Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines. Mammary tumors are the most frequent neoplasia in intact female dogs and often accompanied with high recurrence and metastasis rates . In veteThe Warburg effect was characterized in the early 1920s by Otto Warburg and describes the metabolic energy production of most cancer cells which rely on aerobic glycolysis in presence of oxygen . Hypoxiain vitro and pointed out that non-cancerous cells were not affected [Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase inhibitor  and thusaffected . Dunbar affected  but in caffected . DCA in affected  whereas affected . Higher affected  as well affected .In previous studies we investigated the effect of DCA on canine prostate adenocarcinomas and transitional cell carcinomas in vitro  and founin vitro models to gain more information of cancer independent sensitivity [For evaluation of anticancer drug efficacy in preclinical experiments cell lines represent important sitivity –26. In tThis is the first study evaluating the effect of 10 mM DCA on canine mammary carcinoma as well as canine mammary adenoma cell lines in comparison to a non-cancerous mammary gland derived cell line and a non-treated control. Therefore, the influence on cell counts, viability, apoptosis, mitochondrial respiration and proliferation was examined. Furthermore, the expression of microRNA involved in proliferation and apoptosis was determined.Four cell lines derived from different mammary tissues were used for experiments. MTH53A (non-neoplastic mammary gland), MTH52C (mammary carcinoma) and ZMTH3 (mammary adenoma) were transfected with SV-40 and routinely maintained in the Small Animal Clinic, University of Veterinary Medicine, Hannover, Germany. SV-40 transfection was performed to achieve immortalization as primary cultures derived from mammary tissue  showed limited proliferation capacity. DT14/06T (mammary carcinoma) was established by continuous cultivation in the Small Animal Clinic, University of Veterinary Medicine Hannover, Germany. The cell lines were classified after pathohistological examination of the primary tissue. All tissues used for cell line establishment were collected as part of routine mastectomy and with the owner’s permission. Therefore according to German standards, ethical approval was not required.2 culture flasks  with medium 199  containing 10% fetal calf serum , 2% penicillin-streptomycin  and incubated at 37°C and 5% CO2 in humidified atmosphere. At 85% confluency cells were trypsinized with TrypLETM Express  and were splitted 1:2.All cell lines were routinely maintained in 25 cm2 flasks . DCA was diluted in deionized water and filter-sterilized after pH modulation to 7.4 with NaOH. All cell lines were cultured without DCA and used as non-treated control respectively. The concentration of 10 mM DCA was chosen in order to allow comparability with previous human [in vitro studies. Furthermore, this dosage is applied in an explorative study setting.Cells used in experiments were exposed to 10 mM DCA over 48 hours and cultured with 10 ml medium in 75 cmus human  and canius human  in vitroTM Express  cell number was counted with an automated CellometerTM Auto T4  and compared with untreated control. Cells were washed with PBS, centrifuged at 1000 rpm for 10 minutes (Biochrom) and stored at -80°C for further examinations .After washing with PBS to remove dead cells and trypsinization with TrypLETM BCA Assay (Thermofisher Scientific) as described in manufacturer´s instructions.Lactate release in media was performed by colorimetric determination with Cobas® C311 . Therefore growth media from cell culture was centrifuged at 1000 rpm for 10 minutes to remove floating cells and debris and 1.3 ml were removed to a sodium fluoride vessel . To eliminate influences of phenol red and fetal calf serum, medium was used as negative control and deducted from measurements. Further the lactate content was normalized to intracellular protein concentration to prevent influences of cell numbers and volume. Protein concentration was assessed with Pierce2. Measurements were performed every 24 hours for four days. Therefore medium was replaced and 20 μl MTT  was added and incubated for two hours at 37°C in the dark. Absorbance was determined with a Synergy2 plate reader  and data were analyzed with Gen5TM 1.11 Software (BioTek). Absorbance was reduced by media negative control and in addition normalized to non-treated new seeded cells.Depending on each cell line 3000–6000 cells were seeded in a 96-well-plate  with 200 μl medium 199, 10% fetal calf serum, 2% penicillin-streptomycin, 10 mM DCA and incubated at 37°C and 5% humidified CO5 cells were cultured in a 6-well-plate  as described above. After 48 hours following 10 mM DCA exposure, cells were trypsinized and centrifuged together with supernatant containing non-adherent and dead cells at 1000 rpm for 6 minutes. Cell pellet was resuspended in 500 μl assay buffer and staining was performed as described in manufacturer’s protocol. 104 events were counted with BD FACScaliburTM  and CellQuestTM Pro 6.0 software (BD Biosciences). Annexin and Sytox were detected in FL-1. Data analysis was performed with FlowJo Version 10.0.8r1 . Gates were determined based on positive controls (cells permeabilized with Saponin) and negative controls of each cell line (non-treated viable cells).Apoptosis and quantity of dead cells was analyzed with Annexin FITC and Sytox labeling . Therefore 106 cells using the NucleoSpin Small RNA Kit  as described in manufacturer’s protocol. 35 ng total RNA was used for cDNA synthesis using Taqman® MicroRNA Reverse Transkription Kit  according to manufacturer´s instructions. Following cycle conditions were used: 30 minutes 16°C, 30 minutes 42°C, 5 minutes 85°C. Relative quantification of microRNA expression of treated cells in comparison to untreated control was performed with Eppendorff realplex4 Cycler  using 1.33 μl cDNA in a total volume of 20 μl containing TaqMan® Universal Master Mix NoAmpErase® UNG  and TaqMan® MicroRNA assays for Mir141 (ID 245445_mat), Mir145 (ID 002278), Mir375 (ID 000564) purchased from Thermofisher Scientific. Procedure was maintained as manufacturer´s protocol conducting following conditions: 95°C for 10 minutes subsequently 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Data were normalized to the reference gene RNU6B (ID 001093) and analysis was performed using Rest2009 . Samples with Ct-values >35 were excluded from analysis.Isolation of total RNA was performed with 10TM instrument  and processed with xPONENT 3.1 software (Luminex Corporation). Additionally, samples for PDH measurements were filtered with centrifugal ultrafree filter units with a pore size of 0.65 μm (Merck Millipore) at 7000 rpm for 4 minutes. Values with MFI < background MFI + 2 x standard deviation were excluded from analysis. Quantitation of survivin was performed with ProcartaPlex Human Survivin Simplex Kit  using cell culture supernatant as described in manufacturer´s protocol. Survivin values were normalized to protein concentration . Total PDH, PDH-P and apoptotic proteins (BAD and JNK) were analyzed with multiplex assays from Merck Millipore . For protein evaluation a total protein amount of 10 ng (PDH-P) or rather 25 ng (apoptotic proteins JNK and BAD) was used, which was determined with Pierce BCA Assay according to manufacturer’s instruction (Thermofisher Scientific).Samples for protein expression analysis were prepared as specified in manufacturer´s protocol and measurements were performed with xMAP® Luminex Bead Technology using a Luminex 200Mitochondrial ROS production as indirect marker for mitochondrial activity was determined by staining of mitochondrial derived ROS using 4 μM MitoSox  for 15 minutes. Cells were grown on 8-well μ-dishes  treated with 10 mM DCA for 48 hours. After fixation with 4% paraformaldehyde, μ-slides were washed with Hank´s Balanced Salt Solution (HBSS) containing calcium and magnesium. After staining cell nuclei were labeled with DAPI  for 5 minutes. Fluorescence imaging was performed with an inverted confocal laser scanning microscope  using a 60x water immersion objective (Nikon). Images were taken with EZ-C1 1.80 software (Nikon). The excitation occurred with a diode laser at 408 nm (DAPI) and with a helium/neon laser at 543 nm (MitoSox). Total cell fluorescence of MitoSox deducting background was analyzed with ImageJ and normalized to cell counts.2. After 48 hours incubation 100 μl ATP-solution  was added. Following 10 minutes incubation at room temperature, luminescence measurement was performed with Synergy2 Plate Reader (BioTek). In order to normalize the data with the respective cell numbers, cells were counted in a simultaneously performed experiment by flow cytometry .Mitochondrial respiration was directly shown by measuring cellular ATP synthesis. The cell lines were seeded (5000 cells/well) as triplicate in a 96-opaque-well plate (Greiner Bio-One) with 100 μl medium 199, 10% fetal calf serum, 2% streptomycin-penicillin, 10 mM DCA and incubated at 37°C humidified COCultivation, fixation and washing were performed as described above. After permeabilization using 0.2% Triton X-100 for 20 minutes, cells were exposed overnight to a canine specific rabbit-polyclonal Ki67 antibody . For labeling, a monoclonal anti-rabbit Alexa Fluor® 555 antibody  was incubated for 1 hour (1:250) and cells were counterstained with DAPI (1:1000) for 5 minutes. Fluorescence imaging protocol was the same as described above. Total cell fluorescence was established as described above. For TUNEL staining Apoptag Fluorescein Direct Kit (Merck Millipore) was performed as manufacturer´s instructions. The excitation occurred with an argonlaser at 488 nm and imaging was performed as described above. The amount of TUNEL positive cells was evaluated.Protein expression analysis was performed with Immunoblotting (Western Blot) as described in detail by Wagner et al. . The proStatistical analysis of data was performed with SAS software 7.1 . For comparison of two means, two-tailed t-test was used. The confidence value was set to 5% (p<0.05) and was considered statistically significant.Following 48 hours DCA treatment (10 mM) the cell numbers of both mammary carcinoma cell lines MTH52C (p = 0.0171) and DT14/06T (p<0.0001) decreased significantly in comparison to non-treated control. A comparable significant effect was observed in the mammary adenoma cell line ZMTH3 (p = 0.0131) and could also be constituted in the non-cancerous mammary gland derived cell line MTH53A (p = 0.0039). Although MTH53A showed decreasing cell numbers, a statistically significant difference between the mammary carcinoma cell line DT14/06T (p = 0.0140) and the non-cancerous cell line MTH53A was present. No difference was observed between MTH53A and the adenoma derived cell line ZMTH3 or the mammary carcinoma derived cell line MTH52C .The proliferation, assessed by detection of the proliferation marker Ki67, decreased significantly in all mammary cell lines with exception of the non-neoplastic mammary gland derived cell line MTH53A in comparison to non-treated control and further a significant decrease in proliferation can be observed within comparison of MTH53A and the other cell lines. No difference was assessed between neoplastic cell lines .In comparison to negative control DCA reduced the lactate release into the media in all examined cell lines except in the benign mammary adenoma cell line ZMTH3 (p = 0.1218). No statistical difference was observed within non-cancerous and neoplastic cell lines .In order to assess the effect of DCA on PDK expression, protein analysis of PDK-1 and PDK-3 was performed. As shown in 232 decreased significantly in all cell lines whereas PDH-P at Ser293 was reduced only in the mammary carcinoma DT14/06T (p = 0.0445) (293 in comparison to untreated control. With consideration of PDH-P at Ser300 a significant decrease is apparent between mammary carcinoma derived cell lines MTH52C (p = 0.0263), DT14/06T (p = 0.0282) and non-treated control. ZMTH3 showed visible tendency of PDH-P reduction but did not reach significance (p = 0.0852). No reduction was apparent in all PDH-P residues between MTH53A and neoplastic cell lines.Phosphorylated PDH (PDH-P) and thus inactive PDH was measured with Luminex Magnetic Bead technology to evaluate whether DCA has an effect on pyruvate oxidation. After normalization to total PDH expression phosphorylated PDH at Ser 0.0445) . The mamMitochondrial respiration was evaluated via indirect measurement of mitochondrial derived ROS. As shown in After 48 hours DCA treatment, viability analysis with flow cytometry revealed no changes in apoptosis and amount of dead cells in MTH53A, ZMTH3 and DT14/06T . In MTH5To determine if trypsinization caused negative effects, apoptosis was evaluated with TUNEL method via fluorescence microscopy. No difference in number of TUNEL positive cells was evaluated in any of these cell lines .Further, two proteins  involved in apoptosis were evaluated. The protein expression was performed with Luminex Magnetic Bead technology. After 48 hours of DCA treatment, the phosphorylated and thus activated c-jun N-terminated kinases (JNK) decreased significantly in MTH53A (p = 0.0046), ZMTH3 (p = 0.0228) and MTH52C (p = 0.0131). The inverse case was observed in mammary carcinoma cell line DT14/06T (p = 0.0121) presenting significantly increased active JNK expression .The phosphorylated and thus inactive form of Bcl-2-Antagonist-of-cell-death (BAD) showed significantly increased values in all neoplastic cell lines. In MTH53A (p = 0.2340) no difference was detectable .Decreased survivin expression is apparent in all cell lines but significance is present in cell line DT14/06T (p = 0.0125) in comparison to untreated control as well as in comparison to non-neoplastic mammary gland derived cell line MTH53A (p = 0.0200) .The metabolic activity as reference for proliferation and viability was assessed during 96 hours ongoing DCA treatment . After 9Three microRNAs involved in apoptosis and proliferation were analyzed with quantitative RT-PCR. miR-145 was detectable only in the mammary carcinoma cell line DT14/06T  and showThe initial aim of this study was to determine which metabolic processes occur in canine mammary gland derived cell lines during DCA exposure and if DCA might be beneficial for cancer treatment in dogs bearing mammary gland neoplasia. The results presented illustrate that DCA has anti-proliferative effects on cell growth which is shown in decreased cell number and reduction of the proliferation marker Ki67 expression. The growth inhibiting effect was likewise observed in several human studies –32 incluDue to increased cellular respiration and thus mitochondrial ROS production, it is hypothesized that DCA induces mitochondrial dependent apoptosis by release of cytochrome c and activation of several apoptosis proteins . HoweverApart from this, survivin expression, an apoptosis inhibitor and proliferation promotor , 43, sho232 and Ser300 appear to be the most important metabolic modulation in canine PDH regulation. This supports the assumption of Rardin et al., that PDH phosphorylation at Ser232 is not limited to heart tissue [293 phosphorylation was not significantly affected by DCA [293 is reported to be phosphorylated rapidly by PDK, especially isoform 2 [DCA, a known PDK inhibitor, shifted the energy production from glycolysis towards normal cellular respiration with glucose oxidation . This ist tissue . In contsoform 2 , indicatIn order to evaluate the effect of DCA on cell division and apoptosis, miR-141, miR-145 and miR-375, often deregulated in cancer , were anIn comparison to human mammary cancer cell lines following DCA treatment, canine mammary cell lines are almost consistent , 41. DCAin vivo without severe side effects. But these data indicate that DCA might be supportive in sensitization of cancer cells to other chemotherapeutics [Further the dose of 10 mM DCA is clinically not achievable apeutics . Furtherapeutics .S1 FigIn comparison to non-treated control, no changes in apoptosis were observed. Data are shown as mean ± SD; n = 3 and are presented as percentage of TUNEL positive cells in comparison to negative control. Statistical analysis was performed with two-tailed t-test; *p>0.05.(TIFF)Click here for additional data file.S2 FigWestern Blot analysis of PDK-3 expression in MTH53A, MTH52C, ZMTH3 and DT14/06T. PDK-3 expression was not detectable in all cell lines. GAPDH and α-tubulin were used as loading control.(TIFF)Click here for additional data file.


AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence. A- anLMNA, LMNB1 and LMNB2. Lamins A and C are alternatively spliced isoforms of the LMNA gene. Lamins B1 and B2 are the products of the LMNB1 and LMNB2 genes. Lamins A, B1 and B2 are initially synthesized as precursors, known as prelamins, which are processed into mature lamins through a sequential series of post-translational modifications . G. Gd) [233.2.a).Previous work has shown that A- and B-type lamins form two prominent nuclear meshworks with points of co-localization during interphase . During figure 3b). Double staining of HPFs for AKTIP and either lamin A or lamin A/C showed a partial co-localization of AKTIP with A-type lamins at the nuclear periphery (b). We also observed partial colocalization of AKTIP intranuclear signals with lamin A/C signals (b).We next investigated whether AKTIP co-localizes with A- and B-type lamins in interphase nuclei. The analysis of HPFs stained for both AKTIP and lamin B1 showed that the two proteins have multiple co-localization points, both at the nuclear rim and in the nucleoplasm b. Doubleeriphery b. We als signals b.c–e and electronic supplementary material, figure S4). In all cell types, antibody staining detected specific AKTIP-enriched mitotic structures, which were absent in AKTIP-depleted cells (shAKTIP) . The AKTIP signal was associated with structures at the cell poles that overlapped the spindle microtubules and the centrosomes. In addition, AKTIP concentrated to the midbody, a transient structure that connects the two daughter cells at the end of cytokinesis. The midbody contains microtubules that overlap in a narrow region at the centre of the structure. This region often appears dark after tubulin immunostaining (dark zone), because it is tightly associated with proteins that block antibody binding to tubulin [figure 3d), suggesting a possible role of AKTIP in cytokinesis.To determine AKTIP localization during mitosis, we used a fixative containing 0.25% Triton X-100. This fixative results in a high level of protein extraction allowing a clear visualization of AKTIP, but it does not fully preserve spindle microtubules. We analysed AKTIP localization in BJ-hELT (BJ fibroblasts expressing hTERT and SV40 Large T) and HeLa cells and HPFs (figure 3 tubulin . Remarkae). By contrast, lamin B1 accumulated at the centrosomes and partially co-localized with AKTIP, which was enriched at both the centrosomes and the spindle .To ask whether AKTIP co-localizes with lamins during mitosis, we co-immunostained HPFs for AKTIP and either lamin A or B. As expected from previous work , lamin Afigure 33.3.LMNA gene, leading to a lamin A form deleted of 50 aa and permanently farnesylated [LMNA mutation that replaces an arginine with a histidine at position 527, are characterized by accumulation of prelamin A and progeroid traits [a,b). In unextracted LV-progerin cells expressing both endogenous lamin A and progerin, and more effectively in unextracted HGPS cells, which are heterozygous for the LMNA mutation, the AKTIP signal in the cytoplasm was reduced and the nuclear rim staining was weaker and more discontinuous than in controls (a); the defective AKTIP localization at the nuclear rim of these cells was more evident in preparations extracted with Triton X-100 (b). In MADA cells, homozygous for the LMNA mutation, AKTIP was almost undetectable .Given the connection between AKTIP and lamins, we investigated the functional relationships between these proteins. We first asked whether mutations in lamin genes affect AKTIP localization. We analysed AKTIP localization in HPFs from patients affected by HGPS or MAD type A (MADA). The HGPS mutation exposes a cryptic splice site in the esylated ,36. MADAd traits . In addicontrols a; the deon X-100 b. In MADtectable a,b.Figua–d). In addition, Q-PCR-mediated mRNA quantification in HeLa cells at 7 days after shAKTIP infection showed a significant decrease in the lamin A mRNA level compared to control (e). However, AKTIP deficiency did not affect the level of lamin C mRNA, which is the main alternative splicing product of the LMNA gene. Consistent with these results, lamin A immunostaining in AKTIP-depleted HeLa cells showed a substantial reduction of the lamin A signal, which, however, maintained its normal association with the nuclear rim (f).We next investigated whether loss of AKTIP affects lamin expression. We reduced the AKTIP level by RNAi in early passage HPFs and HeLa cells . Western blotting performed on extracts from both cell types collected at 7 days post-infection showed that AKTIP depletion results in a substantial decrease in lamin A compared with control cells, without affecting lamin C and lamin B1 levels a–d. In a control e. Howevelear rim f.FigureWe have previously shown that AKTIP is required for telomere replication and that its depletion generates telomere-associated DNA repair foci . Depleti3.4.LMNA mutations. Cell labelling with the empirical senescence marker SA-βgal [a) exhibit a significant increase in senescent cells compared with controls . We next analysed K9 trimethylation of histone H3 (trimethyl H3K9), an epigenetic marker of cell senescence that is reduced in prematurely ageing lamin-defective cells [a) displayed higher frequencies of nuclei with diffuse trimethyl H3K9 distribution than passage-matched controls . In addition, Western blotting showed that in AKTIP-depleted early passage (p6) HPFs, the trimethyl H3K9 level was reduced with respect to controls and was comparable to that observed in naturally aged HPFs at p22 (e).Lamin mutations cause defects in nuclear structure, variations in heterochromatic marks and cell senescence . These f SA-βgal  revealedcontrols a,b. We nve cells . Early pve cells ,37,39. Econtrols c,d. In as at p22 e.Figurea) and examined 7 days post-infection cells immunostained with the C20 antibody, which specifically reacts with prelamin A. shAKTIP HPFs at p22 displayed a higher frequency of prelamin A-positive cells than passage-matched control HPFs . We also examined AKTIP-deficient HPFs for distorted nuclei, which have been described in lamin mutant cells [c,d).Previous studies on naturally aged cells have shown that they accumulate unprocessed lamin A (prelamin A) . This acrol HPFs a,b. We ant cells ,14,39. WTaken together, these data indicate that AKTIP depletion results in phenotypic traits that are characteristically associated with lamin A mutations.4.We have recently shown that AKTIP interacts with the PCNA and RPA70 components of the DNA replication machinery and with the shelterin subunits TRF1 and TRF2. Consistent with these results, we found that AKTIP is required for both general DNA replication and telomere replication . Our MS We focused on the relationships between AKTIP and lamins and found that these proteins exhibit similar localization patterns in interphase nuclei, with AKTIP enriched at the nuclear rim, showing partial co-localization with both the A- and B-lamin meshworks. We also found that AKTIP is enriched in structures that partially co-localize with the centrosomes and the spindle of mitotic cells. In addition, AKTIP forms a ring around the midbody during late cytokinesis. Previous studies in different cell types have shown that lamin B is also enriched at the centrosomes and spindle poles ,41,42 asLMNA RNA or by controlling both prelamin A degradation and LMNA gene expression [Indeed, we found that AKTIP knockdown results in a specific reduction of lamin A at both the mRNA and the protein level. A specific downregulation of A-type lamins has been also found in other systems. For example, in mouse brains both lamin A and progerin expression is downregulated by miR-9, a brain-specific microRNA; miR-9 targets prelamin A 3′-UTR but not the lamin C transcript . Lamin Apression . It is apression . The mosWe also found a modest increase (from 3 to 9%) in the frequency of prelamin A-positive cells in late passage samples of AKTIP-depleted cells. This finding is in apparent contrast with the downregulation of lamin A observed in in early passage AKTIP-depleted cells. However, it should be considered that prelamin A accumulation reflects a slowdown in prelamin A processing, a phenomenon that characterizes the senescent phenotype associated with physiological and premature ageing . It is tWe showed that AKTIP is mislocalized in LV-progerin and HGPS cells and in cells from MADA patients that accumulate prelamin A ,14,39. TOur MS analyses, in addition to lamins, identified matrin 3, importin 7 and several mitochondrial proteins as AKTIP partners. The functional relationships between AKTIP and these interacting partners deserve further investigation. In this respect, it should be noted that previous studies have shown that proteins involved in telomere maintenance such as the ATM kinase and the shelterin subunit TIN2 localize to mitochondria and regulate mitochondrial activity ,51.Altogether, our results identify AKTIP as a new lamin interactor, expanding the connection between telomeres and lamins and, more in general, between telomeres and nuclear envelope. In addition, the interplay between AKTIP and both lamin A and its aberrant forms responsible for HGPS and MADA suggests a possible involvement of AKTIP in the pathogenesis of these diseases.


MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 . The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t and strongly expressed  Continuous cell lines have become indispensable tools in leukemia/lymphoma research. Self -renewing and transportable in the frozen state, cell lines provide unlimited supplies of cellular tumor material worldwide. Descendent from single cells of a patient´s tumor, they retain oncogenomic aberrations and gene expression patterns characteristic of the primary tumor. Tumor congruence is a key reason why cell lines are used as models to study the function of aberrantly expressed genes or to test the efficiency of novel anti-cancer drugs ,2.BCL2, BCL6 and MYC [BCL2 and MCL1 are recurrently overexpressed, as result of chromosomal translocations, amplification or other mechanisms [MCL1. Several DLBCL cell lines have been used to validate novel drugs targeting antiapoptotic BCL2 family members [Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults. Various genes are recurrently affected by mutations and chromosomal aberrations therein, including the germinal center oncogenes  and MYC . Express and MYC . DLBCL-d and MYC –9. In co and MYC . Cell li and MYC ,7,10. On and MYC . In DLBCchanisms –14. We d members –18. We pwww.dsmz.de) or were supplied by the originators for research purpose. Detailed references and cultivation protocols have been described previously [BCL2 family inhibitors ABT-263 and A-1210477 were obtained from Selleckchem .Authenticated stocks of cell line U-2946 were grown in RPMI 1640  containing 10% fetal bovine serum (FBS) . Cell lines applied in this study are all held by the DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (eviously . The BCLin situ hybridization (FISH) were performed as described previously [https://genome-euro.ucsc.edu/) guided by the results of combined conventional cytogenetic, SKY and Cytoscan copy number array data (see below). For colocalization experiments commercial chromosome painting probes were mixed 1:1 with labelled BACs. Probe preparation and labelling were as described previously [Cells were harvested and fixed as described previously . Spectraeviously . Tilepateviously . Brieflyeviously .CytoScan HD Array  hybridization analysis was performed to identify numerical aberrations. DNA was prepared using the Qiagen Gentra Puregene Kit . Data were analyzed using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix).Immunophenotyping was conducted on a FACSCalibur . Antibodies (Abs) against CD3 (Leu4), CD5 (UCHT2), CD10 (HI10a), CD19 (Leu12), CD20 (Leu16), CD34 (clone 581), CD37 (M-B371), CD38 (HIT2), CD138 (MI15) and HLA-DR (Tu39) were purchased from Becton Dickinson. Ab CD13 (My7) was from Beckman Coulter . Optimal dilutions of the Abs were determined with positive and negative control cell lines. FITC-labeled Abs against IgG, IgM, Ig kappa and Ig lambda were obtained from Southern Biotech/Biozol .Rearrangement of the immunoglobulin (Ig) heavy chain was determined by genomic PCR using primers described by van Dongen et al. . The PCRFive hundred ng total RNA were used for biotin labelling according to the 3´ IVT Express Kit (Affymetrix); 7.5 μg of biotinylated cDNA were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls . Samples were hybridized to an identical lot of Affymetrix GeneChip HG-U133 Plus 2.0 for 16 h at 45°C. Steps for washing and SA-PE staining were processed on the fluidics station 450 using the recommended FS450 protocol (Affymetrix). Image analysis was performed on GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Data processing and analysis  were performed via R/Bioconductor using limma and affy packages –25.BCL2 (Hs00153350_m1), BCL6 (Hs00153368_m1), MAP2K3 (Hs00177127_m1), MCL1 Exon 1/2 (Hs03043899_m1), MYC exon 1/2 (Hs00905030_m1) and MYC exon 2/3 (Hs00153408_m1) expression levels with TBP as endogenous control. Relative expression levels were calculated using the ΔΔCt method.Quantitative PCR was performed on a 7500 Applied Biosystems real-time PCR system . RNA was prepared using the RNeasy Mini kit (Qiagen). Reverse transcription was performed using the SuperScript II reverse transcriptase kit . TaqMan probes (Applied Biosystems) were used to quantify human BCL2, MAP2K3 and MCL1 were determined by genomic PCR applying the SYBR green assay system (Applied Biosystems) with ABL1 as internal control and NC-NC as diploid reference cell line. DNA was prepared using the High pure PCR template preparation kit . The applied primers are listed in Ploidy levels of Samples were prepared as described previously . Anti BC4 cells (in 100 μl) were seeded in triplicate in 96-well flat-bottom microtiter cell culture plates. Inhibitors were added as 2x concentrated solution in a 100 μl volume. For the last 3 h of the incubation period, 1 μCi 3H-thymidine  was added to each well.Assays of 3H-thymidine incorporation were executed as follows: 1.25 x 10Apoptotic cells were detected and quantified with the annexin-V/propidium iodide method using the TACS Annexin-V-FITC kit  according to the manufacturer’s instructions. Binding of fluorescein isothiocyanate-labeled annexin-V and propidium iodide staining of the cells were determined by flow cytometry on the FACSCalibur (Becton Dickinson).The patient was a previously healthy man, age 52 years who sought medical care due to abdominal pain and tiredness. Computer tomography examination revealed an abdominal tumor. In July 1996 he underwent a right-sided hemicolectomy due to a tumor of the ascending colon. The tumor, 15 cm in diameter, removed macroscopically radically. Pathological examination showed DLBCL. Diagnostic work-up revealed lymphomatous engagement in the right testicle and in paratesticular tissue with a diameter of >10 cm, in lymph nodes and in the bone marrow (stage IVB). International Prognostic Index was retrospectively assigned to 3 ). At diagnosis he also had a palsy of the left N. abducens but examination of cerebrospinal fluid showed no signs of lymphoma and a CT of the brain was normal.9/l. The patient gave informed consent to use the pleural effusion for research including the possible establishment of a cell line. He died of progressive lymphoma three months after diagnosis. The overall picture of the lymphoma was an extremely aggressive disease with fast progression and very limited sensitivity to chemotherapy.He was treated with VACOP-B : a weeklMYC translocation was detected by in situ hybridization.Large blast cells with the appearance of both immunoblasts and centroblasts grew in a diffuse and infiltrative pattern in the intestine. Several mitotic figures and apoptotic cells were present. Immunohistochemical stainings were performed and the lymphoma cells were positive for CD20, CD79a, CD10, BCL-6, MYC, p53 and negative for BCL-2, CD5, CD30, cyclin D1, SOX11 with a high proliferation index of Ki67. Partial expression of MUM1 and FOXP1 was detected. According to the WHO classification and update ,30 the l2 humidified environment. Cells were split every 72 h with an optimal density of 0.3 x 105 cells/ml and a doubling time of 48 h. After one month, the cell line U-2946 was considered established and grew in continuous culture in RPMI 1640 with 10% FBS for another 2 months. U-2946 cells tested EBV-negative excluding a lymphoblastoid origin. May-Grünwald-Giemsa stain of the cell line is shown in Tumor cells from pleural effusion were obtained by Ficoll-Isopaque  gradient separation and cultured in RPMI 1640 containing 10% of FBS, glutamine and antibiotics (100 IU/ml penicillin and 50 μg/ml streptomycin) at 37°C in 5% COWe tested the Ig rearrangement status of cell line U-2946 using previously described primers . The celIGH and MYC loci t, effecting copy number increase of the terminal 8q region just including PVT1, but excluding MYC.The consensus karyotype, performed using classical  and specMYC loci  was resoMCL1, ENSA), while that at 1p11 extending some 461 kbp covered four loci . Coamplification of gene pairs normally arises when a genomically remote gene pair, ectopically juxtaposed or fused by chromosome rearrangement is locally subjected to linear copy number amplification, e.g. following selection. Such copy number aberrations are hallmarks of oncogenes and are thought to arise by conferral of proliferation or antiapoptopic advantage.Cytogenetic analysis also revealed a novel rearrangement, focal co-amplification of 1q21 and 17p11.2 replacing a deleted segment of chromosome 17p . The ca.Expression array analysis allows distinction between two forms of DLBCL, the GC and the ABC forms . Based oBCL2, BCL6 and MYC. These aberrations can occur in the same tumors, which are then called “double-hit” or “triple-hit” lymphoma according to their aberration scores. Cell lines representing double-hit and triple-hit lymphomas have recently been reviewed in detail [BCL2), 3q27 (BCL6) were not detected in U-2946 cells. As shown in MYC on 8q24 was fused to IGH on 14q32. In accordance with the genomic findings, cell line U-2946 was BCL2neg/BCL6low/MYCpos . Accordingly qRT-PCR showed MYC transcript in exon 2 but not in exon 1 suggesting (i) that the genomic break was located in intron 1/2, and (ii) that translation started with the alternative ATG start codon in exon 2 and not with the “internal ribosome entry site” (IRES)-mediated CTG codon in exon 1  were upregulated in U-2946; another 7 genes (15%) lay below the average of B-lymphoma cell lines . High deMAP2K3 (MEK3) was amplified in cell line U-2946  is consistent with the ABC character proposed here for cell line U-2946  or BCL2, BCL-XL and BCL-W (ABT-263). The MCL1 inhibitor  substantially reduced 3H-thymidine uptake and triggered apoptosis in the MCL1pos cell line U-2946, while MCL1neg cell lines were not affected  were responsive to low doses of the BCL2 inhibitor , the MCL1 inhibitor (7.5 μM A-1219477) did not have any effect on U-2932 cells, suggesting that BCL2 was dominant over MCL1 in the U-2932 cell system  had no effect. Other DLBCL cell lines expressed BCL2 and responded to ABT-263. U-2932 was one of these BCL2pos cell lines. This cell line also expressed MCL1, but did not respond to the MCL1 inhibitor. Even with low doses of the BCL2 inhibitor, A-1210477 was inefficient which suggests that the effect of BCL2 is dominant over MCL1, at least in this cell system.Translocations and/or numerical aberrations affecting the germinal center oncogenes BC cases ,14. MCL1proteins ,15–18. Rproteins . Cell lipos/BCL2neg cell line U-2946 promises to be a valuable model for inhibitor studies, useful especially in combination with cell lines expressing other antiapoptotic BCL2 family members. Cell line U-2946 will be available from the public repository DSMZ (www.dsmz.de).We conclude that the MCL1BCL2 family member MCL1. The MCL1 chromosomal region (1q21.3) was involved in a translocation to chromosome 17. The amplified MCL1 gene is highly expressed in the cell line (MCL1pos/BCL2neg). Inhibitor experiments revealed the importance of MCL1 for growth and survival of U-2946 cells. Cell line U-2946 will prove to be an important model system for the functional analysis of small BCL2-family inhibitors, especially in a panel of certified DLBCL cell lines showing other MCL1 and BCL2 expression profiles.We describe the establishment and characteristics of the DLBCL cell line U-2946. According to gene expression data, the cell line clusters together with ABC DLBCL cell lines. The cell line carries an amplification of the antiapoptotic S1 FigA-E) Immunohistochemical stainings of lymphoma cells. Positive: BCL6 (A), CD10 (B), CD20 (C), CD79a, FOXP1, MUM1 , MYC (D), p53 and negative: BCL2 (E), CD3, CD5, CD30, cyclin D1. (F) May-Grünwald-Giemsa stain of cell line U-2946.(TIF)Click here for additional data file.S2 FigCells were stained with Abs against the antigens indicated and analyzed by flow cytometry.(TIF)Click here for additional data file.S3 FigThe 47 genes with strongest expression differences between U-2946 and median of 55 B-lymphoma cell lines in relation to variance –highest differences on the left. Data base on expression array analyses. Red dots, cell line U-2946.(TIF)Click here for additional data file.S4 FigBCL2, MAP2K3 and MCL1. The bars indicate means with standard deviation (n = 3). NC-NC as diploid reference cell line for genomic PCR.Quantitative genomic PCR (upper) and qRT-PCR (lower) detecting correlation between amplification and RNA expression in (TIF)Click here for additional data file.S5 FigMCL1 inhibitor A-1210477 (7.5 μM) inhibits growth of the MCL1pos/BCL2neg cell line U-2946, but has no effect on the MCL1pos/BCL2pos cell line U-2932 –neither alone nor together with suboptimal doses of the BCL2 inhibitor ABT-263 (50 nM). The bars indicate means with standard deviation (n = 3).3H-thymidine uptake after 48 h. The (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableOutliers are listed according to chromosomal position. Note the perfect correlation between genomic imbalances and expression in cell line U-2946, 6/6 hemiploid genes (black and bold) being repressed, 5/5 amplified genes (red and bold) being overexpressed. Up, higher than average; down, lower than average.(XLSX)Click here for additional data file.S3 TableAmplified genes on 1q21.3 are listed from centromere to telomere, genes on 17p11.2 from telomere to centromere. Bold: strongly expressed genes as assessed by expression array analysis.(XLSX)Click here for additional data file.


NOD1 has also been suggested to be a tumor suppressor gene in a model of estrogen receptor-dependent breast cancer. Contrarily, NOD2 polymorphisms are associated with higher risk of breast cancer, with no tumor suppressor role being reported. To better delineate this issue, we investigated NOD1 and NOD2 expression in a panel of breast cancer cell lines, as well as their potential impact in breast tumorigenesis based on in vitro assays.Immune-related pathways have been frequently associated to tumorigenesis. NOD1 and NOD2 are innate immune receptors responsible for sensing a subset of bacterial-derived components, and to further translate these pathogenic signals through pro-inflammatory and survival pathways. NOD1 and NOD2 have been further associated with tumorigenesis, particularly in gastrointestinal cancers. NOD1 expression and the lowest NOD2 expression in our panel. Therefore, we investigated whether NOD1 and/or NOD2 might act as a tumor suppressor in this cell model. Our studies indicate that overexpression of either NOD1 or NOD2 reduces cell proliferation and increases clonogenic potential in vitro. Elucidation of NOD1 and NOD2 effects on tumor cell viability and proliferation may unveil potential targets for future therapeutic intervention.The highly invasive Hs578T breast cell line presented the second highest  Breast cancer is the malignancy with the highest incidence in women worldwide, accounting for 29% of all diagnosed cancers in females . DespiteBreast cancer is classified according to immunohistological detection of protein markers, including receptors for estrogen (ER), progesterone (PR), androgen (AR), and the amplified HER2  receptor . Approxid-glutamyl-meso-diaminopimelic acid) and MDP (muramyl dipeptide), respectively , where Δt is the duration of cell proliferation  in hours, and N0 and Nt are the respective numbers of cells at the beginning and end of this period , 20.2 cells from each population were cultured in 9.5 cm2 wells (6-well plate) for 12 days. Colonies were counted after fixation  and staining . Statistical analysis was performed using two-tailed, unpaired, student’s t-test  (Graphpad Prism V6).2 × 104 cells from each population were seeded in 1.9 cm2 wells (24-well plate) previously covered with 0.5 mL DMEM growth medium containing 0.6% agar. After seeding, a layer of 0.5 mL 0.3% agar DMEM was added and allowed to gellify before addition of 0.5 mL DMEM per well. Cultures were maintained for 14 days. Colonies were fixed in 4% formaldehyde and counted. Statistical analysis was performed using two-tailed, unpaired, student’s t-test  (Graphpad Prism V6).1 × 10NOD1 and NOD2 genes in a panel of breast cancer-derived cell lines, including estrogen-positive (MCF-7 and ZR-75-1) and estrogen-negative  cell lines. We found that NOD1 and NOD2 expression varies among these cell lines, with no clear pattern discriminating estrogen receptor-positive or negative groups. Interestingly, the Hs578T cell line presented the highest expression of NOD1 and the lowest expression of NOD2 in our panel. Due to its specific characteristics, namely, its tumorigenic potential and TNBC origin [NOD1 or NOD2 in Hs578T cells for further functional studies.Figure C origin , we deciGFP, displayed NOD1 and NOD2 expression levels similar to those of the wild-type  Hs578T cell line  and GFP-only control cells . Control populations (wild-type and GFP-transduced cells) displayed statistically identical proliferative rates, with doubling times of 24.9 and 23.9 h, respectively. Intriguingly, the Hs578T/NOD2 population displayed the lowest growth rate, even when compared to Hs578T/NOD1 cells   when compared to the controls. This effect was further enhanced by treatment with 5 μg/mL γ-tri-DAP, a NOD1-specific agonist . Hs578T/NOD2 displayed a non-statistical tendency for increased number of colonies in the absence of any treatment, and a statistically significant increase in colony number upon treatment with 5 μg/mL MDP, a NOD2-specific agonist   [Our in vitro colony formation assays indicated an increased viability and cell growth rate in adherin) , ICAM1 (adherin)  and VCAMadherin) .Given the incidence and high mortality rate of TNBC, decoding sub-pathways through which NOD receptors modulate cell proliferation may offer potential new targets for future therapeutic interventions.NOD1 and NOD2 amounts are usually evaluated by RT-qPCR [A more conclusive assessment of NOD1/2 levels could benefit from western blot detection. However, due to their typically limited protein levels in a vast number of cell models, endogenous  RT-qPCR , 25, 26. RT-qPCR .NOD1 and/or NOD2 would provide a valuable functional corroboration model to complement our overexpression results. Also, determining the sensitivity to apoptotic induction in our overexpressing populations could allow a better understanding of the pathways participating in the cellular effects observed.Additionally, generating Hs578T populations with null expression of


BRCA1/2 mutant tumors , such as disruptive gains in PTEN or FANCD2 and/or losses of ATM, BAP1, BARD1 or CHEK2, were susceptible to talazoparib-induced reduction of cell viability . Consistent with their high sensitivity to talazoparib, MG63 and ZK-58 cells scored positive in a DNA-based measure of genomic instability (i.e. homologous recombination deficiency (HRD)-loss of heterozygosity (LOH) score). In contrast, U2OS cells that carry a heterozygous BRCA2 mutation and therefore most likely have one intact BRCA2 allele left proved to be resistant to talazoparib. Furthermore, we identified TMZ as the most potent chemotherapeutic drug together with talazoparib to synergistically reduce cell viability, as confirmed by calculation of combination index (CI) values, and to suppress long-term clonogenic survival. Mechanistically, talazoparib and TMZ cooperated to induce apoptotic cell death, as demonstrated by activation of BAX and BAK, loss of mitochondrial membrane potential (MMP), caspase activation, DNA fragmentation and caspase-dependent cell death. Genetic silencing of BAX and BAK or pharmacological inhibition of caspases by zVAD.fmk significantly rescued OS cells from talazoparib/TMZ-induced apoptosis. These findings have important implications for the development of novel treatment strategies using PARP inhibitors alone or together with chemotherapy in a subset of OS with features of BRCAness.We recently discovered mutation signatures reminiscent of BRCA deficiency in the vast majority of a set of primary osteosarcomas (OS). In the current study, we therefore investigated the sensitivity of a panel of OS cell lines to the poly(ADP)-ribose polymerase (PARP) inhibitor talazoparib alone and in combination with several chemotherapeutic drugs (i.e. temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin). Here, we identified an association between homologous recombination (HR) repair deficiency and the response of OS cell lines to talazoparib. All OS cell lines with molecular features characteristic of  BRCA1/2 mutations or to mutations in HR pathway genes, such as FANCD2 [BRCA1 or BRCA2 as well as BRCAness have previously been reported to confer sensitivity towards PARP inhibitors, since ineffective HR repair induces a dependency on base excision repair to cope with DNA damage [OS is the most common primary bone cancer in children and young adults . There is FANCD2 . Loss-ofA damage . Thus, bA damage , 6.Currently, several PARP inhibitors  are being investigated in clinical trials . In 2014L and MCL-1), the proapoptotic multidomain BCL-2 proteins BAK and BAX and the proapoptotic BH3-only domain proteins [Programmed cell death such as apoptosis plays an important role in the response of tumors to cytotoxic therapies, as most cytotoxic therapies exert their antitumor activity by engaging programmed cell death . Apoptosproteins .In the current study, we aimed i) to investigate the vulnerability of a panel of OS cell lines to the PARP inhibitor talazoparib, ii) to explore the antitumor activity of talazoparib in combination with chemotherapeutics and to identify the most potent synergistic drug combinations and ii) to elucidate the molecular mechanisms of synergy with a specific focus on cell death pathways.50 values in the nanomolar range  , whereas U2OS cells remained largely resistant   towards treatment with the PARP inhibitor talazoparib. According to the observed response pattern among the OS cell lines, we defined three subgroups. MG63 and ZK-58 cells displayed the highest sensitivity towards talazoparib with IC) Figure . SaOS-2,) Figure .BAP1, FANCA and FANCD2  in four of the tested OS cell lines  that we defined as highly synergistic , intermediately synergistic  and weakly synergistic . In contrast, we found no consistent synergistic interaction of talazoparib with the tested anticancer drugs in U2OS cells, as CI values showed no synergism (CI >0.9) and/or Fa values were below 0.3 with the exception of a few drug combinations Figure .For TMZ in combination with talazoparib we detected a highly synergistic drug interaction in all four responding OS cell lines , as indicated by CI values < 0.2 and Fa values > 0.7 Figure . For SN-Since we identified MG63 and ZK-58 cells to be most sensitive to treatment with talazoparib alone Figure  and in cTo better understand the molecular mechanisms of cell death underlying the synergistic antitumor activity of talazoparib/TMZ combination treatment, we monitored the kinetics of cell death. Talazoparib acted in concert with TMZ to increase DNA fragmentation in a time-dependent manner in both cell lines with a significant increase in cell death upon talazoparib/TMZ cotreatment at 24 hours in ZK-58 cells and at 48 hours in MG63 cells Figure . To moniTo explore whether the addition of talazoparib would potentiate the cytotoxicity of TMZ also in non-malignant cells, we tested the effect of TMZ in the presence and absence of talazoparib on human peripheral blood lymphocytes (PBLs) that were freshly isolated from two different donors. We used PBLs, as hematological toxicity has been described for talazoparib . NeitherNext, we asked whether talazoparib and TMZ cooperate to activate BAK and BAX, two multidomain proapoptotic BCL-2 proteins that control MOMP . To addrBRCA1/2 mutations, including disruptive gains in PTEN and FANCD2 and/or losses of ATM, BAP1, BARD1, FANCA or CHEK2, although none of the tested OS cell lines showed a bi-allelic BRCA1/2 mutation [BRCA2 mutation [BRCA2 allele left.Using exome sequencing, we recently identified mutation signatures characteristic of BRCA deficiency in OS , indicatmutation . In contmutation  and most50 values scored positive in HRD-LOH score, a DNA-based measure of genomic instability [50 values for talazoparib (i.e. SaOS-2 and MNNG-HOS cells) were borderline, respectively positive by HRD-LOH score. Testing of talazoparib monotherapy in the pediatric preclinical testing program has previously shown minimal in vivo activity against OS; however, the BRCAness status of OS was not reported in that study [Importantly, the susceptibility of OS cell lines to talazoparib broadly correlated with their degree of genomic instability, as MG63 and ZK-58 cells that exhibited high sensitivity to talazoparib with nanomolar ICtability . In contat study .PTEN, ATM, BAP1, BARD1, CHEK2, FANCA and FANCD2 have been shown to exert pivotal functions in HR repair [PTEN function has previously been described to cause genomic instability [PTEN-deficient cells were found to be sensitive to the PARP inhibitor olaparib [ATM-deficient lymphoid tumor cells have shown vulnerability to olaparib in vitro and in vivo [R repair . Loss oftability , and PTEolaparib . Also, A in vivo . Further in vivo . CHEK2-m in vivo . Ubiquit in vivo  and the  in vivo . Thus, lFurthermore, among the tested chemotherapeutic drugs we identified TMZ to be most potent for combination treatment with talazoparib in OS, which was confirmed by highly synergistic CI values and by a large fraction of cells affected. In addition, talazoparib and TMZ acted together not only to reduce cell viability, but also to trigger cell death and to suppress long-term clonogenic survival. We and other groups previously demonstrated that the DNA-damaging agent TMZ is particularly suitable for PARP inhibitor-based combination treatment, yielding highly synergistic drug interaction in Ewing sarcoma cells –30. DepeOur molecular studies revealed that the synergistic cytotoxicity of talazoparib and TMZ in OS cells involves the induction of apoptotic cell death. First, we showed that talazoparib/TMZ cotreatment triggered DNA fragmentation and loss of MMP as characteristic markers of apoptotic cell death. Second, talazoparib/TMZ-stimulated activation of BAK and BAX contributed to the induction of apoptosis, since genetic silencing of BAK and BAX significantly reduced talazoparib/TMZ-induced cell death. Third, talazoparib and TMZ acted together to activate caspases, which were required for apoptosis, as the caspase inhibitor zVAD.fmk significantly rescued talazoparib/TMZ-mediated cell death. While our data showing that zVAD.fmk as well as BAK/BAX silencing significantly reduced talazoparib/TMZ-induced cell death demonstrated that caspases and BAK/BAX activation contributed to talazoparib/TMZ-induced apoptosis, additional mechanisms are also likely to be involved, since the protection by zVAD.fmk or BAK/BAX knockdown was only partial.BRCA1/2 mutations or the signature of BRCAness such as ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma, and soft-tissue sarcomas [BRCA1/2-mutated ovarian cancer and castration-resistant prostate cancer [BRCA deficiency [in vivo model of OS. Since the addition of talazoparib did not increase the cytotoxicity of TMZ on non-malignant PBLs, there might be a therapeutic window for the incorporation of talazoparib into treatment protocols for OS. Selective synthetic lethality of the talazoparib/TMZ combination treatment in OS cells might be explained by the fact that cancer cells but not normal cells harbor HR repair deficiency. The doses of talazoparib and TMZ that caused synergistic induction of cell death in our study correspond to clinically achievable plasma concentrations of those drugs , underlOS cell lines  were kindly provided by M. Nathrath  and authenticated by STR profiles. Cells were maintained in RPMI 1640 medium , supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1 mM sodium pyruvate . Cell lines were regularly tested for mycoplasma contamination to ensure that experiments were performed only with mycoplasma-free cell lines. Talazoparib was obtained from Selleckchem . TMZ, SN-38, cisplatin, doxorubicin and etoposide were purchased from Sigma-Aldrich , MTX and carboplatin were purchased from Medac , zVAD.fmk from Bachem . Chemicals were purchased from Sigma-Aldrich or Carl Roth  unless otherwise indicated.Apoptosis was determined by flow cytometric analysis  of DNA fragmentation of propidium iodide (PI)-stained nuclei as described previously . Cell viWestern blot analysis was performed as described previously  using thFor determining caspase activity cells were treated as indicated and caspase activity was assessed by caspase-3/7 staining according to the manufacturer's instructions for CellEvent Caspase-3/7 Green Detection Reagent  using ImageXpress Micro XLS system .Cells were reversely transfected with a combination of targeting siRNAs  using Lipofectamine RNAiMAX reagent  and Opti-MEM medium . After six hours of incubation with transfection solution, the medium was changed and cells recovered for 48 hours before drug treatment.BAK and BAX activation was determined by immunoprecipitation of active conformation by specific antibodies. Briefly, cells were lysed in CHAPS lysis buffer . 500 μg protein was incubated overnight at 4°C with 8 μg mouse anti-BAX antibody  or 0.5 μg mouse anti-BAK antibody  and 10 μl pan mouse IgG Dynabeads , washed with CHAPS lysis buffer and analyzed by Western blotting using rabbit anti-BAX NT antibody  or rabbit anti-BAK antibody (BD Biosciences). Loss of MMP was assessed by JC-1 staining according to the manufacturer's instructions (ThermoFisher scientific).For PBL isolation, blood was drawn from two independent donors and leukocytes were isolated using density centrifugation. Afterwards, leukocytes were treated with indicated concentrations of talazoparib and/or TMZ and cell death was assessed directly after PI staining as described previously .Affymetrix CytoscanHD array data processed with Nexus 8.0 software such that only segments with copy-number changes larger than 100 Kb and with at least 21 probes were considered for analysis using a public algorithm. In brief, HRD-LOH score requires at least 15 sub-chromosomal LOH segments of a size exceeding 15 Mb, which do not affect the whole chromosome and are at least 10 Mb apart. Borderline cases were tumors with 14 LOH events, while negative cases had 13 or less LOH events.Statistical significance was assessed by Student's t-Test  using Microsoft Excel . Drug interactions by CI values and Fa were analyzed by the Chou-Talalay method described by Chou , 35 usin


Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs , and four normal cell lines .NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants.Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers.Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.The online version of this article (doi:10.1186/s12885-017-3514-z) contains supplementary material, which is available to authorized users. Nullomers are the shortest string of monomers absent from a species or species group. In 2007 Hampikian and Andersen  publisheThe current treatment of cancer cannot address many aspects of human tumors driven by their cellular heterogeneity. For example, some cells in a tumor may be designated drug resistant  cancer c2. Passaging of cells was done at 80% confluence after a PBS wash, cells were treated with Trypsin-EDTA 0.25% (Invitrogen).The NCI-60 panel of cancer cells was purchased from Development Therapeutics of the NCI. Cells were thawed in a water bath at 37 °C, and collected by centrifugation at room temperature. For maintenance, the cell were grown in RPMI 1640 supplemented with 10% Hyclone fetal bovine serum (FBS), and 1× penicillin streptomycin  at 37 °C and 5% CONormal cell lines were purchased from the American Type Culture Collection . Human endothelial cell line HUVEC (ATCC CRL-1730) was grown in the ATCC formulated F-12 K medium with 0.1 mg/ml Heparin, 0.05 mg/ml endothelial cell growth supplement and 10% FBS. Human skin fibroblast BJ (ATCC CRL-2522) was grown in the ATCC formulated Eagle’s Minimum Essential Medium supplemented with 10% FBS. Human prostate epithelial cell line RWPE-1 (ATCC CRL-11609) was grown in the Keratinocyte Serum Free Medium  supplemented with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF). Human colon epithelial cell line FHC (ATCC CRL-1831) was grown in the ATCC formulated DMEM-F12 supplemented with 10 mM HEPES , 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone and 10% FBS. Only early cell passages were used for all experiments. No human subjects  has been used in the research reported in this manuscript. All cancer ell lines were obtained from the NCI depository, and all normal cells from the ATCC.Peptides 9R, 9S1R and 124R were made by Pierce Biotechnology . HPLC purified peptides (purity >98%) were delivered in 0.5 mg/tube (lyophilized) and stored at −20 °C. Peptides were solubilized in 1 M Trehalose to produce 50 mM stock solutions. Only freshly prepared solutions were used to treat cells. Trehalose final concentration in all reactions was 1 mM . Sodium Azide (Sigma Aldrich) was solubilized in water at 100 mM.2 incubator. After the 4 h incubation, plates were monitored by a plate reader  at an absorbance of 450 nm. Plate readings were exported to Microsoft Excel and GraphPadPrism software. All the wells were analyzed in triplicates. The statistical analysis was done with GraphPadPrism*. The survival index (SI) is the CCK-8 reading ratio of peptide-treated to untreated cells , multiplied by 100 to yield an index of the percent surviving. A dose response using several concentrations of the peptides  was used for all cancer and normal cell lines. The SI was used for screening in this study at the 50 μM concentration of each peptide.Three thousand–Five thousand normal or cancer cells/well were seeded in 96-well plates. After 24 h incubation, peptides 9R, 9S1R and 124R were added to the wells, with untreated cells as a control. After 48 h exposure to 5 μM, 25 μM, or 50 μM peptides, cell viability was quantified by the addition of 10 μl of cell counting kit  to each well, incubated for 4 h at 37 °C in a 5% COCancer cell lines (one cell line for each organ represented in the NCI-60 panel) and normal cell lines (3000-5000/well) were seeded in 96 well microplates (white plates from Nunc to block luminescence bleeding between the wells), and allowed to attach for 24 h. Then cells were incubated for 3 h with peptides , or 100 mM sodium azide as a control. After 3 h, 10 μl of a single reagent from Cell Titer Glow™ (Promega) was added to cells. For the cellular supernatant ATP content, the supernatant of each well was transferred to a new microplate and 10 μl of a single reagent from Cell Titer Glow™ (Promega) was added to each supernatant. Complete reagent mixing in 96 wells plates required gentle orbital shaking for 2–10 min. The plate reading was taken by a SynergyMx plate reader. Plate readings were exported to Microsoft Excel and Graph-PadPrism software. All the wells were analyzed in triplicates. The statistical analysis was done with the GraphPadPRISM*.In a previous work  peptide The three peptides were prepared as previously published: first solubilized in 1 M Trehalose and then diluted to three doses, 50 μM, 25 μM, and 5 μM final concentration. Cells were incubated for 48 h, and then their viability was assessed by CCK-8 assay . Cell liAs shown in ). Comparison of Nullomer derived peptides (9R and 9S1R) sensitivity with current anticancer drugs approved by the FDA. (XLSX 13 kb)Additional file 4:Results 2.p<0.05,**p <0.01, ***p<0.001. (PDF 276 kb)Effect of 50 μM, 25 μM and 5 μM Nullomer peptides on normal cell growth. Cells (3000-5000 cells/well) were seeded in 96-well plates. After 24 h incubation, peptides 9R, 9S1R and 124R were added to the wells, with untreated cells as control. After 48 h exposure to the peptides, cell viability was quantified by the addition of 10 μl of cell counting kit  to each which was then incubated for 4 h at 37 °C in a 5% CO2 incubator. After the incubation, the plates were monitored by a microplate reader (BioTek) at an absorbance of 450 nm. (A) Endothelial cell line, (B) Skin fibroblast, (C) Prostate cell line, (D) Intestinal cell line. Results are as mean ± SE (standard error) of three different experiments. NS, not significant. *Additional file 5:Table 3ATP depletion of cancer and normal cell lines after treatment with 9R, 9S1R and 124R. ATP content: expressed as % of control. Three-hour incubations with 50uM peptide. (XLSX 9 kb)


Meningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce.Meningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers , and drug treatment with cisplatin or etoposide.Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs.Unsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Collectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.The online version of this article (doi:10.1186/s12935-017-0441-7) contains supplementary material, which is available to authorized users. They acplicated . Genomicr (KLF4) –11.Deregulated molecular pathways identified based on bulk tumor analyses, have been utilized to predict prognosis and help determine appropriate targeted therapy . HoweverA crucial component for the development of targeted therapy is the availability of live tumor models . Early pPreviously we published the gene expression profiles for meningioma patients tissues collected for our cohort . For thiTumor tissue RNA were isolated and processed for microarray and gene expression analysis as previously described , and datg) for 3 min at room temperature, re-suspended in DMEM-F12 (Gibco), 10% FBS (HyClone), 100 U/mL penicillin, and 100 µg/mL streptomycin, and maintained in standard humidified incubators at 5% CO2. After 24 h, non-adherent cells were removed by washing with Phosphate Buffered Saline  and the adherent cells were cultured until they reached confluence, then split (1:2) every 12–30 days dependent on the cell line. For each of tumors Jed62_MN and Jed79_MN, tissues were divided into four representative portions and each was placed into a 25 cm2 tissue culture flasks containing a type of media  with 10% FBS, or DMEM high glucose concentrations of 4500 mg/L (Gibco) with 5% FBS, or DMEM low glucose concentrations of 1000 mg/L (Gibco) with 10% FBS).Meningioma specimens collected between February 2013 and December 2015 were obtained within 30 min of tumor removal and distributed into both RNAlater solution (Life Technologies) for RNA/DNA extraction and cell culture using mechanical and enzymatic dissociation methods . For celCell cultures were observed for a period of 4 weeks with an inverted microscope (Leica DMI6000) to determine their morphologies. Twenty images of cells were taken at 10× magnifications each week using a digital microscope camera (Leica DFC425). The average number of counted cells per cell line exceeded 500 per week. The weekly morphology percentages were calculated as the total number of cells displaying a particular morphology, divided by the total number of cells counted. Morphological profiles for cells were confirmed by immunofluorescence staining with mouse anti-Vimentin . All cell lines and images were reviewed independently at least twice to confirm counts.T test or Chiχ2 values were retrieved using Statistical Package for the Social Sciences (SPSS) Graduate Pack 21.0.For cell lines, cells were seeded on chamber slides and fixed with 4% Paraformaldehyde (PFA). For in situ staining, fresh frozen tissues were cut at 4 μM sections, then fixed with 4% Paraformaldehyde (PFA). The antibodies used for standard immunostaining were rabbit anti-Caspase-3 , mouse anti-CD133 , rabbit anti-Sox2 , mouse anti Sox2 , mouse anti Nestin , rabbit anti-Ki67 , mouse anti-Vimentin , rabbit anti- Frizzled 9 , mouse anti-BMI1 proto-oncogene, polycomb ring finger (BMI1) antibody , and rabbit Anti-Anterior Gradient 2 antibody . For secondary goat antibodies, 488 anti-mouse  and 555 anti-Rabbit  were used. Pictures were taken at 20× magnifications using Leica DMI6000 microscope and Leica DFC425 camera. Photos were edited in Photoshop 7.0 and signal levels were compared to negative controls of secondary only. 50 values for cisplatin and etoposide, preliminary growth inhibition experiments were completed for three cell lines that had fast growth capability ,  or etoposide (in DMSO) at increasing concentrations of drug. Twelve days following treatment, cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. 10% acetic acid was added to cells and absorbance was measured at 590 nm. The survival fractions  were calculated. All assays were performed in triplicate.To determine ICSpalt-Like Transcription Factor 4 (SALL4) (fold change: 3.570) and Wingless-type MMTV Integration Site Family, member 6 (WNT6) (fold change: 2.340) were deregulated. For individual genes, the top four up-regulated molecules in group 1 tumors were Reelin (RELN) , Calbindin 1 (CALB1) , Killer Cell Lectin-Like Receptor Subfamily C, Member 4 (KLRC4) , and Anterior Gradient 2 Homolog (AGR2) . The top four down-regulated molecules in group 1 tumors were Cytochrome P450, Family 4 Subfamily B Polypeptide 1 (CYP4B1) , F-Box Protein 32 (FBXO32) , Mesenteric Estrogen-Dependent Adipogenesis (MEDAG) , and Serpin Peptidase Inhibitor, Clade A , Member 3 (SERPINA3) . Expression for the stem cell related markers Nestin, CD133, Sox2, AGR2 and the known oncogenic BMI1 protein, investigated using in situ tissue immunofluorescence for four additional tumors , support the observed gene expression trends, Additional file Table Consistent with differences observed in the analyzed meningioma tissues, newly initiated primary cell lines appeared to separate into two main groups with differences detect for morphological profiles, proliferation, and invasion in vitro. Following cell lines initiations, distinct cell shapes were observed in all cells lines, Fig. 2: 9.681, P < 0.01; Ki67+ Nestin-, χ2: 9.953, P < 0.01), Fig. 2: 12.156, P < 0.001). The expression of the recently associated stem cell protein AGR2, which was detected earlier to be deferentially expressed in our tissues, was also analyzed in NG type cell lines. Three NG type cell lines were co-stained to detect AGR2 with the oncogenic stem cell associated protein BMI1, Fig. Since stem cell pathways appeared to be differential in tissues, primary G type and NG type cell lines were also investigated for differences in stemness. Immunofluorescence was used to co-stain cells for stem cell markers CD133 Sox2, Nestin Ki67, or Vimentin Frizzled 9 in four cell lines . Interestingly, CSCs stained co-positive for all combinations were present in all cell lines, Fig. To address whether the sub-groups had different drug response, four NG and four G type cell lines were treated with cisplatin (50 µM) or etoposide (100 µM). NG type cell lines survived treatment significantly better than G type cell lines , Fig. To test whether meningioma CSCs survived treatment with cisplatin or etoposide, 12 days following treatment, treated Jed49_MN cells (NG type) were stained with CD133 and Sox2 or Nestin and Ki67. Cells co-positive for CD133+ Sox2+ or Nestin+ Ki67+ were detected following treatment with cisplatin or etoposide, Fig. We examined multiple bio-parameters for meningiomas through analysis of both eight tissue samples and 15 primary cell lines. Whole transcriptome microarray analysis for six tumor samples identified two groups of meningiomas with differential stem cell related pathways and a number of novel stem cell related biomarkers, including AGR2. Biological characterization of primary meningioma cell lines also sub-grouped cell lines into two main types; G type with predominantly glial like cells that grow slowly and were less viable, and NG type that had a mix of neuronal like and glial like cells, grow faster and showed invasive properties. In addition, NG type cells had a significantly higher percentage of cancer stem cells that express CD133+ Sox2+ or AGR2+ BMI1+, and were more tolerant to treatment with the chemotherapeutic agents cisplatin and etoposide. Importantly, drug treatment of NG type cells resulted in the enrichment of CD133+ Sox2+ or AGR2+ BMI1+ cells.Gene expression profiling has been employed to grade, identify and characterize various sub-types within tumors. In this study, unsupervised hierarchical clustering of tumor samples divided tumors into two groups that were primarily different in their stem cell pathways. In addition, three of the top differentially up-regulated genes Reelin, Calbindin 1 and Anterior Gradient 2 Homolog, and two of the most down differentially regulated genes Cytochrome P450 and F-Box Protein 32 are associated with stemness –41. UnfoMost importantly, tumors that formed group 2, with low stem cell gene expression, generated G type cell lines that also had a lower expression of stem cell related markers, while tumors that showed high stem cell gene expression (including Jed45_MN) generated NG type cell lines that showed similar characteristics. This indicated that the generated primary cell lines do represent the essence of their corresponding tumors’ tissues, as previously shown with glioblastoma multiform tumors . Thus fuClear biological features, including morphology which is influenced by the repository of biochemical interactions flowing through the cell signaling system , separatPrevious work has shown the presence of cancer stem cells in meningioma cell lines –31, but Importantly, NG type cell lines showed a significantly higher tolerance of cisplatin and etoposide treatment compared with G type cell lines. Although etoposide has been traditionally thought of as a cell-cycle specific drug, recent data suggests a strong binding of the drug to chromatin and implicates a binding affinity for histone proteins, in particular histone 1, thus contributing to DNA damage regardless of the cell cycle status , 55. NotIn conclusion, analysis of patterns for cellular architecture, drug tolerance and associated resistance makers for primary cell lines in combination with genomic analysis of corresponding tissues of meningiomas, led to the functional sub grouping of meningiomas and the identification of novel stem cells related markers that appear to be associated with drug resistance and are likely to play a role in tumor aggressiveness. Importantly, this work highlights the need to use stem cells molecular markers and early in vitro analysis to classify functional variants of meningioma and predicts tumor aggressiveness and drug resistance.Additional file 1: Figure S1. Growth inhibition assays for A) Cisplatin and B) Etoposide for NG cell lines Jed38_MN, Jed45_MN and Jed49_MN.Additional file 2: Figure S2. H&E slides for studied variants including meningothelial  showing lobules of uniform eosinopholic cells with central nuclei and intranuclear inclusions forming abundant whorls with no evidence of atypia, necrosis or mitosis; fibroblastic  showing spindle cells with indistinct cell boundaries running in fascicle; transitional  with ratios of meningothelial to fibroblastic patterns ranging from 20:80 for Jed04_MN, 50:50 for Jed09_MN, 30:70 for Jed36_MN, and 40:60 for Jed38_MN; rhabdoid (Jed45_MN) showing hypercellular sheets with rhabdoid morphology  and necrosis; psammomatous (Jed43_MN) composed of whorled clusters of spindle cells with numerous psammoma bodies; and angiomatous (Jed12_MN) showing neoplastic growth in the form of nests and whorled of bland- looking polygonal cells with vascular component exceeding 50% of total tumor area and with no evidence of atypia, necrosis or mitosis. All images were taken at 20×.Additional file 3: Table S1. Differentially expressed genes between group 1  versus group 2 .Additional file 4: Table S2. Predicted differential pathways generated by Ingenuity Pathway Analysis Software. The transcriptome comparison was carried out for group 1  versus group 2 .Additional file 5: Figure S3. CSCs markers expression in situ. A) Images for immunofluorescence co-staining of stem cell markers CD133+Sox2+  or Nestin+Ki67+  and AGR2+ BMI1+  in low grade (Jed62_MN) and high grade (Jed45_MN) tumors. B) Mean percentages of co-positive cells. Error bars represent count errors between three independent regions within each tissue. All images were taken at 20×.Additional file 6: Figure S4. AGR2 co-expression with CSCs markers in situ. A) Images for immunofluorescence co-staining of stem cell markers Nestin+AGR2+, CD133+AGR2+, and Sox2+AGR2+  in low grade (Jed62_MN and Jed40_MN) and high grade (Jed49_MN and Jed45_MN) tumors. B) Mean percentages of co-positive cells. Error bars represent count errors between three independent regions within each tissue. All images were taken at 20×.Additional file 7: Figure S5. Average percentages of morphologies counts for Jed62_MN and Jed79_MN. Each tumor was divided into four portions that were grown in either DMEM-F12 +10% FBS (Blue line), or DMEM high glucose concentrations of 4500 mg/L (Gibco) +10% FBS (Grey line), or DMEM high glucose concentrations of 4500 mg/L (Gibco) + 5% FBS (Yellow line), or DMEM low glucose concentrations of 1000 mg/L (Gibco) + 10% FBS (Orange line). Horizontal accesses represent morphologies described in Figure Additional file 8: Figure S6. The expression of AGR2 in G type cell lines. A) Immunofluorescence images for two G Type cell lines (Jed39_MN and Jed40_MN). Images show cells co-stained positively for Vimentin+AGR2+ . B) Average percentages of cells positive for AGR2. Error bars represent errors between three independent counts.