V600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human Braf Using naturally occurring models of animal disease to study complex diseases, such as cancer, allows researchers to better translate their findings, as genetically engineered mouse models have severe limitations when attempting to translate therapy to human patients. Canine IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human cancer, 12. We Human cancer cell lines were maintained in DMEM  or RPMI (Colo829) (Gibco) supplemented with 10% FBS according to recommendations from ATCC. Canine melanoma cell lines CMM3 and CMM5 were obtained from one of the author’s laboratory (MSK), from where they were derived, have been validated to be of canine origin with no inter-species contamination (IDEXX), have been tested to be free of mycoplasma contamination (AZ) and were immunologically typed as being positive for the following melanoma specific markers: Melan-A, S-100, MITF and Tyrosinase. They weGSK1120212 (Trametinib) was purchased from SelleckChem (#S2673) and resuspended in DMSO as per manufacturer recommendations to a stock of 10mM. WW and Scr peptide were obtained from CS Bio Co., manufactured as described previously and reconstituted to a stock of 2.5mM in water[Four primary canine mucosal melanomas and two samples of normal canine oral mucosa were obtained from biopsy tissue collected under IACUC approved protocol 18749 at the University of California Davis School of Veterinary Medicine.5’-GCAATTATTCCCCATGAACG-3’ and reverse, 5’- GGCCTCACTAAACCATCCAA-3’;Human 18s: forward, 5′-TTCGCCACTACCCAGACCTTGTTT-3′ and reverse, 5′-CCTGTCTTGGATGTGGCCTTTGG-3′;IQGAP1: forward, 5’- AAGGACCCCTCGAAGTGTTG-3’, reverse 5’- ACTAAGCAGATGGCCACAGAA-3’Canine HPRT: forward, 5’- AGAAGTGGCCCAGCATTACC-3’, reverse 5’- CAGCTGACTCGTCCTGTGTT-3’Canine IQGAP1: forward, RNA was harvested from melanoma cell lines by treatment using the RNeasy kit (Qiagen). The following primers were used for this study:RT-PCR conditions: 95°C for 5min, 45 cycles of 95°C for 10s, 58°C for 30s, 72°C for 30s. RT-PCR was performed on a Roche Lightcycler 480.For CRISPR-Cas9 infections, cells were infected with PLenti-CRISPR virus with four sgRNAs targeting IQGAP1. sgRNA sequences were based on published sequences for human IQGAP1 sgRNAs and alteAGGACATTCGGAATCAGCGGIQ1: TGGTTGGATGAAATTCAAGGIQ4: GATGGAGTCTCAGACGGGAGAIQ5: GATGGAGTCTCAGACGGGAGAIQ7: sgRNA guide sequences:Immunoblots were performed with the following antibodies: rabbit antibody to ERK1/2 , rabbit antibody to pERK1/2 , rabbit antibody to IQGAP1 , rabbit antibody to RSK1 , rabbit monoclonal antibody to phospho-p90RSK , and mouse monoclonal antibody to β-actin .Cell lines were grown on chamber slides, fixed and permeabilized by incubation 4% paraformaldehyde for 10 minutes followed by blocking in 10% horse serum in PBS for 1 hour. Tissue sections were then incubated with primary antibodies, followed by Alexafluor-488 conjugated goat anti-mouse secondary antibodies (Molecular Probes). The following antibodies were used: IQGAP1 . Tissue was counterstained with Hoescht to visualize cell nuclei.Immunohistochemistry was performed as previously described with mouPercent control growth: 100% * (ATREAT − ACONini) / (ACONfin − ACONini)Percent standard error of the mean: 100% * (SEMTREAT/(ACONfin − ACONini)),where the SEM = (SDEV/ n ^ 0.5), n = number of replicates).Cancer cell lines were seeded in equivalent, low-density cultures in duplicate in 24-well plates. Twenty-four hours after seeding, the medium was removed and replaced with 500μl of a 5:1 mixture of cell medium and cell titer blue reagent (Promega). Cultures were incubated with this mixture for 2h at 37°C in the dark. Fluorescence (560nm/590nm (emission/excitation) for 100 μl in triplicates for each sample was recorded. Values plotted correspond to readings taken while control samples were still growing exponentially. For IC50 assays, cells were seeded into 96-well plates. Twenty-four hours after seeding, serial dilutions of GSK1120212 were prepared in DMSO and added to cells, yielding final drug concentrations ranging from 0 to 30μM. Cells were treated daily and incubated for 72h after the addition of drug. Data from growth-inhibition assays were modeled using a nonlinear regression curve fit with a sigmoid dose response. Cell proliferation curves were generated graphically as % control +/- SEM using the following formula:50) values were generated using Prism 5 (GraphPad).Half-maximal inhibitory concentration  from previously non-treated animals and normal canine mucosal samples for comparison (n = 2) we performed IQGAP1 immunohistochemistry . NegativV600E mutated melanoma line (SK-Mel-28) for comparison are presented in We initially determined that two canine melanoma cell lines responded to ERK1/2 MAPK inhibition similarly to human melanoma cell lines using GSK1120212, a specific MEK1/2 kinase inhibitor that is currently being tested in human cancer clinical trials. These sV600E melanoma cell lines. We compared the RNA and protein expression of canine and human IQGAP1 in our cell lines compared to a panel of human BrafWe next examined ERK1/2 phosphorylation in the context of IQGAP1 depletion in the canine melanoma line that was most sensitive to MEK-inhibition (CMM5). We utilized Cas9-CRISPR targeting IQGAP1 and one of the same sgRNAs we used for the human lines as the target sequence was identical. Immunofluoresence studies indicated that there is a reduction in IQGAP1 protein expression . The proWe assessed proliferation in both canine melanoma cell lines with IQGAP1-targeting Cas9-CRISPR and note a consistent and statistically significant decrease compared to empty vector (EV) controls using four different sgRNAs in three biological replicates . FinallyThis study explores the utility of expanding IQGAP1 targeted therapies to canine models of melanoma. The advantage of characterizing this novel therapeutic target in an alternative cancer model is the ability to better predict and characterize potential human responses to therapy compared to performing pre-clinical testing in conventional rodent models. Treating naturally occurring cancers better models the clinical condition of human patients.For these studies, we use canine mucosal melanomas as these are the most common canine melanoma presentations and currently available cell lines are of mucosal origin. These tumors differ from human cutaneous melanomas in that Braf and Nras mutations are not frequently observed. This is similar to what is found in human mucosal melanomas as well. However, canine mucosal melanomas are reportedly sensitive to MAPK targeted therapies and have upregulated levels of pERK1/2, suggesting that these tumors are reliant on MAPK signaling for tumorigenesis. It is unknown if there are mutations elsewhere in the ERK1/2 MAPK pathway or if the increased pathway activation is primarily due to increased expression of pathway components.We initially characterized the presence and localization of IQGAP1 in primary canine mucosal melanomas, observing that there does appear to be increased IQGAP1 expression, particularly co-localizing with melanocytes as well as at the tumor edge. We additionally examined the expression and localization of IQGAP1 in two canine mucosal melanoma cell lines. While there seems to be a modest difference in the absolute expression of IQGAP1 between human and canine melanoma cell lines (human cell lines express higher levels), the localization to the cytoplasm and the cell periphery is very similar based on immunofluorescence. It is important to note that the antibody used for the immunohistochemistry studies was initially validated in cell lines with IQGAP1 knockdown using CRISPR Cas9 to determine specificity.V600E mutant melanoma lines to up to 174nM for Kras mutant lines[Based on these observations, we then sought to confirm that the cell lines were sensitive to MAPK targeted therapies, as this has generally been a good predictor as to which lines will also respond to IQGAP1 targeted therapy. Western blot analysis, as well as IC50 curves, confirmed that the lines respond to MEK inhibition with a downregulation in pERK1/2 and a significant decrease in proliferation. The calculated IC50 values are higher than for a human melanoma cell line used here for comparison, but still within the range of sensitive lines. It is interesting to note that there is an almost 50-fold increase in IC50 between the human melanoma cell line and the more resistant of the two canine lines tested. Reported IC50s range from 0.48nM to 0.52nm for Brafant lines. MAPK wiant lines.Using a pLenti CRISPR Cas9 system, we were able to successfully deplete the expression of IQGAP1 protein in a bulk population of cell line CMM5 (the more sensitive of the two lines tested). To the authors’ knowledge, this is the first time that CRISPR Cas9 has been used to delete genes in canine cancer cell lines. We confirmed knockdown using immunofluorescence as well as Western blot. The lines in these experiments still exhibit some protein expression because these are mixed cell populations and do not represent clonal populations. In these mixed population cells, we observed an approximately 50% reduction in pERK1/2 compared to WT cells, suggesting that we are, indeed, impacting ERK1/2 MAPK function. In CMM cells with IQGAP1 CRIPSR, we notice a consistent and statistically significant reduction in proliferation.Finally, we performed proliferation assays to determine if treatment with the WW peptide would result in decreased proliferation in the canine lines. This appeared to be a viable treatment strategy based on the observation that the WW domain of IQGAP1 is the same between canines and humans except for two residues in the C-terminal end of the protein. We have demonstrated in human keratinocytes that the four C-terminal residues are not critical to reducing signaling through IQGAP1-ERK1/2. We noted a significant decrease in proliferation with the WW treated lines compared to Scr treated controls. Interestingly, we noted that WW peptide treatment has a greater absolute effect on proliferation combined with the partial knockout using IQGAP1 CRISPR. There may be several explanations for this observation, including incomplete deletion of IQGAP1 in this pooled CRISPR population, potential effects of IQGAP1 deletion on additional signaling pathways, effects WW peptide treatment on other ERK1/2 interactions or other “off-target” effects that have not been fully characterized.These data together provide a preliminary justification for investigating the use of the WW peptide in canine mucosal melanomas, as a model of human disease and for the purpose of treating canine patients. The findings in this manuscript further support canine oral melanoma model as a relevant spontaneous research model for human melanomas.S1 Fig(A) Immunohistochemistry of a canine mucosal melanoma sample from same sections as in (TIF)Click here for additional data file.S2 Fig(A) Quantitation of the qPCR based on Ct values, with house-keeping genes for human and canine shown for comparison. (B) Amplification curves for two canine melanoma lines (CMM) and five human melanoma lines (HM). Housekeeping genes TBP and HRPT1 were used for the CMM lines and 18s was used for the HM lines.(TIF)Click here for additional data file.


Glioblastomas (GBM) are the most common malignant type of primary brain tumor. GBM are intensively treated with surgery and combined radiochemotherapy using X-irradiation and temozolomide (TMZ) but they are still associated with an extremely poor prognosis, urging for the development of new treatment strategies. To improve the outcome of GBM patients, the small molecule multi-kinase inhibitor sorafenib has moved into focus of recent research. Sorafenib has already been shown to enhance the radio- and radiochemosensitivity of other tumor entities. Whether sorafenib is also able to sensitize GBM cells to radio- and chemotherapy is still an unsolved question which we have addressed in this study.The effect of sorafenib on signaling, proliferation, radiosensitivity, chemosensitivity and radiochemosensitivity was analyzed in six glioblastoma cell lines using Western blot, proliferation- and colony formation assays.In half of the cell lines sorafenib clearly inhibited MAPK signaling. We also observed a strong blockage of proliferation, which was, however, not associated with MAPK pathway inhibition. Sorafenib had only minor effects on cell survival when administered alone. Most importantly, sorafenib treatment failed to enhance GBM cell killing by irradiation, TMZ or combined treatment, and instead rather caused resistance in some cell lines.Our data suggest that sorafenib treatment may not improve the efficacy of radiochemotherapy in GBM. Glioblastoma multiforme (GBM), a high-grade glioma (WHO grade IV) is the most common and lethal primary malignant brain tumor in adults, with a median survival of only 16 months. Despite current intensive therapy regimes including surgery, radiotherapy (RT) and temozolomide (TMZ)-based adjuvant chemotherapy (CT), disease progression occurs in almost all patients . TherefoThis includes the promising approach of using the multi-kinase inhibitor sorafenib. Sorafenib has been shown not only to block the members of the MAPK pathways Raf-1 and p38 but also receptor tyrosine kinases like VEGFR, cKit or PDGFR  and it iBecause of the importance of sorafenib for current targeted therapy approaches and the lack of solid data on the effects of sorafenib on X-irradiation and TMZ in GBM we investigated in this study the potential of sorafenib to radiosensitize and chemosensitize GBM cells. This study was performed using six individual GBM cell lines with differences in the p53 status, because the p53 status is known to be important for cell survival. Furthermore, we only used O6-methylguanine-methyltransferase (MGMT) negative cells since the TMZ sensitivity is known to depend strongly on MGMT status .To test if sorafenib is a potential therapeutic drug to improve radio-chemotherapy of GBM we wanted to analyze the influence of sorafenib on cellular radio- and chemosensitivity in various GBM cells lines. To this end, we chose the colony-forming assay, because this assay is able to directly measure the ability of tumor cells for self-renewal (clonogenicity). This is of special importance since effects on proliferation or metabolism might not truly reflect cell inactivation but could be also be caused by prolonged growth arrest. Withdrawal of the inhibitor, re-stimulating events or extended culture times might lead to a restart in growth of solely arrested but not truly inactivated cells.Because the colony forming assay can be influenced by the proliferation rate we first investigated the effect of sorafenib on cell proliferation. In these experiments we observed a strong decrease in the proliferation rate for all cell lines in a concentration-dependent manner Figure .nd day on    Figure . However) Figure .Taken together these results demonstrate that, although sorafenib inhibits proliferation of GBM cells and causes a modest cell inactivation as a single agent, it does not enhance cell inactivation by irradiation, TMZ or combined treatment.In this study, we analyzed the impact of sorafenib on cellular radiosensitivity, chemosensitivity (TMZ) and on the combined treatment of GBM cell lines. To consider potential variations which might affect the outcome of this study, we analyzed six individual GBM cell lines with differences in p53 and PTEN status, but all negative for MGMT expression .Surprisingly, we observed no impact of sorafenib on cellular radiosensitivity in any of the cell lines. Instead, there was a trend towards radioresistance Figure . This reOur results do not agree with data from former studies reporting an effect of sorafenib on the radiation response of GBM cell lines , 23. HowSimilar to the cellular radiosensitivity results we also detected no chemosensitization by sorafenib in combination with TMZ, but again a trend towards resistance Figure . The resSo far there are no other preclinical data available addressing this question. However, in combination with other drugs sorafenib was reported to enhance cytotoxicity in GBM cells but in these studies cellular survival was not analyzed in terms of clonogenicity –30. MoreAlthough we demonstrate here, that sorafenib does not increase radiation- or TMZ-induced cell inactivation we observed a strong inhibition of proliferation which might translate into reduced tumor growth. But when sorafenib was removed after 24 h, proliferation restarted, resulting only in a minor delay of cell growth and a small reduction in clonogenicity for most of the cell lines Figure , which wSorafenib also had no positive effect on the double treatment with irradiation and TMZ. In fact, it instead made two of the six cell lines more resistant, which was significant for Cas-1 cells. These data are in line with recent clinical studies testing sorafenib in the first-line treatment in combination with TMZ after RCT (maintenance therapy) or in combination with TMZ and radiotherapy. These studies also do not support these treatment combinations because they did not seem to improve the efficacy of the treatment but caused increased side effects , 34.Taken together, we have systematically demonstrated here for the first time that for GBM sorafenib induces only minor cell inactivation as a single treatment and has no benefit in respect to cell inactivation when combined with TMZ and/or irradiation. Moreover, sorafenib seems to cause resistance towards TMZ-based RCT as detected by increased cell survival in some cell lines. Therefore, our data do not support the use of sorafenib for GBM with the intention to increase cellular inactivation by RT or TMZ-based RCT.®, Bayer HealthCare), alkylating agent temozolomide , solvent DMSO (Sigma-Aldrich).Small molecule inhibitor sorafenib . Gene sequencing and literature search revealed p53 wildtype expression in LN229, U87MG and DKMG cells and expression of mutant p53 in CAS-1, U251 and BS153 cells. Wildtype PTEN was detected only in LN229 cells  as indicated and the cell number was determined in parallel cultures by counting trypsinised cells using a Coulter counter (Beckman Coulter GmbH) every day henceforward.To analyse proliferation, 1×10Cells were irradiated at room temperature with 200 kV X-rays .® CLx Infrared Imaging System (LI-COR) was utilized for signal detection and quantification.Proteins from whole cell extracts were detected by Western blot according to standard protocols. Primary antibodies: anti-ERK, anti-pERK (T202/Y204), anti-MEK1/2, anti-pMEK1/2 (S217/S221), anti-MGMT  and anti-β-actin (Sigma-Aldrich). Secondary antibodies: anti-mouse and anti-rabbit (Li-COR Biosciences). The OdysseyTo analyse the capacity of the GBM cells for self-renewal (clonogenicity), the colony-forming assay was used. To this end, 200-350 cells (depending on the cell line) were seeded in triplicates 24 h prior to treatment with sorafenib (5 μg/ml), TMZ (10 μM) and/or irradiation. Twenty-four hours after treatment the medium was changed and TMZ was added to the corresponding samples for additional 1-2 days according to the doubling time of each cell line . The celThe experiments were repeated at least three times. The data were presented as mean values (±SEM). Prism software (GraphPad Prism 5) was used for analyzing and graphing the data. Student's t-tests were performed for the statistical analysis. P-values were calculated using unpaired two-sided tests .


Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation.Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis.In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines.Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells. Glioblastoma (GBM) is the most common and biologically aggressive primary brain malignancy in adults, with a median survival of 14.2 months. Current treatment comprises of maximal surgical resection followed by radiotherapy with concomitant temozolomide (TMZ), irinotein vitro.Glioma stem-like cells (GSCs), a multipotent, self-renewing subpopulation of cells within the tumor microenvironment, are believed to be responsible for disease recurrence. These cells are resistant to both radiotherapy and chemotherapy and are in vitro model must be proven in order for it to be useful in the clinic.High content screening (HCS) allows phenotypic changes in cell morphology to be rapidly assessed and analyzed and the resulting multi-parametric readout allows for the build-up of a more comprehensive picture of cell fate when exposed to drug treatments. In the short-term, combining a drug repositioning strategy with HCS could prove a useful tool for matching the best currently available chemotherapy regimen to a particular patient; however, a standardized and physiologically relevant Previous small-molecule screens using GBM cell lines have relied on adherent culture and immortalized cell lines to achieve the standardization needed for such large-scale assays, 9. Throin vivo and have the potential to be rapidly entered into clinical trials.We have therefore established a 384-well high content screen that enabled us to assess the chemosensitivity profiles of patient-derived cell lines in both monolayer and neurosphere culture against a panel of 83 chemotherapeutics, with and without irradiation. Our study puts forward several non-standard chemotherapeutics that show efficacy Patients with pre-operative imaging characteristics in keeping with glioblastoma were identified through the neuro-oncology multi-disciplinary meeting at Salford Royal NHS Foundation Trust. Informed consent was obtained from patients for tissue use, storage and derivation of cell lines. All work was performed with NHS research ethics committee approval (Ref. 68/H1006/38 and 08/H1006/37). Patient demographic features are summarized in In vivo drugs were purchased from Sigma Aldrich, USA and dissolved in DMSO at a stock solution of 80mM and diluted further in sterile saline for administration as indicated. Cell viability experiments were performed via the fluorescent dyes Hoechst 33342  and DRAQ7  and fixed with 1% paraformaldehyde solution .Eighty-three chemotherapeutic drugs  were purPatient cell line derivation was in accordance with Pollard et al.. DissociNeurospheres were cultured over 48 hours then fixed in 4% PFA; 0.1% Triton-X. Neurospheres were then dehydrated through a methanol gradient and incubated in 100% methanol overnight, then rehydrated via the same methanol gradient. Neurospheres were then blocked in 3% fish skin gelatin; 0.1% Triton-X for four hours. Primary antibodies were used at the following dilutions: Nestin 1:1000 (Abcam) and Sox-2 1:250 (Abcam). Secondary staining was performed using Alexa-Fluor 488 and 594 secondary antibodies (Invitrogen), followed by nuclear counterstaining with DAPI (300mM).Images were collected sequentially on a Leica TCS SP5 AOBS upright confocal using a 20x / 0.50 Plan Fluotar objective and 3x confocal zoom. When acquiring 3D optical stacks the confocal software was used to determine the optimal number of Z sections. Only the maximum intensity projections of these 3D stacks are shown in the results.Immunohistochemistry was performed on free floating sections treated with 10% goat serum for 2 hours at room temperature, and then stained with primary antibodies for GFP 1:1000 (Abcam), vimentin 1:200 (Abcam) and human nuclear antibody 1:2000 (Abcam). Primary antibodies were detected using the appropriate secondary antibodies.Patient cell line TS18 was stably transfected with a lentivirus containing an eGFP cassette driven by the SFFV promoter. Transdu4 cells / ml in supplemented NBM +/- laminin. Cells were then dispensed using a Multi-drop liquid dispenser into black 384-well clear, flat bottom plates to achieve a seeding density of 1 x 103 cells in 40μl per well. Neurosphere plates were then incubated for 7 days at 37°C in a humidified incubator with 5% CO2, whilst laminin-treated plates were incubated for 48 hr. under the same conditions. U87 cells were seeded in complete DMEM for adherent culture and NBM (- laminin) for neurosphere culture.Cells were grown to confluence then disaggregated and diluted to 2.5x10Eighty-three chemotherapeutic agents were dosed on to cells according to a 5-point log dosing strategy  using a liquid handling robot (Hamilton Star) and drugs dosed in quadruplicate in a 2x2 pattern. Each plate included 14 positive  and 14 vehicle  controls. Half the plates were then exposed to a single dose of irradiation in a biological irradiator . Plates were then incubated for 96 hours at 37°C in a standard tissue culture incubator before incubation with the fluorescent dyes Hoechst 33342 (1:500) and DRAQ7 (1:1000) for 30 minutes at 37°C. Cells were then fixed in 1% paraformaldehyde (PFA) solution.Image acquisition was performed using the Cellomics ArrayScan VTI HCS Reader (Hamamatsu ORCA®-ER camera with x5 objective lens) and images analyzed using the Cellomics package software . The ArrayScan software algorithm was used to perform compartmental analysis and objects below a certain size excluded from analysis. Regions of Interest (ROI) were identified using the software. DRAQ7fluorescence and ROI were overlaid to determine the amount of DRAQ7 associated with each neurosphere or cell. Hoechst dye incorporation was counted by the software and used count cells and neurospheres.NOD-SCID Gamma Null (NSG) mice between 8 and 12 weeks of age were used in this study. Mice were housed in individually ventilated cages with ad libitum access to food and water, and were kept in a 12-hour light/dark cycle. Female mice were used in this study and housed in groups of 2–5. All procedures were ethically approved by the University of Manchester Ethical Review Process under UK Home Office regulations and project license PPL 40/3658.5) were stereotactically injected into the right striatum of NSG (n = 4 per cell line). Mice were sacrificed when they became symptomatic or suffered significant weight loss (>20% weight loss over 24 hr). After trans-cardial perfusion, brains were harvested, fixed in 4% PFA and cryoprotected with 20% sucrose. Coronal sections were taken with a Hyrax M25 rotary microtome  and prepared on glass slides for immunohistochemical staining.TS1-GFP, GS1, TS9 and TS18-GFP cells (2.5x106). Palpable tumors were established approximately 10 days after cells were injected. Tumors reached 200mm3 approximately 18 days post injection, at which point drug treatment was initiated.Female NSG mice, aged ≥8 weeks, were implanted with subcutaneous xenografts using U87-MG cancer cells  as well as a non-treated control group. Dose, drug delivery method and scheduling of each drug was in accordance with accepted published values to maintain sub-maximal tolerated doses in mice–24.To monitor tumor growth, xenografts were measured (major and minor axis) every two days using calipers, and tumor volume was calculated using the equation:Where a = width and b = length of the tumor. Mice were culled seven days post-treatment and tumors excised. Final tumor volumes were measured and plotted against controls.For monolayer assays cell death was defined as the percentage of cells with DRAQ7 positivity. Defined by setting an upper “circ spot” intensity using the Cellomics compartmental analysis algorithm . Spheroid count was defined as the number of filtered particles identified in neurosphere culture through compartmental analysis. The three-dimensional nature of the neurospheres was taken into account by normalizing the total DRAQ7 intensity against spheroid cross-sectional area. Due to variation and non-normal distribution, the values were incremented by 1 and log transformed. Therefore, cell death in neurosphere assays was defined as:For response space analysis, the average log cell death and spheroid count was calculated across all concentrations for each compound. The y-axis represents the difference in cell death between drug and negative control. On the x-axis, differences were expressed as fold change over DMSO as they represented relative rather than absolute differences over DMSO. 95% confidence limits were calculated using Spotfire  and data collated and visualized using Microsoft Excel . pEC50 values were extrapolated from unambiguous dose-response curves generated from DRAQ7 raw data. Dose response curves, heat maps and Bland-Altman plots were generated using Prism .in vivo . After 96 hours incubation with drug, with or without irradiation, cells were fixed and stained with DRAQ7 and Hoechst prior to imaging and data collection.pEC50 values were generated via percentage cell death or log cell, based on DRAQ7 positivity. DRAQ7 does not cross the membrane of viable cells and thus is a marker of cell membrane permeabilisation, apoptosis, necrosis and dead cells. A heat map was populated using the best-fit pEC50 values to compare the four assay models . A full TS1 was the most sensitive cell line in adherent culture, with 78.3% of drugs with a pEC50 corresponding to less than 100 μM without irradiation, and 60% after a single 5Gy dose. TS9, our one recurrent line, was resistant to most compounds tested in all assay formats, with only 21% of drugs generating a pEC50 below 100 μM over all four assays. This low percentage most likely reflects the fact that the patient the cells were derived from had both first line irradiation plus Temozolomide (TMZ), followed by PCV combination therapy at recurrence.The more commonly used GBM chemotherapeutic compounds, including temozolomide with radiotherapy, showed varying success across cell lines in our screen. None of our assays indicated >10% efficacy at 10μM in TS9 or U87, therefore no pEC50 data could be generated. This lack of response in the U87 cell line is consistent with previous studies , 25, wheVincristine demonstrated good efficacy across all patient-derived lines, with the exception of TS9. GS1 was particularly sensitive, with pEC50s corresponding to low nanomolar efficiency in both monolayer and neurosphere assays. Lomustine and procarbazine were less efficacious alone. Irinotecan, another common second line option, was much more successful, with the majority of drugs demonstrating cytotoxicity in the low micromolar or nanomolar range.In addition to irinotecan, other topoisomerase inhibitors were cytotoxic across the majority of cell lines, with the most consistently efficacious drug being mitoxantrone. pEC50s were obtained in each cell line, ranging between 2.7nM and 87.5μM. Indeed, along with tubulin and proteasome inhibitors, all sub-nanomolar pEC50 values were found in these classes, along with 56% of drugs with an pEC50 corresponding to less than 100 μM.Bland-Altman method comparison plots were used to assess the degree of agreement between the four assays and demonstrated no overall bias for potency between neurosphere and monolayer culture, nor between irradiated and non-irradiated plates . Mean diin vivo studies, pEC50’s for the U87 cell line were ranked and drugs were chosen that either showed high efficacy or ranked markedly different in one assay type over another. In the U87 cell line, bortezomib and mitoxantrone had a high pEC50 value, corresponding to high drug cytotoxicity, in both culture models. Doxorubicin and paclitaxel were more efficacious in the monolayer assay, whilst actinomycin D was ineffectual in the monolayer assay, yet had a high pEC50 in the neurosphere assay. This panel of drugs was additionally validated in an alternative assay read out  with atypical response patterns, such as an increase in cell death with paradoxical increase in spheroid count–a phenomenon we termed ‘spheroid dispersal’. Although the significance of this observation was not clear, it was interesting to note that drugs within the same class (tyrosine kinase inhibitors) tended to cluster together within the Response Space, which also provided indirect evidence that the assay system was capturing a true biological effect. Finally, the Response Space also allows us to immediately identify highly effective drugs, as well as highly resistant cell lines, as exemplified by the rGBM cell line TS9.In this study, we demonstrate the feasibility of using high content screening techniques to generate chemosensitivity data on patient-derived cell lines within a reasonable timeframe of 4 weeks. Although chemosensitivity assays have been performed in GBM previously, 14, ourRadio-resistance of GSCs is well documented; nonetheless, a general increase in efficacy of kinase inhibitors, post-irradiation, was noted. Dasatinib inhibits the PI3K/Akt/GSK3 pathway downstream of BCR/ABL. Dasatinib efficacy was significantly increased in two of our patient lines, corresponding with pre-clinical data demonstrating radio-sensitization of GSCs through Akt inactivation . Thus, tin vivo drug panel was chosen to investigate whether these specific differences were physiologically relevant. Using the U87 cell line, we demonstrate that when pEC50 values are ranked, only the results from our neurosphere assay corresponded to in vivo results. An increased sensitivity to chemotherapeutics in adherent, monolayer cell cultures has been demonstrated before [in vivo tumor growth[Direct comparison of cell lines in monolayer and neurosphere culture demonstrated that the two formats are analogous. However, there were isolated differences with certain drugs. Our d before –29. It ior growth. Thus, oMitoxantrone was the most efficacious chemotherapeutic across all cell lines, demonstrating extremely high levels of cytotoxicity, even at very low concentrations. Mitoxantrone is a type II topoisomerase inhibitor which induces expression of p53 resulting in cellular apoptosis. Mitoxantrone proved to reduce tumor size by an average of 86% compared to controls at sub-maximal tolerated doses in NSG mice. Unfortunately, clinical trials in high grade gliomas in the 1980s using systemic mitoxantrone chemotherapy failed to show similar results.Cisternino et al. have shoin vivo model. Few studies have investigated the efficacy of actinomycin D in high grade glioma, although a prior study has indicated that it is effective in glioma cells through the activation of caspase-2 and -3 mediated apoptosis[Alongside mitoxantrone, several other drugs identified in our study were also ABC-transporter substrates, hence the reason we chose to primarily validate the efficacy of our drugs in a sub-cutaneous apoptosis. Actinomapoptosis. Furtherapoptosis.in vitro has yet to be reflected in clinical trials, with two phase II trials combining bortezomib with tamoxifen[Conversely, bortezomib has been shown to be effective against glioblastoma cells in numerous pre-clinical models and its efficacy has been attributed to various mechanisms, 42. Howtamoxifen and veritamoxifen proving tamoxifen. MeasuraOur HCS platform also demonstrates benefit in the field of rare variants of glioblastoma. We generated chemosensitivity data for a diffuse-pattern glioblastoma cell line (TS1) and a glioblastoma line, diagnosed by a neuropathologist, to have a sarcomatous histopathological appearance (GS1). Robust clinical trial data for these variants is often unavailable, as the scarcity of patients makes trial accrual difficult. HCS therefore could potentially provide a rational basis for therapy selection in these cases.in vivo a number of non-standard chemotherapeutics that have the potential to be effective in the treatment of primary and rGBM, namely mitoxantrone, bortezomib and actinomycin-D. Whilst trials involving systemic administration of mitoxantrone and bortezomib have been unsuccessful, the possibility of directly delivering agents within the tumoral cavity using techniques such as convection enhanced delivery and implantable polymers render the ability to cross the BBB a secondary concern in the selection of effective drugs[This proof-of-principle screen has identified and validated ive drugs. The limIn vivo data suggests that a three-dimensional assay platform gives the most physiologically relevant data; therefore, future work will concentrate on optimizing this model to produce a reproducible and rapid screening model with the potential to generate clinically actionable information.This work represents the initial steps in acquiring, handling and processing chemosensitivity data on an HCS platform. S1 FigHeat Map was populated using best-fit pEC50 values generated from dose-response curves, then color mapped categorically according to the legend. Each heat map color and corresponding number , corresponds to a log change in concentration of drug . Red values of a pEC50 of 9 or above would indicate an EC50 of 1nm or below, indicating high efficacy, whereas any values categorized green-blue or below would likely be poor drug candidates as they correspond to EC50 values above 10μM. Any data Prism was unable to fit was colored black—this was typically due to an inability to generate an EC50 with that drug candidate because of inefficiency at high concentrations.(TIF)Click here for additional data file.S2 FigDose response curves for five non-standard chemotherapeutics identified in the primary HCS plus the standard GBM therapeutic, temozolomide. Data are presented as the mean cell viability compared to mean vehicle control of two replicate assays (six wells per dose) ± standard error of the mean.(TIF)Click here for additional data file.S3 FigEach drug response is separated into change in cell death on the Y-axis (as measured by DRAQ7® intensity/spheroid area), and fold change in spheroid count in the X-axis. Each cell line is treated with drugs only (left), and with irradiation (right). Drug classes are shown below the chart. Gy = Gray.(TIF)Click here for additional data file.S1 TableThe antilog of logEC50s extrapolated from dose response curves were used to populate the table, along with 95% confidence intervals for each EC50. Curves were fitted and EC50 values extrapolated using GraphPad Prism (v7.0).(TIF)Click here for additional data file.S1 FileAdditional methods and description of supporting information figures and tables.(DOCX)Click here for additional data file.


TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC:  Oesophageal cancer is hard to treat. Patients typically present with late-stage disease and respond poorly to conventional anti-cancer therapieset al. have further revealed an association between high intratumour heterogeneity and poor response to NACin-vitro models that represent the mutational burden of the primary tumour.Recent efforts have been directed towards understanding the genetic landscape of OAC with a view to the identification of potentially targetable driver mutations. These studies have revealed the complexity of the genomic aberrations in OAC. The progression from Barrett’s Oesophagus (BE), the only known precursor lesion from OAC, to invasive cancer is characterised by early chromosomal instability, probably due to p53 loss, often including genome doubling and a high frequency of chromothripsis events resulting in considerable genetic diversity followed by a later acquisition of driver mutations at sub-clonal frequencies34et al. found that 3 frequently used lines originated from other tissues Unfortunately there is a paucity of such tools with which to study OAC. This lack of suitable preclinical models is frustrating efforts to understand both the basic biology of the transition from BE to OAC and the development of novel therapies for established cancer9in-vitro models in OAC, such as the immortalisation and malignant transformation of Barrett’s metaplastic epithelial cells by disrupting key signalling pathways and telomerase overexpressionin-vitro modification are valuable tools, such as the recently introduced OANC114in vivoIn an attempt to address these weaknesses a number of groups have used alternative strategies for the establishment of Using complementary genome wide analysis techniques including whole genome sequencing and genotyping of tumour biopsy and matched normal tissue from the oesophagus and peripheral blood we describe the establishment of a new model system in OAC (MFD-1) that retains the mutational profile of the primary tumour, including the identification of novel mutations in recognised cancer associated genes in OAC. Using a whole-transcriptome approach we compare and contrast the expression of recognised OAC associated genes in MFD-1 with established cell lines commonly used to study OAC. We show that MFD-1 is a highly representative model of OAC, it is stable over time and retains the ability to form tumours in xenograft models that respond to microenvironmental stimuli.The study was approved by the Southampton and South West Hampshire Research Ethics Committee . The methods were carried out in accordance with the approved guidelines and written informed consent was obtained prior to the study.The MFD-1 cell line was derived from a 55 year-old white male with locally invasive adenocarcinoma of the oesophagus clinically and radiologically  staged as T3N1M013 pieces that were subsequently digested with trypsin-EDTA  at 37 °C in a 5% humidified atmosphere for 30 minutes. Undigested tissues and debris were removed by sedimentation and the clear supernatant was collected and spun at 600 g for 5 minutes. Cell pellets were cultured in complete DMEM under standard cell culture incubation conditions. The cell line has been deposited in the European Collection of Authenticated Cell Cultures (ECACC), a standard resource for the academic research community. Individual researchers wishing to verify the findings in this study can also approach the corresponding author directly to obtain frozen cell stocks.The specimen was transferred directly from the operating theatre to the research laboratory in complete DMEM media  on ice, washed with 10 ml of phosphate buffered saline 3 times and minced with a scalpel to reach approximately 3 mmGenotyping of genomic DNA from peripheral blood mononuclear cells (PBMC), tumour and adjacent normal squamous oesophageal epithelium, and two early passages of the MFD-1 cell line (P1 & P4) were performed according to manufacturer recommendations Whole genome sequencing (WGS) from snap-frozen oesophageal tumour tissue and germline nucleic acids isolated from peripheral blood mononuclear cells (PBMC) was performed as part of the International Cancer Genome Consortium project and OCCAMS consortium as previously described42 dishes, three replicates for each cell line were grown for 72 hours. RNA extraction was performed following manufacturer recommendations . RNA QC and libraries were processed by Cambridge Genomics Service in a NextSeq500 Illumina Platform. Approximately 20 million reads library per replicate were mapped using Tophat against hg192Counts per Million (logCPM) following the algorithms implemented in EdgeRet al.et al., 2013). Differential expression analysis was carried out using Cuffdiff  alone or epithelial cells (4 million) embedded with normal or cancer associated fibroblasts (2 million). Inoculation of cells proceeded as follows: In the left flank epithelial cells only, in the middle epithelial cells mixed with normal oesophageal fibroblasts and in the right flank epithelial cells mixed with cancer associated fibroblast from the oesophagusμl and analysed by qPCR using the Illumina quantification kit (KAPA Biosystems) in triplicate. The library concentration was calculated using the equation in the KAPA kit instructions which uses the size of the library as calculated by the Tapestation, and multiplied by the dilution factor used to make a final 10 nM stock. ATAC-seq libraries (12 pM) were loaded onto a HiSeq-2500 with a 1% PhiX spike on a Paired End 101 × 101, dual indexed run.ATAC-seq reports on the accessible regions of the genome which are thought to represent areas of “active” chromatin3232Raw fastq files were de-multiplexed and trimmed using Trimmomatic (version 0.32)Analysis of genotypic and Copy Number Variation (CNV) data from the SNP6 platform confirmed the shared, parental origins of DNA from PBMCs, normal oesophagus, oesophageal cancer and two passages (P1 & P4) of the MFD-1 cell line . To alloUsing a Bayesian algorithm in matched paired genomes3To confirm the retention of these potentially important mutations in MFD-1 genomic DNA from the MFD-1 cell line and three derived clones obtained by single cell dilution ring cloning methodPrevious reports have documented the use of various platforms to confirm expression of gene products related to the expected morphology of OAC cells. For instance, verification of the OANC1 cell line was performed using immunohistochemistry for columnar (CK18), glandular (CK7) and intestinal  markers in xenografts. In keeping with the genome wide approach used to document somatic mutations in the MFD-1 parent tumour, we performed RNA-seq of MFD-1, OE33 and FLO1 in culture to compare global gene expression  the aut. This reSixteen of the 33 recurrently mutated genes in OAC had detectable mRNA expression in the 3 cell lines studied . In the KAT6A promoter is more open specifically in MFD-1 cells  analysis32-1 cells , top alt1A cells , bottom.1A cells . To gain1A cells , top. In1A cells , bottom.1A cells .in vivo and respond to microenvironmental signals that are increasingly recognised as determinants of cancer outcomeTo enable the rational design and testing of new therapeutic options for OAC new model systems such as MFD-1 need not only to represent the parent tumour at a genetic level, but furthermore recapitulate tumour growth in vitro assays in response to microenvironmental stimuliWe have previously demonstrated that selected OAC cell lines are invasive in a range of in vitro as primary cell-lines4243454647Research in OAC is hampered by a lack of pre-clinical models. This deficiency is frustrating efforts to understand the basic biology of progression to OAC and the development of novel therapies for established cancerThe majority of cell lines currently used in OAC research are decades old and were not subjected to the kind of detailed genetic authentication that current technologies make possible. For example OE33 was derived from a stage IIa Barrett’s cancer in 1996 and was subjected to karyotyping and cell surface antigen phenotyping, but no analysis of the primary tumour was presented, so the research community cannot know if the mutational burden of OE33 is representative of the tumour of originBy contrast, the strength of the current work is that we have used multiple genome-wide platforms  in the primary tumour and the MFD-1 cell line to confirm the tissue of origin and to accurately document somatic mutations that can be further tested as potential tractable targets. Our initial analysis has concentrated on a panel of genes commonly mutated in OAC constructed by synthesizing data from the WGS studies described by DulakABCB1, DOCK2, SEMA5A and TP53) with relevance to OAC found in the parent tumour by WGS. Interestingly, the mutation observed in the lowest number of reads from the WGS data  is homozygous wild-type in MFD-1, suggesting that the cell line is likely derived from the dominant clone in the tumour. The finding of a mutation in TP53 that has been extensively documented in OAC suggests that MFD-1 is representative of primary OAC tumours. The mutations in ABCB1 and SEMA5A described here have not been previously reported in oesophageal cancer. Importantly, the products of these genes are expressed in MFD-1 making the cell line a useful model to study the biological effects of these mutations. ABCB1 encodes one of a family of ATP-binding cassette membrane transporters that efflux many chemically diverse compounds across the plasma membrane. These transporters are implicated in the development of multidrug resistance in cancer cells treated with chemotherapy  of OAC by Weaver et.al, but the reasons for this association remain unclearABCB1 mutations may indicate a potential mechanism for chemotherapy resistance that was observed in the primary tumour. None of these mutations were found in OE33 or FLO1 and neither of these lines express ABCB1 and SEMA5A together. MFD-1 was generated from a tumour in a patient who had been treated with neoadjuvant chemotherapy. This leads to the potential that some of the mutations in MFD-1 may be chemotherapy induced, however the frequency of mutations observed in SEMA5A and ABCB1 in previous studies of chemotherapy naïve OAC point to these mutations being tumour specific.We describe 5 mutations in 4 genes . This highlights the difficulty in performing research in OAC with the currently available pre-clinical models that may not be representative of tumours seen in the clinic. However, MFD-1 does contain mutations that have been observed in commonly mutated genes in OAC from large, contemporary cohorts34MFD-1 is derived from a single clone from the parent tumour and has to date appeared stable In summary, we describe a pre-clinical model system developed from the oesophageal ICGC project. The availability of genome and transcriptome wide data from the parent tumour and cell line makes MFD-1 perhaps the best-catalogued OAC cell line in existence. Future OAC researchers will have confidence in the integrity of work performed with this cell line and will be able to easily confirm the significance of genetic changes in MFD-1 by reference to the parent tumour by examination of the WGS data. Research in OAC is held back due to a lack of hi-fidelity pre-clinical models and further efforts should be directed to bridge this technology gap.How to cite this article: Garcia, E. et al. Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1. Sci. Rep.6, 32417; doi: 10.1038/srep32417 (2016).


Multiple drug resistance (MDR) of cancer cells is the main reason of intrinsic or acquired insensitivity to chemotherapy in many cancers. In this study we used ovarian cancer model of acquired drug resistance to study development of MDR.We have developed eight drug resistant cell lines from A2780 ovarian cancer cell line: two cell lines resistant to each drug commonly used in ovarian cancer chemotherapy: cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX) and topotecan (TOP). A chemosensitivity assay - MTT was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and immunofluorescence were also performed to determine mRNA and protein expression of genes/proteins involved in drug resistance . Flow cytometry was used to determine the activity of drug transporters.We could observe cross-resistance between PAC- and DOX-resistant cell lines. Additionally, both PAC-resistant cell lines were cross-resistant to TOP and both TOP-resistant cell lines were cross-resistant to DOX. We observed two different mechanisms of resistance to TOP related to P-gp and BCRP expression and activity. P-gp and BCRP were also involved in DOX resistance. Expression of MRP2 was increased in CIS-resistant cell lines and increased MVP expression was observed in CIS-, PAC- and TOP-, but not in DOX-resistant cell lines.Effectiveness of TOP and DOX in second line of chemotherapy in ovarian cancer can be limited because of their cross-resistance to PAC. Moreover, cross-resistance of PAC-resistant cell line to CIS suggests that such interaction between those drugs might also be probable in clinic. MDR1, ABCB1) [ABCG2 gene, cloned for the first time from breast cancer cell line MCF-7 [ABCC1 and ABCC2 genes, respectively [One of the main reasons of low effectiveness of chemotherapy in cancer patients is drug resistance, which is inherent or, more often, acquired during treatment . In most, ABCB1) . Express, ABCB1) . Approxi, ABCB1)  and two , ABCB1)  and doxo, ABCB1)  - are cone MCF-7 . The uprne MCF-7  and ovarne MCF-7  and is kne MCF-7 , 12 and ne MCF-7 , 12. Othectively , 13, 14.ectively . Among mectively , 12.Another protein involved in MDR, but not belonging to ABC drug transporters family, is MVP/LRP major vault protein/lung resistance - related protein . The uprTo better understand the mechanisms of drug resistance development and cross-resistance to different cytotoxic drugs we used the ovarian cancer model, the most lethal gynaecological cancer . OvarianThe current research that improves the knowledge about drug resistance development is based mainly on drug sensitive and resistant cancer cell lines. However, most studies are limited to only one or two resistant cell lines. Therefore, we have developed eight drug resistant cell lines from one parental A2780 ovarian cancer cell line to make model of drug resistance more accurate and effective. Cell lines used in our experiments were resistant to cytotoxic drugs from the first line chemotherapy regimen of ovarian cancer - paclitaxel (PAC) and cisplatin (CIS)  - as welOur study had four main goals: 1. To compare the mechanism of drug resistance to cytotoxic agents used in the first and the second line of ovarian cancer chemotherapy. 2. To determine the expression of the main genes in drug resistant cell lines. 3. To compare the cross-resistance between cell lines resistant to investigated drugs. 4. To determine the differences and similarities between twin cell lines resistant to the same cytotoxic drug.CIS, DOX, TOP, and PAC were obtained from Sigma . RPMI-1640 medium, fetal bovine serum, antibiotic-antimycotic solution, and L-glutamine were also purchased from Sigma . A Cell Proliferation Kit I (MTT) was purchased from Roche Diagnostics GmbH . Goat anti-MRP2 polyclonal (Ab) (H-17), rabbit anti-ABCG2 (BCRP) polyclonal Ab (H-70) were purchased from Santa Cruz Biotechnology . Mouse monoclonal anti-P-glycoprotein Ab (C219) and mouse monoclonal anti-MVP/LRP Ab (MVP 37) were obtained from Alexis Biochemicals .2 atmosphere.The Human Ovarian Carcinoma Cell Line A2780 was purchased from ATCC. A2780 sublines that were resistant to CIS , PAC , DOX , and TOP  were generated by the exposure of the A2780 cell line to incremental increases in the concentrations of the relevant drugs. The final concentrations of each drug were 1000 ng/mL CIS, 1100 ng/mL PAC, 100 ng/mL DOX, and 24 ng/mL TOP. These concentrations were based on the work of Dietel et al. in 1993  and wereThe drug sensitivity of the A2780 cell line and the drug resistant cell lines was confirmed by the MTT cell survival assay. Briefly, all cell lines were seeded at a density of 5000 cells/well in 96-well plates. The cells were allowed to grow for 48 h and subsequently treated with fresh medium supplemented with or without increasing concentrations of drugs and incubated for 72 h at 37 °C. After 72 h of exposure, 10 μL of the MTT labeling reagent was added to the medium , and the cells were incubated for additional 4 h. Following this process, 100 μL of solubilisation solution was added to each well. The absorbance of each sample was measured in a microplate reader at 570 nm with a reference wavelength of 720 nm, according to the manufacturer’s protocol. The negative control was conducted using cell-free culture medium containing both the MTT reagent and solubilisation solution. The experiments were repeated three times, and each concentration in a given experiment was tested in duplicates. Cell viability was expressed as a percentage of the untreated control (means ± SEM).ABCB1, ABCC1, ABCC2, ABCG2 and LRP genes expression in the A2780 and drug-resistant cell lines were examined. Q-RT-PCR was conducted for four independent experiments. RNA was isolated using the Gene Matrix Universal RNA purification Kit (EURx Ltd.), as described by the manufacturer. Reverse transcription was performed using the M-MLV reverse transcriptase (Invitrogen) as described in the manufacturer’s protocol using a thermal cycler . 2 μg of RNA was used to cDNA synthesis. Real-time PCR was performed using the Applied Biosystems PCR System , Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) and sequence-specific primers, as indicated in Table Changes in For amplification, 12.5 μL of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas), 1 μL of each primer   for 10 min at −20 °C, rinsed with PBS and blocked in 3 % BSA for 45 min. Several primary antibodies were used for detection including: MRP2 , P-gp , BCRP  and MVP/LRP  along with the corresponding green dye labeled secondary antibody: anti-goat, anti-rabbit, anti-mouse respectively . Afterwards, the cells were washed three times with PBS and sealed with DAPI-containing mounting medium. The cells were viewed under a fluorescence microscope (Zeiss Axio-Imager.Z1). The expression of MRP2, P-gp, BCRP and MVP/LRP was analyzed using pseudo-colour representations of fluorescence intensity for DAPI at 365 nm excitation and 420 nm emission wavelengths (blue) and for MFP488 at 470 nm excitation and 525 nm emission wavelengths (green).6/ml) were incubated with 1 μg/ml Rho123 or 1 μg/ml H33342 for 1 h at 37 °C in the medium. Next, the cells were washed twice in ice cold PBS with 500 μM verapamil (a known MDR inhibitor), and cellular uptake of Rho123 or H33342 was immediately analyzed using a FACSAria III  with the FCS Express Plus software program. In each analysis, 10,000 events were recorded. The fluorescent emission was collected at 488 nm for Rho123 and at 375 nm for H33342.To assess the efflux activity of P-gp, MRP2 and BCRP in drug sensitive and resistant cell lines, we used the fluorescent dye Rhodamine 123 (Rho123) as an index of P-gp and MRP2 activity and Hoechst 33342 (H33342) as an index of BCRP activity. The cell suspensions , PAC-resistant , DOX-resistant  and TOP-resistant  variant sublines of the A2780 human ovarian cancer cell line were all established by the stepwise selection of A2780 cells cultured in growth media with increasing drug concentrations. To determine the sensitivity of the A2780 and drug-resistant A2780 sublines to CIS, PAC, DOX, and TOP, cells were treated with different concentrations of each drug for 72 h. The concentration- dependent effect of CIS on A2780 and the drug-resistant cell lines was observed Fig. , Table 2We observed high cross-resistance between PAC- and DOX-resistant cell lines Fig. , Table 2The effect of TOP was also investigated. In A2780TR1 and A2780TR2 we observed high level of TOP resistance Fig. , Table 2P < 0.001), both PAC-resistant cell lines (P < 0.01 in A2780PR1 cell line and P < 0.001 in A2780PR2 cell line) and one TOP-resistant cell line A2780TR2 (P < 0.05)  and one TOP-resistant cell line    , both TOP-resistant cell lines (P < 0.01) and both PAC-resistant cell lines A2780PR1 and A2780PR2   Fig. . However.5) Fig. , howeverne) Fig. . Both TO01) Fig. . The LRPly) Fig. , howeverTo confirm the expression of investigated drug transporters at the protein level, we used the immunofluorescence assay. The immunofluorescence analysis validated the transcript expression results. We observed an increased expression of MRP2 protein in the A2780 sublines resistant to CIS Fig. . SimilarTo determine whether expression of MRP2, P-gp and BCRP correlates with their transporter activity or not the fluorescence accumulation was investigated in drug sensitive and resistant cell lines. The MRP2 and P-gp activity was examined with the use of day Rho123 and activity of BCRP with the use of day H33342, respectively.We did not observe increased activity of MRP2 in A780CR1 and A2780CR2 cell lines in comparison with drug sensitive A2780 cell lines Fig. . FurtherIn the present study we investigated the development of resistance to cytotoxic drugs after exposure the ovarian cancer cell line A2780 to cytotoxic drugs used in the treatment of this cancer. The most important cytotoxic drug commonly used in ovarian cancer treatment is CIS , 19. It High level of cross-resistance between PAC- and DOX-resistant cell lines is not surprising because cross-resistance between cancer cells resistant to these drugs has been documented by others , 6 and oAnother drug that is commonly used in many cancers, including second line chemotherapy in ovarian cancer, is topotecan , 21. SimMDR1 gene [The most important MDR protein is P-gp encoded by DR1 gene . We obseDR1 gene , 8, 26. DR1 gene  in curreIt is worth mentioning, that we also observed increased MRP2 transcript level and protein expression in both CIS-resistant cell lines. Expression of MRP2 in CIS-resistant cell lines was also observed by others , 28. AddThe role of BCRP in resistance to TOP seems to be well established , 33 and Among all our resistant cell lines we could observe two different mechanisms of TOP resistance. Both TOP-resistant cell lines showed “classical” mechanism of TOP-resistance based on BCRP expression. It appears in opposition to both PAC-resistant cell lines where resistance to TOP seems to be related to P-gp expression. It has been reported that TOP is a substrate for P-gp  and exprAnother question is about the reason of low level of cross-resistance to DOX in A2780CR1 cell line and total lack of DOX-resistance in A2780CR2 cell line. In A2780CR1 cell line we could not observe any positive expression of P-gp or BCRP. It has been previously reported that MRP2 can also be related to DOX resistance . In our Another protein that is involved in MDR but does not belong to ABC drug transporters family is LRP/MVP . In our All drug resistant cell lines were developed from the same A2780 drug sensitive cell line and, what is more, all twin cell lines were resistant to the same cytotoxic drug but we could still observe some particular differences. From two PAC-resistant cell line only one - A2780PR2 - was cross-resistant to CIS and, similarly, from both TOP-resistant cell lines only A2780TR2 revealed increased level of P-gp and resistance to PAC.Well-described standard response of cancer cells to drugs results in increased expression of typical drug resistance proteins. However, on the basis of our results we can conclude that cancer cells are able to develop an alternative and less specific pathways of response to drug induced stress that leads to cytotoxic drugs cross-resistance. The phenomenon of cross-resistance of cancer cells that has developed effective mechanisms against different types of cytotoxic drugs can directly influence the effectiveness of chemotherapy in ovarian cancer patients.In summary, our results confirm that expression of drug transporters from ABC family is the main mechanism of MDR in cancer cells. It is possible to predict drug cross-resistance when the classical mechanism of MDR based on P-gp expression is involved. We observed two mechanisms of TOP resistance: classical - based on BCRP expression in TOP-resistant cell lines and non classical - related to P-gp expression in PAC-resistant cell lines. Effectives of TOP and DOX in the second line of chemotherapy in ovarian cancer can be limited because of their cross-resistance to PAC. Moreover, LRP/MVP seems to play a complementary role in resistance to cytotoxic drugs. Cross-resistance of PAC-resistant cell line to CIS suggests that such cross-resistance between those drugs is also probable in clinic. Although the main mechanisms of resistance in examined twin cell lines resistant to the same cytostatic were similar, we could still observe some differences between them.


Various organisms exist in the oceanic environment. These marine organisms provide an abundant source of potential medicines. Many marine peptides possess anticancer properties, some of which have been evaluated for treatment of human cancer in clinical trials. Marine anticancer peptides kill cancer cells through different mechanisms, such as apoptosis, disruption of the tubulin-microtubule balance, and inhibition of angiogenesis. Traditional chemotherapeutic agents have side effects and depress immune responses. Thus, the research and development of novel anticancer peptides with low toxicity to normal human cells and mechanisms of action capable of avoiding multi-drug resistance may provide a new method for anticancer treatment. This review provides useful information on the potential of marine anticancer peptides for human therapy. The oceans are a diverse environment comprising about 75% of living organisms . The oceCancer is among the major causes of death with high morbidity and mortality . Cell diIn recent years, novel peptides with antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis, and antiviral activities have been found in marine organisms and studied . Many maThis review focuses on the function and structure of pharmacologically active marine peptides with anticancer activity. Research on peptides derived from marine sources is underway to develop new potential cancer treatments.Many researchers have isolated anticancer peptides from various marine sources, such as, cyanobacteria, fungi, sponges, tunicates, ascidians, mollusks, and fish. Anticancer peptides cause cell death through a unique mechanism involving selectivity to cancer cells and membrane breakdown. The outer portion of the cell membrane of cancer cells has a higher concentration of anionic phosphatidylserine than found in normal cells, resulting in a total negative charge on the cell surface. As a result, the cationic anticancer peptide is attracted to the surface of the cancer cell and binds to the cell membrane, resulting in the collapse of the membrane and rapid cell death ,13. The Cyanobacteria  are an abundant source of established and novel bioactive compounds with powerful therapeutic applications ,16. The Marine fungi produce various secondary metabolites with biological activity and may be structurally unique ,20. IncrHaliclona, Petrosia, and Discodemia .,30.Lyngbectively . On comp at C-35 . In the toxicity ,34.N-glycosylation of endoplasmic reticulum receptors, thereby depleting cancer-associated receptor tyrosine kinases only in cancer cells . Bi. Bi8) is50 value of 40 ng/mL in HeLa S3 cells. Also, for a panel of 39 human cancer cell lines (JFCR39), it showed an average GI50 value of 40 nM. Moreover, a wealth of experimental data demonstrated that the extracellular signal-regulated protein kinase (ERK) signaling cascade recognized its targets through interaction with bisebromoamide . Cr. Cr10) wll lines .The synthetic derivative of cryptophycin, cryptophycin-52 (LY355703), is produced by total synthesis. Cryptophycin-52 induced apoptosis, which is confirmed through the hyperphosphorylation of Bcl-2, cell cycle arrest, and growth inhibition in preclinical trials for the in vitro human non-small cell lung carcinoma (NSCLC) cell line . A clini11) is a cyclic depsipeptide produced by the L. majuscula. It has strong and selective antitumor activity when tested against HCT-116 human colon carcinoma cells as demonstrated by an IC50 of 20 nM and the destruction of cell microfibrils networks (Desmethoxymajusculamide C (networks  45]. In. In11) i12–16) are cyclic depsipeptides containing bis-thiazoline and were isolated from Lyngbya confervoides . Gr. Gr12–1615) and E (16) exhibit potent cytotoxicity against HeLa  and mouse neuro-2a blastoma cells  (Grassypeptolide D (ctively)  47]..15) and 17) is a cyclodepsipeptide with a potent cytotoxicity, isolated from Lyngbya majuscula , and an α-methyl-β-hydroxy acid unit with an alkyne at the C-terminal end. The hydroxyl group is attached on the carbon (C-35) of an unusual hydroxy acid, 3-hydroxy-2-methyloctynoic acid . Ha. Ha17) ioic acid ,50. Hantectively ,50.18) was identified from L. majuscula and showed potent activity in promoting actin polymerization (Hectochlorin (rization  51]..18) was 50) value of 20 μM in PtK2 cells. Among the NCI 60 cancer cell lines, 23 cancer cell lines such as colon melanoma, ovarian tumor, and renal cells showed strong cytotoxicity. Hectochlorin (18) showed cytotoxic activity with IC50 values of 20 and 300 nM for CA46 human Burkitt lymphoma cell and PtK2 cells, respectively . Ho. Ho19) iHCT-116)  with IC5L. majuscula yielded two cytotoxic cyclodepsipeptides: itralamide A (20) and B (21) (21) showed a higher activity than the former, in addition to displaying moderate activity against human embryonic kidney cells 293 . Itralamide A (20) is structurally similar to itralamide B, but has a much lower activity [Caribbean sources of d B (21)  53]. It. ItL. ma22) and B (23) were isolated from the filamentous marine cyanobacterium L. majuscule. These compounds were cytotoxic against P388 murine leukemia cell lines with IC50 values of 6.4–20.5 nM, respectively (The cyclodepsipeptides lagunamide A (ectively  54]. Fu. Fu22) a24) exhibited strong cytotoxicity against P388, A549, PC3, HCT8, and SK-OV3 cell lines with IC50 values ranging from 2.1–24.4 nM . La. La25) iargazole ,58,59,6026, 27) are cyclic peptides isolated from Anabaena laxa. Laxaphycins A (26) and B (27) were re-isolated from an assemblage of L. majuscule, together with Anabaena sp. .,65.28) a29) also exhibited actin polymerization ability at 60 nM; it completely blocked the cellular microfilaments in A10 cells, forming binucleated cells ..Lyngbya 50 values between 120 and 210 nM . Sy. Sy35), 36) and tasiamide B (37) are linear peptides isolated from the cyanobacterium Symploca sp. (36) showed strong toxicity to human nasopharyngeal carcinoma (KB) and human colon carcinoma (LoVo) cells, with IC50 values of 0.48 and 3.47 μg/mL, respectively [Tasiamide (loca sp.  69]. Ta. Ta36) a37) (an octapeptide) contains 4-amino-3-hydroxy-5-phenylpentanoic acid, a unique amino acid moiety, and have cytotoxity against KB cell lines . Ve. VeOscil–1.5 μM) . The incMarine fungi produce biologically active and chemically solitary peptides that have offered research opportunities for effective cancer treatments. However, fungi-derived peptides, like sponge-derived peptides, are mostly secondary metabolites that are difficult to isolate. For this reason, the fungi-derived peptides are less studied than other marine peptides .41) is a hexacyclic dipeptide from Aspergillus insulicola, a Hawaiian marine sediment-derived fungus  and nitrite production (IC50 = 13.70 μM).Azonazine (d fungus  74]. Az. Az41) i42), was isolated from Fusarium sp. living on the marine plant Halodule wrightii (50 = 4.5 μg/mL) . Th. Th42), 5 μg/mL) . Until n5 μg/mL) . Sansalvr growth . In addir growth .43) and B (44) are cyclodepsipeptides isolated from the marine fungi Scopulariopsis brevicaulis and the marine sponge Tethya aurantium (50) [Scopularide A (urantium  77]. Bo. Bo43) aAnticancer peptides from sponges are mostly cyclodepsipeptides with unusual amino acids or non-amino acid parts. These sponge-derived peptides showed a broad range of anticancer or antitumor activity .45), a macrocyclic depsipeptide, was isolated from Dysidea arenaria with extremely strong cytotoxic activity (IC50 = 5 pg/mL) against a human nasopharyngeal carcinoma (KB) cell line (tert-butyl derivative of arenastatin A overcomes this disadvantage and exhibits improved serum stability as well as in vivo antitumor activity.Arenastatin A (ell line  79]. Ar. Ar45), Discodermia sp. containing a macrocyclic ring formed by lactonization of a threonine amino acid unit at the C-terminal and 13–14 common and unusal amino acids-linked structures ..46–53) aGeodia sp. as cyclic forms of peptides consisting of three amino acid residues with a common polyketide unit . Ho. Ho58–6268) was isolated from Jaspis johnstoni as a cyclic depsipeptide with a 15-carbon macrocyclic ring of three amino acid residues  was 50 value of 0.04 ng/mL in P388 murine leukemia cells. It is a bioactive peptide that induces apoptosis in human leukemia cell lines and brain tumor Jurkat T cells by activation of caspase-3 protein expression and decrease in Bcl-2 levels . I. I69) ha70–72) are 17-residue depsipeptides with macrolactone, their IC50 values were inhibited entry from 2.3 to 5.5 μM. (72) was found to have moderate cytotoxicity against the HCT-116 colon cancer cell line with IC50 value of 10 μM. However, koshikamides F–H did not inhibit the growth of Candida albicans, indicating that it is cytotoxic without antifungal activity [Koshikamide F–H ( 5.5 μM.  101]. K. K70–72)73) and B (74) isolated from the Clathria  abietina are linear peptides cyclized via cyclic hexapeptides. Microcionamide A (A) and B (B) showed significant cytotoxicity with IC50 ranges of 125 (A) and 177 nM (B) and 98 (A) and 172 nM (B) in MCF-7 and SKBR-3 cells, respectively  an75) is a cyclic peptide from Theonella sp. with reported cytotoxicity in P388 murine leukemia cells (IC50 = 4.7 μg/mL) and several other melanoma cell lines (Orbiculamide A (ll lines  103]..75) is a76–79), cyclic depsipeptides isolated from Theonella mirabilis and T. swinhoei, are known as the first marine-derived peptides to effectively inhibit the infection of human T-lymphoblastoid cells by HIV-1 in vitro (76) and B (77) blocked the infection with an EC50 value of about 4 ng/mL and papuamide A has an average IC50 of 75 ng/mL, indicating cytotoxicity against a panel of human cancer cell lines. Papuamide E (80) and F (81) were isolated from Melophlus and LD50 values for brine shrimp were 104 and 106 μg/mL, respectively, confirming cytotoxicity . P. PPhakelll lines . Phakell residue . In othe84) and B are two cyclic heptapeptides contained in the Dominican sponge Eurypon laughlini. Rolloamide A (84) showed antiproliferative activity against a panel of histologically diverse cancer cells and showed moderate activity in prostate (LNCap and DU145), breast , ovarian (OVCAE3 and SKOV3) and diverse cell lines with IC50 value range from 0.4 to 5.8 μM, while rolloamide B was largely inactive (Rolloamide A (inactive  109]. R. R84) an85) isolated from Scleritoderma nodosum is a cyclic peptide and showed in vitro cytotoxicity against human tumor cell lines, including HCT116 colon carcinoma (IC50 = 1.92 μM), A2780 ovarian carcinoma (IC50 = 0.94 μM), and SKBR3 breast carcinoma (IC50 = 0.97 μM) (85) has been observed to potently inhibit tubulin polymerization in different cancer cells because of the cell cycle arrest in the G2/M phase . Fo. Fo87) scer drug .A. albicans and exhibits more potent anticancer activity than didermin B . T. T92) an94) is a cyclopeptide with a triazoline ring similar to mollamide, obtained from Lissoclinum  is95–97) were isolated from Diplosoma virens and showed strong anti-apoptotic activity in several cancer cell lines (95) had an IC50 of 2.5 μg/mL for P388 and 10 μg/mL for A549, HT29, and CV1 cells, and inhibited topoisomerase II activity. Both virenamide B (96) and C (97) had an IC50 of 5 μg/mL against P388, A549, HT29, and CV1 cells [The linear cytotoxic tripeptides virenamide A–C (ll lines  133]. V. V95–97)98), a bicyclic depsipeptide isolated from Didemnum cuculiferum and Polysyncranton lithostrotum, is a tubulin-interactive agent, and potentially positive in a cell-based screening for tubulin polymerization inhibitors of purified tubulin (IC50 = 2.5 μM) (98) showed non-competitive inhibition of vinblastine binding to tubulin.Vitilevuamide ( 2.5 μM)  134]. T. T98), aThe colchicine binding to tubulin was tested at concentrations up to 80 μM, and was found to be stabilized in the presence of vitilevuamide. It has also been shown to be weakly affected by the presence of GTP-binding in the presence of vitilevuamide, suggesting that vitilevuamide may inhibit tubulin polymerization through interaction at a unique site .50 values in the range of 6–311 nM, confirming that they were associated with G2/M cell cycle arrest. In mice implanted with P388 lymphocytic leukemia, the intraperitoneal administration of vitilevuamide at doses of 12–30 mg/kg on days 1.5 and 9 after administration resulted in increased life span . D. DDolabecer drug .100) inhibited the growth of L1210 cells (IC50 = 3 nM) and CA46 cells (IC50 = 5 nM) . K. K102) i103), is the potent aurilide-related substance first isolated from Philinopsis speciosa and it is biosynthesized by marine cyanobacteria owing to its structural similarities with the aurilides (103) showed strong antitumor activity against various cell lines, such as P388, SKOV-3, MDA-MB-435, and A-10 (IC50 = 4.2~59.1 nM) . K. K103), 104) present in the venom of Conus magus, has a structure in which 25 amino acid peptides linked by three disulfide bonds  pnal cord .®) was the first marine peptide to receive FDA approval for analgesic use in 2004 and another marine peptide Brentuximab vedotin (trade name Adcetris®) was approved by the FDA in 2011 for drugs that are effective against cancer [It has been shown to have a 1000-fold higher activity than morphine in animal models of nociceptive pain and has a remarkable analgesic character . Ziconott cancer ,160. Vart cancer .105, GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-NH2) is an antimicrobial peptide consisting of 33 amino acids, isolated from the fish Pardachirus marmoratus [Pardaxin (rmoratus . This permoratus . In HT10rmoratus .Pardaxin plays important roles in the scavenging of reactive oxygen species (ROS) to alleviate c-Jun activation. Small interfering RNA-mediated knockdown of c-Jun requiring ROS and c-Jun in pardaxin-induced apoptosis signaling as it abrogates pardaxin-induced caspase activation and cell death .Pardaxin, unlike HT1080 cells, causes caspase-dependent and caspase-independent apoptosis in human cervical cancer cells. Pardaxin also induces >90% inhibition of colony formation in MN-11 cells derived from MC1A fibrosarcoma in male C57BL/10 mice at a concentration of 13 μg/mL . In addi106), an anticancer peptide consisting of six residues derived from the half-fin anchovy (Setipinna taty), has been shown to inhibit the proliferation of prostate cancer cells [50 value of 11.1 μM). It has been confirmed that arginine (R) is an important residue for anticancer activity, but the mechanism for this is still unclear [Tyr-Ala-Leu-Pro-Ala-His (er cells . YALPAH  unclear .Many factors are involved in the discovery and development of anticancer drugs from marine natural products. However, there are various reasons that have ensured the use of marine peptides in the search for anticancer drugs. Marine-derived peptides are chemically diverse, have a wide range of therapeutic activities, and are highly specific to cells or tissues. These peptides can act specifically against cancer cells by either membranolytic mechanisms or mitochondrial disruption . General


Alisertib (MLN8237) is an investigational Aurora A selective inhibitor that has demonstrated activity against a wide variety of tumor types 50 values ranging from 0.06 to > 5 umol/L. Following exposure to alisertib we observed a decrease in pAurora A, B and C in four CRC cell lines. We also observed an increase in p53 and p21 in a sensitive p53 wildtype cell line in contrast to the p53 mutant cell line or the resistant cell lines. The addition of alisertib to standard CRC treatments demonstrated improvement over single agent arms; however, the benefit was largely less than additive, but not antagonistic.CRC cell lines demonstrated varying sensitivity to alisertib with IC50s were calculated. Twenty-one PDX models were treated with alisertib and the Tumor Growth Inhibition Index was assessed. Additionally, 5 KRAS wildtype and mutant PDX models were treated with alisertib as single agent or in combination with cetuximab or irinotecan, respectively.Forty-seven CRC cell lines were exposed to alisertib and ICAlisertib demonstrated anti-proliferative effects against CRC cell lines and PDX models. Our data suggest that the addition of alisertib to standard therapies in colorectal cancer if pursued clinically, will require further investigation of patient selection strategies and these combinations may facilitate future clinical studies. Classical drug development in oncology has often focused on targeting the DNA replication machinery in cancer cells. Of late, the Aurora kinase family has been attracting interest as its members carry out essential roles in centrosome maturation and chromosome segregation . Aurora Many human malignancies, including those of the breast, colon, pancreas, and prostate have aberrant expression of Aurora kinase A. Overexpression of Aurora A is associated with with a poor prognosis and may be associated with resistance to gefitinib, taxol, and cisplatin in various cancers –8. Aurorwww.clinicaltrials.gov). Alisertib demonstrates a 300-fold higher selectivity to Aurora kinase A over B in in vitro assays and 200-fold higher selectivity in the HCT116 colorectal cancer cell line [in vitro [in vitro and in vivo [Alisertib (MLN8237) is a selective Aurora kinase A inhibitor that is currently undergoing Phase I-III clinical development (ell line . Preclinell line . In addi in vivo . Likewis in vivo . Clinica in vivo . More rein vitro and in patient derived xenograft models (PDX), as a single agent and in combination with standard therapies.Based on the novel mechanism of action and its preclinical and early clinical activity, we sought to assess the antitumor activity of alisertib against colorectal cancer models 50s among the CRC cell lines that did not correlate with mutational status of KRAS, BRAF, PIK3CA, or p53. However, Gene Set Enrichment Analysis (GSEA) did show that the cell cycle pathway was upregulated in the more sensitive cell lines, when compared to resistant (data not shown). Core genes in this pathway include CDK1, CDK4, CDK6, CyclinD1, and PLK1. Two sensitive  and two resistant  CRC cell lines, were chosen for further in vitro analysis.We initially sought to determine the relative sensitivity of our CRC panel to alisertib using the CyQuant proliferation assay. As depicted in Figure To determine the effects of alisertib on CRC cell lines, immunoblotting was performed for selected proteins at 8, 12, 24 and 48 hours. As depicted in Figure Flow cytometry was performed to assess the effects of alisertib on cell cycle dynamics/ploidy. CRC cell lines were treated with alisertib (0.1 μmol/L and 1 μmol/L) for 24 and 48 hours. As depicted in Figure To determine the effects of alisertib on induction of apoptosis, HCT116, LS123, GP5D, and COLO678 were exposed to the compound for 24 and 48 hours and cells were stained for Annexin V and propidium iodide, followed by flow cytometry analysis. As depicted in Figure To assess the efficacy of alisertib in mouse models of colorectal cancer, twenty-one CRC PDX models were treated with 30 mg/kg alisertib for at least 28 days and tumor growth inhibition index was calculated. As depicted in Figure MT PDTX models) or cetuximab (KRASWT PDTX models) or the combination for at least 20 days. Cetuximab was used only for the KRASWT, due to no benefit being observed clinically for KRASMT tumors treated with cetuximab. For the combination assessment, alisertib was administered at 10 mg/kg twice daily for both the single agent arm as well as in the combination group. As can be seen in Figure in vivo combinatorial effects of adding alisertib to standard of care agents. In general, the addition of alisertib to standard of care treatments demonstrated improvement over single agent arms; however, the benefit was overall less than additive, but not antagonistic, as can be seen by ψ values being less than 1 but greater than 0  was superior to single-agent alisertib, ten PDX models were treated with either single agent alisertib, irinotecan  and a less than additive effect in one model (CUCRC007 average ψ = 0.5). In fact, out of 60 individual tumors modeled, the combination of alisertib to irinotecan was antagonistic (ψ < 0) in only 7 individual tumors, and additive to synergistic in 23 tumors (ψ ≥ 1). By contrast, the addition of alisertib to cetuximab therapy in KRAS wild-type PDTX models was far more variable with two models demonstrating synergy (CUCRC125 average ψ = 1.3 and CUCRC026 average ψ = 1.4), two models demonstrating a less than additive effect (CUCRC034 average ψ = 0.2 and CUCRC047 average ψ = 0.3) and one model exhibiting antagonism (CUCRC010 average ψ = −0.8). In the Kras wild-type models, a relatively even split between antagonism (18/57), less than additive (16/57) and additive to synergistic (23/57) was observed.In vivo, this agent exhibits modest single agent activity against patient-derived CRC xenografts that was enhanced, to varying degrees, when combined with standard of care agents.Alisertib is an investigational, selective, small-molecule inhibitor of Aurora kinase A. As demonstrated with other Aurora kinase A inhibitors, alisertib has been shown to induce cell cycle arrest, polyploidy and cell death by mitotic catastrophe in a subset of tumor cell lines . The curPrevious preclinical studies have demonstrated that alisertib is effective at inhibiting proliferation of numerous tumor types including esophageal adenocarcinoma, pancreatic, and bladder cancer , 18, 21.In addition to inhibiting proliferation in these models, induction of cell death and the cell cycle arrest was observed. The anti-tumor activity of alisertib exposure is not unexpected, but the results appear to be target dependent. For example, the pan-Aurora kinase inhibitors, PHA-739358 and PF-03814735 demonstrated inhibition of proliferation, induction of apoptosis and cell cycle arrest in multiple tumor types, including melanoma, HCC and SCLC –24. As wWT HCT116 CRC cell line at all time points, however after 48 hours of treatment with alisertib the induction is more pronounced. Conversely, in our p53 mutant model, we observed only a slight increase in p53 at 24 hours and no increase at 48 hours. No increase of p53 was observed in the two alisertib resistant CRC cell lines. These data are somewhat consistent with what has been observed previously in that p53 mutant cells do not upregulate p53 at early time points after exposure to alisertib [WT bladder cell lines demonstrated an increase in p53 following Aurora A inhibition for 48 hours [Other reports demonstrate that Aurora A may directly interact with p53 by phosphorylation, thus affecting its apoptotic activity . For exalisertib . This is48 hours . p21, a 48 hours , however48 hours , 28. By Consistent with previous data, a G2/M cell cycle arrest was observed in all cell lines tested. However following 48 hours of alisertib exposure, an greater increase in aneuploidy was observed in the HCT116 cells compared to the LS123 and COLO678 cells and may be due to the differential effects of Aurora B, which is associated with effects on mitotic slippage and aneuploidy . At highAlisertib has demonstrated efficacy in xenograft models of multiple tumor types, including bladder, multiple myeloma, neuroblastoma and esophageal adenocarcinoma –18, 31. In early Phase I clinical trials in patients with solid tumors, alisertib demonstrated a best response of stable disease of 7.3 months, and stable disease was durable for more than 6 months in 4 patients with colorectal cancer . In addiin vitro work alisertib was dissolved in 100% DMSO at a concentration of 10 mmol/L. For in vivo studies, alisertib was dissolved in a half volume of 20% 2-hydroxypropyl-β-cyclodextrin in sterile water (w/v) and then diluted with a solution of 2% sodium bicarbonate in sterile water (w/v) to provide a final formulation in 10% 2-hydroxypropyl-β-cyclodextrin / 1% sodium bicarbonate.Alisertib was provided by Millennium Pharmaceuticals . For 2. Cells were routinely tested for the presence of mycoplasma . All CRC cell lines used in this study have been fully characterized and authenticated in the University of Colorado Cancer Center DNA Sequencing and Analysis Core.Human colorectal cancer cell lines were obtained from American Type Culture Collection, DSMZ, or the Korean Cell Line Bank. Geo cells, described previously , were kiCells were seeded in 96-well black walled plates at 2000-8000 cells per well, depending on cell line growth kinetics. After 24h, alisertib was added at increasing concentrations and cells were incubated for an additional 72h. Proliferation was assessed by CyQuant assay according to the manufacturer's protocol  and read on a Synergy 2 plate reader .Cells were seeded in 6-well plates and allowed to attach for 24h. Cells were then incubated for 8, 12, 24h or 48h with varying concentrations of alisertib. Cells were then washed with PBS and lysed with RIPA buffer . After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel , electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter . The membrane was blocked and probed overnight with primary antibodies, washed for 10 minutes 3X with TBS/Tween 20, and probed with DyLight secondary antibodies , and imaged using the Licor Odyssey . All primary antibodies were purchased from Cell Signaling Technology  and diluted as per the manufactures' instructions.Cells were seeded in a 6-well plate and allowed to adhere for 24 hours. Alisertib was added at the indicated concentrations for 24 and 48 hours. Following exposure, cells were trypsinized, washed in PBS and stained with Annexin V and PI using the Dead Cell Apoptosis Kit  and analyzed by flow cytometry. For cell cycle analysis, cells were plated and exposed to drug as described above. Cells were trypsinized, washed in PBS and resuspended in Krishan's stain and analyzed for cell cycle and ploidy using flow cytometry. Both analyses were performed by the University of Colorado Cancer Center Flow Cytometry Core.ad libitum. The PDX were generated as previously described [3 pieces, submerged in Matrigel, and implanted subcutaneously into the flank of five nude mice. After tumors were expanded through at least the F3 generation, they were excised, cut, and injected into the left and right flanks of mice for each xenograft study. When the average tumor size reached a volume of approximately 200 mm3, mice were randomized into the four different groups . Mice were monitored daily for signs of toxicity and weighed twice weekly. For single agent treatment alisertib was administered once daily at 30 mg/kg by oral gavage. For combination studies, alisertib was dosed at 10 mg/kg twice daily, irinotecan was dosed at 15 mg/kg weekly, and cetuximab was dosed at 0.4 mg/mouse twice weekly, except where noted differently. Tumor volume (equation for volume = (length x width2) x 0.52) was evaluated twice per week with digital calipers, using the Study Director software package . Tumor growth inhibition index was calculated from average volume of the treated (Vt) and vehicle control (Vvc) groups, with the equation: TGII = 100 × /. TGII values of less than 20% were considered responsive, whereas TGII values of above 20% were considered nonresponsive. At the end of treatment, mice were sacrificed by CO2 overdose followed by cervical dislocation prior to removal of the tumors for further analysis.Five to six week-old female athymic nude mice  were used for all animal studies, caged in groups of 5, kept on a 12 hour light/dark cycle, and given sterile food and water escribed . BrieflyP values<0.05 were considered statistically significant.One-way ANOVA analyses with a Tukey post-test was used to determine statistical significance between multiple groups. Analyses were performed with Prism version 4.02. in vivo study was modeled, similarly to a previously published model, to assess the therapeutic benefit of adding irinotecan or cetuximab to alisertib [Data from the combination lisertib , 37. All