TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains  and losses (chr X), with additional more CNVs found in CDDP-resistant cells . In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g.,  NANOG or the oncogene KRAS. Another typical alteration is the KIT/KITL pathway activation, characteristic for early stages and pre-invasive tumors [Testicular germ cell tumors (TGCT) are the most frequent solid tumors in young adult men aged 18–45 years. The incidence of TGCT has increased over the past decades, but the reason for this observation is unknown ,3,4,5. Ae tumors ,13,14,15e tumors ,16. Moree tumors .Based on the characteristic histological patterns, TGCT are classified into seminomas and nonseminomas. These two main TGCT subtypes show distinct clinical characteristics, differing in the course of the disease and treatment response. Introducing platinum-based regimens into the treatment of TGCT dramatically increased the overall cure rate, with over 90% of patients remaining in long-term complete remission ,19. NeveNANOG, OCT3/4/POU5F1), the cytotoxic stress/DNA damage drives these cells via p53 signaling directly to apoptosis, not allowing for any attempts of damage repair. The loss of this specific type of DDR, e.g. accompanying the differentiation of tumor cells, either spontaneous or induced by the applied chemotherapy, may then result in the establishment of the CDDP resistance [TP53 and MDM2 genes [XRCC2, HMGB4) [PIK3CA, IGF1R) [MAD2g) [BMI1) [The principal cause of the development of cisplatin (CDDP) resistance, which almost inevitably leads to the treatment failure and death of the affected TGCT patients, has not been identified, and the molecular basis of cisplatin resistance is poorly understood. In general, the resistance to cisplatin may arise at any phase of its kinetics and action . Howeversistance ,27,28,29M2 genes . In furt, HMGB4) ,32, onco, IGF1R) ,33, cell [MAD2g)  or epige) [BMI1) .In this project, we generated cisplatin-exposed counterparts of TGCT cell lines and investigated their newly acquired features in comparison with the original cells, to uncover particular genes or pathways involved in the acquired cisplatin resistance. In a group of primary tumor samples of TGCT patients with different stages of the disease, we then looked for similar aberrations and for aberrations that could be related to the disease development.50 approximately 10 times higher than the original sensitive cells (referred to as NCCIT) .NCCIT cell line was cultured with increasing doses of CDDP for 20 months (further referred to as NCCIT_CDDP), achieving a significant resistance to CDDP, with ICp = 0.05), which was accompanied by a decreased number of resistant NCCIT_CDDP cells in S phase . Interestingly, the trend in the cell cycle distribution between parental vs. derived Tera-2 cells was rather opposite. Tera-2_CDDP showed a decrease in G1  and an increase in S phase in comparison with parental cell line . The apoptotic rate of the cell lines was analyzed before and after a short exposure to cisplatin. Apoptosis analysis in the absence of CDDP indicated a good and consistent viability of all analyzed cell lines regardless of their different proliferation rate (p = 0.05) and a dramatic drop in the number of alive cells . In Tera-2 cell lines, mild increase in apoptotic response was also observed in the original CDDP-naive cells, and no change was observed in CDDP-exposed cells after incubation with CDDP.There was a notable difference in the proliferation between the two original cell lines, NCCIT showing much faster proliferation rate than Tera-2. The derived counterparts of both cell lines showed a slower proliferation rate compared with that of the parental sensitive cells, which was nonsignificant and only transient in case of NCCIT_CDDP (around day 6), but profound and permanent in case of Tera-2_CDDP a. To furion rate d. After OPN1LW gene (present in 91% of sequencing reads). Among the acquired variants found in NCCIT_CDDP cells we identified genes involved in cellular pathways crucial for survival, proliferation, metastatic dissemination as well as metabolic set up of cancer cells – e.g., genes involved in DNA damage repair (BCOR), chromatin remodeling , major signaling pathways , metabolic pathways  or maintenance of epithelial-mesenchymal balance (KDR). Interestingly, we found three different missense variants in ATRX gene acquired during the development of CDDP resistance  data of both original and derived TGCT cell lines showed a significantly increased total number of acquired gene variants in the cells that developed CDDP resistance , there PAX5, DAB2, SPINT2, TFDP1, histon methyltransferase NSD1, as well as other genes previously described to regulate proliferation (DLK1), cell signaling , metabolism , cell migration and adhesion . Altered expression was also present in PYCARD gene, which is a key mediator in apoptosis and inflammation. We also detected alteration in the expression of two homeobox genes (SIX6 and EN2).From transcriptome sequencing, a set of 50 most differentially expressed genes between CDDP-resistant (CDDP_NCCIT) and sensitive cell lines (NCCIT and both CDDP_Tera-2 and Tera-2) was identified . UnsuperSBF2/RNF141 and EML4/MTA3, resulting from an in-frame deletion and tandem duplication, respectively. Their presence was also confirmed by Sanger sequencing. In the case of the Tera-2 cell line, three in-frame fusion transcripts were found in both the original and derived counterparts. Two of them (SPG7/CDH15 and ZNHIT6/COL24A) originated from tandem duplication in the coding sequence, while in ZNF160/ZNF415 the coding sequence of one gene was translocated to the 5′ untranslated region of the second gene  analysis revealed large-scale gains and losses in all cell lines. Apart from the obligatory 12p gain, characteristic of TGCT, other CNVs could be identified particularly on chromosome 1, 20, 21 and X. Further acquired CNVs changes were observed in the NCCIT_CDDP cell line comparing with the original NCCIT counterpart  .Tumor mutation burden (TMB) was slightly higher in the CDDP-derived cells than in the original cell lines and did not differ between NCCIT and Tera-2 cell lines  or amplicon sequencing with the Comprehensive Cancer panel , together with patients’ germline DNA as a background control. TFAP2C gene, and another clinically interesting mutation with high variant frequency (30%) within TRERF1 gene. Of the other two patients, one harbored a stop/gain mutation in DOHH gene—metalloenzyme involved in lysine metabolism; and the other was detected with 7 acquired missense variants including genes RIN1—member of RAS signaling pathway, FOXG1—member of FOXO signaling pathway, and DMRTA1—transcription factor. There was no overlap in the affected genes among these 3 patients.In 3 out of 4 patients analyzed in detail by WES, significant acquired somatic gene aberrations were found . The patNSD1, encoding histone methyltransferase, that was altered in 9 of 27 (33%) patients. Interestingly, we also found variations in the SETD2 gene, another histone methyltransferase, in 3 of 27 (11%) patients. Variations in 3 other genes—FANCA, IL21R, and JAK1 were present in more than two patients. The genes with the highest variant frequency  included ATRX, ARID2, FANCA, MDM2, PDGFRA, NSD1, SETD2, and TSHR. The highest number of altered genes (15) was found in the only one patient of this cohort who died of the disease progression. The only gene that was found to be altered in relation to CDDP resistance in TGCT cell lines and was present also in TGCT patients was ATRX gene .In the larger group of patients who underwent amplicon sequencing, a number of gene aberrations were detected, some of the genes being affected in multiple patients . The mosThe comparison with clinical and laboratory data did not prove any relation between the number of detected variants and the histologic subtype (seminoma vs. nonseminoma) but there was a trend towards increasing number of variations in advanced stages of the tumor .The WES-sequenced patient samples were also analyzed for CNV and TMB. Apart from the expected 12p gain, we could observe large scale gains and losses on other chromosomes—e.g., gains on chromosome 17 in 2/4 pts . The oneThe topic of cisplatin resistance is highly relevant as it represents the leading cause of treatment failure in TGCT management. No single major aberration has been so far associated with CDDP resistance in TGCT but increasing number of potential genes, pathways, genomic and epigenetic changes have been emerging from recent experimental and descriptive studies. In order to add further information to the understanding of the basis and conditions leading to cisplatin resistance development, we established two CDDP-exposed cell lines and studied their proliferative and genetic properties. NCCIT and Tera-2 are both non-seminoma TGCT cell lines that contain embryonic carcinoma cells. NCCIT is referred to include also a teratocarcinoma component. Both cell lines showed similar initial sensitivity to CDDP. With the identical approach of intermittent CDDP exposure and resurrection periods, NCCIT developed a significant CDDP resistance  whilIt is well known that cytotoxic chemotherapy is not effective for cancer cells in the G0/G1 phase, and resistant cancer cells are prone to rest at this stage ,39,40. TATRX gene and the alterations of NSD1 gene. ATRX was the gene with the highest frequency of newly acquired mutations found in the CDDP-resistant cell line and the only one mutated also in primary TGCT tumors. This gene is located on chromosome Xq21.1 and encodes an α-thalassemia mental retardation X-linked protein, a member of the SWI/SNF superfamily of chromatin modifiers. It regulates various cellular processes such as replication stress response, transcription or mitotic recombination, and has been shown to play a critical role in sexual differentiation [ATRX sequence or alterations of its expression were found in various cancers including glioma, neuroblastoma, osteosarcoma, colorectal cancer or lung cancer [ATRX mutations, involves apart from thalassemia and mental retardation also gonadal dysgenesis of various degree, in extreme cases even male to female sex reversal [The DNA and RNA sequencing revealed two specific gene aberrations that may have a strong functional relation to TGCT and CDDP resistance development – the mutations in ntiation . Aberratg cancer . They hareversal , and as NSD1 gene were the most common aberrations detected in TGCT patients in this study including one with CDDP-resistant disease. The other CDDP-resistant patient had large scale losses including this gene region on chromosome 5, and downregulation of this gene was also found within the 50 most differentially expressed genes in CDDP resistant cell line comparing to the sensitive cells. NSD1 is located on chromosome 5q35.3, encodes nuclear receptor binding SET domain protein 1, which functions as the methyltransferase H3K36. It has been reported as an oncogene or tumor suppressor depending on cellular context; and its aberrant expression or mutations have been described previously [NSD1 are strongly associated with widespread genome hypomethylation [Mutations of eviously ,45. Altehylation . It is ihylation .ATRX and NSD1 are involved in epigenetic regulations, DNA methylation and chromatin remodeling correlates well with the current understanding to TGCT biological background, where larger genomic and epigenomic aberrations rather than point mutations are supposed to represent the crucial events in TGCT pathogenesis. In accordance with this, repression of H3K27 methylation was associated with acquired CDDP resistance in TGCT in one recent study [The fact that both altered genes nt study .FOXG1 [OPN1LW [EOMES [TFAP2C [DMRTA1 [MDM2, which is the only one gene that have been related to CDDP resistance in TGCT patients repeatedly [Of the other genes found altered in primary TGCT tumors in this study, some have been related to TGCT previously. These include genes related to TGCT occurrence in published genome wide association studies (GWAS) or other genetic association studies, such as FOXG1 , OPN1LW  [OPN1LW , EOMES [W [EOMES ,51, TFAP [TFAP2C , DMRTA1  [DMRTA1 ,53,54; apeatedly ,31,55,56SETD2 and FANCA genes. SETD2 gene, whose variants were found in 10% of patients in our cohort, is another gene with the influence on epigenetic remodeling. It is an important partner of NSD1 gene, acts as a tumor suppressor [NSD1, it has not been described in relation to TGCT, but it seems to be involved in spermatogenesis and its aberrations may be related to male infertility [FANCA gene mutations were also present in 10% of TGCT patients; while this gene is well known for its function in post-replication DNA repair and maintenance of chromosome stability, its high expression levels were found in pachytene spermatocytes (in mice) and its role in the maintenance of reproductive germ cells and in meiotic recombination is suggested [In the primary tumor samples we also identified mutations of several genes that have not been associated with TGCT so far but which role in TGCT could be implied. These include e.g., ppressor ,58,59,60ppressor . Like NSertility . FANCA guggested .MAP2K1, NSD1, TRERF1 and TFAP2C in resistant primary TGCT and several other genes, namely KDR and MAP3K4 in the resistant cell line. TFAP2C gene encodes a sequence-specific DNA-binding transcription factor, which is involved in activation of several developmental genes. It has a significant role in the development of primordial germ cells, and its altered expression was found in some cancer types, including murine germ cell tumors [TRERF1 gene encodes a basal cell cycle regulatory protein that has been shown to play a key role in development of estrogen independence/resistance in solid tumors—e.g., breast cancer [KDR gene encoding a tyrosine kinase protein is necessary for survival, proliferation, and migration of endothelial cells [MAP3K4 gene is an important component of the MAPK pathway, plays a crucial role in epithelial to mesenchymal transition, supports the male gonadal determination, and has been tested as a prognostic factor in other cancer types [CDDP resistance was in our study associated with aberrations in genes l tumors ,65,66,67t cancer . KDR genal cells , testis-al cells , also beal cells . MAP3K4 er types ,73.The signaling pathway analysis revealed an increased number of mutations in the cell cycle control and proteasome pathway in resistant NCCIT_CDDP cells, comparing with the other cell lines. This may underline the previously mentioned role of changes in DDR and cell cycle control, enabling the cell cycle arrest and repair of chemotherapy-induced adducts in the resistant cells.TGCT are tumors with quite low TMB in comparison with other solid tumors of adults. This corresponds with the generally low amount of identified gene mutations among TGCT patients. Interestingly, the CDDP-resistant patient showed an increased TMB, contrary to the remaining patients.In contrast to TMB, large CNVs are expected in TGCT and were also present in our sample cohort. As in the case of low TMB, the abundant CNVs are probably related to the TGCT origin. While the low amount of mutations detected in TGCT may be the result of embryonic DDR leading to apoptosis and not allowing for (aberrant) DNA repair, the germ cell character and abnormalities in cell divisions and mitotic/meiotic switch form strong predispositions for larger genome/chromosome rearrangements. The hallmark of TGCT—the amplification of the short arm of chromosome 12—was clearly present in all analyzed samples, both cell lines and patients. Of other CNV changes, several were present in the majority of samples, while additional ones were found in CDDP-resistant samples only. Of note, many of the newly acquired gains and losses in the resistant cell line and the resistant patient’s sample affected similar chromosomal loci. It again underlines the high functional significance of CNVs and larger genomic changes in TGCT development, only our understanding and interpretation of these very complex alterations is still limited.TGCT samples were obtained after patients’ informed consent and ethical committee  approval . Their characteristics are summarized in Two non-seminoma TGCT cell lines—NCCIT (Lot Number: 59069063) and Tera-2 (Lot Number: 4018167) were purchased from ATCC  , as described in To determine the differences in cell cycle distribution and cell proliferation between original and CDDP-treated cell lines, several staining protocols and measurements were used (details in Nucleic acids (DNA and RNA) from both the cell lines and patients’ frozen samples were isolated with DNeasy Mini Kit and RNeasy Mini Kit . DNA and RNA from FFPE samples were extracted with RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher Scientific) .The sequencing data are available at GEO, accession number GSE136560 , Prism , and R-project .50 values, which were calculated in the R-project [The resistance of the cell lines to cisplatin, as determined by MTS assay, was assessed from the IC-project  using pa-project  and furtAll group comparisons were calculated using non-parametric tests .Principal component analysis was used to determine the genetic relations among the original and derived cell lines.Kegg pathway analysis was made on WES data by counting and visualization of significant variants present in the selected group of pathways that could be assumed to be involved in TGCT development and progression.DMRTA1) or TGCT resistance (MDM2). The ATRX and NSD1 genes, newly depicted in this study, represent strong candidates for the functional role in TGCT and CDDP resistance ethiopathology, though their effect will require further evaluation. Their identification also supports the emerging role of altered epigenetic gene regulation in TGCT biologic background.In summary, our study suggests that the development of CDDP resistance in TGCT is associated with increased number of genetic aberrations on the level of SNVs/TMB as well as CNVs. Some of these aberrations may represent potential driver aberrations, while most of them may be the results of the on-going malignant transformation from a very unique type of tumor with specific embryonic and germ cell features to a malignant solid tumor with more general characteristics and higher genomic instability. Our findings support the hypothesis about the key role of a unique DDR for the high TGCT sensitivity to platinum-based chemotherapy; the acquired CDDP resistance being associated with decreased apoptotic response, changes in the cell cycle control with decreased proliferation and increased cell cycle arrest, allowing the repair of chemotherapy-induced damage. Several genes have been identified to have a potential effect on CDDP resistance development – some of them being previously referred to in relation to TGCT susceptibility (e.g.,


V600E-mutated parent cells (lines A375 and SK-MEL-28) and vemurafenib-resistant cells lines (A375R and SK-MEL-28R) in melanoma. TMB-loaded PEGylated liposomes (TPL) functionalized with nickel-chelated phospholipids were prepared using a modified hydration method. The hydrodynamic diameter and zeta potential values of optimized YTPL were 91.20 ± 12.16 nm and –0.92 ± 3.27 mV, respectively. The drug release study showed TPL did not leak or burst release in 24 h. The hemolysis observed was negligible at therapeutic concentrations of TMB. A differential scanning calorimetry (DSC) study confirmed that TMB was retained in a solubilized state within lipid bilayers. YTPL showed higher intracellular uptake in parental cell lines compared to vemurafenib-resistant cell lines. Western blot analysis and a cytotoxicity study with the EphA2 inhibitor confirmed a reduction in EphA2 expression in resistant cell lines. Thus, EphA2 receptor-targeted nanoliposomes can be useful for metastatic melanoma-specific delivery of TMB.The clinical outcomes of malignant melanoma have improved with the introduction of mitogen-activated protein kinase kinase (MEK) inhibitors. However, off-target toxicities of the MEK inhibitor trametinib (TMB) often result in dose interruption and discontinuation of therapy. The purpose of this study was to anchor a physically stable EphrinA1-mimicking peptide known as YSA (YSAYPDSVPMMS) on TMB-loaded PEGylated nanoliposomes (YTPLs), and evaluate them in BRAF Melanoma is a malignant tumor formed from melanocytes (pigment-producing cells), and is the sixth most common type of cancer in the United States . ApproxiDelivery of anticancer drugs via active and passive targeting has been studied widely using nanoparticulate systems . An enhaV600E and BRAFWT melanomas [V600E-mutated parent and resistant melanoma cell lines.Recent studies demonstrated the pivotal role of overexpressed EphA2 receptors in aggressiveness, metastatic potential, and vasculogenic mimicry in malignant melanoma ,19. EphAelanomas . Compareelanomas . Moreoveelanomas . Thus, telanomas . Moreoveelanomas . To our elanomas ,26,27. Selanomas . Moreoveelanomas . This drTrametinib was purchased from LC Laboratories ; 1,2-Dioleoyl-sn-glycero-3 phosphocholine (DOPC) was purchased from Cordenpharma ; PE 18:0/18:0-PEG2000 was obtained from Lipoid ; cholesterol, chloroform, and a Sephadex G50 column were purchased from Sigma-Aldrich ; 6-histidine tagged PEGylated (PEG) YSA (6His-PEG-YSA) was obtained from GenScript Corporation ; DOGS-NTA-Ni was obtained from Avanti ; Fetal Bovine Serum (FBS) was procured from Atlanta Biologics ; Dulbecco’s modified Eagle’s medium (DMEM) and the BCA protein estimation kit were purchased from ThermoFisher Scientific Inc. ; Penicillin–Streptomycin–Amphotericin B (PSA) was purchased from MP Biomedicals, LLC ; and ALW-II-41-27 was obtained from Cayman Chemical . Other chemicals and solvents were of analytical grade. Melanoma cell lines (A375 and SK-MEL-28) were obtained from American Type Culture Collection .Chromatographic separation of TMB was achieved using Waters e2695 separation module, a 2998 PDA detector instrument equipped with a Hypersil ODS C18 column . Acetonitrile:phosphate buffer pH 3.5 (70:30) was used as the mobile phase with a flow rate of 1 mL/min and an injection volume of 10 µL. The column temperature was kept at 25 °C and the output signal was detected using Empower 3 software. The retention time of TMB was found to be 4.7 min, detected at 248 nm. HPLC standard curve of TMB is provided as 2.Vemurafenib-resistant cell lines (A375R and SK-MEL-28R) were generated by adding 0.2 µM of vemurafenib to the media of A375 and SK-MEL-28 for 2 months. The vemurafenib resistance was confirmed prior to studies. All cell lines were maintained in DMEM, supplemented with 10% FBS and 1% PSA, and incubated at 37 °C with 5% COInitially, liposomes were prepared by different methods. Briefly, TMB:DOGS-NTA-Ni:DOPC:cholesterol:DSPE-PEG 2000 in a 1:0.75:60:16.3:2.1 molar ratio were dissolved in chloroform. DOPC, cholesterol, DSPE-PEG, and TMB were dissolved in chloroform. For thin film hydration, the solution was taken in a round bottom flask under vacuum to form a film, followed by hydration with water at 55 °C and ultrasonication (30% amplitude) for 2 min. For the modified hydration method, the same chloroform solution was added drop-wise to parenteral-grade mannitol (200 μm) with constant stirring at 45 °C and left overnight for evaporation of chloroform. Dispersion of this resultant powder was prepared in water at 55 °C and was sonicated (30% amplitude) for 2 min. For the investigation of the effect of mannitol on stability, mannitol was separated from liposomes using a Sephadex G50 column. For preparation of YTPL, first DOGS-NTA-Ni-loaded liposomes were prepared using the same composition and method described above. Liposomes were incubated with different concentrations of YSA for 30 min to obtain liposomes with varied molar ratios of DOGS-NTA-Ni:YSA.The average size, size distribution by intensity, zeta potential, and polydispersity index (PDI) were measured using a dynamic light scattering (DLS) particle size analyzer . Samples were analyzed using disposable cuvettes at 25 °C with a scattering angle of 173°. The effect of YSA concentration on particle size and zeta potential was evaluated. All the experiments were carried out in triplicates. Entrapment efficiency was estimated using ultrafiltration by Amicon ultra centrifugal filters (50K). The concentration of TMB was analyzed by HPLC. The encapsulation efficiency of TMB was expressed as the percent of drug encapsulated and calculated using the following formula:TPL at different drug loading values  was prepared for the stability study. Total drug content was measured at time zero. Samples were withdrawn at different time points to analyze the amount of precipitated drug by HPLC.TPL with 1% drug loading was used to investigate the effect of concentration of cryoprotectant  on freeze drying. TPL was prepared using the method described above (unfiltered TPL). Further, unfiltered TPL was passed through a Sephadex G50 gel column to separate mannitol. Mannitol-free TPL was refered to as filtered TPL. Briefly, 1-mL aliquots of the liposomal dispersions were filled into colorless glass vials and then stored at −80 °C overnight, followed by lyophilization  overnight in order to achieve a preservable white powder. The same protocol was used for freeze drying of YTPL. Lyophilized liposomes were reconstituted with water. Particle size, zeta potential, and YSA binding efficiency were analyzed before and after freeze drying of YTPL. For YSA binding efficiency, 400 µL of YTPL were filled into Amicon Ultra centrifugal filters (30 kDa) . Free YSA was separated by centrifuging the samples at 10,000 rpm for 10 min. Concentration of free YSA (before and after freeze drying) was analyzed using a BCA protein estimation kit. . The liposomal formulation was dried, and the semi-solid paste was weighed in an aluminum pan and hermetically sealed. The samples were equilibrated at 25 °C for 5 min and were heated at the rate of 10 °C/min from 40 °C to 350 °C. A hermetically sealed empty aluminum pan was used as a reference. TA Instruments Universal Analysis 2000 software (TA Instruments) was used to analyze the data.® 7) were soaked before use in Milli-Q water at room temperature overnight to remove the preservative, followed by rinsing thoroughly in Milli-Q water. Drug release of TMB from TPL was carried out in a dialysis sac with 200 mL of phosphate buffer saline (pH 7.4) containing 0.5% Kolliphor EL at 37 °C with constant stirring. The samples were withdrawn from the release medium at different time intervals. The amount of TMB in the release media was evaluated by HPLC.Drug release from TPL was carried out using the dialysis bag method. Before use, dialysis bags (Spectra/PorRat red blood cells (RBCs) were separated from plasma by centrifugation at 2000 rpm for 5 min. The cell pellet was re-dispersed into an appropriate volume of PBS to achieve the same hematocrit. Then, 1 mg/mL TMB of TPL was added to the RBC dispersion to achieve 100, 50, 10, and 2 μg/mL TMB concentrations. After 30 min of incubation at 37 °C, samples were centrifuged at 2000 rpm for 10 min. Supernatants were diluted with PBS and analyzed. A UV spectrophotometer was used to evaluate the hemoglobin release at 550 nm. PBS was used as the negative control, and sodium lauryl sulfate solution was used as the positive control (100% hemoglobin release). Percentage hemolysis was calculated by following formula:n = 6). Samples were incubated at 37 °C for 30 min and then samples were centrifuged. Plasma was separated from centrifuged samples. Sodium lauryl sulfate was added to half the samples for complete hemolysis. The TMB concentration in haemolyzed blood and plasma was analyzed by HPLC. The plasma-to-blood ratio was calculated by the equation given below:TPL was added to blood to prepare TMB at a concentration of 50 μg/mL (2 for 48 h before treatment. Coumarin-6 loaded PEGylated liposomes (C6PL) and YSA-anchored coumarin-6-loaded PEGylated liposomes (YC6PL) were incubated with cells for 1 h. Afterwards, cells were washed with HBSS and fixed with 3.7% formalin. Uptake of TPL and YTPL in different cell lines was observed for same exposure time using the EVOS FL Auto Cell Imaging System with 40× magnification.Cells were plated in a 96-well plate at a density of 10,000 cells/well and incubated at 37 °C and 5% CO2. Then the medium were removed from wells and MTT-formazan crystals were dissolved by the addition of 100 μL of dimethyl sulfoxide (DMSO) to each well. The quantity of MTT-formazan was determined by 570 nm absorbance as the wavelength reference.Cytotoxicity of TMB, TPL, and YTPL was evaluated in A375, SK-MEL-28, A375R, and SK-MEL-28R cell lines using 3--2,5-diphenyl tetrazolium bromide (MTT) assay. ALW-II-41-27 (an EphA2 receptor ATP-competitive inhibitor), vemurafenib, and vemurafenib with 0.1 µM ALW-II-41-27 were tested in both SK-MEL-28 and SK-MEL-28R. Cells were seeded in 96-well plates at a density of 5000 cells/well and allowed to grow for 24 h before treatments. TMB and TPL were diluted in cell culture medium at different concentrations. After 48 h treatment, cell viability was determined by the MTT assay. Briefly, MTT dye was dissolved at a final concentration of 5 mg/mL in PBS. Cells were incubated with 20 μL of 5 mg/mL MTT solution in each well for 3 h at 37 °C, 5% COv/v NP-40, 0.5% w/v deoxycholate, 0.1% w/v SDS, 10% v/v glycerol, 10 mM NaF, 0.4 mM EDTA, pH 8.0) with protease inhibitors. The lysates were cleared by centrifugation at 10,000 g for 10 min and then reduced with Laemmli buffer containing β-mercaptoethanol, separated on 4–15% MiniProtean TGX gels , transferred to a PVDF membrane, and probed with primary antibodies from Cell Signaling Technology for EphA2 (6997) and β-actin (8457) for chemiluminescence detection using the Azure Biosystems c500 imager .Whole cell protein lysates were obtained from A375, SK-MEL-28, A375R, and SK-MEL-28R cell lines. Briefly, cells were scraped in modified RIPA buffer  and Microsoft Excel. Differences between groups were considered to be significant at a p-value of <0.05. Each experiment has been performed in triplicate and carried out at least three times. All statistical analyses were performed using Student’s The results of TPL prepared using different methods are given in The hydrodynamic diameter of TPL prepared using a modified hydration method was found to be 109.45 ± 9.40 nm with a zeta potential of −35.55 ± 9.60 mV. The zeta potential of DOGS-NTA-Ni-loaded liposomes was lower than TPL. For YTPL, particle size was found to be similar at all the DOGS-NTA-Ni: YSA ratios a. A sligThe precipitation of a hydrophobic drug from liposomes is another issue with respect to long term stability. In order to evaluate the physical stability, TPL with different drug loading values  at a 0.5 mg/mL TMB concentration were prepared. As expected, we observed that lower the drug loading, the lower the percentage of TMB precipitation a. MoreovFreeze drying was carried out to covernt TPL into solid powder for reconstitution. Since TPL was prepared using a modified hydration method that contains mannitol, we evaluated the effect of mannitol on TPL stability. Unfiltered TPL are liposomes without the separation of mannitol. Filtered TPL was prepared by separating mannitol using a G50-Sephadex column while the unfiltered TPL was the liposme which had not undergone filtering of the mannitol. Particle size and zeta potential of unfiltered and filtered TPL before and after freeze drying with various concentrations of trehalose are depicted in DSC endotherms of TMB and TPL were obtained as shown in TPL showed less than an 8% drug release in 24 h at sink conditions , which iNegligible hemolysis was observed even at 100 μg/mL of TMB used , Table 3Coumarin-6 was selected as the fluorescent dye for labelling the liposomes. As shown in 50 values of each of the formulations is given in 50 values for TMB, TPL, and YTPL were similar at around 0.7 nM. Cell viability graphs of TMB, TPL, and YTPL in melanoma cell lines are given in In vitro cytotoxicity of TMB, TPL, and YTPL was evaluated in patent cell lines only. IC50 values of ALW-II-41-27 were 122.4 nM and 177.0 nM for SK-MEL-28 and SK-MEL-28R, respectively. Co-treatment with ALW-II-41-27 did not alter the cytotoxicity of vemurafenib in parent or vemurafenib-resistant SK-MEL-28 cells. Vemurafenib diluted in 0.1 µM ALW-II-41-27 showed similar viability compared to single vemurafenib treatment, which indicates that the EphA2 inhibitor did not change the resistance sensitivity. Thus, EphA2 receptor inhibition is not co-related to cytotoxicity of vemurafenib or vemurafenib resistance in melanoma.To further investigate whether the EphA2 receptor was correlated with vemurafenib-resistance, ALW-II-41-27, an EphA2 ATP-competitive inhibitor, inhibited the growth of SK-MEL-28 and SK-MEL-28R in a concentration-dependent manner . The IC5V600E-mutated melanoma parent cell lines compared to the resistant cell lines.The protein expression of EphA2 was significantly lower in the whole cell lysates from A375R and Sk-MEL-28R melanoma cell lines compared to A375 and Sk-MEL-28 . The resToxicities associated with the use of oral MEKi  severely restrict the targeted therapy of malignant melanoma. The aim of this paper was to investigate whether an EphA2 receptor-directed liposomes could be helpful in targeting parent and BRAFi-resistant melanoma. For the first time, we developed EphA2 receptor-targeted TMB-loaded PEGylated nanoliposomes to minimize the off-targeted side effects of TMB. We further characterized the liposomes for cellular internalization in parent and vemurafenib-resistant melanoma cells.Nanotechnology has had a profound effect on many areas of healthcare and scientific research, the use of tumor-targeted nanocarriers allow for the delivery of low water-soluble cancer medications to selectively bind cancer cells and minimize off-target side effects ,31. The Since there are no previous reports on formulation development of TMB, we explored four different methods for preparation of TPL, which were compared and optimized based on formulation feasibility and physical stability ,39,40. EV600E-mutated melanoma with overexpressed EphA2 receptors. PEGylated nanoliposomes of TMB prepared by a modified hydration method using a chelating lipid, DOGS-NTA-Ni, allow for the simple and one-step attachment of a 6His-PEG-YSA peptide on liposomal surfaces [The enhanced permeation and retention (EPR) effect is known as passive targeting and is effective especially for the parenteral administered nanoparticles. Drug tends to accumulate around tumor tissue due to leaky vasculature and an abnormal lymphatic drainage system . Conventsurfaces . YSA pepsurfaces .Particle size and zeta potential of nanoparticles play an important role, especially for parenteral administration. Different concentrations of 6His-PEG-YSA peptides were used to optimize particle size and zeta potential. The succinyl group of DOGS-NTA-Ni contributes to negative zeta potential of the liposome. Due to the complexation between the YSA target ligand and DOGS-NTA-Ni, the zeta potential changed from negative to positive in a YSA concentration-dependent manner. It was found that 1:2.5 M DOGS-NTA-Ni:YSA showed no further changes in zeta potential, suggesting that YTPL was saturated with YSA. Thus, a 1:2.5 molar ratio of DOGS-NTA-Ni:YSA was used as an optimized ratio for YTPL.® or Myocet®) are available in liquid form [® and Visudyne® are available as lyophilized liposomal powders [®, and Lucentis® are commercially available. Trehalose has an ability to reduce hygroscopicity so that hydrogen bonds can form easily, has low chemical reactivity, and has a high glass transition temperature (Tg) [The application of liposomes in commercial use is restricted by physical stability, even though advantages such as biocompatibility and nontoxicity are very attractive . In thisuid form , whereinuid form . AmBisom powders . Trehaloure (Tg) . Mannitoure (Tg) ,49. Surpure (Tg) . Differeure (Tg) . TMB in The release of liposomes is important to evaluate the quality of the formulation as well as to predict in vivo behaviour of a liposomal drug delivery system. In this study, drug release results did not show burst leaking of TMB from the liposomes. Therefore, we anticipate that TMB will be confined within the liposome and will not show any burst release in the blood. However, it showed promising activity in our in vitro studies, indicating that TMB will be released and act after internalization by cancer cells. We expect liposomes to accumulate at tumor site due to the EPR effect, and for complete release of TMB at the tumor site in vivo due to the active-target effect.Negligible hemolysis was observed even at the therapeutic concentrations of TMB. The blood-to-plasma ratio determines the concentration of the drug in the general circulation and the concentration of the target drug in plasma, which provides an indication of drug the binding to erythrocytes. The result for blood-to-plasma ratios of TMB is around 1 which indicates that TMB is evenly distributed in plasma and red blood cells.50 of the TMB liposome confirmed that TMB was released after internalization of TPL into cancer cells. Moreover, the IC50 values of TMB, TPL, and YSL are similar in A375 and SK-MEL-28, while in-vivo behavior may vary due to drug distribution and the microenvironment of the tumor. Coumarin-6 was used as a fluorescent probe in the uptake study. The intensity of this fluorescence dye can be correlated with the extent of liposome uptake. YTPL showed higher cell internalization compared to non-targeted liposomes (TPL) because of receptor-mediated uptake. Surprisingly, YTPL presented less uptake in vemurafenib-resistant cell lines, indicating that the expression of EphA2 receptor could be lower in vemurafenib resistant cell lines. This finding is contradictory to a previous study that found vemurafenib-resistant melanoma cell lines had higher EphA2 receptor expression compared to parent cells [The very low ICnt cells . Our WesWe have successfully developed YSA-anchored trametinib-loaded PEGylated nanoliposomes (YTPL) by a modified hydration method, with better entrapment efficiency and stability compared to the thin film hydration method. Moreover, YTPL showed compatibility with red blood cells as well as the desired cytotoxicity to melanoma cells. Metastatic melanoma-specific delivery of MEK inhibitors can be achieved by targeting overexpressed EphA2 receptors in vemurafenib-sensitive cells using nanosized EphA2-targeted PEGylated liposomes. Nevertheless, YTPL is not a viable option for the treatment of vemurafenib-resistant melanoma since TMB has cross-resistance to vemurafenib-resistant cell lines. Moreover, according to our studies, resistant cell lines do not internalize YTPL as much as parental cell lines.


BCR-ABL1+ cell lines. To investigate potential resistance mechanisms, ponatinib resistance was generated in BCR-ABL1+ cell-lines by long-term exposure to increasing concentrations of ponatinib. Two cell lines with prior dasatinib resistance demonstrated BCR-ABL1 kinase domain (KD) mutation(s) upon exposure to ponatinib. In one of these cell lines the T315I mutation had emerged during dasatinib exposure. When further cultured with ponatinib, the T315I mutation level and BCR-ABL1 mRNA expression level were increased. In the other cell line, compound mutations G250E/E255K developed with ponatinib exposure. In contrast, the ponatinib resistant cell lines that had no prior exposure to other TKIs (TKI-naïve) did not develop BCR-ABL1 KD mutations. Rather, both of these cell lines demonstrated Bcr-Abl-independent resistance via Axl overexpression. Axl, a receptor tyrosine kinase, has previously been associated with imatinib and nilotinib resistance. Ponatinib sensitivity was restored following Axl inhibition or shRNA-mediated-knockdown of Axl, suggesting that Axl was the primary driver of resistance and a potential target for therapy in this setting.TKI resistance remains a major impediment to successful treatment of CML. In this study, we investigated the emerging modes of ponatinib resistance in TKI-naïve and dasatinib resistant  BCR-ABL1 kinase domain (KD). More than 100 mutations have been identified which either prevent or significantly disrupt the binding of imatinib to Bcr-Abl [BCR-ABL1 T315I mutation (commonly referred to as the gatekeeper mutation), which accounts for approximately 15-20% of clinically observed BCR-ABL1 mutations [Since its clinical introduction over a decade ago, imatinib has revolutionized the treatment of chronic phase-chronic myeloid leukaemia (CP-CML). However, approximately 30-40% of patients fail to respond optimally to imatinib . The bes Bcr-Abl . To over®, Ariad Pharmaceuticals, Cambridge, MA, USA) a third generation TKI, is a potent Bcr-Abl inhibitor approved in the USA and Europe for treatment of CML patients with resistance to other TKIs. The medium human peak and trough plasma levels of ponatinib when dosed at 45 mg once daily are 145 nM and 64 nM respectively [T315I. However, while ponatinib is the only available TKI to target Bcr-AblT315I, the interaction of ponatinib with T315I mutant Bcr-Abl is weaker than its interaction with Bcr-Ablp210 [Ponatinib  –10, incrnd ABCG2 , 11–17, pression , 18–20. activity , suggestactivity –27. WhilM et al. .BCR-ABL1+ cell lines to increasing concentrations of ponatinib. Cell lines were either TKI-naïve, or were previously treated with dasatinib to induce dasatinib resistance to mimic the clinical use of ponatinib in TKI-resistant/refractory CML & Ph+ALL patients, many of which had been on dasatinib with or without the T315I mutation. By characterising the resultant ponatinib resistant cell lines, we identified that in the setting of prior dasatinib exposure, Bcr-Abl dependent mechanisms cause ponatinib resistance. However, in the TKI-naïve setting, Bcr-Abl-independent modes of resistance dominate, and Axl presents as a key mediator of this resistance, suggesting Axl may be a potential therapeutic target in the cases of ponatinib resistance.To investigate potential resistance mechanisms, ponatinib resistance was generated in this study by exposing K562 T315I-R  and K562 DOX 55D-R cells , as higher concentrations of TKI were required to inhibit Bcr-Abl tyrosine kinase activity  compared to the control cell line K562 (7.2 nM). In addition, the K562 DOX-R cells also demonstrated a similar ponatinib IC50  compared to the K562 DOX control (8.9 nM) (K562-R and K562 DOX-R cell lines were able to maintain high viabilities (>75%) in 200 nM ponatinib. These results suggested that Bcr-Abl independent mechanisms were likely involved in the development of ponatinib resistance in these two cell lines.However, as demonstrated in , there w(8.9 nM) . To confBCR-ABL1 mRNA can cause resistance to first and second generation TKIs [BCR-ABL1 transcript number in the four ponatinib resistant cell lines. As expected, substantial increases in the expression level of BCR-ABL1 mRNA were observed during the development of the K562 T315I-R and K562 DOX 55D-R cell lines. There was a significant increase in BCR-ABL1 mRNA from 1206% (relative to %BCR) in naïve cells to 8027% in the K562 T315I-R resistant line   revealed an increase in mRNA expression, peaking at 9034% in the 90 nM ponatinib intermediate, K562 T315I 90 nM PON    demonstrated a further reduction in BCR-ABL1 mRNA expression (1299%), which was not significantly different to the ponatinib naïve control line K562 DOX 55D (1069%)  Figure . Moreove) Figure . During ) Figure . This ov) Figure . This reBCR-ABL1 mRNA levels also increased during the development of K562-R and K562 DOX-R cell lines, however, the gene expression levels were significantly lower compared to the two ponatinib resistant cell lines which had developed prior resistance to dasatinib. As demonstrated in Figure K562 exhibited fluctuations in the BCR-ABL1 mRNA transcript level, from 487% in the K562 TKI-naïve control cells, peaking at 1408% in the 40 nM ponatinib culture . However, the K562-R final (200 nM ponatinib) resistant line did not exhibit a significantly increased BCR-ABL1 mRNA level compared to its K562 control. This result suggested that the overexpression of BCR-ABL1 mRNA may mediate early stages of ponatinib resistance, but it is not the major mechanism that caused resistance in this cell line. Conversely, the BCR-ABL1 mRNA expression level in the K562 DOX-R ponatinib resistant line increased from 266% in the control line to 871% in the final 200 nM ponatinib culture   Figure . These rK562 T315I-R resistant or K562 T315I control cell lines  sequencing was performed on the intermediate cell lines generated during the development of ponatinib resistance and revealed a slow but steady increase in T315I% from 44% in the control line peaking at 66%  and E255K (55%) were present in the K562 DOX 55D-R (200 nM) cell line, however neither the K562 DOX 55D ponatinib naïve control cell line, nor any of the intermediate cell lines, harboured a mutation detectable by Sanger sequencing (limit of detection is 10%).Conventional Sanger sequencing was unable to detect a BCR-ABL1 KD mutation in the K562 DOX 55D cell line. Interestingly, a single mutation E279K and a single mutation E255K were observed in 0.92% and 0.72% respectively in this 100 nM intermediate cell line. In the resistant line K562 DOX 55D-R (200 nM), the compound mutations G250E/E255K (54%) were also confirmed by SMCS   to invesS Figure  and therK562-R and K562 DOX-R demonstrated increased adherence in culture as observed by morphology change, while the cells in the controls K562 and K562 DOX remained in suspension ; a 1.5 fold increase in the K562 DOX-R line compared to the K562 DOX control line .Increased adherence is a hallmark of Axl overexpression which has previously been associated with TKI resistance –27. TherK562-R and K562 DOX-R resistant cell lines was dramatically increased compared to their corresponding control lines. Taken together, these data confirmed the overexpression of Axl in the two Bcr-Abl-independent resistant cell lines.Next, the Axl protein level was assessed in the two resistant cell lines by flow cytometry. As indicated in Figure K562-R  and K562 DOX-R  resistant lines was significantly reduced compared to their corresponding control lines.Since Axl was overexpressed in the two resistant lines, we next determined if Axl inhibition might restore ponatinib sensitivity. A specific pharmacological Axl inhibitor, R428 was employed. R428 is 50 to 100-fold more specific in targeting Axl than the other TAM family members (Mer and Tyro3) –37. AddiTaken together, these results indicate that Axl inhibition restored ponatinib sensitivity in the two Bcr-Abl independent resistant cell lines, suggesting that Axl is critical in mediating Bcr-Abl independent ponatinib resistance in these cell lines.AXL was knocked-down in the K562-R, and K562 DOX-R resistant cell lines by Lentiviral shRNA (with a GFP tag). Stable AXL knock-down K562-R-AXL KD, and K562 DOX-R-AXL KD cell lines were generated, as well as the scramble control lines K562-R-scramble CTL, and K562 DOX-R-scramble CTL. The positive expression of GFP in the AXL knock-down and scramble cell lines indicated the success of the transduction  in viability after incubating with 10 nM ponatinib for 72 hours. This reduction was significantly higher  compared to the reduction in the control line treated with scrambled shRNA (22% reduction). In addition, the level of this reduction was similar to the K562 parental ponatinib naïve control (n=3), suggesting that AXL knock-down restores ponatinib sensitivity in the K562-R resistant cell line to the parental control level.Next, the successfully transduced cell lines were exposed to ponatinib to determine whether AXL knock-down in the K562 DOX-R resistant cell line also demonstrated a significant decrease in viability (53% reduction) compared to the corresponding control line treated with scrambled shRNA (16% reduction)  . This result suggested that AXL knock-down is responsible for restoring ponatinib sensitivity in the K562 DOX-R resistant cell line, to a level that was comparable to that observed in the ponatinib naïve parental control.The ) Figure  after inIn vitro cell line models have been used extensively to characterise resistance to the first and second generation TKIs in CML: our group and others have previously generated imatinib, nilotinib and dasatinib resistance in human and/or murine BCR-ABL1+ cell lines by culturing with gradually increasing concentrations of TKIs in a step-wise manner [e manner , 28, 29.e manner , 28, 29.BCR-ABL1+ cell lines were chosen as in vitro models for ponatinib resistance development, with resistance successfully generated in 4 of the 5 cell lines . The ABCB1-overexpressing K562 variant, K562 DOX, was kindly provided by Prof. Leonie Ashman [et al. [K562 T315I  and K562 DOX 55D which had prior exposure to 200 nM and 55 nM dasatinib respectively  vehicle controls were maintained in parallel.To generate ponatinib resistance, all es Table  were culet al. [5 cells were incubated in the presence of TKI at 37°C (5% CO2) for 2 hours. After incubation, cells were washed and lysed in Laemmli buffer [IC50 experiments for imatinib, nilotinib, dasatinib and ponatinib were determined by measuring the inhibition of the Bcr-Abl substrate CrkL by using the method as outlined by White et al. . In briei buffer . ProteinPonatinib , dasatinib  and nilotinib (Symansis) were dissolved in DMSO (10mM). Imatinib (Symansis) was dissolved in water (10mM). Serial dilutions of all drugs were made immediately prior to use.Axl inhibitors R428 and BMS-777607  were dissolved in DMSO at 10 mM. Serial dilutions were made in DMSO immediately prior to use.BCR-ABL1 quantification was performed as previously described [BCR-ABL1 transcript numbers were reported as BCR-ABL1/BCR%.escribed , 47. In 6 cells, and cDNA synthesized as a template to amplify the KD of BCR-ABL in a long PCR reaction using the Expand Long Template PCR System . Products were purified using ExoSAP-IT  and sent to Molecular Pathology (SA Pathology) for analysis.Sanger sequencing was performed as previously described , 47. BriNext generation sequencing (NGS) termed Single Molecule Consensus Sequencing (SMCS) was performed using previous method and kindly processed by Dr. Wendy Parker .5 cells were incubated with Hanks Balanced Salt Solution (HBSS)  supplemented with 10mM Hepes (Sigma-Aldrich) and with corresponding isotype controls: IgG2aPE/IgG2bPE , IgG1PE ; or PE-conjugated anti-hBCRP1 (ABCG2-PE) , PE-conjugated anti-CD243 (ABCB1-PE) , PE-conjugated anti-hAxl  or PE-conjugated anti-hCD44  antibodies for 40 minutes on ice. Following HBSS wash, cells were resuspended in fixative and analyzed by flow cytometry . Results were analyzed by FCS Express 4 Research Flow Edition software .5 × 10AXL transcript levels were measured by RQ-PCR. GUSB was used as control gene and the expression level of AXL was calculated as a ratio of GUSB transcript levels. The primers used for AXL amplification were as follows:AXL Forward primer: 5′ TGC ATG AAG GAA TTT GAC CA 3′AXL Reverse primer: 5′ TCG TTC AGA ACC CTG GAA AC 3′Samples were amplified in triplicate in the Rotor-Gene 3000 PCR cycler machine  with the following conditions: 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 seconds and 60°C for 1 min.Cells were removed from drug-containing culture medium, washed and re-cultured in fresh media (with testing drug) for 72 hours. Cells then were stained with 7-Amino-actinomycin D (7AAD) and Annexin V-PE. Flow cytometry (BD LSRFortessaTM X-20 with high-throughput platform) was performed to determine cell viability: cells stained positive for Annexin V-PE and 7AAD were considered to be apoptotic or dead respectively. The data were analysed using FCS Express software .HEK 293T cells were transfected by lentiviral shRNA plasmid pGFP-C-shLenti comprised of either scramble control vector (sequence as below) or AXL knock-down (sequence as below) with Green Fluorescense Protein  according to the manufacturer's instructions. The GFP level and Axl expression level were measured after fluorescence-activated cell sorting (FACS) and before each experiment to ensure purity.Scramble control sequence:5′ GCA CTA CCA GAG CTA ACT CAG ATA GTA CT 3′AXL shRNA sequences:5′ GAA CAG GAT GAC TGG ATA GTG GTC AGC CA 3′t-test or Kruskal-Wallis analysis of variance to determine statistical differences. Correlation between 2 groups was compared using Pearson's correlation statistical analysis. The analysis was performed using GraphPad Prism 5 . Statistically significant differences were defined as p<0.05.Comparison between two groups was performed using unpaired 2-tailed Student's


N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the  N-glycosylation, in which oligosaccharides are attached via an N-glycosidic linkage to an asparagine (N) of the consensus sequence -N-X-S/T-  . Ch. ChN-gly cancers ,6,7 and  cancers . FUT1, 2, 3, 4, 5, 6, 7, and 9 and leads to the expression of blood group related Lewis antigens  of the antenna in α1,3- and/or α1,4-linkage, FUT1 and FUT2 are responsible for the addition of a fucose to the galactose  in α1,2-linkage, forming the H-type epitope. FUT2 is further called the secretor gene and polymorphisms leading to an inactive FUT2 lead to the absence of blood type epitopes in saliva and various epithelial cell types, the so-called non-secretor phenotype [FUT8, on the other hand, is the gene encoding for the enzyme mediating the attachment of a fucose-residue to the first GlcNAc of the N-glycan core, the so-called core-fucosylation. More detailed information on the biological role of fucosylation has been given elsewhere [By screening different colorectal cancer cell lines, we previously showed an association of caudal-related homeobox protein 1 (CDX1) mRNA expression with increased multi-fucosylation (more than one fucose), which is indicative for antenna fucosylation . Antennaantigens B, which henotype ,13. FUT8lsewhere .N-, O-, and glycosphingolipid glycans) vs. individual glycan classes. Moreover, core- and antenna-fucosylation are not always distinguished and only a few studies report on stage-dependent changes. While many groups reported a general increase of fucosylation in colon and other cancers [Fucosyltransferases are expressed in a cell type- and organ-dependent manner and altered expression of the enzymes, as well as the produced fucose-containing glycan epitopes, have been associated with various pathological conditions , includi cancers , other r cancers ,19. NotaGMDS), an important player in the fucose biosynthesis pathway, helps cancer cells to escape from natural killer (NK) cell-mediated tumor immune surveillance [GMDS gene have been identified in metastatic lesions of some colon cancers (10%). Also the colon cancer cell line HCT116 bears a GMDS mutation, resulting in almost complete absence of fucosylation. Strikingly, the parental HCT116 cells with GMDS mutation revealed a more aggressive phenotype in tumor formation and metastasis in mice, as compared to the GMDS-rescued HCT116 cells [GMDS-rescued HCT116 were more susceptible to TRAIL-induced apoptosis [The group around Miyoshi and Moriwaki hypothesize that deficiency of fucosylation via mutations of GDP-mannose-4,6-dehydratase , hitherto only very little has been described with regard to CDX1-associated glycosylation profiles. Triggered by our previous findings , which spression  with an FUT3 and FUT6, both involved in antenna fucosylation. Strikingly, other genes associated with fucosylation were likewise differentially expressed between the investigated cell lines with high vs. low CDX1 expression. A higher GMDS gene expression is positively associated with high CDX1 mRNA expression in the tested colorectal cancer cell lines and also transcription factors hepatocyte nuclear factor (HNF)1A and HNF4A, which have been previously shown to regulate antenna fucosylation of human plasma proteins [N-glycomic characterization revealed a decrease in galactosylation and sialylation in cell lines with high CDX1 expression, compared to cells with low CDX1 gene expression.We confirmed a higher degree of multi-fucosylation in cell lines with high CDX1 gene expression, which was supported by higher mRNA expression of fucosyltransferases proteins  and are proteins ,36, show3) were from Merck  and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was from Fluorochem . Peptide N-Glycosidase F (PNGase F) was purchased from Roche Diagnostics . The peptide calibration standard was purchased from Bruker Daltonics . MultiScreen® HTS 96 multiwell plates (pore size 0.45 μm) with high protein-binding membrane (hydrophobic Immobilon-P PVDF membrane) were purchased from Millipore , while the 96-well polypropylene 0.8 mL 96-deepwell plate and 96-well PCR plate polypropylene were from Greiner Bio . All buffers were prepared using ultrapure water generated from an 18.2 MΩ-cm Purelab Ultra system from Elga . Control Visucon-F plasma pool  was obtained from Affinity Biologicals . Cell culture media were purchased from Gibco , fetal bovine serum (FBS) from Autogen Bioclear , and penicillin-streptomycin from Invitrogen . The RNeasy mini kit was purchased from Qiagen .8M guanidine hydrochloride (GuHCl), 1-hydroxybenzotriazole (HOBt) hydrate, 50% sodium hydroxide (NaOH), and super DHB matrix  were obtained from Sigma-Aldrich . HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve . Dithiothreitol (DTT), ethanol, and sodium bicarbonate , based on trypan blue staining.Details of colorectal cancer cell line origin and characteristics can be found in N-glycans were released in duplicate from three biological replicates per cell line using a PVDF-membrane based release protocol followed by linkage-specific sialic acid derivatization. Purification by cotton-HILIC-SPE and MALDI-TOF-MS analysis was performed as described previously [N-glycans were released overnight at 37 °C. Released glycans were derivatized using ethyl esterification allowing for discrimination of N-acetylneuraminic acid linkages  [eviously . Shortlym/z range of 1000 to 5000 for a total of 10 000 shots . Tandem mass spectrometry (MALDI-TOF-MS/MS) was performed for structural elucidation via fragmentation in gas-off TOF/TOF mode.Released, derivatized, and purified glycans (5 µL sample) were spotted on an anchor chip MALDI target plate  and co-crystallized with 0.5 µL of 5 mg/mL superDHB in 50% can, supplemented with 1 mM NaOH. Spectra were recorded in positive-ion reflector mode, after calibration with a Bruker peptide calibration kit, using a Bruker UltrafleXtremeTM mass spectrometer, controlled by FlexControl 3.4 software Build 119 . Mass spectra were obtained over an m/z 0.06, 4 cycles), baseline corrected , and exported to xy-files using FlexAnalysis 3.4 (Stable Build 76). Mean average spectra were generated per technical replicate, which were summed to one spectrum using the open-source software mMass  as calibrants (minimum five used), followed by peak picking in mMass, with cut-off signal-to-noise (S/N) 3. The peaklist was manually revised and analyzed in GlycoWorkbench 2.1 stable build 146 (http://www.eurocarbdb.org/) using the Glyco-Peakfinder tool (http://www.eurocarbdb.org/ms-tools/) for generation of a glycan compositions list. Our novel in-house software, developed for automated data processing, MassyTools version 0.1.8.0 [rd degree function and targeted data extraction of the area under the curve for each individual mass spectrum. To prevent over-estimation of overlapping glycan species, only the first three isotopes were extracted and the area under the curve was corrected based on the theoretical isotopic pattern. The quality of the data was assessed using several quality parameters calculated within the software. Only good quality spectra  as well as analytes  were included for analyses. Raw data after pre-processing is provided in the Supplementary Tables. Finally, the corrected area-under-the-curve values were rescaled to a total relative intensity of 100% for each spectrum. Selected glycan compositions were confirmed by MS/MS and a final peak list as well as MS/MS data is given in Spectra were smoothed . The sphttp://www.r-project.org/) with the significance level α = 0.05 to assess differences in N-glycosylation between CDX1 high and low expressing cells. Bonferroni correction was applied to p-values to adjust for multiple testing . The formulas for calculation are given in  testing . BoxplotTotal RNA was extracted by using the RNeasy mini kit according to the manufacturer’s instructions. Twenty micrograms of RNA of each sample were sent to the Molecular Biology Core Facility of the Paterson Institute for Cancer Research, Manchester, UK, for gene expression microarray analysis using the Human genome U133+2 chips, following the manufacturer’s instructions . Microarray data were analyzed using Partek Genomics Suite software. The data were log2-transformed and RMA-normalized (with GC correction) using quantile normalization with Median Polish for Probeset summarization as optional settings in the software. t-test comparing mean-expression levels of the 8 CDX1high vs. 8 CDX1low cell lines. The significant level was adjusted for multiple testing. Fold changes were calculated for CDX1high and CDX1low cell lines and for significantly different expressed glycosyltransferases, data was visualized as boxplots in Rstudio showing the median and interquartile range. To evaluate the correlation between relative abundances of N-glycans traits based on mass spectrometry data and glycosyltransferase gene expression in the 16 investigated cell lines, linear regression analysis was performed in GraphPad Prism Version 6 . Glycosyltransferase genes  as well N-glycans and CDX1 mRNA expression in a set of colorectal cancer cell lines  [N-glycosylation of an independent set of colorectal cancer cell lines with high  vs. low  expression of CDX1 mRNA. The CDX1high cell lines investigated here have, on average, 65-fold higher CDX1 mRNA expression as compared to CDX1low cell lines , especially in α2,3-linkage, while glycans from HCA46 were characterized by the presence of many fucoses , low sialylation level, and additional N-acetylhexosamines . In total, 221 individual glycan species were identified across all cell lines, from which 81 could be characterized by tandem mass spectrometry  was performed on the relative abundances of all individual N-glycans. This resulted in a model with four principle components (PC) explaining 68.9% of the variation in the data. The score plot of PC1 vs. PC2 showed clear separation of CDX1high and CDX1low cell lines along PC1 (N-glycan profile between the two groups.Our previous data suggested a positive association between the level of fucosylation on protein egative) . To valill lines . Two exetrometry . In ordelong PC1 A in thishigh and CDX1low colorectal cancer cells, derived glycan traits were calculated by grouping glycan species (direct traits) into classes according to glycosylation characteristics, such as sialylation and fucosylation. The relative abundances of derived traits as well as the calculations are given in high and CDX1low expressing cell lines and evaluated using a Mann–Whitney test. The p-values were adjusted for multiple testing (Bonferroni). Labelling of the PC1 vs. PC2 score plots using glycan-derived traits showed that multi-fucosylated glycans , indicative for antenna fucosylation , as compared to CDX1low expressing cell lines  in the eight CDX1high cell lines, compared to eight CDX1low cells  gene expression was 3.7-fold  elevated with high CDX1 expression  = 0.002; p-value (HNF4A) = 0.003; LGALS4), a target gene of HNF4A and a glycan-binding protein, was 23-fold up-regulated in CDX1high cells  to ∅ 31% in CDX1high cell lines  represent glycans with bisecting N-acetylglucosamine (GlcNAc), non-galactosylated antennae, or the addition of N-acetylgalactosamine  and showed a significantly increased expression in CDX1-positive cells in our previous study [high cell lines showed a more than 2-fold increase in the relative abundance of glycans with the feature HexNAc≥Hex, compared to CDX1low cells  was significantly correlated with MS data , and the blood group A epitope Galβ1–3/4GlcNAc). Accordingly, one of the glycosyltransferases adding a GalNAc-residue to a GlcNAc to form LacdiNAc structures on N-glycans, B4GALNT3, was significantly correlated with the MS-based trait HexNAc≥Hex  epitope, the SdA antigen Galβ1–4GlcNAc; NeuAc=xNAc≥Hex  and was,ow cells . Other gow cells .high cells than in CDX1low cell lines  LacNAc repeats , showed a trend towards higher expression with high CDX1 expression in our previous data and were higher expressed in CDX1high cell lines of the new set of cells as compared to CDX1low expressing cell lines, with ∅ 27% vs. ∅ 20% (N-acetylglucosaminyltransferase 3 (B3GNT3) and B3GNT8, both involved in the synthesis of type-1 chains and poly-LacNAc repeats, were around 2-fold up-regulated with high CDX1 expression  that fucosyltransferases FUT3, 5, and 6, all involved in antenna fucosylation, are positively regulated by HNF1A and its downstream factor HNF4A, while FUT8, initiating core-fucosylation, is inhibited [HNF1A and HNF4A resulted in reduced expression of several fucosyltransferase genes involved in antenna fucosylation in HepG2 liver cells and, partly, in pancreatic Panc1 cells, while FUT8, responsible for N-glycan core-fucosylation, was upregulated. Furthermore, GMDS and L-Fucokinase, both involved in two different pathways of fucose synthesis, were drastically down-regulated upon HNF1A or HNF4A knock-down. Publicly available ChIP-seq data of ENCODE show that HNF4A and HNF4G transcription factors bind to FUT2, 3, and 6 genes in cell lines of non-intestinal origin and, also, Lauc et al. confirmed the binding of HNF1A and HNF4A to multiple FUT genes and/or their promotors by ChiP analysis [HNF1A and HNF4A. Boyd et al. identified CDX2 binding sites on CDX1 and HNF1B, both being positively regulated by CDX2, while HNF1B is needed for activation of HNF4A, HNF1A, and HNF3G [HNF4A was also shown to positively influence transcriptional activity [HNF4A was identified as a transcriptional activator for intestinal differentiation and gene expression of HNF4A and its target genes were upregulated in colorectal cancer cell lines with a more epithelial phenotype, as compared to cells with a mesenchymal phenotype [HNF4A is galectin 4, which was described as tumor-suppressor in colon cancer [The transcription factors nhibited . The stuanalysis . At the nd HNF3G . Direct activity . HNF4A whenotype . One tarn cancer ,53, but n cancer , and wasN-glycans is a marker for the epithelial state and that CDX1 is involved in the transcriptional regulation of fucosyltransferases involved in (antenna-)fucosylation, leading to a more differentiated and less invasive phenotype in colorectal cancer cell lines. Taking our findings and the reports from literature, one may speculate that antenna fucosylation of N-glycan phenotype and glycosyltransferase expressions were less pronounced for CDX2 as compared to CDX1 (data not shown). In contrast, a direct involvement of CDX2 in the regulation of FUT2, which revealed potential binding sites for CDX1 and CDX2 and is involved in the generation of LeY/B antigens, was shown in colon cancer cell lines HT29 and DLD-1 [FUT2, could be reduced through treatment with epidermal growth factor (EGF)/bFGF [ST3GAL1/3/4 and FUT3 were transcriptionally up-regulated by c-Myc [high cell lines show a very low expression of α2,3-sialylation, thereby limiting the substrate for specific fucosyltransferases involved in the expression of sialyl Lewis epitopes . The combined expression of α2,3-sialylation and antenna fucosylation, as reflected in the trait CLFa, may be considered a proxy for sialyl Lewis epitope expression. Both groups of cell lines in the present study only showed low levels of CLFa (~4%), indicating low levels of sialyl Lewis epitopes. In the case of CDX1low expressing cell lines, the combination of α2,3-sialylation with overall fucosylation increased , which may likewise reflect increased sialyl Lewis epitope levels. Strikingly, in CDX1high expressing cell lines the expression of multi-fucosylation (indicative for antenna fucosylation) and α2,3-sialylation of N-glycans even showed opposite trends. Though FUT6 seems to have a preference for the sialylated substrate, here, the results point towards the expression of mainly non-sialylated Lewis antigens via action of fucosylytransferases FUT3 (Lewis A) and FUT4 or FUT6 (Lewis X) [high colorectal cancer cell lines analyzed here.Interestingly, in our investigated cell lines, CDX2 mRNA expression correlated with CDX1 mRNA expression (both high or both low), with the exception of the cell line RCM1. Consequently, similar observations as for CDX1-associated glycosylation were made for CDX2, but correlations with the nd DLD-1 . ExpressGF)/bFGF . On the by c-Myc . While sby c-Myc , the CDXLewis X)  in the CN-glycans from different colorectal cancer cells and could not find evidence for a fragment corresponding to an antenna with two or more sialic acids [N-glycans with terminal α2,8-linked sialic acids are expressed in cancer cells, these structures might be expressed at low levels as compared to N-glycans with terminal α2,6- and α2,3-linked sialic acids. Moreover, gene expression of ST8SIA1-5 was not significantly different between the here studied CDX1 high and CDX1 low expressing colorectal cell lines. Sialic acids can further be modified by O-acetyl-groups modulating the ligand function. O-acetylated glycans have been reported to be mainly present in the lower part of the intestinal tract [N-glycans of the same tissues [N-glycans of colorectal cancer cell lines has been shown to preserve O-acetylation [N-glycans in the investigated cell lines.Especially in the context of sialyl Lewis epitopes, the importance to distinguish α2,3- and α2,6-sialylation becomes evident. The derivatization applied here differentially modifies the sialic acids in different linkages resulting in a detectable mass shift allowing the distinction between α2,3- and α2,6-sialylation . Similaric acids . Althougal tract . Furtheral tract . In lineal tract , but nottylation . HoweverCDX1, CDX2, HNF1A, and HNF1B are involved in the transcriptional regulation of B3GALT5, the gene encoding for the enzyme responsible for type-1 chain  expression on glycolipids and glycoproteins, with preferences for O-glycans and glycosphingolipid-glycans [B3GALT5 was found to be down-regulated in colon cancer cell lines, but up-regulated upon CaCo2 cell differentiation [B3GALTs as well as B3GNTs according to observations by Isshiki et al. [N-glycans, O-glycans, glycosphingolipid-glycans, and/or others and changes on different glycan classes can be differently regulated. Therefore, it is striking that the N-glycomic data obtained by mass spectrometry are well in accordance with the transcriptomic data, suggesting that observed changes in, for example, fucosylation and sialylation are mainly attributed to N-glycans or are globally altered, but further investigations on O-glycans and glycosphingolipid-glycans are needed.We further observed a decrease in galactosylation with high CDX1 mRNA expression. Previous reports showed that -glycans . B3GALT5ntiation . Howeveri et al. , while thigh expressing cells. Glycan motifs that may contribute to this increase include LacdiNAc structures as well as the Sda antigen; the latter was shown to be expressed in normal colon tissues and decreased during colon cancer progression [MGAT3 gene expression was up-regulated with CDX1high cells. Several reports describe reduced bisection in several tumors [N-glycan branching by MGAT5 [high cells. Our results further revealed the presence of terminal HexNAc residues to be increased in CDX1gression . LacdiNAgression . Furtherl tumors  and it wby MGAT5 , as wellby MGAT5 , the latlow cells matched largely those described for cells undergoing EMT, which involves the loss of epithelial markers such as E-cadherin and gain of mesenchymal markers such as vimentin. EMT-associated glycan changes have previously been described and include the aforementioned loss of antenna-fucosylation, but also decrease in antennarity of N-glycans and bisection, whereas enhanced levels of high-mannose type glycans, core-fucosylation, and corresponding FUT8 expression, as well as increased α2,6-sialylation and ST6GAL1 gene expression, were observed [FUT3 and FUT6 has been shown to affect TGF-β receptor glycosylation, resulting in decreased fucosylation as well as FUT3/6-associated sialyl Lewis antigens and altered TGF-β-mediated EMT and invasion in colorectal cancer cells [Twist1, Zeb1, and Zeb2 was induced [HNF1A and HNF4A have been described to prevent EMT in liver cancer [Strikingly, observed glycan phenotypes for CDX1observed ,68. Inhier cells . Further induced . Finallyr cancer ,72,73. TN-glycans are still lacking and more research on the integrated regulation, as well as competition of glycosyltransferases, is needed. Also, the role of LacdiNAc structures and other terminal HexNAc epitopes in differentiation and colorectal cancer needs further investigation.While the involvement of transcription factors was shown for fucosyltransferases, reports on sialyltransferases and galactosyltransferases involved in the elongation of HNF4A and HNF1A. However, CDX genes may  influence the expression of fucose-carrying glycoproteins themselves, thereby leading to an N-glycan phenotype with enhanced multi-fucosylation. Taking into account the interaction of the two CDX-proteins with HNF1A, HNF4A, and HNF1B and together with the proven role of HNF1A and HNF4A in the regulation of fucosylation, we hypothesize a cooperation of these transcription factors being involved in the expression of FUT genes and thereby increasing fucosylation of glycoproteins with high CDX1/CDX2 expression  are involved in the regulation of multiple glycosyltransferases, especially fucosyltransferases, likely via interactions with other transcription factors, such as


Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development.TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed.Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 μmol/L. The cell line was inhibited to a maximum of 18% by cetuximab.After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.The online version of this article (10.1186/s12885-019-5469-8) contains supplementary material, which is available to authorized users. In recent years, there has been an increase in the incidence of oropharyngeal squamous cell carcinoma (OSCC) including cancers in the tonsils and the base of the tongue , 2. In SThere is strong evidence that this rise in OSCC incidence is caused by human papillomavirus (HPV) infection  and thatIn many respects, including incidence, survival and treatment response, HPV-positive and HPV-negative OSCC behave differently, and it has become clear that the treatment strategies should differ significantly between the groups and that new regimes should be developed specifically for HPV-positive OSCC.An important tool for testing new drugs or combinations of drugs as well as for the elucidation of the biology of HPV-induced transformation is representative, in vitro and in vivo, HPV-positive OSCC cell lines. So far, a small number of HPV16-positive HNSCC cell lines have been established in vitro but none originating from tonsil tumors and all from patients with a history of smoking and alcohol use , 8. As tFor uterine cervical cancer, the integration process has been shown to be of great importance for the carcinogenic process, though not being an exclusive prerequisite as episomal HPV16 was present in 47% of cervical HPV16-positive cancers, either as pure episomal (27%) or as a mixture of integrated and episomal HPV16 (20%) . MeasureThe aim of the presented work was to establish cell lines in vitro from HPV-positive OSCC tumors that could be used for therapy development and basic research purposes.The study was approved by Lund University regional ethical review board (LU 376–01), and the samples were collected, after informed consent was obtained, at Skåne University Hospital, Lund, Sweden.2. The cells used in the described experiments were passaged less than 18 times after establishment. Mycoplasma tests were performed on a regular basis by GATC Biotech AG  and were negative.Tumor samples were rinsed consecutively in phosphate buffered saline (PBS) and R10 medium , all from GE Healthcare . The tumor samples were cut in pieces and transferred to cell culture flasks with R10 medium and left to attach and grow at 37 °C under a humidified atmosphere with 5% COFor cell doubling determination, cells were seeded in eight 96-well plates. The plates were analyzed by the sulphorhodamine B (SRB) method as previously described  consecutSingle tandem repeat (STR) analysis was performed by Uppsala Genome Center using the AmpFlSTR Identifiler polymerase chain reaction (PCR) amplification kit . Separation of DNA fragments was performed with capillary electrophoresis on the ABI3730XL DNA analyzer  and allele scoring was performed with the GeneMapper 4.0 software package.Experiments on mice were approved by the local ethics committee (488/2017). We used in-house bred, athymic 5- to 8-week-old BALB/c nude (nu/nu) mice. The mice were given food and water ad libitum and treated according to regulations issued by the Swedish Board of Agriculture (SJVFS 2017:40). Cells were injected subcutaneously into the flanks of non-anesthetized animals. To determine the doubling time, tumors were retransplanted to 10 nude mice under ketamine anesthesia, two tumors per mouse. The length and width of the tumors were measured with calipers three times per week and the relative tumor size calculated in relation to day 0 . The data points were fitted to a logarithmic equation in GraphPad Prism. After the measuring period, the mice were euthanized using cervical dislocation and the tumors removed for histological sectioning.Xenografts and cell pellets were sectioned in 3 μm slices and stained with hematoxylin and eosin, and the following antibodies: the CINtec p16-kit , anti-pan keratin, , and anti-p63  all from Ventana Medical Systems , anti-cytokeratin 5  from Leica Biosystems , and p40  from Biocare Medical , All stainings were performed using the BenchMark Ultra platform .Three HNSCC cell lines, previously established in our laboratory, were used for comparison in several experiments: LU-HNSCC-4, LU-HNSCC-5 and LU-HNSCC-6 . They weA 2–3 mm biopsy was immersed in 1 mL saline solution and immediately transferred to the laboratory. The saline was removed and the biopsy was digested in lysis buffer and the DNA extracted with the Total NA-kit using MagNA Pure LC   and stored at − 20 °C. After thawing, 200 μL was used for DNA extraction with the Total NA-kit using MagNA Pure LC (100 μL output).Briefly, the number of viral genomes per cell was quantified by carrying out two separate real-time PCR tests, including appropriate standard curves, to amplify the HPV16 E7 gene and the human beta-globin gene as previously described . The sam> 0.9. The E7 copy numbers were retrieved from the viral load measurement as described above. For quantification of the E2 copy numbers we used a modified version of the PCR described by Peitsaro et al. /44–47,idem,i(8)(q10)[cp3] t was 10–20 times lower than those of the other cell lines.The sensitivities to commonly used treatments were assessed in vitro and compared to three HPV-negative HNSCC cell lines. LU-HNSCC-26 was considerably more sensitive to cisplatin than the other HNSCC cell lines tested Fig. . The IC5max) of the drug instead of the IC50. LU-HNSCC-26 was only inhibited to a maximum of 18%  was 18–26 times lower than those of the other cell lines.As for cisplatin, LU-HNSCC-26 was considerably more sensitive to radiation compared to the other cell lines Fig. . The ID5TP53 gene was wild type and the cell line displayed a relatively simple karyotype including loss of 3p, 4q, and 7p as well as isochromosomes, which are common findings in HPV-positive HNSCC-cases, that often have less complex karyotypes with fewer signs of chromosomal instability compared to HPV-negative ones [In the current work we demonstrated the in vitro establishment of an HPV16-positive cell line, LU-HNSCC-26, derived from a tonsil carcinoma. The cell line manifested episomal HPV16 with active expression of viral mRNA. The ive ones . The douive ones .It is widely accepted that HPV infection plays an etiological role in many oropharyngeal cancers and there is ample evidence that HPV-positivity is coupled to increased survival and better prognosis. As a result of this, there are currently several clinical trials addressing the question whether it is possible to de-intensify the treatment for this patient group to reduce the toxicities while preserving the efficacy . HoweverWell characterized cellular and animal models are valuable tools for efficient treatment development. Such models can provide fast and efficient testing of novel drugs and combinations of drugs or other treatment modalities. So far, no in vitro cell line has been established from HPV-positive tonsil cancer and only few from other head and neck locations , 8. ImpoThe presence of the episomal state of the HPV16 in the LU-HNSCC-26 cell line, as demonstrated by the complete circular genome of HPV 16 and as well as the intact HPV16 E2 mRNA, is similar to that of our previously established HPV16-positive tonsil carcinoma xenograft, which manifested episomal HPV16 and integrated HPV16 simultaneously . OverallTP53, homozygous for arginine at codon 72 of the TP53. Interestingly, this arginine form of the gene product p53 has been shown to be more susceptible to HPV16 E6 mediated degradation than the proline form [The HPV16 positive LU-HNSCC-26 cell line manifested wild-type ine form . In addiine form . Furtherine form . FurtherThe p53 protein is inactivated by the HPV16 E6 protein . Here weIn this work we attempted to set up cell lines from 27 tonsil tumors of which 23 were HPV positive but only one of these actually resulted in an established cell line. This success rate is considerably lower than that for HNSCC in general, previously reported to be around 33% in our lab . In our There are around 14 tonsil cell lines described in the literature  of which50 being 10 to 20 times lower Additional file 2:TP53. The LU-HNSSC-26 is homozygous for the single nucleotide polymorphism chr17:7579472G > C (rs1042522). Partial sequencing results for TP53 mapped against GRCh37 (top) and Sanger sequencing electropherogram (bottom) with the rs1042522 indicated by arrows. (PDF 172 kb)Partial sequencing results for