However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O Primary MEFs can be frozen at early passages to prolong their experimental utility; however, there is concern of the effects that the freeze/thaw cycle has on MEF growth and metabolism. As a result, the short Hayflick limit restricts the amount of experimentation and the total protein lysate that can be generated from a single isolation of MEFs. In addition, a large degree of biological variability is associated with MEFs isolated at different times and from different embryos. Immortalizing primary MEFs with the desired genetic manipulations would reduce the time required for MEF cell line maintenance, enable the generation of large amount of protein lysate, and facilitate the use of biological replicates during experimentation. Two major approaches have been used to immortalize primary MEFs: (i) spontaneous immortalization after a series of passages and (ii) transformation by overexpression of an oncogene. The serial passage protocol, as described by Xu  (ab16879) and Donkey Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150105). Briefly, 4 × 10t-test was applied to the band intensity values to determine if the presence of epithelial cell markers in MEFs from each embryo isolated were statistically different at the 95% confidence interval.The presence of epithelial cells in the primary MEF cell lines was determined by quantifying the relative abundance of three epithelial cell markers via traditional western blotting procedures. The following primary antibodies against epithelial markers were used: Rabbit poly to E-cadherin , Rabbit mono to beta-catenin , Mouse mono to anti-pan Cytokeratine  with secondary antibodies Odyssey Goat Anti-Rabbit 800CW  and Odyssey Goat anti Mouse 800CW  respectively. The following loading controls were used to normalize epithelial marker band intensity: Rab poly to anti-beta-Actin  and Rab poly to Anti-Histone H3 . Blots were imaged with the Odyssey Classic infrared imaging system  with excitation wavelength 778 nm and emission wavelength 795 nm. Band intensities were analyzed with Image Studio Software (LI-COR). Student et al. [Table 1. Genomic DNA was extracted using the DNeasy blood and tissue kit . A master mix with 20 ng of the template genomic DNA, 10 µM forward primer, 10 µ M reverse primer, and fast SYBR green master mix  for the telomere and single copy gene primers. Identical plates were set up for each primer set. The Applied Biosystems 7900HT Fast Real-Time PCR System was used for detection with the temperature profile listed in Table 2. Ct values were automatically generated with the RQ Manager Version 1.2 qPCR software (Applied Biosystems). The average T/S ratio was calculated as follows:Telomere length was determined via the SYBR green qPCR procedure described by Cawthorn et al. . BrieflyAutophagy induction in SV40 transformed versus serially passed MEFs was detected using the Cyto-ID Autophagy detection kit  according to the manufacturer’s protocol. Briefly, SV40 transformed and serially passed MEF lines were plated in biological triplicate on a chambered glass slide  and grown overnight. Then the cells were treated with 500 nM rapamycin (positive control) or an equivalent volume of DMSO (negative control) for 18 h. 60mM Chloroquine was added to each well to enhance fluorescence signal. Post treatment, cells were washed with 1 × assay buffer, then stained with Cyto-ID Green detection reagent and Hoechst 3342 nuclear stain. Following staining, cells were washed with 1 × Assay buffer and fixed with 3% paraformaldehyde. Samples were then mounted with Flouro-Gel  . Fluorescence was detected with LSM 700 Confocal (Zeiss) at 488 nm observed at 40 ×. Fluorescence intensity of images was analyzed with FIJI image processing software.2 and % Viability for MEFs cultured at 37°C with 5% CO2 and either ambient or 3% O2 are shown in Figure 1. At ambient O2, the VCC per cm2 and percent viability declined with each subsequent passage, and the cells reached senescence (Hayflick limit) seven passages post isolation. However, when cultivated at 3% O2, some primary MEFs lines could be proliferated for more than 40 passages with a median doubling rate of 45 ± 55 h (n = 8) while maintaining a median viability greater than 90% (Table 3).Viable cell count (VCC) per cm2, the length of proliferation for each MEF line was highly dependent on the mouse embryo from which the MEF line was derived. As described in section 3.1, MEFs derived from some mouse embryos could be cultivated for more than 40 passages when cultured in 3% O2. However, despite cultivation at 3% O2, MEFs derived from some other mouse embryos still reached their Hayflick limit after seven passages. Liu et al. [et al. [et al. [Despite cultivation at 3% Ou et al.  reported [et al.  reported [et al.  reported2, proliferation could be prolonged for as many as 40 passages by using a media mixture with 75% conditioned media and 5 µM ROCK inhibitor Y-27632 . Cultures supplemented with ROCK inhibitor and conditioned media maintained a median doubling rate of 49 ± 37 h (n = 4) (see Table 3).When MEF cultures reached their Hayflick limit despite cultivation in 3% OFig. 2B and 2D)). Previous studies have reported the utility of ROCK inhibitor to immortalize epithelial cells [Fig. 3).Interestingly, the viable cell count, cell doubling rate, and percent viability of cultures derived from embryos that did not reach senescence after seven serial passages were not affected by ROCK inhibitor supplementation . However, those same MEF cultures derived from embryos a and c when supplemented with ROCK inhibitor showed a stable telomere length as indicated by the constant T/S ratio. MEFs derived from embryos b and d had long life spans without ROCK inhibitor supplementation and showed a comparable average T/S ratio when cultured with or without ROCK inhibitor supplementation . Interestingly, the T/S ratios for MEFs derived from embryos b and d were larger than the T/S ratios for MEFs derived from embryos a and c regardless of ROCK inhibitor supplementation, which suggests that telomere length may not be an accurate marker of replicative senescence in MEFs.The results presented in 2 with less variability between lines (see Table 3). SV40 Ta expression was confirmed via immunofluorescence staining . The short doubling time with low variability and consistent high viability throughout passages make SV40 Ta transformation a highly robust immortalization strategy compared to serial passaging .MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9); a median cell doubling rate that is nearly half that of cell lines immortalized by serial passaging at 3% OFig. 6).Despite the robust immortalized cell lines that SV40 Ta transformation produces, there may be phenotypic and/or molecular differences when compared to wild type lines. For example, SV40 transformed MEFs present a cuboidal cellular morphology compared to the fibril morphology seen with MEFs that achieved spontaneous immortalization after a series of passages  between SV40 transformed and wild type MEFs lines indicates that the SV40 transformed lines can be a useful tool to study the autophagy pathway.In addition to the phenotypic difference in cell morphology, molecular differences may arise since SV40 Ta transformation inactivates tumor suppressor gene p53, an inducer of autophagy. To assess potential molecular differences between SV40 transfected cells and those immortalized via serial passaging, the presence of the autophagy induction marker LC3 was measured. The data shown in 2 and transformation with SV40 Ta. Based on the experimental work presented, transforming MEFs with SV40 Ta generated cell lines with relatively fast doubling rates and consistent high percent viability compared to lines created via serial passing at 3% O2. Despite the robust lines that SV40 Ta transformation consistently produces, we recommend that researchers consider the experimental goals of the immortalized MEFs lines prior to the selection of an immortalization method. Tumor suppressing proteins are inactivated by SV40 Ta. Therefore, SV40 transformation could produce undesired modifications to the endogenous cellular metabolism or phenotype that could potentially skew experimental results. As such, we suggest that SV40 transformed lines be assessed for biological relevance. For instance, we investigated the effect of SV40 transformation on autophagy since tumor suppressing protein p53 is inactivated by SV40 Ta and is known to regulate autophagy. We found no statistically significant difference in the rate of autophagy between SV40 transformed and serially passed immortalized MEFs , indicating that SV40 transformed lines can be a useful tool to study the autophagy pathway.In this work, we compared and evaluated two approaches for immortalizing MEF cell lines: serially passing MEFs at 3% O2 to achieve spontaneous immortalization. One must keep in mind that immortalization via serial passaging is not as robust as SV40 transformation. If cultures seem to be reaching their Hayflick limit despite cultivation at 3% O2, supplementing the culture media with conditioned media and ROCK inhibitor may help to prolong MEF cultivation. In addition to the varying success of spontaneous immortalization after serial passaging, one should also keep in mind that lines immortalized via serial passaging have slower and more variable doubling rates than those MEFs lines immortalized via SV40 transformation. Our recommendations for generating immortalized MEFs are summarized Table 4.In cases where the biological relevance of MEFs transformed with SV40 Ta is a concern, we recommend serially passing at 3% O


Its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. In this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. Finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. Our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.Wound healing assay is a simple and cost-effective  While cancer cells may acquire different phenotypic hallmarks2. Such ‘antimigrastics’, or drugs with ability to prevent cancer invasion and metastasis, can be complementary to the existing cytotoxic drugs that only focus in inducing tumor shrinkage. To identify candidate compounds that target cancer cell invasion, drug screeners often require in vitro assays for scoring therapeutic influence on cell migration. Wound healing assay is one of the most commonly used bioassays for evaluating the therapeutic impact on cell migration mainly due to its simplicity in experimental setup and data analysis at post processing. By scratching cell monolayer to create a wound, one can consistently perform wound healing assays across thousands of treatment conditions. Automated imaging platform with environmental control recently makes possible real-time monitoring of the wound closure, allowing quantification of wound closure rates to be easily assessed. One major challenge in quantifying the therapeutic impacts on cell migration is the interdependent roles of different cellular phenotypes during wound closure3. Prior studies have tried to suppress influence of cell growth on wound closure by pre-treating cells with Mitomycin C (MMC) or growing cells in low serum condition prior to drug treatment6. Cytotoxicity associated with most anti-cancer drugs is another confounding phenotype, which can cause an apparent delay in wound closure7. Because of such complexity, some drug candidates were mistakenly interpreted as cell migration inhibitors, although they were later found to be cytotoxic drugs in different cell lines or other treatment conditions2. An experimental approach that can separate the pharmacological impacts of different phenotypes will help ensure accuracy in screening for cell migration inhibitors while maintaining the simplicity of the conventional wound healing assay.In recent years, pharmaceutical industries have gained more attentions in developing drug candidates that can specifically target molecular mechanisms related to cell motility, cell migration and metastasisTo address this limitation, we present in this study an analytical approach to improve the standard wound healing for accurate quantification of therapeutic impacts on different phenotypes. In addition to the ability to separate cell migration from cell death, our approach can robustly identify cell migration inhibitors across different cell lines at different seeding densities. We demonstrated the use of this new technique for identifying inhibitors of cell migration in cholangiocarcinoma cell lines. Our study offers a simple approach for quantitative scoring of both migrastatic and cytotoxic effects that can be readily scalable for high-throughput drug screening.8. To minimize impact of cell growth when adopting the standard wound healing assay, one often pre-incubate cells with MMC (3–5 hours) to limit cell growth, prior to assessing drug involvement on wound closure  and PD0325901 (potent MEK1/2 inhibitor) on cellular phenotypes during the use of wound healing assay. The KKK-D068 bile duct cancer cell line was chosen for this experiment because MMC pre-treatment could successfully inhibit its proliferation without affecting the baseline wound closure rate Fig. . At 5 μMst-order rate equation and determined the contributions of cell death or cell migration based on the angular contribution at any specific points along the fitted curve, namely the angle between the fitted curve towards the vertical axis for cell death and the angle between the fitted curve towards the horizontal axis for cell migration   across 13 bile duct cancer cell lines and 1 cholangiocyte cell line (MMNK-1), we found that these cell lines could exhibit drastically different wound closure rates, ranging from 2.7 to 34.8 hours for closing 50% of the wound. Interestingly, the observed variability of wound closure rates was found to be independent of growth rate (quantified by doubling time) across cell lines Fig. . With MMp-value > 0.05) consistently across all cell lines  using the experiment endpoint (72 hours) as previously done, B) using the time that the rate of wound closure is declining (slope of the RWD < =0.02 for the no-drug control) and C) using the time where 50% of the wound is filled for each condition. For comparison, we monitored changes of wound closure speed using three different cell lines , each plated at 20,000, 30,000, and 40,000 cells/well  to determine the migration and death scores, we compared the phenotypic bias of six previously developed anti-cancer compounds across five CCA cell lines . We reasoned that different drugs may cause similar phenotypic bias but the strength of the therapeutic impact could potentially be variable  that can be used to classify the two cell line subgroups. In particular, MG-subgroup 1 (RBE and KKK-D068) showed enrichment of genes associating with epithelial-to-mesenchymal transition (EMT), myogenesis and xenobiotic metabolism, while MG-subgroup 2  showed enrichment of genes that are associated with estrogen response, apoptosis, p53, KRAS signaling, and inflammatory response  KKK-D068 and RBE, and (2) HuCCA-1, KKU-213, and KKK-D131 Fig. . We werense Fig. . In addinse Fig. . Similarnse Fig. . The expnse Fig. . These rhttps://sisp.shinyapps.io/MGCalculator/.Wound healing assay has been used ubiquitously for the assessment of cell migration in biomedical research but was also known to be confounded by complex influences of multiple cellular phenotypes in wound closure. We have demonstrated in this study how to modify the standard wound healing assay for accurate scoring of different phenotypic contributions. We showed that the conventional wound healing assay cannot be used to quantify accurately the contributions of different phenotypes, often leading to misinterpretation of phenotypic influences by each compound. By combining the MMC pre-treatment protocol with concurrent monitoring of cell death, we showed that the therapeutic effects on cellular phenotypes can be quantitated based on the established relationship of wound closure rate and changes of cell death at different drug concentrations. Fractional contribution of cell death and cell migration can then be approximated from the slope of the fitted relationship. We demonstrated the use of this simple analytical approach for robust screening of both migrastatic and cytotoxic compounds in cholangiocarcinoma cell lines. In addition to identifying potential drug candidates for this rare cancer type, we demonstrated the heterogeneity of therapeutic influences across cell lines and identified common similarity of cell lines based on basal gene expression profiling. Our GSEA analysis of basal transcriptomics identifies cell apoptosis and cell migration as the major differential hallmarks between the two cell line subgroups, confirming the robustness of our platform in faithfully capturing therapeutic effects on different cellular phenotypes. We have made the developed tool available for public use at 10, and current interest in drug repurposing12. Recent studies began to adopt the standard wound healing assay for screening multi-phenotypic responses of compounds with polypharmacologic effects13. Other studies tried to investigate the effect of compounds or natural extract by integrating results from two independent phenotypic assays including both cell viability assay and wound healing assay15. However, such previous studies still required the primary screening to eliminate false positive compound with cytotoxic effect. The disadvantage in interpreting the therapeutic effects using separate assays is the inability to separate out compounds or natural extract that exhibit multi-phenotypic effects at different drug dosing. In contrast, our technique enable scoring of both migration and death impacts at different drug concentrations, and therefore can avoid misinterpretation of therapeutic effects due to drug toxicity as previously seen with Cytochalasin D or Latrunculin A2. In our own study to identify migrastatic drug candidates for CCA, Cytochalasin D exhibited heterogeneous phenotypic response in different CCA cell lines  Cell Bank, Japan. RBE, SSP-25, and TFK-1 were obtained from RIKEN Cell bank, Japan. KKK-D068, KKK-D131, KKK-D138 were obtained from Khon Kaen University, Thailand4 and 50 mM Ascorbic acid in 0.1 M Tris buffer at pH 8.5. Nuclear staining was performed using DAPI (Thermo Fisher Scientific).Mitomycin C (S8146), Staurosporine (S1421), PD0325091 (S1036), Simvastatin (S1792), Sorafenib (S7397), Anisomycin (S7409), SP600125 (S1460) were purchased from Selleckchem. For Cytochalasin D (C8273) was purchased from Sigma-Aldrich and A443654 (HY-10425) was purchased from MedChemExpress. For drug treatment experiments, we treated cells at eight different concentrations using two-fold serial dilution starting from 10 µM. Each drug was dissolved first in DMSO. All treatment conditions contain the same final concentration of DMSO at 0.5% (v/v). Cell death was quantified using the CellEvent™ Caspase-3/7 Green dye (Thermo Fisher Scientific) at final concentration of 1% (v/v). For cell proliferation assay, cells were pulsed with ethynyl deoxyuridine (EdU) at the final concentration of 10 µM for 90 minutes. After cell fixation, EdU was tagged using click chemistry reaction containing 10 µM of carboxyrhodamine-110 azide dye, 4 mM of CuSO19. After being washed 3 times with 1xPBS, cells were incubated with specified conditions for 72 hours. At each time point , we then fixed the cells with 4% paraformaldehyde for 15 minutes at room temperature and stored in 1xPBS. For quantification of cell proliferation, cells were stained with DAPI (Thermo Fisher) and imaged with the Operetta high-content imaging system (PerkinElmer) at 10x magnification. Image analysis and object count were conducted via Columbus 4.8 (PerkinElmer).Cells were seeded at specified densities and incubated overnight. Cells were then replaced with fresh media containing 5 µM MMC and incubated for 3 hours® ImageLock Plates (Essen Bioscience) and incubated overnight. Growth media were replaced with fresh media containing 5 µM MMC and incubated for 3 hours. After being washed 3 times with 1xPBS and replenished with fresh media, cell monolayers were scratched to create a wound (700–800 micron wide) using the IncuCyte® 96-well WoundMaker Tool (Essen Bioscience). Cells were then washed with 1xPBS three times and replaced with 100 µL fresh media. For concurrent monitoring of cell death, media were supplemented with CellEvent™ Caspase-3/7 Green dye (Thermo Fisher Scientific) at 1% (v/v). For drug screening, 10 µL of compounds were directly added to the cell medium to achieve specified final drug concentrations immediately after labeling with Caspase-3/7 Green dye.Cells were seeded at specified densities on 96-well IncuCyte20 (Essen Bioscience) at 10x magnification. To monitor wound closure rate, the percent RWD was generated at each time point for all treatment conditions. For measurement of cell death using the caspase 3/7 activity dye, integrated signal intensity was obtained from different treatment conditions using the same images acquired for the wound closure quantification.Cells with scratched wound were monitored every 2 hours for 3 days using IncuCyte imaging system2 overnight. Cells were then treated with different drugs or vehicle controls at specified conditions. For activation of ERK, AKT, and JNK signaling pathway, we used 100 ng/mL of EGF, IGF , and Anisomycin, respectively. For inhibition controls, we used 10 μM of PD0325901, A443654, and SP600125 to inhibit ERK, AKT, and JNK signaling pathways, respectively. Cells were treated with drugs for 6 hours followed by fixation using 4% paraformaldehyde for 15 minutes at room temperature and washed 3 times with 1xPBS. Then, the cells were permeabilized with absolute methanol for 1 hour at −20 °C followed by washing 3 times with 1xPBS. Prior to primary antibody staining, the cells were incubated with Odyssey® Blocking Buffer (LI-COR Biosciences™) for 1 hour at room temperature to block nonspecific protein binding. After that, anti-rabbit mAB specific to phospho-p44/42 MAPK (Erk1/2) (THR202/Tyr204) (1:300), phospho-Akt (Ser473) (1:300), or phospho-c-Jun (Ser73) (1:300)  was added to the cells followed by an overnight incubation at 4 °C. On the next day, the cells were washed 3 times with 1x PBST followed by staining with donkey anti-rabbit IgG (H + L)-Alexa Fluor® 647 secondary antibody (1:1000)  for 2 hours at room temperature. Afterward, the excessive secondary antibody was washed out 3 times with 1xPBST and once with 1xPBS prior to nuclear staining using DAPI (1:2000)  and whole-cell staining using HCS CellMask Green stain (1:1000)  for 1 hour at room temperature. After 3 washes with 1xPBS, the cells were filled with 200 μl of 1xPBS and the plate was sealed. Cell images were collected at 10X magnification using CLS high-content imaging system (PerkinElmer) and segmented. The phosphorylation levels of kinases were reported as mean fluorescence intensity (MFI) using Columbus 4.8 software (PerkinElmer). For analysis of phosphorylation activity, MFI of kinases in nuclear compartment was used for p-p44/42 and p-c-Jun whereas MFI of kinases in membrane region was used for p-Akt. IC50 values that inhibit phosphorylation of kinases were calculated from drug concentration versus normalized MFI curve fit using GraphPad Prism version 8.0.1 for Windows (GraphPad Software).KKK-D068 cells were seeded at specified densities on CellCarrier-384 Ultra Microplates (PerkinElmer) and incubated at 37 °C, 5% COTo separate the contribution of cell migration and cell death from the observed delayed in wound closure, we generated the relationship of the cumulative changes of cell death signal and the relative wound density.WCxi), we determined the AUC of the RWD from each drug treatment condition (AUC(RWD)xi) and normalized this value by that from the DMSO control group (AUC(RWD)ctrl), all with MMC pre-treatment, using the following formula.For calculation of “wound closure change” (WCind). We can summarize the relationship between the two types of wound closure by the following equation.It is important to note that some drug cannot completely inhibit wound closure. For example, with PD0325901, the generated wound still close to 50% even at the maximum drug dose , we determined the area under curve from caspase 3/7 activity (AUC(D)xi) at each drug treatment condition and normalized this value by that of the DMSO control (AUC(D)ctrl), both with MMC pre-treatment.For “Death change” , a straight line is used instead. If the fitting of straight line is favorable, we concluded that the observed wound change is dependent only on one cellular phenotype. An example of the linear fit is that from Sorafenib treatment where no cell death is observed at any drug concentration . The summation of the migration score (Migrationxi) and death score (Deathxi) should be equal to the drug-induced wound closure change (WCxi). Mathematically, such relationship can be defined as follows:When the relationship is explainable by 1Migrationxi (as x axis) and Deathxi (as y axis). In this landscape, the diagonal line (y = x) represents all conditions without any phenotypic bias, i.e. no-bias line. To measure the phenotypic bias, one can use the no-bias line as the reference, and calculate 1) the perpendicular distance away from the no-bias line or 2) the angular deviation away from the no-bias line. To calculate the perpendicular distance of each condition away from the no-bias line, we can prove mathematically that this distance is equal to Deathxi − Migrationxi)  at 10 was used to filter data with low quality22. The measurement of gene expression, transcription per million (TPM), is used for only genes that have a median of TPM greater than zero. Gene expression data were log-transformed (log2 (TPM + 1)), and processed with R programming language23. Finally, RNA sequencing data were cleaned by surrogate variable analysis (SVA), before further analysis24. Prior to gene set enrichment analysis, average expression levels of genes in CCA cell lines identified as MG-subgroup 1 or 2 were calculated. We then converted the averaged expression level to fold-change level of gene expression of MG-subgroup 1 over 2, ranked the genes based on the fold-change levels from high to low, and performed GSEA using 50 cancer hallmark gene sets from MSigDB9 (version 6.2) and ‘clusterProfiler’ (version 3.10.1) R package25.Total RNA samples were extracted from CCA cell lines using Trizol-based protocol (Geneaid) and purified by RNA specific column (Invitrogen). The quality of RNA was evaluated by Nanodrop (Thermo Scientific), Qubit RNA BR kit (Invitrogen) and Bioanalyzer 2100 (Agilent Technologies). The RNA sequencing libraries were constructed following standard protocol using Illumina-compatible NEBNext® Ultra™ Directional RNA Library Prep Kit . The libraries were sequenced on an Illumina Hiseq2000 using paired-end reads (150-2 bp) at depth 20–25 million reads per sample. STAR method version v2.5.3a is performed to align RNA sequence26 < 0.05 and FDR q-value27 < 0.05. After identification of significantly enriched gene sets, leading-edge genes, which highly contribute to NES, from each of the gene sets were collected. From this gene list, we identified 20 genes that were also expressed in our cell lines and further considered as potential MG subgroup biomarkers. Principle component analysis was also performed to confirm the correlation between the identified biomarkers and MG subgroups. These data were processed and visualized using ‘pheatmap’ (version 1.0.12) and ‘factoextra’ (version 1.0.5) R packages.As a result, we obtained the cancer hallmark gene sets with normalized enrichment scores (NES) and p-values, representing ‘bird eye-view’ of the transcriptomic profiles of MG-subgroup 1 compared to subgroup 2. Significant cancer hallmark pathways were selected based on Benjamini-Hochberg adjusted p-value29 was used and we collected the enrichment results from ‘GO biological process 2018’ feature. Any GO terms with nominal p-value < 0.05 and FDR-adjusted p-value < 0.5 were excluded and we selected the top 5 GO terms from each MG subgroup.To analyze GO term biological functions of our novel MG subgroup biomarkers, the web-based enrichment tool EnrichrSupplementary information


Here we report there is a frequent loss-of-function of polycomb repressive complex 2 (PRC2) in OS cell lines but it is rare in tumor samples based on genomic sequencing data, western blotting and immunohistochemistry analysis of H3K27me3. U2OS and 143B cell lines have a complete loss of function of PRC2 and several others have partial loss. In OS tumor tissues, only 1 out of 14 has low expression of H3K27me3. Kaplan-Meier analysis indicates that high EZH2, the component of PRC2, is associated with poor metastasis-free survival. Our observations are to raise the alarm that particular caution should be taken when using OS cell line models to study the disease, functional genomics, therapeutic target validation, drug screening, and epigenetic studies. Nevertheless, these cell lines will become useful biological tools to dissect the functions of PRC2.Osteosarcoma (OS) cell lines are widely used in understanding the biological functions of cancer, identification and validation of therapeutic targets, as well as  For example, U2OS is one of the most frequently used cell lines in cancer biology research and Google Scholar had at least 29,000 records for this cell line. Twenty years ago, cancer biologists realized that gene expression and phenotype, including drug response, vary greatly due to culture conditions and passage numbers. These differences are one explanation for variations in biological processes in the in vitro and in vivo settings, which causes difficulties for data reproducibility. Here we report a frequent loss-of-function of polycomb repressive complex 2 (PRC2) in OS cell lines. This observation appears to be a rare event in primary tumor samples, and therefore may be a unique feature of some OS cell lines.Osteosarcoma (OS) is the most prevalent primary bone tumor and has a high propensity to metastasize. Although the 5-year survival rate has increased from about 20% to 65–70% after the introduction of chemotherapy in the 1970s, no significant improvements have been made since then .–21.19–21osarcoma .in vivo in OS. Although it is likely that the genetic/epigenetic loss-of-function of PRC2 in OS cells is due to selection under culture stress or intrinsic genetic instability, there is a third possibility that these cells were derived from rare clones that expanded when the cell lines were established using bulk tumor cells, especially for samples taken from relapsed, metastasized, or refractory disease. In the future, whether PRC2 is involved in metastasis of osteosarcoma warrants further studies. Our observations suggest more validation studies should be done when using OS cell line models to study the disease, functional genomics, therapeutic target validation, drug screening, and epigenetic studies.Despite the importance of PRC2 in cancer, functional studies of PRC2 in osteosarcoma are scarce. Nevertheless, our finding does not mean that the loss-of-function of PRC2 complex does not happen Cell lines without detectable H3K27me3 have rarely been reported. Our discovery that OS cell lines have PRC2 function defects indicate these cell lines might be useful tools for further dissection of regulatory mechanisms of PRC2 mediated and independent functions. EZH2 has been reported to act as a transcriptional coactivator for androgen receptor , which iPlease see


NRAS(Q61) in a set of BRAF(V600E) melanoma lines and vice versa. Controlled expression of a second activating oncogene led to growth arrest (“synthetic suppression”) in a subset of cells, which was accompanied by cell cycle arrest and senescence in several melanoma cell lines along with apoptosis. Through differential gene expression analysis, we identified SPRY4 as the potential mediator of this synthetic response to dual oncogene suppression. Ectopic introduction of SPRY4 recapitulated the growth arrest phenotype of dual BRAF(V600E)/NRAS(Q61) expression while SPRY4 depletion led to a partial rescue from oncogenic antagonism. This study thus defined SPRY4 as a potential mediator of synthetic suppression, which is likely to contribute to the observed exclusivity between BRAF(V600E) and NRAS(Q61R) mutations in melanoma. Further leverage of the SPRY4 pathway may also hold therapeutic promise for NRAS(Q61) melanomas.The underlying forces that shape mutational patterns within any type of cancer have been poorly characterized. One of the best preserved exclusionary relationships is that between BRAF(V600E) and NRAS(Q61) in melanomas. To explore possible mechanisms which could explain this phenomenon, we overexpressed  One of the most commonly activated networks is the RAS-MAPK, which impacts both cancer cell proliferation and survival  − [Vector(+Dox) log2-expression − Vector(-Dox) log2-expression]. The entire expression set in log2 expression levels is shown in Table Cells were seeded 24 h before treatment with or without doxycycline 50–100 ng/ml for a defined time of 4–5 days. RNA was extracted from 2 × 10NRAS* + iBRAF* and GMELBRAF* + iNRAS* but NOT A375BRAF* + iNRAS* (Table https://david.ncifcrf.gov/) functional clustering annotation under the default mode. Per DAVID’s website for functional clustering, “The geometric mean  of member's p values in a corresponding annotation cluster, is used to rank their biological significance. Thus, the top ranked annotation groups most likely have consistent lower p values for their annotation members.” Annotation clustering Enrichment Scores are presented in Tables p value in the cluster.Probes that exhibited >2-fold change (+1.0 or −1.0 log2-fold) for each line Tables -S4 and fS* Table  were subNRASQ61R cells constitutively expressing either control CD516B2-vector or CD516B2-SPRY4 in 0.1 ml 10% DMEM growth medium per flank of mice. Animals were monitored twice weekly for 7 weeks. Body weights and tumor size were measured every week as described previously [N = 8. Tumor histology was confirmed by hematoxylin/eosin staining of formalin-fixed and paraffin-embedded tissue. Animals were maintained in well-ventilated animal facility and tested in accordance with the MGH Animal Care and Use Committee guidelines.Sixteen Nod-SCID-gamma male mice of 6-week-old  were divided into two groups, eight mice per group were injected subcutaneously (s.c.) with 0.2 million SK-MEL-119eviously . Data wel-lysine-coated slides. The assays were performed as described previously [2O2 solution for 15 min at 25 °C followed by protein blocking serum  for 30 min. After this, tissue sections were incubated for 1 h, with primary antibodies such as anti-Ki67 for proliferation, anti-sprouty4 for overexpression, ß-galactosidase for senescence, followed by a secondary alkaline phosphatase antibody, either MACH2 mouse AP-polymer detection  or MACH 2 rabbit AP-polymer detection  for 30 min. The slides were developed with Vulcan Fast Red Chromogen kit2  and counter stained with Mossberg hematoxylin (Vector H-3401). The colorimetric signals were detected with digital slide scanner NanoZoomer-2.0HT and analyzed by NDP.view2 software . (2) H&E, after rehydration hematoxylin and eosin staining was carried out using the Mossberg labs protocol. (3) TUNEL staining was performed using Dead End Fluorometric TUNEL kit (Promega) according to the manufacturer’s instruction.Immunohistochemistry, phosphate-buffered formalin fixed tumor tissues were paraffin-embedded and 5-µM-thick sections were collected onto poly-eviously . BrieflyBRAF* (CONFIRMED), GMELBRAF* (CONFIRMED), WM1361NRAS* (CONFIRMED), SK-MEL-63NRAS* (CONSISTENT) and MGH-CH-1BRAF* (COMPATIBLE). The cell lines were thawed and collected between third or fourth passages for original naive and stably expressing transductant cells. After experiment, the cell lines were either used up or discarded at 15th to 25th day. All used cell lines were mycoplasma tested as instructed by the company (Invitrogen).All cell lines were stratified to three levels of confidence after STR genotyping and MITF expression analysis. Those lines that matched the STR genotyping database were considered “CONFIRMED” as the designated line. Cell lines with no STR hits but had mutational data at melanoma driver loci  were manually compared to public datasets such as COSMIC or individually referenced papers. If the mutational profile of the cell line matched independent public data (i.e. results not from our laboratory), these lines were labeled “CONSISTENT” with the designated line. Lastly, cell lines that were either newly developed or lacked STR information and public domain information were experimentally assayed for MITF levels by RNAseq. Those that showed MITF expression were considered “COMPATIBLE” with melanoma. The following represents the level of confirmation for cell lines in this study: A375p = 0.05, as usual.Data from different experiments were represented as means ± SD from at least three independent experiments. To analyze cell viability, linear regression analysis was performed using GraphPad Prism 7.0 . Significance was established at The mice experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of MGH.Supplementary fig 1supplementary fig 2supplementary figure 3supplementary figure 4supplementary figure 5supplementary figure 6supplementary figure 7supplementary figure 8supplementary figure 9supplementary figure 10supplementary figure 11Materials and Methods-SUPPLEMENTARYKumar et al, NRAS BRAF Oncogene TABLES SUPPLEMENTARY


Mechanistic approaches to modeling the effects of ionizing radiation on cells are on the rise, promising a better understanding of predictions and higher flexibility concerning conditions to be accounted for. In this work we modified and extended a previously published mechanistic model of cell survival after photon irradiation under hypoxia to account for radiosensitization caused by deficiency or inhibition of DNA damage repair enzymes. The model is shown to be capable of describing the survival data of cells with DNA damage repair deficiency, both under norm- and hypoxia. We find that our parameterization of radiosensitization is invariant under change of oxygen status, indicating that the relevant parameters for both mechanisms can be obtained independently and introduced freely to the model to predict their combined effect. Radiation therapy is one of the cornerstones of cancer care, where ~50% of patents receive radiation during the course of disease . RadiobiTo model the response of hypoxic tumor cells to dual treatment, we based our approach on a previously published model of our group . Similar studies ,23. The  studies ,13 assoctin loss  and theytin loss ,25,26. Ital data ,26.Regarding the hypoxic cell population, Carlson et al.  made com context . In lineng rates , the letn et al.  (a functFlanders ) 11]. I. IHRFDSBRSF) that modifies To further extend the model and describe the particular case of hypoxic tumor cells response to dual treatment, radiotherapy, and DDR inhibition, we introduced a so-called radiosensitization factor  exposed to three distinct oxygenation levels  for each of the mutant cell lines, their survival under normoxia was able to be fitted accurately. The numerical values of the parameters can be found in RSF derived for each mutant under normoxia, the survival of the two mutant cell lines under hypoxia could be predicted satisfactorily. However, deviations can be observed at the highest reported doses.Survival data of the CHO cell line and two of its DNA-PK response-deficient mutants (V3 cell line is DNA-PKcs-deficient and xrs-5 cell line is Ku80-deficient) under normoxia and hypoxia were gathered from Cartwright et al.  30]. Th. Th30]. RSFs for the lethality parameters of the isolated lesions  exposed to different concentrations of an ATM inhibitor (ATMi) and irradiated under normoxia and hypoxia . First, Our model provided an excellent description of the survival data of five cell lines and three oxygen levels presented in RSF) for the two DNA-PK response-deficient mutants analyzed. Therefore we decided to introduce the RSF as a free parameter in UNIVERSE, fitted to each mutant cell line. The RSF values found for the two CHO mutants V3 and xrs5 indicate that the probability of an isolated lesion to become a lethal lesion increases through DNA-PK response deficiency in these cell lines by a factor of about 10 and 15, respectively. Even though both RSF factors are related to the functional DNA-PK repair activity, the difference in the RSF of both cell lines might be retraced to the fact, that both are deficient of different enzymes taking part in the DNA-PK response: While xrs-5 cells are deficient of the Ku80 DNA-PK subunit [RSF is coupled to the activity of both proteins. Furthermore, different extents of remaining expression or compensation by other repair proteins might also lead to the observed difference in the RSF values of both mutants. One would have to compare several groups of cell lines with the exact same deficiencies to gain a better understanding of the underlying dependencies. More importantly, we could show that the survival of the mutant cell lines under hypoxia were well predicted by our model by combining the RSF values derived from the normoxic data of each mutant invariant.The idea of increasing the lethality of isolated lesions LE model . They cot et al.  and foun subunit ,31, V3 c subunit ,32. It iRSF for isolated lesions. Further, the values of the derived RSF illustrate the increasing lethality of isolated lesions with increasing drug concentrations, leading to stronger inhibition of the repair, while staying below the value derived for a pathway deficiency. It is also to be noticed that the RSF values for both H460 and H1437 cells are fairly similar. However, more cell lines have to be analyzed to investigate whether this can be extended to a general trend and if the presented method is generally applicable to other repair-inhibiting drugs. Nevertheless, we could show that the RSF values derived from normoxic data can accurately describe the survival in hypoxic conditions by introducing the Since both DNA-PK and ATM are essential molecules for irradiation-induced DNA damage repair, recruited to the DNA damage sites ,34,35, wRSF in our model. Moreover, this modified lethality could stay invariant under change of oxygen supply, while sustaining good predictive capabilities. Thus, the uniqueness of our approach lies in its capability to describe two separate cell response mechanisms in any combination using minimal input parameters, which can be separately derived. Based on this, we believe that our approach has a high potential to implement further cellular mechanisms in order to produce predictions tailored to diverse clinical scenarios.Taken together, we could show that impairment of DNA damage repair, both in repair-deficient cell lines and cells treated with a DDR inhibitor, could be accounted for by a manipulation of the lethality of isolated lesions with an Experimental data used to benchmark the model on survival of genetically DDR-deficient cell lines were taken from . The dat2 atmosphere. Experiments in hypoxic conditions were performed at 0.5 or 1% O2 and 5% CO2 using a custom hypoxic chamber , including an online monitoring controller for O2 and CO2 concentrations . Fifty cells per well in a 96-well format were seeded not more than 16 h before irradiation. Hypoxic irradiation was performed after incubation for 4 h under respective oxygen conditions. Cells were irradiated in the sealed hypoxia chamber with a dose series of photons of 1, 2, 4, or 8 Gy and thereafter incubated under normoxic conditions. The ATM inhibitor was kindly provided by Merck KGaA, and dissolved in DMSO  and diluted in RPMI 1640 medium. The inhibitor was added to H460 and H1437 cells at 100, 200, or 500 nM just before incubation under hypoxia or normoxia started. Controls also contained < 0.1% DMSO. Inhibitors were left in the media for 24 h and then replaced with fresh RPMI 1640 medium and the plates were returned to the incubator for colony formation. After 4 days (A549), 5 days , or 7 days (H1437), plates were imaged by an online microscopy system at 4x magnification . The images were analyzed by the IncuCyte Zoom Software (ver. 2016a)  and colony counts were confirmed by manual curation.For the validation of our model under hypoxic conditions, additional experiments were performed using Renca  and B16-Blue ISG  (B16) cells. Both cell lines were grown in RPMI 1640 Medium  supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% COThe irradiation plan was carried out as a step and shoot intensity-modulated radiotherapy (IMRT) plan, describing the different dose levels as separate target regions. Planning was done with Raystation treatment planning system  based on a CT scan of the hypoxia chamber containing 96-well plates filled with water. Irradiation was performed on a Siemens Artiste (6 MV) .D the applied dose in Gy. The total number of giant loops  is given by [S values obtained from the Monte Carlo algorithm. The lethality parameters can be determined by fitting the result of this routine to survival data.We implemented a Monte Carlo routine in which, at each iteration, the number of total DSB in the nucleus (given by :(3)S= was introduced into the model. The RSF modifies the lethality parameter of isolated damages In order to model the increased cell killing of repair-deficient mutant cell lines or cells exposed to different concentrations of a repair inhibiting drug, a radiosensitization factor  to repair impaired data, while the lethality parameters


In vitro methods using fish cell lines for the assessment of aquatic toxicity are needed for mechanistic studies and as an alternative to in vivo. We describe an in vitro assay to study oxidative stress using zebrafish cell lines. Transfection efficiency of twelve commercially available transfection reagents were tested in the zebrafish cell lines ZFL, ZF4, and Pac2. The most efficient reagent for each cell line was selected for further experiments. Cells were transiently transfected with an Nrf2-responsive luciferase plasmid. The assay was tested using the oxidative stress inducing chemicals tertbutylhydroquinone, hydrogen peroxide, and sulforaphane. Of the transfected cell lines, ZF4 and ZFL showed higher sensitivity. The latter were used to study potential oxidative stress induced by pesticides . Besides known inducers, Nrf2 activity was also significantly induced by diazinon, deltametrin, diuron, and metazachlor. Activation of Nrf2 by metazachlor is a novel finding. The described assay could be a valuable tool for research in toxicology to study the stress response of both pure chemicals and environmental water samples.The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense against oxidative stress and correlated with classical toxicological endpoints.  The nuclear factor erythroid 2-related factor 2 (Nrf2) is a keystone in the cellular defense against oxidants and the regulation of oxidative stress detoxification, metabolism, and induction of related phase-II enzymes (glutathione S-transferase (GST), NADPH-quinone oxireductase 1 (NQO1), heme oxygenase 1 (HO-1), g-glutamylcysteine synthetase (g-GCS), thioredoxin (TRX))2. The antioxidant responsive element (ARE) sequence (“RTGACnnnGC”)4 is located upstream in the regulatory region of the phase-II metabolizing enzymes genes, often coinciding with the xenobiotic response element (XRE)5. Nrf2 occurs ubiquitously under normal physiological conditions in the cytosol where it is bound to its negative regulator Kelch-like ECH-associated protein 1 (Keap1), which marks it for degradation6. Upon conditions of oxidative stress Nrf2 is released from Keap1 and avoids proteolysis. Nrf2 then translocates and accumulates within the nucleus where it heterodimerizes with small Maf proteins and initiates gene expression7 by binding to AREs. The Keap1-Nrf2-ARE signaling pathway is highly conserved within vertebrates10. In ToxCast  high-throughput data on toxicity, e.g. Nrf2 activity, of single chemicals are collected. Nrf2 activity has been shown to be correlated with classical toxicological endpoints in vivo and it has therefore been suggested that oxidative stress response and Nrf2 activity would be an ideal pathway to build in vitro toxicity assays on11.Oxidative stress, with formation of reactive oxygen species (ROS), is a mechanism underlying a number of toxicity processesIn vivo assays are extensively used in assessment of aquatic toxicity of chemicals. The high number of animals used in environmental risk assessment has highlighted the need for novel approaches and development of in vitro assays. Development of fish cell culture-based in vitro assays has been proposed as a promising idea to reduce and replace the use of fish in aquatic toxicity testing, e.g. by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)14. Development of such in vitro models would allow subsequent high throughput screening and application of omics technologies. Cell cultures and embryo tests have emerged as useful alternative approaches in environmental toxicology15.19. However, the use of fish cell lines for reporter gene assays has been very limited and this type of approach is still in its infancy within aquatic toxicology. A requirement for the development of reporter gene assays, is the availability of functioning transfection methods. Most transfection reagents were developed for mammalian cell lines and are based on lipid-fusion mechanisms. However, the latter might be less efficient in fish cells due to lower incubation temperatures20. Furthermore, the transfection efficiency for a specific transfection regent often varies greatly between cell lines21. Thus, in the present investigation cells of different developmental stages and tissue origin were tested.Reporter gene assays in transfected mammalian cells have been shown to be a valuable tool for research in many fields of toxicology22. The established in vitro bioassay might be a useful tool in screening for potential inducers of oxidative stress, both regarding toxicity of pure compounds and for analysis of environmental samples, e.g. for ecotoxicology and environmental monitoring.In this study, we have established a reporter gene assay for detecting oxidative stress by measuring induction of free Nrf2 in zebrafish cell lines Pac2, ZF4, and ZFL. Initially, we tested the transfection efficiency of twelve commercially available transfection reagents for these three cell lines. The established reporter gene assays were tested with potential Nrf2 inducers and six pesticides which are suspected to cause oxidative stress24 (CVCL_5853) was cultured in Leibovitz’s L-15 medium containing phenol red , supplemented with 15% fetal bovine serum , 1% penicillin-streptomycin 100 U/mL , 1‰ gentamicin 50 mg/mL . The cells were cultured in a humidified environment at 28 °C and atmospheric CO2. The cells were passaged weekly in a 1 to 3 ratio, using PBS  for washing and 0.25% Trypsin-EDTA  for detachment.Zebrafish fibroblast cell line Pac 225 (CVCL_3275) was cultured in Dulbecco’s modified Eagle’s medium/Ham’s Nutrient Mixture F-12 (DMEM:F12) containing phenol red , supplemented with 10% fetal bovine serum , 1% penicillin-streptomycin 100 U/mL , 2.5 mM L-glutamine , 15 mM HEPES , 0.5 mM sodium pyruvate , and 1200 mg/L sodium bicarbonate . The cells were cultured in a humidified environment at 28 °C and with 5% CO2. The cells were passaged every 3 to 4 days in a 1 to 3 ratio, using PBS  for washing and 0.25% trypsin  for detachment.Embryonic zebrafish fibroblast cell line ZF427 (CVCL_3276) was cultured in a medium consisting of 50% Leibovitz’s L-15 , 35% Dulbecco’s modified Eagle’s medium , 15% Ham’s Nutrient Mixture F-12 , and phenol red. Additionally, 150 mg/L sodium bicarbonate , 15 mM HEPES , 10 µg/mL bovine insulin , 50 ng/mL mouse EGF , and 5% fetal bovine serum  were supplemented. The cells were cultured in a humidified environment at 28 °C and atmospheric CO2. The cells were passaged every 3 to 4 days in a 1 to 4 ratio, using PBS  for washing and 0.25% Trypsin-EDTA  for detachment.Zebrafish liver cell line ZFLPhotinus pyralis). A plasmid including the cDNA (Rluc) encoding Renilla luciferase reporter gene (Renilla reniformis) was used as a control of transfection efficiency and for normalization.The pGL4.37[luc2P/ARE/Hygro] plasmid was acquired from Promega, Madison, USA. The pGL4.37[luc2P/ARE/Hygro] vector consists of a pGL4 backbone including an ampicillin resistance gene, a gene for hygromycin resistance, and four copies of an Nrf2-sensitive antioxidant response element (ARE) driving transcription of the luciferase reporter gene luc2P (2O2) (CAS 7722-84-1), Sigma-Aldrich, Steinheim, Germany; metazachlor (CAS 67129-08-2), 99.5% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; sulforaphane (SFN) (CAS 4478-93-7), 90% purity, Sigma-Aldrich, Steinheim, Germany; terbutylazine (tBA) (CAS 5915-41-3), 99.5% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; tertbutylhydroquinone (tBHQ) (CAS 1948-33-0), 97% purity, Sigma-Aldrich, St. Louis, USA.The following chemicals were used for exposure studies: atrazine (CAS 1912-24-9), 99.3% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; deltamethrin (CAS 52918-63-5), 99.5% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; diazinon (333-41-5), 99% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; Dimethyl sulfoxide (CAS 67-68-5), 99.9% purity, Sigma-Aldrich, St. Louis, USA; diuron (CAS 330-54-1), 98.8% purity, Dr. Ehrenstorfer GmbH, Augsburg, Germany; hydrogen peroxide  in 100 µl/well. After 24 h incubation at above stated conditions, cells were transiently transfected with the Renilla plasmid only. Varying reagent to DNA mass ratios were applied in quadruplicates according to the manufacturer’s instructions. Mirus, Promega, Roche, and Invitrogen derived reagents were applied in a 1 µg DNA to 2, 4, or 6 µg reagent mass ratio, SuperFect in a 1 µg DNA to 1, 5, or 10 µg reagent mass ratio, Effectene in a 1 µg DNA to 10, 25, or 50 µg reagent mass ratio and jetPrime in a 1 µg DNA to 1, 2, or 3 µg reagent mass ratio. Total background Renilla luciferase induction for the single ratios in comparison to solvent control was considered as the benchmark. Subsequently, for most promising candidates, series of different cell concentrations  were prepared and transiently transfected at the optimal ratio, in order to determine the best DNA-reagent-ratio to cell-density parameters. Induced background luminescence was scored identically to procedures described in the following section, with the only difference that a single luminescence signal was required.Twelve commercially available transfection reagents: TransIT-X2®, TransIT®-LT1, TransIT®-2020 ; FuGENE® HD (FHD), FuGENE® 6, ViaFect™, TransFast™ ; X-tremeGENE HP (XHP) ; Lipofectamine® 2000 ; SuperFect®, Effectene® ; jetPRIME®  were tested for suitability to zebrafish cells and maximal induced transfection efficiency. Cells of all three mentioned cell lines were seeded at a density of 104 cellls/well for Pac2, 1.25*104 cells/well for ZF4, and 2.5*104 cells/well for ZFL in 100 µl/well. After 24 h of incubation, cells reached a confluency of about 80%. Transient transfection using FHD or XHP, respectively was conducted in a 2 µg reagent to 1.2 µg DNA  ratio, according to the manufacturer’s instructions. After 24 h of post-transfection incubation, cells were exposed to postulated Nrf2 inducers or pesticides. Prepared stock solutions  were further diluted using the cell type specific nutrition medium (see section “Cell culture”) supplemented with 5‰ EtOH as a solvent. Seeded cells on 96-well plates were either exposed in technical quadruplicates to increasing concentrations  of postulated Nrf2 inducers tBHQ, SFN, and H2O2 or to increasing concentrations  of the pesticides atrazine, deltamethrin, diazinon, diuron, metazachlor, and terbutylazine. Double quadruplicates of 5‰ EtOH solvent-nutrition medium were used as controls. In parallel with the oxidative stress exposure, increasing DMSO concentrations  were tested as a cytotoxic control. After 24 h incubation, cells were lysed and quantitative Nrf2-dependent luminescence was measured via Dual-Luciferase® Reporter assay according to the manufacturer’s protocol using an auto-injecting Infinite M1000 microplate reader . The luciferase activity was expressed as fold change compared to the controls.Above mentioned cell lines were transiently co-transfected with the pGL4.37[luc2P/ARE/Hygro] plasmid and the Renilla plasmid using FHD  for Pac2 and ZF4, and XHP  for ZFL. Subsequently, after exposure to known Nrf2 inducers or pesticides, Nrf2 induction was analyzed using a Dual-Luciferase® Reporter (DLR™) assay . Cells were seeded into white, clear-bottom 96-well plates  at a density of 10In order to determine cytotoxic concentrations of used compounds within the exposure range, cell viability was examined using an MTS-based [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] CellTiter 96® AQueous One Solution Cell Proliferation Assay  in accordance with the manufacturer’s protocol. Viability tests were conducted in parallel to the dual reporter gene assays, applying the same arrangements and exposure set-ups. Cells were seeded in transparent 96-well plates  and transient transfection was omitted. Simultaneously to transfected cells being lysed for scoring of luminescence signal, nutrition medium of cells being used in viability test was discharged and replaced by PBS containing 17% (v/v) MTS-reagent. After 2 h of incubation, formazan product turnover absorbance was measured at 490 nm using a Wallac Victor2 1420 microplate reader . Relative effects on cell viability were calculated in relation to the vehicle control.28. Thereby, transformed output data was considered as a fixed factor whereas experiments were considered as a random factorial factor within the model in order to account for inter-experimental variation. A p < 0.05 was considered statistically significant. Normality (quantile-quantile-plot) and homoscedasticity  of data sets were checked via graphical analysis of residuals.Results from the dual luciferase assay and the viability assay were processed using R and GraphPad Prism 7 . Graphs and illustrations were designed using GraphPad Prism 7. For both assays data of three or four experiments , each performed with quadruplicate samples for each concentration, were pooled and log-transformed to achieve homoscedasticity, giving a total population size for every exposure group of 10–16 . Data was normalized against internal background within the dual reporter gene assay and against the negative control, giving fold induction as a final output. Significant differences between the control and the exposure groups were analyzed via a mixed-model two way-ANOVA followed by Dunnett’s post-hoc test4 cells/well and a 4 µg FHD to 1 µg DNA ratio . The cells were transfected with a Renilla luciferase plasmid in different reagent to DNA mass ratios, according to the manufacturer’s instructions. The total Renilla luminescence was analyzed as a measurement of transfection efficiency. Additionally, increasing cell densities were tested in the best detected DNA to transfection reagent ratios (data not shown). The following transfection parameters were found to result in the highest transfection efficiency for Pac2, ZF4 and ZFL, respectively: Pac2, plating density of 10tio Fig. . ZF4, pltio Fig. . ZFL, pltio Fig. . Finally2O2 in concentrations of 0.1 to 100 µM for 24 h. DMSO was used as a cytotoxic control. In parallel to detecting Nrf2-activation via the dual reporter gene assays, cell viability was measured within the same experimental set-up using the MTS-assay.All three zebrafish cell lines were co-transfected with an Nrf2-responsive Firefly-luciferase plasmid and a normalizing Renilla-luciferase plasmid. In order to quantify cell line specific responses, cells were exposed to known or postulated Nrf2 inducers tBHQ, SFN, and HIn the Pac2 cell line Fig.  there wa2O2 , as compared to the Pac2 cell line. Therefore, the latter was not further tested since in addition fibroblast-like cells were already covered by ZF4. To test the applicability of the assay, ZF4 and ZFL cells transfected with the Nrf2 sensitive luciferase plasmid were used to investigate the potential oxidative stress potency of six pesticides.In the ZF4 cell line, we found that the Nrf2 activity was significantly increased by the pesticides deltamethrin Fig.  at 50 µMIn the ZFL cell line, statistically significant Nrf2 activation was observed for diuron Fig.  2.8- fo at 12.5 29. Oxidative stress may lead to toxicity e.g. via enzyme deactivation, lipid peroxidation, and DNA damage. These effects may lead to tissue damage, apoptosis or cell necrosis30 and further to developmental toxicity32 and cancer33. So far, in vivo methods for determination of Nrf2 activity have been established in zebrafish strains36, in vitro methods using mammalian cells are described40, and standardized high-throughput assays in human cell lines41 have been developed. Here, we report the development of a reporter gene assay for oxidative stress response in zebrafish cell lines.The Keap1-Nrf2-ARE signaling pathway is a major balancer of oxidative homeostasis, given its role in oxidative stress response but also physiological regulationin vitro assays using transfected zebrafish cell lines.In this study, we have tested the transfection efficiency of twelve commercially available transfection reagents in three different cell lines and found a high variability in efficiency between the transfection reagents. We found that FuGENE HD (Promega) showed the highest transfection efficiency in both Pac2 and ZF4, while X-tremeGENE HP (Roche) and jetPRIME (Polyplus) showed the highest transfection efficiency in ZFL. These are important findings for future efforts to establish 42. In order to test single cell line feasibility, each transfected cell line was first exposed to postulated Nrf2 inducers. The results showed that the Nrf2 activity following exposure varied between cell lines. The Keap1-Nrf2-ARE signaling pathway is not functional immediately after fertilization and the responsiveness to oxidative stress seems to vary during embryogenesis and larval development44. Within 24 hpf, the response to oxidative stress or Nrf2 inducers is quite low, increases up to 48 hpf, exhibits high variability during hatching and post-hatching, and finally stabilizes at around 96 hpf. This could be an explanation for the observed differences in sensitivity between the three investigated cell lines, as they are derived from zebrafish in different developmental stages. In addition, an impact of total serum concentration in the culture medium and therefore less available, non-protein bound compound concentration could be an alternative explanation for the differences in response between these zebrafish cell lines. Pac2 cells are cultured at a higher serum concentration (15%) than ZF4 and ZFL cells (10 and 5% respectively), which might explain the observed differences in sensitivity. Since Nrf2 is ubiquitously expressed in most cell types and acts via a common mechanism, its activity is generally only assayed in a single cell type when using bioassays based on mammalian cells47. However, our result show that cell line selection is crucial for this assay and further research is needed to fully explain the observed differences in oxidative stress sensitivity in zebrafish cell lines.We have analyzed the Nrf2 activity in three zebrafish cell lines; Pac2, ZF4 and ZFL. The Pac2 and ZF4 cell lines are pooled embryonic fibroblasts, whereas the ZFL cell line was established from hepatocytes derived from a single adult individual. In this way, different developmental stages and tissue types are represented in the study. Hepatocytes were chosen since the liver is the primary location of detoxification and show highest abundancy in critical phase II enzymes, such as GSTs2O2 in any of the cell lines studied, although H2O2 is normally metabolized by GSTs which are Nrf2 regulated. However, Nrf2-recruitment failure by H2O2 has previously been described during development in embryos49 and in larvae36. The phenomenon is either interpreted as a systemic breakdown of the Nrf2-Keap1 axis48 or absence of an intermediate “posthatch factor” which is induced by H2O2 and then modulates the Nrf2 response35. On the contrary, Nrf2 induction by H2O2 was reported in mammalian cell lines50. Species differences, methodological aspects, since H2O2 is a very reactive compound with short half-life, and interactions with media components may partly explain why we did not see any Nrf2 induction by H2O2.Surprisingly, we were unable to show an Nrf2 induction by H22 were analyzed. We found that four out of the six analyzed compounds significantly increased the Nrf2 activity in at least one cell line. The dose-effect relationship for the compounds differed, where some compounds showed a clear dose-dependent increase in the Nrf2 activity, while other compounds showed a threshold-like effect with an increased Nrf2 activity only observed within the highest exposure concentration. However, those compounds would need to be tested at higher, non-toxic concentrations in order to exclude dose-dependency. These different patterns may be explained by differing mechanisms by which the compounds are inducing the Nrf2 release from Keap1 and thereby trigger the oxidant stress response. Such a mechanism-related model has been proposed by Kobayashi, Yamamoto, and co-workers52.To test the potential application of the established assay, six pesticides suspected to cause oxidative stressCarassius gibelio)54, spotted snakehead (Channa punctata)56, carp (Cyprinus carpio)57, and bushynose (Ancistrus multispinis)58, whereas other species did not show a similar response to deltamethrin59. We report an effect in ZF4, but no effects were observed in ZFL. Deltamethrin is primarily metabolized by phase I enzymes CYP1A1, CYP1A2, and carboxylesterases61. The lack of effect in the ZFL cell line may therefore be due to a faster metabolism of the compound within hepatocytes, since functional phase I activity has been reported in vitro63 on transcript and protein levels, although also other mechanisms might explain the observed difference in response in ZFL and ZF4. Alterations in antioxidant enzymes, GSH, and LPO levels have also been reported in carp (Cyprinus carpio) after exposure to diazinon65. Beyond that, histopathological endpoints had been identified in bluegill sunfish (Lepomis macrochirus)66. Diuron has been reported to induce oxidative stress after chronic exposure to goldfish (Carassius auratus)67, alternating GST and CAT levels in tropical fish (Astyanax sp.)68, and genotoxicity in rainbow trout liver and gill cell lines 69. An induced Nrf2 activity by diuron and diazinon has also been reported in human liver hepatoma cell line (HepG2) at comparable concentrations11. Metazachlor was the most potent inducer of Nrf2 activity, showing a higher potency than the known inducers used as positive controls. No studies on oxidative stress response by metazachlor were found in the literature. However, regulatory toxicity testing has demonstrated low acute toxicity in rainbow trout (Oncorhynchus mykiss), carp, and bluegill sunfish70. In mammals limited evidence of carcinogenic effects has been reported besides acute toxicity71. To our knowledge, this is a novel finding on the toxicity of metazachlor in vertebrates and prompts further investigation regarding the toxicity of metazachlor.It has been reported that deltamethrin is causing alterations in antioxidant enzymes (glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)), reduced glutathione concentration levels (GSH), and lipid peroxidation levels (LPO) in Prussian carp (We describe the development and application of a novel Nrf2 responsive reporter gene assay to monitor oxidative stress response in various zebrafish cell lines. Further, we have shown that the developed assay is useful for chemical testing by analyzing the effect of postulated Nrf2 inducers and compounds which were suggested to induce oxidative stress in fish. We suggest that this model could be a valuable tool for future research in aquatic toxicology to study the toxicity of both pure compounds and environmental samples.Supplementary Information


Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines . Materials and methods: Cell lines were conditioned in three studied media  + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines , than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times. Long-lived cancer cell lines, which have been derived from patient tumor cells, have been widely used to explore biology of breast cancer and new therapies in the past and currently remain one of the main models for investigation of breast cancer . Still, The complexity of the breast cancer microenvironment with respect to gene expression profiles, signaling pathway activity and drug sensitivity is better recapitulated and modelled by three-dimensional heterotypic culture systems than by adherent cell culture . HoweverGATA3, FOXA1 [TBX3 [HEY1 and HES1, that has been shown to promote luminal differentiation [SOX9, ELF5, FOXM1, FOXQ1, VGLL1, EZH2 [ID3, TBX2. Basal/myoepithelial signature has most often been termed mammary stem cell (MaSC) signature, since basal layer of the mammary gland contain cells with properties of MaSC [Gene expression profiling studies of clinical samples from breast tumors have identified the intrinsic luminal, HER2-enriched, basal, claudin-low and normal breast cancer subtypes ,12,13, a3, FOXA1 ,24, tranA1 [TBX3 , as wellntiation ,27. HER2ntiation . Basal tntiation , while sL1, EZH2 . ClaudinL1, EZH2  with exp of MaSC . Althoug of MaSC . In this study, we assessed the effects of basal culture media and hormones/growth factors used in our laboratory to isolate and culture human breast cancer cells from core biopsies of primary tumors. The set of hormones/growth factors used in the study contained mostly mitogens  supposedly enhancing proliferation of at least some populations of breast cancer cells, and factors  promoting attachment and spread of the breast cancer cells and formation of focal contacts, i.e., promoting epithelial-like features. The studies the concerning role of these hormones and factors in the culture of breast cancer and/or mammary epithelial cells and their influence on the transcriptional expression of basal or luminal phenotype have been performed mostly with individual hormones/growth factors in isolation and the effects of some hormones have been explored comprehensively, while knowledge concerning the mode of action of other hormones/growth factors  in breast cancer cells with the exception of supposedly proliferation and/or attachment enhancing properties is limited.The aim of the current study was to investigate how differences in the culture media with respect to basal medium and hormones/growth factors affect transcriptional expression of breast cancer markers and mammary epithelial cell lineage regulatory transcription factors in reference to breast cancer cell lines MCF7, SkBr3, and MDA-MB-436.® HTB-22TM), SkBr3 (ATCC® HTB-30TM) and MDA-MB-436 (ATCC® HTB-130TM) are indicated in Main clinicopathological features, ER/PR/HER2 status and gene cluster group/transcriptional subtype of the studied breast cancer cell lines MCF7  (DMEM:F12 (1:1)), DMEM:F12 (1:3), and RPMI-1640. The basal media DMEM:F12 (1:1) and RPMI-1640 were chosen as all-purpose media traditionally and widely used for propagation of breast cancer cell lines, while DMEM:F12 (1:3) forms the basis for so-called medium F used to promote proliferation of tumor epithelial cells in combination with Rho kinase inhibitor (Y-27632) and fibroblast feeder cells . NonexhaThe supplements for studied media (hormones and growth factors) and their working concentrations were chosen on the basis of studies performed by Ethier et al. ,32: insuv/v) of fetal bovine serum (FBS). A10 was the basic cell culture medium consisting of DMEM/F12 (1:1) + 10% FBS with added penicillin/streptomycin. Basis of medium A5 was DMEM:12 (1:1), for medium D5—DMEM/F12 (1:3), and for medium R5—RPMI-1640 . All media, except A10, were supplemented as described earlier . For cell line MDA-MB-436 medium A10 was supplemented with 5 μg/mL insulin and 1 ng/mL cholera toxin (Ct).The final formulations for the control medium (A10) and the studied media  are shown in 2. Medium was replaced and cultures were examined in phase-contrast microscope every two days. Total number and viability of cells were determined at each subculture. Cell samples for RNA extraction were obtained after fourth subculture at the studied medium.The studied breast cancer cell lines  were conditioned for growth in the three studied culture media for four passages. Each cell line was also propagated in the control medium A10. Cells were cultured in humid chamber at 37 °C, 5% COTotal number and viability of cells were determined by staining with Trypan blue and manual counting in haemocytometer during each subculture. The length of growth of each subculture was documented. (cells collected)/T(cells seeded), where T is the number of cells; PDL was calculated for each subculture (PDL = Log2(CY)). Average generation time during subculture was estimated as average time for all cells in the population to complete one division, i.e., GT = t/PDL, where t is number of days from seeding to the next subculturing. The level of adaptation of studied cell lines to medium was characterized by the cumulative population doubling levels (PDLs) starting with the first subculture. High levels of adaptation were considered, if the cumulative PDLs at the end of the fourth subculture was at least 15. Data on growth analysis are included in The effects of media on cellular proliferation rate were evaluated by population doubling level (PDL) at the end of fourth subculture, average generation time during fourth subculture and by cell yield (CY). CY was estimated as CY = TGenes for the expression analysis were chosen on the basis of mammary cell tracing experiment by Lim et al. , breast ACTB and RNA285S. Quantitative real-time PCR using 5xHOT FIREPol EvaGreen qPCR SuperMix  was run on Applied Biosystems Viia7 . ACTB, RNA285S and GPDH were used as reference genes. Total RNA was isolated from one million cells with Qiazol Lysis Reagent  according to manufacturer’s protocol. DNase treatment  for all samples was followed by RNA clean-up with NucleoSpin RNA Clean-up XS columns . Two micrograms of total RNA was used for cDNA synthesis . The quality of cDNA was determined by amplification of ACTB1, RNA28S5, GPDH). To analyze differential expression, the difference to the normalized Ct values in between a couple of media conditions for cell line were subjected to a Student’s t-test and evaluated after adjustment of p-values by the method of Benjamini and Hochberg with the alpha for false rate discovery of 5%. The genes were considered to be differentially expressed if the p-value was ≤0.05. Data  values) are shown in Transcriptomic expression analysis was performed in R (version 3.1.2), package “HTqPCR”. The values were normalized using the delta Ct method against three reference genes  and major or moderate differences in the levels of expression.For analysis, it was assumed that the difference in the expression levels was major, if FC of the expression was less than 0.25 or more than 4.00, moderate, if FC was in the range of 0.25–0.50 or 2.00–4.00, and minor, if FC was in range of 0.50–2.00. We considered only statistically significant , while the adaptation to A5 and D5 media was low  A. A5 andClaudin-low MDA-MB-436 cell line achieved high level of adaptation in all studied media  A. All meHER2-enriched luminal SkBr3 cell line achieved high level of adaption in all the studied media  A. All meThe morphological appearance of MCF7 cells in the studied media also changed prominently . In the The growth of MCF7 in A5 and D5 media was characterized by mixture of groups of rounded or irregular cells and fewer, bridge-like clusters with very tightly growing, polygonal cells. The culture showed somewhat detached appearance with increase in free floating or loosely attached cell clusters B,C. BothMDA-MB-436 morphologically is characterized by neuron-type cells, and no noticeable observations were made in addition to the cell density on the same day of subculture .Morphological appearance of SkBr3 culture in A10 medium was characterized by close groups of polygonal cells, large round multinucleated cells growing mostly in isolation and great amount of free floating or very loosely attached round cells A.All studied media decreased numbers of free floating and loosely attached cells in comparison to the control A10 medium. Growth in A5 medium displayed increase in the number of large, round multinucleated cells B. The cup-values for comparisons are shown in The effects of partial substitution of FBS with hormones/growth factors were evaluated by comparison of the levels of expression of studied genes in A5 medium to the expression of studied genes in A10 medium. Statistically significant major or moderate differences in the levels of expression are shown in ID3, luminal markers ESR2 and NAT1, and luminal progenitor regulator FOXQ1. The pattern of changes in gene expression among cell lines overlapped very little, and the only alteration common to all three cell lines was major or moderate increase in the expression of basal marker keratin 6A gene (KRT6A).Our results show that the change of growth factor/hormone environment in the studied cell lines had significant impact on the expression of 35 of the 40 studied genes, and that the only genes whose expression was not significantly affected in any of the studied cell lines were basal regulator TP63, HEY1, IFI16) and markers . The only exception to the trend towards basal phenotype was moderate upregulation of mature luminal regulator PGR. Luminal MCF7 cells displayed regulation mostly towards basal phenotype, as evidenced by statistically significant major and moderate increases in the expression of basal regulators , while the most prominent effect in the basal cell line (MDA-MB-436) was major increase in the expression level of basal regulator TBX2. One of the main events in MCF7, but not SkBr3 cell line, was major increase in the expression level of the basal regulator TP63.Substitution of A10 medium to A5 medium with growth factor/hormone set employed in this study induced more basal phenotype in all studied cell lines. Addition of growth factors/hormones increased levels of expression of basal regulators p-values of comparisons are shown in The effects of changes in the composition of basal medium were evaluated by comparison of the levels of expression of studied genes in D5 and R5 media to the expression in A5 medium. Statistically significant major or moderate differences in the levels of expression are shown in TP63 and moderate downregulation of basal marker PROCR and luminal regulator GATA3. In comparison to A5 medium, growth of MCF7 in R5 medium showed differentiation shift toward less basal and more luminal state—major decrease in the levels of basal regulators HEY1, SNAI2, TP63, basal markers ITGB3, KRT16, KRT6A and luminal marker KRT7, and major increase in the level of luminal marker SLC39A6.As compared to A5 medium, growth of MCF7 in D5 was characterized by minor/moderate shift towards more differentiated state—major decrease in the expression level of ID3 and SNAI2, luminal progenitor regulator FOXQ1, mature luminal regulator HES1 and luminal marker KRT7 and major decrease in the levels of luminal marker ESR2, as compared to A5 medium. Growth of the SkBr3 cell line in R5 medium induced major increase in the levels of mature luminal regulator GATA3 and major decrease in the levels of basal markers KRT17 and KRTAP5-6. The response of SkBr3 cell line to the various media in terms of expression levels of basal- and luminal/HER2 enriched-phenotype associated genes reflected split in the population of cells with the shift towards both—basal and luminal phenotype.The response of the luminal HER2-enriched SkBr3 cell line to D5 medium was somewhat ambiguous with major upregulation of basal regulators KRT6A and KRT16, respectively. Major alterations induced by growth in R5 medium were decrease in the expression levels of luminal progenitor regulator FOXQ1 and luminal marker TMEM45B and increase in levels of expression of basal marker KRT6A. Together, major and moderate changes reflected decrease in luminal-like features and shift of MDA-MB-436 cell population towards basal state. The growth of claudin-low MDA-MB-436 cells in D5 medium showed little changes in comparison to growth in A5 medium. The only observed changes, as compared to growth in A5 medium, were major and moderate increases in the expression levels of basal markers Hierarchical clustering of expression alteration profiles induced by substitution of A10 medium to A5, D5 and R5 media clearly show that, despite the observed changes, cell lines retained their identity in terms of mammary lineage regulator and breast cancer marker expression . InteresTP63 and related gene HEY1, with the most prominent marker change being major increase in KRT6A levels. The main drivers for adaptation of the MCF7 cell line to R5 medium were increased expression of mature luminal regulators ESR1 and PGR and decreased expression of mature luminal regulator TBX3, reflecting the shift of the MCF7 cell population towards strictly luminal population.Clustering also showed, that phenotypical alterations of the MCF7 cell line in A5 and D5 media were driven mainly by increased expression of HES1, TBX3, GATA3, luminal progenitor regulators EZH2, FOXQ1, SOX9, and basal regulators SNAI2, HEY1, ID3, TBX2 with resulting increase in luminal markers KRT7, KRT18, MUC1, TMEM45B and XBP1, and basal marker KRT16. These changes were most distinctively observed in D5 medium. The differences in transcriptional profiles induced by substitution of basal media and hormonal/growth factors in the HER-enriched SkBr3 cell line were numerous and orchestrated by changes in the expression of transcriptional factors of various groups—mature luminal regulators ELF5, FOXA1, and TBX3), luminal progenitor regulators  and basal regulator HEY1.The range of changes induced by the studied media in claudin-low MDA-MB-436 cell line was narrower, and was best displayed during growth of the cell line in R5 medium. It was mostly characterized by downregulation of several mature luminal regulators  suppressed and/or delayed proliferation of luminal MCF7 cell line and claudin-low MDA-MB-436 cell line in terms of average population doubling time and cell yields and enhanced proliferation of HER2-enriched SkBr3 cell line in terms of cell yields, despite shift towards more basal differentiation state as evidenced by changes in transcription profiles. R5 medium, which is composed on the basis of RPMI-1640, showed proliferation enhancing effect on MCF7 cell line with the increase of luminal features in transcription profile, slightly suppressive effect on MDA-MB-436 cell line and was most restrictive to HER2-enriched SkBr3 cell line in terms of proliferation and growth parameters.The response of SkBr3 and MDA-MB-436 to R5 medium, and also to the substitution of A10 with A5 medium, in terms of gene expression level changes, are more difficult to explain, with up- and downregulation of transcriptional programs (and markers) characteristic for both directions—towards more differentiated and towards less differentiated state. Probably this ambiguous response reflects split of the cell population into marked subpopulations with distinctive properties and phenotypes. The existence of distinct subpopulations of cells within claudin-low and basal-like cell lines has been shown before [Generally, the response to the substitution of DMEM:F12 1:1 (A) medium with DMEM:F12 1:3 (D) or RPMI-1640 (R) media probably depends on the initial differentiation state and genomic context of the studied cell lines with luminal MCF7 cell line showing shift towards more luminal phenotype, luminal SkBr3 cell line showing somewhat uncertain alteration with up- and downregulation of both, basal- and luminal-phenotype associated regulators and markers, and claudin-low MDA-MB-436 cell line displaying shift towards mesenchymal phenotype.Luminal MCF7 cell line showed most homogeneous response towards change of growth media, therefore it is a good example for significant changes in phenotype induced by changes in growth conditions. TP63) and markers, and down-regulation of mature luminal and luminal progenitor regulators and luminal markers, including moderate decrease in the expression of lineage defining marker EPCAM and increase in tumor-initiating cell marker PROCR, but also by induction of epithelial-to-mesenchymal like transition observed as increased number of mesenchymal-type cells in A5 and D5 media, as described previously [TP63, HEY1, TBX3 and PGR. Although it has been observed, that high expression of p63 protein is characteristic to breast cancer cells with basal-like features [TP63 as compared to the other studied cell lines. Increased levels of p63 have been shown to be related to the activation of Notch pathway, involved in the maintenance of stem cell population in various tissues and in the regulation of the differentiation of cells. Some of the Notch signaling pathway downstream targets are Hairy/Enhancer of Split related genes  and Notch1 itself [MCF7 displayed most pronounced shift towards less differentiated state in A5 and D5 media. It was showed not only by transcriptional profile with up-regulation of basal regulators  [PGR in ERα-positive breast cancer cells has suppressed estrogen-mediated proliferation and transcriptional activity of ERα [PGR and decreased expression of ESR1 in MCF7 conditioned to A5 and D5 media could at least partially explain suppression of growth of the cell line in A5 and D5 media. Interestingly, in MCF7 cell line partial substitution of serum with hormones/growth factors resulted also in increased expression of  α (ERα) . Additioy of ERα . TherefoTBX3 in breast cancer cell lines has been observed previously, especially in cells with estrogen-receptor positive status, e.g., MCF7 [TBX3 expression, increasing number of stem-like cells [TBX3 expression levels in A5, D5 and R5 medium, as compared to A10 medium, is a good example, how nutrients in basal medium influence the expression of cell differentiation governing transcriptional programs.Overexpression of mature luminal regulator g., MCF7 , and it ke cells . TherefoThe impact of individual nutrients, hormones and factors on the transcriptional expression of various markers in culture of breast cancer and/or mammary cells has been studied before, e.g., reports on the effect of estrogen on transcriptional activity in various cell lines ,45,46,47This study clearly shows that growth conditions employed in various laboratories for propagation of cancer cell lines can have significant major impact on the expression of genes defining the phenotype of the breast cancer cells. Therefore, the possible interference of growth media has to be taken in account in the process of designing and performing various experiments and assays, where phenotype of studied breast cancer cell types is of great importance. The expression of breast cancer cell differentiation phenotypic markers in vitro depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used taking into account this effect and the findings of such studies should always be interpreted in caution. Despite changes in the marker expression levels in the framework of our study cell lines retained their phenotypic identity. The formulation of cell growth media has greater impact on the expression of phenotypic markers in luminal, than basal cell lines. Replacement of fetal bovine serum with hormones/growth factors and substitution of basal medium has significant effect on cellular proliferation rates and cell yields. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.