Mitochondria are dynamic organelles that undergo rounds of fission and fusion and exhibit a wide range of morphologies that contribute to the regulation of different signaling pathways and various cellular functions. It is important to understand the differences between mitochondrial structure in health and disease so that therapies can be developed to maintain the homeostatic balance of mitochondrial dynamics. Mitochondrial disorders are multisystemic and characterized by complex and variable clinical pathologies. The dynamics of mitochondria in mitochondrial disorders is thus worthy of investigation. Therefore, in this study, we performed a comprehensive analysis of mitochondrial dynamics in ten patient-derived fibroblasts containing different mutations and deletions associated with various mitochondrial disorders. Our results suggest that the most predominant morphological signature for mitochondria in the diseased state is fragmentation, with eight out of the ten cell lines exhibiting characteristics consistent with fragmented mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in cell lines with a wide array of developmental and mitochondrial disorders. A more thorough analysis of the correlations between mitochondrial dynamics, mitochondrial genome perturbations, and bioenergetic dysfunction will aid in identifying unique morphological signatures of various mitochondrial disorders in the future. Mitochondria are highly dynamic organelles responsible for a host of cellular functions that include regulation of oxidative metabolism, control of apoptosis, and generation of signaling metabolites, with the production of adenosine triphosphate (ATP) being the most prominent of functions . In liveMuch is still unknown about the role of mitochondrial dynamics in human diseases. However, it has been suggested that in a diseased state, mitochondrial fission and fusion may be modulated as a compensatory mechanism to maintain the pool of healthy mitochondria within cells ,11,12,132Xros (MTR), a dye that localizes in actively respiring mitochondria, were analyzed to quantify mitochondrial morphology in health and disease. We analyzed one control healthy fibroblast and 10 diseased fibroblast cell lines carrying various mitochondrial DNA (mtDNA) mutations and deletions. The diseased cell lines carry some of the most prevalent mutations and deletions involved in mitochondrial disorders, such as: Leigh syndrome (LS), Mitochondrial Encephalopathy, Lactic acidosis, Stroke-like episodes (MELAS), Kearns–Sayre Syndrome (KSS), and Pearson Syndrome (PS) [In this study, we used the mitochondrial network morphology analysis (MiNA) tool , an Imagome (PS) . We analome (PS) . Our resThe patient fibroblast cell lines were obtained from ten different patients with varying developmental and mitochondrial disorders. Three of the patient cell lines carry point mutations in the MTATP6 subunits  of complex V (ATP synthase). Two of the patient cell lines have mtDNA mutations affecting the MTND3 (SBG4: m.10158T>C) and MTND5 SBG5: m.12706T>C) subunits of complex I of the electron transport chain. These mutations affecting complex I and V have been implicated in Leigh syndrome (LS) ,21,22,2306T>C subUnderstanding mitochondrial morphology is important in determining the health status of the cell, with descriptors such as tubular, fragmented, and hyperfused  often usMitochondrial morphology in fibroblast cell lines was determined with the aid of live-cell fluorescent microscopy. To streamline our procedures, we first conducted an analysis with healthy BJ fibroblasts which are expected to exhibit various morphologies balanced between network and fragmented mitochondria a. In addHaving confirmed that the MiNA toolset would detect changes in mitochondrial dynamics in healthy control BJ fibroblasts, we proceeded to analyze differences between healthy control and diseased cell lines carrying mutations in subunits of the Complex V-ATP synthase. We analyzed three patient fibroblast cell lines  carrying mutations that are most prevalent in patients with LS ,38. We ap < 0.0001) mean branch length and mean network size compared to healthy control BJ fibroblasts. FCCP treatment reversed this phenotype, with mean branch length and mean network size within the SBG5 group decreasing by 21% and 14%, respectively, after FCCP treatment. This is consistent with mitochondria fragmentation by FCCP, breaking down the larger networks of mitochondria in this cell line. Unlike what we observed with the SBG1, SBG2, and SBG3 fibroblasts (with mutations impacting Complex V), SBG4 and SBG5 fibroblasts (with mutations impacting Complex I) appear to exhibit different morphologies. These differences could be attributed to the location of the subunits affected by each mutation. The mutation in SBG5 fibroblasts may affect the function of the mitochondria more than that of SBG4 fibroblasts, requiring remodeling of the mitochondria to maintain optimal function of the respiratory chain. We next wanted to determine how mutations in other complexes that impact mitochondrial function affect mitochondrial morphology. We assessed this using two patient fibroblast cell lines with mutations affecting subunits of complex I of the electron transport chain. The SBG4 fibroblast cell line has the m.10158T>C point mutation, while the SBG5 fibroblast has the m.12706T>C point mutation affecting MTND3 and MTND5 subunits, respectively. On average both SBG4 and SBG5 fibroblasts trended towards having fewer actively respiring mitochondria than the healthy control BJ fibroblast, as evidenced by the relatively lower total respiring mitochondria . This dip < 0.001) number of individual, networked, and total respiring mitochondria and mean network size relative to the control cell lines. Similar to the healthy control, FCCP treatment resulted in fragmentation in both SBG6 and SBG7 cell lines. Interestingly, we see a significant increase of 51% in mean network size in the SBG7 cell line after FCCP treatment. It is not fully clear why the network size increased in SBG7 cell lines after FCCP treatment; perhaps this is because of the slightly longer branch length (5%) of SBG7 relative to the healthy control. We next examined mitochondria morphology in patient cell lines with tRNA mutations. One of the cell lines, SBG6, carries an m.3243A>G point mutation. This mutation is present in 80% of MELAS patients ,40. The Finally, we examined the effects of mtDNA deletions on mitochondrial morphology. We used three different patient fibroblast cell lines with varying degrees of mtDNA deletions . Although not statistically significant, we observe a trend towards fission based on an increased presence of fragmented mitochondria in SBG8 and SBG10 fibroblasts, but not in SBG9 fibroblasts . The relp < 0.0001) decrease in MMP by 83% in SBG1-FB (m.8993T>G), 74.13% in SBG2-FB (m.8993T>G), 99.23% in SBG3-FB (m.9185T>C), 80.47% in SBG4-FB (m.10158T>C), and 84.92% in SBG5-FB (m.12706T>C) compared to healthy control BJ-FB cells (p < 0.001) by 53.91% in SBG6-FB (m.3243A>G) and 90.61% in SBG7-FB (m.14739G>A) relative to healthy control BJ-FB cells (p < 0.0001) decrease in MMP by 94.86% in SBG8-FB (10676∆14868), 94.50% in SBG9-FB (7342∆9916), and 96.12% in SBG10-FB (10167∆15568) compared to healthy control BJ-FB cells (p-trifluoromethoxyphenylhydrazone (FCCP) decreased the MMP significantly for most of the diseased cell lines relative to control BJ-FB (p < 0.05), SBG2 was not significantly different from the healthy BJ-FB control  in the diseased and healthy control fibroblast cell lines. We hypothesized that MMP would be perturbed in the diseased cell lines by different mechanisms. MMP generated by proton pumps at complex I, III, and IV is an essential component in the process of energy storage ,42. FlowFB cells a,c. In tFB cells a,c. As eFB cells a,c. Alth control b,d.Mitochondria are dynamic organelles that perform a plethora of cellular processes, including ATP production, regulation of ions, apoptosis, and cellular signaling, processes that depend on tight regulation of mitochondrial shapes and ultrastructure ,45. RecePrevious studies have focused on mitochondrial dynamics in various diseases ,47,48,49MTND5 mutation in this cell line favoring mitochondrial fusion. Although the SBG7 fibroblast cell line also displays an elevated mean branch length, there is no subsequent increase in the mean network size. Therefore, we surmise that the SBG5 fibroblast cell lines exhibit an extreme form of fusion compared to the SBG7 fibroblast cell line, with SBG5 fibroblast cell lines exhibiting hyperfused morphology.After confirming that MiNA is sensitive and can be used to characterize mitochondrial morphology, we used it to quantify the morphology of the diseased fibroblast cell lines containing specific mutations or deletions. Across all ten cell lines with different mitochondrial disorders, about half have the same mitochondrial morphology profile when compared to the healthy BJ control fibroblast . In thesWhen the cell lines were treated with FCCP, we noted that some of the diseased cell lines exhibited the same response to FCCP as the healthy control cell lines, while others did not. We also observed similar trends with basal (oxygen consumption rate) and maximal respiration values using a Seahorse Flux Analyzer. The basal respiration values were significantly reduced for 8 out of the 10 fibroblasts, while only SBG1 exhibited a slight increase and SBG10 exhibited no change when compared with healthy control BJ-FB. Upon addition of FCCP, the maximal respiration values were significantly increased for 5 out of the 10 fibroblasts  and reduced for SBG2, SBG5, and SBG9, while SBG6 and SBG10 exhibited no change when compared with healthy control BJ-FB. Based on ongoing studies in our laboratory, results indicate that variable respiration rates are dependent on specific mtDNA mutations and deletions in the different disease fibroblasts. . A common view of mitochondrial disorders harboring mtDNA mutations or deletions is that there is a direct correlation between disease severity and outcomes to the degree of heteroplasmy. Contrary to this view, our preliminary results based on comprehensive next generation sequencing analysis indicate varying heteroplasmy levels (between 25 and 98%) in the different fibroblasts. These results are consistent with two recent large cohort studies on the phenotypic spectrum of mitochondrial disorders not correlating with the degree of mutation heteroplasmy ,53. Two Our findings support previous results where fragmented mitochondria are the predominant morphology in the diseased state ,54,55,56® CRL-2522™) fibroblasts were obtained from American Type Culture Collection  and the ten patient-derived diseased fibroblasts  were obtained from the Medical University of Salzburg, Austria. These cells were maintained in a fibroblast expansion medium that consisted of minimal essential medium (MEM) , 10% fetal bovine serum (FBS) , and 2 mM l-glutamine (Thermo Fisher Scientific). Fibroblasts were enzymatically passaged in 0.05% Trypsin-EDTA (Thermo Fisher Scientific). All cell cultures were maintained without the use of antibiotics, handled in Biosafety Type II sterile hoods regularly cleaned with UV irradiation and 70% ethanol, and grown in 37 °C incubators at 5% CO2 and 95% humidity. The culture medium was replenished every two days until cells became 80% confluent. Prior to use in experimentation, cells were dissociated using 0.05% trypsin-EDTA (Thermo Fisher Scientific) and 20,000 cells were seeded into 35mm dishes for fluorescence labeling and image analysis.Cultures of healthy control BJ  containing 150 nM Mitotracker Red CM-H2Xros  for 30 min. In the FCCP treatment group, fibroblast cells were incubated with 0.7 µM FCCP for 30 min before the addition of the Mitotracker Red CM-H2Xros. At the end of the incubation period, the cells were washed 3 times with prewarmed Dulbecco’s phosphate-buffered saline (dPBS). The nucleus was stained by further incubating cells with basal medium containing Nucblue Hoechst (Invitrogen) for 15 min. Following this incubation, cells were washed several times with prewarmed dPBS to remove excess dye. At the end of the wash, MEM NEAA basal medium was added to each dish prior to image acquisition.2 incubator until image acquisition. All images of live cells were taken on the same day as the labeling of mitochondria. All live-cell images were exported as TIFF files for further analysis. Ten to fourteen images were acquired per dish and three dishes were stained per trial. Three independent trials were performed for each of the fibroblast cell lines used in the study.Fluorescence images of live cells were acquired using an EVOS FL inverted light/epifluorescence microscope with 40×/0.65 objective and a Sony ICX445 monochrome CCD digital camera. Red fluorescence from Mitotracker Red CM-H2Xros was measured using a 530 nm excitation and a 593 nm emission filter set. Blue fluorescence from Nucblue Höchst was measured using a 360 nm excitation and a 447 nm emission filter set. All live cells were imaged on 35 mm dishes containing phenol-red-free basal medium. Image acquisition was performed one dish at a time with a maximum time of 30 min between dishes. All dishes were stored in a humidified 37 °C, 5% COThe images generated for the human fibroblast cell lines were preprocessed on ImageJ  following steps previously outlined . After pg for 5 min. Cells were then resuspended in basal medium, after which the desired amount of tetramethylrhodamine, ethyl ester  was added . For FCCP and Oligomycin treatment groups, 20 µM and 5 µM of FCCP and Oligomycin were added, respectively for 10 min prior to treatment with TMRE. Cells were incubated with TMRE in a 37 °C, 5% CO2 incubator for 25 min. At the end of the incubation period, cells were centrifuged at 400× g for 5 min. To wash off the excess dye, cells were resuspended in 1× dPBS solution and centrifuged for another 5 min. At the end of the wash, the cells were resuspended in phenol-red free basal medium and transferred to Accuri C6 plus flow cytometer  for data acquisition. A total of 10,000 events were recorded for each cell line. After data acquisition, the data were exported as FCS files and analyzed using FlowJo_v10.6.2 software . To gate for the TMRE-positive population, cells that were not stained with TMRE were used to gate for the TMRE-negative cell populations. Mean fluorescent intensity (MFI) values, a measure of the geometric mean of TMRE-positive cells, were obtained for statistical analysis.In this study, all fibroblasts were maintained in culture following established protocols until the desired passage (p8) was reached. Prior to use in flow cytometry analysis, cells were cultured until 70–80% confluence was reached. On the day of the experiment, cells were enzymatically detached using 0.05% Trypsin-EDTA (Thermo Fisher Scientific) and centrifuged at 400× p < 0.05 was considered significant.To ensure scientific rigor and reproducibility, an ANOVA design accounting for 3 biological and 10–14 images from healthy control (BJ-fibroblast) and diseased  was used to identify any differences with respect to control BJ fibroblasts. Post hoc Tukey HSD test was used to identify differences among specific groups. Data are presented as the mean ± standard deviation (SD) and were analyzed using the GraphPad Prism 8 program . A In summary, we report for the first time the effects of specific mtDNA perturbations (mutations and deletions) on mitochondrial dynamics in the context of specific mitochondrial disorders like LS, KSS, MELAS, and PS. Our results demonstrate that specific point mutations and deletions affecting the OxPhos complexes and translation machinery could upregulate fission dynamics, creating a dysfunction that favors hyperfragmentation of the mitochondria. Although certain mutations and cellular demand cause hyperfused mitochondria, our observations indicate that this is not the predominant morphological adaptation in fibroblast cells with mitochondrial disorders. This study improves our understanding of mitochondrial dynamics in patient fibroblasts with multiple developmental defects and mitochondrial disorders and could lead to a better correlation of mitochondrial dynamics, genetics, and bioenergetics; the specific role of mitochondria in patient diagnosis and prognosis; and focused therapies to treat various mitochondrial disorders.


Repetitive, low-dose  drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/−), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers. PCa in individuals with precancerous, indolent, or slow growing malignant disease can evolve over many years. For those with advancing localized disease the standard treatment includes radical prostatectomy and radiation therapy with or without hormonal manipulation  is limited (<2.0%), neuroendocrine differentiation (NED) following ADT to aggressive treatment-resistant NEPC forms of variant PCa have been estimated to be more than 25% (Prostate cancer (PCa) is the second leading cause of non-cutaneous cancer related deaths in men in the United States (pulation . Howeverpulation . The 202pulation . The useed again . Furthered again . While tthan 25% . Standarthan 25% . Cabazitthan 25%  and incrthan 25% .seer.cancer.gov). Immunotherapy, has been shown to increase OS in patients with mCRPC by 4–5 months (vs Nonblack men (African America (AA) men in general are more likely to progress or be diagnosed with mCRPC, fail to respond to conventional therapies, and show reduced survival compared with other ethnicities  and data from The Cancer Genome Atlas (TCGA). The goal was to determine if the observed gene signatures following METRO-TOPO were present in PCa patients/TCGA, and investigate their influence on PCa patient survival, as well as to identify any ethnic differences. The number and diversity of available PCa cell lines from AA patients is the major limitation of developing treatment specific (METRO) molecular signatures. Currently, there is only one PCa AA cell line (MDa-PCa-2b) available commercially. To overcome this limitation, we confirmed the expression of our treatment specific signatures (METRO) in three additional AA PCa cell lines  from our collaborator’s lab . Overall, our results provide insights into the gene pathway networks governing METRO-TOPO efficacy, as well as identifying pharmacogenomic signatures that may aid in personalizing treatment options for patients with PCa.β-actin was purchased from Sigma-Aldrich; A5316. All glass and plasticware were purchased from VWR .Fetal bovine serum (FBS) and trypsin (0.25% w/v) were purchased from Hyclone . Topotecan (TOPO) was purchased from 21st Century Global E-Commerce Network . Dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), TRIS buffer, acetic acid, ECL western blotting substrate for chemiluminescence were obtained from Bio-Rad . 3--2,5-diphenyltetrazolium bromide (MTT) and RNase A were purchased from Sigma-Aldrich Inc . Mouse anti-human antibodies  and Cell Signaling Technology . Goat anti-Rabbit IgG (H = L) Secondary antibody, HRP, was purchased from Cell Signaling Technology; 345,897 . 2 and 5% CO2 in a humidified cell culture chamber . The RC77, RC165, RC43 cell lines were obtained from Dr. Yates; cell lines were developed from patients that self-identified as African American   and LNC were obtAmerican . Cells wRNA-seq data from prostate cancer patient samples was obtained from Dr. Clayton Yates. Details on patient samples have been analyzed and published previously . Briefly3 cells in 96 well plates with appropriate media with 10% (v/v) FBS supplemented (PCa cell lines were seeded at 4 × 10lemented . Plates lemented . All expn = 5 wells/group), percent change relative to untreated controls was calculated at each drug concentration. Half-maximal inhibitory drug concentration (IC50) values were estimated by nonlinear regression using a sigmoidal dose-response equation (variable slope - three parameters).The effect of TOPO treatment on prostate cell growth and cytotoxicity was conducted by measuring total protein (SRB), mitochondrial enzyme activity (MTT), and examination of cellular morphology. SRB and MTT staining were performed at 48 and 72 h post initial TOPO exposure as described previously . Absorba® 3/7 Assay - Promega Corporation) according to the manufacturer’s protocol. Briefly, a total of 2×104 cells/well were seeded into 96-well plates (triplicates) and treated at the estimated 72 h MTT IC50 (METRO and CONV). Caspase-Glo 3/7 reagent was added and incubated for 2 h, and luminescence was measured using a Synergy 3 plate reader. We normalized the level of apoptosis in each treatment group with the control group (no drug treatment or baseline caspase 3/7 assay luminescence) for each cell line.Caspase-3/7 activation was measured using the Caspase-Glo 3/7 assay (Caspase-Glo3) in 6-well plates and exposed to TOPO at the estimated IC50 for 72 h (MTT) for each treatment protocol (CONV and METRO). Three areas with approximately equal cell densities were identified in each well, and images were captured with a Nikon AZ100 stereo-fluorescent microscope mounted with a Nikon Digital Sight DS-QiMc camera utilizing NIS-Elements image analysis software . Images were recorded in the modes of bright field and phase contrast at ×20 and ×40 magnifications. Further, images were analyzed using ImageJ .Cell morphology was assessed using phase-contrast microscopy. PC-3 and LNCaP cells were seeded  was added to the cells in the control wells, and micrographs obtained at 0, 48, and 72 h. The effect of CONV and METRO dosing of TOPO, at their respective IC50, were applied to the cells in the respective wells, and micrographs of the wound areas were obtained at 0, 48, and 72 h using an EVOS FL digital cell imaging system . Images were recorded in the modes of bright field and phase contrast at ×20 and ×40 magnifications. The initial wound area (at 0 h) and the “gap area” were measured at 48 and 72 h with ImageJ software.Scratch assays were performed by creating a “scratch” in a cell monolayer followed by capturing the images at the beginning and regular interval  during cell migration to close the scratch and comparing the images to quantify the migration rate of the cells. Briefly, PC-3 cells were plated in 6-well plates at 1 × 10® 2.0 Fluorometer , and Agilent 2,100 Bioanalyzer .Total RNA was isolated from cultured cells and patient samples using standard RNA extraction kits (RNeasy Kits–QIAGEN). RNA concentration and integrity were estimated by a NanoDrop 2000 UV-Vis spectrophotometer , Qubit50 of 48 and 72 h TOPO treatment (CONV vs METRO) using the RT2 Profiler™ PCR Array Human Cancer Pathway Finder (PAHS-033A). The qRT-PCR analysis was performed using a LightCycler® 480 II Real-Time PCR System . Three housekeeping genes , reverse transcription control (RTC - Control RNA + primer) and positive PCR control (PPC) were used for assessing PCR efficacy. Gene expression was calculated by ΔΔCt method. Gene expression in treatment groups were normalized to corresponding controls (no drug treatment) and differentially expressed genes (DEGs) were identified.Gene expression in PC-3 and LNCaP cells were assessed at the calculated IC® DNA Library Prep MasterMix Set for Illumina (Details on NGS-based RNA sequencing and data availability have been published earlier . BrieflyIllumina . LibraryIPA (QIAGEN) was used to identify the most significantly affected 1) canonical pathways predicted to be activated or inhibited; 2) upstream regulator molecules like transcription factors, may be causing the observed gene expression changes; 3) downstream effects and biological processes that are increased or decreased; 4) predicted causal networks from our differential mRNA expression analysis .Processed, high-quality RNAseq data PCa patients were obtained from Dr. Clayton Yates’ lab (Tuskegee University) . Detailscancergenome.nih.gov). 717 PCa patients (500 prostate adenocarcinoma) and  had mRNA expression data available. R-based pipeline was created, and UALCAN, an interactive web-portal was used to perform data download, processing, and in-depth analysis of gene expression data files from TCGA’s Genomic Data Commons (GDCs) server and retrieve transcriptome data on target candidate pathway genes from the prostate expression data matrix containing >60,000 transcripts (The mRNA expression of PCa patients was extracted from The Cancer Genome Atlas (TCGA) Data Portal (nscripts .50 for each treatment protocol (CONV and METRO). Post-treatment cells were lysed in cell lysis buffer (Thermo Scientific™ RIPA Buffer). Equal amount of protein loaded onto 4–15% Criterion™ TGX Stain-Free™ Precast Gels. Proteins will be separated under reducing conditions and then transferred to a PVDF membrane using a Trans-Blot Turbo Mini transfer pack from Bio-Rad . Nonspecific binding was limited by incubating the membrane in blocking buffer (2.5% (w/v) casein, pH 7.6, 150 mM NaCl, 10 mM TRIS-HCl, and 0.02% sodium azide). Membranes were incubated with primary antibodies for target protein (1:1000) for overnight and then with the appropriate secondary antibody  at room temperature. Immunoreactivity was detected by Pierce ECL Western Blotting substrate . Images were captured and quantify by Gel Doc™ EZ Gel Documentation System and ImageLab™ Software . Densitometry analysis was performed using standard image analysis software ImageJ.PCa cells were seeded and exposed to TOPO at the estimated 72 h MTT ICp values and further provided False discovery rate (FDR) based on the p-value using Benjamini–Hochberg procedure. All tests were a two-sided and p < 0.05 was considered statistically significant. For analysis of RNAseq data obtained from EA/AA patient cohort, FDR adjusted p-values were used to determine significant associations.All statistical analyses were performed using R statistical programming package (version 3.6.2) and GraphPad Prism (version 7.0). Gene expression profiling (GEP) data were analyzed using Partek Genomics Suite v7.0 software to identify DEGs. Owing to the small sample size, we use limma, an empirical Bayesian method, to detect the DEGs and obtain p ≤ 0.05) time-dependent decreases in the IC50 value, which reflects increased sensitivity after 48 and 72 h compared to CONV treatment  after 72 h. Similar results were observed after 48 and 72 h of treatment and by the SRB assay as shown by a The effect of TOPO administration as CONV and METRO on prostate cancer cells were assessed by measurement of MTT  and SRB reatment . Comparivs CONV) effects on apoptosis were determined by measuring caspase-3/7 activity following treatment. METRO dosing of TOPO induced apoptosis in every cell line  tested as compared to cells with CONV treatment  as well as apoptosis assays. PC-3 and LNCaP cells exposed to METRO dosing regimens showed greater decreases in cellular density (3.6 and 6.2-fold) compared with CONV dosing.Cell morphology studies, as shown in 50 at 48 and 72 h to examine the effect of different dose regimen on cell migration as measured using the scratch/wound healing assay. Results revealed that METRO-TOPO treatment had a greater effect (∼2-fold) in reducing cell migration in prostate cancer cell lines (p < 0.05) (PC-3 cells were exposed to TOPO CONV and METRO IC < 0.05) .vs CONV treatments in androgen-independent vs androgen-sensitive cell lines. GEP data from each treatment group and treatment time point was normalized to baseline gene expression for the corresponding cell line and heatmaps were generated  to identify and compare changes in gene expression following TOPO METRO enerated . Top difaP at 48  and 72 haP at 48 .vs METRO) were CDKN1A, FOS, TNF and SERPINB5 . Among these, 17 were unique to PC-3, while 10 were unique to LNCaP. Seven 7) of these DEGs were common to both cell lines signifying a METRO-TOPO-specific GEP signature for PCa (n = 11) candidate biomarkers  and PTEN (p = 1.53E-13) signaling pathways as targets for METRO TOPO treatment (p = 7.55E-17). IL-8 and p53 signaling pathways were also predicted as targets for METRO-TOPO uniquely in LNCaP cells  . Additioreatment . FurtheraP cells .p ≤ 0.05) (vs EA PCa patient cohorts (p ≤ 0.01)  . THBS1—a ≤ 0.01) .in silico analysis of the top DEGs using patient GEP data available in the TCGA database. We explored the following groups: 1) All men with PCa; 2) AA vs EA men with PCa and 3) high vs low survivors. Differential gene expression between high vs low survival PCa subgroups is depicted by the heatmap in p < 0.05) with TCGA PCa patient’s survival. Additionally, we used TCGA databases to derive K-means clustering to show the top genes expression profile that are beneficially associated with prostate cancer patient survival. Kaplan-Meier plots were derived following K-means clustering (class = 2), showing stratification in survival among TCGA PCa patient clusters based on the top DEGs for METRO-TOPO treatment (p < 0.05) with high patient survival  between AA vs EA PCa patients. However, this analysis was based on one AA PCa cell line and limited number of AA patients within the TGCA. To overcome this limitation, we further compared our signature with gene expression profiles from patient derived PCa cell lines from AA  and EA . In untreated cells, we observed increased expression of SERPINB5, CDKN1A, MMP9, ITGA3, and S100A4 in AA cell lines compared to EA cell lines (p ≤ 0.05)  .in silico analyses, we confirmed protein expression of the top METRO-METRO DEGs via immunoblotting in PCa cell lines. We observed 1) SERPINEB5 and SERPINE1 were downregulated (RNA-seq) in METRO-TOPO; 2) FOS. iii) MMP1 and MMP9—identified as the most important genes by IPA; and 4) ANGPT2 and VEGF. Western blotting results show that protein expression of these top DEGs were concurrently higher in castration-resistant PC-3, and PC-3M compared to hormone-sensitive LNCaP, and 22RV1 . Further, SERPINE1 (-29.6), FOS (-2.25), ANG-2 (-3.18), VEGF (-3.8), and MMP1 (-13.3) were downregulated the most in PC-3 while SERPINE5 (-3.43) and MMP9 (-21.8) were downregulated most in the LNCaP line. In the PC-3M cell line, which is considered an aggressive metastatic mCRPC/NEPC model  in aggressive androgen-independent (NEPC) PCa compared to the androgen-sensitive PCa. SERPINEB5 is a regulator of cell proliferation, differentiation, and transformation. Several studies have reported that higher expression of SERPINB5 is associated with poor prognosis and OS in various cancers (β1 (TGF-β1), which induces SERPINB5 expression in cancer cell lines by either a decapentaplegic homolog (Smad)-dependent pathway or by non-Smad signaling pathways via the intermediate signaling molecules MEK1/2 and p38 MAPK compared to cells that were sensitive to androgen deprivation therapy. These data are interesting and further studies are needed to determine their overall role and importance.Baseline SERPINEB5 expression was lower in the PCa patient group with higher survival and higher in the NEPC cell lines. Further, following METRO-TOPO treatment, SERPINEB5 was downregulated in all PCa cell lines. The downregulation was greatest (3.5 to 7.5 folds  cancers . SERPINB p38 MAPK. In our Urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) complex expression plays a significant role in invasion and metastasis by increasing production of plasmin from plasminogen leading to ECM degradation. uPA reduces tumor growth by downregulating the MMPs (MMP2 and MMP9) associated with metastasis . Serpin Importantly, METRO-TOPO treatment significantly downregulated SERPINE1  and PLAUFOS is a transcription factor known to promote metastatic PCa and to play an important role in PCa progression and aggressiveness . The MAPα, Ang family—Ang1, Ang2, PDGF-BB) and receptors  are involved in angiogenesis  is involved in cell migration and invasion in PCa . MMP9 e. In thisvs EA, and significantly associated with patient’s survival.Taken together, our results showed METRO-TOPO treatment downregulated 34 key cancer pathway genes responsible for cell proliferation, migration, invasion, differentiation, transformation, metastasis, and anti-angiogenesis indicating a probable basis for the increased potency of METRO-TOPO over CONV dosing . Additioin-vitro and ex vivo discovery followed by in silico correlation of molecular signatures associated with METRO response in a diverse panel of PCa, including existing cell lines derived from self-identified AA patients was used. While a variety of PCa cell lines were used, these data represent only a small fraction of genetic and ethnic diversity that is observed clinically. Therefore, larger scale pharmacogenomics studies involving a wider array of patient derived samples are necessary to validate these molecular signatures, as well as in vivo animal studies using a range of patient derived organoids or patient derived xenograft models to evaluate METRO-TOPO activity. Overall, these data suggest that METRO-TOPO treatment was effective against aggressive, metastatic, androgen-insensitive NEPC, where there are limited treatment options.In conclusion, these data suggest that METRO-TOPO treatment may provide a novel strategy to alter the expression of genes that are associated with tumor growth, metastases, and survival in prostate cancers. An integrated approach that combined


Many individuals with head and neck cancer do not survive, even with intense treatment. Patients with HPV-positive tumors generally have better survival; however, for yet unknown reasons, a subset are unresponsive to therapy. One strategy to monitor cancers for progression and recurrence is evaluation of extracellular vesicles, released by tumor cells into the blood and other body fluids. We can also understand differences in tumors and their behavior by comparing the molecules packaged into vesicles that are released from tumor cells. Our study examined differences in the proteins contained within extracellular vesicles released from head and neck cancer cells. We found that key extracellular vesicle proteins differed based on HPV status of the originating cell line and tumor, as well as how responsive the originating tumor was to treatment. Our findings suggest that these extracellular vesicle proteins may be important markers for continued investigation.To identify potential extracellular vesicle (EV) biomarkers in head and neck squamous cell carcinoma (HNSCC), we evaluated EV protein cargo and whole cell lysates (WCL) from HPV-positive and -negative HNSCC cell lines, as well as normal oral keratinocytes and HPV16-transformed cells. EVs were isolated from serum-depleted, conditioned cell culture media by polyethylene glycol (PEG) precipitation/ultracentrifugation. EV and WCL preparations were analyzed by LC-MS/MS. Candidate proteins detected at significantly higher levels in EV compared with WCL, or compared with EV from normal oral keratinocytes, were identified and confirmed by Wes Simple Western protein analysis. Our findings suggest that these proteins may be potential HNSCC EV markers as proteins that may be (1) selectively included in EV cargo for export from the cell as a strategy for metastasis, tumor cell survival, or modification of tumor microenvironment, or (2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. This work demonstrates that our method can be used to reliably detect EV proteins from HNSCC, normal keratinocyte, and transformed cell lines. Furthermore, this work has identified HNSCC EV protein candidates for continued evaluation, specifically tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common and eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity ,2. In 20There are subsets of HNSCC that respond better to therapy; 70–80% of HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) treated with concurrent chemotherapy and intensity-modulated radiation or surgery with lymph node dissection and post-operative radiation respond favorably. This improved outcome suggests that some patients could be treated with reduced-intensity treatments, sparing them the severe morbidities associated with current therapies . HoweverExtracellular vesicles (EVs) are membrane-enclosed particles released from a cell , that contain various biological molecules, including signaling factors, proteins, nucleic acids, and lipids. The International Society of Extracellular Vesicles (ISEV) defines EVs as “particles released from a cell that are delimited by a lipid bilayer and cannot replicate ” . These vEVs are important because they are produced by both normal and tumor cells and have many functions, including roles in the immune response, inflammation, intercellular messaging, and transport ,13. TumoProteins within EVs are protected from degradation in biological fluids, allowing for increased stability of these molecules for liquid biopsy evaluation compared with circulating tumor cells or cell-free circulating tumor molecules . Some caCell culture: This study used 8 representative HNSCC cell lines , one HPVAll cell lines were grown in T-150 flasks in 20 mL standard media containing serum. Upon reaching approximately 80% confluence, the media was removed, cells were rinsed twice with PBS, and exosome-depleted media (EDM) was added . Following 48 h of incubation, the conditioned cell culture media was collected for EV isolation. Three T-150 flasks were used for each HNSCC cell line, and 9 T-150 flasks were used for HOK16b and NOKsi cells.Contamination testing and genetic assessment was performed on all cell lines prior to freezing, upon thawing, and monthly during the course of cell culture experiments. Cell lines were tested for mycoplasma contamination using the Lonza MycoAlert Mycoplasma Detection Kit and genotyped in the University of Michigan Genomics Core using ProfilerPlus, which interrogates 15 tetranucleotide short tandem repeats (STR), to confirm unique genotypes. The cell lines were also tested for known genetic characteristics including key mutations (using exon sequencing) and HPV status/type (using the HPV PCR-MassArray assay).g for 10 min in 50 mL conical tubes, using an Eppendorf centrifuge (Model #5810R) at 4 °C. The supernatants were transferred to new tubes and centrifuged at 2800× g for 10 min with the same parameters as above. Supernatants were then transferred to ultracentrifuge tubes , and were centrifuged at 12,000× g for 30 min at 4 °C . Supernatants were transferred to new tubes  and centrifuged at 110,000× g for 90 min at 4 °C . The supernatants were removed, and the pellets were resuspended in 1 mL PBS and transferred to fresh tubes. The original pellet tubes were washed twice with 1 mL PBS each time, and this was added to the resuspended pellets in the new tubes, for a combined total of 3 mL. The volume was brought to 13 mL with additional PBS before final centrifugation at 110,000× g for 90 min at 4 °C (Type 70 Ti rotor). Supernatants were removed and pellets were stored at −80 °C.EV isolation by ultracentrifugation: Our EV ultracentrifugation method was modified from the isolation strategy described by Thery, et. al. . Brieflyg for 10 min in 50 mL conical tubes using an Eppendorf centrifuge (Model #5810R) at 4 °C. Supernatants were transferred to new tubes and centrifuged at 2800× g for 10 min with the same parameters as above. Supernatants were then transferred to new 50 mL conical tubes and centrifuged at 10,000× g for 30 min at 4 °C .EV isolation by PEG precipitation/ultracentrifugation: This protocol was based on the PEG method from Rider et al. . EVs werg for 60 min, using an Eppendorf centrifuge at 4 °C. The supernatants were removed and discarded, leaving approximately 2 mL on each of the pellets. The pellets were resuspended and transferred to ultracentrifuge tubes ; the precipitation tubes were each washed twice with PBS and the washes were added to the pellet suspensions. The samples were each brought up to 13 mL total with PBS and centrifuged at 150,000× g for 120 min at 4 °C (Type 70 Ti rotor). Supernatants were removed and pellets were stored at −80 °C.Supernatants were combined with an equal volume of 16% PEG and 1 M NaCl, for a final concentration of 8% PEG, 0.5 M NaCl, and incubated overnight at 4 °C. The PEG precipitations were then pelleted at 3220× 2O (3 × 1 min). Grids were then incubated for 5 min in 1% uranyl acetate for negative contrasting. Images were taken using a Jeol JEM-1400 Plus 120 keV transmission electron microscope equipped with C-MOS camera and AMT (Advanced Microscopy Technology) software (ver. 6.02).Electron microscopy: EVs were pelleted as described above and resuspended in 80 µL PBS. An amount of 10 µL of the resuspended pellet was fixed with an equal volume of 5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2. After (10 µL/15 min) application to formvar/carbon coated EM grids (200 mesh copper), they were washed with dHNanoparticle Tracking Analysis (NTA): EV size and concentration for each sample isolation was determined using a NanoSight NS300 instrument  using a Blue405 laser and sCMOS camera. Samples were diluted in PBS, and 100 µL aliquots were measured with three 60 s videos using a syringe pump speed of 100 at a controlled temperature of 25 °C. Videos were analyzed with NTA 3.2 Build 3.2.16 software to calculate particle size and concentration for each sample.Protein extraction: EVs isolated by ultracentrifugation and associated whole cell lysates were prepared using 1% NP40 in PBS for mass spectrometry. EVs isolated by PEG precipitation/ultracentrifugation and associated whole cell lysates were prepared with Roche Complete Mini Protease Inhibitor Cocktail Tablets  for Western analysis, and quantified with the Bradford protein assay.Mass spectrometry: Proteomic analysis was performed on EVs and whole cell lysates from the eight HNSCC cell lines, normal NOKsi cell line, and the transformed cell line, HOK16b. Relative quantitation of proteins using Tandem Mass Tag (TMT 10plex) and LC-Tandem MS was performed by the Proteomics Resource Facility at the Department of Pathology, University of Michigan, using an optimized protocol described by Tank et al. . BrieflyWes ProteinSimple Western: Protein was measured using a capillary-based electrophoresis instrument . Protein amounts (0.25–1 µg) pre-optimized for specific antibodies were denatured using manufacturer-supplied reagents and loaded into multi-well plates. Protein separation and detection were performed via capillary electrophoresis, antibody binding, and HRP-conjugated visualization following the manufacturer’s instructions. Thorough antibody optimization was completed for all proteins for which Wes analysis was performed to determine ideal input protein concentration/denaturation, specific antibody (supplier/clone), and dilution conditions. Antibodies used are listed in 9 particles/mL.EV characterization was based on minimal information for studies of extracellular vesicles 2018 [ PositiveThe coverage—calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the entire protein sequence—of proteins were also taken into consideration for selection of potential protein markers, with higher coverage values considered to be more likely to yield promising candidates. A total of 4945 proteins were identified from the analysis of extracellular vesicles, and 5738 proteins were identified from the analysis of whole cell lysates. HNSCC EV fold-change peptide spectrum matches (PSM) for the EVs from the HNSCC lines compared with the EVs from the normal keratinocyte line (NOKsi) were averaged together. There were 149 proteins identified that were >2; these are represented in the cluster analysis  and listCandidate EV proteins were initially considered based on the proteomic analysis by mass spectrometry , and final candidates were selected by either previously reported involvement in tumors at multiple HNSCC sites, relationship with HPV tumorigenic pathways, or likelihood of EV association, including membrane proteins.Protein results by ProteinSimple Wes analysis were not normalized to an internal control protein. Limitations inherent to working with EVs, recognized by the ISEV, prevent absolute normalization of expression data, due to variations in composition of EVs from different cell lines . The ISESTAT3 A was detβCatenin (βCat) B was detEPHA2 C was detCD59 D was detDespite tenascin-C (TNC) A being dHLA-A B was detE-cadherin (E-Cad) C was detEGFR D was detCytokeratin 19 (CK19) E was detHPV16 and associated proteins, RB, p53, CyclinD1, and p16 are shown in The 112 kDa form of RB protein was detected in all of the whole cell lysates, and none of the EVs, from all of the eleven cell lines tested. The protein was detected at varying levels in the different whole cell lysates. p53 was detected in only whole cell lysates from HPV-negative cell lines UM-SCC-38 (at 52 kDa and 60 kDa) and UM-SCC-17a (at 60 kDa), as well as whole cell lysates from the normal cell line NOKsi (at 48 kDa and 60 kDa). There was also a faint signal in the normal HOKg whole cell lysate sample at 60 kDa. p53 was not detected in any of the EVs tested.CyclinD1 was detected at 40 kDa in all of the whole cell lysates tested with the exceptions of HPV-positive HNSCC cell lines UM-SCC-47 and UPCI:SCC152, where no CyclinD1 protein was found. There was no CyclinD1 protein detected in any of the EVs tested.None of the EVs tested contained detectable levels of p16 protein. We did identify p16 at 24 kDa in whole cell lysates of HPV-positive HNSCC cell lines UM-SCC-47, UM-SCC-104, UM-SCC-105, and UPCI:SCC152, as well as HPV-negative HNSCC line UM-SCC-92. The whole cell lysates of the transformed cell line HOK16b and normal HOKg cell line also contained p16 protein, although the lysate of the normal NOKsi line did not.The proteins detected in the EVs from the HNSCC cell lines were expected to recapitulate the characteristics of the originating cells. This study suggests that this is frequently not the case, as seen in Tenascin-C is an extracellular matrix glycoprotein, expressed during embryogenesis, organogenesis, wound healing and inflammation ,32,33,34Tenascin-C has been implicated in many cancer types: esophageal, breast, colorectal, bladder, Merkel cell carcinomas, glioma, malignant pleural mesothelioma, glioblastoma multiforme, malignant melanoma, and astrocytoma, and has been implicated in a variety of roles: tumorigenesis, angiogenesis, migration, proliferation, maintaining the stemness of cancer stem cells, and metastasis ,39,40,41The signal transducer and activator of transcription 3 (STAT3) is part of the signal transducer and activator of the transcription protein family. STAT3 plays a pivotal role in tumorigenesis through transcription regulation of genes having essential roles in cell proliferation, inflammation, differentiation, apoptosis, angiogenesis, immune response, and metastasis . STAT3 hHLA (human leukocyte antigen) is part of the MHC class I immune system. There are classical forms, HLA-A, HLA-B, and HLA-C, and non-classical forms, HLA-G, HLA-E, HLA-F, and HLA-H, of this protein; both have been identified in cancer with diverse roles ,55,56,57β-Catenin, together with the cadherin family of adhesion proteins, is part of the Wnt signaling pathway, and is thought to have roles in both HPV-positive and -negative HNSCC and be associated to de-differentiation and poor prognosis ,69,70,71E-cadherin is part of the cadherin family of proteins, and functions as a main adhesion protein of the epithelia with key roles in attachment with the formation of E-cadherin/β-catenin complex, polarity, and structure. Repression, downregulation, or loss of E-cadherin is correlated with metastasis and invasion and has been implicated in EMT ,80,81,82EphA2 is a receptor tyrosine kinase that binds to adjacent cells through binding to surface-associated Ephrin (Eph receptor interacting protein) ligands . DysfuncEGFR is a type I receptor tyrosine kinase with a structure that spans the cell membrane and includes an extracellular ligand-binding domain and an intracellular kinase domain. EGFR is expressed in most epithelial tissues, and is involved in multiple pathways, including PI3K/Akt/mTOR, JAK/STAT, and RAS/RAF/MEK/ERK ,102,103.Cytokeratin 19 is part of the cellular cytoskeletal network of intermediate filaments (IF), in the sub-classification of type I epithelial keratins ,123. KerCD59 is a cell surface inhibitor of the membrane attack complex present in most tissues and on all circulating cells ,141. It HPV and associated proteins, RB, p53, Cyclin D1, and p16, are common biomarkers for HPV-positive and -negative HNSCC. HPV16E7 is a viral oncoprotein expressed in the HPV16-positive HNSCC tumors and cell lines. We thought that it might be detected in the EVs from the HPV16-positive cell lines; however, it was identified only in the whole cell lysates, including the whole cell lysate of the HPV16-transformed cell line, HOK16b. Similarly, we did not detect HPV-associated proteins RB, p53, Cyclin D1, or p16 in any of the EVs tested. We will evaluate other HPV proteins  as potential EV markers, or perhaps investigate alternate antibodies, as HPV proteins and several of the other HPV-associated markers have previously been identified in EVs from HPV-positive cell lines . It was We have demonstrated that proteins can be reliably detected in EVs and whole cell lysates from HNSCC, normal keratinocytes, and transformed cell lines, and that EVs released by HNSCC cells contain different proteins based on characteristics of the originating cell, including HPV status. Our work has importantly demonstrated that EVs released by tumor cells do not necessarily recapitulate the complete protein profile of the originating cell, and hypothesize that EV protein cargo is determined by other factors that are advantageous for the tumor cell, such as growth, immune evasion, modification of the tumor microenvironment, cell-to-cell communication, drug resistance, etc. Finally, this work has identified several protein candidates for continued evaluation for HNSCC EV markers, including tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19.


Cytokeratin19 (CK19) was detected as the most related marker for circulating tumor cells, which was assessed in specific cell lines. MCF7, SKBR3, T47D, and MDA-MB-231, and HeLa cell line as negative control were used. CK19 expression was confirmed by using mouse monoclonal anti-human CK19 antibody. CK19 detection in MDA-MB-231 was not observed. CK19 marker expression was compared in T47D, MCF7, and SKBR3 cell lines. T47D and MCF7 belonged to the luminal subtype of breast cancer (BC) that CK19 expression regulated with an ER marker. SKBR3 belonged to the HER2 positive subtype of BC. However, MDA-MB-231 belonged to the claudin-low subtype of BC that lack of CK19 expression strongly is related to negative ER, PR, and HER2. Therefore, there are not only quantitative differences in CK19 expression, but its expression could also link to the other markers of BC that should be considered in the molecular classification of breast carcinoma. Different expression levels related to cell classification could be useful in the prognosis and treatment of cancers with epithelial origins. Malignaand CK19 , 5. Limiferation , 5. HER214, CK17 . This su14, CK17 . The Nor markers . The phe markers . The Cla markers .CKs are the major structural proteins in epithelial cells . They arFurthermore, epithelial cells from initial tumors can be identified in the peripheral blood of breast cancer patients, which these cells are known as circulating tumor cells (CTCs) . CTCs ar+, PR+, and CK19 positive [+, PR+, and express E-cadherin [Additionally, breast cancer cell lines are the reproducible sources widely used to discover biological and clinical functions such as studying tumors, the signal transduction pathways, and modern therapeutic goals . They wepositive . The T47positive . T47D cecadherin . This cecadherin . The SKBcadherin . The MDAcadherin .There is a necessity for finding a common biomarker that could be used in experimental and clinical research. In this study, CK19 marker expression was accessed in characterized cell lines as the most related marker for circulating tumor cells (CTCs), and its relation with other defined markers was considered for the first time. The research has been performed to quantify CK19 expression in different cell lines by flow cytometry used in the research literature. CK19 detection assessment in patients’ blood and cell lines research could support and explain data variation in breast cancer therapy results, which can improve future treatment procedures.2 incubator (5% CO2). Then, cell lines grown in monolayer were harvested by washing once with phosphate-buffered saline (PBS), pH 7.3, and then incubating the cells with trypsin/EDTA (Biosera) for 2–5 min at 37°C. Finally, cells were counted using a hemocytometer.Human breast cancer cell lines  and cervical cancer cell line (HeLa) as negative control were obtained from the cell bank of Pasteur Institute of Iran. Briefly, cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% Fetal bovine serum  and penicillin-streptomycin (Biosera). First, the cells were kept at 37°C in a humidified CO6 cells from each cell lines in 50 μl PBS  1X at 4°C. The protein concentration of the samples was determined using Picodrop. Then, Lysates of cell lines were mixed with Laemmli sample buffer [Cell lysis was performed by resuspending 0.5 × 10e buffer  and incu6 cells from each cell line were fixed with 4% paraformaldehyde (Merck) in PBS 1X for 20 min at room temperature. Then, cells were washed twice with 1% PBS with FBS and were permeabilized with ice-cold 100% methanol (Merck) for 30 min at 4°C. Cells were washed twice with 1% PBS-FBS and blocked with 1% bovine serum albumin (BSA) for 45 minutes at room temperature. Cells were washed twice with 1% PBS-FBS and then were stained with Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CK19 antibody  or FITC-mouse IgG2a isotype antibody  for 1h at room temperature as a negative control. Finally, they were washed twice with 1% PBS- FBS, and detection of bound antibodies was determined by flow cytometry (Cyflow). The results were analyzed with the FlowJo® program.The expression of CK19 in breast cancer cell lines was determined by flow cytometry. In this regard, 1 × 10CK19 expression in the MCF7 and T47D cell lines was also detected by immunofluorescence assay. Cultured cells were assessed on glass slides. They were fixed with 4% paraformaldehyde (Merck) in PBS 1X for 20 min at room temperature. The cells were washed twice with 1% PBS-FBS and were permeabilized with ice-cold 100% methanol (Merck) for 30 min at 4°C. Cells were washed twice with 1% PBS-FBS and blocked with 1% bovine serum albumin (BSA) for 45 min at room temperature. Cells were washed twice with 1% PBS-FBS, and then Immunostaining was done with the FITC-conjugated mouse anti-human CK19 antibody . Then FITC-mouse IgG2a isotype antibody  as a negative control was performed for 1h at room temperature. The cells were washed twice with 1% PBS-FBS, and detection of CK19 protein was determined under a fluorescent microscope (Leitz Laborlux).MCF7, SKBR3, MDA-MB-231, T47D, and HeLa cell lines were purchased from the cell bank of Pasteur Institute of Iran, and after defrosting, they were cultured in the medium. In order to obtain the same amount of cells for western blot and flow cytometry analysis, the cells were cultured several times, and the count was carried out by Neubauer slide.To detect the expression of the CK19 protein in breast cancer cell lines, western blot analysis was performed. In this study, the cervical cancer cell line (HeLa) was also used as a negative control. First, the expression of the β-Actin protein was investigated as a normalizing factor. The nitrocellulose membrane was incubated with the mouse monoclonal β-Actin antibody (primary antibody) and then with the HRP anti-mouse antibody (secondary antibody). Finally, the protein bands were revealed by the ECL technique. β-Actin protein detection showed the same expressions in the cell lines. In order to investigate CK19 expression, we used mouse monoclonal CK19 antibody as the primary antibody and the HRP anti-mouse antibody as the secondary antibody. The CK19 antibody was specific for this marker, and it was from the lgG1 class and BA17 clone. The results obtained from western blot indicated the different levels of CK19 expression in breast cancer cell lines. The SKBR3, MCF7, and T47D cell lines were all positive in terms of expression of CK19, while no expression of this protein was shown in the MDA-MB-231 cell line . In factCK19 expression in cell lines from the Luminal subtypes of breast cancer (MCF7 and T47D) and the HER2 positive subtypes of breast cancer (SKBR3) was detected, while no expression of this protein was detected in cell lines from the other subtypes same as Normal-like/claudin-low (MDA-MB-231). Furthermore, the cell lines from Luminal subtypes of breast cancer had a higher expression of CK19 compared to the cell line of HER2 positive subtypes of breast cancer.6 cells of each cell line were colored with the mouse monoclonal CK19 antibody, linked to FITC, and 1×106 cells of each cell line were colored with the mouse monoclonal IgG2a antibody, linked to FITC (isotype control). The CK19 antibody is specified for this marker, and it is from IgG2a class and A53-B/A2 clone. The obtained results from flow cytometry analysis indicated the different levels of CK19 marker expression in the studied cell lines. CK19 expression in the MDA-MB-231 cell line was not detected, while 37.7, 51.4, and 76.2% expression were detected in SKBR3, MCF7, and T47D cell lines, respectively. In the SKBR3 cell line, the least expression, and in the T47D cell line, the most expression level was detected. CK19 marker expression for the negative control HeLa cell line was not detected by flow cytometry  was considered as the negative control. 1×10ytometry .The microscopic immunofluorescence detection was done to confirm the CK19 expression in MCF7 and T47D cell lines. For each cell line, two cell culture plates were considered. One of the plates was incubated with CK19 monoclonal antibody conjugated to FITC, and the other one was incubated with mouse lgG2a antibody (as the isotype control). The cell lines were investigated by the wavelength of 488 nm with the immunofluorescence microscope .in vitro and in vivo [Breast cancer is a very heterogeneous disease at molecular, histopathological, and clinical levels. In addition, established cell lines derived from the primary or metastatic cancers still provide an important experimental tool for biological cancer studies and genetic modifications  in vivo . The het in vivo . At pres in vivo .et al. studied T47D and MCF7 cell lines by microarray technique and indicated that both cell lines show ER and PR receptors expression and are negative in the expression of HER2 (ER+/PR+/HER2–); hence, they are considered Luminal subtypes [–/PR–/Her-2+) [et al. indicated the CK19 expression in HER2+ tumors and concluded that HER2 influences tumor cells with invasion and metastasis by regulating CK of cytoskeleton’s expression [et al. also indicated that the expression of the CK19 marker in MCF7 and SKBR3 cell lines gives a high metastatic feature to these active cells [et al., with a microscopic investigation of SKBR3, MCF7, and T47D cell lines, indicated that all three cell lines express CK19; but the MCF7 and T47D cell lines show the expression of ER while SKBR3 cell line shows the expression of HER2 [et al. stated that compared to HER2, the complex of ER hormone-receptor has a stronger impact on the expression of the CK19 marker [et al. studied this cell line with microarray technique and indicated that it is negative in terms of the expression of ER, PR, and HER2 receptors (ER–/PR–/HER2–); thus, it is located in the Normal-like/Claudin-low subtype [et al. found that the CK19 marker is expressed in the MCF7 cell line but not in the MDA-MB-231 cell line, and they stated that while both cells have the same origin; their phenotype is different [In this study, flow cytometry was used to quantify CK19 expression in T47D, MCF7, SKBR3, and MDA-MB-231 cell lines. The antibody against CK19 was provided from the A53-B/A2 clone for flow cytometry and the BA-17 clone for western blot. It was indicated that the CK19 marker has no expression in the HeLa cell line; therefore, this cell line was used as the negative control in this study . CK19 masubtypes . The int/Her-2+) –27. Therpression . Using f of HER2 . We show9 marker . CK19 ma subtype . On the  subtype . MDA-MB- subtype . EMT is  subtype . The pro subtype . For theifferent . Up to nifferent .et al. illustrated that CK19 detected in the peripheral blood by flow cytometry is typically an easy and certain method to find the CTCs in patients with breast cancer, the primary metastasis, or disease progress [Different techniques and markers are currently used to identify the cancer cells in blood samples with monoclonal antibodies. However, flow cytometry-based studies provide valuable information for the diagnosis, treatment, and prognosis of breast cancer patients, especially for identifying the tumor cells existing in the blood . Wang etprogress . In 2011progress . CK19 exprogress , 37.The CK19 metastatic potential has been an interest in recent years. Its application as a tumor marker among 30 cancer cell lines such as thyroid, thoracic, lung, pancreatic, cervical, colorectal is important to consider .This is a more comprehensive report of different levels of CK19 expression based on molecular cell lines classification that helps in choosing the right cell line in breast cancer research. In this study, research has been performed to add the quantification of CK19 expression in different cell lines by flow cytometry used in the research literature. Blood specimens were tested by flow cytometry to quantify the expression of CK19. Different expressions of CK19 were studied in human breast cancer cell lines, SKBR3, BT549, and BT474, which are used in most literature articles regarding breast cancer.Another detection technique, western blotting, was also used in this study for confirming the protein expression. CK19 expression in the MDA-MB-231 cell line was not detected, while 37.7, 51.4, and 76.2% mean fluorescence expression was detected in SKBR3, MCF7, and T47D cell lines, respectively. In conclusion, flow cytometry is an easy and reliable method for measuring the level of CK19 marker expression that could indicate the expression regulation of other markers in breast cancer cells. Therefore, it can be concluded that CK19 expression and its regulation by ER hormone or HER2 signaling pathway could be useful in the prognosis and treatment of cancers with epithelial origins.The authors declare that there is no conflict of interest.The approval for this study was obtained from the National Ethical Committee of the Pasteur Institute of Iran . This article is based on a thesis submitted to the graduate office in partial fulfillment of the requirements for the degree of MSc student.The informed consent written form was obtained from each participant.Pasteur Institute of Iran funded the thesis.


CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of  HESCs hf tissue .in vitro and reported karyotype changes in all three cell lines [Recent 10-year research has shown that ESCs may be affected by chromosomal abnormality or genetic change under extended cultures. Rebuzzini et al. performed a cytogenetic analysis of three mouse ESC lines during prolonged passaging ll lines . Draper ll lines . Upon rell lines –7. This ll lines , 5, 8, 9ll lines .Epigenetic aberrations can alter the function of certain genes related to tumorigenesis –14 and aMany methods, such as methylation-specific polymerase chain reaction (MSP), methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), and methylation-sensitive high resolution melting (MS-HRM), have been widely used for the detection of methylation tendency. Among these, MS-MLPA is a fast, inexpensive, and reliable system for epigenetic characterization of tumor suppressor genes , 20. Theβ-mercaptoethanol, 1% nonessential amino acids, 100 units/mL penicillin, 100 μg/mL streptomycin, and 4 ng/mL basic fibroblast growth factor (bFGF); all products from Thermo Fisher Scientific) at 37°C in a humidified 5% CO2 incubator. CHA-hES 20 cells were cultured on human foreskin fibroblast feeder cells with 0.1% gelatin coating in ES-SR medium. CHA-hES 40 and 42 were cultured on human endometrial fibroblast feeder cells with CELLstart  coating in ES-SR XenoFree medium, which was the same as ES-SR medium except that KnockOut SR XenoFree  was used instead of SR. iPS (FS)-1 was cultured in mTeSR1 medium  according to the manufacturer's instructions. Each hPSC line was collected at early passages and late passages for this study.For this study, nine hESC lines  and one hiPSC line (iPS (FS)-1; WiCell, Madison, WI, USA) were used. All hESC lines except H1 were established and characterized in CHA Gangnam Medical Center, CHA University, Korea, after receiving approval from the Institutional Review Board (IRB) of CHA Gangnam Medical Center as previously described , and theμL sample using a NanoDrop™ spectrophotometer . Extracted DNA and RNA were stored at -80°C until further analysis.Genomic DNA was extracted from cultured stem cells using the QuickGene DNA tissue kit  according to the manufacturer's instructions. Total RNA was extracted from cultured stem cells using the RNeasy® Mini kit  according to the manufacturer's instructions. DNA or RNA yield was quantified from a 1 μL of Hi-Di™ formamide  and 0.3 μL of GeneScan™-500LIZ™ Size Standard  and analyzed using a 3130XL Genetic Analyzer . Quantification was performed using the GeneMapper software .DNA methylation status was analyzed by MS-MLPA using the SALSA MS-MLPA ME001-C1 tumor suppressor kit . This probe mix contains 26 probes  for 15-20 min. After incubation, the column was centrifuged at full speed for 30 s, washed by adding 200 μL of M-wash buffer, and centrifuged at full speed (this step was repeated). The converted gDNA was eluted by adding 20 μL of M-elution buffer to the column and spin. DNA samples were stored at -20°C until further use.Genomic DNA was modified using the EZ DNA Methylation-Lighting™ kit  according to the manufacturer's instructions. The samples were subjected to the following steps in a thermal cycler : 8 min at 98°C, 60 min at 54°C, and 4°C for up to 20 h. The DNA was purified and added to a Zymo-Spin IC™ column containing the M-binding buffer and mixed by inverting the column several times. The column was centrifuged at full speed for 30 s, and the flow-through was discarded. The column was washed by adding 200 CASP8, FHIT, CDKN2B, and CHFR genes. Each primer was designed using Pyrosequencing Assay Design Software v2.0 . The primer sequence is listed in Supplementary Data ∗CASP8 and FHIT: 54°C; CDKN2B and CHFR: 50°C). PCR products were confirmed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.We used the bisulfite pyrosequencing method for methylation analyses of The ssDNA template was prepared from a biotinylated PCR product using streptavidin Sepharose® HP beads  following the PSQ 96 sample preparation guide using multichannel pipettes. Fifteen picomoles of the respective sequencing primers were added for analysis. Sequencing was performed on a PyroMark ID system with the Pyro Gold reagent kit  according to the manufacturer's instructions. The methylation percentage was calculated as the average degree of methylation at CpG sites formulated by pyrosequencing.μg of total RNA was reverse transcribed using SuperScript® III First-Strand  according to the manufacturer's instructions. Amplified cDNAs were 1/50 diluted and mixed with BIO-RAD iQ™ SYBR® GREEN Supermix . The primer sequence is listed in Supplementary Data After DNase I treatment, 0.5 Real-time PCR was performed on these samples under the following conditions: 15 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 20 s at 72°C on iQ™5 Optical System Software version 2.0 .ΔΔCt method and normalized to 28S rRNA housekeeping gene expression.Relative gene expression levels were calculated using the APC, DAPK1, IGSF4, RARB, and TIMP3 genes were found to be methylated in cancer cells  , 20. We CASP8 and FHIT genes was detected in a total of 10 cell lines, while that of the CHFR gene was detected at late passage in iPS (FS)-1 cell line , CHA-40 , and CHA-42  cell lines , CHA-31 , CHA-32 , and iPS (FS)-1  cell lines and significantly decreased between early and late passages in CHA-20  and H1  (CHFR gene was detected only in iPS (FS)-1, and the level was significantly increased at the late passage  -1, WiCell). The CGI methylation of ell line . The CGIll lines , while t0.00004) . CGI met= 0.009) .Additionally, MS-MLPA experiments were performed by randomly taking middle passages between early and late passages in 10 previously tested cell lines and H9 hES. The middle passage cell lines were cultured under the same conditions as those used in the previous experiments, and these were randomly selected. As a result, changes in methylation levels occur gradually during extended cell culture , FHIT 528~575 (5 CpG sites), and CHFR 442~478 (3 CpG sites), was designed using the Pyrosequencing Assay Design Software v2.0. The methylation percentage was calculated by the average of the degree of methylation at CpG sites.We performed bisulfite-pyrosequencing analysis for CASP8 was significantly higher at the late passage than at the early passage in CHA-36 , CHA-40 , CHA-42 , and iPS (FS)-1  cell lines , CHA-31 , CHA-32 , CHA-36 , CHA-40 , and iPS (FS)-1 cell lines  and decreased at the late passage in CHA-20 , CHA-42 , and H1 cell lines  (CHFR gene significantly increased only in iPS (FS)-1 cell line   . The met= 0.001) , consistCASP8 was significantly downregulated at late passage in CHA-42  and iPS (FS)-1  compared to their early passage , CHA-31 , CHA-32 , CHA-40 , CHA-42 , and iPS (FS)-1  cell lines as compared to that in early passage. FHIT expression significantly increased at passage in CHA-20  and H1  (CHFR gene significantly decreased at late passage in iPS (FS)-1 cell lines   . The exp= 0.020) .CASP8, FHIT, and CHFR genes and found that the level of methylation changed between early and late passages in some cell lines. We identified that the changes in CGI methylation levels in these genes led to variations in their expression. Except for the methylation of CASP8 and FHIT genes in CHA-36 cells, we reported consistency between MS-MLPA, pyrosequencing, and real-time PCR data , the CGI methylation level of CASP8 or FHIT gene increased at late passage. On the other hand, the level of FHIT in CHA-20 and H1 cell lines decreased at late passage. The results of these two cell lines differed from those of the other cell lines, probably owing to different conditions, such as the type of cell line, feeder cell, or media. According to a recent study by Thompson et al., it was revealed that there is a difference in the methylation change depending on the culture conditions of stem cells [In this study, we attempted to find the methylation changes in the promoter regions of tumor suppressor genes in human ESCs under an extended culture system using the MS-MLPA method. We detected the methylation of PCR data . We confem cells . We condem cells . Based oCHFR was detected in iPS cell line. Previous studies comparing hiPSCs with hESCs have revealed both similarities and differences with regard to the transcriptome, genomic stability, histone modification, and DNA methylation [Unlike hESCs, the CGI methylation of hylation –26. DespCASP8 encodes a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution phase of cell apoptosis. This protein is involved in programmed cell death induced by Fas and various apoptotic stimuli [FHIT plays a role in the induction of apoptosis via SRC and protein kinase B (AKT1) signaling pathways and modulates transcriptional activation by catenin beta 1 (CTNNB1), thereby contributing to the regulation of the expression of the genes essential for cell proliferation and survival, such as CCND1 and BIRC5 [CHFR gene encodes an E3 ubiquitin-protein ligase required for the maintenance of the antephase checkpoint that regulates cell cycle entry into mitosis and, therefore, may play a key role in cell cycle progression and tumorigenesis [The gene  stimuli , 28. FHInd BIRC5 –31. The igenesis , 33.in vitro, further studies on knockout models are needed to assess whether methylation changes in tumor suppressor genes actually lead to the change from stem cells to tumorigenesis.CGI methylation is the main epigenetic mechanism, and changes in this mechanism play an important role in tumorigenesis. The MS-MLPA assay is a fast and sensitive method for detecting changes in the methylation of multiple genes in a single reaction. Therefore, in this study, the MS-MLPA method was applied for the simultaneous analysis of the methylation status of distinct tumor suppressor genes in human ESCs. As this study is limited to cell culture We showed that during extended cultures, human embryonic stem cell lines may not undergo large genomic alterations such as chromosome abnormalities but show alterations in CGI methylation levels of tumor suppressor genes. To use hESC lines for cell therapy, it would be imperative to closely verify structural variations at the chromosome or genome level as well as the epigenetic changes.