FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer  and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.Based on gene mutation patterns and genomic changes we identify lines representative of the Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.The online version of this article (doi:10.1186/s12864-015-1450-3) contains supplementary material, which is available to authorized users. Urothelial bladder cancer (UBC) has a high incidence, with 133,696 new cases and 51,056 deaths from UBC in Europe in 2011 [in situ, and T1) or muscle-invasive . The majority of patients (ca. 70%-80%) present with papillary NMIBC, most of whom have a good prognosis. Patients with high-grade NMIBC, and those with MIBC, have an aggressive disease that can lead to patient’s death, emphasizing the need to better classify these tumor subgroups.UBC displays a high level of clinical and pathological heterogeneity. Morphologically, tumors can show papillary vs. solid growth patterns. A clinically relevant issue is the level of invasion of the bladder wall: tumors are classified as non-muscle invasive  and develop predominantly through the inactivation of the P53 and RB pathways [TERT promoter mutations occur in >70% UBC, regardless of stage/grade [Approximately 70% of NMIBC harbour activating mutations in d in UBC . PIK3CA utations . An addipathways ,8,9. Unlpathways ,10,11; spathways . TERT prge/grade .in vitro. In addition, tumor cells may behave differently in vitro due to the lack of interactions with non-neoplastic cells. Therefore, a thorough knowledge of their genotype and phenotype is essential in order to optimize their use while considering their limitations.Tumor cell lines are invaluable research tools. They are readily amenable to experimental manipulation, providing opportunities for functional analyses and contributing to improved knowledge . Cell liCell lines from primary UBC are commonly used as disease models. It is crucial to identify those lines best suited to answer specific biological questions and to place the studies in the context of patient’s tumors. The genetic make-up of UBC cell lines has been analyzed using array comparative genomic hybridization . High-reWe report here a detailed genomic analysis of a large set of UBC cell lines in order to improve their use as models for the study of this tumor type. Web resources used are listed in Additional file Information on 902,103 autosomal probes covering 2,787 Mb of the genome was analyzed using the waviCGH web server. All of these probes were called as altered (copy number lost or gained) in ≥10 cell lines. An average of 1349 Mb (510-2111 Mb) were altered across the panel: 592 Mb were gained (219-1189 Mb) and 757 Mb were lost (180-1291 Mb). The line showing the lowest fraction of the  genome altered was MGH-U3 . 639V cells showed the highest fraction of gains/losses: 2111 Mb (75.8%) . RT112 and RT4 cells exhibited amplification of a 75 and 79 Mb region, respectively, encompassing FGFR3 and part of the neighboring TACC3. FGFR3 mRNA expression was higher in FGFR3-mutant lines (P = 0.09) . Five of 45 lines (11%) harbored a mutation in both FGFR3 and PIK3CA, comparable with the frequency in COSMIC UBC tissues . Mutations in HRAS (7%), KRAS (8%), NRAS (5%), and AKT1 (5%) were less frequent  both mutations and genomic losses were considered  (n = 7) and homozygous deletions (HD) (n = 20). INK4A mRNA expression was significantly lower in lines with LOH (defined as gene copy number loss) or HD than in wild type lines . Regarding TP53, mutations were significantly more frequent in cell lines than in tumors . LOH was found in 47% of the lines.To assess the status of key tumor suppressors (ed Table A and B. (HD) n = 0. INK4A (HD) n = 0. INK4A INK4A and PTEN loss was similar in cell lines and tumors (P = 0.3) but the frequency of TP53 LOH was higher in cell lines .Figure The grade of the original tumor from which 27 lines were isolated was available . By contrast, TP53 mutant lines showed higher genomic instability   . Large structural alterations were rare: 6 lines showed complete loss of Xp whereas 3 lines showed almost complete gain of Xq. No significant MCRs were identified although peak gains were seen at Xp22.2, Xp11.4, Xp11.23, Xq11.2, Xq12, and Xq25 and peak losses at Xq21.31 and Xp21.3-21.1  or 3p arm (96-1). The number of UPD and their total genome size correlated with the genome size affected by copy number alterations (p = 0.08 and p = 0.09)  and losses  occurred with similar frequency in lines and tumors  whereas the cell lines had a high frequency of both gains and losses of chromosome 9, often in association with UPD affecting either the whole chromosome or its q-arm , ASRGL1 (11q13.4), ATP2B4 (1p12), ITGA3, PRPSAP2, and C17orf39 (17p11.2), all with a fold-change difference between amplified and non-amplified tumors of 1.5-1.9 (p < 0.05).The complete expression dataset is provided in Additional file ANXA10 , ARAP2 , CDS1 , and PTPRG   whereas ANK2, FAT4 and MLL were often lost ; MLL is on chromosome 11 - which is not frequently altered in UBC - and is significantly under-expressed in cell lines with LOH  and UPD (29%); a similar pattern was observed for other tumor suppressor genes. TP53 and EP300 were affected by both LOH and UPD.We analyzed copy number, UPD status, and expression of new driver UBC genes .- We provide novel information on chr. X losses, where new important tumor suppressor genes have been identified  whereas others are more representative of the tumor suppressor-driven pathways . We propose that - as has already been done, in part - these cells be used as models of non-aggressive and aggressive UBC, respectively.- Some cell lines are more representative of the - The UBC lines available cover a wide range of tumor genotypes and phenotypes. While they do not fully represent the spectrum of tumors found in patients and are enriched towards a more aggressive genetic architecture, the main genetic pathways involved in UBC are represented in this panel.- Future efforts should be placed to establish new UBC lines, mainly focusing on less aggressive tumors, as well as collections of patient-derived xenografts.TERT, FGFR3, PIK3CA, KRAS, HRAS, NRAS, p16/INK4A, PTEN, and TP53) was analyzed and complemented/supported through data obtained from COSMIC, CCLE, and IARC public databases [FGFR3, PIK3CA, KRAS, HRAS, NRAS, PTEN, and TP53 in primary UBC tumors with either “papillary” or “carcinoma” histology was retrieved from the COSMIC database. All web resources used in the analysis are listed in Additional file A literature search was performed to retrieve information regarding sex, histology, and stage/grade of the original tumor from which the cell line was established, as well as the original reference. The mutational status of the main genes involved in UBC ; J82, MGH-U3, and RT112 cells were kindly provided by F. Radvanyi ; UM-UC-1, UM-UC-3, UM-UC-4, UM-UC-5, UM-UC-6, UM-UC-7, UM-UC-9, UM-UC-10, UM-UC-11, UM-UC-12, UM-UC-13, UM-UC-14, UM-UC-15, UM-UC-17, and UM-UC-18 were provided by H. B. Grossman  ; HT1197,Cells were cultured in RPMI supplemented with 10-20% FBS and were harvested at 70-90% confluence. DNA from cell lines HT1197, HT1376, HU456, KK47, PSI, UM-UC-2, and VM-CUB-1 was isolated in the laboratory of D. Theodorescu; the remaining cell lines were cultured at CNIO. DNA was isolated using the DNAeasy blood and tissue kit (Qiagen) according to manufacturer’s instructions.Tumor samples (n = 49) came from UBC cases diagnosed with UBC recruited to the Spanish Bladder Cancer/EPICURO study. Informed consent was obtained from study participants in accordance with the Institutional Review Board of the Ethics Committees of participating hospitals that approved the study . The T/G distribution was as follows: Ta (n = 26), T1 (n = 8), T2 (n = 5), T3 (n = 6), and T4 (n = 4); cases were grouped in two categories, non-aggressive (TaG1 and TaG2) (n = 19) and aggressive (TaG3 and T1-T4) (n = 30). Only samples containing >60% tumor cells were used. DNA was isolated using the Puregene kit A (Qiagen) according to the manufacturer’s instructions.FGFR3, PIK3CA, HRAS, KRAS, and NRAS hotspot mutational analysis was performed using ABI PRISM® SNaPshot® (ABI) as previously described [escribed .P‐value based on a permutation test assuming that the alterations found are randomly located in the genome. Consecutive probes with P‐values <0.05 were joined in a common region. Focal copy number alterations are those not involving whole chromosomes or whole chromosome arms.DNA (1.5 μg) was quantified using picogreen and used for array hybridization. The Illumina 1 M Duo array includes 902,103 autosomal probes and 39,779 probes from sex chromosomes. A total of 43 different cell lines were hybridized to the arrays (GSE64572) Genotypes and R values were extracted using the beadstudio software (version 3.1.3.0) and R values were normalized using the method described by Pounds and co-workers  with theThe experimental reproducibility of the gene copy number analysis was assessed using data from DNA isolated in 2 different laboratories (n = 3) or DNA isolated from different cultures in the same laboratory (n = 5). The absolute call concordance rate was between 79.3 and 96.7%. “Gain/loss” type discordances were very uncommon 0.015-0.03%); most discordances were “gain/no-change” (1.4-7.5%) or “loss/no-change” (1.9-15.8%). The replicate with the highest signal to noise ratio was considered the most accurate and selected for subsequent analysis. A summary of the call concordance rate in replicates is provided in Additional file 5-0.03%; FGFR3 (n = 22), PIK3CA (n = 42), KRAS (n = 22), HRAS (n = 2), NRAS (n = 6), INK4A (n = 17), PTEN (n = 49), and TP53 (n = 22).Gene amplification was determined from the copy call results from CGHcall. LOH and HD were determined by combining copy call, B allele frequency (BAF), and genotyping data. Probes with homozygous calls and BAF of either 0 or 1 and a decline in the logR ratio of were classified as LOH and non-called (NC) probes with an abnormal BAF and a decline in LogR ratio were classified as HD. The number of probes representing each gene was: The gender of the patient from whom the cell lines were derived was not always available. The presence of a Y chromosome was considered as a reliable indication that it was of male origin. This, combined with the data from the literature, was used to select cell lines for which we could perform X chromosome gene copy number analysis. The LogR ratio was calculated with the average R value normalized to a pool of control male or female blood leukocytes from the EPICURO Study. For probes representing the X chromosome , or those corresponding to sequences present on both the X and Y chromosomes (395 probes), copy number calls were obtained using the waviCGH software as described for autosomes.Genomic instability was assessed as the fraction of the genome altered (either lost or gained), calculated from call data generated by CGHcall using autosome probes and measured in 3 different ways:(1) total size of the genome (covered by the Illumina array) altered. The size in base pairs (bp) of the segmented regions altered (lost or gained) was calculated from the start and end position of the segments; (2) fraction of probes altered - proportion of probes showing loss, gain, and amplification; (3) number of altered segments identified - the total number of individual altered segments, lost or gained, was determined from the waviCGH segmented call data.FGFR3, PIK3CA, KRAS, HRAS, NRAS, p16/INK4A, PTEN, and TP53 and the original tumor grade with the fraction of the genome altered, calculated by these approaches. The chi-square test was used to assess the difference between the frequency of mutant vs. wild type genotypes and low vs. high grade cell lines in each of the genome instability groups. The Wilcoxon test was used to assess the difference in the mean of the genome instability variable between mutant/wild type and low/high grade cell lines.Cell lines were classified in 3 categories according to fraction of the genome altered  calculated by the 3 different methods; low/medium/high genomic instability groups were thus identified. We compared the relationship between mutational status in Log R ratios from hybridization data were analyzed using the zoo package of the R statistical program in order to identify UPDs . Chromosexprs function with parameters by default.We have used previously reported data corresponding to 28 cell lines (GEO: GSE5845)  and haveGene lists were generated for regions identified as significant MCRs in copy number lost, gained or amplified regions. To determine the relationship between gene copy number alteration and expression, correlation analysis was performed for amplified genes by comparing copy number amplified/gained cases vs. copy number neutral/lost cases. For copy number lost genes, a comparison of copy number lost vs. no copy number loss was performed; the difference in expression between the 2 groups was assessed using the Wilcoxon test. The chi-square test was used to compare the distribution of mutation frequencies in cell lines and tumors.Copy number status and UPD of 44 genes recently shown to be involved in UBC were assessed ,21,23,24A molecular classifier consisting of 1038 genes  was usedThe same approach was applied using the centroids provided by Rebouissou et al.  to classAll genomic data reported in this manuscript have been deposited in GEO. Expression data accession number: GSE64279. SNP array data accession number: GSE64572. Any other information of interest to readers can be requested to the authors.


Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30 % response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC.de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump.We established BC cell lines with acquired or We found that the resistant cell lines showed 7–100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance.Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.The online version of this article (doi:10.1186/s12885-016-2071-1) contains supplementary material, which is available to authorized users. First-line chemotherapy of recurrent breast cancer (BC) is dependent on the type of prior adjuvant treatment but it most often consists of repeated cycles of anthracyclines and/or taxanes, possibly combined with cyclophosphamide, with standard combinations typically associated with response rates of about 50–60 % . Second-http://clinicaltrials.gov/show/NCT01245192).Chemotherapeutic agents that target the Topoisomerase I protein (Top1) are routinely used in treatment of metastatic colorectal cancer  and haveIrinotecan, etirinotecan and topotecan have also been tested in clinical trials in mBC , and iriWith expected objective response rates of approximately 25–30 % in pre-treated mBC patients, it is highly likely that many of the patients developed cross-resistance to Top1 inhibitors during their prior treatment. For example, Top1 and Top2 inhibitors, and taxanes are all known substrates for xenobiotic drug transporters from the ABC-cassette family, which may be up-regulated during chemotherapy treatment . AlternaTOP1 gene [TOP1 gene copy numbers may be a predictive biomarker for response to irinotecan in BC patients [Irrespective of the pre-existence or induction of resistance, a validated molecular drug sensitivity/resistance profile might enable physicians to identify patients who are most likely to benefit from Top1 inhibitor treatment and only offer this therapy option to these patients. Such a strategy would increase the therapeutic index of Top1 inhibitors in mBC. In that respect, it has been shown that around 30 % of BC patients possess amplifications of the OP1 gene  and a clpatients . HoweverWith the aims to search for Top1 inhibitor predictive molecular biomarkers and to identify which drugs are effective in irinotecan resistant BC cells, we have established human breast cancer cell line model systems for resistance to SN-38, the active metabolite of irinotecan. To cover a wide range of potential mechanisms, we have established four SN-38 resistant cell lines through exposure to either gradually increasing concentrations of SN-38 or a single high dose. We describe here potential molecular mechanisms of SN-38 resistance in breast cancer cells and sensitivity to other commonly used chemotherapeutic agents, as well as novel non-camptothecin Top1 targeting drugs.SN-38 and Ko143 , Epirubicin , Cisplatin  and Docetaxel . The indenoisoquinoline drugs LMP776 NSC 725776) and LMP400 (NSC 743400) 776 and L, were prA panel of the 52 breast cancer cell lines was propagated in complete media: RPMI 1640  with 10 % fetal bovine serum  as previously described . Docetax2. SN-38-resistant and DMSO-exposed control cell lines were maintained under the same medium conditions, supplemented with SN-38 or DMSO, respectively. All experiments were carried out in presence of SN-38 except for growth curves, cell cycle analysis and Top1 enzyme activity. The resistant cell lines were designated “acq” for acquired resistance and “de novo” for de novo resistance.The human breast cancer cell line MDA-MB-231 was obtained from American Type Culture Collection . Professor Ole William Petersen (University of Copenhagen) kindly provided the human MCF-7 breast cancer cell line. MCF-7 and MDA-MB-231 cell lines were maintained in DMEM including L-glutamine medium  supplemented with 10 % FBS  for MDA-MB-231 cells and 5 % FBS, 1 % non-essential amino acids  for MCF-7 cells. Cell lines were cultured in the presence of penicillin/streptomycin antibiotics  and incubated at 37 °C in a humidified environment containing 5 % CO50 values for SN-38 were determined by exposing cell lines to a range of SN-38 concentrations using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays to measure the response. To establish acquired resistance, the parental cell lines were exposed to SN-38 concentrations ranging from 5000- to 500-fold lower than IC50 and the highest concentration that only caused minimal effect on the cells was chosen as a starting point. The cell lines with acquired resistance, MDAacq and MCF-7acq, were developed by exposing the parental cell lines to stepwise increasing concentrations of SN-38 (from 3.6nM – 68.2nM for MDAacq and 3.0nM – 36nM for MCF-7acq) over 6 months. SN-38 exposed cell lines were maintained at each drug concentration for three passages. Two resistant cell lines were obtained after a total of 24 passages for MDAacq and 23 passages for MCF-7acq in presence of SN-38. To establish the de novo resistant cell lines we initially exposed parental cell lines to SN-38 concentrations ranging from the low nM range up to 25 μM and selected the cell populations that eventually were able to re-grow with constant exposure to SN-38. Thereby, we selected de novo resistant cell lines, MDAdenovo and MCF-7denovo, which survived constant exposure to 24nM for MDAdenovo and 12nM for MCF-7denovo /ln(c2/c1), where c1 and c2 represent the cell numbers at the beginning and end of the exponential phase during time (h), respectively .Fixation, propidium iodide staining and cell cycle analysis using fluorescence-activated cell sorting  and data analyses by FlowJo software were done as previously described  and analCells were formalin fixed, embedded in agarose and paraffin embedded as previously described .TOP1/CEN-20 probe mix [TOP2A FISH pharmDx™ kit  were applied according the manufactures instructions as previously described [A escribed . This anGene expression dat obtained by Affymetrix U133 microarray (GEO entry GSE41313) were applied . Copy nuacq, MDA-MB-231 parental and MDAacq) using Trizol reagent (Invitrogen). Expression analysis using Agilent Human Gene Expression Microarrays , image quality, background correction and normalization were conducted as previously described [http://www.r-project.org/). Statistical tests were performed using a moderated t-test, and p-values were adjusted for multiple testing by the Benjamin & Hochberg method [p-value < 0.05. For each cell line three biological replicates were analyzed.Total RNA was extracted from three independent passages of 70 % confluent cells for each of the four cell lines  for size, amount and pI pattern of Top1 (28). For size analysis, cell lysates were diluted in MPER lysis buffer with Bicine/CHAPS lysis buffer containing 2 % DMSO inhibitors (ProteinSimple) and 4X master mix/fluorescent standards (Protein Simple) according to the manufacturer’s instructions. The samples were denatured for 10 min at 70 °C and pipetted into a 384-well plate along with primary antibodies (Top1 (Abcam) 1:100, β-actin (Abcam) 1:100) diluted in Antibody Diluent Plus (ProteinSimple) according to the manufacturer’s instructions. For charge analysis, lysates were prepared in Bicine/CHAPS lysis buffer containing 2 % DMSO inhibitors and diluted with this and premix G2 pH 3–10 separation gradient (ProteinSimple) containing 2.2 % ladder 1 (ProteinSimple). Charge analysis samples were pipetted into a 384-well plate along with primary antibodies (Top1 (Abcam) 1:50, β-actin (Abcam) 1:50) diluted in antibody diluent (ProteinSimple) according to manufacturer’s instructions. Data were processed using Compass software (ProteinSimple). The size analyses were repeated four times with two independent biological replicates and the charge analyses were conducted in biological triplicates.The cellular Top1 enzyme activity was analyzed as previously described . BrieflyCells were seeded at 10,000 cells/well in 96-well plates and allowed to adhere for 48 h at 37 °C. Cell lines were exposed to SN-38 with or without Ko143, a specific BCRP inhibitor (Sigma Aldrich) for 72 h. Cell viability was assessed using MTT assays with triplicate determinations from three independent biological passages.T-test assuming equal variance and p-values below 0.05 was considered statistically significant. A non-parametric test  was used to associate the TOP1 copy number (CN)-status with mRNA expression levels with the order of groups being CN loss, CN neutral and CN gain and a two-sided p-value below 0.05 was considered statistically significant.MTT data were analyzed in Excel by two-tailed Student’s TOP1 CN, Top1 mRNA or protein expression or enzyme activity has been correlated to cancer cells’ sensitivity to Top1 targeting drugs [TOP1/CEN-20 probe mixture [TOP1 and CEN-20 CN and the TOP1/CEN-20 ratios in these cell lines  to 5.5 (HCC1419). There was a trend towards association between TOP1 and CEN-20 copy numbers as reflected in a TOP1/CEN-20 ratio close to 1 for the majority of the cell lines  indicating that the TOP1 genes are actively transcribed even in cell lines with many TOP1 copies , we then selected 9 breast cancer cell lines from the panel of the 52 characterized cell lines  and Top1 appeared to be functional because the cellular Top1 enzyme activity could be impaired by SN-38  to the Top1 enzyme activity (data not shown). These 9 cell lines were then tested for sensitivity to SN-38  or the TOP1 mRNA . However, the Spearman’s correlation coefficients between the Top1 activity and the TOP1 CN or the TOP1 mRNA were  or , respectively. This indicates that more TOP1 copies and higher Top1 enzyme activity correlate to increased sensitivity to SN-38.Increased ng drugs , 26–29. TOP1 CN data to IC50 values reported for topotecan [TOP1 CN had non-significant negative Spearman’s correlations to the IC50 values for topotecan  and for camptothecin . Thus, factors beyond TOP1 CN, Top1 expression and enzyme activity appear to be involved in the response to SN-38. Based on the results presented above we selected two cell lines to represent the majority of BC patients likely to be candidates for Top1 targeted therapy, namely the MDA-MB-231 and the MCF-7 cell lines, which represent cell lines with TOP1 CN gain  lower growth rates when compared to their parental and DMSO controls . The doubling times of MDAacq, MCF-7acq, MDAde novo and MCF-7de novo were 35.5, 44.3, 43.9 and 33.2 h, respectively, which was 7.8, 11.7, 16.8 and 6.1 h longer than their respective DMSO controls. The increase in doubling times was significant (p < 0.05) for all cell lines except for MCF-7de novo (p = 0.08).The growth curves , which is not a BCRP substrate [The SN-38 resistant cell lines were assessed for cross-resistance to a range of anticancer drugs  in comparison to their DMSO controls. Resistant cell lines exhibited different and complex patterns of cross-resistance to various anti-cancer drugs, which is summarized in Table ubstrate . For cis400 NSC 74998, whiTOP1 or TOP2A at the gene level, FISH analysis was performed on parental, MDAacq and MCF-7acq cell lines. No differences in the TOP1 or TOP2A CN between the resistant cell lines and the respective control cell lines were detected. Furthermore, neither TOP1/CEN-20 nor TOP2A/CEN-17 ratio numbers were different among the respective cell lines  and the second most deregulated in the MCF-7acq system (4 fold up-regulation)  was not deregulated in the acquired resistance cell lines. Gene Ontology (GO) Molecular Function analysis found that the most prominent common GO term was the category “transcription cofactor activity”, defined by genes that interact selectively and non-covalently with a regulatory transcription factor and also with the basal transcription machinery in order to modulate transcription. Reactome and KEGG pathway analyses identified among the top 10 pathways “Metabolism of xenobiotics by cytochrome P450”, “Nucleosome assembly” and “DNA damage/Telomere stress induced senescence” (data not shown). In the MCF-7acq the MetaCore network analysis of the deregulated genes highlighted two networks of 19 and 10 genes with the most connecting nodes being the transcription factors PR and FUS/DDIT3, the kinase inhibitor p21 and the regulatory protein RIP140 , was the most up-regulated in the MDA140 Fig. . In the 140 Fig. . These nacq, MCF-7acq and MDAde novo cell lines while only a minor decrease in Top1 protein expression was seen in MCF-7de novo. Top2a levels remained unchanged in all resistant cell lines  was evaluated by Western blotting  down regulation of Top1 in MDAacq (0.55x), MCF-7acq (0.35x) and MDAde novo (0.12x) whereas the MCF-7de novo showed a slight increase (1.5x). Post translational modifications (PTMs) of Top1 have been associated with sensitivity to Top1 targeting drugs [acq cells were compared to MCF-7 control cells the pI peak pattern was very similar to the MCF-7 DMSO controls indicating no major changes in Top1 PTMs in the resistant MCF-7 cells. However, the MDAacq cells lost the peak signals in the pI range around 5.5 and 7.5-8.5 but gained peak signals around pI 5 and pI 6.4. These data indicates that Top1 PTMs are changed in the MDAacq cells.We applied nanocapillary electrophoresis for more precise quantification of the Top1 protein. Top1 and β-actin were simultaneously detected and fully separated without background signals  and complete re-sensitization of the MDAacq, MDAde novo and MCF-7de novo cell lines to the level of DMSO control cell line. Although significant (p = 0.01) the MCF-7acq was only slightly re-sensitized to SN-38 following Ko143 co-treatment  proteins that can form heterodimers with other HLH proteins and inhibit its interaction partner in binding to DNA. ID3 is involved in tumor growth, invasiveness, metastasis, and angiogenesis , and wasacq cell line, which had a very strong up-regulation of the MDR1 protein, the cells retained sensitivity to docetaxel. Furthermore, our previously reported docetaxel resistant BC cell lines showed cross-resistance to SN-38 . Remarkably, LMP400 did not display cross-resistance with SN-38 in three of the four SN-38 resistant BC cell lines (Table Taxanes are currently regarded as the most efficient drug class in the management of BC and are commonly used in neoadjuvant and adjuvant therapy of BC and in management of mCB. In spite of showing overall response rates of more than 40 %, patients progress in a relative short time, and in many cases show cross-resistance to second-line anthracyclines and subsequent regimens. Interestingly, none of the SN-38 resistant cell lines had acquired cross-resistance to docetaxel while both of the MDA-MB-231 SN-38 resistant cell lines displayed minor cross-resistance to the anthracycline epirubicin (Table TOP1 CN, TOP1 mRNA, Top1 protein and enzyme activity, we found consistency between the TOP1 CN and protein levels (Additional file TOP1 mRNA levels were significantly associated with increased enzyme activity and non-significantly associated to increased sensitivity to SN-38, which is supported by other studies using fewer cell lines [acq, MCF-7acq and MDAde novo cell lines whereas only a minor decrease was observed in the MCF-7de novo. These results recapitulate data from a BC mouse model of acquired topotecan resistance that suggested reduced levels of Top1 as a mechanism of in vivo resistance [TOP1 CN or gene expression. However, this suggests that acquired resistance to SN-38 might involve a decrease in Top1 protein levels, which is also supported by studies demonstrating a correlation between reduced Top1 levels and reduced sensitivity to Top1 targeting drugs [de novo cell line. Consistently, a recent study suggested elevated levels of Top1 as a predictive biomarker for irinotecan response in MBC patients [acq cells, which may contribute to the resistance to SN-38 (Fig. Clearly, factors beyond ABCG2/BCRP may be involved in resistance to SN-38. To address other possibilities we explored other, known, molecular mechanisms underlying resistance to SN-38. In a panel of breast cancer cell lines with varying levels of ll lines , 43, 44.ng drugs , 46. Thepatients . The Toppatients , 30, 48,patients  and SUMOpatients . These m-38 Fig. .de novo resistance share fundamental characteristics. In particular, up-regulation of the BCRP drug efflux pump, low proliferation rates and down-regulation of Top1 protein are suggested as key mediators of SN-38 resistance in human breast cancer cell lines. These preclinical observations should be clinically validated in breast cancer biopsies derived from clinical studies in which the patients were exposed to Top1 inhibitor treatment to generate level 1 evidence for use of these markers.In conclusion, molecular characterization of the developed SN-38 resistant human BC cell line model system revealed that acquisition of resistance to SN-38 in vitro is multifactorial and that acquired or


Cancer cell transformation entails significant alterations in intracellular metabolic pathways one of which is the aerobic glycolysis phenotype . The oriin vitro and in vivo) of the parental and modified cancer cells was carried out.We used two human breast cancer cell lines (MDA-MB-231 & MDA-MB-453) in this study where we cultured these cell lines in the presence of mitochondrial complex I inhibitor (rotenone) inhibitor for 25 generations thereby creating metabolically adapted cell lines counterparts. Metabolic analysis (In vivo analysis of tumor xenografts revealed that metabolically modified MDA231 cells displayed a ~50% reduction in tumor growth/volume accompanied by a reduced in vivo proliferation rate in comparison with the parental MDA231 cells thereby confirming the physiological relevance of metabolic adaptation in preclinical animal models. In conclusion, long-term metabolic adaptation to mitochondrial complex I modulators can be a unique and novel strategy for achieving tumor control in vitro and in vivo.Analysis of metabolic status in the isogenic parental and metabolically modified adaptive breast cancer cells show an overall improvement in mitochondrial function and a reduction in metabolic switch phenotype.


Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in “reproductive capacity,” i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent—gonads switch between active and dormant states—and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism’s lifespan.Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal—for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the  C. elegans reproductive system as a model organ. We characterize benefits and drawbacks of stem cell cycling—which are keeping worms primed for reproduction, and reducing the number of future progeny worms may bear, respectively. We show that, under specific conditions of reproductive inactivity, stem cells switch back and forth between active and dormant states; the timing of these switches, whose genetic control we start delineating, appears random. This randomness may help explain why populations of aging, reproductively-inactive worms experience an increase in the variability of their reproductive capacity. Stochastic stem cell cycling may underlie tradeoffs between self-renewal and senescence in other organs.Stem cell cycling is expected to be beneficial because it helps delay aging, by ensuring organ self-renewal. Yet stem cell cycling is best used sparingly: cycling likely causes mutation accumulation—increasing the likelihood of cancer—and may eventually cause stem cells to senesce and thus stop contributing to organ self renewal. It is unknown how self-renewing organs make tradeoffs between benefits and drawbacks of stem cell cycling. Here we use the  C. elegans can be simply achieved by a pause in developmental and reproductive activities in the “dauer” state [C. elegans gonad provides a powerful model system to address this question. Previous studies have identified mechanisms by which the “reproductive lifespan”—the period of adulthood over which C. elegans hermaphrodites can bear progeny—can be extended . But thed (e.g. ). That red  with the ~300 stored self-sperm they produce during development, or be cross-fertilized with male sperm transferred during mating, which allows brood sizes of up to 1,200 [fog-1 or fog-2 [fog-2 females were shown to undergo reproductive senescence at roughly the same rate as reproductively-active hermaphrodites [fog-2 females is much reduced [To ask whether reproductive senescence is a simple function of chronological age, or whether it is driven by reproductive activity itself, it is useful to manipulate that reproductive activity (e.g. ). There to 1,200 . Brood sor fog-2 –8. Theseor fog-2 ,10. Fema reduced , suggest reduced , suggestHere, we use genetic, environmental, and pharmacological manipulations to assay the relationship between gonad activity and senescence. We establish a causal relationship between the two. We further characterize germ cell cycle behavior on a gonad by gonad basis, using a new technique we developed, and find intermittent activity. Our results strongly suggest that switching between active and inactive states is stochastic.fog-1 and fog-2 females described above, which have low ovulation rates [inx-22; fog-2 females and spe-8 hermaphrodites—both of which are also sterile (unless mated); loss of inx-22 results in precocious oocyte maturation in feminized gonads even in the absence of the sperm signal [spe-8 preserves stimulation of oocyte maturation by self-sperm that are incapable of fertilizing the oocytes [spe-8 and inx-22; fog-2 females ovulate at a rate close to wild-type , which is in turn significantly higher than that for fog-1 . We mated virgins at either day 0, 1, 2, 3, 7 or 10 of adulthood and assayed total brood size  for two days  Roscovitine. Roscovitine has previously been used to reversibly inhibit mitosis in starfish and sea urchin embryos ; we founThe role that we uncovered for cell cycling in driving reproductive senescence, combined with the role of DNA damage in driving senescence in other systems , led us rpa-1::yfp hermaphrodites taken at day 1 of adulthood, and in rpa-1::yfp hermaphrodites taken at day 4 of adulthood that had been selfed or that had been mated at day 0 of adulthood. We found over ~5-fold more foci per nucleus in the mated group than in either of the selfed groups . While RPA-1 is activated in response to multiple forms of DNA damage –26, it pd groups . Consistd groups . Overallfog-1 and fog-2 females with inx-22; fog-2 and spe-8. Since the former two strains undergo slower reproductive senescence than the latter  and for fog-2 (17%) than for wild-type; it took in excess of 6 h for labeled cells to have appeared in all feminized mitotic zones , which is incorporated by cells in S phase. Although virtually all young, selfed wild-type mitotic zones contained EdU-positive cells following a 30-minute pulse , manic zones . To testfog-2 gonads (the same reasoning applies to fog-1 gonads): some gonads label almost immediately, showing that they are actively progressing through S-phase at the time of label application, while others take up to 8 h and thus do not have cells actively progressing through S-phase during these 8 h. If fog-1 and fog-2 simply cycled continuously, in the same way as wild-type but with uniform slowing down of all cell cycle phases, the distribution of cell cycle phase indices would be unchanged and the number of gonads with at least one cell progressing through S-phase would be the same as in wild-type at any given time—and the continuous labeling time courses would therefore appear identical for all genotypes. Stochasticity can be inferred from the fact that EdU labeling of reproductively-inactive gonads happens immediately for some gonads but takes up to 8 h for others: this shows independent behavior of gonads with identical genotypes, for which stochastic switching between active and dormant states is the most parsimonious explanation. To begin confirming the existence of such states by independent means, we quantified the coefficient of variation (CV) in mitotic index. The CV is defined as the ratio of standard deviation to mean and thus provides a unit-free measure of noise. The fog-1 and fog-2 CVs were 2.3- and 1.7-fold higher, respectively, than the wild-type CV, differences that are significant at a 95% confidence level . Differences in fractions of dormant gonads matched differences in mitotic index CV: the CV for fog-1 was significantly higher than that for fog-2, which in turn was significantly higher than the CV for wild-type, inx-22; fog-2 or spe-8  ≃ inx-22; fog-2 (0.18 cycles / h) > fog-2 (0.13 cycles / h) > fog-1 (0.08 cycles / h), which are equivalent to the rankings for reproductive senescence rates. We do not know the molecular basis for the difference in average fog-1 and fog-2 cell cycle rates—slower oocyte maturation in fog-1 . Dispersion was even stronger in wild-type hermaphrodites at day 3—to the point that cell cycle progression appeared largely randomized. Given that initial progression rates are highly similar across the genotypes and conditions that we tested, the simplest interpretation of these results is that the time at which labeled mitotic zones return to the dormant state is stochastically distributed. To test whether dispersion in the amounts of cell cycle progression after labeling was due to reduced reproductive activity, we measured cell cycle activity in mated young females, and in hermaphrodites mated at day 1 of adulthood and assayed at day 3. In both cases, the increase in reproductive activity caused by mating was accompanied by a switch of cell cycle behavior to that of young adult hermaphrodites  between pairs of mitotic zones from the same worm; this substantial synchrony in gonadal arm states suggests that dormancy may be regulated at the organismal level, which could perhaps occur through neuronal control of TGF-beta signaling  or insulfog-1 females either singled (low density) or as group of 70 individuals (high density) on 35 mm plates, and mated them at day 4 of adulthood to assay reproductive capacity. We found that females that had been kept at low density had a significantly reduced brood size compared to those that had been kept at high density (daf-22(m130) mutation, which abrogates dauer pheromone synthesis [Since population density affects a number of aspects of worm physiology –44, we n density . We then density : 23% of  density . As a fiynthesis , and fou10 bacteria/mL (“high” concentration) had a similar brood size to hermaphrodites kept on solid medium under standard conditions , but brood size dropped more than two-fold  at 1x109 bacteria/mL (“medium” concentration). Further, [9 bacteria/mL and 1x108 bacteria/mL (“low” concentration). At day 1 of adulthood, we transferred wild-type adult hermaphrodites to low, medium or high E. coli concentrations for 24 h, and subsequently performed 1 h continuous EdU labeling. In contrast to mitotic zones from females or older hermaphrodites, those from food-restricted young hermaphrodites were all active (n = 20 each), even at the lowest food concentration. This suggests that intermittent cycling is a specific response to sperm deprivation.Finally, we asked whether intermittent cycling is a behavior that is always associated with reduced overall reproductive activity, or whether it is specific to a state in which worms are well fed but deprived of sperm. Caloric restriction strongly reduces the rate of reproduction as well as total brood size ,46. In oinx-22; fog-2 females at day 3 of adulthood, the rate of viable progeny production increased sharply and remained sustained over the first ~18 h . The trough likely resulted from a drop in the numbers of developing oocytes in diplotene or diakinesis, which was not experienced by inx-22; fog-2  is to the best of our knowledge the first worm mutant reported to have a total reproductive capacity greater than wild-type. What link between cell cycling and reproductive senescence does this mutant suggest? It is possible that an intact DDR is required for full-speed germ cell cycling, and that hus-1(op241) germ cells thus cycle more slowly, leading to delayed reproductive senescence; given that hus-1(op241) does not display slower reproduction as might be expected from slower germ cell cycling, we do not favor this possibility. Another possibility is instead that the DDR, whose intensity according to RPA-1 foci correlates positively with the amount of past cell cycling, could mediate the effects of germ cell cycling on reproductive senescence—through molecular pathways that will require further study. An interesting additional problem will be identifying the downsides of increased reproductive output in hus-1(op241) mutants. We speculate that late-born progeny are more prone to mutation accrual or genomic rearrangements; further study will be required to characterize the fascinating tradeoff between increased brood size and decreased genome quality in progeny.What mechanisms tie reproductive senescence to germ cell cycling? Mutations that delay senescence may provide an entry point to start addressing this question. But although a number of mutations have been uncovered that increase reproductive lifespan, most do not result in an increase in total brood size and many in fact result in a ut (e.g. ). Given We also note that germ cell cycling has been shown by others to shorten lifespan, in addition to reducing reproductive capacity as we report here: mating acts via DAF-12 –a key mediator of lifespan control by the reproductive system –to causeThe role of germ cell cycling in driving reproductive senescence makes intermittent cycling particularly intriguing. Much of this intermittent cycling phenomenon remains to be characterized. The lack of suitable live imaging techniques makes it necessary to infer the existence of dormant and active states and the rules for transition between these states, and certainly leaves open the possibility that, in the future, more fine-grained studies will identify a broader array of sub-states and more complex transitions between them. The existence of at least two states, an active and a dormant one, is strongly supported by EdU continuous labeling studies that show that some virgin females mitotic zones label immediately when placed on EdU while some do not. This is compatible with germ cells being capable of stopping replication within S-phase or significantly lengthening S-phase ,52. FurtUntil cell cycle states are more finely defined at the molecular level, control of transitions between states can only be addressed in qualitative terms. The spread in virgin female mitotic zone cell cycle progression during EdU pulse-chase experiments  suggestsfog-1 and fog-2, both known for their role in germline sex determination, have different cell cycle dynamics: fog-1 undergoes slower average cell cycling that does fog-2. This could be consistent with the previously-established, dose-dependent role of FOG-1 in promoting proliferation [fog-1 and fog-2 exists in that virgin fog-1 ovulates at a slower rate than virgin fog-2; it is possible that a slower loss of cells to oogenesis leads to slower homeostatic mitotic zone cycling, rather than there being a direct role for fog-1 in controlling cell cycling. In line with this idea, it has most interestingly been shown recently that oocyte accumulation inhibits germ cell proliferation [fog-1 and fog-2 differ in the foraging behaviors [fog-1 and fog-2.The molecular controls of intermittent cycling remain to be fully established. It may seem surprising that feration . But a cferation . Lastly,ehaviors , which cfog-1 and fog-2 genes better known for their role in germline sex determination, our results and those from another recent study [daf-22, likely through the dauer pheromone pathway, while the daf-18 and insulin pathways were implicated by [In addition to the nt study  implicatcated by   species, and in older hermaphrodites.Next, we address whether intermittent cycling may occur in the wild in a fashion that is relevant to “fitness.” We focused largely on genetically-feminized older hermaphrodites release a volatile pheromone that attracts males [C. elegans—even if it has lost its direct purpose. Overall, hermaphrodite mating that occurs under conditions of intermittent cycling is likely of relevance to fitness and is thus under active selection, or was in a suitably-recent past.What is the relevance of germ cell behavior in older, sperm-depleted hermaphrodites? Two important questions to address this relevance are 1) how strong a selective pressure applies to hermaphrodites around the end of reproduction and 2) whether hermaphrodite mating is of much relevance in a mostly selfing species. We discuss these two questions in turn. Regarding whether reproduction of older hermaphrodites is under active selection, there is a general expectation that late-life reproduction might only be under weak selection , although the wild ,63 heterthe wild ,64. Lowethe wild , and malC. elegans populations hedge their bets by maintaining individuals that are primed for reproduction at the cost of faster senescence, and individuals whose gonads are dormant and that thus senesce more slowly, helping preserve reproductive capacity of the population over time. Stochasticity in the behavior of individual gonads would then have two roles. First, it would allow individuals to modulate the average rate of germ cell cycling. Second, and more interestingly, stochasticity is a parsimonious mechanism to develop a broad distribution of effective reproductive senescence rates in the population, without those rates being pre-assigned to each individual. Individuals that “take a chance” by cycling allow the population to quickly initiate reproduction if conditions become favorable shortly after they cycle, while individuals that stay in a dormant state avoid the population going extinct if conditions only become favorable after an extended period of time . To our knowledge, such bet hedging would be the first to directly control senescence of a self-renewing organ, and the first to be implemented by integration of stochastic switching between different states. An interesting problem for future studies would be to ask whether bet-hedging strategies are used by other self-renewing organs in which stem cell cycling contributes to maintenance of tissue function, but increases the probability of a cell becoming senescent to avoid cancer [If future reproduction of intermittently-cycling hermaphrodites is relevant to in-the-wild conditions and is specifically selected for, how would intermittency be beneficial? We speculate that uncertainty in the time at which reproduction will become possible could perhaps underlie a bet-hedging strategy. Bet-hedging is a well-established strategy followed by unicellular and multicellular organisms to avoid or spread risks in the face of uncertain environmental conditions ,66, and d cancer .Finally, we note that there may be a number of alternative reasons for intermittent cycling. Intermittent cycling may be an unselected-for, side-effect behavior that derives from the particular structure of the yet-to-be-characterized gene network that regulates it, while the increase in phenotype variability in old age could be explained by phenotypic drift . Rather fog-1(q785) I [fog-2(q71) V [inx-22(tm1661) I; fog-2(q71) V [C. remanei [spe-8(hc50) I [fog-1(q785) I; ced-1::gfp(bcIs39) V (obtained by a cross of MD701 [ced-3(n717) IV; fog-2(q71) V (obtained by a cross of MT1522 [rpa-1::yfp(opIs263) [hus-1(op241) I [Strains used were Bristol N2, JK3743: (q785) I , JK1078:2q71) V , XM1012:2(q71) V , EM464:  remanei , BA784: (hc50) I , CNQ41:  V , XM10opIs263)  and WS22op241) I .E. coli HB101 as a food source. Worms were staged by picking at the L4 stage as identified by visual inspection of vulva shape. Unless otherwise specified, 50 worms were kept per 60 mm plate prior to mating. Mating at “day 0” of adulthood refers to exposure to males for 24 h starting from L4—so that females are exposed to males at the earliest time at which they are sexually mature. More generally, mating at day n of adulthood refers to exposure to males from L4 + n * 24 h to L4 + (n+1) * 24 h. For mating and cell cycle experiments, a sharp increase in cycling intermittency was noted in wild-type selfed hermaphrodites around day 3 adulthood. Therefore, when using day 3 hermaphrodites, special care was taken to use worms at precisely L4 + 72 h. Female mating was performed on 35 mm-diameter plates , at a density of 1 female and 3 young adult males per plate. For C. remanei matings, females were continuously exposed to males, which were refreshed every three days; without this continuous exposure, female reproductive capacity is not exhausted . For brood size scoring, mothers were passaged every day to 35 mm fresh plates, until the end of reproductive activity. Plates on which embryos had been laid were incubated for ~2 days to let progeny hatch and grow in size; progeny were counted before they reached the adult stage. Mothers that crawled off agar were censored from the analysis.Strains were maintained as described  using E.To analyze the early response to mating in fine detail, females isolated 3 days after L4 were each plated with 3 males and any progeny removed from the plate every ~3 h for a total of 21 h. After another 24 h, transferred progeny were scored for viability.For cell cycle inhibition experiments, hydroxyurea (HU) or the CDK inhibitor Roscotivine  were freshly prepared and added to NGM-agar (cooled to 55°C prior to dispensing) at a final concentration of 40 mM for HU  or 50 μMFor starvation experiments, females were picked at the late L4 stage, rinsed 5 times in M9, and starved in complete S-medium . FollowiFor the thermotolerance assay, worms were transferred to HU or control plates at day 1 of adulthood, kept on these plates for 24 h, returned to regular plates, and shifted to 35°C. Every 2 h until all worms had died, plates were removed individually from the incubator and the number of live and dead worms recorded.For the lifespan assay, worms were initially treated as for the thermotolerance assay but without temperature upshift. A count was made of live and dead worms every day until all worms had died. Worms that had desiccated on the side of plates or died due to an exploding vulva were censored from the time of death.8 (low), 109 (medium) or 1010 (high) cells / mL in 24-well tissue culture plates rocked on a nutator. At day 2 of adulthood worms were pulsed for 1 h with EdU resuspended in water and delivered at a final concentration of 0.4 mM and processed as detailed below.Worms were kept on NGM plates until day 1 of adulthood, at which point they were transferred to 500 μl S-medium containing serial dilutions of HB101 at final concentrations of 10E. coli. To prepare labeled E. coli, strain MG1693 was grown in minimal medium supplemented with glucose [To label cells with the thymidine analogue EdU , worms were fed EdU-labeled  glucose  and 75 μ glucose  using 0.EdU continuous labeling image data were assayed manually for the presence of at least one EdU-positive cell in individual mitotic zones. EdU pulse chase data were analyzed as described in For whole germ line imaging, for counts of apoptotic cells detected with the CED-1::GFP reporter or foci detected with the RPA-1::YFP reporter, and for counts of cells in diplotene or diakinesis extruded germ lines were fixed and stained with DAPI, 0.16 μM Alexa 594-conjugated Phalloidin , and 1:1000 anti-GFP . Image stacks were acquired at 0.3–0.6 μm z intervals using a 63x objective. In some instances, several panels were imaged for each gonadal arm that were subsequently stitched .RPA-1 foci were analyzed in the proximal meiotic region . The daThe Wilcoxon rank sum test as implemented by the R project or Matlab was used to test for significance of differences (two-tailed test), unless otherwise stated. A generalization to interval-censored data of the Wilcoxon test implemented by the “interval” R package  was usedS1 Figinx-22; fog-2 and spe-8. For numbers and statistical tests see Remaining reproductive capacity when mated at day 2 of adulthood as a function of capacity when mated at day 0. There is a significant main effect of genotype on reproductive capacity between all genotype pairs except (TIF)Click here for additional data file.S2 FigFor numbers and statistical tests see (TIF)Click here for additional data file.S3 Fig(A–B) HU-treated females have reduced survival in both lifespan (A) and thermotolerance (B) assays. Asterisks indicate significance of p-value computed by applying a log-rank test to the survival curves. For numbers and statistical tests see (TIF)Click here for additional data file.S4 Figfog-2 females after 1 h exposure (n = 30). (B) Older, wild-type hermaphrodites display stochastic cycling with dynamics close to those of C. elegans females. This is rescued upon mating. Graph shows fractions of mitotic zones remaining unlabeled as a function of time on EdU-labeled food (n = 40–55 for each time point). (C—D) C. remanei females undergo reproductive senescence and stochastic cycling similar to C. elegans females. (C) The brood size of C. remanei females mated at days 0, 3, and 7 of adulthood (n = 15–20 for each time point). Error bars represent 83% confidence intervals; asterisks indicate significance of Wilcoxon rank sum test p-value. (D) Fractions of mitotic zones remaining unlabeled as a function of time on EdU-labeled food for virgin or mated C. remanei (n = 40–50 for each time point). (E) Oocyte laying counts for fog-1 or fog-2 females from day 0 to day 6 of adulthood. fog-2 lays significantly more oocytes than fog-1 .(A) Worms ingest food irrespective of reproductive activity. Overlay of transmitted light and fluorescence signal (green), showing bead ingestion by (TIF)Click here for additional data file.S5 Fig(A–C) Overview of image processing for cell cycle analysis (reproduced in part from ). (A) Se(TIF)Click here for additional data file.S6 Figfog-1 and inx-22; fog-2 at times after mating corresponding to the trough in fog-1 progeny production  Number of cells in diplotene (red), and number of cells in diakinesis (green) for (TIF)Click here for additional data file.S1 TableAssociated with (PDF)Click here for additional data file.S2 TableAssociated with (PDF)Click here for additional data file.S3 TableAssociated with (PDF)Click here for additional data file.S4 TableAssociated with (PDF)Click here for additional data file.S5 TableAssociated with (PDF)Click here for additional data file.S6 TableAssociated with (PDF)Click here for additional data file.S7 TableAssociated with (PDF)Click here for additional data file.S8 TableAssociated with (PDF)Click here for additional data file.S1 DatasetNumbers displayed on top of histogram are formatted as “gonad_ID (estimated fraction of cell cycle completion)”.(PDF)Click here for additional data file.S1 MovieEach set of histograms shows DNA content of EdU-positive cells (blue) and EdU-negative cells (red). The movie consists of two repeats of a whole cell cycle period, and shows how DNA content histograms cycle as cells increase in DNA content as a result of S phase, and return to low DNA contents after division.(MOV)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Luminal and ER+ (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple-negative  and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER+ and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all- The higher the ATRA score is, the higher is ATRA sensitivity. Development of this new index was necessary, since determination of standard IC50 values for the definition of sensitivity to the anti-proliferative effect of ATRA was deemed inadequate for at least two reasons. The IC50 is routinely and successfully used to assess cell sensitivity to cytotoxic compounds, while ATRA is predominantly a growth inhibitory and cyto-differentiating agent and it is largely devoid of a direct cytotoxic action  and ER+ (3/14) cell lines is reduced. Group D concentrates ATRA-resistant lines, the majority of which is Basal (10/12). Thus, the ATRA scores indicate that a Luminal phenotype and ER expression are major determinants of cell sensitivity to the anti-proliferative action of ATRA. In contrast, a Basal phenotype represents a negative factor. Indeed, the proportion of Basal cell lines increases as the ATRA score decreases if our panel is divided in tertiles   Fig.Basal lines with a high ATRA score and one of the rare breast cancer lines transplantable in mice  or vehicle on a daily basis for 3 weeks, and tumor growth was followed. A time- and dose-dependent reduction in the tumor volume is evident in mice treated with ATRA  tumors. All the Luminal-A and Luminal-B tumors are characterized by > 70% ER+ cells. ATRA reduces the proliferation of 11 Luminal-A and 10 Luminal-B tumors  , the data confirm that RARA co-amplification results in high levels of RARα  than T3 (ATRA resistant) cell lines . Thus, RARα is likely to be a determinant of ATRA sensitivity in both the total and Luminal fraction of cell lines, while PPARβ/δ may represent a positive factor in Basal cell lines.After grouping all the cell lines in ascending tertiles (T3 to T1) according to the lines Fig. No difflines Fig, RARβ, RARβ/δ Fig than theLuminal cells contain significantly larger amounts of RARα3, RARα1, and RARα4 mRNAs than the Basal counterparts , RARβ (RARβ1-2-5), and RARγ (RARγ1-5) splicing variants are known . Quantitative PCR was used to determine basal expression of these variants in our panel of cell lines. In the majority (40/42) of the cell lines , RARα3 is the most highly expressed RARα mRNA, being at least one order of magnitude more abundant than RARα2 and RARα1. Extremely low levels of RARα4 are generally observed. Only RARα3 and RARα4 show significant co-regulation across the panel , consistent with transcriptional control by the same promoter. -4, RARβ ident Fig. A similBasal relative to Luminal cells. The same is true in ER− versus ER+ cell lines, even if the analysis is restricted to the Luminal/ER− group . Regarding possible associations with ATRA sensitivity, no significant difference in the levels of RARβ1 or RARβ2/5 between the T1 and T3 cell lines is evident before or after stratification for the Luminal/Basal phenotype . Despite regulation by distinct promoters, RARβ1 and RARβ2/5 are always co-expressed . Both RARβ1 and RARβ2/5 are over-expressed in Basal and ER− phenotypes , while only RARγ3 is significantly higher in HER2− than HER2+ cells. In addition, RARγ2 and RARγ3 tend to be over-expressed in the T3 relative to the T1 group . Consistent with regulation by the same promoter, only the RARγ2/RARγ3 couple is characterized by co-regulation across all the cell lines . As the RARβ variants, RARγ2 and RARγ3 show a direct association with the group Fig, supportLuminal cells to ATRA.Taken together, the results point to RARα3 as the principal element of the retinoid pathway mediating the anti-proliferative responses of The profiles of expression of the RAR splicing variants were defined in the primary tumors used to evaluate ATRA sensitivity see Fig. In all Luminal, relative to TN cancers and HER2+ tumors with no co-amplification of the ERBB2 and RARA loci  than in all the other tumor sub-types . In HCC-1428 and SKBR3 cells, only ER50891 blocks the anti-proliferative action of ATRA .To corroborate the results obtained with the RAR agonists, we evaluated the effects of the RARα antagonist, ER50891 , two vector-transfected control (Vect-C1 and Vect-C2) clones, and the parental MDA-MB453 cells (WT) were used in comparative experiments. WT, Vect-C1, and Vect-C2 express barely detectable levels of the RARα protein, while large amounts of the product are synthesized by RARA-C5 and RARA-C7 cells  and void vector-transfected Vect-C6 or Vect-C8 cells . In contrast, ATRA induces the expression of the first three transcripts in RARA-C5 cells. Similar differential effects are observed if the levels of the two retinoid-dependent epithelial differentiation markers, β-catenin and SMAD3 . Conversely, ATRA-dependent induction of the CYP26A1, CYP26B1, BTG2, and RARRES3 mRNAs as well as the β-catenin and SMAD3 proteins observed in Vect-C8 cells is blocked in RARA-Sh18 cells. Thus, RARα is the predominant mediator not only of the anti-proliferative, but also of the transcriptional effects afforded by ATRA in the two breast cancer cells.We evaluated whether modulation of RARα has any effect on ATRA-dependent expression of four direct retinoid target genes. ATRA-dependent induction of CYP26A1, CYP26B1, BTG2, and RARRES3 is not observed in et al, MDA-MB453-derived Vect-C1 cells are unresponsive to the anti-motility action of ATRA . The goal was the generation of ranked lists of genes associated with ATRA sensitivity based on the variable importance scores , it must be noticed that they do not simply stratify the cell lines according to the Luminal or Basal phenotype. To validate the expression results with an independent assay, we performed quantitative real-time PCR on 14 selected genes. The PCR, microarray, and RNA-seq results are concordant .We generated two distinct expression heat-maps of the top 100 RNA-seq Fig, left anCXXC5 is a retinoid-inducible gene  stimulates growth, and it is a biomarker for mammary tumor metastatic spread  knockout macrophages, RARα expression is higher than in the parental counterparts . Some of the 100 top-ranking genes present in both lists may be of interest for the anti-tumor action of ATRA. For instance, Luminal tumors, which are generally responsive to the anti-proliferative action of ATRA, are enriched for elements present in both gene sets  .The results obtained in the cell lines and the short-term cultures of primary tumors support the concept that r see Fig. The cohLuminal/ER+ tumors, but also of TN cancers . A total of 1,702 genes are significantly regulated by ATRA  in either TN or Luminal/ER+ tumors. Approximately 20% of the genes  are similarly modulated by ATRA in both the Luminal/ER+ and TN cases  in tissue slices derived from either Luminal/ER+ or TN tumors , TGM2 (transglutaminase 2), S100A8 , and CYP26A1 (cytochrome P-450 26A1) is significantly higher in Luminal/ER+ tumors. In contrast, THBD (thrombomodulin) is significantly up-regulated only in TN tumors. RARRES3 up-regulation in Luminal/ER+ may play a role in the anti-motility and anti-metastatic effects of ATRA , an important determinant of the sensitivity of breast cancer cells to PI3K inhibitors  with the gene-expression signatures determined for PI3K-inhibitors , which we will conduct in post-menopausal patients suffering from ER+ breast cancer aimed at evaluating the efficacy of ATRA addition to aromatase inhibitors.In conclusion, this work is a first step toward a rational use of ATRA and derived retinoids in breast cancer. The data obtained with both the cell lines and the short-term tissue cultures indicate that ∽70% of https://www.sigmaaldrich.com), AM580 , BMS961 (Tocris), ER50891 (Tocris), CD2665 (Tocris), and UVI2003 . Sulforhodamine was from Sigma-Aldrich Co. A list of the cell lines, their characteristics, and origin is available in Supplementary Table MDA-MB453 cells and knockdown in SKBR3 cells are described below.The following compounds were used: ATRA  was introduced into the pGreenPuro plasmid , using the EcoRI and BamHI sites in the multiple cloning region downstream of the H1 gene promoter. The RARα over-expressing MDA-MB453 clones and the RARA-silenced SKBR3 clones were selected, after transfection with Fugene HD , in the presence of 400 μg/ml G418 and 1 μg/ml puromycin (Sigma-Aldrich), respectively.To generate the RARα plasmid used for the over-expression in et al, Tru-cut procedure were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h in Mammary Epithelial Cell Growth Medium . At the end of the treatment, samples were fixed, paraffin-included, and dissected into 5-μm slices, which were subjected to immuno-histochemical staining with an antibody targeting the Ki67 proliferation-associated marker. The percentage of Ki67-positive tumor cells in the various samples was assessed in a quantitative manner by automatic image analysis, and the results are illustrated. Scoring of Ki67 was blinded as to treatment. Each value represents the mean ± SE of at least five separate fields for each experimental sample. The fresh primary tumor samples used for the short-term tissue slice cultures aimed at assessing ATRA sensitivity were supplied by Fondazione S. Maugeri, Pavia. All the procedures were approved by the internal ethical committee of the Fondazione S. Maugeri, and an informed consent for the donation of the sample was obtained from patients.Tissue cultures of primary breast tumors were performed as described  for 3, 6, and 9 day, and cell growth was determined with sulforhodamine assays (Skehan trans retinoic acid (ATRA) is the primary vitamin A metabolite and a promising agent in the treatment/chemoprevention of breast cancer. Breast cancer is very heterogeneous, being a collection of different diseases. A rational use of ATRA in breast cancer requires the definition of the sensitive subtypes and the molecular determinants underlying retinoid sensitivity.All-ATRA score) defining ATRA sensitivity in a quantitative manner. Luminal and ER+ (estrogen-receptor-positive) cell lines are generally sensitive to ATRA. In contrast, refractoriness or low sensitivity is associated with a Basal phenotype and HER2 positivity. The associations between cellular phenotype and ATRA sensitivity are confirmed using short-term tissue-slice cultures of primary tumors, which also result in the definition of the modifications in the transcriptome afforded by ATRA in Luminal and Basal tumors. Using a retinoid-pathway-oriented approach, RARα is identified as the only member of the retinoid pathway directly associated with the Luminal phenotype, estrogen positivity, and ATRA sensitivity. Studies in selected Luminal and Basal cell lines with RAR-specific agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. In addition, RARα over-expression sensitizes ATRA-resistant cells to the retinoid. In contrast, RARα silencing in ATRA-sensitive cells abrogates the activity of the retinoid. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα mediates also ATRA anti-metastatic effects. Whole-genome gene-expression data allow the definition of two overlapping gene sets expressed in basal conditions and characterized by predictive potential and associated with ATRA sensitivity in breast cancer cell lines and tumors.We examined ATRA sensitivity in a large panel of breast cancer cell lines, recapitulating the heterogeneity of the disease after development of a new parameter  were injected subcutaneously on both flanks of female 6-week-old SCID mice weighing ∽18 g . All the experiments were performed following approval of the internal Ethical Committee on Animal Experimentation and were conducted in compliance with the Italian legislation. Tumor volume was determined with a caliper and by magnetic resonance imaging .et al, et al, et al, et al, Real-time PCR was performed using Taqman assays (Terao Tissue slices were incubated with vehicle (DMSO) or ATRA (0.1 μM) for 48 h. Total RNA was extracted with the miRNeasy Mini kit (QIAGEN), labeled with the Lowinput Quick Amp labeling kit , and hybridized to whole-genome gene expression microarrays (Agilent). Fluorescent signals were determined and quantified with an Agilent microarray laser scanner. The microarray raw data and experimental protocols were deposited in the Arrayexpress database (accession No. E-MTAB-3313).http://www.broadinstitute.org/ccle). RAW sequencing data (Illumina-paired end reads) were derived from two distinct datasets. The first dataset is publicly available in the CCLE project and. BAM files where downloaded through the cgdownload utility from the Cancer Genomics Hub . Sequencing data (.FASTQ files) for those cell lines that were not part of this first set were downloaded from a second GenBank dataset under the accession GSE48216 (GenBank). The heat-maps were generated using the algorithms available in T-Mev (http://www.tm4.org). Further details on the bioinformatic analyses performed on all the gene-expression data are available in the appropriate sections of the Supplementary Information.Gene expression data for the cell lines were derived from the Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (provided by CCLE,


Human breast cancers are a highly heterogeneous group of tumours consisting of several molecular subtypes with a variable profile of hormone, growth factor receptors and cytokeratins  Value of the data•This data shows that the expression pattern of critical growth modulating receptors is dynamic and hence researchers need to confirm the status over progressive passages.•This data provides evidence to the scientific community that gene expression data from cell lines needs to be cross verified with protein detection.•The data highlights the importance of noting the passage number of breast cancer cell lines used for experimental testing of pharmacological agents.1Immunofluorescence was performed for ER, HER-2, AR and EGFR on 4 different cell lines – MCF7; luminal cell line, MDA-MB-231; triple negative cell line, BT-474 and MDA-MB-453; HER-2 amplified cell lines. The data presented includes immunofluorescence images at an early passage (5/8th), an intermediate passage (15/16th) and a late passage (24th to 26th) for the four cell lines.22.12 humidified atmosphere. MDA-MB-231 was cultivated in L-15 (Lebovitz medium) supplemented with 10% FBS and incubated at 37 °C without CO2 humidified atmosphere The cell line MCF7 was cultivated in DMEM-Hi glucose supplemented with 10% FBS. BT-474 and MDA-MB- 453 were grown in RPMI 1640 media. All three cell lines were incubated at 37 °C and 5% CO2.2Mycoplasma testing was done by seeding the cells on glass coverslips and after 24 h, stained with Hoechst 33342 (0.1 mg/ml in PBS) for 10 min. Microscopic analysis was done to detect the presence of mycoplasma. All cell lines were routinely tested for mycoplasma and were found to be negative.2.3Immunofluorescence was done as described previously


Mesotheliomas are aggressive, therapy-resistant tumors that are predicted to increase in incidence at least until 2020. The prognosis of patients with mesothelioma is generally poor because they are typically diagnosed at a late stage and their tumors are resistant to current conventional therapies. For these reasons, improved diagnosis and therapy are urgently required. To address these issues, the aim of our research was to develop novel mesothelioma-specific monoclonal antibodies (mAbs) as diagnostic and therapeutic agents.To develop anti-mesothelioma mAbs useful for diagnosis and therapy, we repeatedly immunized a BALB/c mouse with viable mesothelioma cells, alternating between those from three mesothelioma cell lines. We hybridized the spleen cells from this immunized mouse with P3U1 myeloma cells. We then screened supernatants harvested from the hybridoma clones by assessing whether they bound to a mesothelioma cell line not used for immunization and altered its morphology. We designed this developmental strategy to reduce the risk of obtaining clonotypic mAbs against a single mesothelioma cell line.Our newly generated mouse anti-human mAbs immunostained clinical samples of mesotheliomas. One of the newly generated mAbs did not react with any other tumor cell line tested. Two other mAbs significantly inhibited the proliferation of mesothelioma cells.These newly generated anti-mesothelioma mAbs are potentially useful as diagnostic and therapeutic agents for mesothelioma. Moreover, our novel strategy for establishing antitumor mAbs may facilitate the development of new diagnostic and therapeutic techniques for mesotheliomas and other malignancies. Mesothelioma is an aggressive tumor that develops from mesothelial cells, which line the pleural, peritoneal, and pericardial cavities. The development of mesothelioma is usually associated with chronic exposure to asbestos fibers , 2. The Immunohistochemistry has significantly enhanced the accuracy of cytology, although reaching a diagnosis remains frequently difficult –10. AlthMesothelioma is categorized histologically as epithelioid, sarcomatoid, biphasic, and desmoplastic, among others . The hisAlthough an anti-CD26 mAb is a promising candidate, no effective molecular target for therapy exists , 18. TheDiagnostic or therapeutic mAbs are now typically generated by immunizing mice with synthetic peptides or target antigens that are purified to some extent , 24. ExcFemale BALB/c mice (6–8 weeks of age) were purchased from Charles River Japan  and housed in a specific pathogen-free facility in micro-isolator cages. Animal experiments were conducted following protocols approved by the Animal Care Committee of Juntendo University of Medicine.The human mesothelioma cell lines ACC-MESO-1 and ACC-MESO-4 were purchased from the RIKEN Cell Bank , JMN was a kind gift from Dr. Brenda Gerwin and others  were purchased from the American Type Culture Collection (ATCC) . The lung cancer cell lines , and the HuH-7 (hepatocellular carcinoma), MKN1 (gastric cancer), OVK18 (ovarian cancer), VMRC-RCW  cell lines were purchased from the RIKEN Cell Bank. The MCF7 (breast cancer), KP3 (pancreatic cancer), and HCT 116 (colon cancer) cell lines were purchased from ATCC. All cells were cultured and maintained in RPMI-1640 medium  supplemented with 10 % heat-inactivated fetal calf serum.® 488-conjugated goat anti-mouse IgG was purchased from Invitrogen (CA). Mouse IgG was purchased from Abcam (Cambridge UK). The mAbs for WT-1 (clone: 6F-H2), Calretinin , Podoplanin (clone: D2-40), Cytokeratin 5/6 (clone: D5/16 B4), EMA (clone: E29), Carcinoembryonic Antigen (CEA) (clone: II-7), TTF-1 (clone: 8G7G3/1), and Epithelial-Related Antigen (clone: Moc-31) were purchased from DAKO. The mAb against Mesothelin (clone: 22A31) and anti-GLUT-1 polyclonal Ab were purchased from IBL (Japan). Anti-CD26 mAbs  were established in our laboratory -TdR, for the final 24 h. The incorporation of [3H]-TdR was determined using scintillation counting. Data are expressed as the mean ± standard deviation (SD) of triplicate samples and represent three separate experiments.MSTO-211H cells (1 × 10Mesothelioma NCI-H226 cells were seeded in 6-well plates (Corning) and grown to 90 % confluence in RPMI plus 10 % FCS. The cell monolayer was wounded with the tip of an Eppendorf P200 pipette. After wounding, the wells were washed with media to remove dead cells and debris. The wells were treated with either 3 µg/ml of anti-mesothelioma mAbs (JMAM1–4) or a control IgG (3 µg/ml) and cultured further. The wound closure was observed after 24 h.4 NCI-H226 cells in serum-free RPMI-1640 medium was added to the upper chamber and cultured at 37 °C. After 15 h of incubation, the cells on the upper surface of the filter membrane that had not migrated were gently scraped away with a cotton swab. The invading cells on the lower surface of the filter membrane were fixed with methanol, stained with Diff-Quick™ (Sysmex), and counted as described above. All tests were performed in triplicate.For the cell invasion test, a Corning Matrigel Invasion Chamber  was placed into the wells of 24-well culture plates; RPMI-1640 medium with 10 % serum and JMAM1–4 mAbs (each 10 μM) were added into the lower chamber; then, 2 × 10t test. The results are expressed as the mean ± SD and P values of <0.05 were considered significant. Statistical analyses were performed using SPSS 14.0 software .The data were analyzed using Student’s We found that the newly generated four mAbs reproducibly induced morphological changes in a mesothelioma cell line that was not used for immunization. Light microscopy revealed that the morphology of the NCI-H2452 cells changed from spindle-shaped to round, and the numbers of these cells decreased after incubation with JMAM1–4 mAbs for 72 h compared with control mouse IgG  and sarcomatous  cell lines. In contrast, JMAM4 stained the epithelial cell lines but not the sarcomatous cell lines , anti-GLUT-1, anti-CD25 (BC96), anti-CD26 , anti-C-ERC/mesothelin (22A31)].  was not detectable. In contrast, it bound to the small-cell lung cancer cell lines WA-hT and Lu24. The extent of binding of JMAM2 and JMAM3 mAbs to lung cancer cell lines varied. The JMAM4 mAb did not bind to any of the lung cancer cell lines , HuH-7 (liver cancer), KP3 (pancreatic cancer), MKN-1 (gastric cancer), HCT 116 (colon cancer), OVK18 (ovarian cancer), and VMRC-RCW  cell lines. The JMAM1 mAb only cross reacted with the VMRC-RCW cell line. JMAM4 mAbs did not react detectably with any of these carcinoma cell lines. The JMAM2 mAb slightly or significantly stained all carcinoma cell lines tested. The JMAM3 mAb did not stain the liver or pancreatic cancer cell lines; however, it lightly stained a gastric cancer cell line and strongly stained breast and colon cancer cell lines . The antigens recognized by the mAbs might have been masked or antigenically inactivated by formalin fixation.Body fluid retention is one of the symptoms of many patients with malignant mesothelioma who are often diagnosed with mesothelioma by body fluid cytological examination. Therefore, we investigated whether the mAbs reacted with cytological samples using body fluid specimens that were fixed with ethanol. We analyzed specimens from three patients with malignant mesothelioma and those from patients suspected to have malignant mesothelioma Fig. . All matCase 1 Many cell clusters were present, including tight and loose clusters with flattened cellular borders. Individual cells showed wide variation in shape and size, ranging from small to very large. The JMAM1–4 mAbs stained membranes, whereas the JMAM2 mAb stained the cytoplasm and the membrane.Case 2 The few malignant cells present were judged Class III by Papanicolaou classification. All mAbs stained membranes clearly.Case 3 Present were small to large clusters with knobby borders and a single-cell population. These cells had low nuclear: cytoplasmic ratios, but occasionally showed macronucleoli. The staining of membranes by mAbs JMAM1–4 was distinct. Antibodies against Podoplanin, Mesothelin, EMA, and GLUT-1 stained cell-block specimens, which confirmed these atypical cells as derived from mesothelioma (Table Reactive normal pleura and lung immunoreactive features are shown in Fig. We next tested whether the mAbs inhibited the proliferation of the MSTO-211H mesothelioma cell line Fig. . The JMACell motility is an essential process of tumor metastasis and progression. Therefore, we investigated whether the mAbs would affect the migration of mesothelioma cells using a wound-healing assay. The anti-mesothelioma mAbs JMAM1, JMAM2, and JMAM3 significantly inhibited the ability of NCI-H226 cells to migrate to and close an experimentally induced wound Fig. . These rWe assessed the cell invasion ability of mesothelioma NCI-H226 cells. Significant inhibition of invasion was observed by JMAM mAbs compared with control mouse IgG1 Ab Fig. . JMAM1 mDifferentiating between a mesothelioma and a papillary adenocarcinoma is sometimes very difficult . CliniciIt is now commonly accepted that therapeutic mAbs dramatically improve the treatment of cancer patients. However, after repeated therapy, not all patients respond to therapeutic mAbs  because Therefore, in this study, we investigated reactivity with mesothelioma cells as well as the direct antitumor effect of the newly generated anti-mesothelioma mAbs. We found that JMAM1 and JMAM3 mAbs inhibited the proliferation of the MSTO-211H mesothelial cell line, and JMAM1–4 mAbs inhibited wound closure by NCI-H226 cells to varying degrees. JMAM1–4 also inhibited invasion of NCI-H226 cells to various degrees. Unfortunately, we did not identify the target molecules of JMAM1–4 mAbs. Nevertheless, the promising anti-mesothelioma activities of these antibodies warrant continued studies in vitro and in vivo that will include efforts to identify their targets. Our strategy for generating diagnostic and therapeutic mAbs specific for tumor cells differs from those of conventional methods that employ immunization with peptides or DNA. Specifically, we immunized a mouse with three different mesothelioma cell lines and screened the antibodies using a different mesothelioma cell line to potentially obtain novel mAbs that react with unknown targets on the surface of mesothelioma cells.Newly established anti-mesothelioma mAbs, JMAM1–4, are potentially useful as diagnostic and therapeutic agents for mesothelioma. Moreover, our novel strategy for establishing anti-tumor mAbs may facilitate the development of new diagnostic and therapeutic techniques for other malignancies as well as mesothelioma.