Since microRNAs  have been implicated in oncogenesis, many of them have been identified as therapeutic targets. Previously we have demonstrated that miRNA-10b acts as a master regulator of the viability of metastatic tumor cells and represents a target for therapeutic intervention. We designed and synthesized an inhibitor of miR-10b, termed MN-anti-miR10b. We showed that treatment with MN-anti-miR10b led to durable regression/elimination of established metastases in murine models of metastatic breast cancer. Since miRNA-10b has been associated with various metastatic and non-metastatic cancers, in the present study, we investigated the effect of MN-anti-miR10b in a panel of over 600 cell lines derived from a variety of human malignancies. We observed an effect on the viability of multiple cell lines within each cancer type and a mostly dichotomous response with cell lines either strongly responsive to MN-anti-miR10b or not at all even at maximum dose tested, suggesting a very high specificity of the effect. Genomic modeling of the drug response showed enrichment of genes associated with the proto-oncogene, c-Jun. Systemic treatment options for cancer often include chemotherapy, as a mainstream treatment approach. However, considering the disadvantages of standard chemotherapy, e.g. non-specific delivery, toxicity to healthy tissues, distressing side effects , and the potential for chemoresistance, we envision a future in which chemotherapy is complemented or replaced by alternative approaches to shorten the period of chemotherapy or lower the dose of anti-cancer drugs and minimize adverse symptoms while increasing the survival rate.The alternative that we propose relies on targeting oncogenic microRNAs (oncomiRs) that promote the migration, proliferation, and survival of tumor cells. In our earlier work, we identified miRNA-10b as a master regulator of the viability of metastatic tumor cells. We determined that miR-10b not only promotes the ability of tumor cells to migrate and invade surrounding tissue (become metastatic), as has been described previously  but, mosWe demonstrated that MN-anti-miR10b could completely prevent the formation of de novo metastases  and, wheIn a model of Stage IV metastatic breast cancer, we found that MN-anti-miR10b selectively accumulated in distant  metastases from breast cancer after intravenous injection. A weekly treatment protocol with MN-anti-miR-10b and low-dose doxorubicin demonstrated complete regression of pre-existing lung metastases in 65% of the animals and inhibition of multiple organ metastases in 94% of the animals. This translated into a significant reduction in cancer mortality in animals treated with MN-anti-miR10b and low-dose doxorubicin relative to control groups, including a group treated with monotherapy of standard dose doxorubicin, used to model standard-of-care.miRNA-10b has been implicated in the development of a wide range of human cancers including colorectal, gastric, bladder, pancreatic, ovarian, hepatocellular and brain cancer –8. RecenOur study revealed the association of these responses with gene expression patterns that suggested an effect on transcription, metastatic dissemination, and immune response. Finally, our data implied that the link between miR-10b inhibition and cell viability is not limited to metastatic cells, but also includes cells derived from primary tumors, which is a novel finding. We believe that this finding could have a significant impact on treating both non-metastatic and metastatic cancers.MN-anti-miR10b was synthesized as described in . The LNAAminated magnetic nanoparticles were synthesized following a protocol published previously . Nanoparticles with a size of 20.3 ± 0.6 nm were used for conjugation to the oligonucleotides. The magnetic nanoparticles were conjugated to the heterobifunctional linker N-succinimidyl 3-[2-pyridyldithio]-propionate  and activated oligos sequentially. Briefly, SPDP was dissolved in anhydrous DMSO and incubated with magnetic nanoparticles. The 5’-ThioMC6 of the oligo was activated to release the thiol via 3% TCEP treatment in nuclease-free PBS. The oligos were purified using an ammonium acetate/ethanol precipitation method. After TCEP activation and purification, the oligos were dissolved in water and incubated with the SPDP-modified magnetic nanoparticles overnight. The number of oligos per magnetic nanoparticle was determined as 8.0 ± 0.7 using the electrophoresis analysis method described previously .We have assembled a collection of 624 human tumor cell lines representing the spectrum of common and rare types of adult and childhood metastatic and non-metastatic cancers of epithelial, mesenchymal and haematopoietic origin. We have used this large panel of cell lines in order to better capture the high degree of genomic diversity in cancer and to identify rare mutant subsets with altered sensitivity. All cell lines were obtained from commercial repositories. Cell lines were expanded for less than 3 passages and frozen stocks were created. To further test the quality of the collection post expansion we performed the following: To identify cross-contaminated or synonymous lines, a panel of single nucleotide polymorphisms (SNPs) was profiled for each cell line  and a pair-wise comparison score calculated. In addition, we performed short tandem repeat (STR) analysis  and matched this to an existing STR profile generated by the providing repository. Cells from authenticated stocks were not continuously kept in culture for more than 3 months.Cells were seeded in 384-well microplates at ~15%-50% confluence in medium with 10% FBS and penicillin/streptavidin + high glucose (18–25 mM). The optimal cell number for each cell line was determined to optimize growth during treatment. For adherent cell lines, after overnight incubation, cells were treated with increasing concentrations of MN-anti-miR10b (2-fold dilution series ranging from 0.16 to 10.64 μM as oligo) using liquid handling robotics, and then returned to the incubator for assay at a 72-hr time point. For suspension cell lines, cells were treated with MN-anti-miR10b immediately following plating and returned to the incubator for 72 hrs. Cells were then stained with 55 μg/ml Resazurin (Sigma) prepared in Glutathione-free media for 4 hours. Quantitation of fluorescent signal intensity was performed using a fluorescence plate reader at excitation and emission wavelengths of 535/595 nM for Resazurin. All screened plates were subjected to stringent quality control measures that included having coefficient of variance of control well values below 20% and signal over noise  over 5-fold. Additionally, directionality of drug response was tested by comparing the average of viability values obtained with the two minimum doses and the two maximum doses and rejecting drug response if the ratio was below 1.2 (wrong direction of response). Effects on cell viability were measured and a curve-fitting algorithm was applied to this raw dataset to derive a multi-parameter description of drug response, including the half maximal inhibitory concentration (IC50). Curve fitting was performed using the drexplorer R package .The linear regression Elastic Net  was used to obtain a multivariate model of the drug response across all cell line tested. We used Tissue of origin (Tis), Copy Number (CN), RNA expression data and mutation (MUT) data as input together with IC50s. RNA expression was U219 Affymetrix chip based. To minimize the number of genomic features in the input, the CN data was restricted to 1700 genes for which copy number alterations were translated into RNA expression. Mutation data were from full Exome Seq after algorithmic filtering of germline events (no matched germline sequence available for the cell lines). The EN modeling was run 100 times with 10% of the cell lines randomly left out in each run . To identify biological mechanisms related to the genes identified as response predictor by EN we used the STRING  database  on the fThe sensitivity to MN-anti-miR10b was tested in 624 human cell lines representing metastatic and non-metastatic cancers of the following origin: Biliary Tract, Bone, Brain, Breast, Esophagus, Head & Neck, Intestine, Kidney, Leukemia, Liver, Lung, Lymphoma, Muscle, Nervous System, Ovary, Pancreas, Pleura, Skin, Stomach, Thyroid, Urinary Tract, and Uterus. Representative data are included in Figs The Genomic Modeling results are presented in Genes associated with resistance that were present at a frequency of > 0.7 included:HEXB ;ITPRIPL2 ;SEPW1 (plays a role as a glutathione (GSH)-dependent antioxidant and may be involved in a redox-related process and cell cycle progression); andRPN2 .Genes associated with sensitivity that were present at a frequency of > 0.7 included:VPREB3 (a gene involved in B cell maturation);ZBTB44 (a zinc finger protein involved in nucleic acid binding);BCL7A ;PTPN18 ;VPREB1 (a gene that belongs to the immunoglobulin superfamily and involved in early stages of B cell development);LTB ;SARNP (possibly involved in cell cycle progression);CD79B (encodes the Ig-beta protein of the B cell antigen component); SPIB ;APBB1IP ;ANKLE1 (endonuclease that probably plays a role in the DNA damage response and DNA repair); andLIMD2  .−10) This is supportive of a meaningful functional relation between the genes identified by EN modeling. The list of genes associated with sensitivity  was enriched for genes involved in chromatin organization  and overall for gene products located in the nucleus . The genes associated with resistance were also more interconnected (based on STRING) than expected by chance . The top biological processes enriched were extracellular matrix organization (FDR = 0.0002), and endomembrane system localization . Members of the lysosome pathway (KEGG 04142) were also enriched in the genes more expressed in resistant cell lines (FDR<6.10−5). A full list of enrichment terms and values can be found in Annotation of the genomic modeling output using STRING to identify functional networks and enrichment of biological processes revealed that the genes associated with sensitivity present with more connection in STRING than expected by chance , Copy Number (CN), RNA expression data and mutation (MUT) data as input together with IC50s. Genes are stratified based on sensitivity to MN-anti-miR10b.(XLSX)Click here for additional data file.S2 DatasetThe linear regression Elastic Net results were stratified according to their enrichment score and grouped by annotation to arrive at common biological processes explanatory for sensitivity to MN-anti-miR10b.(XLSX)Click here for additional data file.


PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that  Lung cancer is the most common cause of cancer related mortality worldwide, accounting for approximately 1 in 4 cancer deaths . About 8in vitro and in vivo models of colorectal cancer [in vitro [in vitro and in vivo models of human cancers. Silencing PKM2 specific RNA decreased cell viability and increased apoptosis in several cancer cell lines in vitro and regressed tumor volumes by delivering them in in vivo mouse models of hepatocellular and ovarian cancer [Targeting colorectal cancer cell lines with gefitinib (a tyrosine kinase inhibitor) or oxaliplatin revealed that nuclear PKM2 contributed to therapeutic resistance l cancer , 18. Furin vitro . As manyn cancer . Addition cancer , 21–23. n cancer . As PKM2n cancer . PKM2 isn cancer .shRNA-PKM2) might influence NSCLC growth kinetics. Here, we tested whether SMI and shRNA-PKM2 influence NSCLC growth kinetics in vitro and in vivo. Our results demonstrate that PKM2 is primarily expressed in the cytosol of NSCLC cells and xenograft tumor cells. Both approaches (SMI and shRNA-PKM2) not only inhibited the PKM2 enzyme activity and cell proliferation in NSCL cells, but also decreased PKM2 mRNA expression and ultimately led to cell killing by apoptosis. Additionally, our results showed a unique subcellular compartmentalization of PKM2 in NSCLC cells and xenografts upon treating them with anti-PKM2 therapies in vitro and in vivo indicating that PKM2 expression may serve as a biomarker of therapeutic response in NSCLC.Because patient-derived and established NSCLC cell lines showed overexpression of PKM2 and PKM2 enzyme activities, we hypothesized that inhibiting enzyme activity by small molecule inhibitor (SMI) or gene silencing by shRNA (shRNA (AGAGCCTACCTGTATGTCAAT) and control plasmids (AGAGCCTACCTGTATGTCAAT) were purchased from Qiagen  and transfected by using Attractene transfection reagent (Qiagen) as suggested by the manufacturer.We studied nine patient-derived NSCLC cell lines, which were previously established at the Earle A. Chiles Research Institute, Portland, OR. The two cell lines established at the NCI (H1299 and H358) and 3 normal lung fibroblast cell lines  were purchased from ATCC and were maintained in the ATCC recommended complete medium. These cells lines were purchased from ATCC within first two years of undertaking experiments and if not their identity was confirmed by short-tandem repeats (STR) profiling. The nine cell lines from the Earle A. Chiles Research Institute were cultured in complete medium with 10% FBS. A small molecule inhibitor (SMI) of the PKM2 enzyme inhibitor  phenyl 1–2,5,-dimethylpyrole) was purchased from ChemDiv  and dissolved in PBS for treating the NSCLC and normal lung cell lines. For PKM2 targeting in NSCLC cell lines, 2 incubator using RNA-easy mini prep kit (Qiagen) following the manufacturer’s instructions and amplified using forward; CTATCCTCTGGAGGCTGTGC and backward; CCAGACTTGGTGAGGACGAT primers for human PKM2. β-actin gene specific primers (forward: TGAGAGGGAAATCGTGCGTG and backword TGC TTG CTG ATC CAT ACC ATC TGC) were used to amplify β-actin as a house keeping gene for quantitative expression of PKM2 mRNA [Total RNA from 3 normal lung and 11 NSCLC cell lines was extracted after incubating them with different concentrations of PKM2 inhibitor (1 to 80 μM) for 48 hours at 37°C in a COKM2 mRNA , 28. BriPyruvate kinase activity in control, SMI-treated or shRNA-transfected NSCLC cells was measured by a continuous assay coupled to lactate dehydrogenase (LDH). To assess activity in cell lysates, cells were treated with SMI for the indicated time and lysed in NP40 containing 25 μM Tris/Cl pH 7.4 lysis buffer immediately before measuring pyruvate kinase activity as described previously , 29. BriCell viability of NSCLC cell lines was measured by trypan blue exclusion technique. The number of viable cells was manually counted using a hemocytometer and expressed as % of untreated cells , 31.shRNA targeted cultures was measured in vitro as described previously [shRNA-PKM2 transfected cell cultures.Colony formation of NSCLC cell lines in SMI-treated or eviously . The numshRNA-PKM2 transfected NSCLC cells using earlytox Caspase-3/7 R100 assay kit following manufacturer’s recommendations . The endpoint fluorescence was measured on a SpectraMax M5 plate reader and each value is expressed as arbitrary fluorescence units.Patient-derived and established NSCLC cell lines were seeded in 4 well glass chambered slides and incubated with 0–80 μM of SMI for 5 days. After a brief wash with 1x PBS, the TUNEL positive apoptotic cells were counted as per manufacturer’s instructions . The assay was performed in triplicate and data were shown as mean ± SD. Intracellular caspase3/7 activity was measured from control (untreated), sh-negative control and PKM2 expression in NSCLC cells and xenografts sections was analyzed by ICC and IHC techniques as described previously , 33. ForTwenty million NSCLC or normal lung cells were washed with 1x PBS before processing for subcellular fractionation. Nuclear and cytoplasmic fractions from NSCLC cells and xenografts were prepared using NE-PER nuclear and cytoplasmic extraction reagents . All cytoplasmic and nuclear fractions were stored at -80°C until analyzed for PKM2 or Caspase activity or Western blot analysis.500,000 NSCLC cells were seeded in a 10-cm tissue culture plate and treated with different concentrations of SMI when the cell confluency was between 50–60%. After 48 hours, total cell lysate, subcellular fractions (cytosol/nuclear fractions) were prepared using NE-PER reagents as described above. The supernatant from total cell lysate, nuclear and cytosol fractions were quantitated by Bio-Rad protein assay . Twenty micrograms of protein equivalent lysates were boiled in 4 x SDS sample loading buffer and run on mini-Protean TGX AnyKd gel (Bio-Rad). The proteins were transferred to nitrocellulose and Western blots were performed using rabbit monoclonal anti-PKM2 antibody  and incubated with IR Dye 800CW near-infrared fluorescent goat anti-rabbit IgG secondary antibody . The membrane was washed three times and scanned using Odyssey CLx system .Female nude nu/nu mice aged between 6 and 7 weeks were housed in a temperature-controlled, pathogen-free animal facility with 12-hour light and dark cycles. All animal studies were conducted under approved protocol #1992–42 by the CBER Institutional Animal Care and Use Committee (ACUC) in accordance with the principles and procedures outlined in the NIH Guideline for the Care and Use of Laboratory Animals. All animal experiments were performed under ABSL-2 conditions in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were monitored daily with increased monitoring at a minimum of twice daily during experiments. The analgesics were not used to avoid their possible interference in PKM2 signaling and translocation to tumor cell organelles and in tumor microenvironment. Euthanasia before tissue harvest was by ketamine/xylazine overdose while euthanasia due to body weight loss or termination of study was by carbon dioxide inhalation in a chamber where the carbon dioxide was from a cylinder source delivered by Euthanex equipment .Five million H358 cells were injected subcutaneously into the right flank of each mouse. When tumors reached 5 mm in diameter, H358-tumor-bearing mice were injected with different doses of PKM2 inhibitor  once a day on alternate days for 12 days  by i.t. injection. Tumor size was measured weekly and overall sacrifice time (OST) of the animals was calculated based on the sacrifice of mice when tumors reached 2 cm in diameter, according to NIH animal guidelines. Mice were weighed at day 10 and day 25 after PKM2 inhibitor treatment. Experiments were repeated twice.6shRNA-PKM2 gene silenced or negative control plasmid transfected H358 cells were implanted in six-week-old female nu/nu mice, weighing 17-20g for developing xenografts as described above. Before implanting the cells, they were either PKM2 gene silenced, mock-transfected or negative control plasmid transfected and selected by using G148 antibiotic. The transfected NSCLC cells were tested for PKM2 RNA and protein by RT-qPCR and IFA assays. For these experimental differences of transfection and selection involved in before implanting the cells in mice, the control group of mice was different compared to SMI treated mice, which were labeled as non-silenced mice in which negative control sh-RNA plasmid was used. The therapeutic response and OST were evaluated between these two groups. The xenografts in mice, which responded to anti-PKM2 therapy (either SMI or shRNA-PKM2) were referred to regressing tumors and those not responding were termed as non-regressing tumors.Similarly, 5x10Statistical analysis was performed using Student’s t test for comparison between two groups or ANOVA among more than two groups of mice using Graph Pad Prism program . Survival curves were generated by the Kaplan-Meier method and compared by using the log-rank test. The level of statistical significance was set at a P value of ≤0.05.We observed that all patient-derived and established NSCLC cell lines significantly overexpress PKM2 mRNA ranging from 5.3 to 16.5 times greater than normal lung cell lines  and a marginal decrease in normal lung cell lines (shRNA-PKM2 cell lysates. We observed that no treatment (none) and shRNA negative control lysates showed similar values, and therefore pooled together in Based on RT-qPCR, PKM2 enzyme activities and ICC results, we hypothesized that PKM2 may have a critical role in NSCLC cell growth. As shown in ll lines  compared50 of SMI (concentration of SMI causing 50% inhibition of NSCLC cell line growth) ranged between 30–65 μM  in untreated NSCLC cell cultures. In contrast, treatment of NSCLC lines with SMI for 48 hours or sh-RNA-PKM2 resulted in a decrease in cytoplasmic enzyme activity with a concomitant rise in nuclear PKM2 enzyme activity  in SMI treated or shRNA-PKM2 NSCLC cells compared to respective controls. In contrast, three normal lung fibroblast cell lines failed to show such increase in Caspase 3/7 activities .To investigate whether PKM2 inhibition by SMI or r growth . Howeverr growth . In contshRNA-PKM2 during the course of the experiment. We observed that there was no significant difference in general appearance and body weight among four groups of animals in both tumor models (data not shown).We carefully monitored the general condition and body weight of mice to evaluate the general toxicity of SMI and To determine whether PKM2 inhibition modulates subcellular compartmentalization, we evaluated cytoplasmic and nuclear staining by performing the experiments on two different occasions resulting in four different readings to evaluate the extent of intensity for PKM2 immunostaining by viewing the specimens at 200 x magnification to score percent positive fields in a blinded fashion for statistical significance. PKM2 intensity scores of ≥ 2+ were considered positive, while ≤ 1+ were considered negative. We observed a very high concordance (exact 95% CI = 72.8–91.3) among the four sets of readings in both data sets (83.3%) obtained from each independent experiment, where scores 3+ and 4+ were combined as 3+, while scores 1+ and no score  were considered as 1+. These results demonstrate high precision in the IHC measurement for cytoplasmic and nuclear staining. For the remainder of the analysis, discordant scores were resolved by assigning the majority score and when there was an equal number of discordant scores, the lower score was used. For the rest of the analysis, all discordant cases in PKM2 were coded as negative.shRNA-PKM2 therapy demonstrated a significant decrease in cytosolic PKM2 in shRNA-PKM2 treated mice xenografts (regressing) compared to parental or non-responding tumor xenografts (non-regressing) (P≤0.01). Immunostaining for nuclear PKM2 levels was ≤1+ with ~10% of the positive fields in most of the regressing tumors  showed a statistically significant trend  compared to negative values of PKM2 (≤ 1+) (Nuclear PKM2 was identified as a merged immunostaining of PKM2 (red) with DAPI (blue) staining as described in materials and methods. As shown in g tumors . In contg tumors . But, >92 (≤ 1+) .in vitro. These treatments affected both PKM2 enzyme activity and mRNA transcription, which subsequently led to reduced levels of PKM2 protein, as demonstrated by Western blot analysis. In contrast, three normal lung cell lines, which showed lower enzyme activity and mRNA, did not show such a dramatic response to both types of anti-PKM2 strategies. Further, we observed that PKM2 targeting with SMI or shRNA-PKM2 significantly affected cell viability in all NSCLC cell lines and impaired the ability to form colonies in vitro, suggesting that PKM2 enzyme activity is important for tumor growth in NSCLC cells. The normal lung fibroblast cell lines failed to show such changes after targeting PKM2. Previously published results have shown that PKM2 can interact with mutant EGFR and HSP90 and prolonged the protein half-life of EGFR in lung cancer cells [in vitro and in vivo.We have demonstrated overexpression of PKM2 in patient-derived and established NSCLC cell lines. These cell lines not only overexpressed PKM2 protein but also high PKM2 enzyme activity in >55% of the cells with > 2+ immunostaining intensity. The overexpressed PKM2 in NSCLC is inhibitable in a concentration-dependent manner with either a drug or gene silencing by shRNA er cells . It is aer cells . Our prein situ in the tumor microenvironment [PKM2 in colon carcinoma cell lines led to a decrease in PKM2 activity and an increase in the pyruvate kinase substrate phosphoenol pyruvate, leading to an increased macromolecular biosynthesis required for rapid proliferation; suggesting that PKM2 influences the metabolic state of the cells and has a role in tumor maintenance and growth in vivo [Elevated PKM2 is also associated with disease aggressiveness and poor overall survival of patients with signet ring cell gastric cancer , gall blironment , 38, 39.ironment , 41. Cor in vivo , 43. In  in vivo . PKM2 is in vivo , 29.shRNA-PKM2 affected cell viability and clonogenicity of NSCLC cells in vitro, which confirmed our results that PKM2 is a growth supportive factor We further investigated the mechanism for decreased cell viability and clonogenicity in PKM2 targeted NSCLC cells. We observed that SMI and shRNA-PKM2 both downregulated PKM2 mRNA and decreased PKM2 activities, which correlated inversely with the number of apoptotic nuclei in targeted tumor cell populations. These changes are also associated with increased Caspase 3/7 activities in NSCLC cells upon treating them with SMI or shRNA-PKM2, which is a hallmark of apoptosis suggesting that SMI or shRNA-PKM2 affected the cell viability by inducing Caspase 3/7 mediated apoptosis. Similar observations have been made by Goldberg and Sharp, who showed that targeting PKM2 with encapsulated siRNAs resulted in decreased cell viability and increased apoptosis in 10 members of the NCI-60 panel representing multiple types of cancer cell lines while having little effect in normal fibroblasts or endothelial cells and tumor regression in vivo mouse model [shRNA-PKM2 induced apoptosis in vitro and in vivo human lung cancer xenografts treated with docetaxel, which suggested that PKM2 could, to some extent, reverse chemotherapy resistance to cancer therapy [Our data demonstrated that targeting PKM2 with SMI and se model . It was  therapy , 46.shRNA-PKM2 in vitro. We observed that PKM2 targeting by both strategies had decreased cytoplasmic and increased nuclear PKM2 enzyme activity in patient-derived established NSCLC cell lines, which also corroborated with PKM2 expression examined by ICC assay in vitro. Importantly, our in vivo mouse results also demonstrated such compartmentalization, which could differentiate animals responding to the PKM2 targeted therapy. Interestingly, the regressing xenografts had lower PKM2 in cytoplasm and nucleus while non-regressing tumor specimens showed a significant and dramatic increase in nuclear PKM2.Interestingly, we identified a distinct subcellular compartmentalization of PKM2 enzyme activity and protein expression in cytoplasm and nucleus after targeting PKM2 by SMI or in vitro, PKM2 inhibitor did not mediate significant effects on in vivo tumor growth in two NSCLC tumor xenografts and did not show statistically significant improvement of survival compared to control mice. The reason for lack of SMI activity in vivo is not clear. It is possible that PMK2 inhibitor levels, being a small molecular weight compound of 231.25 Da, were not retained in the tumor bed at a high enough level to exert a measurable anti-tumor effect. In contrast, PKM2 was silenced robustly by shRNA in tumor cells before xenograft development, which showed significant anti-tumor effects compared to non-silenced and mock-transfected xenografts. Because gene silenced and mock-transfected xenograft bearing mice were sacrificed on day 120 per our Institutional Animal Care and Use Committee (ACUC) protocols, the survival curves were found non-significantly separated in mock-transfected and PKM2 silenced group mice were not significant. The percent survival in mock-transfected and non-silenced mice was also non-significant as non-silenced group mice received transfection reagent without any DNA as additional control compared to negative control DNA in mock-transfected group mice. Similar studies of silencing PKM2 in colorectal cancer cell lines modulated PKM2 to the nucleus in response to oxaliplatin treatment sensitivity and characterized chemoresistance [Despite its anti-proliferative activities sistance . It was sistance .in vitro or in vivo models of different types of human cancers. It is likely that PKM2 can have limited role as a biomarker of therapeutic response in certain types of cancer with different pathological grade and clinical stage (TNM). Additional studies in larger sample size of cancer subjects with different pathological grade and clinical stage (TNM) can potentially establish its meaningful role as a biomarker of therapeutic response in clinical settings. Why PKM2 is compartmentalized is not clear. PKM2 has been reported to be localized in the cell nucleus as a dimer that functions as an active protein kinase; whereas, PKM2 in its tetrameric form is localized in the cytosol and is an active pyruvate kinase [The significance of variable PKM2 subcellular distribution is not known. Our results show that when PKM2 is downregulated by SMI or gene silencing, subcellular compartmentalization of PKM2 occurs. It is also not known that other types of PKM2 inhibitors critically play a similar role in subcellular compartmentalization of PKM2 in NSCLC e kinase . It is pS1 FigImmunocytochemical assay was performed to evaluate the intensity of PKM2 immunostaining in NSCLC cells using rabbit monoclonal anti-PKM2 antibody and alexa 595 conjugated goat anti rabbit IgG antibody. The immunostained cells were viewed at 200X magnification.(TIF)Click here for additional data file.S1 TableExtent of ICC was evaluated as ≤ 1+, weakly positive; 2+, strongly positive; 3+ stronger;4+, strongest. Percent positive field was counted after viewing PKM2positive cells at 200X magnification and shown in the parenthesis.(DOC)Click here for additional data file.S2 TableExtent of ICC was evaluated as ≤ 1+, weakly positive; 2+, strongly positive; 3+ stronger;4+, strongest. Percent positive field was counted after viewing PKM2positive cells at 200X magnification and shown in the parenthesis.(DOCX)Click here for additional data file.S3 TableExtent of ICC was evaluated as ≤ 1+, weakly positive; 2+, strongly positive; 3+ stronger;4+, strongest. Percent positive field was counted after viewing PKM2positive cells at 200X magnification and shown in the parenthesis.(DOC)Click here for additional data file.


In MG, highest T-cell responses were noted against recombinantly expressed Epsilon 1-221. Among the synthetic peptides Epsilon 201-215 showed the most prominent T-cell response and represented the peptide with the most remarkable difference between MG and HD. Taken together, prominent T-cell responses against the ε subunit of the human AChR indicate an important role in the pathogenesis of MG.The human acetylcholine receptor (AChR) is well characterized as the target antigen in myasthenia gravis (MG). Pathogenic antibody responses against the AChR alpha-chain have been investigated extensively and are of diagnostic and prognostic value. However, less is known on the pathogenetic relevance of T-cell responses against epitopes of the different AChR chains . Using an enzyme-linked immunospot (ELISPOT) assay we measured T-cell responses against recombinant fragments and synthetic peptides of the  Both antibody responses as well as B-lymphocyte activity have been investigated extensively in MG and are of great diagnostic and prognostic value.The human nicotinic acetylcholine receptor (AChR) composed of five subunits  autoantibody production is T helper cell-dependent. Although MG is considered a prototypic paradigm for an antibody-driven autoimmune disorder, the pathogenetic importance of T-helper cells is well appreciated. Several studies have been performed comparing T-cell responses involving the man AChR –8. The εc thymus . In accoferation , 10. Conxpressed .ε subunit while no significant differences were notable against alpha subunit epitopes.We used an enzyme-linked immunospot (ELISPOT) assay to determine T-cell responses against recombinant fragments and synthetic peptides of the human AChR. In accordance with previous observations we found prominent T-cell responses against the Peripheral blood lymphocytes (PBL) were obtained with informed consent from patients with generalized or ocular MG (n=15) or healthy donors . PBL were isolated by density centrifugation and were either frozen immediately and thawed for analysis or used directly.α and ε subunit polypeptides were synthesized by PCR on cDNA prepared from total RNA of human calf muscle as described elsewhere [ E. coli and purified by SDS/PAGE with a standard protocol [α subunit [ε subunit.Human lsewhere . Recombilsewhere . Fragmenprotocol , 12. AlpPeptides were synthesized by solid-phase Fmoc-chemistry on an automated peptide synthesizer for multiple peptide synthesis as described previously . Epsilonγ-secreting T-cells using an ELISPOT (enzyme-linked immunospot) assay as described previously [γ monoclonal antibody (mAb) . After washing and blocking the plates with culture medium , fresh or freshly thawed PBL from MG patients and HD were incubated for 20 h in duplicate in the presence or absence of human AChR antigens or controls (1 μg/ml). Concanavalin A  was used as positive control. Using biotinylated anti-human IFN-γ mAb , streptavidin-alkaline phosphatase (Mabtech), and BCIP/NBT as substrate (Sigma), antigen-specific IFN-γ secreting T lymphocytes were visualized and counted on a dissecting microscope. Results are calculated and assigned as the numbers of IFN-γ-secreting events among 106 PBL minus the corresponding numbers of events per 106 PBL without antigen.We measured frequencies of interferon (IFN)-eviously . Microti t-test was performed for statistical analysis. A p value of < 0.05 was accepted to be significant.Student'sγ secreting T-cells after stimulation with AChR fragments  were not observed see . In an a subunit . Althougjunction .γ ELISPOT method may provide a valuable tool to measure AChR-specific T-cell responses in MG. In MG patients who tested positive, T lymphocytes specific for epitopes of the AChR ε subunit may be a target for therapeutical intervention in MG.We conclude that the IFN-


Plasmablastic lymphoma (PBL) is a rare aggressive lymphoma. Although it was first described in HIV- (human immunodeficiency virus-) infected patients, PBL has been diagnosed in patients with other immunodeficiencies as well as in immunocompetent patients. PBL immunohistochemically expresses plasmacytic markers and lacks pan B-cell markers. The cells of origin of PBL are considered to be plasmablasts. MYC gene rearrangement and MYC overexpression are frequently found in PBL, but the pathogenesis of PBL is yet to be elucidated. Here, we report a case of composite lymphoma of PBL and diffuse large B-cell lymphoma (DLBCL); that is, PBL in the urinary bladder and DLBCL in the nasal cavity occurred simultaneously. We extracted DNA from the two lymphomas for polymerase chain reaction and sequenced the amplified immunoglobulin heavy variable genes and the complementarity-determining region- (CDR-) 3. The sequence of the CDR3 region of both tumors matched. MYC rearrangement was found in the bladder tumor but not in the nasal tumor. The patient was treated with R-CHOP , and durable remission had been obtained. The results of the DNA analysis indicated that both PBL and DLBCL emerged from common postgerminal B cells. This case may help to elucidate the pathogenesis of PBL. Plasmablastic lymphoma (PBL) is a rare aggressive lymphoma that was first described in 1997 as a new clinical entity related to human immunodeficiency virus infection and is recognized as a variant of diffuse large B-cell lymphoma (DLBCL) .Plasmablasts are considered to be the cells of origin of PBL, but the mechanisms of tumor evolution remain unclear . Here, wA 37-year-old previously healthy man presented with swelling of the right nasal root and frequent urination. Otolaryngologic examination revealed thickened mucosa inside the right nasal cavity. Despite the use of both antibiotics and antihistamines, the symptoms deteriorated and the patient underwent diagnostic biopsy of the nasal mucosa. Hematoxylin and eosin (H&E) staining showed diffuse proliferation of atypical lymphoid cells with a prominent nucleolus and clear cytoplasm. Immunohistochemical staining revealed that the atypical cells were positive for CD20 and negative for CD138 . These cTo evaluate the cause of frequent urination, ultrasonography of the urinary bladder was performed, and it revealed numerous irregular surfaced masses scattered on the wall of the urinary bladder. Biopsy specimens of one of the tumors showed the proliferation of atypical lymphoid cells with abundant basophilic cytoplasm and pleomorphic nucleoli. Immunohistochemical staining indicated that these atypical cells were negative for CD20 and positive for CD138 . These cTo identify the cell type of origin of the two lymphoma subtypes occurring simultaneously in one patient, we extracted DNA from frozen sections of each biopsy sample for polymerase chain reaction and sequenced the immunoglobulin heavy variable (IGHV) genes and the complementarity-determining region- (CDR-) 3, as previously described , 5 see s. The seThis case was diagnosed as composite lymphoma of PBL and DLBCL and classified as stage 4A and IPI high/intermediate . The patient underwent eight courses of R-CHOP  with four intrathecal infusions of methotrexate, cytarabine, and hydrocortisone to prevent central nervous system relapse, achieving a complete metabolic response based on PET/CT. Four years after completion of chemotherapy, he developed a soft tissue tumor on the cheek, and a biopsy specimen revealed recurrence of DLBCL.PBL is an aggressive lymphoma with the distinct morphologic features of large immunoblasts. The characteristic immunophenotype usually seen in PBL is the expression of plasmacytic markers, including CD138, CD38, and IRF4/MUM1, and the absence of pan B cell markers, including CD20 and PAX5. CD45 is weakly expressed or absent, and CD79a is often expressed in PBL . The blaThe cells of origin of PBL are thought to be plasmablasts. Plasmablasts are precursor plasma cells derived from activated B cells. Several studies, including gene expression profiling studies, have investigated the pathogenesis of PBL, but it remains unclear. PBL was first reported in HIV patients, and EBV is detected in tumor cells of 74% of patients with HIV-associated PBL . EBV, huMYC gene rearrangement is detected in up to 60% of PBL cases, and a common partner of the MYC gene is the immunoglobulin gene , 14. GenDNA sequence analysis in our case revealed that PBL in the urinary bladder and DLBCL in the nasal cavity had matched sequences at the CDR3 region of IGHV. These results indicated that both PBL and DLBCL were derived from the same clone. The clonal relation between PBL and low-grade lymphoma has been demonstrated in a case series . Marini R-CHOP is a standard treatment for patients with DLBCL, and two-thirds of nonelderly patients with advanced-stage DLBCL can be cured with 6 to 8 cycles of R-CHOP. A standard treatment for PBL has not been established due to the rarity of the disease and its aggressive clinical course. Median survival time for PBL patients is estimated to be less than 1 year. A standard-dose CHOP-like regimen is considered inadequate because it provides the least possibility of cure . The Nat


TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas.Whole-genome doubling is the second most frequent genomic event, after  A century ago, Theodor Boveri formulated the hypothesis that tetraploid cells are a transient stage during oncogenesis between the diploid and aneuploid states . The mosTetraploid cells may be generated in three ways: endoreplication, cytokinesis failure and cell fusion. Endoreplication is a mechanism in which cells increase their genomic DNA content without dividing  and contThe fusion of myoblasts is the central mechanism for muscle differentiation, leading to a non-proliferating syncytium, thus ensuring the integrity of the genome. We hypothesized that this physiological mechanism could be diverted to proliferation upon activation of oncogenes. This tetraploidization state would lead to rearranged aneuploid genome that characterized pleomorphic sarcomas .CDK4 and hTERT introduction, see Material and Methods) human myoblast cell lines were transfected with plasmid CFP-Hygromycin (A8 CFP) or tdTomato-Blasticidin (A8 Tomato). After 72 h of co-culture, double antibiotic selection was added in order to select hybrids formed by spontaneous fusion. Six hybrid cell lines were established (H1 to H6) from distinct clones, each arising from one fusion event. All hybrids were mononucleated and expressed dual fluorescence, tdTomato and CFP, thus validating their hybrid nature  showed an increase in proliferation capacity compared to the parental cell lines B.Myoblasts are defined by their capacities to fuse to differentiate into myotubes under specific conditions. After 14 days of differentiation, parental cell lines and two hybrids, H1 and H5, had formed multinucleated myotubes C.The other hybrids did not survive in these culture conditions  or formed round multinucleated cells (H4). A8 Tomato, H1, H2 H4 and H6 formed colonies in soft agar. However, apart from H4, hybrids did not form more colonies than the parental cell lines in non-adherent conditions D. In regTo investigate the DNA content of hybrids, we first evaluated their ploidy  to 4N (H6) and was heterogeneous in some clones . To test whether the DNA content could evolve with passages, we also re-evaluated the ploidy at later passages  and PS100 (melanoma marker) and had a strong expression of desmin and a focal expression of myogenin . All tumResults above show that LP cell lines developed tumors faster than EP ones and that hybrid cell lines evolve in culture conditions leading to cell lines with recurrent and specific chromosome number variations. Our hypothesis was that the genome of these LP cells is fit for oncogenesis, while EP cell lines need to acquire more instability to become competent for it. To test this, we studied the genome of tumors developed from EP and LP hybrid cell lines.As expected, LP tumors did not have a different genomic index than the cell lines they derive from, so the genome did not harbor extra chromosomic rearrangements after in vivo engraftment A. In conSince chromosomic alterations tended towards the same pattern after in vivo experiments with EP and LP cell lines, we hypothesized that all the cells moved to a specific genomic remodeling, resulting in a tumorigenic transcriptomic program. To test this hypothesis, we performed RNA sequencing of parental, EP and LP hybrid cell lines and three tumors from LP cell lines. PCA analysis separates samples into four groups: parental cell lines, EP cell lines, LP hybrids and cell lines derived from three tumors . Then, wGenome remodeling upon fusion was then analyzed by whole genome sequencing (WGS) in three samples. This highlighted three types of structural variants in addition to CNVs A: (i) inDMD in 82% of cases (DMD deletions were homozygous. Since DMD inactivation by deletion has been reported to be a driver event in sarcomas with myogenic differentiation [DMD deletion was detected in 2/3 samples subjected to WGS (H2-LP-Tumor1 and H2-LP-Tumor3), so we were able to define three fusion points in DMD and to validate these deletions by PCR and Sanger sequencing  did not express Dp427, or only faintly, in proliferation conditions, whereas the expression increased in conditions of muscle differentiation. Second, tumors with the DMD deletion (H2-LP-Tumor1) did not express Dp427 in proliferation and differentiation conditions. The third group was composed of tumors that displayed a DMD deletion  and maintained a weak expression of Dp427 in differentiation conditions. In this group, a heterozygous deletion (H4-LP-Tumor3) and/or a heterogeneous representation of DMD deletion in the tumor could be an explanation for the weak expression of Dp427.Protein analysis  showed tDMD deletions did not target the Dp71 ORF and this isoform has been demonstrated to be essential for proliferation in myogenic cancers [ cancers ,35. Our DMD deletion), Dp427 (green) was detected by immunofluorescence on the FFPE tumor at the membrane of the cells as well as all the other isoforms (red)  B. In sam nucleus .DMD deletions suppressed Dp427 isoform expression and all the other isoforms, including Dp71, were detected in the nucleus (H2-LP-Tumor1).To sum up, when Dp427 was strongly expressed, it was located to the membrane with all DMD isoforms (H4-LP-Tumor1). In contrast, DMD deletion is thought to occur specifically in sarcomas with myogenic differentiation including leiomyosarcomas (LMS), we looked at the localization of the different isoforms of DMD in LMSs  and in vivo (for the early passage hybrids). It is likely that these specific losses and gains are necessary for tumor development in this specific myogenic context . All theDMD deletions, likely related to expression/replication conflict under in vivo pressure selection. This kind of alteration has been described as “replicative stress” [The third phase of genome evolution might correspond to the acquisition of punctual focus alterations, i.e., 5′- stress” ,39. The  stress” ,40. Prog stress” . Genes a stress” ,43 and cDMD structural alteration (deletions). DMD is one of the longest human genes (2.2 Mb). Spatial and temporal overlap of transcription and replication machineries in the late S phase triggers DNA break spots called common fragile sites (CFSs), particularly in long genes [DMD deletions are then detected because the locus fits these features and deletions have a tumor suppressor impact on oncogenesis [The development of myoblast hybrid tumors is associated with highly frequent ng genes . This inng genes . We showogenesis .DMD abrogates the expression of the Dp427 isoform, always preserving Dp71. Even if we cannot definitively rule out the possibility that cell cycle status could account for Dp71 delocalization this is unlikely since we observed, both in hybrid cell lines and in human tumors, that Dp427 loss is strongly associated to Dp71 nucleus relocation. Dp427 is a tumor suppressor [DMD deletion is associated with metastatic evolution [Deletion of ppressor  whereas ppressor , demonstppressor ,35. In eppressor . Dp71 isppressor  and is lppressor .One of tIn conclusion, cell fusion, the event involved in myoblast differentiation is a way to induce in a single event genomic remodeling and genetic aberrations leading to tumor development. Together with oncogene activation upon mutation, myoblast fusion generates a model reflecting the evolution of the genome following whole genome doubling and could account not only for the aneuploidy commonly found in pleomorphic sarcoma but also, and more specifically, for the targeted genomic alterations specific to histotypes.CDK4 and hTERT) were kindly provided by Bénédicte Chazaud. Immortalization of human myoblast were performed as previous described [2. Parental cell lines were generated by lentiviral infection using hygromycin-CFP and blasticidin-tdTomato plasmids (kindly provided by Dr. Richard Iggo). Tumor cell lines obtained from xenografts were obtained after 24 h of collagenase treatment and then cultured in the medium described above. Passages of cell lines described as “early passages hybrid” range from passages 13 to 20 after selection of clones; passages of cell lines described as “late passage hybrids” range from passages 25 to 32.Immortalized human myoblast cell lines  and hygromycin . To validate hybrid selection, dual fluorescence was evaluated by microscopy  and flow cytometry .Cell lines were resuspended in PBS 1X and fixed with 70% ethanol overnight at −20 °C. Then, cells were washed and a solution of propidium iodide  and RNAse A  was added for 30 min at room temperature. DNA content was quantified with propidium iodide fluorescence intensity by flow cytometry  and analyzed using FlowJo software .® VYB, Miltenyi, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (BD Bioscience).In triplicate, 1000 cells were seeded in a 96-well plate. From day 1 to day 9, cells were harvested and collected in 200 µL of FACS buffer (PBS 1× supplemented with 5% FBS and 2mM EDTA). The number of viable cells was determined by flow cytometry  and transferrin . Medium was changed every two days during the first week and then every day. At 14 days, cells were fixed with 4% PFA and images were taken with Axio Vert.A1 (Zeiss). For western blot and immunofluorescence in muscular differentiation conditions, cells were cultivated for 6 days in differentiation medium.Cells were diluted in 0.35% agar medium solution in order to seed 5000 cells per well. A mix of agarose and cells was left on the top of pre-coated 0.5% agarose. Medium was changed once a week. After 6 weeks, colony number for one field was manually counted using an Axio Vert.A1  at 4x magnification. Each cell line was seeded in triplicate and two fields were counted for each well.Animals were maintained under specific pathogen-free conditions in the animal facility of Bordeaux University, and under opportunist and pathogen-free conditions in the CREFRE US006, Toulouse. Experiments were performed in conformity with the rules of the Institutional Animal Care and Use committee  and all efforts were made to minimize animal suffering.2)/2. Once tumors reached 2000 mm3, mice were sacrificed by cervical dislocation. Tumors were formalin-fixed and nitrogen-frozen, and one part was also used to establish cell lines from the tumor. Survival curves were plotted with Graph Pad Prism ) and statistical analysis was performed with the Log-rank Mantel-Cox test.One million cells in 100 µL PBS 1× were subcutaneously injected into 8-week-old NSG mice. For early passage cell lines, five mice were injected and for late passage cell lines six mice were injected. Tumors were measured every week using a digital caliper and tumor volume was calculated using the formula V =  was performed using the mouse monoclonal antibody against human protein S100 , myogenin , desmin  and pan-cytokeratin . Pictures were captured using a Zeiss Cell Observer Microscope.A 4-µm formalin-fixed paraffin-embedded tissue section was de-paraffinized in three xylene baths for 5 min and then rehydrated in successive baths of ethanol from 100% to 70%. For heat-induced epitope retrieval, slides were incubated for 20 min in a microwave oven in Dako Target Retrieval pH6. Then, they were incubated with primary antibodies for 1 h at room temperature in a humidity chamber and after 1 h with secondary antibodies. Slides were then mounted with Vectashield mounting medium plus DAPI . Images were acquired on a Zeiss Cell Observer Microscope or a LSM 780 confocal microscope (Zeiss). Primary antibodies used for Dp427 were NCL-DYS1  and ab15277 for all the dystrophin isoforms . Secondary antibodies used were Alexa Fluor 488  anti-mouse and Alexa Fluor 594 anti-rabbit .https://github.com/obigriffith/biostar-tutorials/blob/master/Heatmaps/heatmap.3.R). Finally, six groups of genes were identified and a functional enrichment analysis of gene ontology was performed using Bioconductor packages goseq and org.Hs.eg.db.Total RNA from cell lines was extracted with TRIzol reagent (Life Technologies) and purified with the RNeasy MinElute Cleanup Kit  according to the manufacturer’s instructions. RNA concentration was evaluated with Clariostar and quality-checked with an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA sequencing was performed by the Centro Nacional de Análisis Genómico  to obtain more than 20 million paired-end reads with a length of 75 bp. Subsequent CNAG RNA sequencing steps, ERCC spike-in sequencing control quality check and bioinformatics analysis pipeline have already been described . ThereafDNA extractions were performed on cell lines and frozen tumor samples. Genomic DNA was extracted using the standard phenol-chloroform method and quantified using Clariostar. DNA arrays were performed using Affymetrix CytoScan HD Arrays  according to the manufacturer’s instructions. Analysis was performed using the Chromosome Analysis Suite software (Affymetrix) with genome version GRCH37 (hg19). Genomic index was calculated as follows: GI = A²/C where A corresponds to the total number of alterations and C to the number of chromosomes affected by these alterations. To compare genomic index of the different groups, a Mann-Whitney test was performed. Losses and gains of each sample were defined with an average log2(ratio)  ≤ −0.2 or ≥ 0.2, respectively. Thus, penetrance plots were obtained for four subgroups: EP hybrids, LP hybrids, EP tumors and LP tumors.http://broadinstitute.github.io/picard/). Thus, genomic rearrangements were established with GRIDSS [https://samtools.github.io/bcftools/bcftools.html) v1.6 with RAS > 0, AS > 0 and QUAL > 0 criteria.DNA of H2 LP Tumor 1, H4 LP Tumor 1 and H4 LP Tumor 3 were sequenced at CNAG. Whole genome sequencing characteristics were paired-end sequencing with 150 bp reads and an expected coverage of 45×. Sequenced reads were aligned on Human GRCh38/hg38 genome with BWA mem v0.7.17  and PCR A Circos plot  was perfhttp://frodo.wi.mit.edu.proxy.insermbiblio.inist.fr/primer3/). The touch-down (TD) 60 °C program was used . PCR was performed on 50 ng of DNA using AmpliTaqGold® DNA polymerase (ThermoFisher Scientific).For PCR, primers were designed using the Primer 3 program  supplemented by phosphatase/protease inhibitor cocktail . Proteins were collected in supernatant after 15 min of centrifugation at 13,000g and quantified by DC protein assay kit, Biorad. 40 µg of total proteins were loaded on Mini Proteane TGX stain free 4–15% . Transfers on PVDF membrane were performed using a dry transfer system  and membranes were blocked in 5% non-fat dry milk in 0.1% PBS-Tween solution. Membranes were incubated overnight with the primary antibody: mouse anti-Dp427  or Rabbit anti-Dp71  at 4 °C overnight. After wash, membranes were incubated for 1 h at room temperature with appropriate secondary antibodies: anti-rabbit HRP  and anti-mouse HRP . After incubation with chemiluminescent substrate , signals were detected using PXi .In conclusion, cell fusion is a way to induce in a single event genomic remodeling and genetic aberrations leading to tumor development and metastasis progression. This tetraploidization mechanism, with oncogene activation, permits to reflect the evolution of myogenic sarcoma, from aneuploidy to genomic alterations specific to this histotype.


Colon cancer constitutes 33% of all cancer cases in humans and the majority of patients with metastatic colon cancer still have poor prognosis. An important role in cancer development is the communication between cancer and normal cells. This may occur, among others, through extracellular vesicles (including microvesicles) (MVs), which are being released by both types of cells. MVs may regulate a diverse range of biological processes and are considered as useful cancer biomarkers. Herein, we show that similarity in the general chemical composition between colon cancer cells and their corresponding tumor-derived microvesicles (TMVs) does exist. These results have been confirmed by spectroscopic methods for four colon cancer cell lines: HCT116, LoVo, SW480, and SW620 differing in their aggressiveness/metastatic potential. Our results show that Raman and Fourier Transform InfraRed (FTIR) analysis of the cell lines and their corresponding TMVs did not differ significantly in the characterization of their chemical composition. However, hierarchical cluster analysis of the data obtained by both of the methods revealed that only Raman spectroscopy provides results that are in line with the molecular classification of colon cancer, thus having potential clinical relevance. Colon cancer is one of the most common cancers worldwide and in the last 15 years, the number of patients with this type of cancer has increased by 60% ,3. In caIn this study, by using Raman and FTIR spectroscopy, we determined the general chemical composition of four colon cancer cell lines: HCT116, LoVo, SW480, and SW620, which differ in their aggressiveness/metastatic potential ,24. In pTMVs released by four colon cancer cell lines HCT116, LoVo, SW480, and SW620 were isolated from culture supernatants by differential centrifugation. Cell lines differed in malignancy potential, as HCT116 was described as fast growing, SW480-having low metastatic ability, SW620-high metastatic ability, and LoVo-undifferentiated . The isoFor the size distribution measurements, nanoparticle tracking analysis (NTA) was used. As shown in By using two complementary spectroscopic techniques, FTIR and FT-Raman, the general chemical composition of the four colon cancer cell lines and their corresponding TMVs was determined. In the obtained FTIR  and Rama−1 to 3000 cm−1. In the case of TMVSW480 and SW480 cells  and between 3000 cm−1 and 3500 cm−1, were noticed when we compared the TMVsHCT116 and the HCT116 cell lines. When comparing LoVo . Moreover, in the Raman spectra of SW480 cells and TMVSW480, differences in the shape of the spectra and peak shifts originating from DNA, phospholipids, and protein vibrations (800–1100 cm−1) were visible. Furthermore, in the Raman spectra of TMVHCT116 and TMVSW620, absence of the peak corresponding to the C–H groups from cholesterol and cholesterol ester were noticed in comparison to their respective cell lines. The peaks originating from the stretching vibrations of the CH, NH, and OH groups were not observed in the Raman spectrum of the LoVo cell line in comparison with the Raman spectrum of the TMVLoVo. The identified peaks are described in detail in The acquired Raman spectra showed differences in the intensity values as well as in the shape of the spectra between the respective cell lines and TMVs released by them. In the spectra obtained for the HCT116 cells, the Raman intensities in the analyzed range were higher than for their TMVs a. Moreoving LoVo b and SW6ing LoVo d cells weir TMVs c, higher−1), proteins (~1250–1700 cm−1), and lipid (~2700–3000 cm−1) vibrations, respectively.To obtain information about the differences in the structure between cell lines and the corresponding TMVs released from them, the second derivative of the FTIR spectra was calculated . HoweverHCT116, TMVsSW480, and TMVsSW620 were compared with those of their respective cell lines. Furthermore, structural changes in the IR region corresponding to DNA and phospholipid functional group vibrations were observed when we compared the second derivative of the FTIR spectra obtained for the SW480 cell line and its respective TMVs. Indeed, it is well known that in the IR region between 1700–1600 cm−1, there is a vibration of the C=O group, which participates in the formation of intramolecular and intermolecular hydrogen bonds. In this case, the stretching vibrations of the C=O bands in the IR spectra are very sensitive to the formation and changes in the hydrogen bonds between peptide groups [−1; for structures of the β-form, the frequencies are in the range between 1640–1620 cm−1, while for β-sheet structures, it was 1670–1690 cm−1 [−1). This analysis enabled the determination of the protein secondary structure by quantifying the percentage of α-helix and β-sheet structures.While comparing the respective cell lines and their TMVs, the most visible, however very small, structural changes occurred in the IR region corresponding to lipids. Changes in the protein structure were also noticed, when the second derivatives of the FTIR spectra of the TMVse groups ,45. Intr690 cm−1 . To deteHCT116, one component band less was detected than in the HCT116 cells. Moreover, when comparing LoVo cells and the TMVs released by these cells, the TMVLoVo showed one component band less than the parental LoVo cells. Additionally, the component band areas corresponding to the α and β structures were different when comparing cells with their TMVs. The peak area values, percentage, and ratio of α and β secondary protein structures obtained for the analyzed cell lines and their TMVs are described in Similarity in the value of α and β secondary protein ratio structures in tumor cell lines and corresponding TMVs is presented. High similarities in the ratio of α-helix/β-harmonica in HCT116, LoVo, and SW480 cells and their corresponding TMVs have been detected. A similar trend was also observed for the SW620 cells and corresponding TMVs, however, it was less significant.To determine the similarities in the general chemical composition between the analyzed cell lines and their TMVs, we performed hierarchical cluster analysis (HCA) .LoVo (Euclidean distance around 0.25). When we compared Euclidean distance in HCA obtained for Raman and FTIR data, the lower value of this parameter was in FTIR HCA analysis , which showed that the samples included both exosomes and MVs. This heterogenic composition was further confirmed by western blot analysis and the expression of proteins characteristic for both exosomes and MVs. The FTIR and Raman spectra of the HCT116  confirme solvent . The sol2), we cannot observe any IR absorption lines since the molecule cannot change its dipole moment. It has been observed that molecules with a strong dipole moment are typically hard to polarize [HCT116, which could result in the observed distance between the cells and TMVs in the HCA analysis.Interestingly, in the HCA analysis , we founpolarize ,51. AddiMoreover, in the obtained spectra, some vibration peaks were absent while the peaks corresponding to functional groups within the measured samples have been identified. Therefore, using these two complementary methods, a whole chemical structure of analyzed samples was obtained. Differences in the peak-shifts as well as the absence of some peaks were observed in The two isogenic colorectal cancer SW480 and SW620 cell lines were derived from the same patient, the first originating from the primary tumor, whereas the latter was from a metastatic lesion to the lymph node, therefore similarities between these cells and their corresponding TMVs could be expected, as evidenced by several papers ,53,54,55On the other hand, Pablo et al. used Raman spectroscopy to distinguish different colon cancer cell types including SW480 and SW620. They showed that these two cell lines could be distinguished by ratio of α:β secondary protein structure. Furthermore, the PCA-LDA analysis showed that by using this approach, it is possible to distinguish different types of colon cancer cell lines with an accuracy of 92.4 ± 0.4% . These rTo summarize, in this report, through the use of FTIR and Raman spectroscopy, we showed that the chemical composition of TMVs is similar to the composition of the cells from which they are released. We suggest that Raman as well as FTIR spectroscopies could find a potential application for the first rapid chemical identification of circulating TMVs, reflecting the composition of their parental cancer cells. It seems however, that only the data obtained from the Raman analysis were in line with the molecular classification of colon cancer, having thus a potential clinical relevance. Further studies involving other tumor cell lines and their respective TMVs using Raman and FTIR spectroscopies would provide additional information on the usefulness of these methods, which could potentially translate into the clinical setting .TM, Corning, NY, USA) in a 37 °C humidified atmosphere with 5% CO2. All media were supplemented with 10% fetal bovine serum  and gentamicin (50 µg/mL), . The cells were cultured by bi-weekly passages and regularly tested for Mycoplasma sp. contamination with the PCR-ELISA kit , according to the manufacturers’ instructions.Colon cancer cell lines  were obtained courtesy of Prof. Caroline Dive, Paterson Institute for Cancer Research, University of Manchester. Human colon carcinoma cell line HCT116 was obtained from the American Type Culture Collection  and maintained according to the ATCC’s instructions. Briefly, HCT116 cells were cultured in McCoy’s 5A medium , while LoVo, SW480, and SW620 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose content  for 1 h at 4 °C. Pellets were washed twice in PBS to remove FBS and finally re-suspended in serum-free medium. Quantification of TMVs proteins was evaluated by the Bradford method . TMVs were tested for endotoxin contamination by the Limulus test, according to the manufacturer’s instruction  and stored at −20 °C until use. To simplify, TMVs were named according to their cell line of origin  containing the protease inhibitor cocktail (Roche). The concentration of samples was measured using the Bradford kit (Bio-Rad) as per the manufacturer’s instructions. A total of 20 μg of suspended TMVs was mixed with NuPAGE LDS Sample Buffer (4×) and NuPAGE Sample Reducing Agent (10×) . Samples were heated  and electrophoresed in 12% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Next, electrophoresed samples were transferred onto the polyvinylidene fluoride membrane . Then, after blocking for 1 h at room temperature in Tris buffered saline (TBS) with 0.1% Tween-20  and 1% bovine serum albumin , the membranes were incubated overnight at 4 °C with mouse mAb anti-Alix (3A9), rabbit mAb anti-CD9 (D801A), and anti-Flotillin-1 (D2V7J)  diluted 1:1000 as well as rabbit polyclonal Ab anti-CD63 (SIGMA) diluted 1:2000. As a loading control, rabbit anti-GAPDH antibodies  diluted 1:5000 were used. After incubation, the membranes were washed in TBS supplemented with BSA and Tween-20 and incubated for 1 h in room temperature with appropriate secondary antibody: goat anti-mouse and goat anti-rabbit  conjugated with horseradish peroxidase (Santa Cruz Biotechnology). The protein bands were visualized with the SuperSignal West Pico Chemiluminescence Substrate kit (Pierce), according to the manufacturer’s protocol and analyzed with ChemiDoc system (Bio-Rad).Average, modal size, and size distribution of the TMVs were obtained using the NANOSIGHT LM10-HS488FT14 Nanoparticle Characterization System . Briefly, 1 μL of the TMV suspension was diluted 1000× in filtered (0.22 μm) PBS to obtain the total sample volume of 1 mL. Next, approximately 700 μL of the sample was loaded manually into the measuring chamber using an insulin-type syringe, after which the syringe was mounted onto the pump and the sample was delivered at a constant flow rate of 80 units. Next, three one-minute videos were recorded by the sCMOS camera for each sample and used later for analysis using the NanoSight NTA 3.0 analytical software .8 cell/mL were centrifuged for 5 min at 3000 rpm. Subsequently, the cells were washed three times in isotonic solution  to ensure complete removal of trypsin and culture medium. Next, dense cell suspensions were placed onto an attenuated total reflection (ATR) crystal (FTIR spectroscopy) or CaF2 slides (Raman spectroscopy).For FTIR and FT-Raman spectrum acquisition, cells in the concentration 10−1. Each spectrum was obtained by averaging 64 scans recorded at a resolution of 4 cm−1. For statistical validation, three samples for each of the cell lines were measured. Moreover, each sample was measured three times. Baseline correction and vector normalization of the obtained spectra were performed using OPUS 7.0 software .All measurements were carried out on an EXCALIBUR FTS-3000 spectrometer  at room temperature. All spectra were recorded by attenuated total reflection (ATR) with a ZnSn crystal. A total of 0.5 mL of cell containing solution was deposited on the ATR ZnSn crystal. FTIR spectra were recorded between 4000 and 500 cm−1 with a laser power of 1 W. Unfocused laser beam was used with a diameter of approximately 100 μm and a spectral resolution of 8 cm−1. Raman spectra were processed by the Omnic/Thermo Scientific software based on 128 scans. The obtained spectra were normalized using vector normalization in OPUS 7.0 software . Moreover, each spectrum was smoothed using the Savitzky–Golay algorithm. The number of smoothing points was nine.FT-Raman spectra were recorded using a Nicolet NXR 9650 FT-Raman spectrometer  equipped with an Nd:YAG laser (1064 nm) and a germanium detector. Measurements were performed in the range from 150 to 3.700 cmThe secondary protein structure was analyzed by means of curve fitting using MagicPlot software . First, the second derivative spectra were calculated based on the ATR-FTIR spectra to determine the initial peak positioning for the curve fitting. The peaks were fitted using a Gaussian function and the area under the curve was considered at 100%. Each component was expressed as its percentage after fitting.−1–1700 cm−1 and between 2700 cm−1–3000 cm−1. PCA-LDA results had been verified by the leave-one-sample out cross-validated method, which involves leaving all spectra from a single sample out of the model once before assessing performance. This analysis was done using the OriginPro 2015 software . Moreover, to determine similarities between HCT116, LoVo, SW480, SW620 cell lines, hierarchical cluster analysis (HCA) with Euclidean distance was performed in Past software (developed by Oyvind Hammer). HCA analysis was done for the fingerprint region (between 800 cm−1 and 1800 cm−1) for both the Raman and FTIR spectra of the cells and TMVs. For this purpose, 518 points from the FTIR spectra and 259 points from the Raman spectra were analyzed using the paired group (UPGMA) algorithm.For all obtained spectra, vector normalization and baseline correction were applied. These operations were performed using OPUS 7.0 software. Moreover, in each FTIR as well as Raman spectrum, vibrations corresponding to nucleic acid, phospholipids, proteins, and lipids were analyzed. The number of obtained data from FTIR and Raman spectra was very large, therefore, to determine the similarity between the cancer and control cells, principal component analysis-linear discriminate analysis (PCA-LDA) was performed. First, PCA was undertaken, which reduced the dimensionality and the number of data variables, while maintaining as much variance as possible. Next, with the use of the LDA method, a suitable model for grouping data based on independent variables (wavelengths) was developed. Then, the PCA-LDA procedure was performed based on the selected spectral regions: between 1250 cm


Treatment of uveal melanoma (UM) is generally successful, with local primary tumour control being at 90–95%. Localized radiotherapy in the form of plaque brachytherapy or proton beam radiotherapy is the most common treatment modality in the UK. However, the basic mechanisms of radiation response, DNA repair and tissue reactions in UM have not been well documented previously. We have investigated the comparative radiosensitivity of four UM cell lines in response to exogenous radiation sources (both X-rays and protons), and correlated this with DNA repair protein expression and repair efficiency. We observed a broad range of radiosensitivity of different UM cell lines to X-rays and protons, with increased radioresistance correlating with elevated protein expression of ataxia telangiectasia mutated (ATM), a protein kinase involved in the signaling and repair of DNA double strand breaks. The use of an ATM inhibitor in UM cell lines enhanced radiosensitivity following both X-ray and proton irradiation, particularly in cells that contained high levels of ATM protein which are otherwise comparatively radioresistant. In proton-irradiated compared with non-irradiated primary enucleated UM patient samples, there was no significant difference in ATM protein expression. Our study therefore suggests that ATM is a potential target for increasing the radiosensitivity of more resistant UM subgroups. In the adiation ,10. ConsPhoton (X-ray) and proton radiotherapy are effective at inducing the formation of DNA damage, such as DNA double strand breaks (DSBs), which contribute to their therapeutic effect in cancer cell killing. Despite this, there is still a significant biological uncertainty with protons, particularly due to increases in LET at and around the Bragg peak, where the majority of the radiation dose is deposited . Cells aIn this study, we examined the radiosensitivity of UM cell lines following photon (X-ray) and proton irradiation, and identified that the most radioresistant cell lines contain increased ATM protein levels. These UM cell lines can be radiosensitized using an inhibitor targeting ATM. These results suggest a potential therapeutic strategy employing ATM inhibitors, which could be exploited for more effective treatment of UM by radiotherapy.β between the cell lines  polymerase-1, PARP-1; X-ray repair cross-complementing protein 1, XRCC1, polynucleotide kinase phosphatase, PNKP and AP endonuclease 1, APE1) and DNA DSB repair  in extracts from four UM cell lines derived from primary (Mel270 and 92.1) and metastatic (OMM1 and OMM2.5) tumors. Variability was observed particularly in the levels of ATM, XRCC1, PNKP and Pol ll lines . HoweverWe subsequently analyzed the comparative radiosensitivity of the cells to photon (X-ray) and proton irradiation by clonogenic survival assays. OMM2.5 and Mel270 were ~3.5–6.2-fold (across the dose response) more radioresistant to X-ray irradiation than OMM1 and 92.1 B,C. The We analyzed the comparative kinetics of DNA DSB repair in UM cell lines in response to photon and proton irradiation using neutral comet assays. We observed in response to photons that, surprisingly, all the four UM cell lines analysed  were able to largely repair DSBs within a 4 h period post-irradiation A,C. The n = 32) or non-irradiated . Examples of the degree of staining (from negative up to 65% staining) observed in cells from UM tissues are shown (p = 0.57).Given the range (~1.7–6.0-fold) of different expression of ATM protein in UM cell lines, and apparent links with modulating cellular radiosensitivity to photons and protons A–D, we are shown . There wre shown . Additiore shown . ATM stare shown . Finallyp = 0.33–0.72; one-sample t-test). We consequently analysed the impact of targeting ATM on the radiosensitivity of UM cell lines in response to both photon (up to 4 Gy) and proton (up to 8 Gy) irradiation. We observed that ATM inhibition resulted in increased radiosensitivity of both the OMM2.5 cells (by ~1.7-fold across the dose response) and, more dramatically, the Mel270 cell lines (by ~16-fold), but not the 92.1 cells, to photon irradiation  and proton beam therapy for UM, we have utilized both X-rays and protons to investigate the comparative radiobiology of four UM cell lines.The radiobiology of UM has only been subject to limited investigation. Early studies using single UM cell lines suggested that these are generally radioresistant ,4,5, butAs demonstrated in previous studies, our data support the variability in radiosensitivity between UM cell lines, and that there is no correlation with the UM cell line derivation, be it from a primary or a metastatic tumor. A novel finding of our study is that this degree of variability between UM cell lines is observed following both photon (X-ray) and proton irradiation. In fact, the OMM2.5 and Mel270 cell lines were consistently the most radioresistant, whereas the OMM1 and 92.1 were the most radiosensitive following both X-rays and protons. We also showed that cellular radioresistance appears to correlate largely with the levels of ATM that co-ordinates the signaling and repair of DNA DSBs. Indeed, an ATM inhibitor was able to enhance the effects of photons and protons in reducing clonogenic cell survival in a range of UM cell lines. This suggests a potential therapeutic strategy to enhance the impact of radiotherapy (photons and protons) in UM cell killing. Targeting ATM is currently being investigated to enhance the radiosensitivity of other tumor types, particularly glioblastoma ,19,20, bWe analyzed the expression of ATM in UM tissues, but did not observe any difference in immunohistochemical staining between proton-irradiated and treatment-naive UM, nor any correlation of ATM staining with known poor prognostic parameters or clinical outcomes. Given the small sample size, it is possible that our study was not powered sufficiently to identify any statistical difference. The phosphorylation of ATM and the relocation of the protein into the nucleus have been suggested to act as predictive biomarkers of radiotherapy response ,24,25 an106 brachytherapy but which have subsequently been successfully treated with protons.Interestingly, our analyses found no differences in the kinetics of DSB repair through neutral comet assay analysis in UM cell lines following X-rays, suggesting that there is no direct correlation with DSB repair efficiency and radiosensitivity. This could indicate that the inherent photon (X-ray) radiosensitivity is largely determined by other factors, such as chromatin structure, cell cycle progression, and cell survival mechanisms including apoptosis, autophagy and senescence. This requires further investigation in UM. However, in response to protons, there was a correlation with UM cells displaying reduced DSB repair capacity post-irradiation (OMM1 and 92.1), and their increased radiosensitivity compared to the other cell lines examined . This could alternatively suggest a different spectrum of DNA damage induced by proton rather than photon irradiation, or that the DSB repair pathways utilized  are distinct in their response to the different radiation modalities. Therefore, a comprehensive analysis of the induction and repair of DNA base damage and single strand breaks , and DSBs  following photon and proton irradiation is necessary to further understand this. It was noticeable, though, that lower doses of X-ray radiation (0.5–2 Gy) appeared to have a greater impact on reducing cell survival than protons, when comparing the response between OMM2.5 and Mel270 with OMM1 and 92.1. Given the lack of correlation with DSB repair efficiency, the mechanism through which reduced cell survival  is triggered in response to X-rays at these lower doses should be investigated in more detail. It should be noted that our proton experiments utilized low-LET protons at the entrance dose of a pristine beam, and therefore we would not expect LET, and subsequently DNA damage complexity, to play a role in the differential cellular response compared to photons. Nevertheless, this was recently discussed in the context of head and neck cancer cells , and reqThe following antibodies for immunoblotting were used. APE1, Pol β and XRCC1 antibodies were kindly provided by Dr G. Dianov. PNKP antibodies were also kindly provided by Prof M. Weinfeld. ATM (sc-23921), DNA-Pk (sc-9051), Ku-86 (sc-9034), PARP-1 (sc-53643) and RAD51 (sc-8349) antibodies were from Santa Cruz Biotechnology ; ATR antibodies (ab2905) were from Abcam ; 53BP1 (A300-272A) antibodies were from Bethyl Laboratories ; and actin (A5441) antibodies were from Sigma-Aldrich . 2 at 37 °C, and were authenticated in our laboratory by STR profiling. Cells were irradiated using a CellRad X-ray irradiator , or at the entrance dose of a passive scattered horizontal proton beam line at 60 MeV maximal energy (~1 keV/µm), as previously described [UM cells  were routinely cultured as monolayers in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1× penicillin-streptomycin and 1× non-essential amino acids. All cells were cultured under standard conditions in 5% COescribed .Whole cell extracts were prepared as previously described . BrieflyCells were seeded in 35 mm dishes to ~70% confluence at the time of irradiation. Following irradiation, cells were trypsinized, counted and seeded into 6 well plates, and colonies were allowed to grow for 10–14 days, prior to fixing and staining with 6% glutaraldehyde and 0.5% crystal violet for 30 min. Plating efficiencies for the non-irradiated cells were as follows: OMM1 (~25%), OMM2.5 (~15%), Mel270 (~35%) and 92.1 (~40%). Dishes were washed, left to air dry overnight, and colonies were counted using the GelCount colony analyzer . Relative colony formation (surviving fraction) was expressed as colonies per treatment level versus colonies which appeared in the untreated control. For inhibition experiments, media containing 10 µM ATM inhibitor  or DMSO as a vehicle only control were added onto the monolayer cells for 1 h prior to irradiation. The irradiated cells were then trypsinized, counted and seeded into 6 well plates containing 10 µM ATM inhibitor for a further 24 h. Following this, fresh media only was added until the colonies formed. Statistical analysis was performed using the CFAssay for R package .5 cell/ml, and 250 µl aliquots of the cell suspension were placed in the wells of a 24 well plate. For inhibitor experiments, media containing 10 µM ATM inhibitor (KU-55933) or DMSO as a vehicle only control was added to the cells 1 h prior to trypsinization. Cells in suspension were irradiated with 4 Gy X-rays or protons, and then embedded on a microscope slide in 1% low melting point agarose  in PBS. Slides were incubated for up to 4 h in an humidified chamber to allow for DNA repair, and subsequently cell lysis was performed in buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl pH 10.5, 1% N-lauroylsarcosine, 1% DMSO and 1% (v/v) Triton X-100 for at least 1 h at 4 °C. DNA was allowed to unwind in cold electrophoresis buffer  for 30 min in the dark, before separation of the DNA DSBs by electrophoresis at 25 V, ~20 mA for 25 min. Slides were washed three times with 1 × PBS before being allowed to air dry overnight. The agarose was subsequently rehydrated for 30 min in water (pH 8.0), stained for 30 min with SYBR Gold  diluted 1:10,000 in water (pH 8.0), and air dried again overnight. Cells  were analyzed from the dried slides using the Komet 6.0 image analysis software , and three individual biological replicates were performed.Neutral single cell gel electrophoresis (comet) assays for the measurement of DNA DSBs were performed as previously described ,29,30. Et-test.Sections (4 µm thick) from FFPE UM were deparaffinized and underwent heat induced epitope retrieval at 96 °C, pH 9.0 for 20 min on the Leica Bond RXm, followed by staining using the Leica Bond polymer refine red detection kit, according to the standard manufacturer’s protocols. A mouse monoclonal anti-ATM antibody  was used at 1:100 dilution. Sections were counter-stained with hematoxylin, dehydrated and mounted using a solvent based mountant. Negative controls used mouse IgG1 at the same concentration as the primary antibody. Staining was assessed and scored by three individuals . Statistical analysis was performed using a one sample We have demonstrated that UM cell lines display a broad range of radiosensitivity to photon (X-ray) radiotherapy and proton beam therapy, with an apparent correlation of increased radioresistance with elevated levels of ATM. Whilst no correlation between ATM protein expression in UM tissue samples and an adverse patient outcome was found, inhibition of ATM in vitro was shown to be effective in potentiating the effects of photons and protons in reducing clonogenic survival. Our data therefore suggest that targeting ATM, in combination with radiotherapy, can be a more effective strategy for the treatment of UM.


The drug resistance of first-line crizotinib therapy for ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion non-small cell lung cancer (NSCLC) is inevitable. Whether the administration of immune checkpoint inhibitor (ICI) therapy is suitable for ROS 1 fusion NSCLCs or after the development of crizotinib resistance is still unknown. In this study, five different crizotinib resistant concentration cell lines (HCC78CR1-5) from primary sensitive HCC78 cells were cultured. Ba/F3 cells expressing crizotinib sensitive ROS1 fusion and crizotinib resistant ROS1-G2032R mutation were used to explore the relationship between ROS1 fusion, ROS1-G2032R mutation and programmed death-ligand 1 (PD-L1) expression and the clinical potential of anti-PD-L1 ICI therapy. The signaling pathway net was compared between HCC78 and HCC78CR1-5 cells using RNA sequencing. Anti- PD-L1 ICI therapy was performed on mouse xenograft models with Ba/F3 ROS1 fusion or ROS1-G2032R mutation. HCC78CR1-5 showed more immunogenicity than HCC78 in immune-related pathways. The PD-L1 expression level was remarkably higher in HCC78CR1-5 with ROS1 fusion upregulation than HCC78 primary cell. Furthermore, the expression of PD-L1 was down-regulated by RNA interference with ROS1 siRNAs and up-regulated lower in Ba/F3 ROS1-G2032R resistant mutation than ROS1 fusion. Western blotting analysis showed the ROS1–SHP2 signaling pathway activation in HCC78CR1-5 cells, Ba/F3 ROS1 fusion and ROS1-G2032R resistant mutation. Mouse xenograft models with Ba/F3 ROS1 fusion showed more CD3+PD-1+ T cells both in blood and tissue, and more sensitivity than the cells with Ba/F3 ROS1-G2032R resistant mutation after anti-PD-L1 therapy. Our findings indicate that PD-L1 upregulation depends on ROS1 fusion more than ROS1-G2032R mutation. We share our insights of NSCLCs treatment management into the use of anti-PD-L1 ICI therapy in ROS1 fusion and not in ROS1-G2032R resistant mutation. At present, molecular targeted therapy is an important strategy for the treatment of non-small cell lung cancer (NSCLC) , 2. DrivROS1 fusion NSCLCs are sensitive to crizotinib, but their development of drug resistance is inevitable. As a new drug class, immunology checkpoint inhibitor (ICI) therapies, such as those involving anti-CTLA4 , anti-prCurrently, the pembrolizumab (anti-PD-1) is approved for use as first- and second- line therapy in patients with advanced NSCLC whose tumors express PD-L1 in immunohistochemistry analysis , 13. Nivin vitro and in vivo experiments.The mechanism of the regulation of PD-L1 expression in ROS1 fusion primary tumor and acquired resistance to crizotinib is still unknown. Although many options are available for molecular targeted therapies of primary sensitive or secondary mutation of ROS1 tyrosine kinase domain drug resistance mutation, it remains unclear about the clinical potential of anti-PD-L1/PD-1 ICI therapy on ROS1 fusion and ROS1-G2032R crizotinib resistant mutation. Our aims of this study were 1) to explore the relationship between ROS1 fusion, ROS1 G2032R crizotinib resistant mutation and PD-L1, and 2) the clinical potential of anti-PD-L1 ICI therapy in ROS1-driven and drug-resistant ROS1 fusion G2032R mutation cell lines and mouse models. In this research, we share our insights related to ICI therapy by examining the role of ROS1 fusion and ROS1 G2032R resistance mutation on PD-L1 expression in NSCLCs by http://www.cobioer.com/). The cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  with 10% fetal bovine serum (FBS). Crizotinib (Cat NO.: S1086), Lorlatinib (Cat NO.: S7536), TPX-0005 (Cat NO.: S8583) and SHP099 (Cat NO.: S8278) purchased from Selleckchem. The HCC78 cell line was maintained in RPMI-1640 medium that contained Crizotinib at a starting concentration of 100 nM until a ﬁnal concentration of 2 mM over 10 months. Then, the resulting resistant cells were maintained in RPMI-1640 medium with 0.5 mM crizotinib and were designated as HCC78CR1, -2, -3, -4, and -5 cell lines. HCC78 and HCC78CR1-5 cell lines were authenticated by detecting the SLC34A2–ROS1 fusion using next-generation RNA sequencing. All cells were maintained under a humidified atmosphere of 5% CO2 at 37°C. For in vitro studies, crizotinib was dissolved in dimethyl sulfoxide (Sigma Aldrich) at 10 mM and stored at -80°C. Ba/F3, Ba/F3 ROS1 fusion harboring SLC34A2-ROS1 (Cat NO.: CBP73191) and Ba/F3 ROS1-G2032R harboring both ROS1 fusion and G2032R mutation (Cat NO.: CBP73192) were also obtained from Cobier Company. The Ba/F3 ROS1 fusion, Ba/F3 ROS1-G2032R harboring SLC34A2-ROS1 with G2032R crizotinib resistant mutation and IL-3 producing WEHI-3 cell lines were cultured in RPMI 1640 medium  with 10% FBS. The IL-3-dependent mouse pro-B cell line, Ba/F3, was cultured in 10% FBS and 10% WEHI-3-conditioned medium as a source of IL-3. To obtain the WEHI-3-conditioned medium, WEHI-3 cells were seeded into culture medium (107/100 mL) in the T75 culture flask and cultured for about 4 days until the color of medium turned almost yellow. The IL-3 containing medium was harvested, centrifuged, filtered with a microfilter system (0.2 μm), and kept frozen at –80°C.HCC78 (Cat NO.: CBP60100) and IL-3 producing WEHI-3 (Cat NO.: CBP60532) cell lines were obtained from Cobioer Company (Homo sapiens genome and transcriptome (gencode v19) using RNA STAR software (v2.4.0). The RNAseq datasets presented in this study can be found in online repositories. The names in the National Omics Data Encyclopedia and Project ID OEP000975 can be reached.Libraries for transcriptome sequencing (RNA sequencing) were constructed with NEB Next Ultra RNA Library Prep Kit for Illumina (NEB). Poly(A) tailed mRNA molecules were enriched from 1 μg total RNA with NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit. The mRNA was fragmented into approximately 200 base pair pieces. The first-strand cDNA was synthesized from the mRNA fragments reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized using DNA polymerase I and RNase H. The end of the cDNA fragment was subjected to an end repair process that included the addition of a single ‘A’ base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit  and an Agilent 2100 Bioanalyzer. After quantitative reverse transcription-polymerase chain reaction (RT-qPCR) validation, libraries were subjected to paired-end sequencing with pair end 150-base pair reading length on an Illumina HiSeq sequencer (Illumina). First, the sequenced reads in raw fastq format data were mapped to the hg19 The HCC78 cell lines were plated at 40% to 50% confluence in 12-well plates and incubated for 24 h before transient transfection for 72 h with siRNAs mixed with Lipofectamine reagent (Invitrogen). The siRNAs specific for ROS1 mRNA  and a nonspecific siRNA (5′-GUUGAGAGAUAUUAGAGUU-3′) were obtained from RUIBIO Inc.Cells were stained with biotinylated monoclonal antibodies against human PD-L1 (Biolegend) and with phycoerythrin-labeled streptavidin (eBiosciences) for flow cytometric analysis with Cytoflex instrument (Beckman coulter). The data were analyzed using FlowJo software.Western blotting analysis was performed as most described. Protein estimation was performed by using Thermo Pierce™ BCA Protein Assay Kit (cat NO.: 23225). Rabbit polyclonal antibodies against human phosphorylated ROS1 (Y1604), phosphorylated EGFR (Y1068), phosphorylated ERK, phosphorylated signal transducer and activator of transcription 3 (STAT3), and glyceraldehyde-3-phosphate dehydrogenase were obtained from Cell Signaling Technology. All antibodies were used at a 1:1000 dilution. Horseradish peroxidase-conjugated goat antibodies against rabbit immunoglobulin G were obtained from Abcam. Immune complexes were detected with the use of Pierce Western Blotting Substrate Plus (Thermo Scientific) and Las-mini 4000 system (GE Inc.).2×0.5 was used to calculate tumor volume (mm3). For the analysis of the effect of ICI therapy on ROS1 fusion or ROS1 G2032R resistance mutation, tumor-bearing animals were treated with a single dose of 100 µg per mouse by intratumoral injection.Ba/F3 Tumor Model: Ba/F3 ROS1 fusion and Ba/F3 ROS1-G2032R resistant mutation cell lines were implanted subcutaneously into the right flank of female C3H mouse  buffer). Mice were treated once daily by oral gavage with vehicle (PBS buffer) or crizotinib (25 mg/kg) was administered orally to tumor-bearing mice daily for 28 consecutive days, and tumor volumes were monitored every 2 days for 4 weeks. L×WIn this study, HCC78 cell line was selected for its SLC34A2–ROS1 fusion gene, whereas the other lung cancer cell lines are wild type for ROS1. To investigate the effect of ROS1 fusion on PD-L1 expression, we conducted two cell experiments by transfecting HCC78 cells with siRNA specific for SLC34A2–ROS1 fusion or treating HCC78 cells with crizotinib. Immunoblot analysis with antibodies of the total forms of ROS1 confirmed the downregulation of ROS1 and PD-L1 expression after using siRNA specific for SLC34A2–ROS1 . PD-L1 expression in HCC78 and HCC78CR1–5 was analyzed by flow cytometry  and immunoblotting  to confirm the cell surface and total expression, respectively. The HCC78CR1–5 sublines showed remarkably higher levels of PD-L1 than their parental HCC78 cell line. HCC78CR1–5 cell lines showed higher levels of PD-L1 protein compared with the HCC78 cell line, as shown by Western blotting, along with the upregulated expression of ROS1 fusion. These data overall suggest that the increased expression of PD-L1 is associated with ROS1 fusion.To further explore the relationship of PD-L1 expression and ROS1 fusion in crizotinib resistance, we cultured five HCC78 crizotinib resistant cell lines (HCC78CR1–5). Crizotinib resistance of HCC78CR1–5 was detected by Cell Counting Kit-8 with half maximal inhibitory concentration (CCK8 IC50) . HCC78CR1 harbored the most genes, of which 234 were upregulated, and 466 were downregulated. HCC78CR1–5 shared 145 genes, which were speculated to be related to the expression of PD-L1 in acquired drug resistance without resistant mutation. Enrichment analysis of the pathway showed that the HCC78CR1–5 cell lines are more immune sensitive to PD-1 signaling, cytokine signaling in immune system, and immune system compared with the HCC78 cells . Western blotting analysis showed that after the ROS1 fusion group was treated with crizotinib and anti-PD-L1, the expression of PD-L1 in the tumor immune micro-environment decreased, whereas the expression was up-regulated in the G2032R group , and T-cell immunoglobulin mucin-3 (TIM3) on Th and Tc cells was performed in blood and tissue  is the standard ; combinavia oncogenic signaling pathways, such as RAS/RAF/MEK/mitogen-activated protein kinase-ERK , which is hosted by the Chinese Academy of Sciences (CAS), under the Project ID OEP000975.Data Access: All the RNA-sequencing data of HCC78 and HCC78CR1-5 have been deposited at the National Omics Data Encyclopedia , the National Natural Sciences Foundation Key Program (81630071), the National Natural Science Foundation of China (81771689 and 81900194), the Ministry of Education Innovation Team Development Project (IRT-17R10), the CAMS Key Lab of Translational Research on Lung Cancer (2018PT31035), the CAMS Innovation Fund for Medical Sciences (CIFMS 2016-I2M-3-008), the Aiyou Foundation(KY201701), and the Wujieping Foundation(320675018477).The 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.