The emergence of the role of microRNAs (miRNAs) in exacerbating drug resistance of tumours is recently being highlighted as a crucial research field for future clinical management of drug resistant tumours. The purpose of this study was to identify dys-regulations in expression of individual and/or networks of miRNAs that may have direct effect on neuroblastoma (NB) drug resistance.Individual subcultures of chemosensitive SH-SY5Y and UKF-NB-3 cells were rendered chemoresistant to doxorubicin  or etoposide (SH-SY5Y). In each validated chemoresistance model, the parental and subcultured cell lines were analysed for miRNA expression profiling, using a high-throughput quantitative polymerase chain reaction (RT-qPCR) miRNA profiling platform for a total of 668 miRNAs.A unique expression signature of miRNAs was found to be differentially expressed (higher than 2-fold change) within all three NB chemoresistance models. Four miRNAs were upregulated in the subcultured chemoresistant cell line. Three miRNAs were found to be downregulated in the chemoresistant cell lines for all models.Based on the initial miRNA findings, this study elucidates the dys-regulation of four miRNAs in three separate NB chemoresistant cell line models, spanning two cell lines (SH-SY5Y and UKF-NB-3) and two chemotherapeutic agents (doxorubicin and etoposide). These miRNAs may thus be possibly linked to chemoresistance induction in NB. Such miRNAs are good candidates to be novel drug targets for future miRNA based therapies against aggressive tumours that are not responding to conventional chemotherapy. The utilisation of RT-qPCR based miRNA profiling can nowadays be employed for serving such research objectives.2.1Eight separate NB cell line models were implemented in this study. Each model consisted of two cell lines, a parental chemosensitive together with a sub-cultured cell line that was rendered chemoresistant to a single conventional chemotherapeutic agent through repeated exposure see .2.2Prior to miRNA profiling of all NB cell lines, validation of their chemosensitivity status was paramount to ensure that the subcultured chemoresistant cell line in each model retained its drug resistance properties over several culturing passages without further drug exposure.The method adopted for validating chemoresistance status in each model consisted of luminescence-based cell viability analysis, through the utilisation of the Cell Titer Glo assay  on a Fluorostar Optima luminescence plate reader platform , according to the manufacturer's protocol. Due care was applied in maintaining a seeding cell population across all culture wells utilised in the functional assay, in order to avoid false positive viability readings.2.2.1l-glutamate 200 mM  respectively. Prior to a three-minute step within T25 cell culturing flasks , versene solution  was used for washing all adherent cells within the culture flasks. The trypsin solution utilised consisted of 0.05% trypsin with EDTA . Harvesting of cell suspensions for RNA extraction was performed by adding 0.7 mL Qiazol solution .The growth medium utilised for culturing of all cell lines in this study consisted of RPMI 1640  treated with 10% foetal calf serum  and 1% by volume of 1:1 penicillin/streptomycin mix 10,000 U/mL, kanamicin 10 mg/mL and 2.2.2For each chemoresistance model investigated in this study, both the parental chemosensitive and subcultured chemoresistant NB cell lines were grown in culture and simultaneously harvested and transferred to a sterile 50 mL tube . The cell population density for each cell line was counted by extraction of a 20 μL sample (following vigorous pipette mixing) and analysed on a Cellometer Auto T4 cell counter platform  according to manufacturer's protocol. Both cell cultures where then diluted as necessary with sufficient growth medium to produce a cell culture with a cell population density of approximately 10,000 cells/95 μL. Consequently, 95 μL aliquots from each cell line were transferred (following vigorous pipette mixing) to an opaque Nunclon 96 well microtitre plate .2 for 24 h, to allow cell adhesion on well surface.For each cell line, 18 wells were utilised for consequent drug treatment, and nine wells were to serve as non-treated NB negative controls . A further six wells were treated with 195 μL growth medium only, in order to act as negative controls (drug exposure only). All remaining wells were treated with 200 μL growth medium. Finally, the microtitre plate was placed in an incubator at 37 °C and 5% COFollowing the 24-hour incubation, the microtitre plate containing adherent cells was retrieved and treated with the appropriate chemotherapeutic agents for each chemoresistance cell line model. The varying drug dilutions see  were preOnce retrieved from the incubator, the plate was placed inside a laminar flow cabinet and all wells designated for treatment with the appropriate chemotherapeutic agent were exposed. A 5 μL aliquot of the appropriate drug and dose was pipetted, following vigorous pipette mixing of the drug solution, into the appropriate triplicate wells. An extra 5 μL aliquot of each drug dose was applied to a single well containing only growth medium, in order to obtain background luminescence data from a drug/growth medium solution for future use following cell viability analysis. The plate was then re-incubated for a further 36 h.Following this time period, the plate was collected and a cell viability assay was performed on all wells, by using the Cell Titer Glo assay  according to manufacturer's protocol.2.2.3Following the assay, the Excel sheet containing all raw luminescence data was collected for analysis, with outliers being identified and discarded. The chemosensitivity profiles, based on the cell viability analysis data set results, were plotted by application of the Graph Pad Prism software package . No detailed statistical analysis was performed on the resultant data sets as long as the inhibiting concentration for 50% cell viability (IC50) demonstrated distinct values, reflecting the chemosensitivity profiles.2.32.3.1All cell lines from each validated NB chemoresistance model were cultured and harvested accordingly. Following trypsin incubation step, each T25 flask was subjected to horizontal mechanical shock, necessary for dislodging all cells from the flask surface. The resultant cell suspension from each flask was transferred to an individually labelled, sterile 15 mL collection tube  and centrifuged at 1500 rpm for five minutes to allow all cells to form a pellet at the base of the collection tube. The supernatant from each collection tube was aspirated by disposable, sterile glass pipette with due care to avoid inadvertent aspiration of cell pellet. Consequently, the remaining cell pellet was treated with 700 μL of Qiazol solution  in order to induce cell lysis. This step was performed within the confines of a fume cupboard and thorough pipette-mixing was applied for ensuring a resultant homogenous cell lysate suspension. Immediately after cell lysis induction, all cell lysate suspensions were snap frozen in liquid nitrogen solution and transferred to a − 80 °C freezer until RNA extraction was performed.2.3.2All NB cell line lysates were allowed to thaw to room temperature prior to miRNA extraction. Consequently, all lysates were treated with the miRNeasy  miRNA extraction protocol, within the confines of a fume cupboard. The resultant 30 μL volume of extracted miRNA from each NB cell line lysate was quantified through utilisation of the Nanodrop UV spectrophotometry platform . The extracted miRNA samples were then stored at − 80 °C until further use. The RNA quality was not tested following extraction, as previous experience from earlier studies repeatedly demonstrated that the RNA quality derived from NB cell lines was always of an excellent nature , thus such a step was not performed.2.3.3All miRNA samples were allowed to thaw prior to further treatments. The protocol utilised in this step was a validated, high throughput RT-qPCR based miRNA profiling method 2.3.4All raw Cq values obtained from each individual run were corrected by inter-run calibrators  and consequently normalised against the average Cq value obtained from the total quantity of miRNAs assayed within the same run Ultimately, all miRNAs dysregulated in all three chemoresistance models' miRNA shortlists were identified and selected for further investigation.2.3.5The putative miRNAs identified from miRNA profiling to be dysregulated in the chemoresistant cell lines were further analysed by multiplex RT-qPCR.Taqman miRNA RT kit  was utilised for the reverse transcription step. Reverse transcriptase (RT) primers specific for the relevant target miRNAs  were diluted (5 nmol) with 250 μL nuclease free water , and 10 μL aliquots from each individual primer solution were pooled in a 1.5 mL Eppendorf tube. The primer pool was then diluted with nuclease free water or concentrated by vacuum centrifugation in order to obtain a final volume equivalent to 20% of the total RT reaction volume utilised in this step. The RT reaction volume (per well) consisted of 4 μL stem-loop primer pool, 0.4 μL of 100 nM dNTPs, 4 μL MultiScribe reverse transcriptase , 2 μL of 10 × RT buffer, 0.25 μL RNase inhibitor  and 0.65 μL nuclease free water. The total reaction volume was then placed in a thermal cycler and subjected to 30 min at a temperature of 16 °C, followed by 30 min at 42 °C, 1 s at 50 °C, 5 min at 85 °C and 5 min at 4 °C respectively. The finalised reaction volume was diluted by a factor of five, prior to RT-qPCR step.The TaqMan® miRNA assay for RT-qPCR quantification was utilised in this step for all miRNA assays. Assays for hsa-mir-99b, hsa-mir-125a and hsa-mir-425 were also prepared as reference miRNAs for post-run data normalisation and analysis. All miRNA assays for each cell line sample cDNA were performed in triplicate, with water — containing negative controls. The reaction volume (per well) contained 2.5 μL TaqMan Master mix, 0.125 μL TaqMan miRNA probe and primers, 2 μL of sample cDNA and 0.375 μL nuclease free water. The cycling protocol was run on the ABI 7900HT qPCR platform  and consisted of an initial holding stage of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s/60 °C for 1 min, ending with a final holding stage of 37 °C for 5 min. Following the qPCR run, the data was exported and analysed, including statistical analysis, on the qBasePlus software package .33.1Prior to performing miRNA profiling for each individual cell line within the varying NB chemoresistance models, it was essential to confirm the chemosensitivity profiles. This was implemented by performing a luminescence based cell viability assay, following a pre-determined exposure period by the cell lines to the relevant chemotherapeutic agent. However, the luminescence detection platform also required preliminary analysis to confirm optimum performance.From the eight NB chemoresistance cell line models available, cell viability analyses only confirmed three models to still have the necessary chemosensitvity profiles required for investigating miRNA expression profiling see . The thrEach NB chemoresistance cell line model was analysed to confirm chemosensitivity status of its constituent cell lines. The cell lines that confirmed chemosensitivity status were re-analysed with a second, identical cell viability analysis in order to ascertain such status prior to miRNA profiling.Following the set of cell viability assays on each NB chemoresistance model, only three out of the initial eight cell line models were confirmed to have maintained their chemosensitivity profiles, namely the SH-SY5Y/DOXO, SH-SY5Y/ETOPO and UKF-NB-3/DOXO NB chemoresistance models. The extracted RNA from constituent cell lines, from each of the three cell line models, were consequently eligible for miRNA profiling.3.2The expression profiling of 668 miRNAs for each constituent cell line of the three validated and selected NB chemoresistance cell line models demonstrated that approximately 50% of all miRNAs are expressed above the 35 Cq value see . AdditioThe results of the miRNA expression profiling revealed that a putative chemoresistance signature expression of seven miRNAs was present in all three NB chemoresistance cell line models see . All sevAll identified miRNAs deemed to be involved in NB chemoresistance were consequently eligible for downstream validation studies.3.3Once the short list of putative chemoresistance miRNAs was elucidated, it was deemed necessary to validate such findings by re-confirming the degree of dysregulated expression of each individual putative miRNA.Since the miRNA profiling step did not utilise technical replicates for each individual miRNA RT-qPCR assay (one replicate/miRNA), it was essential to re-confirm the dysregulated expression status for each individual putative NB chemoresistance miRNA within each constitutive NB cell line of each chemoresistance model utilised in this study. This was implemented by performing a secondary RT-qPCR assay, with three technical replicates for each miRNA assay, in order to ascertain the specific miRNA expression levels.Following re-analysis of RNA from each of the three NB chemoresistance models' cell line constituents by multiplex RT-qPCR miRNA assay, only four out of the original seven-member putative chemoresistance miRNA signature were pursued for functional validation see .4Initial performance results of the luminescence reader platform proved to be successful in achieving specificity of luminescence analysis, with a standard deviation of approximately 1%. Additionally, the dynamic range of the luminescence platform was also deemed optimal, with the lower end of the platform sensitivity spectrum denoted at a cell suspension density of 12,500 cells/mL.From the eight NB chemoresistance cell line models at disposal in-house, only three models  demonstrated distinct chemosensitivity profiles for the constitutive cell lines.Analysis of the three Kelly NB chemoresistance models highlighted incongruencies in the chemosensitivity profiles of the component cell lines and was therefore excluded from the study.For the UKF-NB-3 chemoresistance models, the UKF-NB-3/vincristine model was also excluded due to inability to confirm the projected chemosensitivity profiles for the two component cell lines, over two separate cell viability assays post-vincristine exposure. However, the UKF-NB-3/DOXO cell line reproducibly demonstrated to confirm the predicted chemosensitivity profiles, with the doxorubicin-resistant component cell line proving to be resistant to the drug exposure dose on comparison with the chemosensitive counterpart cell line by a factor of ten, on comparing the doxorubicin doses required to induce 50% decrease in cell viability. A peculiar observation was also denoted within the UKF-NB-3/DOXO model, in that at the elevated doxorubicin doses the cell viability in both component cell lines seems enhanced. This observation was found to be reproducible across two separate cell viability assays and was solely noticed within this specific NB chemoresistance model (UKF-NB-3 cell line exposed to doxorubicin). Possible explanations for such an anomaly would be technical interference with the luminescence assay, though the actual source thought to cause such interference is still unclear. Consequently, for the following transient transfection validation assays concerning the UKF-NB-3/DOXO model, the doxorubicin dose range utilised had to be reduced accordingly.Analysis of the SH-SY5Y chemoresistance models revealed that the SH-SY5Y/cisplatin NB model had lost its chemoresistance phenotype, due to the observation of near-identical chemosensitivity profiles by both components of the NB model and therefore was excluded from the study. For the SH-SY5Y/DOXO and SH-SY5Y/ETOPO chemoresistance models, both met the predicted chemosensitivity profiles required for inclusion in the study, although it was also denoted that the SH-SY5Y/DOXO chemoresistance model demonstrated to be more robust. This was due to the enhanced chemoresistance properties highlighted by the doxorubicin-resistant cell line model component, on comparison with its etoposide-resistant counterpart cell line. Such phenotypes were confirmed for both NB chemoresistance models, following secondary cell viability assays.The utilisation of RT-qPCR technology in a high throughput manner for the purpose of quantitatively analysing the miRnome in clinical and scientific studies is, of late, a rapidly developing tool for investigating miRNA expression. The essential reason for such popularity lies in the fact that such a method combines the high-throughput efficiency of the platform to simultaneously analyse almost 700 miRNAs from a single biological sample, though retaining the robustness and quantitative analytical advantage of RT-qPCR techniques. Such a technique was utilised in this study for identifying dysregulated expression of individual and/or networks of miRNAs within the selected NB chemoresistance cell line models.Within the context of this study, pre-determined criteria were applied for the purpose of identifying putative chemoresistance miRNAs. All Cq values obtained for the investigated miRNAs, observed at a value above 35, would lead to the interpretation that such miRNAs were either not expressed at all, or expressed at very low levels in the corresponding biological sample. This specific Cq threshold value was thus utilised in order to eliminate false positive expression data inclusion due to quantification noise, which is normally introduced due to natural biological variances or also due to the technical setup of the RT-qPCR assay Such a fold change threshold range is deemed to be more restrictive as a selection strategy utilised for identifying relatively elevated levels of dysregulated expression by the individual miRNA. It is important to realise that such restrictive selection criteria might have excluded minor miRNA dysregulations (ie. less than two fold up-regulation or less than two-fold down-regulation) that may still prove to induce a tangible phenotypic influence (eg. fold-change × 1.8).However, the fold change threshold was implemented in this study at such cut-off points for practical reasons, with the assumption that only those miRNAs falling within the dysregulation thresholds were deemed to have possible influence on NB cell line model chemoresistance properties.Consequently, it must be emphasised that the threshold fold-change levels are entirely subjective and therefore a careful balance must be attained whereby reliable hits can be selected, though without elevating the stringency levels of the threshold fold-change values to the point at which identification of even a low quantity of hits would prove to be a challenge. Since the minimum miRNA fold change acceptable for scientific publications is a threshold of 1.5-fold, it would be recommended to set the least stringent miRNA fold-change selection criteria at this level (+/− 1.5 fold-change) for all studies of this nature The overview analysis of miRNA expression profiling of all three NB chemoresistance models demonstrated that approximately 50% of the miRNAs analysed in this study had a raw Cq value at/above a value of 35. Those miRNAs having a Cq value above the value of 35 were expressed at low/single molecule level and excluded from further analyses. The notion that half of all miRNAs are expressed at low levels (or not expressed) can be attributed to the fact that in most cancer conditions there exists an overall down-regulation of miRNA expression In addition, the scatterplot analysis of normalised Cq values for each individual miRNA, obtained from each constitutive NB model component cell line, revealed overall low levels of data spreading. This implies that only a minute fraction of the entire miRnome may be deemed to be grossly dysregulated in such investigated cell line models.For the three NB chemoresistance models investigated in this study and following implementation of the above selection criteria, a total of seven putative chemoresistance miRNAs were observed to be dysregulated across all three NB models see . From suFollowing miRNA expression profiling, it was necessary to confirm dysregulation of the selected putative chemoresistance miRNAs by multiplex RT-qPCR techniques. This step was of importance for confirming the results obtained from miRNA profiling, since the latter RT-qPCR assays were performed using one sole technical replicate for each individual miRNA. Consequently, confirmation by multiplex RT-qPCR analysis utilising three technical replicates allowed for such validated putative chemoresistance miRNAs to be further validated by functional assay implementation.Results from the multiplex RT-qPCR assays fully confirmed the up-regulated expression profile for miR-188-5p across all three NB chemoresistance models. Two additional putative miRNAs were also of major interest, namely miR-125b-1# and miR-501-5p, due to confirmation of dysregulated miRNA expression in at least two NB chemoresistance models. The fourth putative miRNA was miR-204. However, the down-regulated expression of miR-204 was only confirmed within the UKF-NB-3/DOXO chemoresistance model. This suggests that, due to the mutated p53 status present in UKF-NB-3, this dysregulation is cell line dependent and is totally absent in wild type p53 status NB cell lines such as SH-SY5Y The remaining miRNAs failed to confirm their dysregulated expression profile within the majority  of the three NB chemoresistance models. The main reasons for such results stems from the RT-qPCR technique implemented for miRNA profiling. A typical step utilised for sample preparation prior to miRNA profiling is the pre-amplification of RNA collected from the biological sample Therefore, any miRNAs identified as expressed following miRNA profiling with a raw Cq value between 30 and 35 might eventually not be equally identified on multiplex/singleplex RT-qPCR analysis, since the latter is applied on non-pre-amplified RNA samples solely. Ultimately, such low expressed miRNA profiling expression values must be investigated with due attention and awareness of such technical influences Following the results of this study, exact mechanistic links between miRNA dys-regulation levels and chemoresistance phenotype cannot be concluded. However, it certainly is of interest to highlight the possible involvement of such miRNAs in affecting the most common means of chemoresistance-inducing cellular pathways within such cell lines. A typical example would be the effect of such miRNAs on the ABC transporter system and its member genes that regulate drug efflux properties of the cell Such mechanistic links have been identified within other cancer models. The study conducted by Ma and colleagues Future research focusing on the influence of miRNA effects on NB chemoresistance properties may be highly varied and of equal importance. One specific research option would be to monitor the miRNA expression profile of individual NB cell lines during prolonged exposure to specific conventional chemotherapeutic agents, until such cell lines develop sustainable chemoresistance properties. Such a study would provide great insight onto which miRNA networks are most affected by prolonged cytotoxic drug exposure, through evaluation of miRNA profiles for dysregulated miRNA expression across the varying phases of the drug exposure period. Consequently, the miRNA profiles across the entire timeline for rendering a specific NB cell line chemoresistant to individual or combinations of chemotherapeutic agents may provide a wealth of information regarding which miRNAs could possibly have key roles in orchestrating such a phenotypic shift at the cellular level.Furthermore, the prospect of performing miRNA profiling of NB cell lines having combinations of differing NB oncogene expression, as described above, could also shed more light on the effect of NB oncogene expression on miRNA expression profiles. Downstream miRNA dysregulations correlated to exacerbated oncogene expression may consequently be investigated for the identification and validation of key miRNAs deemed to act as intermediate effectors of chemoresistance.The implementation of a systems biology approach might also be of valuable use for enhanced mapping and characterisation of miRNA networks with the capacity to interact and regulate complex gene networks directly affecting the various biological processes within the neuroblast that ultimately lead to the attainment of pronounced chemoresistance properties in vitro evaluations folowing our findings, further in vivo murine model research could be enacted, consisting of NB xenograft implants bearing constructs with individual putative chemoresistance miRNAs, which are actively expressed on oral administration of tetracycline by the individual mouse, thus minimising distress to the animal. Following miRNA induced expression, the animals would be exposed to cytotoxic agents and tumour progression is monitored accordingly. Induced expression of the putative chemoresistance modulating miRNA would be expected to lead to NB tumours having resilience to chemotherapy.Ideally, should any miRNA have be successfuly validated within Clinical relevance of the identification of individual/networks of miRNAs directly linked with NB chemoresistance properties is two-fold.Firstly, the identification and proper validation of hallmark miRNAs which are proven to be key players in conferring individual and/or multi — drug chemoresistance properties to NB tumours may be utilised for the development and implementation of novel diagnostic miRNA expression profile and screens Similar diagnostic screens have already been designed for accurate diagnosis of other severe conditions such as human papilloma viral infectious strains, though such bespoke microarray technology could be implemented for the recognition of specific miRNA sequences present in any clinical sample e.g. chemoresistance, cell proliferation, cell differentiation).However, the validation methods for confirming individual candidate biomarkers may be time consuming. This is a particular problem for exome sequencing approaches, due to the vast array of data that is commonly generated and thus requires thorough bioinformatic analysis prior to even identifying candidate exome sequence biomarkers for consequent validation assays. In distinct contrast, the possibility of identifying novel body fluid biomarkers, such as free-circulating miRNAs For the validation of miRNAs associated with chemoresistance, transient transfection of miRNA antagonists remains the mainstay method due to the rapidity of the assay (72–96 h). Unfortunately, this method also has the drawback that direct quantification of candidate biomarker miRNA by RT-qPCR cannot be performed, unless the miRNA in question is known to down-regulate specific target genes (which have been previously confirmed in other studies).Following the acquisition of additional information from these novel biomarkers, the therapeutic avenues chosen by the clinician will be decided with the inclusion of pre-emptive strategies against chemoresistant tumours, based on the additional awareness of the chemosensitivity status of the individual NB tumour. The therapeutic options applied will thus maximise the chances of survival of the patient, with minimal suffering due to chemotherapy-induced adverse effects.Secondly, such miRNAs are good candidates to be novel drug targets for future RNAi based therapies against aggressive tumours that are not responding to conventional chemotherapy


We havef lipids . Since mde novo lipogenesis, proliferation, growth, and cell survival.We used a panel of breast cancer cell lines with various mutations resulting in constitutive mTORC1 activation, as well as MCF10a cells transformed with oncogenic KRAS or PIK3CA to explore the role of SREBP in mTORC1-driven cancers. We used chemical inhibitors and siRNAs to assess the dependence of our breast cancer systems on the mTORC1-SREBP pathway for de novo lipogenesis in an SREBP-dependent manner in cancer cells. In addition, we found that the mTORC1-stimulated activation of SREBP2 promotes cell growth, proliferation, and survival of breast cancer cells. We are in the process of identifying the essential functions of SREBP2 in breast cancer and assessing the dependence of breast cancer cells on SREBP2 in vivo. These findings emphasize the therapeutic potential of targeting lipid metabolism in cancers, particularly those with activated mTORC1 signaling.Using both the panel of breast cancer cell lines and the isogenic MCF10a cell lines, we have revealed an essential role for SREBP in driving lipogenesis and proliferation in tumor cells. Our results indicate that SREBP and its lipogenic targets are activated in cell lines with oncogene-induced mTORC1 signaling, and this activation was ablated by mTOR inhibitors. mTORC1 signaling promotes


Yet no predominant wildlife reservoir has been identified, and gaps in our knowledge of RVFV permissive hosts still remain. In North America, domestic goats, sheep, and cattle are susceptible hosts for RVFV and several competent vectors exist. Wild ruminants such as deer might serve as a virus reservoir and given their abundance, wide distribution, and overlap with livestock farms and human populated areas could represent an important risk factor. The objective of this study was to assess a variety of cell lines derived from North American livestock and wildlife for susceptibility and permissiveness to RVFV. Results of this study suggest that RVFV could potentially replicate in native deer species such as white-tailed deer, and possibly a wide range of non-ruminant animals. This work serves to guide and support future animal model studies and risk model assessment regarding this high-consequence zoonotic pathogen.Rift Valley fever virus (RVFV) causes disease outbreaks across Africa and the Arabian Peninsula, resulting in high morbidity and mortality among young domestic livestock, frequent abortions in pregnant animals, and potentially severe or fatal disease in humans. The possibility of RVFV spreading to the United States or other countries worldwide is of significant concern to animal and public health, livestock production, and trade. The mechanism for persistence of RVFV during inter-epidemic periods may be through mosquito transovarial transmission and/or by means of a wildlife reservoir. Field investigations in endemic areas and previous  Phlebovirus in the family Bunyaviridae, and is a vector transmitted pathogen that causes endemic disease across Africa and the Arabian Peninsula  belongs to the genus eninsula . Periodieninsula . RVFV theninsula . Humans eninsula . Human peninsula . The humeninsula .The potential of RVFV spreading to the United States (US) or other countries worldwide, whether by accident or with intent, is of significant concern . SeveralWild ruminants in North America, such as deer, could represent an important risk factor given their abundance, wide distribution, and overlap with livestock farms and human populated areas . AlthougThe attenuated RVFV MP-12 strain was provided by the United States Army Medical Research Institute for Infectious Diseases. The virulent Saudi Arabia isolate SA01-1322  was provXenopus laevis) kidney cell line was obtained from Jane Homan at the University of Wisconsin, and is commercially available through American Type Culture Collection (ATCC), Manassas, VA, USA. The sheep , calf , pig , American pronghorn (Antilocapra americana), elk (Cervus elaphus), mule deer (Odocoileus hemionus), white-tailed deer (O. virginianus), and coyote (Canis latrans) cell lines were initiated and established by the Arthropod-Borne Animal Diseases Research Unit (ABADRU), Manhattan, KS, USA from tissues derived from specimens donated to ABADRU or the Wyoming State Veterinary Diagnostic Laboratory. Briefly, primary lung and kidney cells were harvested by manual homogenization, and then pressed through tissue sieves. Subsequent cells were washed and resuspended in 199E media  supplemented with 10% gamma-irradiated fetal bovine serum  and 1x antibiotics (penicillin and streptomycin)-antimycotic . Non-adherent cells were removed and media replaced at weekly intervals. Cell lines were screened for bacteria, fungi, and mycoplasma contamination by cultivation methods or using the MycoTect kit . Specific tests for the presence of bovine viral diarrhea and bluetongue viruses were also performed. Certain cell lines were additionally screened for other viral contaminants by electron microscopy using negative staining techniques. All of the cell lines used for this study were negative for contaminants included in the screening processes mentioned. Lung cultures were a mixture of fibroblastic and endothelial like cells. All brain cell lines used in this study were prepared similar to the lung and kidney cell lines as described above and then transformed. Transformation consisted of transfection of primary cultures with an expression plasmid carrying the simian virus 40 genome  using Lipofectamine 2000 reagent  per the manufacturer’s instructions. Medium was changed 48 h later to OptiMEM  and 10% FBS, and the cells were grown for an additional 1 to 2 weeks, when clusters of cells showing loss of contact inhibition and an increased rate of cell division were selected using cloning cylinders. All cell lines were maintained at 5% CO2, 37°C with the exception of the frog cell line which was kept at 28–30°C.The African green monkey Vero cell line is a clone from the Middle America Research Unit (MARU). The frog (Overnight cell cultures (∼90% confluent) were infected at 0.1 multiplicity of infection (MOI) with either the MP-12 or SA01-1322 RVFV strains. Virus was adsorbed for 1 h then monolayers were washed and fresh media added. Time course samples were collected at 0, 6, 12, 24, 36, and 48 h post infection. Cells and culture supernatant were collected separately for intracellular and extracellular virus titration, respectively. Total virus was measured by collecting both cells and culture supernatant simultaneously. Samples were subjected to three freeze/thaw cycles and stored at -80°C until analysis. Standard plaque assays on Vero MARU cells were performed to calculate titers in plaque forming units per ml (pfu/ml).Table 1 and locations of the primer and probes on the RVFV L segment are shown in the Supplementary Figure Total RNA was extracted from virus time course samples using TRIzol-LS reagent  and the magnetic-bead based capture MagMAX viral RNA isolation kit . Briefly, 100 μl of aqueous phase was added to 90 μl of isopropanol and 10 μl bead mix. Total sample RNA was washed four times and eluted in 30 μl of elution buffer by use of the automated KingFisher Magnetic Particle Processor . Sequences of primers and probes directed toward a region of the large (L) genome segment of MP-12 or field strains of RVFV have been published previously and were used to estimate viral RNA levels . Beta ac5 and 107 pfu/ml . Replication kinetics of MP-12 were more rapid in the calf, white-tailed, and mule deer cell lines, with titers nearly 102 to 103 pfu/ml higher at 6 hpi compared to titers from sheep, pronghorn, and elk cell cultures . Replication kinetics of MP-12 in the pig brain cell line was significantly delayed compared to the ruminant brain cells. Expansion of MP-12 in the pig brain cells did not occur until after 12 hpi and only a titer around 103 pfu/ml was achieved by 48 hpi which was more than 102 pfu/ml lower than the lowest titer observed at the same time point among the ruminant-derived brain cell lines . Conversely, replication kinetics and titers of the SA01-1322 strain were more consistent among the white-tailed deer, sheep, and pig brain cell lines tested . Titers averaged around 103 pfu/ml by 6 hpi and then peaked around 105 to 106 pfu/ml between 24 and 36 hpi, with the white-tailed deer cell line supporting slightly higher virus titers .A total of nine different animal-derived cell lines indigenous to North America were evaluated, including domestic ruminant , wild ruminant , and non-ruminant  species. In both animals and humans, RVFV typically targets the liver and spleen, and can be neuroinvasive; however, it is also known to replicate in a variety of other tissues and cells . Cell liFigure 1C); however, high titers were still achieved in these cell lines. Titers of MP-12 in the white-tailed deer and sheep kidney cell lines reached 107 pfu/ml, while the pig and frog kidney cell lines supported MP-12 titers of 105 and 106 pfu/ml, respectively . Similar replication kinetics and titers in the range of 105 pfu/ml were observed for the SA01-1322 strain among the white-tailed deer, sheep, and pig kidney cell lines . Compared to the brain cell line, SA01-1322 peak titers were about 101 pfu/ml lower in white-tailed deer kidney cells .The replication kinetics of the MP-12 strain were slightly slower in the kidney cell lines compared to the brain cell lines with titers peaking after 36–48 hpi . Infectious virus was detected by 12 hpi in the white-tailed deer lung cells and by 24 hpi in the coyote lung cell line . The pig lung cell line had the most delayed viral kinetics with virus not detected until 36 hpi . Overall, the SA01-1322 strain viral kinetics and titers were relatively consistent with little variation among the cell lines tested, while the MP-12 strain exhibited more variability among species and the tissues the cell lines were derived.Although RVFV is not typically associated with respiratory disease, results from this study demonstrated that MP-12 was capable of replicating in three different lung cell lines. Lung-derived cell lines consisted of a heterogeneous mixture of cells showing epithelial and fibroblast-like morphology. Titers in the lung cells peaked around 10Figures 1 and 2). For example, SA01-1322 RNA levels in the brain cell lines  were lower than SA01-1322 RNA in kidney cells . Levels of MP-12 RNA were generally higher than the RNA levels produced by the SA01-1322 strain . This observation could be explained by more efficient primer binding and amplification of the MP-12 RNA template compared to the SA01-1322 strain, although no mutations were detected in the primer or probe binding sites by sequencing of the original virus stock used in these experiments , while the inverse was observed in cell lines with more delayed viral kinetics such as the coyote lung and pig kidney cell lines . The difference between intra- and extracellular viral titers was not as clearly observed with the viral RNA levels . While lower MP-12 titers were observed in the coyote cell line compared to the sheep cells , the viral RNA was higher in the coyote cells than the sheep cells .In order to determine the ability of RVFV to be efficiently released from infected cells, extracellular virus from the culture supernatants and intracellular virus from cells was also compared. Although not statistically significant, there were some observed differences between the two fractions from certain cell lines that were evaluated. In particular, the brain cell lines derived from sheep, calf, and white-tailed deer which supported efficient viral replication had slightly higher extracellular virus titers compared to intracellular  were detected in nasal swabs from some of the infected animals; however, no signs of viremia were detected from any of the uninfected control animals housed alongside the challenged animals throughout the 21 days study (unpublished data). Although it may be possible for airborne transmission to occur, it is more likely to occur through direct contact with infected tissues and bodily fluids. Nonetheless, creation and exposure to aerosols of RVFV should be avoided due to the severe pathology associated with this route of infection. Further investigation is necessary for understanding better the potential risk of aerosol transmission of RVFV.Finally, it is interesting to note that three different lung cell lines were permissive to RVFV infection, even though RVFVis is typically not associated with respiratory disease. Infection by aerosol transmission of RVFV has occurred in the laboratory environment; however, no human-to-human transmission has ever been documented . Intranain vivo host competency studies and guide statistical risk modeling for this high-consequence zoonotic pathogen. The results presented here suggest RVFV could potentially infect a wide range of domestic livestock and wildlife native to North America. All of the cell lines tested in the current study were permissive and susceptible for RVFV replication, especially ruminant-derived cell lines. Based on these results and serology surveys of wild ruminants in endemic countries, deer and other wild ruminant species should be given high priority for further investigations. It is currently unknown whether these animals are competent hosts for RVFV, or what role they could play in the dissemination of this virus. In the event that RVFV were introduced to the US and wildlife was capable of becoming reservoirs for this pathogen, controlling the spread of RVFV would be difficult, if not impossible. Not only is RVFV a threat to both human and animal health, but the establishment of this pathogen would have costly economic impact related to livestock production and trade. Thus, expanding research to investigate competence of potential wildlife hosts in the US remains critical in order to plan effective response and control strategies if an introduction were to occur.The objective of the current study was to build a foundation for future NG, SI, and PB contributed to the acquisition of data. NG, JR, and WW contributed to experimental design, and the analysis and interpretation of data. All authors contributed to the drafting and final approval of the manuscript for submission, and take responsibility for the work presented.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Melphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen  is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma.Cytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice.50-values in the submicromolar range (0.011-0.92 μM) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 μM) and an increased ratio vs. melphalan . Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals.Melflufen showed activity with cytotoxic ICThis study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases. The ma model . Intravema model –9. It watoxicity . MelphalMalignant lymphomas are a group of tumors originating from cells of the lymphatic system. The lymphomas are classified according to the WHO-classification into B-cell lymphomas, T-cell lymphomas and Hodgkin lymphoma (HL) and further into many different entities. Of the B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL) is the most common subtype. Systemic chemotherapy is the mainstay of therapy for all lymphomas and approximately 60–70 % of DLBCL patients are cured. In HL even higher cure rates are possible, and the chemotherapy may be combined with radiotherapy. Poor responders and/or early relapse patients at young age and good physical condition are often considered for high dose chemotherapy and stem cell transplantation. Still a significant proportion of lymphoma patients relapse, and there is a need for new drugs to further improve the results and reduce the toxicity of treatment , 12.para-fluoro-L-phenylalanine. The drug is susceptible to hydrolysis by aminopeptidases, like aminopeptidase N  for which melflufen is a substrate [50-value of melflufen significantly lower than for melphalan (10-600-fold lower in various in vitro cell systems) [Melflufen  is a derivative of the classical alkylating agent melphalan. Chemically melflufen may be described as the ethyl ester of a dipeptide consisting of melphalan and ubstrate , providiubstrate . It has ubstrate , 16. Thesystems) , 17, 18.systems) , 20.Increased expression of various hydrolytic enzymes like peptidases, esterases and proteases has been described in several types of human malignancies, especially those characterized by fast-growing and aggressive phenotypes . Among tThis study was undertaken to characterize the activity of melflufen against lymphoma cells in vitro and in vivo.Cytotoxicity was assayed in a panel of twelve human lymphoma cell lines. A brief description of the cell lines is presented in Table All lymphoma and CCRF-CEM cells were cultured in RPMI 1640 cell growth medium  supplemented with 10–20 % heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin . Cells were grown at 37 °C in a humidified atmosphere containing 5 % carbon dioxide, split twice weekly and harvested in log-phase for experimental use.Patient tumor cells from 16 patients with different lymphoma subtypes were also analyzed for in vitro sensitivity Table . The samMelphalan Fig.  was obta2 for 24 h followed by the addition of drugs (2.5 nl portions/well of DMSO drug stock) using Echo 550 Liquid Handler (Labcyte). The plates were further incubated for 72 h. Patient lymphoma cells were handled similarly or seeded  into drug containing (5 μL/well) 384-well microtiter plates. The plates were incubated at 37 °C, in humidified atmosphere containing 95 % air and 5 % CO2 for 72 h. Each drug concentration was tested in duplicates. Each 384-well microtiter plate had three columns without drugs serving as controls, and one column with medium served as blank. The cells were subsequently washed with PBS and supernatant aspirated leaving 10 μl in each well, before the addition of 50 μL, 10 μg/mL FDA. Plates were incubated for 60 min before measurement of fluorescence (485/538 nm for excitation and emission respectively) in a Fluostar Omega. The fluorescence generated is proportional to the number of cells in the well with an intact plasma membrane, and data are presented as Survival Index (SI %), defined as the mean fluorescence of the test wells expressed as a percentage of control cultures, with blank values subtracted.Cell viability after drug exposure (cytotoxic efficacy) was analyzed with the fluorometric microculture cytotoxicity assay (FMCA) which has been described in detail previously , 31. TheQuality criteria for a successful assay included > 75 % starting viability (judged by Trypan blue exclusion), a control signal more than ten times the blank, and finally, a coefficient of variation in control and blank wells of < 30 %.Cell cycle distribution analyses were performed on melflufen treated  and control cells from the KM-H2 and SU-DHL-10 cell lines in which the cytotoxic activity of melflufen during the 72 h FMCA assay was calculated as 0.92, 0.22, and 0.71 μM respectively . The animals had FELASA  SPF-status and the housing and changing systems were designed to assure that the SPF-status was preserved during the study. Diet (Altromin 1324) and water was administered ad libitum.6 DOHH-2 cells into the right flank. Appearance of tumors was carefully monitored, and the tumors were scored (if not large enough to be measured) or measured three times a week. Tumor diameters were measured in two dimensions using a digital caliper and the volume was estimated by the following formula: L × W × ½ W (Length × Width × ½ Width). Measurements/observations began at day 0, i.e. the day of inoculation. After randomization on day 21 the animals were allocated to groups and dosed with intravenously injected vehicle , vincristine , or melflufen . Treatment continued for two weeks, and mice were terminated by cervical dislocation at the end of the experiments or at humane endpoints .Female C.B-17 Scid female mice (nomenclature: C.B-lgh-1b Prkdcscid/lcrTac), 5–7 week of age at arrival, were supplied from Taconic-Europe, Denmark, and after 7 days of acclimatization inoculated subcutaneously with 5×1050-vaules (inhibitory concentration 50 %) for the drugs were determined from log concentration-effect curves in Graph Pad Prism  using non-linear regression analysis. Statistical considerations and comparisos between melphalan and melflufen groups were based on logIC50-values and made with two-sided paired t-test, and for three groups one-way ANOVA was used. For comparison between survival curves log-rank test was used. In all tests p < 0.05 was considered significant.The cytotoxic IC50-values varied almost 100-fold among the cell lines, identifying the DLBCL cell lines Ly-3 as the most sensitive cell line  and DB as the most resistant (44 and 0.92 μM). Melflufen was consistently more potent than melphalan throughout the panel, and the average ratio of IC50-values was 49-fold . The pattern of sensitivity among the cell lines was similar for all standard chemotherapeutic drugs. As expected, the correlation coefficients of log IC50-values between the alkylating agents was high  in this panel of lymphoma cells. Interestingly, bortezomib deviated from this pattern, and all cell lines appeared sensitive to this drug with comparably small variations in IC50 (range 2.0–7.1 nM).The cytotoxic activity of melflufen in human lymphoma cell lines is presented in Table 50 range from 2.7 nM to 0.55 μM. The efficacy of melflufen corresponded to a 13- to 455-fold potency improvement (p < 0.001) compared to melphalan , between two strands (interstrand) or between DNA and proteins, are usually more active than monofunctional agents. Although covalent adduct formation is the mechanism of cytotoxicity common to all the antitumor alkylating agents, these drugs have widely different potency, toxicity and disease selectivity. Differences in the non-alkylating portions of these molecules probably lead to different biodistribution and normal tissue toxicity of these drugs . New repThe clinical use of most chemotherapeutic agents is limited by the associated toxicity, often originating from organs with high proliferation (e.g. bone marrow). Many attempts have been made to, more or less, specifically target the tumor cells or the tumor microenvironment, for example, the use of a prodrug approach where the prodrug is selectively activated in tumors overexpressing the prodrug-activating enzyme(s) . Many suAPN/CD13 is commonly expressed in hematopoietic malignancies of myelomonocytic origin and has less commonly been described in lymphoid neoplasms. However, effects of the aminopeptidase inhibitor bestatin on lymphoma cells have been shown in vitro , and the50-values in the range of 11 nM to 0.92 μM. A similar pattern was found in patient cells (IC50 of 2.7 nM to 0.55 μM) clearly suggesting that lymphoma is a possible clinical diagnosis for future use of melflufen. The in vivo xenograft model study indicated modest activity of melflufen in the dose and regimen used (3 mg/kg on a twice weekly schedule), but on the other hand, without any signs of toxicity the dose and/or intensity of the treatment schedule could most probably be increased. Animals in the positive control group suffered severely from toxicity, and albeit excellent tumor control, some of them had to be prematurely sacrificed due to treatment associated weight loss.The results presented here show that melflufen is indeed an active drug both in Hodgkin and non-Hodgkin lymphoma cell lines in vitro, yielding ICThe conclusion of this study demonstrates that melflufen is an active drug against lymphoma in vitro and in vivo and further evaluation in this diagnosis seems warranted.The xenograft study was performed at Pipeline Biotech A/S, Trige, Denmark and was approved by the national authority “Danish Animal Experiments Inspectorate” (2012-15-2934-00051 C1).The use of patient samples was approved by the regional Ethics Committee of Uppsala University (Ns 2008/246 and 2014/233).The use of the patients’ samples in research was approved without written informed consent of the patients. The reasons why the ethical committee waived informed consent were that the patients had approved storing of samples in the Uppsala biobank and that these samples could be used for research after ethical approval without contacting the patient if the committee found the research of good quality and not compromising integrity of the patient. Furthermore, all patients included in this study have given an oral informed consent but that was not a requirement of the ethical committee.The datasets supporting the conclusions of this article are included within the article.


Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor, Y15, was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines, profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878, and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines, TPC1, BCPAP and K1, and 380, 109, and 74 genes were significantly >2-fold changed with Y15 treatment, respectively. Common up-regulated genes were involved in apoptosis, cell cycle, transcription and heat shock; down-regulated genes were involved in cell cycle, cell-to-cell interactions, and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes >4-fold (p<0.05). Among genes changed 4 fold, 11 were shared between the treatments, including those involved in metabolism, cell cycle, migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib, Sorafenib, Pazopanib, and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor, detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines, demonstrated differential response changes with Y15 and PF-04554878 treatment, and showed the synergy of Y15 with PF-04554878, Cabozantinib, Sorafenib, Pazopanib, and Sunitinib. Over 60,000 new cases of thyroid cancer are diagnosed each year in the United States, comprising over 95% of endocrine malignancies. It is the fastest increasing cancer, with rates rising 5-7% every year  in the UIn order to overcome the current radioiodine resistance within thyroid cancer, identifying and targeting other proteins of interest may work in tandem to effectively treat thyroid cancer. Focal Adhesion Kinase (FAK) is one of these targets. FAK is expressed in all cells at a low basal level, however it is significantly overexpressed in a majority of solid tumors, including papillary carcinomas, with even higher levels of expression in metastatic tumors . The focin vitro resulted in decreased cell viability, increased detachment, and increased apoptosis in colon cancer cells  with protease inhibitors  for 30 min. Lysed cells were spun in a centrifuge to collect protein samples which were stored at −80 °C. Protein samples (15 μg) were separated using electrophoresis and transferred onto an Immobilon-P PVDF membrane (Millipore), then incubated for one hour in 1% BSA/0.01% Tween-20 in PBS. The membranes were washed three times with 0.01% Tween-20 in PBS and then were incubated with primary antibody for one hour. The membranes were again washed three times with 0.01% Tween-20 in PBS before and after being incubated in secondary antibody for one hour. The membranes were then developed with Western Lightning Plus-ECL Enhanced Luminol Reagent Plus (PerkinElmer). Photoshop and Illustrator were used to prepare images from scanned films.Cells were seeded onto 96-well plates . Twenty-four hours following inhibitor treatment, 20 μL of Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega) was added to each well. After two hours of incubation with reagent and the plate was read with the Gen 5 1.07 microplate reader at 490 nm. Each sample was analyzed in triplicate.Two 6-well plates were seeded with TPC1 and K1 cells at a concentration of 500 cells per well in 2 mL of media. BCPAP cells were plated at 1500 cells and 2mL of media per well. After overnight incubation , each well was treated with a different Y15 doses and left to incubate. After 14 days the media was aspirated from each well and a solution of 1% methylene blue and 50% methanol was added. The cells were incubated in this solution for 30 minutes to fixate and stain them. The colony number in each well was counted in duplicate plates.Six-well plates were seeded with TPC1, K1, BCPAP and TT cells in respective media at a concentration of 2 × 10^6 and allowed to incubate overnight to attach to the plate. After 24 hours, each well was treated with a specific Y15 dose. After an additional 24 hours, the media from each well was collected and the number of unattached cells in each well was counted three times with a hemocytometer. The attached cells were collected with trypsinization and counted in the same manner. For each treatment condition the average percent of detached cells was calculated by dividing the number of detached cells by the number of total (attached plus detached) cells.Apoptosis assay was done with PE Annexin V Apoptosis Detection Kit (BD) to identify apoptotic nuclei. One hundred millimeter plates were seeded with TPC1, K1, BCPAP and TT cells at a concentration of 1 × 10^6 cells in normal growth media. After overnight incubation, plates were treated with one of several doses of Y15 or left untreated. After another 24 h of incubation, cells were collected with trypsin, and following centrifugation, were washed with 1× PBS. Again, cells were centrifuged, PBS was removed and cells were resuspended in binding buffer and filtered through BD mesh tubes. Annexin V and 7-AAD were added to the solution, vortexed and incubated for 15 minutes in the dark. Binding buffer was added and the cells were analyzed by flow cytometry within 1 hour.GraphPad Prism 6 software was used to graph cell viability data. Raw data was matched to nonlinear regression dynamic fitting curves for dose inhibition. Statistical analyses were done using this program and inhibitory concentration at 50% were calculated using 3 or 4 parameter variable slope log inhibitor response curves.6 cells of TT cells. After incubating 24 hours to allow cell attachment the plates were treated with 10 μM Y15 and 10 μM PF-04554878 in duplicate and two plates were left untreated. The plates were allowed to incubate for 24 hours and then the cells were collected. The cells were submitted for gene expression profiling to the gene microarray facility. HumanRef 8 whole genome gene expression array and direct hybridization assay (Illumina) was used to derive cDNA from 500ng total RNA. The Ambion Illumina Total Prep RNA Amplification Kit (Ambion) was used to make biotin-labeled cRNA via in vitro transcription of the cDNA. Illumina HumanRef-8 v3 Bead Chips were made through labeling and hybridizing probes at 58 ° C overnight and then the fluorescence of each probe was measured; the Bead Chips were analyzed with the Illumina Bead Array. The data were entered into the NCBI Database with GEO accession numbers GSE55619 and GSE55603.Microarray assay was used to detect the changes in gene expression between cell lines and with Y15 and PF-04554878 treatment. One hundred millimeter plates were seeded with 1×10The microarray data were analyzed by the R-based Bioconductor package. The expression intensity was converted to a log2 scale and then the Quantile normalization algorithm was used to normalize; according to the lumi model. Next, the limma program was used to calculate the degree of expression for each gene. A significant difference in gene expression was represented by p-value <0.05 and fold change at least 1.2.Student's t-test in Excel was used to calculate p-values for determining statistical significance between values for MTS, detachment, and apoptosis assays. Threshold for p-value was set to <0.05.At first, we compared the level of expression of FAK in untreated thyroid cancer cell lines Figure . The medIn order to investigate the effect of Y15 on autophosphorylated and total FAK expression Western blotting was performed with Y397-FAK and FAK antibodies in TPC1, BCPAP, K1 and TT cell lines. In each cell line Y15 inhibited pY397 and total FAK expression in a dose-dependent manner. TT was the most sensitive cell line with effective inhibition of pY397 expression by 3 μM Y15 Figure . TPC1 ceWe also tested the effect of the Pfizer inhibitor PF-04554878 (now VS-6063) on thyroid cancer cell lines to compare with Y15 inhibitor. In TT, TPC1, K1, and BCPAP cells PF04554878 did not change the expression of Y397 FAK and FAK as much as Y15 Figure . Thus, PNext we evaluated the effect of FAK inhibitor, Y15, on cell viability. MTS assay was completed using a range of Y15 doses on all cell lines .Y15 inhibited cell viability in a dose-dependent manner across all thyroid cell lines evaluated Figure . The med50 values of each cell line were calculated as described in Materials and Methods and are shown in Figure 50 values allow determination of sensitivity by cell viability of these cells to Y15: in decreasing sensitivity order TT, TPC1, BCPAP, and K1 that was consistent with decreased Y397-FAK levels by Y15 in these cells.IC50 concentrations were also calculated for PF-04554878. IC50 values for TT was 1.98, TPC1 was 23.04, K1 was 10.34 and BCPAP was >50 μM , clonogenicity assay was performed with Y15 treatment. Y15 caused decreased colony formation in each cell line in a dose-dependent manner Figure . K1 demos Figure . Thus, YIn addition, we tested the effect of PF-04554878 inhibitor on clonogenicity in TPC1 and K1 papillary thyroid cancer cells. In both cell lines PF-04554878 decreased colony growth. Complete colony inhibition was found in 6 μM and 8 μM in TPC1 and K1, respectively Figure . Thus, PNext, we analyzed cell death effects of Y15 in thyroid cancer cell lines by Annexin staining. Cells were treated with increasing doses of Y15 and apoptosis assay was performed with Annexin V/7AAD staining by flow cytometry. The necrosis increased in a dose dependent manner. As demonstrated previously in the MTS assay, the medullary cell line, TT, was most sensitive to Y15 treatment as seen in the lower doses of Y15 that were used. These results demonstrated an increase of cell death even at the lowest dose of Y15 (5 μM), inducing cell death in 45.33% of TT cells Figure . ConversIn order to detect and compare gene changes in response to Y15 treatment between cell lines, microarray assay was performed in untreated and Y15 treated papillary thyroid cell lines. The heat map of significant gene changes by Y15 in TPC1, BCPAP and K1 cell lines are shown in Figure Since Y15 and PF-04554878 had similar sensitivities in TT cells by MTT assay, but different levels of Y397FAK and FAK expression in response to these inhibitors Figure , we usedSince we detected different gene expression changes in response to Y15 and PF-04554878 inhibitor in TT cells, we tested the combination of Y15 and PF-04554878 for synergistic effect Figure . PF-0455Since we detected that the K1 cell line was more resistant to Y15, we used combination of Y15 with other targeted therapeutics approved for clinical trials  in this cell line . To inveFAK has previously been shown to be up-regulated in many cancers, including thyroid . Our labIn each thyroid cancer cell line FAK and Y397 phosphorylated FAK were expressed in different levels. In each cell line pY397 FAK expression decreased with Y15 treatment in a dose-dependent manner, but to different degrees, consistent with data in colon cancer . We obseThe degree to which each papillary thyroid cancer cell line decreased in cell viability with Y15 treatment was correlated with the levels of pY397 FAK. The higher decreased level of Y397-FAK and FAK correlated to a higher sensitivity to Y15 treatment. Therefore, TT was the most sensitive and K1 the least, with BCPAP and TPC1 showing intermediate inhibition of cell viability with Y15. Because our small molecule inhibitor targets the Y397 phosphorylation site, it is consistent that it would cause more of an effect with a relatively higher amount of phosphorylated FAK. The medullary cell line was highly sensitive to both Y15 and PF-04554878 suggesting different signaling versus papillary cell lines in this type of thyroid cancer. Two out of the three papillary cell lines, TPC1 and BCPAP, were more sensitive to Y15 than PF-04554878; the last papillary cell line, K1, was more sensitive to PF-04554878. This indicates the two FAK inhibitors effect cancer cells through a different mechanism. Y15 was also shown to increase detachment and decrease clonogenicity in each cell line in a dose-dependent manner. PF-04554878 decreased clonogenicity in two papillary thyroid cancer cell lines to a greater degree than Y15. This can be explained by a slower short-term mechanism of action for PF-04554878 that does not show as much effect in 24 hour experiments, but appears in a two week clonogenicity assay.The mechanism by which Y15 exerted these effects was shown to be through a combination of apoptosis and necrosis. Necrosis was detected at the highest doses, but a lower highest dose likely would have shown continued increase in apoptosis. Likely at the highest dose, the necrosis is induced as was shown in breast cancer cell line BT474 . Y15 incTo investigate the mechanism of Y15 in the papillary thyroid carcinoma cell lines a microarray gene profile was obtained and compared between cell lines. There were some gene overlaps between genes changed with Y15 in the three cell lines, but none over 4-fold in all three lines. This indicates that each cell line has different protective mechanisms in response to Y15 treatment or the common responses induce small gene changes. BCPAP, TPC1, and K1 cell lines are genetically distinct cell lines from papillary thyroid tumors with varying mutations . For exaA member of the heat shock family, HSPAIB, was up-regulated 3.32 fold in TPC1 cells. Heat shock proteins are a major part of the stress response and were previously also found to be increased with Y15 treatment in colon cancer cells . This inTo compare and contrast the mechanisms of action for Y15 and PF-04554878 we identified common and specific genes changed by both in the medullary thyroid TT cell line. Multiple kinesins were down-regulated by both drugs, including KIF11, KIF20A, and KIF 4A. Kinesins are involved in multiple cellular processes, including mitosis and intracellular transport, and drugs targeting kinesins are in clinical trials for treatment of cancer . MultiplBoth Y15 and PF-04554878 also up-regulated encoding of AEN, apoptosis enhancing nuclease, in TT cells; Y15 by two-fold and PF-04554878 by three-fold. AEN is directly induced by p53 and subsequently acts to induce apoptosis . Up-reguAlso among genes involved in apoptosis, GADD45A, GADD45B, and GADD45G, were up-regulated with Y15 in the TT cell line. These genes were up-regulated significantly 2.73 to 3.42 times, while they were not changed significantly with PF-04554878 treatment. GADD45G is similarly decreased in anaplastic thyroid cancer cells and expression of the gene induces apoptosis . GADD45 The expression of BIRC5 gene that encodes survivin was decreased with Y15 treatment. BIRC5 was decreased 0.49-fold in Y15 treated TT cells, while it was statistically significantly increased 1.26-fold by PF-04554878. Survivin is expressed highly in thyroid cancer tumors and associated with metastasis and a worse prognosis [22]. Survivin blocks both the mitochondrial apoptosis pathway and the death receptor pathway for an anti-apoptotic effect. Again, this indicates Y15 works through multiple genes that PF-04554878 does not to induce apoptosis. These genes changes signify Y15 and PF-04554878 work through a distinct mechanism, but the similar classes of genes changed and shared genes could explain the synergy with combination of the drugs.We also demonstrated synergy of Y15 and PF-04554878 in TT cells. PF-04554878, now known as Defactinib, is currently undergoing clinical trials in multiple cancers including mesothelioma, ovarian and lung. PF-04554878 was shown to decrease tumor volume in a chemotherapy-resistant ovarian tumor model through decreased AKT and YB-1 phosphorylation . FAK is In this report, for the first time, synergy was demonstrated with Y15 and Cabozantinib, Sorafenib, Pazopanib, and Sunitinib in the most resistant cell line, K1. Papillary thyroid cancer cell line K1 has a BRAF mutation, a PI3KCA mutation, and a p53 mutation . The preThis study indicates that FAK inhibition through Y15 causes dose-dependent decreases in cell viability, clonogenicity, and attachment through an apoptotic and necrotic mechanism that synergizes with current and investigational therapeutics and this inhibition is related to the relative amount of active FAK in each cell line. The study identified common and unique pathways in response to Y15 in three cell lines and detected differences and common pathways between Y15 and PF-04554878 inhibitor. Future experiments are needed to further characterize the mechanism through which Y15 causes its apoptotic effects.


Here we used a well-characterized panel of lung cancer cell lines to develop the most comprehensive view of cancer cell metabolism to date.Cancer cells display oncogene-driven rewiring of metabolism to produce energy and macromolecules for growth. Inhibition of growth-promoting metabolic pathways may prove to be a useful therapeutic strategy in cancer. We previously identified distinct metabolic platforms that enabled cancer cells to produce macromolecular precursors from glucose and glutamine, the two most abundant nutrients. However, neither the full breadth of cancer cell metabolic diversity, nor the complement of mechanisms by which tumor mutations elicit metabolic reprogramming, are known. Because metabolic flux can be analyzed in vivo.81 non-small cell lung cancer cell lines were analyzed for a set of well-defined metabolic parameters. These cell lines were also subjected to extensive genomic, epigenetic, and gene expression analysis, and tested for sensitivity to chemotherapeutic agents and genome-wide siRNA screens. These rich data sets will streamline the establishment of novel correlations between metabolism and molecular/cell biological features. All cell lines were characterized for nutrient utilization, nutrient addiction, and focused flux assays to trace the metabolism of isotope-labeled glucose and glutamine in identical culture condition. Ongoing experiments are assessing metabolism of the same cells grown The 81 cell lines displayed surprising metabolic heterogeneity. Although all cells used glucose and glutamine to produce biosynthetic precursors, contributions of these two nutrients varied considerably, as did the pathways used. For individual isotopic labeling patterns, differences of >30-fold were observed across the panel. Unsupervised clustering produced two metabolic superfamilies differentiated by relatively glucose carbon contribution into precursor pools, as well as numerous subfamilies which may correlate to different metabolic pathways. Although KRAS-mutant cells were distributed across panel, the LKB1/KRAS co-mutation cells aggregated into the low-glucose m0 superfamily. Ongoing work will examine the ability of metabolic phenotyping to predict combinatorial mutations and vulnerabilities to drugs and siRNAs.Focused metabolic assays can produce a highly informative view of the metabolic phenotyping among large panels of cell lines. It describes an unparalleled view of the connections between genetics, drug sensitivity and cell-autonomous metabolism in NSCLC.


Determination of response to chemotherapy is a major requirement of personalized medicine. Resistance, whether developed or native, critically affects a treatment’s success. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer.We treated human breast and colon cancer cell lines with melphalan, cisplatin, mechlorethamine or doxorubicin, as monotherapies. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types.The comet assay technique can provide reliable and quick results with minimum requirements and is applicable to a wide variety of drugs. Personalized medicine requires that therapy should be customized to individual patients, using genetic or other information . PredictSingle Cell Gel Electrophoresis (SCGE), also known as comet assay can measure DNA damage in individual eukaryotic cells. The principle of the comet assay is that unfragmented DNA maintains a well-organized structure in the nucleus, but becomes disrupted when the cell is damaged. It detects both single-strand and double-strand breaks, and has a simple and inexpensive setup. Comet assay is therefore a promising technique for predicting response to drugs that are affected by DNA structure .The present study evaluated the predictability of response to widely used chemotherapy drug from comet assay results in established human breast and colon cancer cell lines. We tested nitrogen-mustard alkylating agents  and doxorubicin in breast cancer, and tested cisplatin in colon cancer cell lines.p set to be 0.05, we estimated the ranges of DNA percentage in untreated and treated cells; where these ranges overlapped, the cells were rejected. In the MCF-7 and MDA-MB 231 cell lines, we clearly observed functional activity for all drugs. The T47D cells showed functional activity for melphalan and doxorubicin, but not for mechlorethamine. All the colorectal cancer cell lines except LoVo, were also successfully affected  and in “tail” . We examined ≥ 100 cells for each combination of cells and drugs. With Comet visual scoring can be also calculated based on a five-grade scale from 0 to 4. In a grade 4 comet cell, the entire DNA is in the “tail”, while in grade 0 the entire DNA is in “head”. These values given to comets can be summarized and provide a quantitative measure for 100 cells on a scale from 0-400. These data are presented in Figure Breast cancer is the most lethal malignancy in women . ColorecMelphalan in combination with other drugs, or as monotherapy, is highly active in patients with advanced breast cancer. It attaches an alkyl group to the 7′ nitrogen atom of the imidazole ring, in the DNA guanine base. Melphalan has several side effects including leucopenia, thrombocytopenia and anemia, nausea, vomiting and diarrhea . MechlorMany techniques to predict chemotherapy response are available. Assays based on PCR can show genes for enzymes that affect metabolism of these drugs, and can therefore indicate response to a given agent , 21. HowThis particular work contains numerous cancer cell lines, which represent two widespread types of cancer. For each cancer type cell lines with different properties were tested. Also there is a study of several most common chemotherapeutic drugs.The experimental data demonstrated that personalized treatment is essential in cancer treatment. The data from colon cancer cell lines indicated that cisplatin does not affect each cell line to the same grade. Also, in some cases, like LoVo, the cisplatin is probably ineffective. Between HCT-15 and LoVo, which both represent colon adenocarcinoma, completely different data were observed. However, it is remarkable that LoVo derived from a metastatic tumour. Recent literature data, indicate that cisplatin in combination with other agents is more effective than monotherapy in this particular cell line .Among the breast cancer cell lines, the T47D cells carry receptors for a variety of steroids, while MCF-7 cells express oestrogen receptors. The variety between these receptors may explain the different response to chemotherapy. MDA-MB 231 established from a pleural effusion of a 51 year old woman with metastatic breast cancer, indicating that mechlorethamine might be more effective in metastatic breast tumour.Comet assays are a sensitive means of detecting both single strand and double strand DNA damage, using only a few cells. Our results here reinforced the heterogeneity of cancer by showing how the effectiveness of each drug varied in each cell line, even when derived from the same cancer type. Potentially, comet assays could allow physicians to evaluate patient response in advance, thus avoiding inappropriate therapies with undesired effects. SCGE is a relatively quick versatile, simple-to-perform technique with a few requirements.Tailor-made therapy is a promising theme in translational cancer research. However, identifying optimal treatment for an individual patient can be difficult, time-consuming and expensive. Here, we evaluated the effectiveness of comet assays on four commonly used chemotherapeutic drugs, and found that this technique can predict responses of different cancers to the subject drugs and generally to drugs with the same mechanisms of action. Further studies with a wider variety of tumor cell types and drugs are required to establish the clinical utility of comet assays in selecting cancer treatments.2 flasks  at 37°C in a 5% CO2 atmosphere, in the recommended media supplemented with the appropriate amount of heat-inactivated fetal bovine serum  and 2 mM L-glutamine .We used human breast cancer cell lines MCF-7, MDA-MB 231 and T47D, and colon cancer cell lines HCT-116, HT55, HCT-15 and LoVo, which came from the European Collection of Cell Cultures . Cells were cultured in 75 cmAccording to literature data, the optimal concentration of each drug varies by cell type and cancer type. Cells were cultured in 12-well plates  in different concentrations (0.01-50 μM) for each drug. Their viability was analyzed with propidium iodide staining after 24 hrs of incubation . Concentrations that were chosen for further experiments were 0.1 μM for mechlorethamine , 1 μM for melphalan , 1 μM for doxorubicin  and 1 μM for cisplatin .2 flasks , one for each drug and one with no drug added. After 24 hrs of incubation, cells were detached by trypsinization  and 20000 cells were used for single cell gel electrophoresis with IKZUS comet assay kit . Alkaline lysis of the cells was performed according to manufacturer’s instructions. Slides were observed under a UV microscope. Data were analyzed with Comet Score software .Cells were divided into 25 cm