TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors  or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic  Ovarian cancer is the fifth cause of cancer-related deaths in the Western world, the second most common gynecological cancer and the leading cause of death from gynecological malignancies . The mosTP53 tumor suppressor gene is one of earliest mutational events in HGS as nearly all cases harbor a somatic mutation in this gene as confirmed by a recent molecular genetic profiling of over 300 HGS samples by The Cancer Genome Atlas Research (TCGA) network [TP53 mutations and BRCA1/BRCA2 deficiency likely leads to incompetent DNA repair which in turn contributes to chromosomal instability, resulting in severely aberrant karyotypes. In general, HGS EOCs are highly heterogeneous having widespread inter- and intra-tumoral mutation profiles, although common patterns of chromosomal regions of gain and loss have been observed [As a group HGS tumors account for over 60% of all EOCs and are characterized by high genomic instability and chromosomal anomalies with cells exhibiting complex karyotypes, which include intrachromosomal breaks and aneuploidy , 11. It  network , 13. Fur network , or thro network , 14, 15,observed , 16-18.BRCA1 or BRCA2 mutations have been described [The study of processes or pathways relevant to HGS EOC biology, treatment or etiology has been facilitated by cell-based models -22. Howeescribed , 25, 26.escribed , and thein vivo and in vitro growth characteristics of six new EOC cell lines all derived from HGS cases. Two of these cell lines were derived from patients harboring a germline BRCA1 or BRCA2 mutation. The molecular genetic characterizations of these cell lines suggest that they exhibit features consistent with HGS tumors. Their unique profiles, which include different mechanisms of inactivation of TP53 and BRCA1, reflect the heterogeneity observed in HGS tumors. Therefore, these new cell lines offer a significant contribution to the list of HGS EOC cell lines available for research as they present distinct features characteristic of this multiple-faceted disease.Here we have described the generation, molecular characterization, and The clinical characteristics of the patients 866, 2978, 3041, 3291, 4453, and 4485 from which the cell lines were derived are shown in Table The cell lines were derived from samples collected at diagnosis or at the time of relapse, from either solid tissue (TOV) or ascites (OV) Table . The TOVAfter 50 passages, the cell lines appeared homogeneous in their morphology, exhibiting a cobblestone appearance characteristic of epithelial cells Figure . NotableBRCA1 and BRCA2 confer susceptibility to breast and ovarian cancers. BRCA2 and BRCA1 germline mutations were identified in patients 4453 and 4485, respectively, as a consequence of genetic testing in hereditary cancer clinics due to their breast and ovarian cancer family history (see BRCA2:G6085T (c.5857G>T) mutation was identified in a screen of the most common BRCA1 and BRCA2 mutations found to recur in French Canadians due to common ancestry [BRCA2 mutation was found in the homozygous state in OV4453 cell line  mutation. This mutation was also found in OV4485 cell line (Table BRCA1:IVS14-1G>T mutation is unknown. This unique variant has not been reported in either of the Breast Information Core mutation database (research.nhgri.nih.gov/bic/) or the LOVD-Leiden Open Variation Database (chromium.liacs.nl/LOVD2/cancer/home.php?select_db=BRCA1), nor the Exome Aggregation Consortium (ExAC) database (exac.broadinstitute.org). However, an investigation of this mutation in the OV4485 cell line suggests that it probably results in the inactivation of BRCA1 protein function . However, it is not known if these predicted transcripts are translated into truncated proteins or are eliminated by other mechanisms such as nonsense-mediated decay. In the OV4485 cell line, no evident BRCA1 protein band was detected  cell line was derived. These results are consistent with our findings from whole exome sequencing analysis, which also revealed no further evidence for BRCA1/BRCA2 mutations in these cell lines , and the two splice TP53 mutations are predicted to affect the translation of the encoded protein [KRAS and BRAF mutations affecting common mutation sites in these genes were identified in the cell lines.In our initial study of the EOC cell lines, we performed a targeted mutation analysis of selected genes as C tumors , 32-34.  protein . Using aTP53 locus at 17p13, the BRCA1 locus at 17q12, and the BRCA2 locus at 13q13.SNP array genotyping analysis of each EOC cell line was performed to infer genome-wide chromosomal abnormalities as HGS tumors and cell lines exhibit a high frequency of chromosomal instability as displayed by copy number anomalies (CNA) across the genome , 23, 35.MYC, MECOM, CCNE1, KRAS, ALG8, SOX17, and TACC3, as regions containing these genes have been shown to exhibit amplification (in order of descending frequency) and for the NFI, RB1 and PTEN loci as regions containing these genes have been shown to exhibit homozygous deletions (in order of descending frequency) in HGS tumors [MYC and MECOM loci were observed in all of the EOC cell lines (Table KRAS and CCNE1 loci in the OV866(2) cell line akin to classical gene amplification . Although this amino acid alteration is predicted to be tolerated by SIFT (http://sift.jcvi.org), it is predicted to be probably damaging by PolyPhen-2 (genetics.bwh.harvard.edu/pph2/), based on computational tools that assess the functional effects of amino acid substitutions. The amino acid substitution occurs in a highly conserved region of the gene based on Genomic Evolutionary Rate Profiling (GERP) for mammalian alignments (genome.ucsc.edu). Finally, we also reviewed PIK3CA, CTNNB1, and ARID1A, as well as KRAS and BRAF, for somatic mutations in these genes have been reported to occur in EOC subtypes and corresponding cell lines most often representing other non-HGS subtype cancers [We have performed whole exome sequencing analysis to evaluate the presence of somatic anomalies in  cancers . None ofIn order to investigate the epithelial origin of the tumors and corresponding cell lines, cytokeratin expression was investigated by immunohistochemistry (IHC) and Western blot (WB) analyses, respectively. IHC was not performed on tissue samples from patient 4453 since no tumor cells were found in a review of the histology. In this case, surgery was performed during the chemotherapy treatment cycles. It is possible that the tumor mass had regressed having already responded to therapy by the time of surgery. HGS diagnosis of this patient was determined from a pre-chemotherapy omentum biopsy. For the available tumor samples, HGS histopathology was confirmed by hematoxylin-eosin staining and all of the cytokeratins investigated  were present in the tissue sections Figure . CytokerTo further characterize the presently described cell lines, expression of WT1 and PAX8, which are markers specific for the classification of the HGS EOC subtype -39, was Protein expression of the human epidermal growth factor receptor-2 gene, HER2, which has been implicated in malignant transformation, was also used to characterize the cell lines, as overexpression has been reported in 10-20% of serous EOC tumors , 41. HERTP53 mutated HGS samples, where missense alleles expressed stable mutated protein in contrast to other mutation types which resulted in no detectable protein  [TP53 mutations (splice) of unknown function (based on TP53 IARC Mutation database). While, p53 mRNA and protein are expressed in the OV4453 cell line (TP53:c.376-1 G>A mutation), no mRNA or protein was detectable in lysates from the TOV2978G cell line (TP53:c.920-2 A>G mutation)  . Proteinn) Table . Our reses Table . Our RT-e Figure , which se TOV21G  that alsCDKN1A, PMAIP1 and MDM2, which encode the cyclin-dependent kinase inhibitor 1A (p21), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) and, E3 ubiquitin protein ligase MDM2 proto-oncogene, respectively [TP53 wild-type TOV21G cell line even 2h after gamma-irradiation  of other HGS EOC cell lines [The growth characteristics of the new cell lines were compared to other HGS EOC cell lines previously established in our laboratory , 27. Thell lines , 27. HowThe ability of each cell line to form spheroids was measured using the hanging droplet method as previously described . ObserveWe next measured the ability of the cell lines to grow in an anchorage independent environment by culturing the cells in soft agar. Only the OV866(2) and OV4453 cell lines were able to clearly form colonies in soft agar Figure , Table 3The migration potential of the cell lines was measured using an established scratch migration assay , 27, 48.50 (32 μM) among all the tested cell lines (Table 50 = 6 μM), making it a good model to study new therapeutic strategies for platinum resistant BRCA mutated HGS EOC cases.The carboplatin response of the cell lines was measured by a clonogenic assay. Our results indicate that the OV866(2) cell line was strongly resistant to carboplatin, exhibiting the highest IC50 2 μM amon50 2 μM amonin vivo growth potential of all the cell lines was determined by subcutaneous injection into SCID mice (n = 5 mice for each cell line). Only the HGS cell lines OV4453 and OV4485, which were derived from BRCA2 and BRCA1 mutated cases, respectively, formed tumors at subcutaneous injection sites in all the SCID mice (5/5 mice)  as early as 98 and 138 days for the OV4485 and OV4453 cell lines, respectively, and reached endpoint limits allowable for tumor growth (2500 mm3) at 170-230 and 190-320 days, respectively. Figure 3 in 265 days, and this was observed only in two of five mice  Figure , Table 3e Figure , Table 3We also investigated the capacity of the tumorigenic cell lines, OV866(2), OV4453 and OV4485, to form tumors and produce ascites at intraperitoneal injection sites in SCID mice. The experimental endpoints for these experiments were within ethical limits and included palpable peritoneal tumors, bloated abdomen and stress behavior. The survival curves are shown in Figure TP53 mutations, BRCA1/BRCA2 germline/somatic mutations or silencing of gene expression in approximately 33% of the tumors, and somatic mutations in CSDM3, NF1, CDK12, and RB1 that each occurred at low frequency [PIK3CA, PTEN, KRAS, BRAF, ERBB2, CTNNB1 and ARID1A) that have been reported to occur more often in other EOC subtypes. Few cell lines examined in their study exhibited the molecular genetic features characteristic of HGS EOC samples. In contrast, we have shown that all of our six newly derived cell lines exhibit genetic and genomic features characteristic of HGS samples, suggesting that they have a high probability of being of HGS EOC tumor origin. Namely, that they have high copy number alterations and/or harbor TP53 mutations. Some of the cell lines were derived from patients with germline BRCA1/BRCA2 mutations. Moreover, some of these cell lines had somatic mutations in CSDM3, RB1, and CDK12, genes that have been shown to exhibit a low but recurrent mutation frequency in HGS EOC tumors. Although we have shown that the molecular genetic spectrum of each of our EOC cell lines is unique, collectively they reflect the heterogeneity of HGS EOC disease. These novel HGS EOC cell lines provide novel research tools for investigating the most common and lethal form of EOC.The genomic instability and aberrant karyotype of HGS EOC tumors has been well described and is distinct from that observed in the other EOC histotypes, which often exhibit a more stable genome , 50. Therequency . As critBRCA1/BRCA2 mutation harboring cell lines (OV4485 and OV4453) also each have a somatic TP53 mutation, and they were the only cell lines that exhibited robust tumorigenic potential in SCID mice. These genetic features makes them attractive models to study novel therapeutics targeting HGS EOC patients harboring BRCA1/BRCA2 mutations, such as the inhibitors of poly (ADP-ribose) polymerase, a class of drugs currently in phase II/III clinical trials in EOC [BRCA-mutated platinum-sensitive HGS EOC [BRCA1 mutation located at the intron/exon boundary of exon 15 (IVS14-1 G>T). The establishment of the OV4485 cell line made it easier to characterize this mutation and we showed that it induced alternative splicing as well as instability of the BRCA1 mRNA  found in the OV4453 patient and corresponding cell line is a pathogenic germline mutation that has been found to recur in the French Canadian population [BRCA1/BRCA2 mutations from EOC samples had been described in the literature [BRCA mutated HGS EOC cell lines were reported [BRCA2 mutated) cell line is tumorigenic in xenografted mice [BRCA1 and BRCA2 mutated cell lines (OV4485 and OV4453 respectively) form tumors in mice. To our knowledge, the OV4485 cell line is the only BRCA1 mutated HGS EOC cell line with in vivo tumorigenic properties in SCID mice. This cell line also showed resistance to carboplatin  cell line was the only line expressing markers of epithelial-mesenchyme-transition (EMT), as reflected by the absence of cytokeratins  and E-cadherin expression, and strong vimentin expression Figure . This ces Figure . ResearcTP53 mutation was not detected by our mutation screening. However, we have demonstrated the absence of both p53 mRNA and protein as well as the impaired p53 function in this cell line. Therefore, although TP53 mutation analysis suggests that it harbors a wild-type allele, this cell line has features characteristic of a p53-null mutation. The p53 protein functions as a transcriptional factor with a crucial role in cellular stress response . Owiiewed in , 67). Fuunstable -70. Our silenced , 32, 71.BRCA1 mutations. These cell lines would then represent HGS EOC with silenced BRCA gene (possibly by epigenetic mechanisms), which is another very common feature of this disease [BRCA1 is expected given the apparently high frequency of BRCA1 promoter hypermethylation, estimated as 12% of HGS EOC tumors observed by the TCGA study [TP53 mutation, a complex CNA pattern and highest ploidy of the six cell lines, and is the only one to have a CDK12 mutation. Moreover, we have demonstrated that all of our cell lines do not have a functional p53, a feature characteristic of HGS EOCs.Interestingly, the two p53-deficient cell lines (TOV2978G and TOV3041G) also did not express BRCA1 mRNA or protein , Division of Gynecologic Oncology. The study was approved by the Comité d'éthique de la recherche du CHUM, the institutional ethics committee. Stage was determined at time of surgery by a gynecologic oncologist. Histology and tumor grade were determined by a gynecologic-oncology pathologist using criteria consistent with the International Federation of Gynecology and Obstetrics (FIGO) classification .2, and 5% CO2 and grown in complete OSE medium, which includes OSE medium , 10% FBS, 0.5 μg/mL amphotericin B (Wisent) and 50 μg/mL gentamicin . The solid ovarian tumor (TOV)-derived cell lines  were established using the scrape method as previously described [In total, six cell lines were derived from samples from six patients, 866, 2978, 3041, 3291, 4453 and 4485. All cell lines were maintained in a low oxygen condition of 7% Oescribed , 76. Briescribed , 76. The3 dishes (2 × 105 cells per dish). The complete OSE media was replaced every three days for the duration of the experiment. Three times a week from day one to 14, cells were trypsinized, resuspended in media and counted using a hemocytometer. Saturation density was defined as the mean maximum number of cells counted at confluence. Each experiment was performed in triplicate, and repeated twice. Doubling times were determined using a publically available algorithm (www.doubling-time.com).Growth rates were assessed as previously described , 42. BriWB and IHC analyses were performed using the following antibodies: p53 ; HER2/ErbB2/Neu  Ltd., Etobicoke, ON, for IHC); Cytokeratin (CK) 7 ; CK8 ; CK18 ; CK19 , WT1  Ltd.), BRCA1  Ltd.), Vimentin , E-cadherin , PAX8  and beta-actin .In general , 30 micrograms of total protein extracts were electrophoresed in 10% SDS-polyacrylamide gels. Proteins were then transferred onto nitrocellulose membranes using the Trans-Blot Turbo Transfer System  and membranes were blocked with a specific Odyssey® blocking buffer  for immunofluorescence detection. Membranes were then probed with primary antibodies in 1% BSA-Phosphate buffered saline (PBS)-Tween at the following dilutions: 1/10000 for beta-actin, 1/1000 for CK8, 1/2000 for CK7 and CK18, 1/500 for p53, E-cadherin, and vimentin, 1/200 for HER2, 1/5000 for PAX8 and 1/250 for WT1. Primary antibody labeling was detected with an IR-Dye-fluorochrome conjugated secondary antibody and visualized with the LI-COR Odyssey apparatus (Mandel Scientific). Antibody against beta-actin was used as a loading control.For BRCA1, 60 micrograms of total protein extracts were electrophoresed in 4%-15% pre-cast gels (Bio-Rad). Proteins were then transferred onto nitrocellulose membranes at 400mA  using the conventional blotting system. Membranes were then blocked with 5% milk PBS-Tween and probed with primary antibody diluted 1/1000 in 1% BSA PBS-Tween . Antibody labeling was detected with a HRP-conjugated secondary antibody and visualized by the enhanced chemiluminescence (ECL) method.Tissue sections (4μm) were stained with the Benchmark XT automated stainer . Antigen retrieval was obtained using Cell Conditioning #1 or #2  depending on the protein target see . Pre-dilTP53, and the common mutations occurring in either exon 2 of KRAS or exons 11 and 15 of BRAF. Mutation analysis was performed using PCR-based assays followed by bidirectional sequencing using the 3730XL DNA Analyzer system  at the McGill University and Genome Quebec Innovation Center  as previously described [TP53), NM_004985.3 (KRAS) and NM_004333.4 (BRAF), and the genomic structures available from the February 2009 GRCh37/hg19 assembly of the human reference genome. Sequence variants were compared with those reported in the SNP Database (www.ncbi.nlm.nih.gov/SNP). In addition, TP53 variants were evaluated based on information in the International Agency for Research on Cancer (IARC) TP53 Database (www-p53.iarc.fr).Mutation analyses were performed using DNA that was extracted from the cell lines (passages 52 to 108) as described previously . Mutatioescribed . SequencBRCA1 and BRCA2 mutations due to common ancestors [BRCA1 (C4446T and 2953delGTAinsC) and BRCA2  using established PCR-based screening assays [BRCA1/BRCA2 mutations (including the above recurrent mutations) that represent the majority of pathogenic mutations identified in the French Canadians of Quebec as described [Analysis®, a comprehensive mutation analysis for mutations in BRCA1 and BRCA2 (www.myriad.com). All variants were verified by bidirectional Sanger sequencing as described above, and compared with BRCA1 U14680 or BRCA2 U43746 GenBank sequences (www.ncbi.nlm.nih.gov), and the GRCh37/hg19 assembly of the human reference genome. The Human Genome Variation Society (HGVS) (www.hgvs.org/mutnomen/) designation is provided for all mutations investigated and identified in this study. However, for historical reasons, the original BRCA1/BRCA2 mutation nomenclature is referred to throughout in cases where a recurrent mutation was identified. The variants were also investigated in the patient-matched cell line.The ovarian cancer specimens were obtained from French Canadian women, a population known to have specific recurrent ncestors . DNA froescribed , 77. Thrwww.usadellab.org/cms/?page=trimmomatic), BWA  (bio-bwa.sourceforge.net/), GATK  (www.broadinstitute.org/gatk/) and Picard  (broadinstitute.github.io/picard/) software programs. Variant calling was performed using Samtools and Bcftools (www.htslib.org/), and annotated using SnpEff (snpeff.sourceforge.net/) and dbNSFP (sites.google.com/site/jpopgen/dbNSFP). Additional variant filtering included: minimum read depth of 10 reads, minimum allele frequency of 20%, and a minor allele frequency (MAF) of less than 2% in European Americans from the Exome Variant Server (evs.gs.washington.edu/EVS/). The sequencing reads for the variants in candidate genes were also reviewed manually using the Integrative Genomics Viewer (IGV) (www.broadinstitute.org/igv).Libraries for whole exome sequencing were prepared from 500 ng of DNA from the six cell lines using the NimbleGen SeqCap EZ Human Exome Library v3.0 kit , followed by paired-end, 100bp sequencing on the HiSeq 2000  at an equivalent of three samples per lane, according to recommended protocols at the McGill University and Genome Quebec Innovation Centre. Alignment to the human genome (NCBI37/hg19) and variant calling was performed using Trimmomatic (to remove adaptors and trimming reads to minimum phred score of 30) (TP53 or beta-actin amplification) or not (for BRCA1 amplification) was subjected to standard polymerase chain reaction (PCR) using the Phusion High-Fidelity PCR kit , according to the manufacturer's instructions. Two pairs of primers were used for BRCA1 amplification, one covering exons 13 to 15: forward, 5′-Gactcttctgcccttgagga-3′ and reverse, 5′-ctgttgctcctccacatcaa-3′; and another covering exons 14-16: forward, 5′-Ggcctttctgctgacaagtt-3′ and reverse, 5′-aattctggcttctccctgct-3′. Primers for TP53 were: forward, 5′-GGAAGACTCCAGTGGTAATCTA-3′ and reverse, 5′-TTGGGCAGTGCTCGCTTA-3′, which amplify all TP53 variants; and for the beta-actin control gene: forward, 5′-ACTCTTCCAGCCTTCCTTCC-3′ and reverse, 5′-GTACTTGCGCTCAGGAGGAG-3′. PCR products of BRCA1 amplifications were subjected to bidirectional Sanger sequencing , and sequence chromatograms were compared with BRCA1 U14680 GenBank sequence (www.ncbi.nlm.nih.gov), and the GRCh37/hg19 assembly of the human reference genome.Total RNA was extracted from all six cell lines by the TRIzol Reagent  followed by the RNeasy kit . One microgram of total RNA was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen Inc.). One microliter of the reverse-transcribed product either diluted . An image file was created for inferring genomic rearrangements based on the allele frequency (BAF) and copy number (LogR ratio) for each marker assayed. Homozygous deletions were inferred based on Log R ratios less than −2 for at least five adjacently mapped markers. Normalized SNP intensity files were also analyzed by the allele-specific copy number analysis of tumors (ASCAT) algorithm , modified to allow for 100% aberrant cell fraction [Genome-wide chromosomal anomalies, such as chromosomal breaks, CNA and LOH, were inferred in the six cell lines using the HumanOmni2.5Exome BeadChip . This BeadChip assays 2,567,845 markers, with an average spacing of 1.14 Kb per marker (median spacing is 0.57 Kb). Genotyping, using 200 ng of DNA, and scanning were performed at the McGill University and Genome Quebec Innovation Centre. All samples had call rates  within the range of 0.945 and 0.993 (average 0.968). Genotyping analysis was performed using the Genome Viewer module in GenomeStudio software . The software aligns genotyping data for each marker with genomic map coordinates based on the NCBI37/hg19 assembly of the human reference sequence  followed by the RNeasy kit (Qiagen Inc.). One microgram of total RNA was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen Inc.). One microliter of the reverse-transcribed product was diluted (1:10) and subjected to Q-PCR using sequence-specific primers (400 nM) and the SYBR Select Master Mix . Sequence primers for target genes were: p21, forward, 5′-ACCCTAGTTCTACCTCAGGC-3′, reverse, 5′-AAGATCTACTCCCCCATCAT-3′; NOXA, forward, 5′-AGAGCTGGAAGTCGAGTGT-3′, reverse, 5′-GCACCTTCACATTCCTCTC-3′; MDM2, forward, 5′-ATCTTGGCCAGTATATTATG-3′, reverse 5′-GTTCCTGTAGATCATGGTAT-3′; and GAPDH, forward, 5′-GGGAAGGTGAAGGTCGGAGT-3′, reverse, 5′-TTGAGGTCAATGAAGGGGTCA-3′. The Q-PCR was achieved with the Applied BioSystems® Step One Plus apparatus. Thermocycling conditions were one UDG activation cycle at 50°C for 2 min and one AmpliTaq activation plus denaturation cycle at 95°C for 2 min followed by 40 cycles at 95°C for 15sec, 60°C for 1 min and 72°C for 30 sec. Gene expression values were normalized to the GAPDH gene. Two or three independent experiments were performed in duplicate.Cells were seeded on 35 mm2, 5% CO2, with spheroid formation of the cell lines being classified concordantly with previous research [A spheroid assay was performed to determine the ability of cell lines to generate three-dimensional structures in the form of aggregates, as previously described , 79. Briresearch , 47.4 cells in a semi-solid media containing noble agar. Cells suspended in 0.33% w/v agar in complete OSE medium were applied over a base layer comprised of 0.66% w/v agar in complete OSE medium [Cell lines were assayed for their ability to grow in anchorage independent conditions by culturing 2 × 10E medium , 76. CelMigration potential was evaluated using the scratch assay method as previously described , 27, 48.50 values were determined using Graph Pad Prism 5 software . Each individual experiment was performed in triplicate and repeated three times.Carboplatin sensitivity of cell lines was assessed using a clonogenic assay , 80. Bri6 cells resuspended in 100 μl of cold D-PBS  and 100 μl of either Matrigel  for sc. injections or D-PBS for ip. injections. The animals were housed under sterile conditions in a laminar flow environment with ad libitum access to food and water. Tumor formation was assessed twice a week for over 200 days. Animals were sacrificed before neoplastic masses reached limit points established by the CIPA according to the Canadian Council on Animal Care.All animal studies were approved by the Institutional Committee on Animal Protection (CIPA) protocol according to the Canadian Council on Animal Care. The tumorigenic potential of cell lines was assessed based on their ability to form tumors in 6-week old female SCID (severe combined immunodeficiency) mice  at subcutaneous left gluteal injection (sc.) or intraperitoneal sites (ip.). A volume of 200 μl was injected in each mouse and consisted of 5 × 10


Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study. Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor . The stain vitro and in vivo experimentation. These models are utilized to study the basic cellular and molecular biology of tumors and are the basis for preclinical testing of new treatment modalities. Cell lines used for GBM research can be broadly subdivided into two groups depending on their in vitro growth conditions. First are adherent cell lines, which have been used widely in neuro-oncology research. These cell lines grow adherently on culture plates and are generally grown in serum .Oncospheres were triturated into a single-cell suspension and then counted using Trypan Blue and a hemocytometer. Between 5,000 and 15,000 cells/cmIn vivo tumor formation was assayed using 5- to 6-week-old female athymic nude mice  as described previously [5-1x106 cells were injected stereotactically into the striatum of two to four animals. Animals were monitored weekly until development of neurological deficits, at which point they were euthanized or until they passed away from natural causes. Brains were removed, fixed in formalin for at least 24 hours, and stained with hematoxylin and eosin (H&E). Each mouse brain was examined and characterized by board certified neuropathologists (BAO and CGE). In cases from patients with GBM variants, xenografts were also compared to H&E slides from the original patient tumors.eviously . All aniAll 12 patients from whom cell lines were established carried a diagnosis of either a GBM or GBM variant. Clinical data from these patients are summarized in Cell line JHU-0879 was derived from a female patient with a tumor in the left parieto-occipital lobe. The lesion was found to be a GBM with areas of PNET features . After sCell line JHH-66 was derived from a female patient who presented with a GBM in her right occipital lobe. She demonstrated radiographic progression within 1 month of surgery and passed away 3 months after her tumor resection.Cell line JHH-75 was derived from a female patient with a GBM in her left frontal lobe. After resection, the patient received concurrent radiotherapy and temozolomide followed by 6 cycles of adjuvant temozolomide. After progression, the patient received a second resection followed by 1 cycle of BCNU, an alkylating agent. She progressed through BCNU and underwent a third resection. At that point, the patient enrolled in a clinical trial with a PDGF monoclonal antibody, IMC-3G3. After 4 cycles she progressed and underwent a fourth resection with placement of Gliadel wafers, surgically-implanted, BCNU-impregnated, biodegradable wafers. Tumor regrowth led to a fifth resection, at which time MGMT was reported to be methylated, which was followed by concurrent radiotherapy and temozolomide. She subsequently had a sixth resection for progression. The patient received no additional therapies and died 43 months after her diagnosis of GBM.Cell line JHH-136 was derived from a male patient with a GBM in the right frontal lobe. He was lost to follow-up but, according to the Social Security Death Index, died 17 months after his initial surgery.V inhibitor. The patient progressed after his third cycle and was removed from trial. He then received one dose of bevacizumab, an anti-VEGF antibody, and passed away 12 months after surgery.Cell line JHU-0937 was derived from a male patient with a GBM in the left temporal lobe. Following surgery, the patient received adjuvant radiotherapy and temozolomide in addition to going on a clinical trial for veliparib, a PARP inhibitor. He continued to take veliparib during his adjuvant temozolomide for four cycles. After his fourth cycle, the patient progressed and enrolled in a clinical trial with cediranib, a VEGF inhibitor, and cilengitide, an integrin αCell line JHH-211 was derived from a male patient with a tumor in the left frontal lobe. The tumor was diagnosed as an anaplastic astrocytoma by biopsy. Upon resection one month later, the tumor was diagnosed as GBM. Tissue for culture was obtained from the resection. After surgery, the patient received concurrent radiotherapy and temozolomide followed by one cycle of adjuvant temozolomide. The patient died 9 months following resection.Cell line JHH-227 was derived from a male patient with a right periatrial GBM. He received concurrent radiotherapy and temozolomide following surgery. The patient was lost to follow-up 6 months after surgery.Cell line JHH-245 was derived from a male patient with a GBM in his right occipital lobe. He received Gliadel at surgery and afterwards received concurrent radiotherapy and temozolomide. Following this, he received 2 cycles of adjuvant temozolomide before progressing. The patient then underwent an additional resection at which time tissue was removed for a clinical trial involving Vitespen, a heat shock protein-peptide complex-96 based vaccine. After his second surgery the patient was put on bevacizumab in addition to the vaccine treatments. He received this treatment for 4 months before progressing and died 14 months after his initial surgery.Cell line JHU-1014 was derived from a male patient with a recurrent GBM in the left temporal lobe. The initial diagnosis of GBM was made following surgical resection 12 months prior to obtaining tissue. In the interval, the patient received 6 weeks of radiotherapy with concomitant temozolomide and bevacizumab. Following seven cycles of temozolomide and bevacizumab, progression prompted additional surgical intervention. Tissue obtained from this procedure, which was notable for extensive treatment effect on histopathological analysis, was used for cell line establishment. The patient died 5 months later.Cell lines JHU-1016A and JHU-1016B were derived from a male patient with a GBM in his right temporal lobe. The specimen obtained from surgery was split into two pieces, which were used to create two separate cultures. After resection the patient received concurrent radiotherapy and temozolomide. The patient then received adjuvant temozolomide with bevacizumab for 9 cycles before progressing. He then switched to CCNU, an alkylating agent, and bevacizumab for one cycle. He died 18 months after his initial surgery.Cell line JHH-505 was derived from a male patient with a GBM with oligodendroglioma component in his right parietal lobe. Molecular studies reported partial deletions on chromosomes 1p and 19q. The 1p deletion did not include FUBP1 but the region of loss on 19q did include CIC, both oligodendroglioma-implicated genes . The patCell line JHH-520 was derived from a female patient with a right frontal GBM. She received her initial diagnosis from a recent resection at an outside hospital. Tissue was obtained from her second surgery, 9 days later, at which time the diagnosis of GBM was confirmed; MGMT was not methylated. The patient passed away 2 months after surgery.in vivo orthotopic tumor formation . Multiple additional tissue samples resulted in short-term suspension cultures; however, these cultures did not grow past passage four and were not analyzed. Each cell line was prepared using a fixed protocol . We evalormation .To ensure cell line independence, we performed STR profiling . An overAll 13 cell lines demonstrated growth of spheres beyond passage four . We obsein vitro differentiation assays on 9 lines . In addition to hypercellularity and neovascularization , this prDespite recent advances in treatment modalities, GBM remains a cancer with a very poor prognosis . Cell liThere are numerous reports describing the establishment of oncosphere lines from GBM tumor samples , 15–17. in vitro. An additional consideration is the location of the tumor tissue harvested. There are likely differences in the ability to form cell lines from tissue obtained from central necrotic regions, contrast enhancing regions, and in the periventricular region where the stem/precursor cells likely reside. Interestingly, although JHU-1016A and JHU-1016B were established from the same patient, they had dramatically different growth properties, highlighting the importance of intratumoral heterogeneity. JHU-1016A was a fast growing line in vitro, and while both lines generated tumors in all animals injected, JHU-1016A xenografts resulted in an average survival half that of JHU-1016B. Additionally, harvest techniques, specimen transport, and processing and tissue culture specifics, which vary greatly, also likely impact the establishment rate. These issues highlight some of the challenges in establishing cell lines from heterogeneous tumor tissue.It is interesting to note that we were able to establish oncosphere lines that met all of our criteria in only a quarter of the tumors processed. There are numerous possible explanations and sources of bias underlying this finding. GBMs are heterogeneous molecularly, genetically, pathologically, and biologically. It is possible there is something specific regarding the genetic and molecular properties from these 12 patients that allowed their tumor cells to grow in vivo orthotopic tumor formation. To date, 8 of the cell lines have met all four criteria.We used four criteria to evaluate these cell lines including 1) genetic uniqueness using STR profiling; 2) oncosphere formation/self-renewal past passage four; 3) lineage capacity as demonstrated by differentiation; and 4) et al. showed that in a commercial cell line repository, 18% of the cell lines were cross-contaminated [Our first criterion was verification of genetic uniqueness. Cross-contamination or incorrect identification of a cell line occurs with unfortunate frequency. Cross-contamination can happen both among human cell lines and between species. MacLeod aminated . Misidenaminated –32. Use aminated . The kitin vitro three-dimensional model for tumor research. A growing body of research on areas such as metabolism and drug resistance has been done on the physiologic relevance of the three-dimensional sphere structure [et al. investigated the glycolytic rates in multicellular tumor spheres and compared them to adherent monolayers [Our second criterion was growth as cancer derived oncospheres. These oncospheres provide an tructure –36. Rodret al. explored hypoxia-inducible factor 1α (HIF-1α) and its effects on tumor cells independent from angiogenesis by using a spheroid model [Leek id model . They deet al. examined drug resistance in a murine breast carcinoma line, culturing spheroids from in vivo tumors [et al. proposed that multicellular resistance contributed to tumor drug resistance and that to study it in vitro required spheroid tumor models [Translational research in oncology has benefited from spheroid cultures as they may be a better model for drug resistance. Kobayashi o tumors . While tr models . The layin vivo tumor formation to begin to further evaluate the stem-like nature of our oncosphere lines. Multipotency, our third criterion, was evaluated in the majority of cell lines. Similar to other studies, our work demonstrates such cultures can be differentiated to express different lineage markers [et al demonstrated that not only did treating GBM xenografts with ionizing radiation enhance the cancer stem cell proportion, those cancer stem cells showed higher activation of DNA damage checkpoint proteins [-/CD44+ enriched population growing as spheres, showed increased resistance to irradiation when compared to a non-enriched, monolayer culture [-/CD44+ enriched population of breast cancer cells [The ability to form spheroids in and of itself it not necessarily a characteristic of cancer stem cells and in this study we investigated multipotency and  markers , 9, 18.  markers , 38. Witproteins . These r culture . Similarer cells .in vivo tumor formation. In vivo tumor models play a critical role in cancer research, particularly in new drug testing [in vivo tumors that have closer histological characteristics to GBMs than adherent cell lines. Lee et al. showed that tumors formed by oncosphere cell lines more closely resembled GBMs than tumors formed by adherent cell lines [in vivo tumorigenicity, providing a counterpoint to the xenograft-forming cell lines.Our fourth criterion was  testing . Oncosphll lines . Importac-Myc amplification. Patient JHH-505 had a GBM that contained an oligodendroglioma component. This cell line efficiently formed intracranial xenografts with characteristics of GBM-O. Model systems of these GBM variants are rare in the literature [Of note, in our study we were able to establish oncosphere cell lines for patients with GBM variants. One patient, JHU-0879, had PNET features in the tumor. These features were also found in the xenograft, including increased cell density and a large number of apoptotic bodies, which was suggestive of terature . The creWe report our protocol for establishing suspension cell lines directly from intraoperatively obtained tumor tissue. Using this protocol, we established 13 primary human oncosphere cell lines derived from patients with GBM and GBM variants. A large body of research has demonstrated that suspension cell lines are more applicable models for GBM and a valuable resource in GBM research.S1 Table(DOCX)Click here for additional data file.


Cell culture is essential across cancer cell biology laboratories. Cancer cell lines that are attached to the culture vessel as monolayers are routinely passaged to new vessels in order to produce a large number of cells for carrying out various experiments. Cell detachment from the extracellular matrix can activate numerous cell signaling pathways. For example, enzymatic cell detachment using trypsin activates the HIPPO pathway, resulting in phosphorylation and inhibition of YAP within 10 min after cell detachment . It is lDrosophila melanogaster almost a century ago  of non-small cell lung cancer (NSCLC) cell lines. Even when unstained cells were passed through a cell sorter without being sorted for a specific population (sham-sort), the protein levels of cleaved, activated Notch1 were increased 17-fold, as compared to monolayer cells Figure . We reasWe next explored if Notch1 activation is caused by cell detachment. As shown in Figure Notch1 signaling regulates a wide range of processes in cancer, including cell proliferation, cancer stem cell self-renewal, cell survival, and epithelial–mesenchymal transition , 18, 19.2 (0.5 mM) was added to the trypsin and Hes1 mRNA levels were still increased (Figure We further checked whether adding calcium to trypsin solution during cell detachment would prevent Notch1 signaling pathway activation. CaCld Figure , or cellWe then surmised that repeated Notch1 activation caused by cell detachment can be avoided by propagating cell lines as tumorspheres. H1299 and H358 lung cancer, MCF7 breast cancer, and LnCap prostate cancer cell lines were expanded as tumorspheres, as described . TumorspIn summary, we found that detachment of adherent cells using various cell enzymatic, calcium depletion, and mechanical methods results in transient activation of the Notch1 signaling pathway across various cell lines. Caution should be exercised when studying the Notch1 pathway or pathways interacting with Notch1 signaling after routine subculture of cells. It would be interesting to know if Notch2, 3, or 4 are activated similarly to Notch1. Notch4 was suggested to not be activated by EDTA , and theThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The imatinib-resistant cell lines GK1C-IR and GK3C-IR showed imatinib resistance with IC50 values of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively. The phosphorylation status of key cell signaling pathways, receptor tyrosine kinase KIT (CD117), platelet-derived growth factor receptor alpha (PDGFRA) and downstream signaling kinases: serine-threonine kinase Akt (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) or the non-receptor tyrosine kinase: proto-oncogene tyrosine-protein kinase Src (SRC), was analyzed in established cell lines and ERK1/2 phosphorylation was found to be increased compared to the parental cells. Nilotinib demonstrated significant antitumor efficacy against GIST xenograft lines and imatinib-resistant GIST cell lines. Thus, nilotinib may have clinical potential for patients with GIST or imatinib-resistant GIST.Despite the benefits of imatinib for treating gastrointestinal stromal tumors (GIST), the prognosis for high risk GIST and imatinib-resistant (IR) GIST remains poor. The mechanisms of imatinib resistance have not yet been fully clarified. The aim of the study was to establish imatinib-resistant cell lines and investigate nilotinib, a second generation tyrosine kinase inhibitor (TKI), in preclinical models of GIST and imatinib-resistant GIST. For a model of imatinib-resistant GIST, we generated resistant cells from GK1C and GK3C cell lines by exposing them to imatinib for 6 months. The parent cell lines GK1C and GK3C showed imatinib sensitivity with IC Gastrointestinal stromal tumor (GIST) is a mesenchymal cell neoplasm with an annual incidence of 10–20 cases per million people. While GIST rarely recurs locally after initial surgical resection, advanced disease is characterized by distant metastasis to the liver and serosal surfaces of the abdomen Advances in the understanding of disease pathogenesis and molecular characteristics resulted in the use of novel tyrosine kinase inhibitors (TKIs), a class that includes imatinib  and sunitinib  in vitro and in vivo to inhibit the auto-phosphorylation and proliferation of cells transformed with activating mutations of KIT or platelet-derived growth factor receptor-alpha (PDGFRA) tyrosine kinases, which are the kinases that are the key oncogenic drivers in GIST The disease control time by imatinib is limited due to intolerance or resistance. Nilotinib  is a second-generation TKI that is expected to show enhanced clinical efficacy against GIST. Nilotinib is a potent TKI that has been shown both Here we showed that nilotinib can potently inhibit the growth of GIST xenograft lines  and cell lines (GK1C and GK3C) and imatinib-resistant GIST cell lines (GK1C-IR and GK3C-IR). Significantly, nilotinib effects on tumor growth were seen in both GIST and imatinib-resistant GIST, suggesting that treatment with nilotinib may represent a promising approach for overcoming the significant clinical challenge of imatinib resistance in GIST.Imatinib mesylate  and nilotinib hydrochloride  were provided by Novartis Pharma AG . For in vitro studies, a stock solution of 10 mM was prepared in 100% DMSO and then diluted to final concentration in medium. For peroral administration, the compound was dissolved in N-methyl-2-pyrrolidone (NMP) at 100 mg/mL and diluted 1∶10 (10 plus 90 v/v) with polyethylene glycol 300 (PEG300) to final 10 mg/mL.2.The human GIST cell lines GK1C and GK3C were established and characterized in our previous study nu/nu mice. Mice bearing GK1X, GK2X and GK3X tumors (6–8 mice per group) were treated daily with vehicle or 40 mg/kg imatinib or nilotinib for 4 weeks. Tumor volume (TV) was determined from caliper measurements of tumor length (L) and width (w) according to the formula LW2/2. TV was determined every two to three days and on the day of evaluation. Mice were sacrificed and the percentage of tumor growth inhibition (TGI) was calculated as follows: TGI (%)  =  [1– /]×100.All animal experiments were approved by the Laboratory Animals Center in Keio University, School of Medicine (Permit number: 09198). The GIST xenograft lines GK1X, GK2X and GK3X in nude mice were established from GIST patients as described in our previous study Cells were plated in 96-well microplates and cultured for 12 h before exposure to imatinib (1–100 µM) or nilotinib (1–100 µM) for 72 h. The cells were quantified by the WST-8  assay as described by the manufacturer . The optical density (OD) was determined with Sunrise rainbow . The rate of inhibition was calculated as follows: % of inhibition  =  (OD of treated group – blank)/(OD of control group – blank) ×100%. The concentration of tested drugs resulting in 50% growth inhibition (IC50) was calculated.GK1C-IR and GK3C-IR cell lines, and GK1X, GK2X and GK3X xenograft lines were stained by CD117 antibody  to assess KIT protein expression as described previously For an apoptosis assay, the supernatant was aspirated and cells were resuspended in 150 µL binding buffer, before staining with 5 µL Annexin V-FITC and 5 µL PI at room temperature for 25 min in the dark. After incubation, cells were processed as directed by the kit instructions  and analyzed using a FITC signal detector and PI detector using a flow cytometer  and Cell Quest software (Becton Dickinson).6 cells) and incubated on ice for 15 min. After blocking on ice for 10 min, cells were then washed and incubated with primary antibodies against phospho-KIT (Tyr719), phospho-PDGFRA (Tyr754), phospho-Src (Tyr416), phospho-AKT (Ser473), and phospho-ERK1/2 (Thr202/Tyr204)  for 30 min at room temperature. Cells were washed with PBS before incubation for 30 min with Alexa Fluor 488 donkey anti-rabbit IgG antibody . Then, each sample was analyzed using a FACSCalibur (Becton Dickinson). Cell distribution was analyzed using FlowJo software .Cells were washed in PBS and fixed with 2% paraformaldehyde (PFA) in a 37°C water bath for 10 min. Then, cells were washed with PBS and pelleted by centrifugation (800×g) for 5 min, and the supernatant was removed. Cells were mixed to disrupt the pellet and permeabilized by adding 500 µL 90% methanol  . Tumor v50 of 4.47±0.97 µM and 11.15±1.48 µM, respectively. In contrast, the resulting cell lines GK1C-IR and GK3C-IR demonstrated in vitro imatinib resistance with IC50 of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively (P<0.01)  protein expression was determined by immunocytochemistry on the human GIST cell lines GK1C-IR and GK3C-IR . The parectively , which r(P<0.01) .The profiles of the main cell signaling activity of phospho-KIT (Tyr719), phospho-PDGFRA (Tyr754), phospho-SRC (Tyr416), phospho-AKT (Ser473) and phospho-ERK1/2 (Thr202/Tyr204) were analyzed by flow cytometry in GK1C-IR and GK3C-IR cells and compared to that of the parent cells. The phosphorylation status was detected as the mean of fluorescence intensity (MFI) by analysis software (FlowJo). Interestingly, there was little difference in the phosphorylation state of KIT, PDGFRA, AKT and SRC in the parent and imatinib-resistant cells. In contrast, the phosphorylation of ERK1/2 showed about a 2.50 and 3.57 fold increase in resistant cells, respectively , which c50 values of parent GIST cell lines being 3.15±0.31 µM for GK1C and 3.32±0.18 µM for GK3C (n.s.), and the imatinib-resistant cell lines showing IC50 values of 4.10±0.46 µM and 3.96±0.19 µM for GK1C-IR and GK3C-IR (n.s.), respectively. These results indicate that nilotinib inhibited the proliferation of both imatinib-resistant and parent GIST cell lines, with IC50 values not varying significantly between the individual cell lines.We evaluated whether the tyrosine kinase inhibitor nilotinib could inhibit the proliferation of parent GIST cell lines (GK1C and GK3C) and imatinib-resistant GIST cell lines (GK1C-IR and GK3C-IR). Parent and imatinib-resistant GIST cell lines showed sensitivity to nilotinib in a concentration-dependent manner  with theGastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. Prior to the availability of imatinib mesylate (imatinib), the prognosis of some advanced patients with unresectable or recurrent GIST was poor c-kit and PDGFRA mutations and secondary resistance against imatinib is often acquired by a secondary mutation or amplification of c-kit or PDGFRA expression c-kit or PDGFRAc-kit or PDGFRA expression. As a cause of other secondary resistance, there are a report to be due to the overexpression of drug efflux pump such as MDR1, the report to the low activity of OCT-1 in connection with the intracellular taking-in of imatinib in vitro with IC50 of 11.74±0.17 µM and 41.37±1.07 µM, respectively. In an apoptosis assay, these imatinib-resistant cells (GK1C-IR and GK3C-IR) showed a decrease in the size of dead/apoptotic populations. Unlike in the parent cells, phosphorylation of KIT or PDGFRA or its downstream intermediates such as AKT, and the non-receptor tyrosine kinase Src in imatinib-resistant GIST cell lines remained unchanged even in the presence of imatinib. Moreover, phosphorylation ERK1/2 was accelerated following imatinib treatment. While patients with GIST usually respond to imatinib, most eventually experience disease progression with the reactivation of KIT tyrosine kinase and its downstream signaling pathways Resistance to chemotherapy remains a major obstacle for the treatment of GIST. As such, understanding the molecular mechanism of resistance is important to the development of effective new therapies for treatment of this disease. The effect of imatinib is different for various types of 6 cells were transplanted into the back of SCID (C.B-17/IcrHsd-Prkdc scid) mice (each 10 mice). However, cells did not grow in mice, we could not acquire imatinib-resistant GIST xenografts. In many other studies, it has not yet been reported for imatinib resistant GIST cell lines in vivo drug assay In the past few years, several tyrosine kinase inhibitors (TKIs), including nilotinib, dasatinib and sorafenib were investigated for use in treating imatinib- or sunitinib-resistant GIST. We evaluated the inhibitory efficacy of nilotinib against GIST xenograft lines  and imatinib-resistant GIST cell lines (GK1C-IR and GK3C-IR). These survival assays demonstrated that nilotinib inhibited the proliferation of both the xenograft lines and imatinib-resistant cell lines. Moerover, to investigate the antitumor effect of nilotinib in vivo, we have tried for imatinib-resistant GIST xenografts. Imatinib resistant GIST cell lines (GK1C-IR and GK3C-IR) were prepared and expanded, and 2×10ENESTg1  Phase III study, the clinical utility of nilotinib did not demonstrate the superiority of Tasigna over imatinib. On the basis of the trial results, nilotinib is not generally recommended for GIST therapy. Nevertheless, this new TKI may merit to patients with high risk GIST or imatinib-resistant GIST.


Klotho is a discovered aging suppressor gene, and its overexpression in mice extends the life span of the animal. Recently, Klotho is also identified as a tumor suppressor gene in variety of tumors; however, the potential role and the antitumor mechanism remain unclarified in liver cancers.RT-PCR and western blotting analysis were used to detect the expression of Klotho, β-catenin, C-myc, and Cyclin D1. MTT assay was used to detect the survival rates of HepG2 and SMMC-7721 cells. Colony formation assay was used to test the proliferation ability in Klotho transfected cells. FACS was used to detect the cell apoptosis rate in different groups.The results showed that lower expression of Klotho were found in liver cancer cell lines than the immortalized liver cell L02. Also, MTT assay results found that overexpression or recombinant Klotho administration suppressed the proliferation of liver cancer cells HepG2 and SMMC-7721. Moreover, the colony formation assay results showed that the number of colonies was significantly lower in the cells with transfection with pCMV-Klotho than the controls. Thus, functional analysis demonstrated that Klotho expression inhibited the proliferation of liver cancer cells and Klotho worked as an important antitumor gene in tumor progression. Next, the mechanism was partly clarified that Klotho expression induced cell apoptosis in HepG2 and SMMC-7721 cells, and this phenomenon was mainly involved in the Wnt/β-catenin signaling pathway. The western blotting analysis revealed that overexpression or recombinant administration of Klotho obviously decreased the expression levels of β-catenin, C-myc, and Cyclin D1 in HepG2 cells. Most importantly, the antitumor mechanism for Klotho due to that overexpression of Klotho not only decreased the endogenous β-catenin levels but also inhibited the nuclear translocation of β-catenin to delay the cell cycle progression.Klotho was a tumor suppressor gene, and overexpression of Klotho suppressed the proliferation of liver cancer cells partly due to negative regulation of Wnt/β-catenin signaling pathway. So, Klotho might be used as a potential target, and the study will contribute to treatment for therapy of liver cancer patients. Liver cancer is one of the most lethal cancers in the world, and its mortality ranks the third in cancer deaths , 2. EachKlotho was a new identified aging suppressor gene, which is highly conserved between human and mouse, with 86 % amino acid identity . RecentlHowever, the relationship and association between the expression of Klotho and primary liver cancers is not clarified till now . In the 2 in a humidified atmosphere.Recombinant human Klotho (rKlotho) is a 65–70 kDa glycoprotein containing 516 amino acid residues. The protein of rKlotho was obtained from Sigma Corporation (cat. SRP3102). The myc-tagged Klotho expression vector (pCMV6-Klotho) and the control vector (pCMV6) were designed and obtained from OriGene corporation . The liver cancer cell lines HepG2, SMMC-7721, and BEL-7404 were obtained from American Type Culture Collection . The normal liver cancer cells L02 was purchased from Junrui Coporation . The liver cancer cells were cultured in DMEM medium  supplemented with 10 % fetal bovine serum and cultured at 37 °C with 5 % COCell proliferation is tested by MTT assay. Generally, the human liver cancer cells HepG2 and SMMC-7721 cells were planted into 48-well plates. Then, the cells were cultured for 8 h and transfected with p CMV6-Klotho and p CMV-6 vector for 24, 48, 72, and 96 h. Finally, MTT agent was added into the medium, and the purple crystals were dissolved with DMSO. The liquid was transferred into 96-well plates and read on a microplate reader at a test wavelength of 490 nm and a reference wavelength of 570 nm.The RNApure kit  was used to extract total RNA, and reverse transcriptase (RT) SuperScript III  was used to transcript the cDNA. The primer sequences were as follows: for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAAGGTGAAGGTCGGAGTC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense) and for Klotho, 5′-ACCTGGTGGCGCACAACC-3′(sense) and 5′-TTGGCAAACCAACCTAGTACA-3′. The Klotho PCR reaction condition is that 94 °C 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and finally, elongation is 72 °C for 10 min. The GAPDH reaction condition is similar to Klotho but for the annealing temperature it is 55 °C for 30 s.The liver cancer cells were transfected with pCMV6-Klotho and pCMV-6 vector for stable cell lines. For colony formation assay, the cells were plated into 6-well plate. After 8 h, the cells transfected with Klotho or control vector were cultured for 14 days, and the medium was refreshed every 3 days. Then, methanol was used to fix the colonies (≥50 cells per colony). They were stained with 1.25 % crystal violet and counted under a light microscope.Annexin V-FITC/PI dual staining analysis is used for detection of the apoptosis rates in human lung cancer cells according to the kit protocols . Briefly, the liver cancer cells (4 × 105 cells/well) were plated into 6-well plate and transfected with pCMV6-Klotho and pCMV6 vector for 48 h. Then, the cells were washed with ice PBS buffer for three times and resuspended in binding buffer with HEPES–NaOH 10 mM pH 7.4, 25 mM CaCl2, and 144 mM NaCl. At last, the staining dye of Annexin V (0.1 μg/μl) and PI (0.05 μg/μl) were added for incubation in the dark for 30 min on ice, and the cells were subjected to FACS analysis.The liver cancer cells were plated into 48-well plates and cultured for 8 h for adherence. Then, the cells were transfected with pCMV6-Klotho and p CMV-6 vector for 48 h. The pCMV6 vector was used as the control. The other treatment was that the cells were treated with rKlotho or BSA at the concentration of 250 ng/mL. BSA was used as the negative control. Next, the cells were washed with ice-cold PBS buffer, and cell lysates were prepared for PAGE. The primary antibodies used were included; the anti-beclin 1, anti-LCI, anti-LC-II, anti-C-myc, anti-Cyclin D1, and anti-GAPDH were all purchased from Santa Cruz biotechnology. The primary antibodies of β-tublin (ab6046), lamin B1(ab16048), and Anti-Klotho (ab18131) were obtained from Abcam corporation. The horseradish peroxidase-conjugated goat anti-mouse secondary antibody was purchased from Abgent Corporation.t test was used to assess the significance of the data, and all of the data are showed as mean ± SD. P < 0.01 means that there is significant difference.All of the data was analyzed using SPSS software. All of the experiments were repeated three times in duplicates. Student’s To explore the Klotho expression of Klotho in liver cancer cell lines, we analyzed Klotho levels in a panel of four liver cancer cell lines by RT-PCR and western blotting analysis. Low mRNA levels and protein levels were detected in liver cancer cell lines, such as Bel-7404, HepG2, SMMC-7721 etc., compared with the immortalized liver cell L02 , and the cells were cultured for 10 days. As shown in Fig. P < 0.01), and this was consistent with the situation in SMMC-7721 cells.It is proved that overexpression or recombinant Klotho administration could inhibit the proliferation of liver cancer cells. In order to identify whether cell apoptosis is induced in cancer cells with overexpression of Klotho, FACS analysis was used by dural staining with PI and Annexin V-FITC. As shown in Fig. It is reported that Wnt/β-catenin signaling pathway is abnormally expressed in liver cancer cells. In order to explore the relationship between the tumor suppressor, Klotho and Wnt/β-catenin signaling pathway, the HepG2 cells were transfected with p CMV6-Klotho and control plasmid p CMV6. The expression levels of Klotho, β-catenin, C-myc, and Cyclin D1 were detected by western blot. As shown in Fig. The activation of Wnt/β-catenin signaling pathway is that β-catenin was translocated into the nucleus. Next, we wanted to know whether overexpression of Klotho would affect the translocation of β-catenin. Thus, we detected the distribution of β-catenin in cytoplasm and the nucleus. As shown in Fig. Primary hepatocellular carcinoma is a common malignancy in the world . With thKlotho has been newly identified as an antiaging gene that is involved in the suppression of several aging phenotypes –25. But Then, we further explore the mechanism of Klotho involving in the progression of liver cancers. It has reported that overexpression of Klotho inhibits growth and invasion through inhibition of IGF1R-mediated PI3K/AKT pathway in colon cancer cells . In lungHere, we also detected the association of Klotho expression with Wnt/β-catenin signaling pathway. The western blotting analysis showed that high levels of Klotho were associated with decreased levels of β-catenin. Importantly, the nuclear translocation of β-catenin was also suppressed in HepG2 cells. Additionally, the target genes of the downstream (C-myc and Cyclin D1) were downregulated. All of the data demonstrated that overexpression of Klotho suppressed the proliferation of liver cancer cells and promoted cell apoptosis partly due to negative regulation of Wnt/β-catenin signaling pathway. Thus, Klotho might be used as a potential target, and the study will contribute to treatment for therapy of liver cancer patients.Klotho was a tumor suppressor gene and overexpression of Klotho suppressed the proliferation of liver cancer cells partly due to negative regulation of Wnt/β-catenin signalling pathway. So, Klotho might be used as a potential target and the study will contribute to treatment for therapy of liver cancer patients.


Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders. Type 2 diabetes mellitus (T2D) is a metabolic disease currently affecting 9% of the global adult population467891011hIAPP is co-secreted with insulin by pancreatic β-cells and largely contributes to glycemic control1516et al. proposed that β-cell viability is only reduced when hIAPP concentration is high enough to mediate fibrillation, and identified amyloid-membrane contact triggering apoptosis as the primary mechanism of toxicityet al. screened a number of amyloid peptides in PC12 and B12 cells for production of ROS with 2,7-dichlorofluorescin diacetate, and determined that hIAPP was correlated with ROS production and subsequent loss of cell viability, while no such species were measured with the non-amyloidogenic rat-derived IAPP252627Early research of the past two decades favoured amyloid as a causative agent of β-cell failure, thought to be mediated through physical association between the plaques and the cells, leading to membrane perturbation, production of reactive oxygen species (ROS), and/or apoptosis23et al. observed that hIAPP oligomers mediated a disruption in islet architecture ex vivo, impairing cell coupling and insulin secretion and inducing apoptosiset al. and Meier et al. described cell membrane permeabilisation induced by hIAPP oligomers, while monomeric hIAPP or amyloid fibrils showed no adverse effectset al. refuted this, revealing that rifampicin had no effect on hIAPP fibrillation and demonstrating that the ligand can interfere with ThT fluorescenceIn more recent years, however, the focus has shifted to the soluble oligomeric form of hIAPP as the main toxic species, but considering similar mechanisms of toxicity as were postulated for hIAPP amyloids. In light of the biophysical and biochemical connections between hIAPP and Aβ fibrillation, the working paradigms concerning hIAPP toxicity have been influenced by the mechanistic studies of Alzheimer’s disease, an approach which remains to be validated. Ritzel In the present study, we sought to further investigate fresh and oligomeric hIAPP, as well as mature amyloid as toxic agents to pancreatic β-cells , and att3031333435This paper is arranged as follows. We first present a characterisation of hIAPP in aqueous and at micromolar concentrations, rendering in three states as fresh and oligomeric hIAPP (stabilised by resveratrol) as well as mature amyloids, using high-throughput dynamic light scattering (DLS). We then show our ratiometric imaging of MIN6 pancreatic cells resulting from their exposure to hIAPP of the three states, quantified by generalised polarisation (GP) of the cell membranes partitioned with a Laurdan dye. Finally we compare our GP measurement with the assays of MIN6 cell viability and ROS generation induced by hIAPP of the three states and draw conclusions of the present study. Implications of understanding protein aggregation for research in neurological disorders and T2D are discussed at the end.High-throughput DLS was utilised to observe hIAPP amyloid fibrillation in aqueous solution. In order to investigate the specific properties of oligomeric hIAPP, polyphenol resveratrol was utilised as a fibrillation inhibitor. As shown in lo, predominantly fluorescent at 430~470 nm) and the liquid disordered phase , were determined by generalised polarisation  and/or resveratrol (20 μM) to obtain the three stages of the polypeptide. After 24 h treatment, cells were labelled with the DNA binding dyes Hoechst-33342 and propidium iodide to discriminate viable cells from dead cells. Hoechst-33342 labels dsDNA blue and is able to diffuse freely through intact and damaged membranes. Propidium iodide labels dsDNA as red, however due to its molecular properties, is impermeable to cells with intact membranes, thus staining only dead cells. Cell death was quantified by blue-red fluorescence or by fragmented nuclei, to the exclusion of viable cells identified by intact blue nuclei. We observed that treatment of MIN6 cells with fresh hIAPP for 24 h resulted in the greatest induction of cell death  . TreatmeWe then determined whether ROS production correlated with cell toxicity dependent on the aggregation state of hIAPP. To analyse this MIN6 cells were stained with 2,7-dichlorofluorescein diacetate (DCFDA) and subsequently treated with the three different forms of hIAPP. DCFDA is a fluorogenic dye that diffuses readily into the cell and is then deacetylated by cellular esterases to a non-fluorescent compound. After exposure to ROS, DCFDA is oxidised into 2,7-dichlorofluorescein (DCF). DCF fluorescence can be measured by flow cytometry through excitation at 488 nm, and is detectable at 535 nm. After 2 h and 4 h treatments with the three different forms of hIAPP we observed that treatment with mature amyloid fibrils resulted in the highest induction of ROS inside cells   was obtained from Abcam in lyophilised powder form. The hIAPP was weighed on a Cubis MSE balance  and dissolved in Milli-Q water to form a 1 mg/mL (256 μM) stock solution immediately prior to (freshly-dissolved monomers) or around two weeks before (pre-formed/“mature” amyloid fibrils) commencing measurements. Resveratrol  was obtained from Sigma Aldrich and dissolved in Milli-Q water to form a 120 μM (0.027 mg/mL) stock solution. Laurdan dye  was obtained from AnaSpec and maintained as a 4 mM stock solution (1.4 mg/mL) in DMSO, with a 500 μM solution in Milli-Q made up immediately prior to addition to wells. Hoechst-33342 was obtained from Sigma Aldrich and propidium iodide was obtained from Life Technologies, both were made to stock concentrations of 10 mg/mL in PBS. The DCFDA cellular ROS detection kit was purchased from Abcam.The hydrodynamic sizes of hIAPP and hIAPP-resveratrol were acquired at room temperature over 430 min using an automated, high-throughput DLS device , with samples plated in quadruplicate in black 384-well plates (Thermo Fisher). Each well contained a total volume of 20 μl of either 10 μM hIAPP or resveratrol (20 μM) mixed with hIAPP at a 1:2 molar ratio in aqueous solution. To ensure good mixing, samples were spun for 1 min at 1,000 rpm/164 RCF  prior to the start of the experiment. 20 data points for each sample well were collected by an optical module while scanning through all samples continuously. Processing of the acquired data was automated through the Dynamics 7.1.7 software.2O, with excess drawn off as previously described. The grids were stained with 5 μL 1% uranyl acetate for 30 s, with excess stain drawn off. Grids were air-dried as needed. Electron microscopy was undertaken utilising a Tecnai G2 F30 Transmission Electron Microscope , operating at a voltage of 300 kV. Images were recorded using a Gatan UltraScan 1000 (2k × 2k) CCD camera  using Gatan Microscopy Suite control software.Mature amyloid fibrils were visualised by TEM as follows: a 5 μl aliquot of amyloid hIAPP  was pipetted onto a copper grid  and allowed 60 s of adsorption. Excess sample was then drawn off using filter paper and the grids washed using 10 μL ddH2 incubator. Prior to imaging, old media was removed and replaced with serum-free Opti-MEM (Life Technologies), with cells washed in between using Opti-MEM. Laurdan dye was added to each well to a final concentration of 50 μM and allowed to equilibrate with the cell membranes for at least 30 min. Cells were transferred to a Leica SP8 inverted confocal fluorescence microscope housed in a humidified, 37 °C, 5% CO2 environment. Appropriate areas of each well, containing at least 10 viable cells, were identified. Freshly dissolved hIAPP monomers (10 μM), pre-formed amyloid fibrils (10 μM), resveratrol (20 μM), and fresh hIAPP monomers + resveratrol in a 1:2 molar ratio were added to respective wells. Imaging was undertaken at 1~30 min timepoints for 2 h with a 20 × /0.70 dry objective. Laurdan dye integrated into cell membranes was excited along the 405 nm laser line and emission read at 430~470 nm (representing the lipid membrane at the gel/liquid ordered phase) or 480~550 nm (representing the lipid membrane at the liquid disordered phase). To calibrate dye background levels, a well containing dye only was excited on the 405 nm laser line using 0.5 and 2 × laser power. An additional control of mixing individual hIAPP species with Laurdan abiotically did not show any noticeable effect on the dye fluorescence in aqueous solution , containing 10% fetal calf serum onto 8-well slide plates  coated with poly-D-lysine. Cells were allowed to attach overnight in a humidified, 37 °C, 5% COsolution . Post-exet al. (2011)The acquisition of GP images was performed using the ImageJ  software and custom-written macro by Owen I represents the intensity of pixels in the areas of interest in the image acquired in the ordered (430~470 nm) and disordered (480~550 nm) spectral channels. The sample pool for GP analysis was an average value of 25 cells (each cell 960 ± 330 pixels) from each image, totalling ~25,000 data points for each established histogram. GP shift was observed by subtraction of the GP distribution peak maximum of each sample with 2 h of incubation time from the GP value of the image taken at the beginning of the experiment (0 h).where The insulin-producing MIN6 cell line was cultured in DMEM  supplemented with 10% fetal calf serum. The percentage cell death of MIN6 was determined by labelling cells with the DNA dyes Hoechst-33342 (10 μg/mL) and propidium iodide (5 μg/mL) and counting at least 600 cells per experimental condition via inverted fluorescence microscopy. Cells were treated with 10 μM of fresh hIAPP, 10 μM of stabilised oligomeric hIAPP (with resveratrol 20 μM), 10 μM of preformed amyloid fibrils, or 20 μM of resveratrol for 24 h in Opti-MEM media (Life Technologies).hi (DCF+) population.ROS detection was performed using a DCFDA cellular ROS detection kit (Abcam). Mixing individual hIAPP species with DCFDA abiotically did not show any effect on the dye fluorescence in aqueous solution . Single Data are represented as means ± SEM. Given the paired nature of the experimental design comparisons between treated groups were made by analysis of variance (ANOVA). A p value < 0.05 was considered statistically significant.How to cite this article: Pilkington, E. H. et al. Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species. Sci. Rep.6, 21274; doi: 10.1038/srep21274 (2016).


Purpose. We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM). Materials and Methods. Mononuclear cells obtained from MM patient's bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM. Results. After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC. Conclusion. Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma. Multiple genetic and microenvironmental changes  lead to https://www.atcc.org/).By the way, multiple myeloma has correlation with plasmacytoma, which is a mass of plasma cells found outside of bone marrow  that neeMany clinicians are curious about the adequate treatment strategy of plasmacytoma . And theFor this aspect, we present, in this study, the establishment of two human multiple myeloma cell lines, called SNU_MM1393_BM and SNU_MM1393_SC from a patient with aggressive multiple myeloma using an animal model. SNU_MM1393_BM cell line is derived from bone marrow of a mouse, and SNU_MM1393_SC is derived from a subcutaneous plasmacytoma. Here, we characterized phenotypic, genetic, and functional properties of these cell lines. Also, we further investigated the response to cytokines and chemotherapeutic agents of these cell lines. RB1 deletion, trisomy 1q, IgH rearrangement, and IgH 3 copies. His myeloma cell secreted monoclonal protein of immunoglobulin A, kappa chain. He received radiotherapy for vertebral plasmacytoma. After radiotherapy, four cycles of thalidomide/dexamethasone chemotherapy were given to the patient, which yielded in partial response (PR). Stem cell collection was performed in preparation for autologous stem cell transplantation after PR to thalidomide/dexamethasone chemotherapy. We used bone marrow cells at the time of stem cell collection for this experiment. The karyotype was sustained to be normal by the time of this stem cell collection. He is still in PR after autologous stem cell transplantation with high-dose melphalan conditioning with progression free survival time of 8.3 months. He did not receive either bortezomib or panobinostat.In February 2012, a 63-year-old male patient visited Seoul National University Hospital for back pain and tingling sense on the trunk below nipple. He was diagnosed as multiple myeloma with spinal cord compression due to osseous plasmacytoma on the third thoracic vertebrae. His disease stage was 3 by Durie-Salmon staging and 2 by International Staging System. Karyotype of this patient was normal, but fluorescent in situ hybridization (FISH) revealed that the disease had trisomy 9,6 mononuclear cells suspended in a total volume of 300-microliter PBS. After 14 weeks, bone marrow specimens were obtained from mice, and isolated bone marrow cells were cultured in RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 g/mL) . They were cultured in a highly humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was exchanged every 3-4 days depending on the rate of cell growth.Bone marrow specimens were obtained from a patient diagnosed with multiple myeloma under a protocol approved by the Seoul National University Hospital Institutional Review Board. Mononuclear cells were separated by Ficoll-Hypaque density sedimentation. Eight-week-old NRG/SCID mice were injected intravenously via the dorsal tail vein (i.v.) with 1 × 10Histological sections of bone marrow from NOD/SCID mice were prepared and stained with haematoxylin and eosin (H&E) using standard methods. Cell morphology was examined using light microscopy.Metaphase chromosome spreads from peripheral blood and the cell line were prepared and G-banded according to standard procedures. The karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN) (2009) .Cell proliferation assay was performed using Cell Counting Kit-8  according to the manufacturer's instructions. Means and standard deviations were generated from three independent experiments. Absorbance values were normalized to the values obtained from control group to determine the value for % of survival.μg of whole cell protein extracts was boiled for 5 min, and the proteins were resolved in 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes . The membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk for 1 h at room temperature and incubated with the appropriate primary antibody for 2 h. Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibody  and enhanced chemiluminescence reagents .The cells were treated with indicated reagents for the indicated time periods, washed once in ice-cold phosphate buffered saline (PBS), and resuspended in lysis buffer (20 mM MOPS (pH 7.0), 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mM b-glycerophosphate (pH 7.2), 20 mM sodium pyrophosphate, 1 mM sodium, orthovanadate, 1% Triton X-100, 1 mM PMSF, aprotinin, leupeptin, and pepstatin 1 mg/mL). The protein concentration of lysate was measured, 30 6 cells/mL) were incubated with 20 μL of phycoerythrin-conjugated anti-CD138 and anti-CD45, respectively  for 30 min at 4°C, washed, and then fixed with 2% paraformaldehyde. Then, the samples were analyzed by FACSCalibur flow cytometer (Becton Dickinson) and inbuilt software.Cells  as a control experiment in our study. The used cell lines include U266, U266_SC, IM9, and RPMI8226 cell lines.We used commercially available cell lines which were bought from DSMZ  . Myelomaraploidy . Hence, As shown in When we evaluated the cytokine response in these cell lines, differential response was noted between the two cell lines. We used cytokine interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R), which is well known to regulate the biologic behaviors of myeloma cells in the progression of multiple myeloma. It is well known that sIL-6R potentiates the IL-6 mediated signaling . Based oIn myeloma cells established from bone marrow of a mouse (SNU_MM1393_BM), phosphorylated form of Erk (p-ERK) was increased when treated with IL-6 only and combination of IL-6 and sIL-6R, but phosphorylated form of Akt (p-AKT) was not. On the other hand, in myeloma cells established from subcutaneous plasmacytoma (SNU_MM1393_SC), the induction of p-ERK was not as evident as in SNU_MM1393_BM. Increase in p-AKT was not evident in SNU_MM1393_SC either .Since IL-6 acts in an autocrine manner , we measP < 0.05). Meanwhile, SNU_MM1393_BM seemed to be more sensitive to panobinostat compared to SNU_MM1393_SC (P > 0.05). However, when compared to well-known myeloma cell lines (U266 and U266_SC), SNU_MM1393_SC and SNU_MM1393_BM were more resistant to bortezomib  (We tested the cytotoxic effect of proteasome inhibitor bortezomib and pan-HDAC inhibitor panobinostat in these two cell lines. Both SNU_MM1393_SC and SNU_MM1393_BM were more sensitive to panobinostat when compared to U266 (e <0.05) . ex vivo cultured cells via tail vein into NRG/SCID mouse. We used three kinds of cells: (1) ex vivo cultured myeloma cells of a patient's bone marrow, (2) cells from SNU_MM1393_BM, and (3) cells from SNU_MM1393_SC. When this was performed, we found tumor growth at both bone marrow and subcutis with ex vivo cultured myeloma cells. However, when cells from SNU_MM1393_BM were injected into a mouse, growth at subcutis was not noted. Only engraftment at bone marrow was observed. Lastly, when SNU_MM1393_SC cells were injected into a mouse, a mouse died in 7 weeks after injection. Tumor at subcutis was not noted at the time of death.To determine the tumorigenicity of these cells, we reinjected theseIt is known that the establishment of multiple myeloma cell line is notoriously difficult . Here, w in vivo and ex vivo. As an in vivo report, Yuan et al. [ ex vivo data, Balsas et al. [We observed several interesting findings during the establishment of these cell lines. First, there was a difference between patient's original myeloma cells and established cell lines. While patient's karyotype was a diploidy, karyotype of SNU_MM1393 was tetraploidy. This reflects a clonal evolution of a myeloma cell during cell line establishment. In fact, there are reports regarding the clonal evolution to near-tetraploidy bothn et al.  reporteds et al.  reporteds et al. . In a cos et al. . We thinFrom the above, we conjecture that aggressive myeloma cells might have been selected through cell line establishment using animal model. And, as expected, SNU_MM1393_SC had greater lethality than original myeloma cells of the patient. Mouse injected with SNU_MM1393_SC died within 7 weeks, while mouse injected with the original myeloma cells survived more than 14 weeks. One interesting finding in this experiment (regarding the tumorigenicity of established cell line) is that SNU_MM1393_BM was not as lethal as SNU_MM1393_SC. This coincides with the results that SNU_MM1393_SC showed more prominent resistance to bortezomib and SNU_MM1393_SC had more chromosome 13 loss which is a well-known negative prognostic marker in multiple myeloma . VariousSecond, there were differences between SNU_MM1393_BM and SNU_MM1393_SC in various aspects . In an eLastly, interesting findings were when drug response was investigated in these cell lines. Most importantly, newly established cell lines showed relative resistance to bortezomib compared to U266. This is an unexpected phenomenon, because the myeloma cell of our patient is bortezomib naïve. Considering relative indolent clinical course of our patient, it is unusual that a myeloma clone of our patient is resistant to bortezomib. When used with dexamethasone, response rate of bortezomib in treatment-naïve myeloma patients is 88%. Hence, we conjecture that this resistance to bortezomib shown in our newly established cell line would originate from clonal evolution during cell line establishment. We suggest that biologic behaviors of myeloma cells could be altered in the course of clonal expansion and this alteration would contribute to the chemotherapeutic resistance.Whether the above findings could be generalized to myeloma cells in bone marrow and plasmacytoma is questionable. Not many researches have been performed regarding the comparison of bone marrow myeloma cells and plasmacytoma, and we think our study results should be recognized as exploratory ones. Comparison with future researches focusing on plasmacytoma biology is necessary to adapt our findings to clinical practices. We are also willing to supply our established cell lines to other researchers for further studies.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma and its chemotherapeutic responses.