-319-specific TCR-transgenic T cells were thawed and expanded by adding 100 U/mL rhIL-2 and 2 ng/mL rhIL-15. T2 cells primed with influenza and CHM1 peptides served as negative and positive control groups, respectively. For the interferon-319/HLA-A*02:01-specific T cells and 20,000 target cells were used. For granzyme B ELISpot assays, effector to target ratios were titrated from 2.5:1 to 0.156:1. All experiments were performed in sextuplicates for interferon-CHM1® WT Expression Kit) which was fragmented and labelled following the Affymetrix® GeneChip® WT Terminal Labelling and Hybridization protocol prior to its injection onto a GeneChip® Probe Array. Alterations in gene expression were compared and analyzed by gene set enrichment analysis and visualized using iSEE (Bioconductor). Affymetrix-based RNA expression analyses were performed in order to evaluate RNA signatures induced by DC medium conditioning in A673 and SBSR-AKS EwS cell lines. Gene expression analyses were illustrated using iSEE [RNA from A673 and SBSR-AKS cells cultivated in supplemented–versus control medium was isolated using the extraction reagent TRIzol and the Zymo Research Direct-zol™ RNA MiniPrep Kit. The extracted RNA served as a template for cDNA synthesis  was used to calculate mean and standard deviation of the mean (SEM). Differences between two groups were determined using the student’s Heatmap analyses showed an increase of CD83 and ICAM-1 expression in both DC medium supplemented versus control A673 and SBSR-AKS cell lines . SBSR-AKOn day seven of cell mediator treatment , we asse2 and IL-1β treatment alone, respectively. Correspondingly, FACS analysis of A673 treated with three out of four cytokines showed a varying increase in CD83 surface expression in all groups , 0.625 (p = 0.04) and 0.156 (p = 0.03), confirming stronger anti-tumor effects when A673 was supplemented (We evaluated the effect of CD83 upregulation on the cytotoxic potential of CHM1 T cells  directedere used A. The grependent . A673 sulemented B. These lemented .According to a recent analysis performed by Gröbner et al., pediatric cancers can be genetically subdivided into two classes: entities with low mutation rates and entities with structural/copy-number variants . In the 319/HLA-A*02:01-specific T cells. Therefore, we treated EwS cell lines with different cytokines routinely used for DC generation of CD14+ monocytes. Interestingly, we observed a wide spectrum of upregulated immunogenic genes and surface markers upon treatment associated with immunogenicity towards T cells, such as CD83, ICAM-1 and MHC class I upregulation both on RNA level and protein level. Given these observations, we sought to identify the cytokine responsible for inducing such immunogenicity in EwS.In the present study, we sought to identify cytokines with the potential to render EwS more susceptible to treatment with CHM1ICAM-1, also known as CD54, is a transmembrane glycoprotein of the Ig superfamily and is most prominently known for its co-stimulatory function enhancing the interaction between DCs and T cells . ICAM-1 As indicated previously, several types of cancer downregulate their cell surface expression of MHC class I over time as an immune escape mechanism by which they dampen the immune response directed against tumor tissue . Studies+ and CD8+ T cell responses. In immature DCs, CD83 is preformed intracellularly and is rapidly expressed on cell surface upon activation and maturation [Membrane-bound CD83 is a surface protein suggested to be a key immunological player in the regulation of CD4turation ,24. Furt319/HLA-A*02:01-specific T cells in ELISpot assays.Among the implemented cytokines of the DC-supplemented EwS culture media in this study, TNF was associated with most prominent CD83 upregulation. These findings are in line with previous reports identifying TNF as an activating agent for increased CD83 expression on monocyte-derived DCs and therefore a potent driver for DC maturation ,26. More319-specific TCR-transgenic T cells.In the past, studies conducted with TNF as an anti-tumor agent have yielded heterogeneous results. As a systemic agent, simultaneous application of TNF and other anti-cancer agents resulted in an increased uptake and enhanced anti-tumor efficacy. However, TNF has also shown to induce severe toxicity at doses even lower than the predicted efficient dose ,28,29. Hh as EwS ,33. Inteh as EwS ,35. Varih as EwS ,36. ConsThe present results imply that it is possible to induce tumor immunogenicity in primarily non-immunogenic EwS via exogenous stimuli. Neo-adjuvant sensitizing of therapy-refractory EwS induced by triggering a local inflammation in the tumor area, for example by local irradiation or by delivery of inflammatory mediators, may be a further step towards the improvement of successful T cell-based immunotherapy. Rübe et al. showed that increased biologically active TNF levels can be induced in a dose-dependent manner in a majority of EwS cell lines by local irradiation in both in vitro and in xenograft mouse models . Our obs319/HLA-A*02:01-specific T cells may significantly enhance anti-EwS responses.In the present analysis, we demonstrate that EwS can be altered to become more susceptible to the effect of adoptive T cell therapy using proinflammatory cytokines conventionally used for the generation of DCs. In the future, the induction of immunogenicity prior to adoptive transfer of CHM1


Listeria effect. This manuscript evaluates the intestinal cell monolayer invasive capacity of Listeria monocytogenes strains obtained from human, salmon, cheese, and L. innocua treated with two WPP (WPP-N and WPP-C) of different polyphenol contents using Caco-2 and SW480 cells. The invasion was dependent of the cell line, being higher in the SW480 than in the Caco-2 cell line. Human and salmon L. monocytogenes strains caused cell invasion in both cell lines, while cheese and L. innocua did not cause an invasion. The phenolic contents of WPP-N are characterized by high levels of anthocyanin and stilbenes and WPP-C by a high content of phenolic acids. The inhibitory effect of the WPPs was dependent of the strain and of the degree of differentiation of the intestinal cells line. The inhibition of Listeria invasion by WPPs in the SW480 cell line, especially with WPP-C, were higher than the Caco-2 cell line inhibited mainly by WPP-N. This effect is associated with the WPPs’ ability to protect the integrity of the intestinal barrier by modification of the cell–cell junction protein expression. The gene expression of E-cadherin and occludin are involved in the L. monocytogenes invasion of both the Caco-2 and SW480 cell lines, while the gene expression of claudin is only involved in the invasion of SW480. These findings suggest that WPPs have an inhibitory L. monocytogenes invasion effect in gastrointestinal cells lines.Red wine pomace products (WPP) have antimicrobial activities against human pathogens, and it was suggested that they have a probable anti- Listeria monocytogenes is a Gram-positive pathogenic bacterium, responsible for serious systemic infections such as septicemia, gastroenteritis, and meningitis, among others, both in animal species and in humans, with a high lethality rate [L. monocytogenes is difficult due to its ability to tolerate a wide range of temperatures (−1.5–45 °C) and pH (4.3–9.1), salt concentration, and lower water activity. Furthermore, it is a facultative microorganism, resistant to alkaline media and to preservatives, such as sodium nitrate [inlA and inlB, respectively, are the main invasion factors, which bind to the receptors present on the surface of the host cell E-Cadherin and Met, allowing adherence and, therefore, the invasion of bacteria after the destabilization of the cell membrane [Microbial cross-contamination is a problem in the food industry, and listeriosis outbreaks are a focus of special interest ,2. Listeity rate ,4. Accortures −1.–45 °C an nitrate . Due to  nitrate , associamembrane ,9.To prevent microbial cross-contamination and increase foodstuff safety and shelf lives, the food industry uses antimicrobial agents—commonly, synthetic or “chemical” additives. However, in the last decade, the demand for using natural products with antimicrobial properties has increased. The activity of natural products derived from plants against microorganisms has been mainly associated with the presence of phenolic compounds with antimicrobial activities ,11. PhenL. monocytogenes has been described in previous works [Listeria activity could be attributed to the wine pomace flavonoids that interact with soluble extracellular proteins and the bacterial cell wall [L. monocytogenes intestinal invasion [Wine grapes are very rich in phenolic compounds, and most of them are not extracted during the winemaking process. Thus, the wine industry generates large amounts of grape pomace rich in phenolic compounds that have been considered as an alternative for obtaining sustainable functional ingredients, which may be used in the food industry with different purposes ,16,17, aus works . The antell wall ,20,21. Iinvasion ,23,24.Listeria monocytogenes could then be used as a functional ingredient in the food industry. Therefore, we investigated the invasion of L. monocytogenes strains treated with WPP using Caco-2 and SW480 intestinal cell lines and the expression of adheren and the tight junctions involved in the mechanism of invasion. Our hypothesis is that wine pomace products that have antimicrobial activity in vitro might also have invasion potential capacity against different L. monocytogenes strains.Wine pomace products that have antivirulence effects on different strains of v/v), iron (III)-chloride acid (FeCl3), iron (II)-sulphate (FeSO4), sodium acetate (NaC2H3O2), and sodium carbonate (Na2CO3) were purchased from Panreac Quimica S.L.U. . Malvidin-3-glucoside was obtained from Estrasynthese .Saline peptone water, Brain Heart Infusion (BHI), Tryptic Soy Broth (TSB) supplemented with Yeast, Plate Count Agar (PCA), Tryptone Sulphite Neomycin (TSN), Dextrose Sabouraud (DS), and Mannitol Egg Yolk Polymyxin (MYP) mediums were obtained from Merck KGaA, . Acetonitrile; amphotericin B solution (250 μg/mL); 2,2′-Azinobis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS); Fetal Bovine Serum (FBS); glacial acetic acid; hydrochloric acid; 6-hydroxy 2,5,7,8-tetramethyl-2-carboxylic acid (Trolox); L-glutamine solution (200 mM); MEM ; methanol; penicillin ; pure phenolic compounds ; streptomycin solution ; and 2,4,6-Tris (2-pyridyl)-S-triazine (TPTZ) were purchased from Sigma-Aldrich Chemical Co. . Folin–Ciocalteu (FC) reagent, 70%  and WPP-C (red wine pomace product from the center winery), were made by applying previously described processes with minor modifications. Briefly, each collected wine pomace was dehydrated in an oven at 60 °C. Each WPP was made from seedless dried wine pomaces by milling and sieving (particle size <0.250 mm), and the microbiological stabilization was done by heat treatment (90 °C/90 min).Red wine pomaces were collected, from the same vintage, in two different wineries of two Spanish wine regions, one cited in the north and other in the center of Spain. Both wineries made red wines mainly with Clostridium spp., 1 mL was poured on PCA and TSN, respectively, and incubated at 30 °C for 72 h for TVC and 37 °C for 24 h in anaerobic conditions for Clostridium spp. One hundred microliters were streaked on DS and MYP to determine the molds and yeast and Bacillus spp., respectively, and incubated at 25 °C for 5 days for the molds and yeast and 30 °C during 24 h for Bacillus spp. Microbiological assays were carried out in triplicate.One gram of each one of the WPP (N and C) was added to 9 mL of different culture media  and incubated for 24 h at 37 °C. To determine the total viable counts (TVC) and Extractable phenolic compounds of each WPP were quantified by HPLC after extracting using acidified methanol. Rehydrated powders were extracted with methanol/HCl (97:3) at 25 °C for 24 h and continuous stirring. Then, supernatants were decanted and filtered through a VWR cellulose filter with 8–12-µm particle retention . Each supernatant was raised to a known previous volume to be analyzed. Extracts were made in triplicate. Different phenolic compounds were analyzed using analytical reverse-phase HPLC on an Agilent 1100 series HPLC system  coupled to a diode array detector. Hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols, and stilbenes were analyzed using a spherisorb3 ODS2 reverse-phase C18 column  using the chromatographic conditions previously published . RetentiQUENCHER (Q-) methods better reproduce the real circumstances in which WPP solid products, and not their extracts, are used. Q-methods are based on the corresponding classical methods, but they were adapted to measure the TAC  of solid products. QUENCHER versions of three classical TAC assays: Folin–Ciocalteu index, ABTS , and FRAP (Ferric Reducing Antioxidant Power), were selected to evaluate the TAC of the WPP . QUENCHEL. monocytogenes strains used in this study were either previously isolated from food products in the food technology area at the University of Burgos  or from human cases  obtained from the International Life Sciences Institute (ILSI) North America Listeria monocytogenes strain collection [L. monocytogenes PCR-serogrouping was performed for the salmon-derived strain using a multiplex PCR as previously described [Listeria strain was grown on Tryptic Soy Broth supplemented with yeast extract (TSB-Y) at 37 °C overnight to obtain 9 log cfu/mL. Then, the culture was diluted to inoculate 7 log cfu/mL in BHI supplemented with 40 g/L of each WPP (WPP-N and WPP-C) and incubated for 24 h and 48 h at 37 °C. The quantity of WPP supplemented was decided considering previous satisfactory results [The llection . L. monoescribed , using Iescribed . Bacteri results . To cons2. Cell monolayers were infected with the different strains of L. monocytogenes with and without a previous treatment with WPP-N and WPP-C, and a multiplicity of infection (MOI) of 25:1 was used. After 1 h of incubation at 37 °C, the cell monolayers were washed with PBS and incubated in MEM and 10% of fetal bovine serum, containing gentamicin (100 µg/mL) for 4 h (intracellular growth). Half of the samples were lysed with 1 mL of 0.1% Triton X-100, and counting was performed by plating on TSAYE agar. The other half of the samples were tripsinized and centrifuged, and the pellets were frozen until the q-PCR analysis. All assays were independently repeated four times.For the in vitro virulence assay, human adenocarcinoma cell lines Caco-2 and SW480 from ECACC  were used, and the assays were performed as previously described . Caco-2 ® Green Supermix . All the procedures were performed according to the manufacturers’ protocols. The sequences of the primer sets (forward and reverse) were, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCTCTCCAGAACATCATCCC-3′ and 5′-GTCCACCACTGACACGTTG-3′; claudin, 5′-GTGGAGGATTTACTCCTATGCCG-3′ and 5′-TCAAGGCACGGGTTGCTT-3′; E-cadherin, 5′-ATGCTGAGGATGATTGAGGTGGGT-3′ and 5′-CAAATGTGTTCAGCTCAGCCAGCA-3′; and occludin, 5′-TCAAACCGAATCATTATGCACCA-3′ and 5′-AGATGGCAATGCACATCACAA-3′. Amplification efficiencies were calculated for each pair of primers, and efficiency-corrected quantification was performed using 2−∆∆Ct with GAPDH as the reference gene. Relative gene expression was expressed as the folds of change compared to the control (control cells uninfected with L. monocytogenes).Caco-2 and SW480 pellets were resuspended in TRI Reagent solution . After treatment with DNase I , 1 µg of total RNA was reverse-transcribed using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific), and, finally, amplified using an iQ™ SYBR® Centurion XVI, version 16.2.04 . ANOVA analysis and the Student’s t-test were used to determine the statistical significance, with a p-value of <0.05).Representative data were expressed as the mean ± standard deviation of independent experiments. Statistical analysis was performed using StatgraphicsL. innocua, showed in vitro [The antioxidant and antimicrobial properties of wine pomace products (WPP) are of interest to the food industry, and they confer the possibility of considering the role of WPP as food preservatives. In previous studies, it was observed that the WPP obtained from red wine pomace could be used as natural alternative additives due to their antioxidant activity, their ability to improve microbial stability, inhibiting spoilage bacteria growth in foods with low contents of salt , and thein vitro .L. monocytogenes treated with two types of WPP, together with their effect on intestinal E-cadherin as an InlA cell receptor and on tight junction proteins occludin and claudin for their role in the maintenance of intestinal paracellular permeability.In this study, we evaluated the possible association between the bactericide effect of WPP observed previously and their antivirulence effect in intestinal cells. Intestinal epithelial cells are the first line of defense against pathogens that can breach this barrier. The implication of cell junction proteins (adherens and tight junctions) is that they are critical for the maintenance of the intestinal barrier. Therefore, this study evaluated the intestinal invasion of L. monocytogenes virulence, and to avoid contamination from possible microorganisms contained in WPP, the microbiological quality of each WPP was evaluated. The results revealed that both WPP (N and C) did not show any microbial load.Before studying the WPP effect in Furthermore, extractable phenols were analyzed as the principal antimicrobial agents of WPP, and the antioxidant capacities were considered as an index of additional interesting properties of the WPP also associated to the phenol content.The levels of the different phenolic compounds analyzed  showed sThe largest differences were detected between the anthocyanin levels, which were significantly higher in WPP-N than in WPP-C . On the contrary, the levels of the phenolic acids (hydroxybenzoic and hydroxycinnamic acids) were statistically higher in WPP-C than in WPP-N. The total content of the flavan-3-ols was similar in both WPP, but stilbenes, like anthocyanins, were statistically higher in WPP-N than in WPP-C. These results evidenced the possible large variability that can occur between the compositions of the wine pomaces collected in different regions and wineries, which derived from pedologic-climatic conditions, cultural vine practices, and oenological processes, among other factors.The results of the total antioxidant capacity (TAC) of both WPP also highlighted the differences between the products . The Q-FL. monocytogenes can cause two forms of listeriosis: noninvasive gastrointestinal listeriosis and invasive listeriosis [L. monocytogenes strains isolated from humans ; salmon (S11); and cheese (E10.652), together with one L. innocua strain, was evaluated after their serotyping.teriosis ,2. The iThe serotyping results showed that the salmon strain belonged to the serogroup 1/2a, 3a, as well as the cheese strain and ILSI18. ILSI9 belonged to the serogroup 1/2b, 3b, 7 and ILSI17 to the 1/2c, 3c serogroup. It is well-known that serogroups 1/2a, 3a and 1/2c, 3c are related with the food environment, and 1/2b, 3b, 7 and 4b, 4d, 4e are related with human cases and outbreaks. Besides, the 1/2a, 1/2b, and 4b serotypes are responsible for 98% of listeriosis cases .Listeria strains was evaluated using two cell lines: Caco-2 and SW480, with different cell structures and growth rates. The Caco-2 cell line is a model of differentiated cells that retain their morphology and most of the enterocytes function, resembling the intestinal epithelium [The invasion capacity of the ithelium . The SW4ithelium . Cell adithelium . ChangesL. monocytogenes is due to the capacity to adhere, invade, and multiply within cells, adhesion to the intestinal epithelium being the first step in the pathogenesis that precedes the invasion, and consequently, the inhibition of adhesion can prevent colonization and can limit opportunities for systemic infection [Listeria strains’ invasive potential must also be considered [It is known that the virulence of nfection . Therefonsidered .Listeria strains was evaluated at two times of bacterial growth (24 and 48 h). Monolayer cultures of the Caco-2 and SW480 cells were inoculated with each Listeria strain cultured for 24 and 48 h. The results of the invasion produced by the different strains were expressed in cfu/mL after the lysis of the host cells. The obtained results showed the invasive capacity of ILSI9, ILSI7, ILSI18, and S11 at 24 h and 48 h in both cell lines, while E10.652 and L. innocua did not invade in either cell line  and was higher in the SW480 than in the Caco-2 cell line  among the invasion capacity of each Listeria strain, which were more numerous in the case of the more differentiated cell model, the Caco-2 cell line. ILSI17 was the most invasive Listeria strain in the two cell lines.In general, the invasion of ell line . The datL. monocytogenes invasive capacity observed between both intestinal lines could be a consequence of the different characteristics of each type of intestinal cell line. The Caco-2 cell line exhibits characteristics of enterocytes with a polarized epithelium with a homogenous distribution of microvilli. However, SW480 cells correspond to nondifferentiated cells, and their surface protein expression differs from the Caco-2 cells with irregular microvilli and with high losses of the tight junction, indicative of highly disorganized epithelia [Listeria Adhesion Proteins (LAP) binding Hsp60 activates NF-κB signaling in Caco-2 cells, facilitating the myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of the tight junction proteins (claudin-1 and occludin) and adheren junction protein (E-cadherin) [Listeria surface [L. monocytogenes into the intestinal barrier [L. monocytogenes incubation using L. monocytogenes strains.The difference in the pithelia . Furtheradherin) . E-cadhe surface , and thi barrier . TherefoListeria strains (E10.652 and L. innocua) did not induce any changes in the expression of TJ proteins (claudin and occludin) or of the adheren (E-cadherin) compared with the noninfected cells (control) . This fact can explain the incapacity of these strains to invade the cells (both Caco-2 and SW480) (The noninvasive d SW480) . Howeverp < 0.05)  A. Beside < 0.05) . This fa < 0.05) . The mosell line ,48, the Listeria invasive strains after their incubation with WPP-N and WPP-C. (L. innocua and E10.652 were excluded from the study due to their lack of invasion capacity). The cell invasion capacity of each WPP-treated Listeria strain was expressed as the percentage of invasion with respect to the control group (cell lines inoculated with Listeria strains grown in the absence of WPP). L. innocua, a nonpathogenic Listeria spp. found in similar environments to L. monocytogenes [Phenolic compounds are efficient bacteriostatic agents, and different studies have considered their uses as anti-invasion agents of bacteria . Hence, ytogenes , was incListeria strains . Quantitatively, the values were higher, ranging between 3.7 and 1.8 times, in the case of the gene expressions of occludin  and ranged between 2.9 and two times, respectively , keeping the cell permeability and reducing the bacteria capacity to infect the cells. It is also remarkable the inhibitory effect of the incubation with WPP-C, raising the 100% of invasion inhibition of the Caco-2 cells for the strains ILSI18 and S11 (p < 0.05), the results were also notable since this strain corresponds to the serogroup 1/2b, one of the most related to human cases.With respect to the Caco-2 cell line, both WPP, in general, induced a high inhibition of the invasion capacity A,E, alth ILSI17;  showed toccludin C. In theectively D. No sigectively B. The la and S11 E. The in and S11 G,H. Leve and S11 F. FurtheListeria strains incubated with both WPP induced high invasiveness inhibition of the four strains ; then, some levels of gene expression were higher than those of their control (L. monocytogenes strains were treated with WPP-N respect the corresponding control. These results reflect the involvement of this tight junction protein and the protection of WPP against L. monocytogenes invasion of SW480 cells. In this case (SW480 cell line) results showed a significant effect of WPP incubation on the levels of claudin. Gene expression levels of this protein were higher than the control , noting a higher level of protection, mainly in the case of ILSI17 and ILSI18 incubated with WPP-C (L. monocytogenes invasion involved their interactions on the expressions of both TJ and AJ proteins.With respect to SW480 cell line, the invasion with  strains A,E, alth control B,C,F–H. th WPP-C B,F. AfteL. monocytogenes studied strains. This fact could be due to some of the components of WPP, especially some phenolic compounds. Some previous papers reported the anti-listeria activity of various natural products, among them flavonoids [L. monocytogenes invasion. Some flavonoids as chalcone inhibited SrtA activity [L. monocytogenes [Listeria strains.Altogether, the results revealed a significant effect of WPP on the virulence of four avonoids ,50 that activity . Then, aactivity , and othytogenes . Both flL. monocytogenes, probably responding to the degree of differentiation of each cell line and the level of expression and maturation of their cell-cell junctions. In this aspect, the TJ protein occludin is involved in the L. monocytogenes invasion of both Caco-2 and SW480 cell lines while claudin is only involved in the invasion of SW480. By the other hand, E-cadherin is also implicated in this invasion, mainly in the cases of the most invasive Listeria strains and more clearly in the case of Caco-2 cell line.The data suggest that there are different mechanisms involved in the invasion of Caco-2 and SW480 cells by L. monocytogenes strains studied. The capacity of WPP to reduce or inhibit the invasion of the Listeria strains is associated with their ability to modify the gene expression of adherens and tight junction proteins, which play a notable role in maintaining the integrity of the intestinal cell membrane. Therefore, the results of this study suggest an inhibition of intestinal cell invasion by L. monocytogenes treated with a wine pomace product.Wine pomace products, with rich contents of the bioactive compounds’ polyphenols, are effective natural products against the invasion of the four invasive


The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM. Glioblastoma multiforme (GBM) is the most common and most aggressive brain tumor in adults. It remains an incurable disease with a markedly diminished life expectancy despite multimodal therapy comprising surgical resection, chemotherapy, and radiation (RTX) ,2. RecenRTK-AXL is a transmembrane receptor with a C-terminal intracellular domain and an N-terminal extracellular domain. RTK-AXL belongs to the TAM family of kinases and has a specific ligand, which is the growth arrest-specific protein 6 (GAS6) ,14. BindIn GBM, RTK-AXL is widely expressed and activated in primary and recurrent diseases . The actFortunately, there is now a variety of AXL TKIs available whose effectiveness can be studied in detail in vitro and in vivo ,22,23. TTo evaluate the role of RTK-AXL in acquired and intrinsic therapy resistance and to provide a robust rationale for future study designs of clinical trials investigating TKIs in GBM, we aim to: (i) characterize the regulation of RTK-AXL in response to RTX and temozolomide (TMZ) treatment, (ii) investigate whether endogenous RTK-AXL expression is related to therapy response; and (iii) evaluate if the co-administration of the AXL-specific TKI R428 to standard GBM therapy, consisting of TMZ and RTX, augments treatment effects.p = not significant, First, we investigated whether the treatment with TMZ modifies the RKT-AXL expression in glioma cells. SF126 and U118MG cell lines were exposed to different TMZ concentrations, and the corresponding AXL mRNA levels were determined. Only minor changes (less than 1.5-fold) of AXL mRNA levels were observed in both cell lines following exposure to therapeutic doses of TMZ (10 µM) after 24, 48 and 72 h, and was most pronounced at 48 h (Ordinary one-way ANOVA p < 0.0001). This dose-dependent, opposed regulation of FL-AXL and CT-AXL could be interpreted as a post-translational receptor modification in response to TMZ treatment. To prove this observation, we aimed to measure the N-terminal RTK-AXL domain, also known as soluble AXL (sAXL), in the supernatant of the TMZ-exposed cells. In accordance with the increasing CT-AXL expression levels, we detected an increased activity of the tumor necrosis factor-alpha converting enzyme (TACE/ADAM17), a member of the disintegrin-metalloprotease (ADAM) family that is involved in proteolytic shedding of the RTK-AXL releasing sAXL following TMZ treatment [To simulate the clinical situation of prolonged TMZ exposure, we now exposed cell lines to increasing doses of TMZ on five consecutive days. Interestingly, FL-AXL decreased and CT-AXL increased in a dose-dependent manner E. Quantireatment . This inTo determine RTK-AXL regulation in response to RTX, the cell lines SF126 and U118MG were exposed to increasing dosages of RTX ). On mRNA level, no significant regulation of AXL mRNA was observed with the most pronounced however not significant changes 24 h after RTX A,B. CorrTo simulate a radio-resistant microenvironment, we cultivated the cells under hypoxic conditions resulting in an increased expression of hypoxia induced factor 1 , a known trigger of radio-resistance ,27,28. WTo assess RTK-AXL phosphorylation, downstream pathway activation and activation of alternative RTKs in response to TMZ and RTX, Western blot with activated Akt (pAkt) and STAT (pSTAT)  and Protein phospho-kinase array was applied. No significant changes of RTK-AXL down-stream pathway activation or phosphorylation of alternative RTKs were observed in response to the therapy .t-test SF126 vs. SF126-TR: ** p = 0.006; paired t-test U118MG vs. U118MG-TR: ** p = 0.025, p = ns, U118MG ** p = 0.0069). To study the efficacy of TKI R428 in the established TR-cells, we treated SF126-TR and U118-TR cells with R428 and TMZ and performed a CFA. Interestingly, the CFA revealed a significant reduction in the colony formation capacity in both SF126-TR and U118-TR cell lines with R428 given alone (Student’s t-test comparing SF126 with SF126 + R428: ** p = 0.0015 and U118MG with U188MG + R428: ** p = 0.0028). The combination of TMZ and R428 further increased this effect .Irrespective of RTK-AXL modifications in response to therapy and hypoxia, we aimed to determine whether simultaneous RTK-AXL inhibition with TKI R428 and TMZ/RTX could increase therapy response and if TKI R428 could aid in overcoming therapy resistance. Therefore, we established two TMZ-resistant cell lines (SF126-TR and U118MG-TR) in addition to the TZM naïve cell lines (SF126 and U118MG), to study the efficacy of the combination treatment. To create the TR cell line, SF126 and U118MG cells were treated with gradually increasing concentrations of TMZ (10–150 µM) for 10 to 12 weeks. Resistance to TMZ was based on a stable colony formation capacity in SF126-TR and U118-TR cells under TMZ treatment, compared with a significant decrease of viability in naïve SF126 and U118MG glioma cells (p < 0.0001). A similar trend was observed for the U118MG cell line; however, the results were not statistically significant (p = ns). These results indicate that the application of R428 alone in TR cells seems to be effective, whereas the combination of R428 with TZM or RTX leads to an improved therapeutic response even in TMZ resistant cell lines. This in turn supports the hypothesis that RTK-AXL inhibition might increase sensitivity towards TMZ and RTX and has the capacity to resolve mechanisms of therapy resistance.We set out to test the radio-sensitizing capacity of R428 by exposing SF126 and U118MG cell lines to RTX and R428 and quantified cell survival using CFA. The addition of R428 to RTX treatment resulted in a highly significant decrease in colony formation capacity in the SF126 cell line (p < 0.0001). With this data we show that elevated endogenous expression of the functional RTK-AXL is associated with less radiosensitivity, whereas sensitivity to TMZ remains unaffected.Lastly, we aimed to test the hypothesis whether endogenous RTK-AXL expression levels are related to treatment response. Therefore, the following modified cell lines were exposed to TMZ and RTX: SF126-DN: overexpressing the RTK-AXL lacking the intracellular domain (AXL-DN) and SF126-WT: overexpressing the functional RTK-AXL . SF126, With this study we showed that RTK-AXL undergoes post-translational degradation in response to repeated TZM exposure and under hypoxia, representative for a radio-resistant microenvironment, whereas these effects were not observed following a single fraction of RTX or single dose of TMZ. Further, we showed that high endogenous levels of FL-AXL reduced therapy response to RTX. However, therapy resistance may be overcome by combining AXL TKI R428 to standard treatment, even in TMZ-resistant GBM cell lines.Post-translational receptor modifications are considered a relevant mechanism of therapeutic resistance to TKIs ,29. AddiIn the clinical context, the question arises as to the optimal timing for the use of TKIs and appropriate combinational therapies. Our data shows that TKI R428 is acting as a therapy sensitizer to TMZ. The same has been observed in cell lines with acquired resistance to TMZ. When combining TKI R428 and RTX, we observed a significant improved therapy response of the SF126 cell line. These findings indicate that TKI R428 increases sensitivity towards TMZ and RTX and has the capacity to resolve mechanisms of therapy resistance. Since these results could not be reproduced for the combination of RTX and TKI R428 in the U118MG cell line, there might be a rationale for individual testing of patient-derived cell lines in future GBM studies to generate substantial rationales for applying AXL TKIs as single agents or to standard therapy consisting of fractionated stereotactic RTX and TMZ.Regarding patient stratification for the use of AXL TKIs in clinical trials, we addressed the role of endogenous RTK-AXL expression in GBM. Although we could not establish a correlation between high RTK-AXL expression and the TMZ treatment response, there seems to be a correlation between endogenous AXL expression and radiosensitivity, which corresponds well with previously published studies . We, aloIn summary, our data show that RTK-AXL appears to play a critical role in both intrinsic and acquired resistance to standard GBM therapy. Monitoring RTK-AXL expression as well as shedding products as indicators of therapy resistance and tumor progression could be a promising approach for individualized therapy management as it is currently being studied for malignant melanoma and hepatocellular carcinoma patients ,31.Human high-grade glioma cells U118MG were obtained from American Type Culture Collection (ATCC). Human high-grade glioma cells SF126 were obtained from the JCRB Cell Bank. Both were primary tumor cell cultures derived from surgical specimens of human GBM, WHO Grade IV. Cell authentication was carried out with LGC Standards Cell line Authentication service in June 2014 with 16 loci service of short tandem repeat profile. Further, we used a truncated form of human RTK-AXL lacking the intracellular RTK-bearing domain into SF126 cells (AXL-DN). SF126 naïve cell line served as control. Western blotting confirmed expression of the wild-type and truncated receptor in AXL-DN cell clones .2, 95% humidity in a tissue culture incubator. Growth medium encompassed Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (penicillin/streptomycin). Each experiment was conducted under the same conditions and repeated in three independent runs. Cell lines were cultivated to 80% confluence before starting an experiment. Therefore, cells were cultivated with 10–15 mL of cell culture medium in a T75 flask or with 5 mL of cell culture medium in a T25 flask. Cells were seeded at a density of 7 × 105 cells per mL medium. The cell culture medium was replaced 24 h after thawing the cells and was then replaced twice a week. Cell counts and cell viability were assessed with CASY® Cell Counter TT (OLS). For hypoxia experiments, cells were cultured under 1% O2 for 3, 6, 12, and 24 h accordingly to reports from the literature [Tumor cells were maintained as monolayer cultured in tumor growth medium at 37 °C, 5% COterature ,39.A known number of cells (250–1000) were plated into a 12-well plate and treated with either TMZ, RTX or combined treatments starting after one day in culture. After a growth phase of 12 to 14 days cells were fixed with 70% ethanol and stained with Giemsa. Surviving cell colonies were counted visually and microscopically. A colony was considered >50 cells. Survival fractions were graphed in relation to vehicle control.® Reverse Transcription Kit . Quantitative analysis was performed using QuantiTect® SYBR Green gene expression master mix  and QuantStudio™ 6 Flex Real-Time PCR System . Each reaction compound was composed of 5 µL SYBR Green master mix, 4 µL cDNA (10 ng/mL), 0.2 µL ROX lL, 0.2 µL of forward and reverse primers, respectively and 0.4 µL H2O yielding a total volume of 10 µL. Expression levels of the housekeeping gene 18S were used to normalize RTK-AXL expression levels and RQ values were graphed in relation to vehicle control. We consider a RQ significant when there is a minimum of two-fold change. The relative mRNA levels were calculated based on the cycling threshold (Ct) values. Each experiment was repeated in biological triplicates.Following isolation of total mRNA using the PureLink™ RNA Mini Kit  according to the manufacturer’s instructions, the concentration and purity of extracted mRNA was measured with Tecan Infinite Pro 200 microplate reader . Reverse transcription was carried out using QuantiTecthttps://www.ncbi.nlm.nih.gov/tools/primer-blast/ accessed on 20 January 2017) and according to Roux et al. [Primer design was done with the NCBI primer design tool . Santa Cruz: Gas6 antibody (C-20). Sigma Aldrich: mouse monoclonal β-actin antibody (clone 1A4). Immune complexes were visualized using a second HRP-conjugated anti-rabbit or anti-mouse antibody (Pierce Biotechnology). Pierce™ ECL Western Blotting Substrate  was used to develop blots and ImageJ Software was used for densitometry analysis. Reported values were first normalized to the loading control and then multiplied by a constant to reach the lowest whole integral. Each Western blot was carried out with a negative control consistent of sample diluent or beads incubated with antibody and sample diluent only. Western blot quantification was carried out using ImageJ software  according to the user guide IJ1.46r.After lysis in Radioimmunoprecipitation assay (RIPA) buffer, samples were frozen and stored in −80 °C. Protein concentrations of the samples were measured using the BCA Protein Assay as instructed by the provider . Experiments were run with 20 µgr protein. Western blots were repeated with protein samples from 2–3 independent experiments. Protein samples were boiled for 5 min with Laemmli sample buffer (Bio-Rad Laboratories) and loaded into pre-poured Tris-HCl-glycine SDS-PAGE gels . Gels run at 150 V for 1.5 h following transfer to a polyvinylidene difluoride membrane  at 400 mA constant current for 2 h. Blots were blocked with 5% BSA in 1xTBST, primary and secondary antibodies were dissolved in TBST. Following primary antibodies were used. R&D Systems: human phopho-AXL (Y779) mAb (Clone 713610), Cell Signaling: anti-AXL C89E7 rabbit mAb, abcam: anti-AXL (EPR21107) ab215205 mAb, phospho-MET (Tyr1234/1235) rabbit mAb (D26) XPTACE assay , was carried out as previously described according to manufactures instructions. Cells were lysed in lysis buffer as previously described and total protein content was measured using Pierce™ BCA Protein Assay Kit  . 50 ug pThe Proteome Profiler™ Array Human Phospho-RTK Array Kit  was used according to the manufacturer’s instructions. Protein was harvested in lysis buffer and 300 ug total protein was loaded onto specialized membranes coated with multiple human phospho-kinase antibodies. Detection was performed with GeneSnap program shooting for 20 min with 1 min running time between captures.TZM for use in cell culture was acquired commercially from Sigma Aldrich . To establish TMZ-resistant glioma cell lines, SF126 and U118MG cells were exposed to gradually increasing concentrations of TMZ (5–150 µM) in culture media for 10 to 12 weeks. TMZ was prepared freshly every day and added as 1% DMSO composite of total volume to fresh tumor growth medium. Vehicle control cells were fed daily with fresh tumor growth medium containing 1% DMSO. For two weeks cells were exposed to low-dose concentrations of 5 µM TMZ. Then, concentrations were increased prior to every other passage when cells had reached 85% confluence. Cells were passaged in half to ensure sufficient survival. Later during high-dose treatment (>80 µM) cells were given multiple passage cycles before rising in treatment concentration. The generated TMZ-resistant cell lines were named SF126-TR and U118MG-TR. Cells were assessed regularly for sufficient viability using colorimetric microtiter (MTT) assay and microscopy .® Cell Counter TT (OLS) in 12-well plates for CFA or 25 cell culture flasks for Western Blot and PCR experiments and received different RTX doses  after 24 h in culture. Applied RTX doses were determined according to other studies investigating RTX effects in glioma cells [Human high-grade glioma cells SF126 and U118 were plated at a defined density measured with the CASYma cells . Cell dama cells .RTX dose was applied at room temperature using the Xylon Maxi shot  at 200 KV, 10 mA at a rate of 1 Gy/1:08 min. Irradiated cells were further used for Western blot analysis or real time PCR as described above 24 or 48 h after RTX. Radiosensitivity was assessed using the CFA.R428  was purchased from Selleckchem  and was dissolved in dimethyl sulfoxide . Stock solutions of 1 mM were prepared according to the manufacturer’s instructions, and R428 was diluted in Dulbecco’s modified Eagle medium (DMEM) to final concentrations of 0.5 µM, 1 µM, and 2 µM. Human glioma cells were treated with a single dose of R428. t-Test and one-way ANOVA combined with Bonferroni’s multiple comparison test. Significance level was set at alpha = 0.05 . The level of significance was set at p < 0.05. Data were expressed as mean +/− standard deviation. Quantitative and qualitative statistical analyses were performed using GraphPad Prism 5 .Each group consisted of at least triplicates. Statistical analysis was performed using GraphPad Prism, Version 7.0c. Statistical significance was measured using Student’s


Arabidopsis thaliana. Analyses of multiple homozygous lines depleted of phytochromes in stomatal guard cells indicated elongated hypocotyls specifically in red and far-red growth conditions. Furthermore, rosette leaves of adult plants with guard cell-specific phytochrome deficiencies showed enhanced serration compared to the wild-type Col-0 parent. Thus, we demonstrate that guard cell-localized phytochromes impact the inhibition of hypocotyl elongation, as well as leaf margin morphology of adult rosette leaves in A. thaliana.Using a previously established transgenic approach to inactivate phytochrome chromophore synthesis in specific organs or tissues, we used a guard cell-specific promoter to induce phytochrome deficiencies in guard cells of  Light actively inhibits elongation in the hypocotyl, but promotes the growth and development of cotyledons, leaves, and roots. Distinct responses in specific tissues are maintained to some extent through the differential accumulation of photoreceptors such as phytochromes in distinct tissues and at different times during development . Specific phytochromes have been shown to impact stomatal development and function, with phyB promoting the stomatal index, or the percentage of stomata cells out of the total number of epidermal cells, and regulation of stomatal opening in red light . The photoreceptor phyA stimulates production of stomata in far-red light . Notably, phytochromes are localized in guard cells themselves , and thus stomatal responses can potentially be controlled by photoreceptors locally or through intercellular communication.Stomata are specialized epidermal cells that occur in the epidermis for all above-ground tissues and serve to facilitate gas exchange and water uptake central to photosynthesis. Stomata respond to environmental signals, including light  expression to explore phytochrome-dependent roles in guard cells . Expression of PHYB in guard cells demonstrated that phyB accumulation in these cells was sufficient to rescue a defect in photoregulation of the stomatal index in a phyB mutant . These plant lines with guard cell-specific PHYB expression also exhibited larger leaves, potentially indicating more extensive growth-related impacts of guard cell-specific phytochrome function. Prior investigations also correlated stomatal regulation with control of additional whole plant growth phenotypes, including correlating the regulation of stomatal opening with vegetative development in terms of the hypocotyl length of seedlings , and the rosette and flowering time at the adult stage .Tissue-specific expression of et al., 1997; Montgomery et al., 1999; Montgomery et al., 2001; Warnasooriya and Montgomery, 2009; Costigan et al., 2011). We used the promoter pGC1 (At1g22690), which drives high-level, guard-cell specific gene expression and has low expression activity in mesophyll cells , to generate plant lines with guard cell-localized, plastid-targeted BVR (pBVR) accumulation to induce guard cell-specific phytochrome depletion. We used these lines to examine the roles of guard cell-localized phytochromes in distinct aspects of photomorphogenesis.To investigate the function of phytochromes in guard cells, we used a previously verified transgenic approach for expressing a phytochrome chromophore-inactivating enzyme, biliverdin reductase  to regulate accumulation of photoactive phytochromes in planta . We observed significant enrichment of BVR expression in GCEEP compared to MC in several GC::pBVR lines, confirming guard-cell enriched BVR expression driven by the GC promoter. To confirm the reliability of our MC and GCEEP fractions, we tested RNA fractions for expression of an MC-enriched marker gene, i.e., CALMODULIN-BINDING PROTEIN  , and guard-cell marker genes, i.e., POTASSIUM (K+) CHANNEL IN ARABIDOPSIS THALIANA 1   and MYB DOMAIN PROTEIN 60  . As expected, we detected preferential expression of CBP in the MC RNA enrichment and of KAT1 and MYB60 in the GCEEP RNA enrichment . Additionally, we assessed protein extracts from GCEEP for BVR enzymatic activity using a previously described specific activity assay . Whereas no BVR activity was detected for GCEEP protein extracts from WT and a CAB3::pBVR line, we detected BVR activity in the range of 0.2 – 2.8 (I.U. mg−1) × 103 for GCEEP extracts from GC::pBVR lines.We successfully isolated four lines with guard-cell enriched expression of BVR, i.e., GC::pBVR1 to GC::pBVR4 as verified by qRT-PCR analyses with RNA extracted from mesophyll cell (MC) and guard cell enriched epidermal peel (GCEEP) fractions and compared to MC and GCEEP fractions from the CAB3::pBVR representative line with MC-localized et al., 2011). The disruption in light-dependent inhibition of hypocotyl elongation only under R and FR light for GC::pBVR lines is notable as these are wavelengths specifically associated with phytochrome-regulated functions. Although not previously associated specifically with light-dependent regulation, prior analyses of plants with altered ABA-regulated stomatal function showed a correlation between higher rates of stomatal closure and elongated hypocotyls .We assessed light-dependent growth of GC::pBVR seedlings compared to Col-0 WT seedlings under constant blue (Bc), red (Rc), far-red (FRc) and white (Wc) light. We observed significantly elongated GC::pBVR seedlings under both FRc and Rc illumination . GC::pBVR seedlings were not significantly different relative to WT seedlings in the dark or under either Bc or Wc light , which is distinct from phenotypes observed due to depletion of phytochromes in mesophyll cells as CAB3::pBVR lines were previously reported to have elongated hypocotyls in Wc, FRc, Rc, and Bc . In Arabidopsis, leaf heteroblasty can be indicated by leaf margin serration. As the major disruption at the adult leaf stage for GC::pBVR lines was an alteration in leaf serration or leaf margin morphology defects, these plants may exhibit alterations in heteroblasty correlated with guard-cell phytochrome deficiency. Relatedly, prior analyses with tissue-specific expression of phyB also indicated a correlation between a role for guard cell-localized phyB and leaf phenotypes. The leaves of phyB mutants are significantly different from WT with less leaf area and elongated petioles . When a phyB mutant was specifically complemented with a guard cell-localized PHYB construct, i.e., SPCH::PHYB, the leaf phenotype was largely complemented, in addition to the stomatal index phenotype . Together, these associations of the regulation of leaf phenotypes with stomatal phytochrome pools indicate key roles of GC-localized phytochromes in regulating whole leaf phenotypes.For adult plants, GC::pBVR plants exhibited altered leaf shape  under long-day conditions of W light at 100 µmol mPlasmid constructionBVR fragment for plastid-targeted BVR protein accumulation from the previously described CAB3::pBVR construct pBIB/CAB3-TPBVR  and guard cell-specific GC promoter . Specifically, the guard-cell specific, plastid-targeted BVR construct (GC::pBVR) was made by isolating the transit peptide-BVR (pBVR) fragment from pBIB/CAB3-TPBVR using XbaI and SacI and subcloning into XbaI and SacI digested pORE_O3_GC plasmid, which contains the GC1 promoter [pGC1 (At1g22690)], by ligation to create the pORE_O3_GC/TPBVR plasmid.We constructed plant transformation vector pORE_O3_GC/TPBVR using a TP-Plant transformation and growth conditionsArabidopsis thaliana (Columbia or Col-0 ecotype) plants were used as wild-type (WT) and for transformation of all constructs. We used both pBIB/CAB3-TPBVR and pORE_O3_GC/TPBVR constructs in Agrobacterium tumefaciens strain GV3101. We isolated multiple single insertion homozygous lines that were screened for BVR expression, including enrichment in the mesophyll or guard cells for the CAB3::pBVR4  and GC::pBVR lines, respectively.For hypocotyl growth analyses, surface sterilized seeds were plated and grown on MS medium containing 1% (w/v) sucrose and 0.7% (w/v) Phytoblend agar  at 22°C for 7 d under the indicated light conditions. Light sources used included blue , far-red , red , and white (W) as previously described . Light intensities were measured using a LI-COR LI-250A Light Meter with inline LI-COR quantum sensor for R, B and W light, and using a StellarNet EPP2000 spectroradiometer (Apogee Instruments) for FR light.Phenotyping-2 s-1 and 22°C/20°C day/night temperature cycles.We determined light-dependent hypocotyl elongation of seedlings under specified growth conditions by scanning images of seedlings and quantifying hypocotyl length using ImageJ software as previously described . For phenotypic observation of rosette leaf morphology, plants were grown in a long-day growth chamber with 16 h/8 h day/night light cycle at a white light intensity of ~100 µmol mGene expression analysiset al., 2007) or guard cell-enriched epidermal peels from adult plants grown in a short-day growth chamber with 8 h/16 h day/night light cycles at a white light intensity of 100 µmol m-2 s-1 and 22°C/20°C day/night temperature cycle, according to the protocol of . Quantitative RT-PCR (qRT-PCR) was performed essentially as described . Expression of BVR and tissue-specific marker genes was assessed, including mesophyll cell-enriched marker CBP (At4g33050)and guard cell marker genes KAT1 (At5g46240) and MYB60 (At1g08810). The control genes used for qRT-PCR analyses were PP2A (At1g13320) and ACT2 (At3g18780). Primers for each gene are listed in Table 1.RNA samples were extracted using the RNeasy® Plant Minikit  as previously described  from whole plants, mesophyll cells from ~15 g of 5 to 6 week-old plant leaves (Yoo Table 1. Primers for quantitative reverse transcriptase PCR (qRT-PCR)


Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) ( •Searches >16,000 stem cell lines in Europe, Japan, and US major databases•Data formats standardized by minimum items in MIACARM guidelines•Searches specific stem cell lines according to disease, donor, tissue, etc.•User-friendly website accesses >6,000 diseased stem cell lines from 36 countries This new and largest stem cell data portal can retrieve >16,000 cell lines from four major stem cell data resources in Europe, Japan, and the United States, is automatically updated to provide the latest cell line information, and, through its integrative search engine, can collect cell line information from specific donors with rare diseases worldwide. Since the first report of human induced pluripotent stem cells (iPSCs) , there hhttp://icscb.stemcellinformatics.org/ or, as a slim version by removing cell line redundancy as much as possible, through the SHOGoiN  homepage at http://shogoin.stemcellinformatics.org/.In this paper, as our next step toward the unification and utilization of the stem cell line data in the world, we report our new database portal, Integrated Collection of Stem Cell Bank data (ICSCB), which was designed using MIACARM guideline items and formats to serve as an entrance “port” to individual data repositories. The main objectives of ICSCB are (1) to establish an integrated stem cell database portal that can cover the majority of stem cell resources in the world and (2) to offer users minimum but efficient access to information on stem cell lines based on MIACARM guidelines. Currently, ICSCB provides data on more than 15,000 stem cell lines registered in four major stem cell line databases: hPSCreg , SKIP K, RIKEN BICSCB was designed for researchers searching for available cell lines to conduct various studies, such as regenerative medicine and disease analysis. Covering as many diverse cell lines as possible was the first priority when deciding which resources to include in ICSCB. Sharing cell line information between different stem cell banks and registries has been problematic due to different cell naming methods, different policies on cell assessment in different registries, unclear data sources, and so on. ICSCB is a collection of cell lines from four major and reliable cell line data resources based in Europe, Japan, and the United States. ICSCB updating is regularly performed for new SKIP and eagle-i stem cell line data as well as automatically performed for hPSCreg and RIKEN BRC data in a synchronized manner. Users can retrieve all related stem cell line information by using a free text search. Detailed information on a specific cell line can be accessed by clicking on the stem cell ID, which is linked to the information page in the original resources . The resICSCB covers more data than any other stem cell line repository available. The integration of all major data resources allows us to check the current state of stem cell research in the world . Althoughttps://stemcellinformatics.org/). SHOGoiN is a repository for accumulating and integrating diverse human cell information to support a wide range of research using cell-related data. The database consists of several modules that store cell lineage maps, transcriptomes, methylomes, cell conversions, cell type markers, and cell images with morphology data curated from public as well as contracted resources based on sophisticated cell taxonomy. Collaboration between ICSCB and SHOGoiN makes it possible for users to directly use free text searches for stem cell line data on the SHOGoiN homepage. The ICSCB easy search module in SHOGoiN supports a simplified ICSCB search with keywords, and the advanced search is designed to redirect users to the ICSCB homepage with full functions. Results from the SHOGoiN homepage have the same structure as in the ICSCB homepage.ICSCB also has a quick and easy search module on the SHOGoiN homepage  SKIP , (2) hPSCreg , (3) RIKEN BRC , and (4) eagle-i  . These data resources were selected because they had the highest number of registered cell lines and a large diversity, which would provide a good regional balance of cell sources to reduce redundancies in cell line entries. RIKEN BRC basically collected cell lines from Japanese institutions, SKIP contained data mostly from other Japanese and Asian institutions, hPSCreg collected data mainly from European institutions, and eagle-i collected data mostly from the United States. Details of the data sources are listed in Since our previous research on the listed stem cell banks 4 years ago , the numTo provide fast and easy access to the latest and accurate cell line information, we built an automatic updating system that adds newly released cell lines to ICSCB as soon as they become available in any of the four data resources. Data from eagle-i and SKIP are directly collected and stored in the MySQL database with the terms required for the MIACARM modules. Data from hPSCreg and RIKEN BRC are collected on the fly per request using a web application programming interface (API) provided by the respective sites. RIKEN BRC also uses SPARQL language for data retrieval requests .SOX2) such that the results page will display relevant entries by showing the indicated keyword in the extra field below (To simplify the search process, ICSCB provides an easy-to-use and mobile-friendly web application. The goal of the application is to help users find the desired stem cell lines as quickly as possible. The interface of the search engine is designed with the 16 MIACARM terms , except ld below B. Furthein vitro, morphology data, marker gene expression/surface antigen expression data, karyotyping assay results, copy number variation, residual exogene detection results, genome profiling, transcriptome profiling, and epigenome profiling data are accessible from ICSCB.ICSCB also provides a quality control panel based on MIACARM, thereby supporting customized searches according to quality control results. At present, assays for teratoma formation, differentiation ability https://github.com/YingChen-bio/ICSCB.The original data and R source code for creating figures and supplementary figures and tables are available at Y.C. and Y.P. drafted the manuscript. S.M., T.M., H.O., J.D., S.S., A.K., H.M., Y.N., M.S., J.S., and W.F. provided and facilitated the stem cell data. W.F. and K.S. conceptualized the research. A.K., G.S., and W.F. led the project.H.O. is a founding scientist of SanBio Co., Ltd., and K Pharma, Inc.