Characterization studies revealed that all analogues of HW-155, as well as HW-155, were effective inhibitors of the fibril formation by hIAPP.The abnormal folding and aggregation of functional proteins into amyloid is a typical feature of many age-related diseases, including Type II diabetes. Growing evidence has revealed that the prevention of aggregate formation in culprit proteins could retard the progression of amyloid diseases. Human Amylin, also known as human islet amyloid polypeptide (hIAPP), is the major factor for categorizing Type II diabetes as an amyloid disease. Specifically, hIAPP has a great aggregation potential, which always results in a lethal situation for the pancreas. Many peptide inhibitors have been constructed from the various segments of the full-length hIAPP peptide; however, only a few have their origin from the screening of combinatorial peptidomimetic library. In this study, based on HW-155, which was previously discovered from a one–bead–one compound (OBOC) library to inhibit Aβ There is overwhelming evidence of the useful role of soluble amylin in glucose metabolism. However, the insoluble aggregates resulting from the misfolding of this protein are implicated in type II diabetes4. Just like the amyloid deposits formed in neurodegenerative disorders 5 and progressive diseases 7, amylin aggregates consist of stacked protofilaments, leading to a structural architecture known as the cross-β structure10. The oligomerization of amylin into amyloid fibrils has been suggested to be a stepwise process: first, the aggregation of monomers into colloidal spheres (nucleation units), which stop growing after reaching a threshold diameter; second, the association of the spheres to form linear chains of toxic, mature fibrils of pore-like morphology8. The extent of this oligomerization process is strongly correlated to pancreatic β-cell dysfunction and death in Type II diabetic patients13; therefore, an impairment of the initial monomer aggregation process is an appealing therapeutic approach to diabetes drug discovery and development.The human islet amyloid polypeptide (hIAPP), also known as amylin, is a unique amyloidogenic precursor peptide, and a critical pathogenic biomarker of Type II diabetes. Amylin is co-secreted with insulin by the β-cells of the pancreas, and co-stored in the secretory granules14. Hence, there is an urgent need for potent inhibitors of the process producing these pathogenic fibrils. In line with this, some inhibitors, including the peptides derived from the various secondary recognition elements along the amylin peptide chain17, engineered peptides20, constrained peptides27, small molecules29 and natural products32, have been reported. Many of these inhibitors are specific in their activity; however, a good number of them have exhibited dual inhibitory activity such that they can inhibit amyloid aggregation of multiple classes of proteins non-specifically35.To date, there is no cure for pancreatic amyloidosis, and there are only limited approved therapeutic strategies for its prevention37. γ-AApeptides contain N-acylated-N-aminoethyl amino acid units, and are derived from γ-PNAs38. Each monomeric unit of γ-AApeptides is equivalent to a dipeptide motif in a conventional α-peptide; thus a γ-AApeptide can project an identical number of functional groups as an α-peptide of the equal length. γ-AApeptides have been shown to form well-defined helical structures41, target bacterial membranes44, and specifically bind to protein surfaces to modulate protein functions and cellular activities48.In order to advance the application of peptidomimetics in chemical biology and drug discovery, we have recently introduced a new class of peptidomimetic scaffold called γ-AApeptides Fig. 36,37. γ-40 aggregation, HW-155, which was identified from an OBOC (one-bead-one-compound) γ-AApeptide combinatorial library  with little structural similarity to KLVFF, the core amyloid-forming unit of Aβ. Inspired by the findings on molecules that show cross-inhibition toward multiple amyloidosis35, and based on the fact that HW-155 was identified from an OBOC library, we hypothesized that HW-155 could be a non-specific amyloid inhibitor, and as such it may exhibit inhibitory activity toward the aggregation of hIAPP. To test our hypothesis, we synthesized HW-155 and eight (8) analogues (1–8) with methyl group substitution on each position , and investigated their ability to prevent the fibrillization of amylin (hIAPP), as well as to disrupt preformed hIAPP aggregates. Furthermore, we studied the aggregation behaviour and morphological characteristics of hIAPP in the presence and absence of various concentrations of HW-155 and its eight (8) analogues using transmission electron microscopy (TEM), time-dependent Thioflavin T (ThT) fluorescence assay, and atomic force microscopy (AFM). To further explore the biological potential of HW-155 and its analogues  alone . Using HW-155 as a lead sequence, we designed and performed a functional analysis of eight (8) analogues of HW-155, namely γ-AApeptides 1–8 , we substituted each of the N-side chains in HW-155 with an acetyl group, while alanine scanning was performed on the chiral side chains (5–8).Inspired by the preliminary data, we sought to understand the relationship between the structure of 1–8 Fig. . For fou1–8 was monitored by time-dependent ThT assay. In an initial attempt, we studied the anti-aggregation activity of 2.5-, and 10-fold molar excesses of 1–8 against hIAPP aggregation. Interestingly, peptides 2–8 were able to suppress the ThT fluorescence induced by hIAPP aggregation by about 50% or more, while, peptide 1 suppressed ThT fluorescence induced by hIAPP aggregation by at least 43%  in the presence of 0 µM in t3, 4, 5, and 6  improved the aggregation inhibitory activity. In the achiral side chain truncation series, peptide 4 emerged with the best anti-aggregation activity with almost 2-fold decrease in hIAPP aggregation across the concentrations tested compared to HW-155.Intriguingly, we identified that peptides  6 Table  could ine 5 Fig.  reduced HW-155 into methyl groups (5–8) gave rise to a better or similar inhibitory activity than HW-155. However, unlike acetylation, which seems to be more critical in inhibition near the N-terminal end, mutation to methyl side chain played a more important role near the C-terminal end, as evidenced by 5 and 6. As a matter of fact, for all the synthesized analogues, 5 exhibited the best anti-aggregation potential with almost 3–4 fold inhibition against hIAPP aggregation.Interestingly, conversion of the chiral side chain (derived from α-side chain) in 2, 3, 4, 5 and HW-155 with hIAPP.Given the promising inhibition study by ThT assay, we next carried out TEM experiment to further confirm the ability of the few most potent compounds to inhibit hIAPP aggregation. This is done by pre-incubating nearly equimolar amount of peptides 49. However, no such fibril was observed in the presence of 2, 3, 4, 5, and HW-155, indicating a significant prevention of fibril formation by hIAPP. A prevention of fibril formation by 2 and 5 resulted in monomers as observed in the TEM images. Likewise, oligomers seen in 3, 4, and HW-155 were a result of the inhibition of mature fibril formation.The TEM micrographs for hIAPP revealed a dense network of intertwined mature fibrils with linear, and thread-like morphology Fig. , indicat5 was able to disrupt preformed hIAPP fibrils at a tested concentration of 100 µM  h. Interestingly, only  µM Fig. .Figure 55 on hIAPP preformed aggregates, we allowed 10 µM hIAPP to age for 10 h before treatment with a 10–fold molar excess of 5. AFM showed a disappearance of mature fibrils . However, at a peptide concentration of 5 µM, peptides 3, 4, 5, and HW-155 reduced the cell viability by at least 10%, while peptide 2 showed a significant cell viability reduction of about 20%. At both concentrations tested, peptide 2 exhibited a mild cytotoxic effect on NIH-3T3 cells.Based on our findings of the inhibition of hIAPP fibrillization by lls Fig. . At 1 µM2, 3, 4, 5, and HW-155 to modulate the hIAPP amyloid–induced cytotoxicity, we treated NIH-3T3 cells with hIAPP (10 µM) and 5 µM peptide as described in the general information section. Results showed that cells treated with only hIAPP exhibited a significant reduction in survival, whereas treatment with 5 µM peptides 2, 3, 4, 5, and HW-155 was able to rescue the cells from the hIAPP amyloid cytotoxicity  capping reaction with 500 uL Acetic Acid. After capping, the beads were washed and alloc-deprotection reaction was done by reacting the beads with Pd (PPh3)4  and Me2NH BH3  (10 mins each time). The beads were then washed with DCM and DMF, followed by the reaction of the deprotected beads with acyl chloride  and DIPEA  in 3 mL DCM for 1 h or with the carboxylic acid , HOBt , and DIC  for 6 h (x2).Peptides were synthesized on the rink amide resin (0.6 mmol/g) on a Burrell Wrist-action shaker. 200 mg resin was treated twice (15 mins each time) with 3-mL 20% Piperidine in DMF to deprotect the Fmoc group. The beads were then washed twice with 3 mL each of DCM and DMF. The desired red Fig.  and are The above steps were repeated to assemble the desired sequence on the resin Fig. . After t50. 3.1 mg of hIAPP was dissolved in minimal amount of HFIP (50 uL) to remove aggregated hIAPP and the HFIP was removed over a gentle stream of Nitrogen gas. The process was done twice and 5 mL of Milli Q pure water was added to the disaggregated hIAPP film. The solution was then divided into 25 aliquots of 200 µL each. The 25 aliquots were lyophilized and reconstituted in 800 µL of PBS prior to use to obtain a hIAPP stock solution of 40 µM . hIAPP stock solution (40 µM) was also prepared as described above. The peptide, hIAPP and 15 µM ThT were added into a 96-well black plate. Equal volume of hIAPP solution was added into the 96-well black plate to make the final concentration of hIAPP in each well to be 10 µM. Time-dependent fluorescent change was monitored for 24 h by a Biotek Synergy H1 hybrid plate reader at excitation and emission wavelengths of 435 and 490 nm, respectively.Fi is the fluorescent intensity of the hIAPP treated with 5 at time t, Fh is the Fluorescent intensity of the hIAPP treated with PBS at time t.Similar to the protocol described above for the inhibition studies, hIAPP and ThT were added to PBS in a 96-well black plate and at concentrations of 10 and 15 µM, respectively. Time dependent fluorescent change was monitored for 10 h, after which 5 uL peptide was added to make a final peptide concentration of 100 µM. 5 µL PBS was also added for control wells. Each test well containing the peptide has a control well to which equal volume of PBS was added. The fluorescent change was then monitored for another 12 h. The relative Fluorescence, Fi/Fh was then plotted against the time (Hr),where Sample preparation was similar to ThT assay. Sample solution containing 10 µM hIAPP and 12.5 µM of the respective peptide were incubated at 30 °C for 24 h. 10 µL aliquot of the incubated solution was placed on a carbon-coated 200-mesh copper grid. After 10 mins, excess solution was wicked away and the grid was allowed to dry. This is then followed by the addition of 2% uranyl acetate solution (10 µL), which was allowed to float for 5 min. The excess solution was then removed using the blotting paper. The copper grid was left to dry at room temperature before imaging with a FEI Morgagni 268D TEM operated at 60 kV.A droplet (10 µL) of the sample solution was placed on a freshly cleaved mica disk and dried in open air at room temperature. Imaging was performed using a Multimode 8 AFM  and a Nanoscope V controller in the Peak-Force quantitative nanomechanics (QNM) mode (in air). A silicon cantilever probe with a spring constant of 2.8 N/m was used for imaging at room temperature (scan rate of ~1 Hz). The acquired images were leveled by subtracting a linear background.The percent aggregation was calculated using the formula below:Percent aggregation = (F in the presence of inhibitor/F in the absence of inhibitor) × 100%, where F is the average of the ThT signals  from 600 min onwards. These time points were chosen because the maximum ThT signals were observed at these points in the uninhibited hIAPP control experiment.4 cells/well. When the cells reached 60% confluence, the cell culture medium in each well was then replaced with 100 μL of cell growth medium containing peptides 2, 3, 4, 5, and HW-155 alone at concentrations of 1 or 5 µM peptide with 10 µM hIAPP.Mouse embryonic fibroblasts NIH-3T3 cell were purchased from Lifetechnoglies. The NIH-3T3 cell were grown at 37°C and 5% CO2 humidified atmosphere in DMEM medium, respectively, supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin . Cell proliferation was determined using the CCK-8 cell counting kit (Sigma- 41 Aldrich). Cells were seeded in 96-well plates at 1 × 10After incubation for 24 h at 37 °C, the peptides were washed with PBS three times. Then, 10 μL of CCK-8 dye and 100 μL of DMEM cell culture medium were added to each well, and the cells were incubated for another 1.5 h at 37 °C. The absorbance at 450 nm was measured by a Synergy H1 Hybrid Reader . Untreated cells served as controls with 100% viability. The results are presented as the mean ± SD of three measurements.Supplementary materials.


Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate  for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential  and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models  injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma. Osteosarcoma is the first primary malignant bone tumor that predominantly occurs during adolescence ,2. StandThe aim of the study was to establish and characterize osteosarcoma models resistant to common chemotherapeutic agents and analyze their behavior comparatively to their parental counterparts in-vitro and in-vivo in a bone orthotopic setting.In order to establish resistant cell lines, a panel of six osteosarcoma cell lines was exposed to increasing concentrations of methotrexate (MTX) or doxorubicin (DOXO). Parental and resistant lines were assessed for their response to chemotherapy in terms of cell viability, migration ability, and resistance index. Acquired resistance to MTX developed in 5/6 lines (83%) up 1 µM for HOS, Saos-2, Saos-2-B and 143B after 3 months, and up to 0.03 µM after nine months exposure for MG-63 A. Half-mNo differences in morphology, growth rate or migration ability were detected between the MTX-resistant cell lines and their parental counterparts . In contWe then evaluated the stability of this acquired chemotherapy resistance by culturing resistant lines with or without the compound for at least nine weeks, and determined the resulting IC50 values. Under continuous drug pressure (Drug-ON) all lines exhibited high Resistance Index (RI) values for MTX (>37), while Drug-OFF cell lines exhibited variable behavior A. HOS-R/The possibility of a cross-resistance to other compounds was evaluated in resistant cell lines in Drug-ON and Drug-OFF culture conditions A,B. CellCross-resistance was not detected with any tested compound in the MTX-resistance lines Drug-ON or Drug-OFF, except for MG-63-R/MTX A. SurpriFor HOS-R/DOXO, cross-resistance was detected with ETOP (RI = 276), VCR (RI = 172), and MTX (RI = 12) B. The coBased on copy number abnormalities (CNA) by Oligonucleotide Comparative Genetic Hybridization Array (aCGH) and gene expression (GE) profiles by RNA sequencing (RNAseq), the resistant lines clustered according to their genetic background rather than the resistance mechanisms  Comparison of CNA-acquired changes of each MTX-resistant line to their respective parental counterpart showed common acquired CNA in MTX-resistant lines issued from similar genetic backgrounds , in regions which appeared to contain genes involved in MTX metabolism and resistance. We further analyzed the differential expression of these genes by RNAseq between each parental and corresponding MTX-resistant line Drug-ON and Drug-OFF and validated some findings (dihydrofolate reductase (DHFR) and reduced folate carrier (RFC/SLC19A1)) at protein level A–C.Chromosome 5q region containing Dihydrofolate reductase (DHFR)  was gained in HOS-R/MTX and 143B-R/MTX, amplified in MG-63-R/MTX, and not modified in the other lines compared to their parental counterpart . While up value 1,60×10-15). In Drug-OFF conditions compared to parental lines, the most significant enriched pathway was histone acetyltransferase . Among the 100 most significant fold changes, we found genes implicated in osteosarcoma oncogenesis (RTN1) [To explore other potential mechanisms of resistance common to all MTX-resistant lines, we performed a differential GE analysis between all MTX-resistant lines Drug-ON versus their parental counterpart, and Drug-OFF versus their parental counterparts . DiffereHAT; ID120435. Amos (RTN1) , down-res (RTN1) ,10. Enris (RTN1) .p value 6.63×10-100) followed by MMP13 . Several other regions with acquired CNA containing multiple multidrug resistance genes were also up-regulated in the resistant line in GE  and has been implicated in osteosarcoma oncogenesis [p value 6,45×10-04), while none of the cancer stem cell markers  [In the unique doxorubicin-resistant line HOS-R/DOXO, the acquired gained CNA translated in an increased mRNA GE level . The mosne in GE C. Chr7q2ogenesis ,13. Decrd ABCG2)  were modThese drug-induced changes suggest a broader impact of the acquired resistance in tumor cell behavior, especially in an in-vivo context.To explore the impact of the in-vitro acquired resistance on the in-vivo tumor behavior, the parental and resistant HOS and Saos-2-B lines stably transduced with Luc/mKate2 vector to express luciferase (above 90% Luc/mKate2 positive cells)  were injBone engraftment rates of HOS-R/MTX and parental counterpart were maximal (100%), whereas the HOS-R/DOXO-CDX had a slightly lower engraftment rate (66%) .The HOS-resistant lines had more difficulties to adapt to the in-vivo bone environment, with an initial decrease in in-vivo bioluminescence (up to 27 days), followed by a slightly faster growth compared to the HOS-parental-CDX A.Bone engraftment rate of Saos-2-B-R/MTX and parental-CDX were comparable  . Both paThe different models revealed tumor-bearing tibia bone structure abnormalities similar to those observed in osteosarcoma patients.The resistant models retained the primary tumor-induced bone abnormalities of their parental counterpart in the Computed Tomography (CT scan) B. The slMetastatic foci were detected by in-vivo bioluminescence in all CDX models as early as 30 days after intratibial injection A except After mice sacrifice, cells isolated from primary tumors of each CDX model (no in-vivo treatment was performed) were cultured for two in-vitro passages. All resistant-CDX-derived cells grew in-vitro. Drug response was assessed. RI of in-vitro resistant-CDX-derived cells were either lower , higher , or stable  compared to the matched RI of the in-vitro initial resistant cells .Resistance to chemotherapy and metastatic phenotypes are the two main problems in osteosarcoma patients that lead to recurrence and death. We developed six new in-vitro osteosarcoma models, resistant either to MTX  or DOXO (HOS), by continuous in-vitro drug exposure, adding new models to those previously described ,18,19,20High level of in-vitro acquired resistance was obtained with MTX (5/6 cell lines), while only one of the six lines developed resistance to DOXO. This is consistent with observation in other models derived from patient samples which showed resistance to MTX while still sensitive to DOXO and CISP . ContinuFor the unique, highly DOXO-resistant line HOS-R/DOXO line RI = 224), we observed a multi-drug resistant phenotype, with cross-resistance to other agents used in osteosarcoma treatment and substrates of PgP , associated with ABCB1/MDR1 gain translated in high MDR1 mRNA and PgP protein expression. The DOXO-resistant line gained several chromosomic regions associated with mRNA expression up-regulation on the chromosome 7 region containing ABCB1/MDR1 and ABCB4, and several other regions encoding various multiple multi-drug resistance genes or genes implicated in the apoptotic response to doxorubicin. Up-regulation of MDR1 has been associated with chemo-resistance development of osteosarcoma tumor-initiating cells 4, we obs,24,25. AFor our MTX-resistant lines, high level of resistance was obtained (RI > 37) and persisted after drug removal although at lower level, except for MG-63 which lost MTX-resistance. Higher resistance has been observed in patient-derived cell lines . All ourSeveral more general cellular and biological pathways were modulated in the chemo-resistant lines related to cell adhesion/motility, extracellular matrix organization/degradation/composition, and cellular microenvironment. These processes, not fully assessed in-vitro, might affect the resistant cell behavior in-vivo. In DOXO- and MTX-resistant osteosarcoma cells, Fas expression was increased, suggesting a decreased in-vivo metastatic potential . SimilarWe developed in-vivo orthotopic intratibial bioluminescent parental and resistant-CDX models in NSG mice, with the experimental procedure used previously . The difOur resistant-CDX models can substantially help to evaluate new drug efficacy in osteosarcoma and complement the few pre-existing osteosarcoma models. Future work will include the comparison of our models to the PDX models that we are currently generating issued from relapsed osteosarcoma and the human relapsed osteosarcoma cohorts that might be available in the future .v/v) fetal bovine serum  at 37 °C in a humidified atmosphere (5% CO2 and 95% air), under mycoplasma free conditions.Human osteosarcoma cell lines HOS, 143B, Saos-2, Saos-2-B, MG-63 and IOR/OS18  with difDoxorubicin (DOXO), methotrexate (MTX), cisplatin (CISP), etoposide (ETOP), vincristine (VCR), and verapamil (VER) were purchased from Sigma Aldrich , mafosfamide (MAF) from Toronto Research Chemicals Inc. , and cabozantinib (Cabo) from LC Laboratories . All compounds were solubilized in dimethyl sulfoxide , except cisplatin, solubilized in N, N-dimethylformamide , at 10 mM stock solutions, and stored at −20 °C.Cells were seeded into 6-well plate at 100,000 cells/well  or 120,000 cells/well  in DMEM supplemented with 10% FBS, then exposed continuously to an initial concentration of 0.01 µM DOXO or 0.07 µM MTX. In parallel, vehicle (DMSO) treated cells  were seeded at the same conditions and used as controls. The medium was changed twice a week, and passages (1:10) were performed when cells had reached 80% confluence. The MTX concentration was progressively increased up to 0.3 µM for MG-63 and 1 µM for all other lines; DOXO concentration was raised up to 1.3 µM for HOS. The resistant lines were maintained in culture with the maximal tolerated drug concentration and named as “Drug-ON” cell lines. Drug pressure was stopped after resistance confirmation, and resistant cells were cultured in drug free medium for minimum nine weeks, designed as “Drug-OFF” cell lines. The same experiments were performed with MAF without success .The cell lines that did not developed resistance, died under drug continuous exposure. Therefore, no experiments could be done.Parental and resistant derived HOS, 143B, MG-63 and IOR/OS18 lines were seeded at 5000 cells/well, and cells derived from Saos-2 and Saos-2-B at 10,000 cells/well in a 96-well plate in DMEM supplemented with 10% FBS for both assays.Cell proliferation rate and doubling time were assessed using the IncuCyte live-cell imaging system . Phase-contrast photographs were collected for 72 h.The day after seeding, cells were incubated in the presence of a range of drug concentrations for 72 h . Verapamil was used at 5 μmol/L and cabozantinib at 0.1 μmol/L, to revert PgP function. Cell viability was evaluated using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS assay) , according to the manufacturer instructions. The half-maximal inhibitory concentration (IC50) was determined using the GraphPad Prism5 software .Cells were seeded at 10,000 cells/well , or at 20,000 cells/well  in 96-well ImageLock tissue culture plates  in DMEM supplemented with 10% FBS. The day after, cell layers were scratched with the WoundMaker™, washed once and then incubated in the presence of the indicated drugs  or DMSO (control). The wound-healing was monitored using IncuCyte™ system for 48 h. Data analyses were performed using Graphpad Prism5 Software.Parental and resistant HOS and Saos-2-B cells were stably transduced with Luc/mKate2 vector as previously described .Animal experiments were approved by the CEEA26, Ethics Committee and the French Ministry of Research (APAFIS#1648-2015090713516480) and performed under the conditions established by the European Community (Directive 2010/63/UE).6 cells in matrigel solution at 4 mg/mL), as previously described [The parental cell lines  and their resistant counterparts to either MTX  or DOXO (HOS-Luc/mKate2/DOXO) were established into 7-week-old immunodeficient NSG mice by unilateral intratibial injection  in DMEM supplemented with 10% FBS.In-vivo and ex-vivo images were acquired using IVIS SpectrumCT  as previously described . BrieflyFormaldehyde-fixed-paraffin-embedded (FFPE) tissue sections (4 µm) were either stained with hematoxylin-eosin-safranin (HES) for morphology, or processed for immunohistochemistry as described before . PrimaryDNA and RNA from cells and CDX samples were isolated using AllPrep DNA/RNA mini kit (Qiagen) according to manufacturer’s instructions. Quantification/qualification were performed using Nanodrop 2000 spectrophotometer  and Bioanalyzer DNA 7500 .http://www.agilent.com). 500 ng of tumor and reference DNAs were fragmented with AluI and RsaI  and labeled with Cy5-dUTP and Cy3-dUTP, respectively. Hybridization was carried out on SurePrint G3 Human CGH Microarray 4 × 180K  arrays for 24 h at 65 °C in a rotating oven  at 20rpm and followed by appropriate washing steps. Microarrays were scanned with an Agilent G2505C DNA Microarray scanner at 100% PMT with 3 µm resolution at 20 °C in low ozone concentration environment. Data were extracted using the Feature Extraction software , along with protocol CGH_1105_Oct12. All further data manipulations were performed under the R statistical environment . Raw intensities were normalized according to their dye composition (Cy3 fitted over Cy5). Data were transformed to log2(Test/Ref) and normalized according to their local GC content through a lows regression. Resulting profiles were segmented with the CBS algorithm [Sex-matched normal DNA from a pooled human DNA  was used as a reference. Oligonucleotide aCGH processing was performed according to manufacturer’s protocol version 7.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped with Salmon v0.8.1 [RNAseq analysis was performed as previously described . RNAseq n v0.8.1  using GRn v0.8.1 .http://bioinfogp.cnb.csic.es/tools/venny/). Toppfun website was used for functional enrichment analysis (https://toppgene.cchmc.org/enrichment.jsp).Differential mRNA expression lists were compared using Venny diagram produced by Venny 2.1.0  was reversely transcribed into cDNA using M-MLV Reverse Transcriptase , according to the manufacturer’s recommendations. Amplifications monitored with StepOnePlus PCR System , were performed using Maxima SYBR Green/ROX qPCR kit , and with a first step at 95 °C for 10 min followed by 40 cycles with 95 °C for 15 s and 60 °C for 1min. Melting curve was performed at the end of the PCR  to identify unique PCR products. The sequences of the specific primers are described in Parental and resistant Drug-ON and Drug-OFF cell lines were seeded into 100*20mm culture dish at 130,000 cell/dish in DMEM supplemented with 10% FBS. Cells were collected at 80–90% confluence, followed by a PBS wash and re-suspended in lysis buffer  and stored at −20 °C. Proteins were extracted and measured with Pierce BCA Protein Assay Kit .Western-blot analysis was performed using specific primary antibody against topoisomerase-II alpha/beta (TOPO2) , anti-dihydrofolate reductase (DHFR) (1:1000), anti-reduced folate carrier (RFC) (1/200), anti-methylenetetrahydrofolate reductase (MTHFR) (1/1000), all purchased from abcam  and β-Actin (13E5) Rabbit mAb . Appropriate secondary antibodies  at 1:5000 dilution were used, followed by visualization with the enhanced chemiluminescence ECL reagent  and imaged with ChemiDoc™MP image system . Signal intensities were quantified with the Image Lab version5 .®5 Software . The one-way ANOVA analysis was used to compare the groups. p < 0.05 was considered to indicate a statistically significant difference.Data were shown as the mean ± standard error of mean (SEM) of three independent experiments performed using Graphpad PrismIn-vitro acquired resistance to MTX and DOXO, induced CNA and possible epigenetic changes that involved specific but also more general mechanisms of resistance that might influence OS cell behavior in their microenvironment. These resistant-CDX models can help to evaluate new drug efficacy in osteosarcoma and complement the few pre-existing osteosarcoma models ,18,19,20


Furthermore, XN, DXN, and TXN induce extensive apoptosis in all these carcinoma cell lines. Finally, TXN induces G0/G1 cell cycle arrest in the colon carcinoma cell line HT29. Our findings suggest that DXN and TXN could show promise as therapeutic agents against colorectal and liver cancer in preclinical studies without the drawback of metabolism into a phytoestrogen.Xanthohumol (XN), a prenylated flavonoid found in hops, inhibits growth in a variety of cancer cell lines; however, its use raises concerns as gut microbiota and the host’s hepatic cytochrome P450 enzymes metabolize it into the most potent phytoestrogen known, 8-prenylnaringenin (8-PN). The XN derivatives dihydroxanthohumol (DXN) and tetrahydroxanthohumol (TXN) are not metabolized into 8-PN and they show higher tissue concentrations in vivo compared with XN when orally administered to mice at the same dose. Here we show that DXN and TXN possess improved anti-proliferative activity compared with XN in two colon  and two hepatocellular  carcinoma cell lines, as indicated by their respective IC Humulus lupulus [First discovered at the turn of the twentieth century by Power et al., XN is a simple prenylated flavonoid found in the female inflorescences, or hop cones, of the hop plant  lupulus . Hops ar lupulus ,6,7,8,9. lupulus .2-extracted hops, a byproduct of the hops industry, which provides a less expensive alternative to synthesis [Synthesis of XN from phloracetophenone is inefficient ; therefoynthesis . Furtherynthesis . In humaynthesis .Eubacterium limosum metabolizes IX via O-demethylation , which is the first step in the metabolism of XN  than XN in these cell types. While XN and DXN do not appear to induce cell cycle arrest, HT29 cells treated with TXN arrest in the G0/G1 phase of the cell cycle. These findings demonstrate that the two non-estrogenic XN derivatives are attractive alternatives to test along with XN in future preclinical studies using mouse models of colorectal and hepatocellular carcinoma.Here we demonstrate the anti-proliferative and cytotoxic effects of XN in two human colon adenocarcinoma cell lines (HT29 and HCT116) and two human hepatocellular carcinoma cell lines (HepG2 and Huh7). Furthermore, we show for the first time that DXN and TXN possess similar effects, and in most cases, a lower half-maximal growth inhibitory concentration (IC50) of DXN and TXN for two colon carcinoma and two hepatocellular carcinoma cell lines was determined using the SRB colorimetric assay for cytotoxicity screening, as previously described [50 values experimentally determined for XN, DXN, and TXN in the colon carcinoma cell lines HCT116 and HT29, and the hepatocellular carcinoma cell lines HepG2 and Huh7. DXN and TXN (except HepG2) exhibited lower IC50 values than XN. For the cells most sensitive to DXN and TXN, the decrease in IC50 values was approximately 30% compared to XN  to XN treatment than HCT116, but showed a similar sensitivity to the derivatives DXN and TXN as HCT116  7-AAD(−)), indicating caspase activation without membrane alterations (early- to mid-stage apoptosis). In addition, there was a statistically significant increase in doubly stained SR-VAD-FMK(+) 7-AAD(+) cells, indicating cells in late apoptosis , while TXN induced slightly greater apoptosis (+7.1%) than XN .1 phase . The human liver carcinoma cell line HepG2 was provided by Dr. Tori Hagen . We purchased Huh7 from the American Type Tissue Collection . The other cancer cell lines were originally purchased from ATCC. All cancer cell lines were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) without sodium pyruvate  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin . All cell types were maintained in 5% COXN and TXN were provided by Hopsteiner Inc., while DXN was synthesized as described previously . The purCells were seeded into 96-well plates . After 24 h, cells were treated with either XN, DXN, or TXN, as described above. Cell viability was evaluated using a sulforhodamine B (SRB) assay , as described previously . AbsorbaAt various times after treatment with XN, DXN, or TXN, adherent and floating cells were collected for analysis. Apoptosis was assessed using flow cytometry-based annexin V  and a multicaspase assay kit . The annexin V assay is based on measurement of externalization of phosphatidyl serine, a common characteristic of cells undergoing apoptosis. The multicaspase assay is based on measurement of caspase enzymes activated during apoptosis. Cells were trypsinized, washed in Dulbecco’s Phosphate Buffered Saline (D-PBS), and stained with Annexin V, sulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone (SR-VAD-FMK), and 7-amino-actinomycin D (7AAD) according to the manufacturer’s instructions. Cell populations were quantified using a Guava personal cytometer .g, washed with PBS, and re-suspended in cellular DNA staining solution containing 40 mg/mL propidium iodide  and 100 mg/mL RNase A (Sigma-Aldrich) in PBS. After a 15-min incubation at room temperature, cell populations were quantified using a Guava personal cytometer (Guava Technologies). At various time points after treatment with XN, DXN, or TXN, adherent and floating cells were collected and fixed in 70% ethanol at one million cells per aliquot. Samples were centrifuged at 500× p ≤ 0.05.Statistical analyses were performed with the GraphPad Prism 7 software . Group differences were assessed with a one-way ANOVA, followed by a Sidak’s post hoc test. The Sidak’s post hoc test was used to compare every mean with either the control mean, or XN mean. Statistical significance was set at


Gallbladder cancer is the most common malignant neoplasm of the biliary tract, responsible for 80–95% of cases. Appropriate models are required for investigating the molecular pathogenesis of gallbladder cancer.In this study, we aimed to establish a gallbladder cancer cell line from primary tumour. Single cell RNA sequencing, Light and electron microscopy, DNA content analysis, cytogenetic analysis, short tandem repeat (STR) DNA fingerprint analysis, immunophenotypic characterization, and xeno-transplantation were utilized to characterize the novel ZJU-0430 cell line in vitro and in vivo.The cell line showed multiple cell shapes and characteristic epithelial morphologies under the microscope, but no too much heterogeneity by scRNA-Seq, with a population doubling time (PDT) of 19.81 h, which was shorter than that for GBC-SD cells. An immunophenotypic analysis revealed that ZJU-0430 cells were positive for CD24, CD44, CD29 and CD133 expression, and partially positive for CD184, and CD326 expression, and negative for CD34, CD90, CD117, and CD338 expression, similar to the primary cancer cells. A pathological analysis confirmed the origination of cell line from gallbladder tumour. ZJU-0430 cells had higher migration, invasion and proliferation properties than GBC-SD cells in vitro, and showed in vivo tumorigenicity in nude mouse xenograft settings.The results confirm the potential utility of ZJU-0430 cell line as a representative model of gallbladder cancer and suggest that it could be used in the in vitro and in vivo studies of gallbladder cancer pathogenesis and to develop new therapeutics.The online version of this article (10.1186/s12935-019-0911-1) contains supplementary material, which is available to authorized users. Gallbladder carcinoma (GBC) is an invasive adenocarcinoma that originates from the epithelial linking of the digestive system . GBC is Despite the progress in therapeutic strategies, the overall survival rate has remained poor, mainly due to late diagnosis, early metastasis, ineffective surgical resection, and insensitivity to chemoradiation , 6–8. ThIn this study, a novel gallbladder cell line derived from a primary GBC, referred to as ZJU-0430, was successfully established. All our data together confirmed that it as a potentially useful model for the further study of this disease.This study was performed in accordance with the Declaration of Helsinki of 1975, and the official recommendations of Chinese Community Guidelines, and was approved by the Ethics Committee and Institutional Review Board of the Sir Run Run Hospital. Written informed consent was obtained from the patient.A 74-year-old male patient with pain in the upper abdomen was admitted in our centre. Gastroscopy showed the presence of a gastric antral ulcer (stage S2), and irregular deep concave ulcer at the gastric angle, and cancer priority. Abdominal computed tomography (CT) also found a 1.5-cm thick wall at the bottom of the gallbladder. A radioimmunoassay showed that the patient’s serum levels of a variety of biomarkers were normal  except for ferritin, which was high . A radical resection of stomach and gallbladder were performed and a pathological examination showed that gastric carcinoma was a poorly differentiated adenocarcinoma derived from signet-ring cell carcinoma, whereas the primary gallbladder cancer was a well differentiated adenocarcinoma.GBC-SD cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences , and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented by 10% fetal bovine serum (FBS) , and 1% penicillin/streptomycin and amphotericin B (Invitrogen).3), and transferred into a gentleMACS™ C tube containing the buffer from the Tumor Dissociation Kit (human), and subjected to a gentleMACS™ Tissue Dissociator. Cells were cultured in complete growth RPMI 1640 medium (containing additives) at 37 °C in a humidified incubator containing an atmosphere of 5% CO2. Fibroblasts were removed by differential trypsinisation as described previously   assays were performed. As shown in Fig. A colony formation was also performed to examine the ability of ZJU-0430 cells proliferate. As shown in Fig. In order to investigate their in vivo tumorigenic capacity, ZJU-0430 cell suspensions were injected subcutaneously into the flank of nude mice. Tumour development was observed in all of mice and no obvious metastatic lesions were observed. Gross examination revealed that the tumours were round or oval shaped, and often showed mucus production. The atypia of tumour cells was obvious, when arranged with a small strip of cord and viewed under a microscopy they formed different cavities that included foam cells, cancer cells and normal epithelial cells Fig. d, e.To characterize the original ZJU-0430 cell lines, the original GBC, and the original gastric cancer, a series of markers were examined by IHC technology. As shown in Fig. To interpret the characteristic observed during the experimental investigation, we do single cell RNA sequencing analysis on ZJU-0430 cell line. By using Seurat pipeline, we identified 3 main cell clusters Fig. b. All thHuman GBC is an extremely malignant disease owning to its relative rarity, histological heterogeneity, and proximity to vital structures. Surgical resection, and chemoradiotherapy are ineffective , 8, 40, Cell lines derived from primary tumours are more likely to reflect the characteristics of the original primary tumour more accurately. Based on this idea, here we report the establishment of a GBC cell line designated as ZJU-0430, which was derived from a 74-year-old male patient who had some serum ferritin abnormalities, and GBC that was a well-differentiated adenocarcinoma. The cell grew in adherent monolayer pattern with at least two morphologies that co-existing. scRNA-Seq results revealed that ZJU-0430 cell line was no too much heterogeneity although the morphological difference. Optical microscopy, ultrastructural examination, immunostaining of epithelial markers for positive  and negative mesenchymal expression (vimentin), and H&E staining of cells displayed features that were typical of malignant epithelial cells. The STR genotyping analysis showed the ZJU-0430 cell line was of human origin and that it differed from other established cell lines. A mycoplasma examination confirmed ZJU-0430 to be free of mycoplasma. The PDT was approximately 19.81 h, which was shorter than GBC-SD cells. Taken together, these results suggest that ZJU-0430 cell line is a representative of the disease and may be used for pre-clinical investigation of the pathogenesis and treatment of GBC.Recently, Devendra Chaudhary, et al. reported that 46.7% of GBCs have an abnormal karyotype . In the Mycoplasma can impede a whole range of cellular properties and functions and hinders the use of cells and their derived products in pharmacological area . The culAlthough numerous GBC cell lines have been documented, including G-415, NOZ, KMG-A, GBK-1, FU-GBC-1, FU-GBC-2, PTHrP-GBK, GB-d1, TGBC1TKB, TGBC2TKB, OCUG-1, TUGBK-1, HAG-1, GBC-SD, SGC-996, EH-GB1, EH-GB2 and TJ-GBC2 , 47, 48,GBC is highly aggressive in patients ; therefoAn immunoblotting assay showed ZJU-0430 cells to be positive for biliary tract cancer epithelial marker , which are found to be positive at high rates in real GBC specimens. The mesenchymal marker vimentin was also tested, but it is not found to be expressed. Since the patient (source of ZJU-0430) had been diagnosed with gastric carcinoma before GBC, gastric carcinoma markers were also explored on the xenograft tumour. The gastric carcinoma markers MUC2, MUC5AC, and MUC6 were expressed weakly or not at all. Since the colon and liver are contiguous with each other, the origin of ZJU-0430 cell was a little unclear. In this study, liver markers  and colon markers  were used for staining of xenograft-derived tumours, with the result that all markers were either expressed only weakly, or were not express at all. In addition, the colon cancer marker CDX2 protein were also not found to be expressed on original GBC. Therefore, ZJU-0430 cell line was verified to be originated from the gallbladder primary site.We have successfully established a human GBC cell line referred to as ZJU-0430 that is identical to the original surgical specimen. This well-characterized GBC cell line should be helpful in the investigation of the pathogenesis of GBC, and may help in developing early diagnostic tools as well as potential targeted therapies.Additional file 1. STR Profile Report for ZJU-0430 at P100.Additional file 2. STR Profile Report for ZJU-0430 at P0.Additional file 3: Figure S1. Immunophenotypic analysis for ZJU-0430 as determined by flow cytometry. The representative graph of CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184 CD326, CD338 protein expression were detected on primary cells and P100 cells of ZJU-0430.Additional file 4: Figure S2. IHC for original gastric cancer sections. ZJU-0430 original gastric cancer tissues positively expressed most gastrointestinal tract markers , and negative expressed β-catenin and SATB2.Additional file 5: Figure S3. Characteristic of ZJU-0430 cell line in by scRNA-Seq data. (a) tSNE plot of ZJU-0430 cell line clusters defined by Seurat pipline before merging similar sub cell clusters. (b) UMAP plot of ZJU-0430 cell line clusters as showed in (a). (c) Dot heatmap of CD24, CD44, CD29, CD133 expression in each cell clusters. Heatmap showed the expression pattern of genes associated with Wnt signaling pathway (d), extracellular exosome (e), apoptotic signaling pathway (f) and nuclear transcribed mRNA catabolic process nonsense mediated (g).


AbstractRattusnorvegicus   embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Among studied cell lines, rESCs showed the lowest level of telomere dysfunction, while the riPSCs demonstrated an elevated level on early passages of cultivation. In cultivation, the frequency of dysfunctional telomeres has increased in all studied cell lines; this is particularly true for dysfunctional telomeres occurring in G1 stage in riPSCs. The obtained data are mainly discussed in the connection with the specific structure of the telomere regions and their influence on the differential DNA damage response in them.We studied the level of spontaneous telomere dysfunction in  Telomeres are specialized structures on animal chromosome ends; they preserve chromosomes from degradation and end-to-end fusions contributing to genome stability and play an important role in the maintenance of chromosome integrity. The lagging strand of telomeric DNA consists of repeat arrays of TTAGGG and are ended by G-rich overhang, which inserts into double strand telomere region forming t-loop. As a result, the chromosome ends are no longer recognized as DSBs (Double Strand Breaks). In addition, telomeres are protected from inappropriate recombination by a specialized protein complex shelterin consisting of six proteins , 2018. TSorexgranarius   fibroblasts without of hallmarks of growth crisis  signaling containing not phosphorylated CHK2 (Check point 2 protein) and partial telomere deprotection with sufficient TRF2   . Moreovecomplex) . In thisγ-H2AX is usually used for detection of both manifest and invisible non repaired DNA breaks and also for visualization of deprotected telomeres . γH2AX iRattusnorvegicus  embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Most of the metaphases of these cell lines showed dysfunctional telomeres occurring before and after DNA replication, the last prevailed. The rESCs on the early passages of cultivation showed the lowest level of telomere dysfunction, while the riPSC lines demonstrated an elevated level. The level of telomere dysfunction increased in all studied cell lines after prolonged cultivation, especially noticeable increase in the level of telomere dysfunction arisen after G1. In addition, we have revealed a preferred accumulation of such dysfunction telomeres in riPSC lines than in rESCs. Note also the significant heterogeneity of the level of spontaneous telomere dysfunction between lines in the studied groups.Here we study the level of spontaneous telomere dysfunction in R.norvegicus 12-day embryos of laboratory strains Wag (RNFM1 and RNFF1 lines) and Brattleboro Wag (RWF1 and RWM1 lines). Three rESC lines  were isolated from blastocysts of different Wag rats. Three riPSC lines  were established as clonal lines from RNFF1 on 6 passage of cultivation by transduction. Briefly, rEFs were seeded at 104 cells/cm2 in a six-well plate. One hour before transduction, the growth medium was supplemented with 4 mg/ml hexadimethrine bromide  and the virus particles diluted in culture medium were added to wells for 18 hours .Four rEF lines were obtained from 18 hours . Then th18 hours . The rES18 hours . The rEFFISH (Fluorescent In Situ Hybridization) procedure with probe to telomeric DNA. Preparation of metaphase spreads for experiment was performed as described in FISH procedure. FISH with a high sensitive telomere PNA (peptide nucleic acid) probe (CCCATT)3 conjugated with FITC (Applied Biosystems) was performed as previously described , equipped with filter sets No. 49 (Zeiss),SP101 FITC and 103v1 (Chroma Technology), CCD-camera  and running ISIS5 software (METASystems GmbH). Microscopy was performed in the Microscopic Centre of The Federal Research Center Institute of Cytology and Genetics of SB RAS, Novosibirsk, Russia. Statistics were performed using the t-test in STATISTICA 10. The results are presented as mean ±SD.R.norvegicus telomere status was based on the accumulation of γ-H2AX histone in telomeres of metaphase chromosomes (Meta-TIF). A fluorescent signal located on one of the two sister chromatids demonstrates the chromatid type of dysfunction (Meta-TIF of chromatid type) that has arisen after DNA replication while signals located on both sister chromatids on the one chromosomal arm confirm the chromosome type dysfunction (Meta-TIF of chromosome type) arose in prereplication period R.norvegThe obtained data are presented in Table rEFs (97 percent) on 5–9th passage of cultivation contained Meta-TIFs  between lines on 5–9th and 23–29th passages, correspondingly. At the same, there observe wide disparities between studied lined in both groups on early passages and after prolonged cultivation. For instance, an average number of Met-TIFs on metaphase were 1.46 in RNFF1 on 9th passage of cultivation and 4.53 in RWM1, on 5th passage, and 2.66, in RWF1 on 29th passage.The on Table , Fig. 2.The standard method of embryonic fibroblasts generation suggests a presence of fibroblasts at different stages of differentiation, as well as the other types of cells in primary cultures; as a rule, further cultivation leads to changes in cell content. These factors appear can affect the level of telomere dysfunction in the lines.rPSCs)The level of spontaneous telomere dysfunction in rat pluripotent stem cells . In rESCs, the average number of Meta-TIFs of chromosome type per metaphase was 0.14±0.04 an average, and 1–5 percent of metaphases contained five and more of such Meta-TIFs. During cultivation, the number of Meta-TIFs on metaphase has increased. RES28 showed 9 percent metaphases containing five and more Meta-TIFs of chromosome type on 29th passage and 5 percent on 9th passage. RES27 on 42nd passage contained 52 percent of such metaphases, whereas on 10th passage only 2 percent. The maximum number of such Met-TIFs on metaphase was 18.Similar to the ed Table , Fig. 2.riPSCs were established from RNFF1 on 6th passage of cultivation. This line had the least number of dysfunctional telomeres among the rEFs  and 0, correspondingly; and in rECSs, 0.91±0.10 on average and 1–5, correspondingly.Fs Table . HoweverriPSCs has increased during cultivation. Some indicators characterized Meta-TIFs of both types differed significantly (P<0.05) in riPSCs on 4–6th and 15–22nd passages of cultivation  in the rESCs and the riPSCs; and one indicator, the number of metaphases with Meta-TIFs of chromosome type, was significantly higher (P<0.05) in the riPSCs, than in the rEFs. In general, the pattern of telomere dysfunction in the riPSCs is looking closer to those in the rEFs than in the rESCs.Similar to the other studied cell lines, the level of telomere dysfunction in the in vitro pluripotent stem cells showed low levels of spontaneous telomere dysfunction at the early stages of cultivation. The least level of telomere dysfunction characterized the rESCs whereas the riPSCs demonstrated elevated level. This applies to both types of dysfunction, chromatid and chromosomal, but increasingly to the chromosome type. For instance, the mean number of metaphases containing five and more Meta-TIFs of chromosome type in riPSCs exceeded that in normal human cells while the mean number of such Meta-TIFs on metaphase did not  telomeres, containing 213 kb of telomeric repeats on average . However, such telomeres activate DDR signaling and recruit ɤ-H2X (TRF2 deficit takes place, the activation of NHEJ, chromosome ends fusion with the following genome instability, a cell growth crisis, and cell death occur. Unlike whole genome, only five arisen in G1 partially deprotected telomeres with DDR signaling are able to induce replicative senescence in human cells and a more such telomeres can be accumulated without impact on growth cells in the absence of p53 signaling (TRF2. Since ALT cells have larger telomeres and, as a rule, inactive p53 the partially deprotected telomeres may be involved in the occurrence of telomere dysfunction in them (A special protein complex shelterin protects telomeres from inappropriate recombination . In thisit ɤ-H2X . If TRF2ignaling . The tel in them  Possibly in them . These fSpontaneous telomere dysfunction or telomere signaling are associated with the special mode of telomeric DNA replication and with a functioning of telomere safeguard complex shelterin, contributing to repression of NHEJ activation to avoid a numerous chromosomal rearrangements. They are involved in regulation of chromosome length and maintaining of genome stability.rEFs and riPSCs, the rESCs showed a reduced frequency of spontaneous telomere dysfunction on early passages of cultivation while the riPSC lines demonstrated an elevated level; moreover, the level of dysfunction was very different in studied riPSC lines. As far as cultivation, the number of dysfunctional telomeres has increased in cells of all lines; this is especially true for riPSCs. riPSCs are a model system, the study of which shows how important in personalized medicine the deeper monitoring of human iPSC attributes including the level of spontaneous telomere dysfunction before clinical implementation.Compared with


These Ds-containing DNA aptamers were generated by a method called cell-ExSELEX to target three breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. Aptamer 14A-MCF7, which targets MCF7 cells, specifically binds to MCF7 cells, but not other cancer cell lines. Aptamer 07-MB231, which targets MDA-MB-231 cells, binds to a series of metastatic bone and lung cancer cell lines. Aptamer 05-MB231 targets MDA-MB-231 cells, but it also binds to all of the cancer and leukemia cell lines that we examined. None of these aptamers bind to normal cell lines, such as MCF10A and HUVEC. In addition, aptamers 14A-MCF7 and 05-MB231 are internalized within the cancer cells, and aptamer 05-MB231 possesses anti-proliferative properties against most cancer cell lines that we examined. These aptamers and the generation method are broadly applicable to cancer cell imaging, biomarker discovery, cancer cell profiling, anti-cancer therapies, and drug delivery systems.The potential of genetic alphabet expansion technologies using artificial extra base pairs  has been rapidly expanding and increasing. We present that the hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5- The creation of unnatural base pairs (UB pairs or UBPs) that exhibit high fidelity in replication as a third base pair has led to novel biotechnologies. The UBP technologies provide new material production platforms, such as semisynthetic organisms that generate novel proteins containing unnatural amino acids15 different sequence species by an evolutionary engineering method called SELEX .22DNA aptamers are single-stranded oligonucleotides that specifically bind to target molecules and materials of interest. They are isolated from a nucleic acid library containing ∼10b]pyridine) and Px (2-nitro-4-propynylpyrrole)  A.24, 25 Here, to explore the versatility and further availability of Ds-DNA aptamers, we applied ExSELEX to Ds-DNA aptamer generation by targeting three representative breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. From each cell-ExSELEX used with the respective cell line, we obtained several Ds-DNA aptamers with unique specificities, from target cell specific to broadly cancer cell specific, and/or with some biological activities, such as internalization within the target cells and anti-proliferative activity. These results indicate the high potential of the Ds-DNA aptamers obtained by cell-ExSELEX for a wide range of applications toward cancer diagnostics and therapeutics and also suggest that genetic alphabet expansion increases the diversification of DNA aptamer generation and functionalization.In this cell-ExSELEX B, we use15 or 1.2 × 1015 different sequence combinations. After repetitive rounds of cell-ExSELEX, each DNA sequence in the enriched libraries was determined with an Ion PGM sequencing system (Life Technologies). In the sequencing system, the Ds bases in each DNA were replaced with the natural bases by replacement PCR, and the Ds positions in each DNA sequence were identified by the barcode embedded in each sub-library.We chose three breast cancer cell lines—MCF7, MDA-MB-231, and T-47D—as the targets and started cell-ExSELEX for each target using the Ds-predetermined DNA library with 0.6 × 10The cell-ExSELEX conditions are summarized in The enriched sequences of each library were determined by standard deep sequencing with the Ion PGM system, followed by the barcode identification of the Ds positions.To optimize each aptamer sequence and to predict its secondary structure,We chemically synthesized the selected aptamers—08B-MCF7DsDs51), 14A-MCF7(DsDs53), 07-MB231(DsDs43), 05-MB231(DsDs49), 23-MB231(DsDs49), and 03-T47DDsDs33)—and their variants to assess each aptamer’s binding affinity. First, we replaced one or two Ds bases in each aptamer with adenine to identify which Ds bases are essential  for bind1, 14A-MC—and theiWe assessed the binding affinities of each aptamer and its variants by flow cytometry, using its respective target breast cancer cell line . We usedAll of the aptamers originally contained two Ds bases, which existed within the initial Ds-predetermined sub-libraries. From the examination of Ds → A variants, one or two Ds bases strongly affected the target binding of each aptamer. For example, for 08B-MCF7  and S14,D = 1–3 nM) by flow cytometry , 14A-MCF7(DsDs42mh), 05-MB231(Ds45mh), 07-MB231(DsDs52mh), 23-MB231(DsDs58mh), and 03-T47D(DsDs42mh)—that target each breast cancer cell line are shown in We confirmed the high affinities of the aptamers 08B-MCF7, 14A-MCF7, 05-MB231, and 07-MB231 to their target cell lines  B and 3C We tested the binding specificity of each Alexa 488-labeled aptamer to the three breast cancer cell line targets and other cancer and leukemia cell lines , as well as normal cell lines (MCF10A and HUVEC), with fluorescent microscopic imaging . AptamerWe confirmed the binding of 14A-MCF7(DsDs38mh), 07-MB231(DsDs52mh), and 05-MB231(Ds45mh) to each target cell by flow cytometry at 4°C and 37°C . AlthougWe also examined the stabilities of 14A-MCF7(DsDs38mh), 07-MB231(DsDs52mh), and 05-MB231(Ds45mh) in human serum at 37°C, in comparison with AS1411-26 . AS1411 We performed competition experiments between high-affinity aptamer pairs to determine whether each aptamer binds to different targets from the others  and S23.For further exploration of the specificity of each Ds-DNA aptamer to each cancer cell line, the binding properties of each aptamer-cell combination were analyzed by flow cytometry assays using six Ds-DNA aptamers (14A-MCF7(DsDs42mh), 08B-MCF7(Ds54mh2), 07-MB231(DsDs52mh), 23-MB231(DsDs58mh), 05-MB231(Ds45mh), and 03-T47D(DsDs42mh) , AS1411-26, and Cont26, with 15 cancer cell lines and two normal cell lines. The binding pattern panels of each DNA sample to each cell line are summarized in These cancer cell panels can be classified into four categories, based on each aptamer’s binding tendency : categorWe found that categories 1–3 each have a common aspect. The category-1 cell lines are metastatic cancers of the bones (MDA-MB-231 and PC-3) and lungs (A549). The category-2 cell lines, except for T-47D, are low cell adhesive. The category-3 cell lines are related to gastrointestinal or gynecological cancers of endocrine organs. Thus, a new type of cancer profiling might be possible, with classifications using several Ds-DNA aptamers that have different binding specificities to cancer cell lines.Furthermore, the differences in the fluorescent intensities of each category might reflect the unique expression level of each aptamer target on the cell, when the aptamer’s affinity is sufficiently high. For example, the fluorescence intensity of the aptamer 05-MB231 binding to T-47D cells was much higher than that to MDA-MB-231 cells . Thus, hThe cytostatic activities of five Ds-DNA aptamers, 08B-MCF7, 05-MB231, 07-MB231, 23-MB231, and 03-T47D, were examined by a WST-8 assay using several cancer cell lines. The activities were compared to those of AS1411-26 and Cont26, as a positive and a negative control, respectively. In addition, other anti-cancer drugs, 5-fluorodeoxyuridine (FUdR) and Paclitaxel (PTX), were also used as positive controls , and S2750 = 120 ± 30 μM against CCRF-CEM, 56 ± 39 μM against HT-1080, and 190 ± 140 μM against WI-38).Salmonella typhimurium  and Escherichia coli (WP2uvrA) in an Ames test (data not shown).The anti-cancer activity of 05-MB231 does not result from the Ds nucleoside or nucleotide itself. The Ds nucleoside exhibits slight toxicity against both cancer and normal cell lines . An analysis of the mechanisms by which 05-MB231 exhibits the cytostatic activity might lead to the development of a better anti-cancer drug and synergistic combinations with other types of anti-cancer drugs.Based on the binding and profiling data and the competition experiments of each aptamer, the target antigen for 14A-MCF7 and 08B-MCF7 would be the same, while the antigens of 14A-MCF7, 05-MB231, and 07-MB231 would be different. It is notable that the expression level of each antigen could be predicted from the binding amount (fluorescent intensity) of each labeled aptamer when high-affinity aptamers are used, as in the case of 05-MB231 .D = 30 nM), generated by cell-SELEX using their UBP, Z-P.D = 2.6–108 nM) that target MDA-MB-231 cells by conventional cell-SELEX, and these aptamers specifically bound to MDA-MB-231 and T-47D cells.D = 5.9–138.2 nM) that targets the metastatic colorectal carcinoma LoVo bound to only the target cells.D = 1–5 nM measured by flow cytometry) will be required.As shown here, ExSELEX could provide a new cancer-profiling method, using a series of Ds-DNA aptamers for personalized medicine to select appropriate anti-cancer drugs.The cell-ExSELEX method could provide valuable information for cancer research and pharmaceutical applications toward individualized cancer medicine. In addition, cell-ExSELEX can be used to target other types of cells, including stem cells and induced pluripotent stem cells (iPSCs).The unnatural nucleoside triphosphates, dDsTP and dPxTP, and the Ds phosphoramidite were synthesized as described previously.The DNA library used for cell-ExSELEX was prepared by mixing 24 different single-stranded DNA sub-libraries 93-mer; . Each su2 atmosphere.The human breast cancer cell line MCF7 (RCB1904) was provided by the RIKEN BioResource Center (BRC), through the National Bio-Resource Project of the MEXT, Tsukuba, Japan. The MCF7 cell line was maintained in Minimum Essential Media (Life Technologies) supplemented with 10% fetal bovine serum , non-essential amino acids, and 1 mM sodium pyruvate (Life Technologies). The MCF10A cell line (CRL-10317), used in the counter-selection in cell-ExSELEX, was purchased from the American Tissue Culture Collection (ATCC). The base medium  for MCF10A was prepared with the additives provided in an MEGM kit (Lonza). Other human breast cancer cell lines, T-47D  and MDA-MB-231 , were maintained according to the supplier’s instructions. For the specificity profiling of the obtained aptamers, other types of cell lines—MDA-MB-453 , HCT-116 , A549 , MKN45 , NIH:OVCAR-3 , PC-3 , MIA PaCa-2 , Tohoku University, #TKG0227), Panc-1 , PLC/PRF/5 , HeLa , KG-1 , CCRF-CEM , and HUVEC —were also cultured according to the suppliers’ instructions and used for evaluations of the aptamers’ binding. These cell lines were cultured in a 37°C incubator under a 5% CO2, 0.1 mg/mL tRNA, and 1 mg/mL BSA, in D-PBS (−)) and incubated with approximately 5 × 106 target cells in a culture dish at 4°C for 60 min with gentle agitation. Unbound DNA species were removed by washing with ice-cold washing buffer (4.5 g/L glucose and 5 mM MgCl2 in D-PBS (−)) and then 0.5 mL water was added to the culture dish. To collect the cell-bound single-stranded DNA (ssDNA) species, the cells were scraped off from the culture dish with a cell scraper. The recovered cell-DNA solution was then heated at 95°C for 10 min, followed by centrifugation at 14,000 rpm for 20 min to pellet the cell debris. The supernatant, which contained the eluted DNA, was then subjected to PCR amplification, as described previously.The initial single-stranded DNA library (1 or 2 nmol), diluted in Dulbecco’s PBS (D-PBS (−)), was denatured by heating at 95°C for 5 min and then kept at room temperature for 20 min. The folded DNA library solution was diluted in binding buffer  values were calculated by curve fitting, using the OriginPro 9 software (OriginLab) according to the following formula: Y = BmaxX/(KD + X), where X is the DNA concentration, and Y is the geometric mean fluorescence intensity (MFI). The MFI without DNA was subtracted from that with each aptamer, and these values were plotted and used for curve fitting.The enrichment process of the DNA library during the cell-ExSELEX, as well as each aptamer’s binding capability, was analyzed by flow cytometry. The cultured cells were dissociated by an incubation with 0.2% EDTA, after washing with D-PBS (−). After washing with ice-cold washing buffer twice, the cells were incubated with Alexa 488-labeled DNA  in 100 μL binding buffer at 4°C for 30 min. After the incubation, the cells were washed three times with ice-cold washing buffer, re-suspended in 200 μL binding buffer, and then filtered with a 40-μm cell strainer  to remove clumped cells. The fluorescence intensity of the DNA bound to the cells was monitored with a cell sorter, S3e (Bio-Rad), by counting 10,000 events. The obtained data were analyzed with the FCS Express software (De Novo Software). Dissociation constant  and purified by denaturing gel electrophoresis. Each Alexa 488-labeled aptamer was subjected to a binding analysis with the S3e cell sorter, as described earlier.For further optimization and characterization of each aptamer candidate, partially randomized, doped libraries corresponding to 08B-MCF7, 14A-MCF7, 05-MB231, 07-MB231, 23-MB231, and 03-T47D were prepared, as described previously.To visualize and compare the binding specificity of each selected aptamer, fluorescence images of various cell lines bound with the aptamer were observed by fluorescence microscopy  or confocal laser microscopy . Each cell line was cultured on an 8-well chamber slide (Corning) and incubated with 250 nM Alexa 488-labeled aptamer, either on ice or at 37°C for 30 min. After the incubation, the supernatant was removed and the cells were washed with ice-cold washing buffer three times. The cells were fixed with 4% formaldehyde  for 10 min at room temperature, stained with DAPI , and mounted with VECTASHIELD mounting medium . In the confocal imaging, prior to the treatment with the aptamer, the cells were incubated at 37°C for 30 min in the presence of 75 nM LysoTracker Red DND-99 (Molecular Probes).4 cells per milliliter for cancer cell lines, except for KG-1, and 4 × 104 cells per milliliter for HUVEC and MCF10A cells) were plated into each well of a 96-well microplate, 1 day before the addition of DNA aptamers. In the case of KG-1 cells, 90 μL cell solution was plated into each well, 4 hr before the DNA addition. Each DNA aptamer was folded in D-PBS (−), similar to the DNA library used for cell-ExSELEX, at a 0.1mM concentration. The folded DNA aptamer, as well as FUdR , was used as a positive drug control and was diluted with D-PBS (−) and the medium used for each cell line, and added to the cells at final concentrations of 1, 2.5, 5, or 10 μM for DNA and 10 or 100 nM for FUdR in 10% D-PBS (−)/media (0.1 mL per well). As another positive control, PTX  in DMSO was also used for the analysis and added at the final concentration of 10 nM in 0.1% DMSO/media (0.1 mL per well). After incubation for 96 hr, 10 μL Cell Count Reagent SF  was added to each well, and the absorbance of the formazan product obtained by WST-8 reduction was measured at 450 nm with an ARVO multilabel counter (PerkinElmer).The cytostatic activity of representative aptamers against each cell line was examined through WST-8 assays, as follows. Aliquots (0.1 mL) of each cell solution


The use of EGFR inhibitors on oral squamous cell carcinoma (OSCC) as monotherapy yielded modest clinical outcomes and therefore would benefit from biomarkers that could predict which patient subsets are likely to respond. Here, we determined the efficacy of erlotinib in OSCC cell lines, and by comparing sensitive and resistant lines to identify potential biomarkers. We focused on the 4717C > G polymorphism in periplakin (PPL) where the CC genotype was associated with erlotinib resistance. To validate this, erlotinib-resistant cell lines harbouring CC genotype were engineered to overexpress the GG genotype and vice versa. Isogenic cell lines were then studied for their response to erlotinib treatment. We demonstrated that overexpression of the GG genotype in erlotinib-resistant lines sensitized them to erlotinib and inhibition of AKT phosphorylation. Similarly, the expression of the CC genotype conferred resistance to erlotinib with a concomitant increase in AKT phosphorylation. We also demonstrated that cell lines with the CC genotype generally are more resistant to other EGFR inhibitors than those with the GG genotype. Overall, we showed that a specific polymorphism in the PPL gene could confer resistance to erlotinib and other EGFR inhibitors and further work to evaluate these as biomarkers of response is warranted. It is most prevalent in India, Bangladesh and Pakistan due to the practice of known risk habits such as smoking, excessive alcohol consumption and betel quid chewing2. Patients diagnosed at early stage can be treated by surgery or radiotherapy alone while concurrent radio-chemotherapy is often used in patients with locally advanced disease3. About a third of the patients will progress into metastatic stage, with palliative chemotherapy as the only therapeutic option. Recently, pembrolizumab4 and nivolumab5 have been approved for patients with metastatic OSCC with disease progression during or after chemotherapy. Despite this advancement, therapies for advanced OSCC remains limited and targeted therapies are actively being explored to improve the survival of OSCC patients.Oral squamous cell carcinoma is one of the top ten cancers among men in the world6. Increased activity of EGFR results in activation of downstream signalling cascade such as PI3K/PTEN/AKT, ERK, and Jak/STAT pathways to promote cell proliferation, invasion and metastasis. Hence, increased protein expression of EGFR is a prognostic marker for poor survival in OSCC patients6. To target EGFR for therapeutic purposes, inhibitors have been developed and several of these have been tested in OSCC7. The success of cetuximab, a recombinant monoclonal antibody targeting EGFR in extending the progression-free survival (PFS) in patients with recurrent/metastatic OSCC, resulted in its approval by US Food and Drug Administration (FDA) in 20067. Whilst this is encouraging, this success has not been recapitulated with small molecule inhibitors targeting EGFR. One of these small molecule inhibitors is erlotinib or known as OSI-774 or Tarceva. Erlotinib is an orally active small molecule that blocks EGFR-mediated intracellular signalling by binding competitively to the ATP binding region8. It is approved for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with stable disease after standard platinum-based first-line chemotherapy9. A clinical study showed the efficacy of erlotinib by tumour shrinkage in 9 out of 35 locally advanced OSCC patients in a neoadjuvant setting before surgery10. However, a further phase II clinical trial on OSCC patients from 2006 to 2011 did not demonstrate a significant increase in PFS when erlotinib is combined with cisplatin and radiotherapy11. Similar results were also shown using another EGFR inhibitor, gefitinib12. Despite the conclusions, detail analysis showed that 52% of patients treated with erlotinib and cisplatin had a complete response as compared to 40% of patients who responded to cisplatin alone11; for gefitinib, 12.5% of patients who received docetaxel and gefitinib showed response as compared to 6.2% for patients treated with docetaxel alone12.OSCC has been characterized by high expression of epidermal growth factor receptor (EGFR)13 and olaparib for breast cancer patients who are HER-2 negative and carrying BRCA mutations14. Studies in NSCLC showed that 60–80% of the patients with EGFR mutations respond well to erlotinib, but it was evident that patients without these mutations also benefited from erlotinib15, suggesting that EGFR is not a reliable biomarker that could predict for drug response. Furthermore, EGFR mutations are not frequently observed in OSCC and hence may not be a useful biomarker in this context16. Further biomarker analysis examining the mutational status of EGFR and KRAS, copy number of EGFR and protein expression of EGFR, cMET, HER2, HER3 and PTEN from the TORCH trial in patients with advanced NSCLC was not able to identify other biomarkers for erlotinib17 that could be further tested in the OSCC setting. In OSCC, Martins and colleagues attempted to investigate the efficacy of erlotinib on human papillomavirus (HPV) positive tumour by examining p16 expression, but the test was performed in less than 50% of the patients, rendering it difficult for further analysis11. Taken together, there is currently no biomarker identified for EGFR inhibitors for OSCC, and this has in part stalled the advancement of these targeted therapies in OSCC7.Recent clinical trials on small molecule inhibitors were proven to be more effective when the patients were stratified based on biomarkers. For example, the approval of trametinib and dabrafenib for melanoma patients with BRAF V600 mutationsIn order to progress with further testing of EGFR inhibitors, it is pivotal to identify biomarkers that could predict the response to these drugs. In this study, we combined genomic information on cancer cell lines with drug response data to identify potential biomarkers that could predict response to EGFR inhibitors. Focusing primarily on the EGFR inhibitor erlotinib, we demonstrated that a single nucleotide polymorphism (SNP) in PPL could inhibit AKT phosphorylation and modulate response to erlotinib.50 at sub-micromolar concentrations (0.15 ± 0.02 µM to 0.62 ± 0.14 µM) and 6 cell lines were resistant with IC50 from 1.39 ± 0.05 µM to >4 µM. A431 with IC50 less than 1 µM was used as sensitive control18 whereas MCF7 was used as a resistant control with IC50 of more than 4 µM19  were sensitive to erlotinib with ICM19 Fig. .Figure 1p = 0.019) and 78.3% in ORL-214 (p = 0.011); Fig. Further, we determined the cytostatic effect of erlotinib on 2 representative cell lines from the erlotinib sensitive and resistant groups respectively by measuring the active DNA synthesis using the EdU assay. We showed that the proliferation rate of sensitive cell lines  reduced significantly by >75% (p < 0.001) when treated with 1 µM erlotinib for 24 hours compared to the control (0.01% DMSO). Meanwhile a large proportion of the cells in the resistant group were still proliferating at 1 µM  were significantly associated with increased sensitivity towards erlotinib treatment (p < 0.001 for both). Previous studies have suggested that the linker domain of PPL could bind to AKT21. Therefore, we decided to focus on rs2037912 harbouring homozygous GG at codon 4717, coding for glutamine, or homozygous CC, coding for glutamate located in this domain. The polymorphism was confirmed using Sanger sequencing on the cell lines ; ORL-214 (p = 0.043); Fig. , demonstrating that PPL is very likely to be important in modulating response to erlotinib.In order to validate the importance of the PPL gene in modulating the response of OSCCs lines to erlotinib, transient knockdown using siRNA was carried out in erlotinib-resistant lines (ORL-153 and ORL-214). The success of PPL knockdown was validated by western blotting as shown in Fig. Since cell lines harbouring the GG genotype in PPL are more sensitive to erlotinib, we went on to confirm the effects of this genotype in response to erlotinib. To do this, we overexpressed GG in ORL-153 and ORl-214 which originally carry the CC genotype. The same lines were also transfected with the vector control (VC) as a control and the plasmid expressing the CC genotype to ensure that our observations were not due to the transfection. The success in transfection was confirmed with Sanger sequencing using DNA from the transfected clones Fig. . With th50 of ORL-153 (VC) at 29.02 ± 1.7 µM, the IC50 of ORL-153 (GG) was reduced by ~3 fold to 11.67 ± 2.1 µM (p = 0.015). Similarly, IC50 of ORL-214 (GG) was ~2-fold lower than ORL-214 (VC), at 13.1 ± 1.4 µM and 27.6 ± 1.9 µM respectively . Overexpressing the CC genotype in ORL-153 and ORL-214 did not alter the IC50 significantly in comparison to VC as these lines originally harbour the CC genotype ; ORL214 (p = 0.040). Biochemical analyses were done to determine the changes of expression in PPL and the molecules within the EGFR signalling pathway in VC, GG, and CC clones after being treated with erlotinib . In Cal27, overexpressing the CC genotype increased the IC50 of VC by 4.4-fold, from 0.62 ± 0.1 µM to 2.7 ± 0.4 µM . Consistently, the CC genotype conferred resistance to erlotinib where EdU assays demonstrated that in ORL-115, EdU expressing cells were higher in CC than VC clones, albeit not statistically significant. For Cal27, the cells expressing CC were less responsive to erlotinib, as evidenced by higher proliferating cells as compared to VC . Western blot analyses were carried out to determine the biochemical changes in EGFR signalling in VC, GG, and CC after being treated with erlotinib. Protein expression of PPL was increased by 2.3-fold and 1.5-fold respectively in VC and GG clones of ORL-115 when treated with erlotinib. For Cal27, PPL expression increased by 4.3-fold and 2.6-fold respectively in VC and GG clones upon erlotinib treatment. Erlotinib treatment did not alter the level of PPL in CC clones. Phosphorylation of AKT levels were not changed in the CC clones of ORL-115 and Cal27 despite erlotinib treatment as compared to VC and GG clones where pAKTser473 were reduced as clearly demonstrated in the western blot analysis . Both pERK1/2 and pS6 expression levels were reduced in VC, GG and CC clones upon erlotinib treatment . To validate the hypothesis that genotype GG and CC would modulate the response to EGFR TKIs in other head and neck cancer (HNSC) cell lines, we looked into the Genomics of Drug Sensitivity in Cancer (GDSC)22. We were able to analyse data on HNSC cell lines treated with afatinib and gefitinib from the database. As for afatinib, the mean IC50 of cell lines with GG genotype was 12.36 µM ± 18.76, lower compared to cells harbouring CC genotype with IC50 at 17.37 µM ± 27.6 for afatinib treatment even though it was not statistically significant  and GDSC facilitates the identification of putative biomarkers as drug responses can be correlated to the well-characterized genomic features50 remained relatively high. Although these data suggest that PPL could modulate sensitivity to erlotinib, the modest difference could be due to the fact that the level of PPL knock-down was not sufficient to sensitize cells to erlotinib to the IC50 levels seen in inherently sensitive cells. Perhaps, the level of PPL may not be the only factor driving erlotinib sensitivity, and other factors such as its ability to interact with other proteins depending on its genotype could also be an important factor. Notwithstanding, our data is supported by Tonoike and his colleagues27 who showed that knocking down PPL reduced cell proliferation in pharyngeal cancer. This demonstration that the disruption of endogenous PPL levels could affect response to erlotinib suggests that PPL is involved in EGFR signalling. To examine this in more detail, we identified and validated the role of a polymorphism at rs2037912 on PPL in modulating sensitivity to erlotinib. We focused on rs2037912 where cell lines with the CC genotype (a glutamine at amino acid 1573) were significantly associated with resistant to erlotinib compared to cell lines with a glutamate (GG) at the same position. This polymorphism is particularly interesting in the context of EGFR signalling because previous immunoprecipitation studies have indicated that codon 4717 resides in a section of the PPL protein that binds to AKT21. To demonstrate whether the PPL polymorphism would directly affect erlotinib sensitivity, we engineered the GG genotype in erlotinib-resistant cells endogenously carrying CC genotype and showed that the change could sensitize cells that were originally resistant to erlotinib. This response was accompanied by a reduction in the expression of pAKTser473 rendering the GG clones more sensitive to erlotinib treatment. Meanwhile, overexpression of the CC genotype into erlotinib responsive cells endogenously harbouring GG genotype sustained the expression of pAKTser473 and cells were less responsive to erlotinib. Our data clearly demonstrated that this particular polymorphism in PPL directly affects response to erlotinib and this is likely due to modulating the activation of AKT, a key signalling molecule downstream of the EGFR. We noted that although overexpressing GG into erlotinib-resistant cells significantly reduced the IC50 when treated with erlotinib, however the IC50 was still relatively high and this could be possibly due to the effects of the second polymorphism (rs1049205), which changes the amino acid position at 589 from arginine (GG) to glutamine (AA) that was also significantly associated with erlotinib sensitivity. We did not test the effect of both polymorphisms simultaneously in this study so that the effect of each polymorphism can be delineated independently for their effects on drug sensitivity. Both of the loci have high LOD score and codon change from GG to AA at codon position 1766 occur together with CC change to GG at codon position 4717 in cell lines sensitive to erlotinib  response29. PPL which interacts with AKT, acts as a localization signal to AKT or a shuttle for delivery of AKT to the various cellular compartments21. For example, when PPL localizes to the cell membrane, AKT becomes activated and translocated to other compartments in the cell30, further underscoring that fact that PPL could play an important role in affecting the treatment response by EGFR TKIs.In addition to erlotinib, we also found the CC genotype at rs2037912 to be associated with resistance to other EGFR inhibitors including afatinib. Afatinib is approved for metastatic NSCLC with common EGFR mutations as well as L861Q, G719X, and/or S768I32. Whilst one of the factors is that Asian populations have a higher mutational rate in EGFR33, this could also be due to other genetic factors. Looking at the PPL polymorphism in the HapMap study of Asian and Caucasian populations (available from dbSNP website)34, ~47% of Asians including Japanese and Han Chinese in Beijing have the GG genotype as compared to only ~27% of Europeans. By contrast, ~23% of Caucasian have CC genotype in comparison to ~8.5% in Asians. The effect of this polymorphism in PPL between Asian and Caucasian populations could provide some additional insights into the differential response to EGFR inhibitors and should be investigated further.Clinical trials on NSCLC patients have demonstrated a better response to EGFR inhibitors amongst patients of Asian ethnicity compared to Caucasian patientsin vivo models to further investigate the role of 4717C > G polymorphism in PPL in modulating sensitivity to erlotinib. Cal27 overexpressing CC or GG in PPL were transplanted into NOD.CB17-Prkdcscid/NCrCrl mice but at the end of the study, genotyping showed that overexpression was not retained. Future investigations on establishing a suitable model to overexpress GG or CC stably and how the polymorphism alters the structure of PPL and affect binding to AKT is warranted. Nevertheless, our data add to the array of possible biomarkers for EGFR targeted therapy and suggest that other proteins that interact with molecules within the EGFR signalling pathway could confer resistance to treatment. In conclusion, our data provide evidence that a polymorphism in PPL could play a role in conferring resistance to EGFR inhibitors by affecting the activation of AKT. This finding is significant because many other tyrosine kinase receptors signal through AKT, and PPL could also play a role in conferring resistance in these pathways. We show that proteins such as PPL that interacts with signalling molecules could confer resistance to kinase inhibitors and affords an opportunity to investigate PPL as a biomarker for cancer therapeutics that directly or indirectly inhibits AKT phosphorylation.One of the limitations of the study is the lack of suitable Y1068 (#2234), AKT (#2938), pAKTser473 (#4060), ERK1/2 (#9102), pErk1/2 (#9101), S6 ribosomal protein (#2317), pS6 ribosomal protein , periplakin  and α-tubulin . The secondary antibodies goat anti-rabbit IgG-HRP (SB4010-05) and goat anti-mouse IgG-HRP (SB1010-05) were purchased from Southern Biotech. 3--2,5-diphenyltetrazolium bromide  solution was prepared at 5 mg/ml in phosphate-buffered saline (PBS).Erlotinib HCl  was dissolved in dimethyl sulfoxide  at a stock concentration of 10 mM and diluted in culture medium for use. Afatinib  was dissolved in DMSO at a stock concentration of 40 mM, and further diluted in culture medium to the required concentrations. Primary antibodies used in these studies were EGFR (#2232), pEGFR20. These cell lines were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient mixture F-12 HAM’s medium  supplemented with 500 ng/ml hydrocortisone (Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum . The breast cancer cell line MCF7 was cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 100 IU Penicillin/Streptomycin. Cal27 and A431 were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose  with 10% heated-inactivated FBS and 100 IU Penicillin/Streptomycin. Cell line authentication was carried out by comparing the STR profiles of cell lines to the matched blood or tumour from the patients for 16 ORL lines20 or to ATCC database for other lines. Cell lines were routinely checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). All cultures were incubated in a humidified atmosphere with 5% CO2 at 37 °C.The ORL cell lines used were established in our laboratory as described previously4 cells were seeded per well in 96-well plates and treated with erlotinib at ~1 nM to 4 µM. 72 hours after treatment, MTT solution was added to each well and incubated at 37 °C for 3 hours. Next, 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan and the optical density was determined using Biotek microplate reader set at 570 nm. IC50 of the cell lines was presented as the mean ± SEM of three independent experiments with DMSO (0.01%) as the control. MCF7 which is resistant to erlotinib19 and A431 previously reported to be sensitive18 to erlotinib were used as resistant and sensitive controls.Cell viability of OSCC lines was determined using MTT assay as described previously. Briefly, 2–5 × 104, Alexa Fluor azide, reaction buffer additive) for 30 minutes in the dark. After washing (3% BSA), cells were incubated with 1X Hoechst 33342 solution (5 μg/mL) for 30 minutes. Coverslips were mounted with VECTASHIELD mounting medium (Vector Laboratories Inc) after washing with PBS. Olympus IX81 imaging system equipped with XcellencePro imaging software (Olympus) was used to capture EdU positive cells with Alexa Fluor 647: excitation 650 nm and emission 667 nm while live cells stained with Hoechst were captured using a filter with excitation at 360–370 nm and emission at 420 nm at 20x magnification. Images were merged using XcellencePro imaging software. Unmerged images were analyzed using Quick Count® to determine the number of Edu positive cells and live cells . Percentage of EdU positive cells was calculated using the following formula: (EdU positive cells/number of DAPI cells) × 100. The percentage of EdU-positive cells was normalized against vehicle control. The proliferation rate of cell lines was presented as the mean ± SEM of two independent experiments with DMSO (0.01%) as the control.Two OSCC cell lines shown to be resistant to erlotinib in this study (ORL-214 and ORL-153) and two other sensitive lines  were subjected to cell proliferation assay using Click-iT EdU Alexa Fluor 647 Imaging Kit (Invitrogen) according to manufacturer’s protocol. Briefly, cells were plated on coverslips in 12-well plates and treated with erlotinib at 1 µM for 24 hours. Following EdU incubation at 10 µM for 2 to 4 hours at 37 °C as optimized previously for each cell line, cells were fixed with 3.7% (v/v) formaldehyde in PBS for 15 minutes and permeabilized using 0.5% (v/v) Triton-X in PBS for 20 minutes at room temperature. Cells were washed with 3% (w/v) BSA in PBS. Next, the cells were stained with the Click-iT reaction cocktail  identified, the SNV was transformed into a two-way contingency table with two categories (i) sensitive and (ii) resistant. Cell lines ORL  were grouped as sensitive and ORL  were grouped as resistant. Fisher’s exact test was performed in R (version 3.20) using the function “fisher.test”. SNV with p value < 0.05 was considered significant.Identified mutations from the OSCC cell lines were tested for statistical significance between the erlotinib sensitive  or non-targeting control (Sigma-Aldrich) using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were collected 48 hours post-transfection to be plated for MTT assay and western blotting.The pcDNA3.1 FLAG plasmids construct carrying the PPL CC or GG genotype respectively at position 4717 were purchased from GenScript. Plasmids were transfected into the ORL-153, ORL-214, ORL-115 and Cal27 using Turbofect transfection reagent (Fermentas) according to the manufacturer’s instructions. Control cells were transfected with the pCDNA3.1 FLAG vector. Transfected cells were selected using G418 (Sigma-Aldrich) at 400 µg/ml for 7 days.2), 100 µM of each dNTP, 50 µM of each primer and 1.25 U of Taq DNA polymerase (Promega) using 200 ng of gDNA template. PCR was performed using Veriti DNA thermocycler (Applied Biosystems) with an initial denaturation at 95 °C for 3 minutes, followed by 30 cycles of denaturation at 95 °C for 30 seconds, primer annealing at 55 °C for 30 seconds and DNA extension at 72 °C for 30 seconds. The PCR product was subjected to 1% agarose gel electrophoresis and viewed using FluorChemTM HD2 imaging systems (Alpha Innotech). The 462 bp fragment was purified using QIAquick Gel Extraction Kit (Qiagen) and sequenced using Sanger sequencing.Genomic DNA (gDNA) was extracted using the Blood and Cell Culture DNA Kit (Qiagen) from the following samples: blood genomic DNA from donors of the ORL cell lines, ORL cell lines, Cal27, ORL-214, ORL-153, ORL-204, ORL-115 transfected with PPL[G/G], PPL[C/C]. Polymerase chain reaction (PCR) was conducted to amplify exon 22 of the PPL gene using the primers: PPL-seq-F 5′ACCGAGCAAGAGATCCAGAG3′ and PPL-seq-R 5′TGTGTCAGGGTGGATGACTACG3′. PCR was set up in 50 µl containing 1X amplification buffer (containing 1.5 mM MgClTM HD2 imaging systems (Alpha Innotech). Blots were exposed for 1 minute to detect the bands except for PPL, pEGFRY1068, pS6 and S6, where blots were exposed for 3 minutes. To normalize the loading, tubulin was used as loading control. Densitometry analyses of protein bands were performed using Image J36 and normalized by α-tubulin. Fold-change of band intensity was normalized by comparing to DMSO-treated control.Cells were lysed on ice using RIPA lysis buffer , 1 mM HALT proteinase and phosphatase inhibitors (Thermo Scientific). Proteins were collected from untreated cells to establish basal levels of protein expression as well as treated cells. Treated cells were serum starved for 16 hours before treatment with 1 µM of erlotinib (or DMSO control) in serum-free medium for 1 hour before the cells were washed and stimulated with 10 ng/ml of recombinant human EGF (R&D Systems Inc) for 10 minutes before harvesting. Protein concentration was measured using BCA protein assay (Thermo Scientific). 20 µg of protein was resolved in 8% SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane  in a tank transfer apparatus for 2 hours at 100 V . The membrane was blocked with 5% (w/v) skim milk in Tris-buffered saline with 1% (w/v) tween 20 (TBST) for 1 hour and probed with primary antibodies at 1:1000 in 1% BSA in TBST overnight at 4°C. Blots were washed in TBST (x3 for 5 minutes). The blots were then incubated with secondary antibodies conjugated with horseradish peroxidase (dilution 1:10000). After washing using TBST, detection was performed by ECL™ detection reagents  and viewed using FluorChemp value was indicated as p < 0.05, p < 0.01 and p < 0.005.Statistical analyses were performed using Graph Pad Prism (GraphPad Software). Data are presented as mean ± SEM of at least 2 independent experiments conducted in triplicate. Unpaired t-test with two-tailed was used to calculate statistical significance between control and treatment groups. ANOVA Kruskal-Wallis test was carried out to determine the significance between control and more than one treatment groups. Significant Supplementary information