Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community. Ovarian cancer (OC) is the most lethal gynecological malignancy and is the 5th leading cause of cancer related deaths among women in the United States of America . While mUntil recently, a major issue with identifying good experimental models has been a lack of knowledge regarding the molecular and mutational profiles that are most characteristic of OC . High-grThe Cancer Genome Atlas Research Network (TCGA) study  revealedThe realization that the most commonly used cell lines in OC research are not representative of HGSOC will steadily lead to a shift towards using the more HGSOC-like cell lines. In order to be useful, these HGSOC cell lines need to be well characterized and should accurately represent the disease . Since mRecent studies have implicated EMT as a driver of OC metastasis –13. An ain vitro functional assays, their sensitivity to cisplatin and their expression of epithelial and mesenchymal markers. The absence of published reports of such consolidated data hampers effective transition to the use of these HGSOC cell line models for ovarian cancer research. We believe that our data will be very beneficial to the field and will serve as a guide to optimize assay and treatment conditions for various mechanistic, drug development and screening studies. It will enable researchers to extensively use these to more accurately model OC.We report the characterization of a panel of these HGSOC cell lines for commonly used in vitro to study the role of genes or effect of treatments on metastasis [p < 0.0001). OVCAR5 and OVCAR4 were not different from each other but were different from all other cell lines. OVCAR8, CAOV3, COV362, and HeyA8 were not different from each other (with the exception of HeyA8 being different from OVCAR8), but were different from all other cell lines. Kuramochi was significantly different from all other cell lines. SNU119 and OVSAHO were not different from each other but were significantly different from all other cell lines. Since each cell line had a different propensity to migrate, the number of cells seeded per insert had to be varied between cell lines in order to obtain quantifiable migrated cell numbers. The migration was then normalized to the number of cells seeded and ranked accordingly . OVCAR5 and HeyA8 were not different from each other but were different from all other cell lines. OVCAR8 was different from all other cell lines, Kuramochi was not different from OVCAR4 but was different from all other cell lines. OVCAR4, COV362, and CAOV3 were not different but were different from all other cell lines. The independent experiments with each cell line are presented in In addition to the ability to migrate towards chemoattractants, cancer cells need to have the ability to invade through the extracellular matrix surrounding cells and the basement membrane. Therefore, the ability of these cells to invade through matrigel was studied by using transwell invasion assay , 17. Thep < 0.0001). HeyA8 was different from all other cell lines. OVCAR5, OVCAR8 and CAOV3 were not different from each other but different from all other cell lines. COV362, Kuramochi, OVCAR4 and SNU119 were not different from each other (with the exception of COV362 being different from SNU119) but were different from all other cell lines.Having studied the ability of the OC cells to migrate and invade in response to chemoattractants, we proceeded to test their proliferation and clonogenicity. Both these traits are essential for tumor progression and are extensively used to study the effects of therapies, role of specific regulators or genes in OC , 19. Forp < 0.0001). Both OVCAR8 and OVSAHO were different from all other cell lines. Kuramochi and OVCAR4 were not different from each other but were different from all other cell lines. OVCAR5, HeyA8, COV362, SNU119, and CAOV3 were not different from each other . The time required for forming colonies ranged from 7 days for HeyA8 cells to 4 weeks for OVSAHO cells  and CAOV3 was the most sensitive (IC50 dose of 3.14 μM) to cisplatin  were much less potent in the functional assays. These in vitro functional assay results mirrored their ability to form mouse xenograft tumors reported previously [The present study is the first to systematically compare the eviously . HeyA8 ceviously . Similareviously , 18, 23.eviously . These neviously . This shThere was no clear correlation between genetic resemblances to HGSOC and the EMT phenotype. All the cell line models were found to be relatively sensitive to cisplatin, which agreed with the previous report on COV362 and CAOV3 . Since mIn conclusion, these HGSOC cell line models can be used as better representatives of the most common form of the disease. However, there are significant variations in their functional abilities, their EMT status as well as their sensitivity to cisplatin. Therefore, it is imperative that the assay conditions be adapted to the fact that some of these are less aggressive in the common functional assays tested compared to the OC cell lines that have been predominantly used. The standardized conditions reported herein would be useful as an appropriate starting point.Dulbecco's Modified Eagle's Medium (DMEM) , MEM vitamins , MEM nonessential amino acids , Penicillin-Streptomycin  and trypsin  were purchased from Corning. FBS was purchased from Gibco . Giemsa stain  and crystal violet  were obtained from Sigma Aldrich. Thiazolyl blue tetrazolium bromide was from Acros Organics  and 4% paraformaldehyde solution was purchased from Fisher Scientific (NC9245948).All cell lines were grown in DMEM with 10% FBS along with 1% MEM vitamins, MEM nonessential amino acids and Penicillin-Streptomycin. CAOV3, HeyA8, OVCAR5 and OVCAR8 were obtained from Ernst Lengyel, University of Chicago and OVCAR4 from Joanna Burdette, University of Illinois at Chicago. OVSAHO was obtained from the Japanese Collection of Research Bioresources and SNU119 was from the Korean Cell Line Bank. COV362 (Sigma), and Kuramochi (Japanese Collection of Research Bioresources). All cell lines used were genetically validated and also confirmed to be mycoplasma free using respective services from Idexx BioResearch . The genetic validation was done using the CellCheck 16 (16 Marker STR Profile and Inter-species Contamination Test) and mycoplasma testing was done using Stat-Myco.2 incubator (5% CO2) and then seeded in the upper chamber in 500 μl of serum free DMEM and allowed to migrate at 37°C. The numbers of cells seeded per insert and migration times are included in Table Transwell migration assays were conducted using 8 μm pore size inserts . OC cells were trypsinized, recovered for 30 min at 37°C in the CON = 200,000/number of cells seeded for the cell lineSimilarly, the baseline migration time was set at 8 h and the migration time adjustment factor (T) was calculated as follows:T = 8/migration time for the cell lineThe final adjusted cell count took into account the cell number adjustment factor (N) and the migration time adjustment factor (T) and was determined by:Adjusted cell count = average number of cells per field × N × T2 incubator and then seeded in the transwell insert in 500 μl serum-free DMEM. The number of cells seeded per insert are included in Table Cellular invasion through the extracellular matrix proteins was assayed using growth factor reduced matrigel coated transwell inserts  in 24-well plates. OC cells were trypsinized, recovered for 30 min at 37°C in the COAdjusted cell count = average number of cells per field × NWhere N = 200,000/number of cells seeded for the cell line.OC cells were seeded in 96-well plates in 8 replicates and allowed to grow for 5 days. To identify the optimal number of cells for comparison between cell lines, 1000, 2000, 5000 and 10000 cells of each cell line were seeded per well. On the fourth day MTT -2,5-diphenyltetrazolium bromide) Assay was conducted to measure proliferation of the OC cells. 20 μl of MTT solution (5 mg/ml in sterile PBS) was added per well, mixed and incubated at 37°C. The incubation times for the OC cell lines are listed in Table OC cells were seeded in 6-well plates  and allowed to form colonies. Medium was changed every fifth day. Once visible colonies were formed  reaction was done for E-cadherin and vimentin using TaqMan gene expression assays as described previously . BrieflyWestern blotting was done as described previously . BrieflyCell diameter was analyzed using the Z2 coulter Particle Count and Size Analyzer  using a 100 μm aperture size. Briefly, cells were diluted 1:10 in diluent (Isoton II) and 0.5 ml was loaded into the counter. All particles over 12 um were counted and measured. Between sample readings, the counter was flushed with the diluent.p-value from the overall F-test is reported. Tukey's method was used for pair-wise comparisons. The overall error rate per outcome was controlled at 5%.ANOVA was used to compare mean migration, invasion, proliferation, and colony formation by cell line. The


We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines.Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of  Another melanoma model includes the Tm1 and Tm5 melanoma cell lines, which were established by submitting the melanocyte cell line Melan-a to sequential cycles of substrate-adhesion impedimentin vivo and a lower doubling time in vitro101H NMR spectroscopy allied to multivariate analysis. We also investigated the effect of mitogenic agents on the metabolic profile of the melanocyte cell line.Melanoma presents a major challenge in medical practice, due to difficulty in diagnosis, poor response to available therapies and high incidence in western populations61H NMR spectra of the aqueous extracts obtained from the four cell lines showed many overlapping signals 1H NMR spectra obtained from the cell extracts. The 4 cell lines were separated into 4 distinct groups on the scores plot  with PMA present in the media during the entire time (MA), 2) with the cell line cultivated in a medium without PMA for 24 hours (MA-24), and 3) where the cell line was cultivated for 72 h without the presence of PMA in the medium (MA-72). ¹H NMR and principal component analyses were then carried out on the lipid extracts of the cells. The first principal component explained 45% of the variance, the second 72%, and the third 84%, cumulatively. Even the explained variance in the first PC not being ideal, the 3 conditions were well discriminated on the scores plot of both PC1 vs PC2 , and the Melan-a cell line is no exception. Stranzl In summary, the present study successfully applied NMR techniques in order to identify and determine the relative amounts of aqueous and lipid-soluble metabolites of both melanocyte and melanoma murine cell lines. Through PCA analysis it was possible to identify the main contributors which differentiate the 4 cell lines. The results showed that the metabolite which most contributes to differentiation in the aqueous extract obtained from the 4 cell lines on both PC1 and PC2 was creatine, as confirmed by gene expression assays. Therefore, we can state that there is an imbalance in creatine metabolism , such asFurthermore, this study demonstrated that the metabolomic profile of the melanocyte cell line is influenced by the use of mitogenic agents, such as PMA. Although not so evident as the concentration changes observed for aqueous metabolites, lipid metabolism changes between the Melan-a cell line grown with and without PMA added on culture media could be revealed by the use of multivariate analysis techniques, showing that the main alterations were related to the metabolic pathway of cholesterol. The 2D multiplicity-edited HSQC NMR fingerprints proved to be a useful tool for identifying and measuring different expression patterns of metabolites in both water soluble and lipid extracts, enabling us to identify potential progression biomarkers and thus contribute to the development of methods for the prognosis of melanoma.2 tissue-culture flasks and maintained in RPMI medium supplemented with 5% (v/v) fetal bovine serum (FBS), both from Gibco , 200 nM 12-O-tetradecanoyl phorbol-13-acetate - PMA , 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) in a humidified atmosphere containing 5% CO2 at 37 °C. Melanoma cell line B16-F10 was cultured under the same conditions, without PMA. The cells were grown until 75–80% density and then collected using 0.1% trypsin in PBSA containing 1 mM EDTA and centrifuged at 4000 rpm for 20 minutes (4 °C). The cell pellet was washed thrice with phosphate buffer, the supernatant discarded and the pellet subjected to the extraction process. We standardized a quantity of 15 mg for each cell line, which corresponded to approximately 4 × 75 cm2 tissue-culture flasks.Mouse melanoma cell lines  and the immortalized murine melanocyte cell line Melan-a were kindly provided by Dr. Roger Chammas . Melan-a, Tm1 and Tm5 cells were grown in 75 cm3:MeOH 3:1 (v/v), being vortexed for 1 minute and then kept for 30 min at room temperature. After this, it was centrifuged at 10000 rpm for 20 min, the supernatant collected and the solvent dried under a stream of nitrogenThe cell pellets were subjected to lysis, being frozen with liquid nitrogen and returned to a 37 °C water bath several times. After being freeze-dried overnight, 15 mg of each cell line, was weighted and transferred to a glass tube with screw cap and Teflon (volume of 4 mL). The pellet was extracted using acetonitrile:water 1:1 (1 mL), vortexed for 1 minute and then kept for 10 min at room temperature (this procedure was repeated thrice), and then centrifuged at 10000 rpm for 6 min. The supernatant was collected and lyophilized. The pellet from the previous aqueous extraction procedure was then subjected to lipid extraction using CHCl3, Deuterium oxide  and 3-trimethylsilyl-2H4-propionic acid sodium salt (TSP) were purchased from Cambridge Isotope Laboratories, Inc.  and from Sigma-Aldrich . The samples obtained from the aqueous extraction were deuterium-exchanged by repeated dissolution in D2O, and the samples obtained from the lipid extraction were deuterium exchanged by repeated dissolution in MeOD-D2O (2:1). Both were freeze-dried overnight. Their spectra were obtained from solutions in D2O (580 μL plus 20 μL of TSP 1 mg/mL) or CDCl3-MeOD  at 30 °C, using TMS (Tetramethylsilane) or TSP as a reference (δ = 0.00 ppm). Spectra were obtained on a Bruker 400 MHz AVANCE III NMR spectrometer with a 5 mm BBI inverse probe and a Bruker 600 MHz ASCEND equipped with a QXI probe . 1D 1H-NMR was carried out using enough scans to give a Signal/Noise (S/N) ratio of at least 2000/1 . Experiments were performed without tube rotation and with the TMS or TSP signal at a medium width ranging from 0.8–1.0 Hz. Water suppression was obtained by presaturation, using the 1D zgpr pulse sequence, applying a low power continued wave irradiation before the first 90° pulse on channel 1, using a power level for presaturation of 44.98 -dBW (3.17 × 10−5 Watts). For multivariate analysis and estimation of metabolites concentration, the spectra were acquired using the noesygppr1d.2 pulse sequence, with 128 transients and a delay of 10.0 s . 2D NMR experiments were carried out using multiplicity-edited 1H-13C HSQC, heteronuclear correlation via double inept transfer with decoupling during acquisition, using trim pulses in inept transfer with multiplicity editing during the selection step (hsqcedetgpsp.3), TOCSY, total homonuclear correlation via Hartman-Hahn transfer using MLEV17 sequence for mixing with a mixing time of 0.06 s (mlevphpr.2), and 1H-13C/1H-31P HMBC, heteronuclear correlation via zero and double quantum coherence optimized on long range couplings (hmbcgplpndqf and hmbcgpndqf) pulse sequences. The 2D experiments were recorded for quadrature detection in the indirect dimension, HSQC spectra were acquired using 128 scans per series of 2 K × 400 W data points with zero filling in F1 (4 K) prior to Fourier transformationMeOD, CDCl18 primers, according to manufacturer’s instructions. RT-qPCR reactions were performed in a StepOne Plus  thermal cycler, using SYBR Green PCR Master Mix (Applied Biosystems) and specific primers for the genes of interest. The equipment’s default cycling program was used (15 s at 95 °C and 1 min at 60 °C for 40 cycles). Amplification efficiency and data normalization were performed as previously describedGAMT (Guanidine-Acetate Methyl Transferase - EC 2.1.1.2) mRNA expression levels were quantified in murine cell lines Melan-a and B16-F10. For this, cDNA were synthesized using the ImProm II Reverse Transcription Kit (Promega), from 1 μg of total RNA, and using Oligo d(T)Melan-a cell line were grown in RPMI medium containing 200 nM of PMA during four passages. The medium was then removed and replaced with a medium which did not contain PMA. The cells were left for a further 24 and 72 hours of growth.2OD resonances (δ 3.33–3.38) was removed from all data sets prior to normalization and multivariate data analysis in order to remove deviations due to water suppression efficiency or solvent multiplicity inhomogeneities. Following a preliminary PCA analysis, the region corresponding to (CH2)n (δ 1.20–1.40) and N+ (CH3)3 (δ 3.18–3.25) resonances was also removed from all datasets, mainly because minor peak shape deformations in this region interfered with PCA analysis, due to the high intensity of signals. All remaining frequency regions of the spectra were analyzed without scaling, in order to preserve natural differences in intensities from the datasetsMultivariate analyses were performed on the spectra obtained from both aqueous and lipid extractions. Spectra obtained from 3 to 5 independent experiments, each one with 3 or more technical replicates were assembled to build the bucket tables. For spectra obtained from aqueous extractions, the spectral width considered was from 0.50 to 10.00 ppm. After NMR spectral phase and baseline correction, each spectrum was data-reduced to 1183 regions of equal width (0.005 ppm) using the AMIX  software package, version 3.8 . The confidence level was 95.00%. For spectra obtained from lipid extractions, the spectral width considered was from 0.50 to 6.00 ppm. Each spectrum was data-reduced to 453 regions of equal width (0.005 ppm). The spectral region close to CHDHow to cite this article: Santana-Filho, A. P. d. et al. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery. Sci. Rep.7, 42324; doi: 10.1038/srep42324 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.DOI:http://dx.doi.org/10.7554/eLife.24179.001 Like a person who is dependent on coffee to be productive, cancer cells are dependent on the products of certain genes in order to dominate their environment and grow. Cancer cells will stop growing and die when the activity of these gene products is blocked. These genes are known as cancer dependencies or “addictions”. As a result, researchers are constantly looking for cancer dependencies and developing drugs to block their activity.It was previously believed that a gene called MELK was an addiction in certain types of breast cancer. In fact, pharmaceutical companies had developed a drug to block the activity of MELK, and this drug is currently being tested in human patients. However, Lin, Giuliano et al. have now taken a second look at the role of MELK in breast cancer, and have come to a different conclusion.Using a gene editing technology called CRISPR/Cas9, Lin, Giuliano et al. removed MELK activity from several cancer cell lines. This did not stop cancer cells from multiplying, suggesting that MELK is not actually a cancer addiction.Additionally, when breast cancer cells that do not produce MELK were exposed to the drug that is supposed to block MELK activity, the drug still stopped cell growth. Since the drug works when MELK is not present in the cell, the drug must be binding to other proteins. This suggests that MELK is not the actual target of the drug.Lin, Giuliano et al. suggest that, in the future, CRISPR/Cas9 technology could be used to better identify cancer dependencies and drug targets before cancer drugs are given to human patients.DOI:http://dx.doi.org/10.7554/eLife.24179.002 Tumors of the breast can be divided into five distinct subtypes based on characteristic gene expression patterns. These breast cancer subtypes are referred to as Luminal A, Luminal B, Her2-enriched, normal-like, and basal . Basal bIn recent years, significant progress has been made in the treatment of certain malignancies by targeting cancer cell ‘addictions’, or genetic dependencies that encode proteins required for the growth of specific cancer types . Drugs tThe Maternal Embryonic Leucine Zipper Kinase (MELK) has received substantial attention as a potential cancer cell addiction and promising target for drug development. MELK was first identified as an AMPK family member expressed in the mouse pre-implantation embryo , and hasAs part of a project in our lab to characterize genes whose expression is associated with patient prognosis in cancer , we idenOver a dozen previous publications have reported that MELK is a cancer dependency, as blocking MELK with RNAi or small molecules inhibited the proliferation of cell lines derived from multiple tumor types . HoweverTo assess the efficacy of our CRISPR system, we purified GFP+ populations from cell lines harboring each individual guide RNA, and then we analyzed the targeted loci by Sanger sequencing. TIDE analysis, which decomposes raw sequencing traces into linear combinations of indel mutations , revealeWe next set out to determine whether MELK was required for cancer cell fitness. To test this, we measured cell proliferation over 15 days in culture in the 30 independent lines of A375, Cal51, and MDA-MB-231 that we had generated . SurprisIn order to assess whether a wider range of cancer cell lines were dependent on MELK for viability, we performed individual GFP dropout experiments in 13 Cas9-expressing cancer cell lines . In thesAs negative controls in this experiment, we utilized three gRNA’s targeting Rosa26, and as positive controls we designed six gRNA’s targeting the essential replication genes RPA3 and PCNA . We firsTo extend these observations, we repeated the GFP dropout experiments in six Cas9-expressing cell lines  from other cancer types previously suggested to require MELK expression. Consistent with our observations in the triple-negative breast cancer cells, guides targeting Rosa26 exhibited minimal dropout over five passages, while guides targeting RPA3 or PCNA were depleted up to 90-fold . HoweverSeveral laboratories have conducted genome-wide or kinase-focused screens to identify novel cancer addictions. If these unbiased screens indicated that cancer cell lines required MELK expression to proliferate, then that would bolster the contention that MELK could be a therapeutic target in cancer. We therefore examined data from four recent screens: a kinome-wide siRNA screen in 117 cancer cell lines , a genomIf MELK is not a cancer cell dependency, then drugs that inhibit MELK must either be ineffective at stopping cancer cell division or they must also act on other cellular targets. We therefore assessed the efficacy of the MELK inhibitor OTS167 , a therapeutic agent being tested in several clinical trials. We treated a variety of cancer cell lines with 7-point serial dilutions of OTS167, and we observed that OTS167 did in fact impede cell proliferation at nanomolar concentrations . As OTS167 was able to inhibit growth despite the non-essentially of MELK, we considered the possibility that OTS167 acted through an off-target effect. To test this, we set out to determine whether MELK expression was actually required for OTS167 sensitivity. We calculated the GI50 value of OTS167 in A375, Cal51, and MDA-MB-231 cells that harbored gRNA’s targeting either Rosa26 or MELK, and we found that cell populations with wild-type or mutant MELK displayed equivalent sensitivity to the drug . For insTo further explore this observation, we tested the efficacy of OTS167 against a panel of triple-negative or receptor-positive breast cancer cell lines. MELK is significantly up-regulated in triple-negative tumors relative to receptor-positive tumors , and oneLastly, to confirm that OTS167 treatment fails to phenocopy MELK mutations, we examined their effects on cell cycle progression. We found that treatment with OTS167 blocked cytokinesis in a dose-dependent manner, resulting in populations harboring 13% to 60% multinucleate cells. In contrast, cells transduced with either Rosa26 or MELK gRNAs progressed through the cell cycle without gross mitotic defects, and exhibited no significant difference in the frequency of multinucleate cells . These rTo unambiguously demonstrate that MELK is dispensable for the proliferation of certain cancer cell lines, we used CRISPR to generate clonal cell lines that lack MELK protein. We transduced the MDA-MB-231 triple-negative breast cancer cell line with sets of 2 guide RNAs and then expanded clonal populations from single cells . RecombiThe MDA-MB-231and Cal51 MELK-KO clones exhibited robust proliferation, demonstrating that MELK is fully dispensable for the growth of these cancer cell lines . In factAs a mitotic kinase highly expressed in many cancer types, MELK has been identified as a promising target for therapeutic intervention. However, through the use of CRISPR/Cas9-mediated mutagenesis, we have demonstrated that MELK is dispensable for growth in 13 out of 13 cancer cell lines tested, and that a MELK inhibitor currently in clinical trials blocks cell division by inhibiting another target. We believe that our results highlight the importance of using CRISPR/Cas9 technology to study and validate preclinical targets in cancer drug development.RAD51 and MAD2 are unusually sensitive to off-target RNAi inhibition  that failed to reveal an effect of MELK loss on cell fitness, further strengthening our conclusion that MELK is dispensable for cancer cell proliferation.CRISPR mutagenesis can also assist in the pharmacological study of potential drugs. Several lines of evidence indicate that OTS167 does indeed inhibit MELK: for instance, a crystal structure of OTS167 binding to the MELK kinase domain has been reported . HoweverOur results leave open the question of what role, if any, MELK plays in mammalian biology and cell cycle progression. While MELK is up-regulated in diverse tumor types, it is also expressed in several normal cell lineages, including embryonic cells, hematopoietic cells, and neural progenitor cells . MELK ma2. Cell counting was performed using the Cellometer Auto T4 system .The identity of every human cell line utilized in this paper was authenticated using STR profiling . Cell lines were also confirmed to be negative for mycoplasma contamination using the MycoAlert Detection Kit . Cell lines HCC70, HCC1937, MDA-MB-453, MDA-MB-468, and ZR-75–1 were grown in RPMI 1640 supplemented with 10% fetal bovine (FBS), 2 mM glutamine, 1% Nonessential Amino Acids , and 100 U/ml penicillin and streptomycin. HCC1143 and NCI-H1299 were grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. T24 was grown in McCoy’s 5A media supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cal51, A375, MDA-MB-231, HCT116, Cama1, JIMT-1, and U118-MG cells were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. T47D cells were grown in RPMI supplemented with 10% FBS, 6.94 μg/ml insulin , 2 mM glutamine, and 100 U/ml penicillin and streptomycin. MCF7 cells were grown in DMEM supplemented with 10% FBS, 0.01 mg/ml insulin, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cell lines were kindly provided by the individuals thanked in the acknowledgments. All cell lines were maintained in a humidified environment at 37°C and 5% COGuide RNAs targeting protein domains in MELK, PCNA, and RPA3 were designed with assistance from Osama El Demerdash (manuscript in preparation). Oligonucleotides were ordered from IDT and then cloned into the LRG 2.1 vector [a gift from Jun-Wei Shi (University of Pennsylvania) and Chris Vakoc (Cold Spring Harbor Laboratory)] using a BsmBI digestion . PlasmidHEK293T cells were transfected using the calcium-phosphate method . SupernaTo measure cell proliferation, 100,000 cells of each strain were plated on a six well plate and then allowed to grow for 72 hr. Cells were then trypsinized, counted, and 100,000 cells were re-plated in fresh media. Cells were passaged five times, and cumulative population doublings and doubling times were calculated at each passage.Solutions of 1.0% and 0.7% Difco Agar Noble in sterile water were autoclaved and then allowed to equilibrate at 42°C or 37.5°C, respectively. The 1% agar solution was then mixed 1:1 with 2 ml of the appropriate media, supplemented with 2X the concentration of serum, glutamine, and penicillin/streptomycin. 1 mL of this mixture was then plated in one well of a six well plate and allowed to solidify at room temperature for 30 min, resulting in a 0.5% agar base layer. Cells of interest were then trypsinized and re-suspended in their appropriate media. 20,000 cells were diluted in 1 ml of 2X media and then mixed with 1 ml of the 0.7% agar solution. 500 μl of this mixture was then plated on the solidified base layer, resulting in 5000 cells in a 0.375% agar suspension. The agar was allowed to solidify at room temperature for one hour before being transferred to an incubator. Fresh 1X media was added to the surface of each well 24 hr after plating, and then were re-fed every 3 days. After 21 days of growth, colonies were scored under 10x magnification. All experiments were plated in triplicate and performed twice.Cells were lysed with RIPA buffer . Lysates were quantified using the Pierce BCA Kit (Thermo Scientific), and equal amounts of protein were denatured and loaded onto an 8% SDS-PAGE gel. The protein was transferred onto a polyvinylidene difluoride membrane using the Trans-Blot Turbo Transfer System . The membrane was blocked in 5% non-fat milk-TBST and then incubated with anti-MELK  at a 1:3000 dilution, or blocked in 10% BSA-TBST and then incubated with anti-MELK  at a 1:1000 dilution. Anti-alpha-tubulin  was used as a loading control at a 1:3000 dilution. All primary antibody incubations were performed overnight at 4°C. Following incubation, the membranes were washed and then incubated in either anti-rabbit secondary  at 1:50000 for MELK or anti-mouse secondary  at 1:10000 for Tubulin for 1 hr at room temperature.Genomic DNA was extracted from transduced cell lines using the QIAmp DNA Mini kit . Loci targeted by guide RNAs were amplified using the primers listed in For every cell line of interest, 10,000 cells were plated in 100 μl of media in an 8 × 4 matrix on a flat-bottomed 96-well plate. Cells were allowed to attach for 24 hr, at which point the media in every well was changed. 500 nM of OTSSP167  was added to one row of cells, and then 6 3-fold serial dilutions were performed. After 72 hr of growth in the presence of the drug, cells were trypsinized and counted using a MacsQuant Analyzer 10 . The fraction of cells recovered at every drug concentration, relative to a row of untreated cells, was determined. GI50 values were calculated using a four-parameter inhibition vs. concentration model in Prism 7 . Sensitivity experiments in Cells were transduced on day 0 with sgRNA lentiviral supernatant, which was then replaced with fresh media on day 1. On day 3, the baseline percentage of GFP+ cells was measured using a MacsQuant Analyzer 10 (Milltenyi Biotec). Cells were then passaged every 3 or 4 days, according to their growth rate and confluence, and the percentage of GFP+ cells was measured at every split. Dropout values represent the fold decrease in GFP+ cells at each passage, relative to the GFP+ percentage on day 3. In preliminary experiments with A375 and MDA-MB-231, replicate dropout assays were highly reproducible across independent replicates. For that reason, GFP dropout experiments in the 13 tested cell lines were performed once.10,000  or 20,000 (MDA-MB-231) cells of interest were plated in 250 μl of media in a flat-bottomed 24-well plate and allowed to attach for 24 hr. Then the media was replaced, and OTS167 or Cytochalasin B  were added to control wells. Following an additional 24 hr period of growth, cells were stained with 2.5 μg/ml of Hoechst dye  for 30 min and imaged using appropriate filters. DNA staining experiments were performed twice. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Tony Hunter (Senior Editor) and two reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "CRISPR/Cas9 mutagenesis invalidates a genetic target of clinical trials in cancer" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Lin and colleagues report their CRISPR-based analysis of the candidate oncology drug target, MELK, a kinase that has been previously implicated in cancer in numerous publications, and for which a pharmacologic inhibitor is currently being evaluated in early clinical trials. There has been particular interest in its role in triple-negative breast cancer. In short, they made use of CRISPR cutting technology to generate MELK mutant alleles in a panel of cancer cell lines, including several in which MELK dependency has previously been reported. In cell culture assays including competitive cell growth, proliferative potential, and anchorage-independent growth, they used these cells to demonstrate that, in contrast to previous claims, MELK disruption did not seem to significantly affect the oncogenic potential of any of the tested cell lines. Furthermore, after generating single cell-derived MELK-deficient clones of one of these lines, they showed that the clinical small molecule MELK inhibitor retained its growth inhibitory properties, suggesting that this inhibitor displays off-target activities that are responsible for the observed effects on cell growth. They also examined published datasets associated with independently performed CRISPR and RNAi screens that included MELK-targeted agents and concluded that MELK dependency was not observed in any of those unbiased screens. Based on these findings, the authors conclude that previously reported MELK dependency in cancer is likely to reflect an experimental artifact, and that the MELK inhibitor that is currently being evaluated in the clinic may be functioning through a distinct, currently unknown mechanism.Overall, this is a well-executed and well documented study. Although these are essentially "negative results", the fact that others are still pursuing MELK as a candidate therapeutic target, and that clinical trials focused on MELK inhibition are underway, justifies publication of the findings.Essential revisions:1) The authors need to demonstrate that the MELK protein is substantially depleted in cell lines following CRISPR engineering. They have shown this for one cell line by western blot, but it should be shown for a few of the other lines as well to rule out the possibility that in some (or even many) of these lines, a small amount of "residual", non-mutated, expressed MELK is sufficient to maintain normal proliferation.2) The authors have not mentioned one of the most likely possibilities for the MOA of OTS167, which is polypharmacology. It could even be that OTS167 inhibition of MELK + Target X explains how it kills cells. Most kinase inhibitors, including the prominent approved drugs, Gleevec (bcr-abl + ckit), dasatinib (Bcr-abl + src family kinases), Sutent (many) work in this manner. The single gene knockout studies here would not show differences in this case. This should be acknowledged explicitly.3) The authors should also address other possible explanations of the difference between genetic perturbation (compensation could take place by upregulation of other kinases during the time it takes to deplete the protein after gene editing) and pharmacology . These differences can be acute in the setting of oncogene addiction/cancer cell signaling. Essential revisions:1) The authors need to demonstrate that the MELK protein is substantially depleted in cell lines following CRISPR engineering. They have shown this for one cell line by western blot, but it should be shown for a few of the other lines as well to rule out the possibility that in some (or even many) of these lines, a small amount of "residual", non-mutated, expressed MELK is sufficient to maintain normal proliferation.To address the reviewers’ concern that residual MELK protein may remain in our cell lines, we derived a second set of MELK-knockout clones, this time from the Cal51 triple-negative breast cancer cell line . We confWe also conducted additional western blot analyses on sorted populations of A375 and Cal51 cells transduced with MELK gRNAs . We obseIn total, our manuscript presents functional evidence that our CRISPR system is active (dropout of gRNAs targeting PCNA and RPA3), sequencing evidence that our MELK gRNAs efficiently induce indel mutations in the MELK gene, immunoblot evidence that these indel mutations result in the loss of MELK protein expression in multiple independent cell lines, and we confirm that no residual MELK protein is left in six MELK-knockout clonal lines.2) The authors have not mentioned one of the most likely possibilities for the MOA of OTS167, which is polypharmacology. It could even be that OTS167 inhibition of MELK + Target X explains how it kills cells. Most kinase inhibitors, including the prominent approved drugs, Gleevec (bcr-abl + ckit), dasatinib (Bcr-abl + src family kinases), Sutent (many) work in this manner. The single gene knockout studies here would not show differences in this case. This should be acknowledged explicitly.In light of the apparent promiscuity of OTS167 , we agree with the reviewers that polypharmacology is a likely MOA of OTS167. We have added a discussion of this possibility in the fourth paragraph of the Discussion section.3) The authors should also address other possible explanations of the difference between genetic perturbation (compensation could take place by upregulation of other kinases during the time it takes to deplete the protein after gene editing) and pharmacology . These differences can be acute in the setting of oncogene addiction/cancer cell signaling.We agree that one potential drawback of CRISPR is the lag time between the introduction of a gRNA, the loss of the targeted protein, and the ability to analyze a resulting phenotype. In particular, during the periods of growth required to generate and analyze pure GFP+ populations or MELK-knockout clones, it is possible that cells could “adapt” to the loss of MELK. However, we believe that the cell competition assays that we also performed give us insight into the more immediate consequences of MELK loss, as cells are under observation from the time in which the gRNA is introduced onward. A transient loss of cell fitness, followed by adaptation, would be detectable as a decrease in GFP+ cells in passage 2 or 3 of this assay, followed by stabilization of the GFP+/GFP- ratio. In total, we conducted 91 independent cell competition assays (7 MELK gRNAs in 13 cell lines) that could have revealed a transient fitness defect, but we failed to detect any evidence that this was occurring. It’s true that we cannot rule out the possibility that immediate adaptation is taking place, e.g. cells are able to compensate for MELK loss in such a way that not even a transient fitness defect is evident, but we believe that our experiments have given us a good chance to detect anything short of perfect compensation. To clarify these points, we have added a discussion of this issue in our manuscript .


Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents  in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated  In vitro anti-leukemic activity of BTZ against ALL was firstly reported in 2000 [in vitro when combined with standard chemotherapeutic agents [Bortezomib (BTZ) is a proteasome inhibitor approved for the treatment of multiple myeloma (MM) . Recentl in 2000 . Subsequ in 2000 , and, in in 2000 . However in 2000 . In contc agents . Based oc agents , and a fc agents : 16 (73%Carfilzomib (CFZ), a second-generation proteasome inhibitor, demonstrated more potent and more specific proteasome inhibition against the chymotrypsin-like activity of the 20S proteasome in a stable and irreversible fashion –12. CFZ in vitro BTZ sensitivity and possible cross-resistance of BTZ with conventional chemotherapeutic agents using a large panel of ALL cell lines. We also tested the in vitro anti-leukemic activity of CFZ in ALL cell lines as a possible substitute for BTZ in BTZ combination chemotherapy for refractory ALL.In the present study, we investigated the association of cytogenetic abnormalities with 2 at 37°C.Seventy-nine BCP-ALL cell lines, nine T-ALL cell lines, and two MM cell lines listed in 5 cells were plated into a 96-well flat-bottom plate and assays performed in triplicate in the presence or absence of seven concentrations of each drug. The cells were cultured for 44 h for sensitivities to BTZ, CFZ, DNR, and VCR, or 68 h for sensitivities to Dex and L-Asp. After a 6 h additional incubation with alamarBlue, absorbance at 570 nm were monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of treated wells to that of untreated wells as a percentage. The concentration of agent required to reduce the viability of treated cells to 50% of untreated cells was calculated, and the median of three independent assays was determined as IC50 for each cell line.To determine the 50% inhibitory concentrations (IC50s) of BTZ, CFZ, Dex, daunorubicin (DNR), VCR, and L-Asp, an alamarBlue cell viability assay  was performed. 1–4 x 10To detect apoptosis, cells were cultured in the absence or presence of BTZ or CFZ for 17–18 h, stained with a fluorescein isothiocyanate (FITC)-conjugated Annexin-V and 7AAD . To verify combination of P-glycoprotein inhibitors, cells were pretreated with verapamil or nilotinib for 2 h, and subsequently cultured in the absence or presence of BTZ or CFZ for 17–18 h. Cell surface expression of P-glycoprotein was analyzed with a FITC-conjugated anti-P-glycoprotein antibody . For the functional assay of P-glycoprotein-mediated efflux of calcein-AM , cells were incubated with 0.25 mM of CAM for 10 min at 37°C in the presence or absence of verapamil or nilotinib. To detect apoptosis in P-glycoprotein knockout HALO1 cells, cells were stained with a FITC-conjugated anti-P-glycoprotein antibody and a phycoerythrin (PE)-conjugated Annexin-V . The stained cells were analyzed by flow cytometry .3, 1 mM phenylmethylsulfonyl fluoride, 100 μM sodium vanadate). The lysates were separated on a SDS-polyacrylamide gel under reducing conditions and then transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibody at 4°C overnight, and then with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. The bands were developed using an enhanced chemiluminescence detection (ECL) kit . Anti-CHOP  and anti-NOXA  mouse monoclonal antibodies were purchased from Cell Signaling Technology  and abcam , respectively. Horseradish peroxidase-conjugated anti-mouse IgG  was purchased from MBL .Cells were solubilized in lysis buffer . The relative copy number, obtained after normalization against controls, was used to determine genomic copy number of each gene. Values between 0.75 and 1.3 were considered to be within normal range. Values below 0.75 or 0.25 indicated monoallelic or biallelic loss, respectively.SALSA multiplex ligation-dependent probe amplification (MLPA) kit P335-A4 was used according to the manufacturer’s instructions  . The kitABCB1 gene using the CRISPR design tool . We selected 5’-tttggctgccatcatccatgg-3’, which showed the highest off-target hit score, and the synthesized oligomers were cloned into CRISPR/Cas9 vector . Three days after electroporation of the ABCB1-targeting CRSIPR/Cas9 vector using Neon electroporation transfection system (Thermo Fisher Scientific) into HALO1 cells, CD4-positive cells were selected using CD4-microbeads  and expanded for further analyses.To knock out P-glycoprotein  expression with CRISPR/Cas9 system, we screened exon 3 of the IKZF1 deletion and biallelic loss of CDKN2A in 65 Ph-negative BCP-ALL cell lines using Excel software.We applied Mann-Whitney test for comparison of drug sensitivities and chi-square test for comparison of incidence in gene deletion. We applied a paired t-test for comparison of two means that were from the same objects and Pearson’s correlation analysis for correlation between paired data. All analyses were performed using Excel software. A multivariate analysis of log IC50 value of BTZ was performed to evaluate the effects of in vitro sensitivity to BTZ using 79 BCP-ALL, 9 T-ALL, and 2 MM cell lines. Based on dose-response curves determined by the alamarBlue cell viability assay using seven concentrations of BTZ that ranged from 1.25 nM to 80 nM -ALL, 4 t-ALL, and 3 t-ALL cell lines  and t-ALL cell lines . No statistically significant difference was observed between Ph+ALL cell lines and t-ALL cell lines . Further, t-ALL cell lines tended to be more sensitive to BTZ than t-ALL cell lines (p = 0.098).We compared the sensitivity of 79 BCP-ALL cell lines with different types of translocations to BTZ, which included 14 Ph+ALL, 11 ll lines . The remIKZF1 [CDKN2A/CDKN2B [IKZF1, CDKN2A, and CDKN2B, while 11 (16.9%), 36 (55.4%), and 40 (61.5%) out of 65 Ph-negative BCP-ALL cell lines carried deletions of IKZF1, CDKN2A, and CDKN2B, respectively (IKZF1 (p<0.00001 by chi-square test), CDKN2A (p = 0.0014), or CDKN2B (p = 0.0035) was significantly more common in Ph+ALL cell lines than in Ph-negative BCP-ALL cell lines. Regarding deletion of CDKN2A/CDKN2B genes, biallelic loss of CDKN2A was significantly more common in Ph+ALL cell lines (64.3%) than in Ph-negative BCP−ALL cell lines , while there was no statistically significant difference in the incidence of biallelic loss of CDKN2B between Ph+ALL cell lines (42.9%) and Ph-negative BCP-ALL cell lines   .IKZF1 was significantly associated with higher BTZ sensitivity  was significantly lower than that of 54 BCP-ALL cell lines not carrying the deletion  , respectively, were almost similar, and those of 54 and 25 BCP-ALL cell lines with or without CDKN2B deletion , respectively, were identical  was significantly lower than that of 57 BCP-ALL cell lines with monoallelic or no loss of CDKN2A   was almost similar to that of 57 BCP-ALL cell lines with monoallelic or no loss of CDKN2B  (data not shown).Among the nine genes or region tested, only deletion of sitivity . The IC5ey test) . Althougdentical . When we= 0.012) , while tIKZF1 deletion and/or biallelic loss of CDKN2A. Among 65 Ph-negative BCP-ALL cell lines, 11 cell lines carrying IKZF1 deletion  were significantly more sensitive to BTZ than 54 cell lines without the deletion   were significantly more sensitive than 52 cell lines with monoallelic or no loss of CDKN2A  (p = 0.032)  and biallelic loss of CDKN2A (p = 0.007) were independently associated with higher BTZ sensitivity.The above observations suggested that higher BTZ sensitivity of Ph+ALL cell lines may be associated with ey test) , and 13 = 0.032) . Among 1f CDKN2A , suggestll lines . Of noteTo investigate cross-resistance of BTZ to the chemotherapeutic agents that were used in the BTZ combination therapy in the TACL study , 9, we ein vitro anti-leukemic activity of CFZ, a second-generation proteasome inhibitor with irreversible activity [We next investigated the activity . InductiMLL+ALL , t-ALL , t-ALL , and B-other ALL cell lines . Although deletion of IKZF1 was not associated with higher CFZ sensitivity in 79 BCP-ALL cell lines including Ph+ALL cell lines, 11 Ph-negative BCP-ALL cell lines carrying IKZF1 deletion  were significantly more sensitive to CFZ than 54 Ph-negative BCP-ALL cell lines without the deletion  . Finally, we examined the cross-resistance of CFZ to the four drugs that were used in the TACL study regimen using 79 BCP-ALL cell lines  . With rell lines . There wIn general, the anti-leukemic activity of CFZ was higher than that of BTZ, and the IC50 of CFZ was significantly lower than that of BTZ in 79 BCP-ALL cell lines and 9 T-ALL cell lines (p<0.000001 by paired t-test) . The medTo further verify a possible involvement of P-gp expression in specific resistance to CFZ, we examined the sensitivities to CFZ and BTZ in the presence or absence of verapamil  or nilotTo directly confirm an association of P-gp expression with specific resistance to CFZ, we first analyzed P-gp-positive subline of 697 cell line  that wasABCB1-specific CRISPR/Cas9 vector that contains cDNA of Cas9 fused to human CD4 cDNA via a 2A peptide sequence was electroporated into HALO1 cells. 3 days after electroporation, CD4-positive cells were harvested using anti-CD4 antibody and subsequently expanded for further analysis. Flow cytometric analysis of P-gp expression Click here for additional data file.S2 FigShade indicates fluorescence intensity of anti-P-glycoprotein antibody, and line indicates that of control antibody. Relative fluorescence intensity of each cell line is indicated in the middle of each panel, and ratio of bortezomib IC50 value to carfilzomib IC50 value of each cell line is indicated at the top of each panel.(TIF)Click here for additional data file.S3 Fig(A) Flow cytometric analysis of CAM staining in P-glycoprotein-positive HALO1 cells (left panels) and P-glycoprotein-negative RS4;11 cells (right panels) cultured in the presence or absence of P-glycoprotein (P-gp) inhibitors (0.8 μM of nilotinib or 5 μM of verapamil). Geometric mean (GeoMean) of CAM staining is indicated in each panel. (B) Effect of P-glycoprotein (P-gp) inhibitors on CAM staining of P-glycoprotein-positive HALO1 cells (left panel) and P-glycoprotein-negative RS4;11 cells (right panel). The vertical axis indicates GeoMean of CAM staining. Mean ± SD of triplicated experiments are indicated. Asterisks indicate significance (**p<0.01) in a paired t-test.(TIF)Click here for additional data file.S4 Fig(A) Flow cytometric analysis of CAM staining in parental 697 cells (left panels) and 697R cells (right panels) cultured in the presence or absence of P-glycoprotein (P-gp) inhibitors (0.8 μM of nilotinib or 5 μM of verapamil). Geometric mean (GeoMean) of CAM staining is indicated in each panel. (B) Effect of P-glycoprotein (P-gp) inhibitors on CAM staining of parental 697 cells (left panels) and 697R cells (right panels). The vertical axis indicates GeoMean of CAM staining. Mean ± SD of triplicated experiments are indicated. Asterisks indicate significance  in a paired t-test.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.


Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro.Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls . Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines  were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR.p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response.A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology  contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 mutations, who have received three or more prior lines of chemotherapy [Epithelial ovarian cancer (EOC) represents 90% of ovarian cancers, being also associated to very heterogeneous clinical presentation, histological features and therapeutic response . The higotherapy . MoreoveNonetheless, further advancements in the management of recurrent or persistent disease are still required, especially for drug-resistant EOCs that generally show poor responsiveness to additional cytotoxic therapy. Transcriptional profiling represents a useful tool to identify tissue-specific therapeutic targets that impact on clinical outcome.Several studies show that the transcription factor Forkhead box M1 (FOXM1) is widely expressed in solid tumors , acting In the present investigation, we generated the mRNA signatures of tumor specimens belonging to two EOC patients’ cohorts representing the less common cancer subtypes (endometrioid and clear-cell), and of their normal counterpart (endometrium samples). Next, we explored the trend in FOXM1 expression at mRNA and protein level across clear-cell, endometrioid and HGS EOCs compared to their supposed normal tissue of origin . We investigated the association between FOXM1 protein and patients’ survival in relation to their tumor histology and sensitivity to platinum-based therapy. We determined the functional activity of FOXM1 in EOC cells in terms of cell proliferation, cell-cycle regulation, colony formation, and invasion ability by transient FOXM1 knockdown, using two in-house derived EOC cell lines expressing FOXM1, as in vitro models that accurately represent a metastatic HGSC and a primary clear-cell EOC. HGSC is the most frequent, lethal and extensively studied EOC subtype , whereasSince FOXM1 is an emerging master regulator of response to DNA damage , in thisThis study was performed on 90 cases of EOCs diagnosed and treated at the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Brescia, Italy, between November 2001 and September 2013, following the Declaration of Helsinki set of principles, and with approval by the Research Review Board- the Ethics Committee- of the ASST Spedali Civili, Brescia, Italy (study reference number: NP1284). Normal control tissue samples were obtained from 45 patients undergoing surgery for benign pathologies. Written informed consent was obtained from all patients enrolled.Patients’ details are reported in Table Disease Specific survival (DSS) was defined as the time from surgery to death from disease or the last follow-up. Progression Free Survival (PFS) was calculated from the time of surgery until the first clinical recurrence/progression. Cancer progression was defined according to RECIST 1.1 . The PFINinety EOC specimens were collected at the time of primary debulking and immediately frozen as previously described . Normal Ten normal luminal fallopian tube epithelial cells (TEC) and 6 pools of ovarian surface epithelial-cell (OSE) brushings obtained from 20 patients, were separately collected by scraping in 1 ml of physiological saline solution immediately after surgery, as previously described .Two new EOC cell lines (named OSPC2 and EOC-CC1) were derived in our laboratory from fresh clinical samples. Source-patient characteristics and full method of cell line characterization  DNA profiling, BRCA1/2 sequencing and growth rate analysis) are described in Additional file The following Silencer® select pre-designed siRNAs  were employed for in vitro transient gene knockdown: FOXM1 (ID # s5248), negative control no.1 (AM4636) and KIF11 (Eg5) (ID # AM4639). Based on cell lines’ growth-rate analysis  previously verified to be free of any neoplastic pathology, and from EOC cell lines after siRNA transfection. For both tissues and cells, TRIzolⓇ Reagent (ThermoFisher) was used according to manufacturer’s instructions. Total RNA extraction and quality control were performed as previously reported .N = 26) and clear-cell histology (N = 14), as well as NE samples (N = 15), were labelled and hybridized to GeneChip® Human Genome U133 Plus 2.0 oligonucleotide microarray chips  following the manufacturer’s protocols. Microarray experiments on scramble- (siControl) and FOXM1-siRNA transfected (siFOXM1) OSPC2 and EOC-CC1 cell lines were performed using the commercially available G4851B human whole GE Microarray kit (SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray Kit Agilent Technologies) according to manufacturer’s instructions.EOC samples of endometrioid  EOC tissues , stored in the Department of Pathology at the University of Brescia, Italy. As controls, specimens obtained from normal ovaries, fallopian tubes and endometria were used. FOXM1 immunostaining was performed using a primary antibody  diluted at 1:160 after pretreatment with microwave in citrate buffer at pH 6.0 (3 cycles of 5 min at 750 Watt). Immunoreactivity for FOXM1 was considered positive in tumor cells showing a nuclear staining, with or without cytoplasmic staining. FOXM1-immunostained slides were digitalized using an Aperio ScanScope CS Slide Scanner  at 40× magnification, and analyzed using Tissue Studio™ 2.0 workstation . Subsequently, a quantitative scoring algorithm was customized for FOXM1 using commercially available templates from Aperio Technologies and Definiens, and applied in order to quantify the percentage of FOXM1-positive tumor cells and to assess FOXM1 nuclear-staining intensity according to mean brown chromogen intensity. The overall FOXM1 expression “positive index” was determined by multiplying the percentage of the positive cells and the intensity score, then a weighted sum was calculated for each tumor samples. A final immunoreactive score (IRS) from 2 to 90 was calculated.One μg of extracted RNA was reverse-transcribed using random hexamers according to the SuperScript TM II protocol (Invitrogen). The qPCR reactions were performed on CFX96 Touch™ Real-Time PCR Detection System  using the TaqMan Universal PCR master mix and the following Taqman gene expression assays: Hs01073586_m1 (FOXM1), HS99999905_m1 (GAPDH), Hs00188166_m1 (SDHA), Hs00259126_m1 (CCNB1), HS00244740_m1 (CDC25B), Hs00411505_m1 (ASPM), Hs01118845_m1 (CENPF), Hs00921424_m1 (FOXO3), Hs00959834_m1 (XRCC1), Hs00254718_m1 (XRCC4), Hs00998500_g1 (CYR61), Hs00172214_m1 (TOP2A), Hs01037414_m1 (BRCA2), Hs00947967_m1 (RAD51), Hs00766186_m1 (MACC1), Hs01070181_m1 (CEP55), Hs01026371_m1 (CDK6), Hs01548894_m1 (CDK2), Hs00976734_m1 (CXCR4), Hs01548727_m1 (MMP2). Reaction and thermal cycling conditions were performed as previously reported [FOXM1 isoform-specific RT-qPCR was performed using iTaq Universal SYBR Green Supermix (BIO-RAD) and the following primer pairs: FOXM1c F caattgcccgagcacttggaatca, FOXM1c R tccttcagctagcagcaccttg, FOXM1b F ccaggtgtttaagcagcaga, FOXM1b R tccttcagctagcagcaccttg . An anneWhole protein extracts from cells at 24 h following siRNA transfection or untransfection were lysed in NaCl, 1% Nonidet‑40, 50 mM Tris‑HCl (pH 7.5) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and protein concentrations were determined using a Bio-Rad protein assay system (Bio-Rad). Equivalent amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris-buffered saline (TBS) containing 5% non-fat milk, the blots were incubated with the following primary antibodies: anti-FOXM1 , and anti-β-actin ((20–33), 1:200 dilution) , at 4 °C for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary (anti-mouse or anti-rabbit) IgGs at room temperature for 1 h. Signals were detected on BioSpectrum® Imaging System  with the LiteAblot® EXTEND (EuroClone). Images were processed with VisionWorks® LS Image Acquisition and Analysis software .Fixed cells were stained with propidium iodide (PI)  at times 0, 24, 48, and 72 h after transfection with scramble siRNA or specific siRNA targeted for FOXM1 and KIF11. Tests were performed in triplicate, and cell-cycle percentages were measured .For accurate cell-cycle analysis, FOXM1- or scramble-treated cells up to 72 h were exposed to 30 μM bromodeoxyuridine (BrdU) and fixed in 70% ethanol into Matrix™ small tubes. Nuclei were isolated using pepsin/0.1 M HCl mixture and subsequently treated with 2 N HCl for 20 min; BrdU detection was performed after DNA denaturation using FITC Anti-BrdU mAb (BD Biosciences) for 1 h and DNA-content staining using PI (2 μg/ml) for 30 min at room temperature . BrdU anFollowing 24 h siRNA transfection, cells were seeded in 6-well plates in 2 ml medium and grown for 7–9 days. Colonies were fixed and stained with 20% ethanol, 1% crystal violet solution and automated counted using the Entry Level image analysis system . A background correction was performed and the control-cell colony size (≥50 cells) was established as the minimum for setting the cut-off point .4 (OVCAR-3), 1 × 105 (OSPC2), 1.4 × 105 (EOC-CC1) cells/well in serum-free medium. RPMI 10% FBS was applied to the lower chamber as chemoattractant. Following 72 h incubation, the invading cells on the lower surface of the membrane were fixed with methanol, stained with 0.5% crystal violet, photographed and counted in 3 randomly selected fields per well, and the mean number of cells was recorded. The experiments were done in triplicate and repeated four times.After 24 h siRNA transfection, cells were seeded on the upper chamber of the BioCoat Matrigel Invasion Chamber (BD Biosciences) at a density of 2 × 10) was dissolved in DMSO (Sigma).Carboplatin, Cisplatin and Doxorubicin hydrochloride (Sigma) were diluted in culture medium immediately before addition to cell lines. Olaparib  , according to manufacturer’s instructions. Absorbance was measured by a 96-well plate reader  at a reference wavelength of 490 nm. Each experiment was repeated a minimum of three times. The effect of drugs on cell growth inhibition was assessed as percent cell viability, where vehicle-treated cells were taken as 100% viable.The prognostic value of FOXM1 was evaluated on serous and non-serous EOC patients’ subgroups. The survival analyses were performed using Cox’s proportional-hazards models  with FOXProliferation data were modelled as normalized counts to a reference and transformed to a logarithm scale. The model was fit by a linear mixed model to account for the variation of technical replicates between independent experiments. Time was expressed on a 24 h scale.All gene chip analyses are explained in detail in Additional file From differentially expressed genes, network reconstruction was carried out in siFOXM1 EOC-CC1 and OSPC2 cell lines, respectively, with the Ingenuity Pathway Analysis software .The differentially expressed genes in each cell line were assessed for “enrichment” or over-representation in a set of 111 invasion-related genes  or SAS v9.4 software , and employed significance levels of alpha = 0.05 except where previously indicated.The correlation between microarray and RT-qPCR data for FOXM1 gene expression was evaluated by Spearman rank correlation. The statistical significance of differences between two groups was evaluated by Mann–Whitney Comprehensive gene expression profiles of 40 EOC specimens of two different histological patterns (26 endometrioid and 14 clear-cell EOCs), and of their relative normal counterpart  were generated using high-density oligonucleotide microarrays, with the aim to identify subtype-specific biomarkers.A total of 1462 genes (468 up-regulated and 994 down-regulated) were found significantly expressed in 26 endometrioid EOCs when compared to 15 NEs , and the 12th-most up-regulated gene in clear-cell EOCs compared to NEs .FOXM1 was the second-most up-regulated gene in endometrioid EOCs compared to NEs , endometrioid EOCs vs NE . The negative values of the correlation coefficients come from the fact that higher Ct means lower expression.FOXM1 expression measured by genechip platform was highly correlated with FOXM1 expression measured on the same samples via confirmatory RT-qPCR: clear-cell EOCs vs NE , and advanced FIGO stage (p = 0.004), as shown in Table FOXM1 protein overexpression was significantly correlated to serous histology  factor and patients were categorized into the high and the low FOXM1 expression groups, respectively. Thresholds for FOXM1 categorization were computed both for DSS and PFS based on time-dependent ROC curves using 24 and 12 months, respectively, as time points that correspond approximately to 75% survival in each outcome. The estimated best thresholds for grouping FOXM1 expression as high versus low were 10.7 for DSS and 13.3 for PFS. DSS was regressed on FOXM1 expression groups via Cox regression with additional covariates being the FIGO stage, platinum resistance, and histotype dichotomized as serous versus non-serous. Given a significant departure from the proportional-hazards assumption, platinum response was entered as a stratification variable. Moreover, an interaction term for FOXM1 and histotype was entered in the model.The effect of FOXM1 expression group on PFS was similarly modelled via Cox regression, with additional covariates again being the FIGO stage, platinum resistance and serous histotype. An interaction term was added for FOXM1 and platinum resistance.p-value = 0.042) in non-serous histotypes   Fig. .Fig. 3Kap-value < 0.001) in partially sensitive/resistant subjects   Fig. .BRCA status and growth rate analyses is reported in Additional file We derived two new cell lines from clinical samples collected from either solid tumor (EOC-CC1) at first surgery or ascites (OSPC2) at disease progression, respectively. A detailed description of their immunocytochemical features, genetic fingerprint, Since it is well known that alternative splicing of the Va (A1) and VIIa (A2) exons gives rise to three distinct FOXM1 variants, FOXM1a, FOXM1b, and FOXM1c , we firsp < 0.001). Similarly, we estimated a FC = 0.78  and FC = 0.89  for OVCAR-3 and OSPC2 cell lines, respectively  and fold difference (2-fold) in each cell line  software. Functional analysis and annotation revealed a unique highly significant network in each cell line, respectively .By enrichment analysis on a gene set of 111 invasion-related genes, we found that 4 genes (3.6%) were differentially expressed in siFOXM1 OSPC2 cells, namely, CXCR4, EPHB2, TIMP3, and TNFSF10. This constituted statistically significant enrichment compared to OSPC2’s background rate of 1.1% (387 out of 36.226) differential expression in genes unrelated to invasion  and clear-cell (EOC-CC1) histology, to evaluate and compare its functional role in two different EOC subtypes. First of all, we demonstrated that both newly established cell lines retained the histopathologic characteristics of the original clinical samples, thus representing a new platform to study the role of FOXM1 in a case of progressive HGSC, the most common and lethal form of EOC, and in a rarer clear-cell EOC. In agreement with FOXM1 isoforms’ expression in a panel of commercially available HGSC cell lines , we founIn agreement with previously published studies –19, our A cell line-specific FOXM1-dependent gene-expression pattern was successfully validated by RT-qPCR for each cell line, respectively. Of interest, in FOXM1-depleted EOC-CC1 cells, we confirmed the down-regulation of cyclin-dependent kinase 2 (CDK2) gene, known as an ideal target to suppress in order to reduce the peritoneal spread of clear-cell EOC in xenograft mice and thereby to prolong their survival , 43. OthThe molecular profile of FOXM1-depleted OSPC2 cells revealed some common and other unique altered genes when compared to the profile of EOC-CC1. Despite the unexpected findings of invasion assay, we assessed by RT-qPCR that some FOXM1 downstream targets involved in metastasis, cancer progression, and cisplatin resistance, such as MMP2  and MACCBeyond altering tumor phenotype, we demonstrated that FOXM1 silencing conferred a greater sensitivity to standard first-line chemotherapeutic drugs, carboplatin and cisplatin, in our novel cellular models. Our data are consistent with those previously reported for commercially available HGSC cell lines , 18 and BRCA1 and BRCA2, since their absence results in unrepaired lesions, cell-cycle arrest and cell death in cancer cells exposed to cytotoxic chemotherapy [BRCA1/2 status is not the only useful biomarker to predict PARP-inhibitor effectiveness [BRCA1/2 genes, it is likely that their responsiveness to this PARP inhibitor might be associated to other mild genetic, epigenetic or molecular HR defects, and we verified that FOXM1 inhibition enhanced olaparib cytotoxicity in both cell lines.The efficacy of PARP inhibitors has been ascertained in HR-deficient breast and ovarian cancers as single agents or in combination with cytotoxic chemotherapy or ionizing radiation . Essentiotherapy . Howevertiveness . In the tiveness , as a sitiveness . Since oFinally, we demonstrated by RT-qPCR the significant overexpression of FOXO3 mRNA in FOXM1-depleted EOC cells of both subtypes. FOXO3, like FOXM1, is a forkhead transcription factor, a typical tumor suppressor and a functional antagonist of the oncogene FOXM1 . Since FThis is the first report proving evidence of a consistent overexpression of FOXM1 among the three major EOC subtypes, and demonstrating its prognostic value in non-serous EOC patients and in platinum-resistant cases, regardless of their histotype. A full description of the tumorigenic properties of FOXM1 has been reported for a clear-cell and a metastatic HGS EOC cell lines, accurately representing their tumors of origin. Importantly, our data suggest that FOXM1 might be considered a valid target for combinatorial anticancer therapy, despite molecular and histological tumor heterogeneity. Consistent with this view, FOXM1 small-molecule inhibitors  and otheAdditional file 1:Table S1: Clinical features of patients and tumor characteristics of samples used to derive cell lines; Table S2: Antibodies features and detailed staining protocols; description of short tandem repeat (STR) DNA profiling; description of BRCA1/2 sequencing; determination of cell lines’ growth rate. (DOCX 23 kb)Detailed protocols for the establishment and the characterization of the EOC cell lines. Additional file 2:Table S3: Genes related to tumor invasion-metastasis used for the Gene-Sets Enrichment Analysis (GSEA). (DOCX 18 kb)Description of microarray analysis on EOC tissue specimens and cell lines. Additional file 3: Table S4.Differentially expressed genes in endometrioid EOCs compared to normal endometrium. (XLSX 327 kb)Additional file 4: Table S5.Differentially expressed genes in clear-cell EOCs compared to normal endometrium. (XLSX 337 kb)Additional file 5:Table S6: Panel of immunocytochemical stains in EOC cell cultures; Table S7: STR profiles of EOC-CC1 and OSPC2 biopsies and derived cell lines; Table S8: List of BRCA1 and BRCA2 sequence variants in OSPC2 cell line; Table S9: List of BRCA1 and BRCA2 sequence variants in EOC-CC1 cell line; Figure S1: Immunohistochemical stain for FOXM1 in original clinical tumor samples and in derived cell lines. Figure S2: EOC cell lines growth curves; Table S10: Optimal cell densities for seeding different cell lines in culture. (DOCX 552 kb)Results of the characterization of OSPC2 and EOC-CC1 cell lines; Additional file 6: Figure S3.Flow cytometric and BrdU analyses of DNA content in siFOXM1 EOC cells. (DOCX 1136 kb)Additional file 7:Figure S4: Volcano plot displaying differential expressed genes between siFOXM1 and siControl EOC-CC1 cells. Table S11: List of down-regulated genes in siFOXM1 EOC-CC1 cells. Table S12: List of up-regulated genes in siFOXM1 EOC-CC1 cells. (DOCX 160 kb)Additional file 8:Figure S5: Volcano plot displaying differential expressed genes between siFOXM1 and siControl OSPC2 cells. Table S13: List of down-regulated genes in siFOXM1 OSPC2 cells. Table S14: List of up-regulated genes in siFOXM1 OSPC2 cells. (DOCX 260 kb)Additional file 9:Figure S6: Volcano plot displaying differential expressed genes between siFOXM1 and siControl OVCAR-3 cells. Table S15: List of down-regulated genes in siFOXM1 OVCAR-3 cells. Table S16: List of up-regulated genes in siFOXM1 OVCAR-3 cells. (DOCX 345 kb)Additional file 10: Table S17.Differentially expressed genes in siFOXM1 EOC cells compared to siControl. (XLSX 100 kb)Additional file 11: Figure S7.Functional analysis of the genome-wide transcriptional response in FOXM1-silenced EOC cells. Putative network reconstruction on EOC-CC1 and OSPC2 cells. (PDF 178 kb)


CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties.Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen  and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 , ACHN , HKCSC (putative positive control), and ASE-5063 .CD105 and/or CD133 expression with the enhancement of stem-like properties.In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines. Renal cell carcinoma (RCC) is the seventh most common tumor and is associated with high mortality . SeveralIn the case of RCC, putative CSCs markers were proposed, including CD105 , CXCR-4 2) /cell-free area (0 h) × 100. Each scratch was performed in triplicate.After passage, the cells were collected as described above and seeded at 500 cells per 15 μL drop of appropriate medium on the inner side of a 100 mm Petri dish lid. The lid was turned upside down and placed on top of the dish filled with 10 mL of PBS. Cells were observed daily for 10 days to detect the creation of aggregates. Photos were taken using an inverted light microscope . For quantitative analysis, areas of formed aggregates were counted from three representative microscope fields (drops) by two independent experimenters using CellSens software .2 cell culture flasks as described above. After 72 h incubation, cells with approximately 80% confluence were collected with Accutase, and suspended in PBS. Then cells were centrifuged and re-suspended in 4% PFA for 10 min at room temperature. PFA was removed by centrifugation, and cells were placed in ddH2O onto ICC SuperFrost microscope slides  and allowed to dry. The staining was performed in a Flex Autostainer instrument  and with the use of EnVisionFlex kits . Antigen retrieval was performed with 5 min proteinase incubation for CD105 or 1 h incubation in a target retrieval solution (pH = 6) for CD133. After the blockade of endogenous peroxidase activity, the slides were incubated for 1 h with primary antibodies: mouse anti-human CD105 , or rabbit anti-human CD133 . After washing, the slides were incubated with appropriate secondary antibodies labelled with HRP. For CD105 staining, HRP activity was amplified with the FLEX+ Mouse Linker. Visualization was performed with Flex DAB+ chromogen. Slides were counterstained with hematoxylin , coverslipped with Pertex  and observed in a light microscope .Cells were cultured in 75 cmP-value was less than 0.05 in the Student’s t-Test calculated in MS Excel (Microsoft), and were designated with an asterisk on graphs (P < 0.1 is designated with a double asterisk).Data are presented as the mean values ± SD or ratios (hypoxia/normoxia). Each experiment was performed at least three times with three technical replicates. The differences between the groups studied were considered significant when the Among an evaluated panel of established RCC cell lines, including primary tumor derived and metastasis derived Table , the CD1For further analyses, HKCSCs, ASE, Caki-2 (high CD105 expression), and ACHN (low expression) cell lines were selected. Caki-2 and ACHN cell lines were recently evaluated as derivatives of papillary RCC –34; therA high number of CD105+ cells in Caki-2 were confirmed in ICC staining—one-third of the cells were positive for this marker Fig. —and CD10The CD133 receptor as the RCC progenitor cell’s putative marker –37 was aoct4 and sox2 genes—markers of embryonic, adult, and CSC—was evaluated, but only oct4 expression was significantly higher in CD105-high cells (Caki-2). The expression of oct4 and sox2 was also detected in ASE cells, but not in HKCSCs , only ACHN and Caki-2 cells were viable Fig. . ACHN ceCell–cell cohesion properties were screened in a hanging drop assay, and Caki-2 and ASE cells formed the aggregates, while HKSCSs created a compact structure with firm edges Fig. . Through2 would promote stemness features. Both pRCC cell lines Caki-2 and ACHN overexpressed hif1, in comparison to tested normal renal ASE cells, but the level of the hif2 gene was lower than in the ASE cells  subpopulations were previously effectively isolated from established cell lines including RCC, mostly with the side-population isolation approach  , 59, 60.2 tension develops in tumors in vivo [oct-4 and sox2). TF expression was observed both in ACHN cells where growth was suppressed by low O2, and in Caki-2 that proliferate more extensively in hypoxia. The above mentioned TFs are known to be up-regulated by HIF2 [hif2 expression simultaneously with stem-related TFs in hypoxia. This is in accordance with pRCC showing lower hif2 expression than ccRCC [111. Cancer cell movement in the wound healing assay corresponds to the cell’s ability to exit a primary tumor, to penetrate into blood vessels, and to finally spread in vivo. In many cancer types, it has been proven that internal tumor hypoxia induces cancer migration potential both in vivo [2. On the other hand, Caki-2 cells, which proliferate more rapidly in hypoxia, simultaneously exerted more stem-like features and grew in large spheres, but not in semi-soft agar while remaining in aggregates in a hanging drop culture. Cumulatively, in vitro assays performed to test cell line aggressiveness and tumorigenicity [2 tension, promotes vascularization, and protects cells from hypoxia-induced apoptosis [vhl status [tp53/vhl wild-type.The next aim of the study was to unravel the role of hypoxia in pRCC invasiveness and the promotion of stem-like features and marker expression. It is well described that low O in vivo , 65 and  in vivo . Our stu by HIF2 ; howeveran ccRCC . As a rean ccRCC , 69, 70.an ccRCC . Cancer  in vivo  and in v in vivo , 74, altgenicity  suggest poptosis . Howeverpoptosis  nor on vl status , as bothWe did not observe a positive correlation of CD105 level and colony formation and clonogenicity in tested ACHN and Caki-2 cell lines, neither in normoxic nor hypoxic conditions. The Caki-2 pRCC cell line, characterized as CD105high, had weaker stem features when compared to the CD105low ACHN cell line with a more aggressive phenotype. The differences in stem potential of those cells describe the characteristics of primary and metastatic cells rather than the contribution of CD105+. Also, in contrast to other studies, we have shown that in the case of pRCC, hypoxic conditions in vitro diminish the stem-like properties of cell lines –85.


Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.in vivo for the treatment of most aggressive medulloblastoma variants.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics  Angiogenesis is critical for tumor growth and metastasis, therefore receptor tyrosine kinase inhibitors targeting tumor blood vessel formation by VEGFR inhibition increasingly gain access to multimodal therapeutic approaches against brain tumors , 2. ReceThe efficacy of Vandetanib has been suggested to rely not solely on inhibition of angiogenesis but also on direct attenuation of tumor growth by blockade of its target receptors namely vascular, endothelial growth factor receptor 2 and 3, epidermal growth factor receptor (EGFR) and c-RET expressed on the cancer cells themselves. –11. VEGFHere we present first evidence that Vandetanib has profound cytotoxic activity in pediatric medulloblastoma cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma –25. In kin vitro and in vivo potential for the clinically available PI3K inhibitor GDC-0941 for medulloblastoma therapy [In our latest study we already documented promising  therapy . In thisIn a dose-response analysis we evaluated the anti-carcinogenic potency of Vandetanib in Daoy, a cell line modeling SHH-TP53-mutated medulloblastoma, and in the MYC-amplified Non-WNT/Non-SHH medulloblastoma derived cell lines MEB-Med8-A, D283 Med and D341 Med,  in comparison to control (CN: 164±23 and ACS: 1028±29p2). At the same concentration in Daoy we only documented a reduction in colony size (1618±900p2) compared to control (3723±1000p2) but not in colony numbers. Increasing the dose to 4 μM enhanced the observed effects in both cell lines and additionally resulted for Daoy in a profound decline in the number of colonies (173±45) in comparison to control (300±35).At 2 μM, a concentration typically achieved in patient plasma, MEB-Med-8A showed significant reduction of colony numbers and colony size  or patient plasma (10 μM) levels results in enhanced anti-neoplastic efficacy , 36. ForIn the SHH-TP53-mutated cell line Daoy and the MYC-amplified cell lines D283 Med and D341 Med single application of Etoposide at cerebral spinal fluid levels of 1 μM displayed anti-neoplastic efficacy with 30-60% residual cell viability. In contrast MEB-Med-8A, which also models MYC-amplified tumors, was resistant at 1 μM of Etoposide and displayed only moderate susceptibility to 10 μM Etoposide corresponding to patient plasma levels with 74±4% residual cell viability.We have previously show additive anti-neoplastic efficacy for the concomitant application of Etoposide and the PI3K-antagonist GDC-0941 in medulloblastoma cell lines . Here weWith regards to concomitant application of Vandetanib and GDC-0941 in the SHH-TP53-mutated cell line Daoy we determined in our previous experiments a trend of higher anti-neoplastic efficacy in comparison to single drug application. Here in a cumulative assay determining anti-proliferative and cytotoxic drug effects by means of the metabolic state of the residual cell population this trend could be confirmed. Furthermore, we posed the question, whether inhibition of receptor tyrosine kinases and their downstream signaling molecules could enhance medulloblastoma standard treatment regimes. We treated the investigated cell lines therefore concomitantly with Vandetanib and GDC-0941 in the presence of the chemotherapeutic Etoposide. Indeed, in the SHH-TP53-mutated cell line Daoy as well as in the MYC-amplified cell lines D283 Med and D341 Med the application of the Vandetanib-GDC-Etoposide combination augmented the cytotoxic effect regardless of the applied concentration of Etoposide in comparison to single or dual drug application. In detail, in Daoy and D283 Med at 1 and 5 μM of Etoposide respectively the concomitant application of Vandetanib, GDC-0941 and Etoposide resulted in complete loss of viable cells while in D341 Med at Etoposide concentrations of 1 and 5 μM residual cell viability plateaued 13.6±6 and 15.3±4% respectively. Applying higher Etoposide concentrations in combination with the applied kinase inhibitors did not lead to further enhancement of anti-neoplastic efficacy. In line with above described Etoposide resistancy of MEB-Med-8A concomitant application of Vandetanib and GDC-0941 was sufficient to reduce cell viability completely.In this study we report that Vandetanib exerts anti-neoplastic activity against a panel of four pediatric medulloblastoma cell lines. MEB-Med-8A, D283 Med and D341 Med exhibit distinct characteristics such as MYC-amplification and occurrence of isochromosome 17 of the most aggressive group of tumors within the Non-WNT/Non-SHH group of medulloblastoma –23. In c0/G1-Phase of the cell cycle , was still highly sensitive to single treatment with Vandetanib and GDC-0941 respectively. Moreover, concomitant application of Vandetanib and GDC-0941 resulted in de facto complete loss of cell viability. Whether this finding, although promising, can be translated to refractory tumors therapy in patients needs further evaluation.The development of rational and personalized cancer therapy depends on the identification of valid biomarkers. Expression of target structures might serve as markers for the integration of TKI such as Vandetanib into standard treatment regimes of medulloblastoma. However, in other cancers microRNA expression patters are discussed as promising indicators of eligibility for patient treatment with Vandetanib .In general, our findings underscore the notion that combined inhibition of specific oncogenic tyrosine kinases together with important downstream signaling nodes - such as the PI3K that integrates signals of multiple oncogenic kinases - can enhance anti-neoplastic efficacy in combination with standard medulloblastoma chemotherapeutics.In conclusion, our study demonstrates that Vandetanib, a clinically available angiogenesis inhibitor, displays direct anti-neoplastic activity against pediatric medulloblastoma cell lines modeling the clinically most aggressive SHH-TP53-mutated and MYC-amplified tumor variants. Furthermore, our study documents that combined application of Vandetanib and GDC-0941, a clinically available PI3K/AKT pathway inhibitor, results in fortified anti-neoplastic responses against SHH-TP53-mutated and MYC-amplified medulloblastoma. Furthermore, we delineate that Vandetanib in combination with the standard chemotherapeutic Etoposid displays additive anti-neoplastic efficacy that can be further enhanced by PI3K inhibition. Therefore, our findings provide a rational to further investigate Vandetanib alone, in combination with PI3K/AKT pathway inhibitors and standard chemotherapeutics for the treatment of medulloblastoma variants that despite of intensive multi-modality treatment are currently still associated with poor prognosis.Vandetanib and GDC-0941 were obtained from LC Laboratories. The primary antibody pSTAT3 , STAT3 (124H6), pAKT (Ser473), AKT (11E7) and GAPDH (D18H11) were purchased from Cell Signaling while secondary antibodies were purchased from Dianova. Carboxyfluoreszein-Succinimidyl Ester (CFSE) was purchased from Invitrogen, while Hoechst 33258 was provided by Sigma.The human medulloblastoma cell lines, Daoy (HTB 186), D283 Med (HTB-185) and D341 Med (HTB-187) were obtained from American Type Culture Collection (ATCC). The medulloblastoma cell line, MEB-Med-8A, was generated by Prof. T. Pietsch. The medulloblastoma cell lines Daoy, D283 Med and MEB-Med-8A were maintained in complete medium, namely Dulbecco's Modified Eagle Medium  with L-glutamine supplemented with 1 mM sodium pyruvate (PAA), 1% penicilline/streptomycine (Invitrogen) and 10% fetal bovine serum . The medulloblastoma cell line D341 Med was maintained in DMEM with L-glutamine supplemented with 1 mM sodium pyruvate, 1 % penicilline/streptomycine and 10 % Human Serum .3 Daoy, 6×103 MEB-Med-8A, 104 D283 Med and 104 D341 Med cells respectively. After overnight culture in complete medium, the cells were treated with 1, 2, 4 and 10 μM of Vandetanib alone or in combination with 0.1, 0.5, 1, 5 and 10 μM of etoposide and 1 μM of GDC-0941. The vehicle Dimethylsulfoxid (DMSO) served as control. After 48h of MKI treatment, MTS was added according to the supplier's protocol and the absorbance was measured at 490 nm using an ELISA plate reader . Cell viability was calculated in percent of control.Cell viability was assessed with CellTiter 96 Aqueous One Solution Cell proliferation Assay (Promega) that contains 3--5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To ensure a linear growth curve over 48h for assessment of MKI-effects, each well of 96-well plates was seeded with 2.5×10Medulloblastoma cells were stained with CFSE according to the instructions of the supplier. Daoy, MEB-Med-8A, D283 Med and D341 Med cells were seeded in 6-well cell culture dishes in complete medium. After overnight culture, the cells were treated with 2 μM of Vandetanib for 48h. Thereafter floating and attached cells were collected, resuspended in 200 μl, stained with Hoechst33258 and analysed by flow cytometry. The number of viable cells was assessed for 120 sec in same volume and at constant speed. Proliferation was traced by CFSE staining and normalized to the control DMSO, while cell death was determined by Hoechst33258 staining.in vitro scratch assay was performed as described by Liang et al. [The g et al. . BrieflyA total protein concentration of 25 μg derived from medulloblastoma cell lines was separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1h at RT in 1x Tris-buffered saline containing 0.1% tween-20 (TBST) supplemented with 5% BSA. Thereafter, the membranes were incubated with the primary antibodies overnight at 4°C and subsequently with the respective secondary antibody for 1h at room temperature. Immunoreactivity was detected by chemiluminescence and quantified by means of a ChemiDoc XRS Imaging System (Bio-Rad).2 were excluded from the analysis since these represented stain artefacts, cell detritus or non-proliferating single cells.The cell lines Daoy (200 cells/well) and MEB-Med-8A (1000 cells/well) were plated in six well cell culture dishes. The cells were allowed to adhere and spread properly for 12h at 37 °C. Thereafter the cells were exposed to 1, 2 and 4 μM of Vandetanib. After 48h of exposure the cells were washed with standard medium to remove any trace of the inhibitor and cultured for another week. Colony numbers, and colony size was assessed by IMAGEJ. Particles smaller than 40 pixelt-test was applied to determine statistical significance between groups. p<0.05 (*), was considered as statistically significant. Values stated within text and figures represent mean ± standard deviation.The two-sided Student's