Cancer chemotherapy agents are assessed for their therapeutic utility primarily by their ability to cause apoptosis of cancer cells and their potency is given by an IC50 value. Chemotherapy uses both target-specific and systemic-action drugs and drug combinations to treat cancer. It is important to judiciously choose a drug type, its dosage and schedule for optimized drug selection and administration. Consequently, the precise mathematical formulation of cancer cells' response to chemotherapy may assist in the selection process. In this paper, we propose a mathematical description of the cancer cell response to chemotherapeutic agent exposure based on a time-tested physical model of two-state multiple-component systems near criticality. We describe the Ising model methodology and apply it to a diverse panel of cytotoxic drugs administered against numerous cancer cell lines in a dose–response manner. The analysed dataset was generated by the Netherlands Translational Research Center B.V. (Oncolines). This approach allows for an accurate and consistent analysis of cytotoxic agents' effects on cancer cell lines and reveals the presence or absence of the bystander effect through the interaction constant. By calculating the susceptibility function, we see the value of IC50 coinciding with the peak of this measure of the system's sensitivity to external perturbations. Cytotoxicity refers to killing or damaging a viable cell by chemical compounds or pathogens. Toxic agents usually damage molecular targets such as metabolic sites, signalling proteins or DNA, which are essential to the cell's reproductive ability and survival. The result is dose-dependent cell death or inhibition of its proliferative potential. Unlike radiotherapy, which specifically targets cancer cells in a tumour, chemotherapy is typically applied systemically and is not site-specific unless combined with target-specific antibodies or via special drug delivery strategies. Hence, it may also affect metastasized cells distant from the primary tumour site and also the entire body of the patient with concomitant detrimental side effects –4. BesidR is the receptor, n  is the number of ligands L and Kd is the dissociation constant. Despite its simplicity, the Hill equation is not always physically plausible and the Hill coefficient n can only be accurately estimated for extremely positive cooperative interactions among multiple ligand-binding sites. Even for a reaction with a high degree of positive cooperativity, e.g. binding four oxygen molecules to haemoglobin, the Hill coefficient ranges from 1.7 to 3.2 rather than 4 [Cytotoxicity mechanisms are commonly analysed using the Hill model representing an empirical sigmoidal fit describing the binding equilibria in ligand–receptor interactions  based onr than 4 . Therefor than 4 . The mair than 4 . The sysr than 4 –21.Our aim is to implement the spin-1/2 Ising model of phase transitions as an elegant and powerful mathematical approach to study cancer cells exposed to cytotoxic chemotherapeutic agents  ,22–41. R2.ith cell and ith cell. Following the radiation-induced bystander effect [ij indices are summed over all nearest-neighbour cell pairs at site i and j, i and j (namely the strength of the bystander effect) and ih represents the potency of the external agent at location of is. i and j, so Z, and the order parameter, M, are found after several steps of calculations as M, and control parameter, h, are equivalent in the case of cancer cells to the death rate, R, and the logarithm of concentration, log(C), respectively, we rewrite equation (2.3) asC is the drug concentration and MC is a Michaelis constant representing the drug concentration associated with reaching the half-maximal inhibition effect [α shows the slope of the dose–response curve and depends on the system's heterogeneity, drug-binding efficacy and diffusion of drug molecules. It can be estimated by fitting the solution to experimental data points. In cytotoxicity assays, MC is usually denoted as IC50. As shown in the next section, equation (2.5) provides excellent agreement with the cytotoxicity experiment data. Equation (2.5) has a critical value for λ, at which a discontinuity in the death rate emerges and can be solved numerically for given values of α and λ. Figure 1a–c illustrates the death rate, R, as a function of α and λ, respectively. Figure 1a shows the result for uncorrelated cells, which presents similar behaviour to that found in the Landau theory of phase transition  [figure 1a–c, increasing the interaction strengths between cells for R, with respect to the λ = 0, using equation (2.6), we show that the susceptibility function maximum occurs at ‘zero-field’ or α. The susceptibility of the Ising model was extensively studied by Fisher [α and λ, respectively. In the case λ = 0, figure 1d, the susceptibility maximum occurs at zero-field, while by increasing λ it peaks at concentrations below MC. Interestingly, by increasing α in the non-zero λ case, the maximum shifts towards zero-field. This demonstrates that there is a competition between α and λ, where larger values of α provide solutions similar to λ = 0. This is observed in the parameter values found using the experimental data.Similarly to the Ising model with two spin states, up and down, the effect of chemotherapeutic drugs on cancer cells is associated with two possible outcomes: either survival or death states of cancer cells, respectively. For simplicity, we assign two states as r effect ,6, we inλ=J/2kBT . Using tn effect . The pardetails) –35,38. Aarameter ,46, whicy Fisher ,46. Lowe3.R, is plotted in terms of the logarithm of anti-cancer drug concentration, a, b, c and d are the best-fit parameters. As an example, figure 2a shows the results for Bortezomib acting on a melanoma cell line (A375). Based on the cell response-Ising model, the IC50 value marks the drug concentration at which the phase transition from a live system to a dead one occurs. In figure 2a, dashed lines represent the best-fitting function and a red star and a purple circle represent the experimental and predicted IC50 values, respectively. Taylor expansion of the fitted function around IC50 is shown using a green solid line in figure 2a. Note that the solid line in cyan shows the susceptibility in equation (2.6) and its highest value coincides with the IC50 concentration, which provides a rationale for the thus far arbitrary use of IC50 as a significant parameter for cytotoxicity estimates. These findings also demonstrate good agreement between our model and the Landau mean-field theory of phase transition . Figure 2b illustrates a similar behaviour for Paclitaxel applied to other cell lines, namely: 769-P (kidney), A-172 (blood), A-375 (skin), A-427 (lung), BxPC-3 (pancreas), BT-549 (breast) and Colo-205 (colon). All findings in this section demonstrate good agreement between the proposed model and the experimental data.In this study, we applied the Ising model methodology to better understand and more accurately describe cancer cell response to chemotherapy agents. Inhibition profiles of 13 diverse anti-cancer compounds were analysed from proliferation assays performed on 66 cancer cell lines provided by Oncolines, Inc., The Netherlands ,43. The d of the fitting function. For those cytotoxic drugs, which follow the model, the difference between the predicted and experimental values of IC50 is very minor, of the order of 10−3. Comparing the fitted curve with equation (2.5) derived from the cell response-Ising model in λ = 0 in equation (2.5)). a, b, R2 andc and d values. To study the possibility of the bystander effect, we use equation (2.5), derived using the Ising model, with an assumption that α = 1. The obtained values are fairly consistent among all the drugs . It emerges that drugs with higher correlations to our model have high susceptibility values as well. The correlation coefficient includes the cell lines with R2 > 0.5 and the susceptibility One of this model's advantages is its ability to predict a precise value of IC50 by finding the parameter In addition, In the original dataset, there are some cell lines for which the corresponding IC50 value was not reported. This is due to it exceeding the maximum tested concentration (less than 31 600 nM). At this concentration, the compounds still show less than 50% inhibition of cell proliferation. R2 < 0.5, exhibiting insufficient cytotoxic dose, showing a bizarre reversal effect (i.e. increasing the dose decreases the cells' death rate), showing efficacy of less than 40%. Figure 4a shows the number of the cells exposed to different drugs, which kill less than 40% of the cells. It demonstrates the cells that either were not responding well to the cytotoxic drugs or the drug was not cytotoxic enough. Comparing figure 4a and Figure 4b illustrates the cell lines for which the dosage used was insufficient to cause the death of these cell lines. Also, figure 4c shows the efficacy of the drug on these cancer cell lines. Busulfan has lower efficacy in more than 90% of the cell lines. The efficacy value is 35% and 33% for Tazemetostat and Idelalisib, respectively, and 1.6% for Irinotecan and Methotrexate. Finally, figure 4d shows that some of the cell lines show a toxicity reversal effect when exposed to Tazemetostat, Busulfan and Trametinib such that increasing the cytotoxic dosage decreases the death rate, although the number of such cases is negligible.λ have also been considered. The same datasets have been fitted to equation (2.5) and the implicit equation for log (C) was solved. The interaction coefficients were assumed to be positive, c of the fit should be less than 1 and λ should be less than the critical valueλ, changes between 2.1 and 6.1 on average. In fact, since λ is greater than cλ, the cell–cell interactions are not significant here  two-dimensional and (b) three-dimensional and Nitazoxanide (c) two-dimensional and (d) three-dimensional. It can be seen that in the case with no cell–cell interactions, λ = 0, the two cytotoxic drugs have a better correlation than in the interaction case. In the presence of the interaction term, λ in the curves a to d, are 1.32, −3.01, 0.74 and 0.05, respectively. These values show that the model does not work well for the Mitomycine three-dimensional experiment, while for Nitazoxanide three-dimensional, the λ value shows that the cell–cell interactions are very weak . However, in the case of the two-dimensional experiments for both drugs, we see a better correlation in the cell–cell interaction cases . We can, therefore, tentatively conclude that spatial dimensionality does indeed affect the response of cell cultures to cytotoxic agents but a more in-depth analysis of larger datasets is required to develop an appropriate mathematical model that captures these complex systems' behaviour better than the present Ising model.Until now, all the cases analysed in this paper involved typical well-plate cytotoxicity assays, in which cell culture grows within a plane and is then exposed to toxic chemotherapy agents. Spatial dimensionality of physical systems undergoing phase transitions plays a crucial role in the response of the system to control parameter changes. In order to explore whether this can also be seen in biological systems such as cancer cells, we have found some experimental data in the literature that provide examples of dimensionality dependence. on cases a,c. We c4.In this study, the physical concepts developed for the theory of phase transitions occurring in bistable systems were for the first time applied to describe the effects of various chemotherapeutic agents on cancer cell lines. Specifically, we adopted the Ising model of a spin system and applied it to the survival plots of cancer cells at different concentrations of the various chemotherapeutic agents these cells were exposed to. This model was originally proposed for a spin system in a uniform external field with a constant interaction parameter and a variable temperature. In the case of cancer cells, the external field is analogous to the logarithm of the drug concentration, while the interaction parameter describes the cell–cell interactions and hence accounts for the bystander effect. Unlike in physical systems, temperature is kept constant for cancer cells in the reported assays. It should be noted that this model has been successfully applied to both interacting and non-interacting cells depending on the underlying biological situation. We have tested the model on a consistently produced dataset of 66 cancer cell lines exposed to 13 different cancer chemotherapy drugs. The results show good agreement between the cell response-Ising model and the biological data. Using the bistabiliy concept in the Ising model, IC50 values can be very accurately determined with an error on the order of 1 nM by one of the parameters of the fitting function in the non-interacting case. The cell–cell interaction was also applied to the experimental data, although in our case, most of the cell lines tend to be non-interacting. Nonetheless, the presence of interactions can be determined using our fitting procedures, and it offers a clear biological insight that bare experimental data do not reveal. We have additionally introduced the thermodynamic concept of the susceptibility function and found its peak to closely coincide with the value of IC50. Further studies should be performed considering a non-constant interaction term as well as non-uniform fields in the Ising model applied to cytotoxicity assays with both two-dimensional and three-dimensional geometries and various cell concentrations. In addition, spatial dimensionality of the cancer cell culture was shown to affect the response to cytotoxic agents, which requires a future study to gain insight into how two-dimensional culture may not be an appropriate proxy for tissue-based studies. This approach is expected to introduce a high level of consistency in cytotoxic data analysis and hence better confidence in the preclinical data assessment for cancer chemotherapy and related applications.


Wnt signaling plays key roles in oncogenic transformation and progression in a number of cancer types, including tumors in the breast, colon, ovaries, liver, and other tissues. Despite this importance, no therapy targeting the Wnt pathway currently exists. We have previously shown that the anti-mycobacterium drug clofazimine is a specific inhibitor of Wnt signaling and cell proliferation in triple-negative breast cancer (TNBC). Here, we expand the applicability of clofazimine to a set of other Wnt-dependent cancers. Using a panel of cell lines from hepatocellular carcinoma, glioblastoma, as well as colorectal and ovarian cancer, we show that the efficacy of clofazimine against a given cancer type correlates with the basal levels of Wnt pathway activation and the ability of the drug to inhibit Wnt signaling in it, being further influenced by the cancer mutational spectrum. Our study establishes the basis for patient stratification in the future clinical trials of clofazimine and may ultimately contribute to the establishment of the Wnt pathway-targeted therapy against a diverse set of cancer types relying on the oncogenic Wnt signaling. Translation of advances in technology and enhanced scientific knowledge of human diseases to novel therapeutics still faces high costs, long development time, and high attrition rate. Therefore, drug repositioning  is considered an attractive strategy for identifying new uses of an approved or investigational drug that are outside its original medical indication , 2. VariWnt signaling is a highly evolutionary conserved pathway playing major roles in the control of cell fate, proliferation and migration during organism development, but in healthy adult organs, it remains relatively silent . The abein vitro. In the in vivo mouse xenograft models of TNBC, clofazimine efficiently suppresses tumor growth through inhibition of the Wnt signal transduction. Clofazimine is also well compatible with doxorubicin, exerting additive effects on tumor growth suppression, producing no synergistic adverse effects. Together with the well-characterized pharmacokinetic profile and lack of serious adverse effects at therapeutically effective doses, this makes clofazimine a prime candidate for repositioning clinical trials , an aggressive subtype of breast cancer (BC) currently lacking targeted therapy , 9. Clofl trials , 9.Colorectal cancer (CRC) is reported to account for 10.2% of all diagnosed cancers and 9.2% of deaths caused by cancer. It is considered as the second most common cancer worldwide . HepatocThis current work continues and extends the exploration of the potential of the Wnt inhibition and of the anti-proliferative properties of clofazimine for other Wnt-associated cancer cell types, including panels of CRC, HCC, OC, and glioblastoma cell lines. Upon treatment with clofazimine, we observed a correlation between cancer cell survival and the Wnt pathway levels; mutational spectrum of the cancer-associated genes is also found to influence the sensitivity to the drug. These data uncover the potential for expansion of the anti-cancer indications of clofazimine beyond TNBC and will be instructive for patient stratification.50 μl of the indicated cell lines re-suspended at 60000 cells/ml for SW620, SW48, HepG2, Hep3B, PEO1, OVCAR3  were added into each well of a transparent 384-well plate. The cells were maintained in DMEM containing 10% FBS for DLD1, SW620, SW48, LS174T, HT29, HCT116, Hep3B; RPMI without glucose containing 20% FBS and 10 µg/ml insulin for OVCAR3; RPMI without glucose containing 10% FBS, 2 mM sodium pyruvate, and 2 mM Glutamine for PEO1; RPMI without glucose containing 10% FBS, non-essential amino acids and 5 µg/ml insulin for KURAMOCHI; RPMI without glucose containing 10% FBS for OVSAHO; RPMI with 10% FBS for SNU398; DMEM without glucose containing 10% FBS and 1 g/L glucose for Huh7; DMEM without glucose containing 10% FBS, 1 g/L glucose and 25 mM HEPES for U87, U118, and U251; MEM without glucose containing 10% FBS for HepG2 and incubated at 37°C, 5% CO2 overnight. The medium of each well was replaced by 100 μl fresh medium the next day containing the indicated concentrations of clofazimine (Santa Cruz) or MSAB (Sigma-Aldrich). After incubation for 4–7 days (depending on the growth rate of line), the medium in each well was replaced by 50 μl of 0.5 mg/ml Thiazolyl blue (Carl Roth) solution in 1xPBS. The plates were incubated for 3 h at 37°C. Then the solution was removed and 50 μl DMSO was added into each well. Absorbance at 570 nm was measured in a Tecan Infinite M200 PRO plate reader.The Wnt-induced and basal luciferase activity was analyzed as described , 20. BriFor comprehensive analysis of the relevant mutations in all the cell lines used, we have employed Broad Institute Cancer Cell Line Encyclopedia (CCLE). Since DLD1 and LS174T lines were absent in CCLE, we have used similar dataset generated in . The genEach cell line was seeded at 100,000 cells/well in 24 well plates. The next day, the medium was replaced with fresh medium with or without clofazimine at the indicated concentration pre-warmed at 37°C. After further 24 h incubation, the medium was removed, followed by washing with 500 μl of 1x PBS twice per well. The cells were lysed in the well by addition of 30 μl of ice-cold RIPA buffer  and incubated on ice for 10min. The cells were resuspended and then centrifuged at 18,000 g at 4°C to remove debris. 5 μl of the supernatants each were further analyzed by Western blot with antibodies against β-catenin , α-Tubulin , and c-Myc  at 1:1000 dilutions.In our current investigation aiming at broadening the scope of repositioning of clofazimine against Wnt-dependent cancers, beyond TNBC previously analyzed by us , 9, we tAs expected , a generSimilarly, the mutational upregulation of the Wnt pathway is seen in three out of the four HCC lines, with pathogenic activating mutations in CTNNB1 [S37C in SNU398 and in-frame deletion of amino acids 25–140 in HepG2 ] or lossIn contrast, no genetic mutation-associated upregulation of the Wnt pathway can be observed in the OC and glioblastoma lines in our study Table 1,50 around 3–8 µM (50 for CRC is from ca. 2 µM (SW48) to ca. 5 µM (HT29) and 8–9 µM for the remaining four lines , making CRC cells comparable to TNBC cells regarding their sensitivity to clofazimine.In our previous work, we demonstrated that clofazimine has anticancer properties against six TNBC cell lines. MTT survival assay with clofazimine demonstrated an antiproliferative effect with ICd 3–8 µM . Concent50 of 1–2 µM, whereas the other two—Huh7 and HepG2—are one order of magnitude less sensitive with IC50s of 13 and 17 µM, respectively  reveal poor sensitivity, with the IC50 ≤ 10 µM) to strong sensitivity to clofazimine (IC50 ≤ 2 µM), on one hand, and those displaying poor sensitivity (IC50 > 10 µM). This cutoff was based on our prior extensive studies of the effect of clofazimine on TNBC growth in vitro and in vivo  to clofazimine, we grouped these 17 cell lines into those displaying good  between the basal Wnt level and the sensitivity of the cell line to clofazimine, so that the lower the basal level of Wnt pathway activation, the lower the observed sensitivity to clofazimine , we do not see any correlation between these induced pathway levels and the sensitivity to clofazimine (data not shown).50 value in the cell proliferation assay  on the Wnt3a-induced response in the TopFlash assay in the panels of CRC, HCC, OC, and glioblastoma cell lines  in repressay see Figure 1A certain tendency of a correlation (R = 0.3697 after log transformation) could be seen between the extent of the Wnt pathway suppression by clofazimine and the sensitivity to the drug in terms of the cell growth inhibition, so that the stronger the ability of clofazimine to inhibit Wnt signaling, the more apparent is the growth sensitivity to it sulfonyl]amino}benzoate. Interacting with β-catenin and promoting its degradation , MSAB acThus, we conclude that subsets of cancer lines—and by inference, cancer cases—across different types of solid tumors reveal sensitivity to the Wnt pathway inhibition. The differences in the sensitivity to clofazimine we identify across the cancer panel can serve as the basis for future patient stratification in the precision oncology applications and are further considered in Discussion.M. tuberculosis was considered insufficient. Instead, clofazimine demonstrated to be more potent against another mycobacterium, M. leprae (https://apps.who.int/iris/handle/10665/64636). Clofazimine’ efficacy in treating leprosy results not only from its antimicrobial activity, but also from the anti-inflammatory activity  how high the basal level of the Wnt pathway in the cancer cell line is, and ii) how strongly clofazimine inhibits the Wnt signaling in these lines. Despite this general predictivity matrix, deviations still exist. We envision that these deviations can result from a) mutational and/or epigenetic portrait of each cancer line/case, and b) other mechanisms of action of clofazimine apart from its ability to block the Wnt pathway. We discuss these below.Analysis of the mutational spectrum in the cancer lines we study Table 1 It is also intriguing to note that among the four HCC lines analyzed in our work, both clofazimine-sensitive ones (Hep3B and SNU398) originate from Hepatitis B virus (HBV)-positive HCC, while the two lines poorly sensitive to the drug (HepG2 and Huh7) are negative for HBV DNA Table 1.Another basis for the differential sensitivity of cancer lines to clofazimine, deviating from that to be expected based on the analysis of the Wnt pathway in these lines  and pancTo summarize, we have established the broad sensitivity of cancer lines across different cancer types to clofazimine. This sensitivity grossly correlates with and can be predicted by the basal levels of Wnt signaling and by the ability of clofazimine to inhibit the Wnt pathway in the cancer lines. This work lays the ground for continued repositioning of clofazimine against a broad range of Wnt-dependent cancers and creates the basis for patient stratification in eventual clinical trials.The original contributions presented in the study are included in the article/JX performed experiments and analysis, and wrote the manuscript. AK performed and supervised experiments and analysis, and wrote the manuscript. VK led the project, supervised experiments and analysis, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The work was funded by the Novartis Foundation for Medical-Biological Research grant #17C153 to VK. JX is a recipient of the Theodor et Gabriela Kummer fellowship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gastric cancer is the fifth most common malignancy and the third leading cause of cancer‐related death worldwide. Activation of c‐MET increases tumour cell survival through the initiation of the DNA damage repair pathway. PARP is an essential key in the DNA damage repair pathway. The primary role of PARP is to detect and initiate an immediate cellular response to single‐strand DNA breaks. Tumours suppressor genes such as BRCA1/2 are closely associated with the DNA repair pathway. In BRCA1/2 mutations or deficiency status, cells are more likely to develop additional genetic alterations and chromosomal instability and can lead to cancer. In this study, we investigate the role of c‐MET and PARP inhibition in a gastric cancer model. We exploited functional in vitro and in vivo experiments to assess the antitumour potential of co‐inhibition of c‐MET (SU11274) and PARP (NU1025). This leads to a reduction of gastric cancer cells viability, especially after knockdown of BRCA1/2 through apoptosis and induction of γ‐Η2ΑΧ. Moreover, in AGS xenograft models, the combinatorial treatment of NU1025 plus SU11274 reduced tumour growth and triggers apoptosis. Collectively, our data may represent a new therapeutic approach for GC thought co‐inhibition of c‐MET and PARP, especially for patients with BRCA1/2 deficiency tumours. As a result, PARP inhibition leads to DNA double‐strand breaks (DSBs) that are the most deleterious form of DNA damage.BRCA1/2 proteins are necessary for the HR progression as the cells are susceptible to PARP inhibition when the BRCA1/2 protein is deficient.It has been shown that c‐MET stimulation is necessary to develop resistance to the DNA damaging agent.In the current study, we try to explore the combination of c‐met and PARP inhibition in GC cell lines models (AGS and HS746T). In more detail, co‐treatment of GC cell lines with NU1025 and SU11274  decreased cell viability through induction of apoptotic cell death in BRCA1/2 deficiency manner. Furthermore, in vivo experiment in AGS xenograft mouse model, co‐inhibition of c‐MET and PARP decreases tumour volume mass. Collectively, we proposed that co‐treatment of PARP and c‐MET inhibitors had a beneficial effect in the BRCA1/2 deficiency GC model and are a putative therapeutic approach for GC patients.22.1The c‐MET inhibitor SU11274 (#S9820) and PARP inhibitor NU1025 (#N7287) were obtained from Sigma‐Aldrich. Both inhibitors were dissolved in DMSO and stored at − 80°C.2.2Hs746T and AGS GC cell lines were obtained from American Type Culture Collection (ATCC) and American Type European Collection of Authenticated Cell Cultures (ECACC).2. The experiments were done with the approval of the Ethics Committee of our University.All cell lines used in this study were grown in RPMI Medium 1640—GlutaMAX™ (#61870‐010 Life Technologies Carlsbad) supplemented with 10% foetal bovine serum (FBS), penicillin and streptomycin antibiotics . Cells were maintained at 37°C in a humidified incubator containing 5% CO2.3For the siRNA transfection experiments, we use siRNA for stable knockdown of c‐MET (sc‐35924), BRCA1 (sc‐29219) and BRCA2 (sc‐29825) and control (sc‐37007) from Santa Cruz Biotechnology and Lipofectamine 3000 (#L3000‐15 Invitrogen Corp.).2.4Cell growth and viability were confirmed by MTT assay. Approximately 3,000 cells were placed in a 96‐well plate with 200 μL culture medium. At the end of treatment time, cells were incubated for 4 hours with 0.8 mg/mL of MTT, dissolved in a serum‐free medium followed by DMSO (1 mL) and gentle shaking for 10 minutes to achieve the complete dissolution. Finally, the absorbance was measured at 560 nm using the microplate spectrophotometer system (SpectraMax 190‐Molecular Devices). Results are presented as percentage of the control values.2.5As described in detail previously,2.6For the 2D culture experiments, cells (5000 cells/well) were grown on coverslips in 24‐well plates in medium, at 37°C. After knockdown of BRCA1/2 and treatment with NU1025 or SU11274, alone or in combination, for 24 hours cells were fixed in 4% paraformaldehyde, then permeabilized and then blocked with 0.5% BSA/PBS‐5% 22 Triton X‐100. Next, cells were treated with the primary H2AX antibody and then incubated with an antimouse fluorescence‐labelled secondary antibody (#20014). Cells were examined using an Olympus FV1000 confocal microscope with an Olympus digital camera. The nuclei were stained with Dapi No. 33 342.2.7HS746T GC cell line (40 000 cells/well) was grown in a 12‐well plate and after knockdown of BRCA1/2 with siRNA transfection, cells were incubated with 5 μmol/L NU1025 and/or SU11274, alone or in combination, for 24 hours. At day 0, we formed the wound with a yellow pipette tip. After 24 hours of incubation, cells were photographed utilizing computer‐assisted microscopy. We measured the gap distance of the wound on day 0 and after 24 hours using Image‐Pro Plus software.2.8HS746T GC cell lines were plated into a 6‐well plate. After knockdown of BRCA1/2 with siRNA transfection, cells were incubated with 5 μmol/L NU1025 and/or SU11274, alone or in combination, for 14 hours. We renew the inhibitors every 2 days. Following 14 days of incubation, colonies were fixed with methanol: acetic acid (3:1) solution and stained with haematoxylin. Cells were subsequently washed with PBS, dried and imaged.2.9For the in vivo experiments, we used NSG MICE. All scid mice, housed in micro isolator cages, were used between 6 and 8 weeks of age. All procedures were carried out in accordance with the guidelines for animal experimentation following the European Union of the National and Kapodistrian University of Athens, Medical School Bioethics Committee in agreement with the European Union . For injection of cell suspensions, we used 1‐5 million cells in 100 μL into the right flank of each mouse and allowed to grow for approximately 3‐4 weeks to reach a tumour size of 100 mm3. The mice were randomly divided into groups (n = 5 per group) for each treatment, control, NU1025 (1 mg/mouse), SU11274 (1 mg/mouse) and NU1025 + SU11274. Inhibitors were injected intraperitoneally every 4 days. The mice were killed, and solid tumours were measured and excised after 20 days of treatment. The tumour volume was calculated using the following formula: 1/2(length × width2).2.10The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions  for 2 weeks prior to experimentation. Intragastric gavage administration was carried out with conscious animals, using straight gavage needles appropriate for the animal size . All animals were killed by barbiturate overdose  for tissue collection.2.11P < .05, **P < .005, ***<0.0005.The results are representative of three independent experiments and expressed as mean values ± SD (standard deviation). For sample size, we used G*Power software version 3.1. For the calculation of tumour volume, we used Microsoft Excel 10. The results were evaluated by t test. Error bars indicate ± SD. *33.1Different gastric cancer cell lines were examined regarding their protein levels of BRCA1, BRCA2 and c‐MET using Western blot analysis. In detail, the protein levels of BRCA1, BRCA2 and c‐MET were decreased in the AGS cell line as compared to HS746T  in an increasing dose‐dependent manner (0‐40 μmol/L) for 48 hours with MTT assay. We used HS746T and AGS GC cell lines to exhibit high and low protein levels of c‐MET, respectively Figure . Althoug3.3In order to identify whether the expression of BRCA has differential drug sensitivity, we continue with the silence of BRCA1/2 by siRNA in the HS746T cell line Figure . Then, HAdditionally, in order to identify the primary mechanism of expression of BRCA impact on c‐MET, we continued with the silence of c‐MET. The knockdown of c‐MET increases the sensitivity of BRCA‐deficient HS746T cells to NU1025 after 48 hours Figure . BRCA1/23.4In the light of previous results that silence of c‐MET and/or BRCA1/2 sensitizes AGS and Hs746T cells to NU1025 treatment, we continue with the investigation of an additive effect in co‐inhibition of c‐MET (5 μmol/L SU11274) and PARP (5 μmol/L NU1025) inhibitor in order to further reduce the proliferation of GC cell lines. When Hs746T cells (control and siControl) were treated with SU11274 or NU1025 alone or combined, the additive effect was not observed after 48 hours  cell lines with PARP and c‐MET inhibitors reduces cell viability through apoptosis and attenuated tumour growth in xenograft models. Our results highlight the impact of BRCA1/2 deficiency on gastric tumorigenesis. Moreover, we identified a mechanism that involves γ‐H2AX foci formation, which is well‐known DNA damage marker.Over the last few years, a plethora of studies have identified c‐MET as a potential target for GC tumours. Several small molecules have been developed against the c‐MET pathway with promising results in clinical studies.In our experiments, in order to identify the impact of c‐MET in PARP inhibition, we use two different GC cell lines, HS746T and AGS, with high and low protein levels of c‐MET, respectively, in the presence of NU1025 (PARP inhibitor).The inhibition of PARP did not cause significant inhibition on cell viability of both cell lines. Knockdown of c‐MET with siRNA sensitizes GC cells, especially AGS, in PARP inhibition. These results partially recognize c‐MET as a resistance mechanism to PARP inhibition in the GC cell line model.Several studies highlight the vital role of BRCA in malignancy. Huang et alIt is well known that c‐MET activation increases the DNA repair function of PARP1.Du et alPARP1 is a crucial protein for sensing single‐stranded (ss)DNA breaks (SSBs).It is known that BRCA deficiency causes problems in the DNA damage repair mechanism. Tumours with mutant BRCA genes are more sensitive to PARP inhibition.An assay for histone γH2AX generally reflects the presence of double‐strand breaks in DNA. In our experiments, the detection of cl. Caspase‐3 and PARP in co‐inhibition of PARP and c‐MET does not correlate with the protein levels of γ‐H2AX by Western blot analysis.There were some limitations to this study. First, the sample size of in vivo experiments may not have been large enough. Second, an in‐depth analysis of signalling pathways that are controlled by c‐MET has not been tested. Third, the relationship between BRCA1/2 and c‐MET needs further tests; for example, mutations on BCA1/2 and c‐MET can change the effect of NU1025 and SU11274 inhibitors. Nevertheless, these limitations may point to the future direction along the lines of this study.5Our results collectively evaluate the additive effect of c‐MET and PARP inhibition in the GC cell line model. Both inhibitors (NU1025 and SU11274) trigger DNA damage response and apoptotic cell death, resulting in the reduction of cell viability of GC cell lines, especially with BRCA deficiency status. Furthermore, the xenograft mouse model supports our hypothesis that NU1074 plus SU11274 displays a significant reduction of tumour growth in a cell line with low levels of BRCA1/2 and c‐MET. Our results evaluate that the co‐administration of NU1074 and SU11274 has an additive effect. Based on these findings, further clinical testing of this combinatorial scheme is suggested in patients with locally advanced and/or metastatic gastric cancer.The authors declare no potential conflicts of interest.Evangelos Koustas: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology . Michalis V. Karamouzis: Conceptualization ; Data curation ; Formal analysis . Panagiotis Sarantis: Data curation (supporting); Investigation (supporting); Methodology (supporting). Dimitrios Schizas: Data curation (supporting); Formal analysis (supporting). Athanasios G Papavassiliou: Conceptualization ; Data curation ; Formal analysis .All procedures were carried out in accordance with the guidelines for animal experimentation of the National and Kapodistrian University of Athens, Medical School Bioethics Committee in agreement with the European Union .All authors confirm that any aspect of the work covered in this manuscript that has involved either cell lines or animal models has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.All authors consent for the publication of the manuscript.Figure S1Click here for additional data file.


Malignant peripheral nerve sheath tumors (MPNSTs) are a rare subtype of soft-tissue sarcoma, derived from a peripheral branch or the sheath of the sciatic nerve, brachial plexus, or sacral plexus. The clinical outcomes for MPNST patients with unresectable or metastatic tumors are dismal, and novel therapeutic strategies are required. Although patient-derived cancer cell lines are vital for basic research and preclinical studies, few MPNST cell lines are available from public cell banks. Therefore, the aim of this study was to establish cancer cell lines derived from MPNST patients.We used tumor tissues from five patients with MPNSTs, including one derived from a rare bone tissue MPNST. The tumor tissues were obtained at the time of surgery and were immediately processed to establish cell lines. A patient-derived xenograft was also established when a sufficient amount of tumor tissue was available. The characterization of established cells was performed with respect to cell proliferation, spheroid formation, and invasion. The mutation status of actionable genes was monitored by NCC Oncopanel, by which the mutation of 114 genes was assessed by next-generation sequencing. The response to anti-cancer agents, including anti-cancer drugs approved for the treatment of other malignancies was investigated in the established cell lines.We established five cell lines  from the original tumors, and also established patient-derived xenografts (PDXs) from which one cell line (NCC-MPNST3-X2-C1) was produced. The established MPNST cell lines proliferated continuously and formed spheroids while exhibiting distinct invasion abilities. The cell lines had typical mutations in the actionable genes, and the mutation profiles differed among the cell lines. The responsiveness to examined anti-cancer agents differed among cell lines; while the presence of an actionable gene mutation did not correspond with the response to the anticipated anti-cancer agents.The established cell lines exhibit various characteristics, including proliferation and invasion potential. In addition, they had different mutation profiles and response to the anti-cancer agents. These observations suggest that the established cell lines will be useful for future research on MPNSTs. TP53 [CDKN2A [SUZ12 and EED, which are subunits of the Polycomb repressive complex 2 (PRC2) [Malignant peripheral nerve sheath tumor (MPNST) is a rare subtype of soft-tissue sarcomas representing approximately 10% of all soft-tissue sarcomas , 3. MPNSTP53  and CDKN [CDKN2A  are obse2 (PRC2) . The los2 (PRC2) –12. In f2 (PRC2) , PI3K/AK2 (PRC2) , and Raf2 (PRC2)  was repo2 (PRC2) . Current2 (PRC2) ; however2 (PRC2) , sorafen2 (PRC2) , imatini2 (PRC2) , dasatin2 (PRC2) , and ali2 (PRC2)  have not2 (PRC2) , 24, andhttps://web.expasy.org/cellosaurus/), 24 MPNSTs cell lines have been reported in previous studies. However, only five are available from a public cell bank such as American Type Culture Collection (ATCC) [Patient-derived cancer models such as cell lines and xenografts are indispensable tools for furthering our understanding of the molecular mechanisms of carcinogenesis and cancer progression. We can investigate the functions of novel genes and proteins and determine the efficacy of novel drugs using cell lines. Furthermore, cell lines allow high-throughput screening of the anti-tumor effects of investigational drugs  and analn (ATCC) , 28. ConIn this study, we established six patient-derived MPNST cell lines from five patients who exhibited unique clinical features and investigated the morphological and functional characteristics as well as anti-cancer drug responses of these established cell lines.This study included tumor tissues from five patients with MPNST, who underwent surgical resection at the National Cancer Center Hospital, Tokyo, Japan. This study was approved by the ethical committee of the National Cancer Center, and all patients in this study provided written informed consent.PrkdcscidIl2rgtm1Sug/Jic, also known as NOD/Shi-scid IL-2Rγnull or NOG; Central Institute for Experimental Animals, Kanagawa, Japan) using a 13-gauge transplant needle. Tumor size was measured using a digital caliper  and tumor volume was calculated as pi/6 × length × width × thickness [3. After two passages, the tumors were cryopreserved using Cell Banker 1 plus  in liquid nitrogen. All animal experiments were performed in accordance with the guidelines for Animal Experiments of the National Cancer Center and approved by the Institutional Committee for Ethics of Animal Experimentation.MPNST tissues were obtained at the time of surgery and were subcutaneously grafted into the bilateral hind flanks of 6–12-week-old female mice with a severe immunodeficient phenotype .g, and seeded in tissue culture plates . The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum , 100 U penicillin G, and 100 µg/mL streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO2. The tissue culture medium was refreshed every several days, and when the cell monolayers reached sub-confluence, the cells were dispersed with 0.1% trypsin–EDTA (Gibco) and seeded into other culture plates.The tissues of original tumors and PDXs were minced with scissors and passed through an 18-gauge needle. Cell aggregates and tissue fragments were removed with a 70-μm nylon mesh . Cells were collected by centrifugation for 5 min at 200 xThe cell lines were authenticated by analyzing short tandem repeats (STRs) in ten loci using the GenePrint 10 system  according to the manufacturer’s instructions. In brief, genomic DNA was extracted from tumor tissues or cultured cells using AllPrep DNA/RNA mini kits , quantified using a NanoDrop 8000 instrument (Thermo Fisher Scientific), and stored at − 80 °C until further use. Genomic DNA (500 pg) was amplified and examined using a 3500xL Genetic Analyzer . The digital data were analyzed using the GeneMapper software (Applied Biosystems), and the results were compared with the STR profiles available in public cell banks such as ATCC, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and Japanese Collection of Research Bioresources (JCRB).Mycoplasma in the established cell lines was also determined. Briefly, DNA was recovered from culture supernatants when the cells reached 70–90% confluence, heated at 95 °C for 10 min, and amplified using the e-Myco Mycoplasma PCR detection kit . The amplified DNA was separated using electrophoresis on 1.5% agarose gels, stained with the Midori Green advanced stain , and analyzed using an Amersham Imager 600 .Possible contamination with 3 cells per well  or 12 and 16 × 103 cells per well (NCC-MPNST3-C1 and NCC-MPNST3-X2-C1 lines), incubated at 37 °C, and analyzed for growth at 24, 48, 72, and 96 h. At the end of each time point, the cell counting kit (CCK)-8 reagent  was added to the cells and incubated for 2 h, and the absorbance at 450 nm was measured using a microplate reader . Growth curves were constructed by plotting the absorbance value (y-axis) versus culture time (x-axis) and were used to calculate the doubling time for each cell line. All experiments were performed in triplicate.MPNST cells were seeded into 96-well culture plates at densities of 2, 4, and 8 × 105 cells for NCC-MPNST1-C1, NCC-MPNST2-C1, NCC-MPNST3-C1, NCC-MPNST3-X2-C1, and NCC-MPNST4-C1, and 1 × 106 cells for NCC-MPNST5-C1 were seeded into 6-cm plates  in DMEM containing 10% FBS and incubated in a humidified atmosphere of 5% CO2 at 37 °C. The presence of spheroids was confirmed by microscopic observation . All assays were performed in duplicate.To explore spheroid formation, 1 × 105 cells were plated in the upper chamber in serum-free DMEM, whereas the medium in the bottom chamber contained 10% FBS, and the plates were incubated for 48 h. The tumor cells invading the bottom surface were stained with Diff-Quick staining kit  according to the instruction manual, and their number was counted in nine separate areas under a microscope at the magnification of 200×.The invasive ability of the cultured cells was assessed using the Transwell assay and BD Biocoat Matrigel invasion chambers  according to the manufacturer’s instructions. In brief, 1 × 104 cells were seeded into the RTCA upper chamber. The cells were starved in serum-free medium for 24 h. Then, the complete growth medium was added to the RTCA lower chambers as a chemo-attractant. The cells invading from the upper chamber through the Matrigel membrane into the bottom chamber contacted and adhered to the electronic sensors on the underside of the membrane. The attached cells affected the electrical impedance of the membrane; the impedance correlated positively with the number of cells on the bottom of the membrane [RTCA Cim-16 plates  in a label-free real-time setting was used for RTCA invasion assays according to the manufacturer’s protocol. In brief, Matrigel at a protein concentration of 8.95 mg/mL  was subjected to the upper sides of the RTCA membrane, and 4 × 10membrane . The impAlterations, including mutations, amplifications, and homozygous deletions in the coding region of 114 genes  for 72 h and analyzed for survival using the CCK-8 reagent according to the manufacturer’s protocol. The list of anti-cancer agents is provided in Additional file Cells were seeded in a 384-well plate at 5 × 10Mapping of NGS reads to the human reference genome was performed with the Burrows-Wheeler Aligner  and the Cluster analysis was performed using the R software version 3.4.0 . The disCell lines were established from tumor tissues of five patients with MPNSTs. The overall design of the study, including cell line designation, is illustrated in Fig. Case 1: The donor was a 37-year-old woman with NF1, who had a first-degree relative with NF1. She had a painful mass on the right posterior thigh detected using magnetic resonance imaging (MRI) Fig. a, which Case 2: The donor was a 54-year-old man without NF1. He presented a left femoral bone tumor with a pathological fracture  was designated as NCC-MPNST3-X2-C1. The patient had a local recurrence after 3 months, and progression of other metastatic lesions was observed in the final follow-up 13 months after the surgery despite chemotherapy with doxorubicin and pazopanib; representative MRI and CT images are shown in Fig. Case 4: The donor was a 48-year-old man without NF1. The original tumor developed in the posterior aspect of the right thigh Fig. g. The paCase 5: The donor was a 9-year-old girl with NF1 who had wide bilateral excisions in her thigh after two surgeries. The surgical margin of the left thigh was positive, and radiotherapy of 50 Gy was performed postoperatively. Almost 1 year later, a rapidly growing mass at the left popliteal fossa was detected using MRI and positron emission tomography-computed tomography Fig. i, j, andThis study included five patients to establish the patient-derived cancer models. The clinical characteristics of the donor patients are presented below. Cell lines as well as their corresponding donor patients and samples are summarized in Table Surgically resected tumor tissues were subcutaneously inoculated into immunodeficient mice; after in vivo propagation, they were serially transplanted twice. HE staining of tumors from different PDX transplantation passages indicated histopathological characteristics that were consistent with those of their original tumors Fig. a, b. TheWe determined the statuses of ten microsatellite STRs in the original tumor tissues, PDXs, and cell lines Table . All theThe growth rates of the cell lines were assessed by seeding a different number of cells and calculating the doubling time, which was 40, 57, 66, 58, 77, and 33 h for NCC-MPNST1-C1, NCC-MPNST2-C1, NCC-MPNST3-C1, NCC-MPNST3-X2-C1, NCC-MPNST4-C1, and NCC-MPNST5-C1 cells, respectively (Fig. The in vitro invasion assays revealed that the established cell lines exhibited different invasive abilities Fig. a, b. TheWe examined the status of genetic mutations in the established cell lines using NCC Oncopanel, which consists of the examinations of 114 actionable genes (Additional files Finally, we assessed the sensitivity of the established cell lines to treatment with known chemotherapeutic agents and quantitatively evaluated cell viability (Additional file Patient-derived cancer models are useful tools not only for basic research but also for preclinical studies. Various cell lines obtained from donors with different clinical background possess distinct in vitro characteristics, thereby representing a unique tool for clinical research. In addition, the patient-derived cell lines may retain original characteristics of the tumor tissues, making them adequate resources for the study of disease etiology and tumor cell origin. Here, we describe the establishment of six cell lines and one PDX from tumor tissues of five MPNST patients with different clinicopathological features. These cell lines may reflect the clinical diversity of the disease and can be used to further our understanding of various MPNST manifestations. The comparative study between the features of the tumor in patients and the characters of cell lines and the PDX is intriguing. To perform such studies, we need enough number of patient-derived cancer models, considering the diversity of the disease.The clinical backgrounds and characteristics of the cell lines used in this study were diverse. Cases 1, 3, and 5 were diagnosed with NF1, however, Cases 2 and 4 were not, and the tumor in Case 1 originated in the bone , whereasWe observed that the proliferation potential of tumor cells in PDX models varied and depended on the passage number Fig. . Thus, oThe responses of the established cell lines to treatment with anti-cancer agents were also intriguing. According to their sensitivity to the agents, cell lines were classified into two groups; the first consisted of NCC-MPNST1-C1, NCC-MPNST4-C1, and NCC-MPNST5-C1, and the other of NCC-MPNST2-C1, NCC-MPNST3-C1, and NCC-MPNST3-X2-X1. The examined anti-cancer agents were also grouped into two categories according to the effects on the MPNST cells, such as those in Cluster A and Cluster B. The molecular backgrounds of MPNST cells about the sensitivity to anti-cancer agents are worthy of further investigation./in vivo models can predict the clinical response of MPNST patients should be addressed using a statistically adequate number of cases in the prospective studies. Second, the molecular mechanisms underlying the described in vitro observations should be investigated to confirm the utility of patient-derived cancer models using comprehensive experimental approaches such as genomics, proteomics, and proteogenomics. Third, the further characterization of established cell lines should be required to establish the similarities between the cell lines and original tumor cells. Further, many factors can affect cell line characteristics, therefore, it is worth investigating the typical genetic and epigenetic aberrations of MPNST, including NF1 gene mutations [The present study had certain apparent limitations. First, the number of examined cases and established cell lines used in this study were small. Hence, the observed correlation trends between clinical characteristics and cell behavior in vitro should be validated using more patients and cell lines. The general question of how faithfully the established patient-derived in vitroutations , and losutations , which iWe successfully established PDXs and cell line models of MPNST with different genomic backgrounds and in vitro characteristics, such as drug responses. As our cell lines exhibited different phenotypes, probably reflecting those of their original tumors, they will be useful research tools for studying the clinical diversity of MPNST. Continuous efforts for developing and analyzing patient-derived cancer models will contribute to the improvement of clinical trials and the development of effective therapies for MPNST and other cancers.Additional file 1: Table S1. Genes examined by NCC Oncopanel.Additional file 2: Table S2. Anti-cancer agents examined in this study.Additional file 3: Table S3. Summary of detections by NCC Oncopanel.Additional file 4: Table S4. Detection of SNV and indels by NCC Oncopanel.Additional file 5: Table S5. Detection of CNV by NCC Oncopanel.Additional file 6: Table S6. Detection of fusion by NCC Oncopanel.Additional file 7: Table S7. Detection of Mapping by NCC Oncopanel.Additional file 8: Table S8. Detection of depth by NCC Oncopanel.Additional file 9: Table S9. Cell viability after treatment with anti-cancer agents.


Ovarian cancer remains a significant challenge in women worldwide. Tumors of the high-grade serous carcinoma (HGSC) type represent the most common form of the disease. Development of new therapies for HGSC has been hampered by a paucity of preclinical models in which new drugs could be tested for target engagement and anti-tumor efficacy. Here, we systematically assessed in vivo growth of ovarian cancer cells, including six validated HGSC cell lines, in highly immunocompromised NSG mice by varying the injection site. We found that, with the exception of OVCAR3, HGSC cell lines COV318, COV362, KURAMOCHI, OVCAR4, and OVSAHO, generally demonstrate poor growth as either subcutaneous or intraperitoneal xenografts. Intrabursal injections performed with KURAMOCHI and COV362 cells did not improve tumor growth in vivo. Additional analysis revealed that OVSAHO and COV362 express moderate levels of estrogen receptor (ERα), which translated into improved growth of xenografts in the presence of 17β-Estradiol. Surprisingly, we also found that the growth of the widely used non-HGSC ovarian cell line SKOV3 could be significantly improved by estrogen supplementation. By describing successful establishment of estrogen-sensitive HGSC xenograft models, OVSAHO and COV362, this work will enable testing of novel therapies for this aggressive form of ovarian cancer. Classification of the ovarian epithelial cancers is based on histology, with the five main types being: clear cell, endometrioid, mucinous, low-grade serous, and high-grade serous tumors3. High-grade serous carcinoma (HGSC) accounts for 70% of all ovarian cancers and is also one of the more lethal forms of ovarian cancer due to its rapid growth and early spread to other organs in the peritoneal cavity4. Genome instability is a prominent feature in this cancer type, with aneuploidy widely reported across HGSC tumors5. Concomitantly, the tumor suppressor TP53 is mutated in nearly 100% of this cancer8. Additionally, a low number of mutations are found in NF1, BRCA1, BRCA2, RB1, and CDK12 genes. Consistently, mice with Cre-mediated deletion of RB1 and TP53 in the ovarian bursa develop ovarian cancer with serous histology9.S1Ovarian cancer remains one of the leading causes of cancer-related deaths in women worldwide, with the all-stage 5-year relative survival rate across the globe of only 30–40%10. A subset of HGSC patients with BRCA1/2 mutations is now treated with PARP inhibitors , which were recently approved by Food and Drug Administration (FDA). Their use demonstrates the power of targeted therapy, which could significantly increase survival rates in women with ovarian cancer11. Development of new therapies for the HGSC has however been hampered by scarcity of easy-to-use, high-fidelity preclinical tumor models.The current therapy landscape for HGSC is dominated by surgery and platinum-based chemotherapy, which has been standard of care for almost 40 yearsKRas) or conditional deletion of relevant tumor suppressors  in ovarian surface epithelium16 or fallopian tube20. However, while they are highly relevant for in vivo studies involving immunomodulation, their use also entails a number of disadvantages, including dissimilarity to the human disease, relatively long latency and high cost. On the other hand, the use of more affordable human cancer cell-line derived xenografts, with their own limitations , remains in the mainstream of drug development. For a long time, human ovarian cancer cell lines of a non-HGSC origin had been used as xenografts until a dedicated genetic analysis identified those with the likely HGSC genotype21. Consequently, two studies in 2015 investigated the growth of a panel of validated HGSC cell lines in vivo and revealed significant hurdles in establishing ovarian cancer models as either subcutaneous or intraperitoneal xenografts in immunocompromised mice. Mitra et al. found that, of 11 HGSC cell lines tested in nude mice, approximately a half  formed intraperitoneal tumors with HGSC histology, while far fewer cell lines (OVCAR3 and OVCAR5) gave rise to usable subcutaneous xenografts22. Elias et al. managed to grow small subcutaneous xenografts from two additional HGSC cell lines, KURAMOCHI and OVSAHO, using the highly immunodeficient NSG (NOD scid gamma) mouse strain23.There is a growing number of transgenic mouse models of ovarian HGSC, based on conditional expression of oncogenes  and estrogen receptor (ER), which correlate with the survival in some ovarian tumor entities. For example, strong PR, but not ER, expression was associated with improved survival in HGSC patientsHere, we report a study in which we systematically assessed and compared the growth of HGSC  and non-HGSC  ovarian cancer cells, as well as their luciferase (Luc)-transfected variants, in highly immunocompromised female NSG mice. We describe their growth characteristics as subcutaneous, intraperitoneal, and intrabursal models and identify OVSAHO and COV362 as estrogen-sensitive subcutaneous xenograft models suitable for preclinical drug development. We also show that the very well established non-HGSC cell line SKOV3 shows sensitivity to estrogen supplementation for its growth as a tumor xenograft, suggesting a broader role of hormone signaling in modeling different types of ovarian cancer in vivo.2. Authentication of the cells was established in-house by short tandem repeat (STR) profiling, with results being matched to online databases, e.g., https://www.dsmz.de/fp/cgi-bin/str007.cgi with an 80% acceptance rate. Cells were regularly screened for Mycoplasma using a PCR-based assay and were cultured for no longer than 8 weeks. Cell lines that expressed firefly luciferase (Luc) were transduced with a Luc-encoding lentiviral system. The open reading frame (ORF) encoding a firefly Luc was taken from pGL4.1 (Promega) as a NheI/XbaI fragment and ligated into the multiple cloning site (MCS) of pCDH-CMV-MCS2-EF1-Hygro (System Biosciences Inc). Cells were then selected using hygromycin B (Thermo Fisher 10687010), with the optimal concentration having been determined for each individual cell line from kill curves obtained previously on the parental lines. The transduction efficiency was high with these cell lines (apart from OVSAHO), so no colony selection was required, and all transduced cells were used for expansion. Cell lines were checked for luminescence on the In Vivo Imaging System (IVIS) Lumina X5 (Perkin Elmer) prior to implantation in vivo.Cell lines Table  were obt6 cells were seeded in 10-cm dishes and incubated overnight, after which the cells were rinsed with ice-cold Ca2+–Mg2+-free PBS  and lysed in 500 μl ice-cold lysis buffer . Lysates were gently passed three times through a 27G needle, after which they were cleared by centrifugation at 11,000g for 15 min at 4 °C. Supernatants containing soluble proteins were collected, and protein content was determined using the Pierce BCA Protein Assay Kit . Proteins were resolved on a NuPAGE 4–12% Bis–Tris protein gel  and transferred onto Immobilon-P PVDF membrane . Immobilized proteins were detected using respective primary antibodies: rabbit anti-mouse estrogen receptor 1α , rabbit anti-mouse GAPDH ; and a secondary antibody [HRP-linked anti-rabbit IgG ].Approximately 2–3 × 103 cells/well in 96-well plates  in charcoal-absorbed media , supplemented with 10% activated charcoal-absorbed FBS  and, if required, 500 pg/ml 17β-Estradiol , and then incubated for the indicated period of time. CellTiter-Glo  was used to assess cell proliferation as directed by the manufacturer. Alternatively, cell growth was observed using the IncuCyte S3 CMP cell imager (Essen BioScience). Cell counts were performed in triplicates.For the analysis of cell proliferation in 2D conditions, cells were seeded at 4 × 103 cells/well in Ultra-Low Attachment (ULA) 96-well plates  in charcoal-absorbed media, supplemented with 10% activated charcoal-absorbed FBS and, if required, 500 pg/ml 17β-Estradiol, and then incubated for the indicated period of time. Spheroid growth was visualized every day for 8 days, using the Celigo Imaging Cytometer (Nexcelom Bioscience), while the medium was replenished every second day. Individual spheroids from six wells per cell line per condition were analyzed.For 3D spheroid assay, cells were seeded at 4 × 1027. All cell lines were tested in 6–7-week-old, female non-obese diabetic (NOD), severe combined immunodeficiency (scid), IL2 receptor common gamma chain mice, NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ), bred at the Institute of Cancer Research (ICR) London.All animal studies and breeding were approved by the institutional Animal Welfare Ethical Review Body (AWERB) and carried out in accordance with UK Home Office Regulations under the Animals (Scientific Procedures) Act 1986 and national guidelines6 cells were mixed with 50% Matrigel  and implanted subcutaneously into either one or two flanks. Once visible, tumors were measured across two diameters, and volumes were calculated using the formula V = 4/3π [(d1 + d2)/4]3. Body weights were also recorded. Once tumors approached Home Office License limits (i.e. maximum mean diameter of 1 cm), or experiments exceeded 150 days, mice were culled and tumors were weighed and snap or viably frozen, if present.For subcutaneous (SC) tumor xenograft studies, 5 × 107 cells were mixed with 50% Matrigel and implanted into the peritoneal cavity. Mice injected with parental cell lines were weighed and palpated weekly to assess for signs of tumor burden. Mice injected with cell lines expressing the Luc, in addition to the above methods of monitoring, were imaged weekly using the in vivo imaging system (IVIS) Lumina X5 (PerkinElmer) after an intraperitoneal injection of Luciferin  at 150 mg/kg body weight. Total flux was measured using the Living Image software.For intraperitoneal (IP) tumor xenograft studies, 1 × 104 cells in 5 µl) in both ovarian bursae of the isoflurane-anesthetized mice, to see if the growth rate could be enhanced. Animals were weighed and palpated weekly. Mice implanted with the Luc-expressing cells were also imaged on the IVIS once weekly as above.For intra-bursal models, COV362 and KURAMOCHI cell lines, both parental and transduced with the Luc, were implanted . Cell implantation and tumor monitoring were initiated 24–48 h post pellet implant.For all studies, once a substantial weight gain was witnessed (i.e. ascitic fluid build-up), a high-flux reading was observed on IVIS, or a large mass could be palpated, mice were culled, and tumors collected for analysis.28, allows engraftment and growth of human tumors with an unprecedented efficiency29. We tested a panel of ten ovarian cancer cell lines  alongside the parental cell lines revealed reduced tumor growth for the genetically altered cell lines of the non-HGSC type . Luc-transduced COV318 and COV362 did not produce any subcutaneous tumors similarly to the parental cell lines (data not shown).In order to study ovarian tumor growth orthotopically, we also produced Luc-expressing versions of the ovarian cell lines, which we first tested for their growth in a subcutaneous location. All cell lines, with the exception of OVSAHO and OVCAR3, could be established to express Luc stably Table . InjectiThus, unlike the non-HGSC xenograft models of ovarian cancer, only very few HGSC cell lines could be grown as subcutaneous tumors in the advanced immunocompromised NSG strain. They can be represented in the order of the efficiency with which they show tumorigenicity at a subcutaneous location: OVCAR3 > OVCAR4 > OVSAHO > KURAMOCHI > COV362 > COV318. With the exception of the OVCAR3 cell line, HGSC xenografts generally require a long engraftment time and result in smaller tumors. NSG mice tolerated the subcutaneous engraftment of ovarian cancer cells well, as judged by positive body weight dynamics  produced small tumors throughout the abdomen  formed rapidly growing intraperitoneal tumors with ascites , while revealing the potent ascites-forming capacity of many cell lines across the spectrum of ovarian cancer models  of parental and Luc+ cells, COV362 and KURAMOCHI, were injected into the surgically exposed bursa of the female NSG mice, and the animals were monitored for general well-being, change in body weight, and ascites. Luc+ cell lines were also monitored by IVIS. The results of this study are summarized in Table Given the limited success of the intraperitoneal models, we also tested several of the ‘more difficult’ HGSC cell lines as orthotopic models using the ovarian bursa of the mice, which may arguably represent a better environment for the autochthonous growth of some ovarian cancers. Small numbers 5 × 10 cells of+ line failed to develop tumors (data not shown). On the other hand, both COV362 and COV362 Luc+ tumors could be observed approximately 100 days post injection in some animals  detected in SKOV3 and the HGSC cells, OVSAHO and COV362  prompted us to test whether estrogen can help establish HGSC xenografts in the highly immunocompromised NSG mice. We injected COV362 and OVSAHO cells subcutaneously into the flank of ovariectomized mice that had also been implanted with a 60-day 17β-Estradiol pellet. MCF7 cells were used as a well-described positive control of an estrogen-dependent xenograft model, while OVCAR4 were used as a negative control , we investigated the growth of ovarian cancer xenografts in the highly immunodeficient NSG mice. We confirmed that the unmodified  non-HGSC cell lines, SKOV3, JHOC5, IGROV-1 and OVK-18, readily form tumors when injected either subcutaneously or intraperitoneally . This pleiotropic effect of estrogen will therefore potentially cause an altered response to a therapy on the part of ovarian tumors grown in the animal model. While a number of ER+ breast cancer models, such as MCF-7 that require in vivo estrogen supplementation (reviewed in41), are routinely used in drug development, future work will need to investigate the effect of estrogen supplementation on ovarian cancer cell biology and response to targeted therapeutics.Another potential caveat of hormone supplementation in preclinical models in vivo will undoubtedly be its physiological and supraphysiological effects on tumor cells. Ligand-bound ERα recognizes over 70,000 estrogen-response elements (EREs) in the human and mouse genomesSupplementary Information 1Supplementary Information 2


GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.Exposure to physiological estrogens or xenoestrogens  increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes ( Fusarium sp., which is one of the most economically important fungal genera. They frequently infest crops in the field or during storage and thus ZEA is one of the most frequently isolated mycotoxins in cereals, mainly in maize [Cancer is the second leading cause of death worldwide. Among women, ovarian cancer is considered to be the fifth most common cause of cancer death and it is the most lethal form of gynecological malignancy . It is gin maize ,7,8. BPAin maize ,10. BPA in maize ,10. Bothin maize ,12,13.Estrogens contribute to the development of cancer by affecting the expression of several genes involved in cellular proliferation, apoptosis or DNA repair. Furthermore, estrogens support metastasis formation through promoting tumor cell migration by inducing epithelial-to-mesenchymal transition (EMT), where tumor cells lose their epithelial features and gain a mesenchymal phenotype ,14. EstrAlthough the carcinogenic effect of estrogens is a well-known phenomenon in breast cancer , limitedGAPDH). The expression of ERα was not detectable by qPCR in the A2780 cell line in our measurements, which is in good agreement with previous results [Two human epithelial ovarian cell lines were used in the study. PEO1 is considered to be an estrogen-responsive cell line that highly expresses ERα ,27. ERα  results ,26. TherIn the proliferative assay, cell counts relative to the non-treated controls were compared. As Cell migration was studied by wound-healing scratch assay where wound closure % was used to indicate the rate of cell migration. The migration rate of the PEO1 and A2780 cell lines proved to be comparable 24 h after scratching in the non-treated control wells: wound closure % was 18.7 ± 3 in the case of the PEO1 cell line and 16.5 ± 2.6 in A2780. It is important to note that wound closure % was significantly higher in the A2780 cell line (36.1 ± 5) than in the PEO1 (24.3 ± 4) 48 h after scratching, which suggests a higher migrative ability of A2780. In order to compare the migrative effect of E2, ZEA and BPA molecules on the two cell lines, wound closure % was estimated 24 h after scratching because of the comparable migration rate of the two cell lines at this time point in the non-treated control wells. The addition of E2 and BPA increased the rate of migration in case of the PEO1 cell line at 10 nM concentration. ZEA proved to have the strongest migrative effect since migration was induced at both 10 and 100 nM doses . None ofIn order to characterize the toxic effect of these molecules to ovarian cells, apoptosis and cell lysis were studied. The decrease of mitochondrial membrane potential is an early marker of apoptosis that was determined by the DilC1(5) assay. According to our results, the rate of apoptosis was not comparable after the addition of E2, ZEA or BPA with the rate measured in case of the positive control in the tested cell lines . This suGREB1 is a highly estrogen-responsive growth regulator [DEPTOR is an mTORC1 target [CA12 codes for an anhydrase [RBBP8 is involved in the regulation of DNA replication, transcription, in G1 and G2 checkpoint control and DNA repair [CDH1 and ZEB1 was also studied. CDH1 codes for E-cadherin and its downregulation contributes to tumor cell invasion by promoting EMT of the tumor cells [ZEB1 is involved in the inhibition of CDH1 expression and induces EMT [The effect of E2, ZEA and BPA on gene expression was also compared. Based on previous data and biological significance, four estrogen-responsive genes were selected for gene expression analysis: egulator ; DEPTOR 1 target ; CA12 conhydrase ; RBBP8 iA repair . All of A repair ,33,34. Bor cells . ZEB1 isuces EMT .GREB1, CA12 and RBBP8 was comparable in the estrogen-responsive PEO1 and non-responsive A2780 cell lines. However, the relative expression of DEPTOR, CDH1 and ZEB1 genes differed in the tested cell lines where the deviation in CDH1 expression proved to be the most significant , miR34a, miR34b and miR203a are considered to play important roles in tumor cell migration by the regulation of and CDH1 ,37,38. Mand CDH1 . As FiguWe also observed changes in miRNA expression in response to E2, ZEA and BPA exposure in the case of the PEO1 cell line . Estrogep < 0.001), the same as in the A2780 cell line (r = 0.71 (p < 0.01)).The cell-free expression of miR200s, miR34a, miR34b and miR203a was also determined. According to our results, all the tested miRNAs were detectable in the supernatant of both cell lines, although the miRNA content of the supernatant was lower than what was observed in the cell lysates (ΔCT (supernatant-cell lysate) = 7.9 ± 1.9). A similar phenomenon was reported previously . The lowThe effect of E2, ZEA and BPA treatments on the expression of cell-free miRNAs was also determined. In the case of the ZEA and BPA treatments, a similar tendency was observed in the medium as to what was observed intracellularly, i.e., a higher miRNA content was detected at 12 h which decreased 24 h after the treatment . NotablyThe high basal expression level of miR200b and miR200c suggests their high biological relevance in PEO1. We also investigated whether the cell-free counterparts of these miRNAs are able to influence the microenvironment through their transport into neighboring cells. In these experiments a co-culture assay was performed where PEO1 cells were seeded to a transwell chamber that was placed on the top of the A2780 cultures. Intracellular miR200b and miR200c levels of A2780 was determined by qPCR 24 and 48 h after co-culturing. According to our results the miR200b and miR200c levels of co-cultured A2780 cells increased significantly compared to the non-co-cultured cells in a time- dependent manner .GREB1 and CA12 genes was determined by qPCR. According to our results the effect of E2 was significantly decreased in the case of the miR200 family members and miR203a, as well as in the case of GREB1 and CA12, after the addition of MPP (We aimed to investigate the ERα dependence of miR200s and miR203a expression. For this reason we added methyl-piperidino-pyrazole (MPP), a well-known ERα-selective antagonist , to the n of MPP .GREB1 and CA12 protein coding genes, whose induction in response to E2 treatment was confirmed previously (MIR200B-MIR200A-MIR429 locus (2) chr12:6943920–6983920, covering the MIR200C-MIR141 locus and (3) chr14:104097437–104137437, covering MIR203A locus , a method used to map protein–DNA interactions genome-wide, was applied to identify such putative cis-regulatory elements. We used publicly available ChIP-seq data for this purpose. At the time of our study, ERα ChIP-seq from human ovarian cells was not available, therefore, a ChIP-seq performed in endometrial Ischikawa cell line  was usedeviously . The add3A locus . The mulse genes , Table 1 for ERα . We concmic loci .GREB1, CA12, DEPTOR, RBBP8) after E2 exposure in PEO1 also supports the idea that the observed phenotype is due to gene expression changes induced by E2, which were not observable in A2780. ZEA and BPA were also able to induce cell proliferation and migration in several cell lines [The role of estrogens in the development of breast cancer is a well-known phenomenon . Howeverll lines ,48,49,50ll lines ,52,53.CDH1 and low ZEB1 expression. This specific expression pattern is well explained by the fact that ZEB1 is targeted by miR200s that promotes EMT by inhibiting E-cadherin expression [CDH1 and enhanced migratory ability in response to estrogens in PEO1. The addition of ZEA and BPA also altered miRNA expression although these changes were not as remarkable as in the case of E2. Our results are in agreement with previous studies where BPA and ZEA altered miRNA expression in several other cell lines [MIR200A-MIR200B-MIR429 locus [We also investigated whether the expression of miR200 and miR34 family members and miR203a is influenced by estrogens in human ovarian cells. These miRNAs are involved in the regulation of EMT according to previous studies, but they have an influence on other processes as well, including the cell cycle and angiogenesis ,38,54,55pression . A similpression . Unexpecpression , was higpression ,59. It ill lines ,61,62,63ll lines ,65. It w29 locus ,66. HoweIt is generally accepted that cell-free miRNAs might play prominent roles in cell-to-cell communication and influence the tumor microenvironment that favors tumor growth ,23. Our 2 in RPMI1640  supplemented with 10% FBS , 1% l-glutamine, 100 µg/mL streptomycin and 100 U/mL penicillin. In order to study the effect of E2, ZEA and BPA, exponentially growing cells were harvested by trypsinization and plated to 96- or 24-well plates in RPMI1640 medium supplemented with 10% FBS. After attachment, the medium was removed and replaced with PRF-RPMI1640  supplemented with 5% DCC-FBS . Cells were incubated for 24 h then E2, ZEA and BPA  were added to the cells in different concentrations . This time point was designated as 0 h. In some experiments, 1 nM MPP was also added to the cell cultures together with E2.Human epithelial ovarian cell lines were used in the study. PEO1 was purchased from Merck (ECACC) and A2780 was kindly provided by Katalin Goda . Both of the cell lines were routinely cultured at 37 °C, 90% humidity and 5% COIn order to determine the proliferative effect of the E2, ZEA and BPA treatments, cells were harvested when the non-treated control wells reached ~90% confluency (48 h after estrogen treatment in the case of A2780 and 72 h after the treatment in the case of PEO1) and were counted in a Bürker chamber. Relative growth rate was calculated from the results of 4 independent experiments where the cell count of treated wells was presented relative to the cell count of non-treated wells (1).Cell migration was determined by wound-healing scratch assay. Cells were plated to 24-well plates as previously described. After reaching 100% confluency a scratch was made with a pipette tip and the cells were treated with E2, ZEA and BPA. Three photos were taken from each well in the time of scratching (0 h) and 24 h later. Open wound area was determined by the TScratch software . The ratThe decrease in mitochondrial membrane potential was used as a marker of apoptosis that was determined by the DilC1(5) Assay as described elsewhere . FluoresThe rate of cytotoxicity was determined by measuring LDH activity in the supernatant of cell cultures by the CyQUANT LDH Cytotoxicity Assay Kit  according to the manufacturer’s instructions. LDH activity was determined at 490 nm by a Multiskan sky microplate reader . Cell lysis was induced by the addition of lysis solution in the case of the positive control.In order to determine the ability of cell-free miR200b and miR200c to influence the miRNA level of neighboring cells, a co-culture assay was performed . PEO1 ceCells were plated to 24-well plates and treated with E2, ZEA and BPA molecules as previously described. The cells were treated in 10 nM concentration according to the phenotypic studies where both proliferation and migration were induced significantly at this concentration. Then, 24 h later, cells were harvested with a cell scraper and total RNA was isolated by RNeasy Mini Kit  according to the manufacturer’s instructions.ESR1, GREB1, DEPTOR, CA12, RBBP8, CDH1 and ZEB1 was detected by the QuantiTect SYBR Green RT-PCR Kit . The PCR reaction mixture contained 100 ng total RNA. RNA was reverse transcribed at 50 °C for 30 min, then the reaction was denatured at 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 54 °C for 30 s and 72 °C for 30 s. Finally, a melting curve was generated by taking fluorescent measurements from 65 °C until 97 °C to ensure the detection of a single PCR product. All measurements were performed in triplicate. Relative expression was determined relative to GAPDH expression from 4 independent experiments (∆CT method). Relative expression ratios were determined in response estrogen treatments relative to the non-treated control samples (Ratio: relative expression level in treated samples/relative expression level in non-treated samples). Primer sequences are listed in The expression of g, 4 °C) in order to pellet detached cells and cell debris, then total RNA including small RNAs was extracted from a 200 µL sample with the miRNeasy Serum/Plasma Kit  according to the manufacturer’s instructions. The concentration of extracted miRNA was determined by miRNA-specific fluorometric assay using a Qubit® 2.0 Fluorometer .Cells were plated to 24-well plates and treated with E2, ZEA and BPA molecules in 10 nM concentration as previously described. Then, 12 and 24 h later cells were harvested with a cell scraper and total RNA including small RNAs was isolated by a miRNeasy Kit  according to the manufacturer’s instructions. Cell-free miRNAs released from the cells were also extracted from the supernatant of the cultures as follows. The supernatant was centrifuged in these experiments  was applied for the detection of mature miRNAs. Reverse transcription of mature miRNAs was performed by the miScript II RT Kit . The expression of mature miR200a-3p, miR200b-3p, miR200c-3p, miR141-3p, miR429-3p, miR34a-5p, miR34b-3p, miR34c-3p and miR203a-3p was detected by Lightcycler 96 instrument  with the miScript SYBR Green PCR Kit  using miRNA specific probes . The PCR reaction mixture contained 500 pg reverse transcription products. The reaction samples were first denatured at 95 °C for 15 min, followed by 50 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Finally, a melting curve was generated by taking fluorescent measurements from 40 °C until 85 °C to ensure the detection of a single PCR product. All measurements were performed in triplicate. MiR103-3p was used as a housekeeping miRNA that proved to be a reliable marker in the case of the plasma samples previously ,76 and sThe following ChIP-seq fastq files were obtained from the SRA database: SRR6653434 (vehicle control) and SRR6653432 (E2-stimulated) . The prit-test. Multiple analysis was performed by one-way ANOVA with Dunnet’s test as post hoc analysis. Pearson’s correlation was used to determine the correlation between the expression values of miRNAs in the cell lysates and supernatant of non-treated samples. In these calculations the expression values of miRNAs were grouped in the non-treated samples and correlation was determined between the ΔCT values of intracellular and cell-free miRNAs. Statistical analysis and figures were made by GraphPad Prism 7.Statistical significance was studied by Student’s Cell-free miRNAs represent a highly promising but understudied area of miRNA biology which might open new avenues in cancer research. Accumulating evidence suggests that cell-free miRNA expression correlates with the occurrence of several malignancies, and thus they are considered to be promising biomarker candidates in liquid biopsy ,80. Howe


SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of  Rothenburger et al. combine the analysis of data from large pharmacogenomics screens with cell line experiments to elucidate mechanisms behind nelarabine resistance in acute lymphoblastic leukaemia (ALL). They identify low expression of the dNTP hydrolase SAMHD1 as a determinant of nelarabine sensitivity, suggesting the enzyme as a biomarker. Among ALLs, T-ALL is responsible for ~15% of paediatric ALLs and 25% of adult ALLs3. Nelarabine displays selective activity in T-ALL over B-ALL and is used for the treatment of relapsed and refractory T-ALL but not routinely for the treatment of B-ALL13. However, the molecular mechanisms underlying this difference remain elusive. Moreover, nelarabine therapy can be associated with irreversible life-threatening neurotoxicity14. Hence, biomarkers indicating patients who are most likely to benefit from nelarabine therapy are needed.Acute lymphoblastic leukaemia (ALL) cells originate from precursor lymphoid T- (T-ALL) and B-cells (B-ALL). In children, ALL is the most common cancer associated with high cure rates of about 85%. In adults, ALL accounts for 15–25% of acute leukaemias and is associated with a less favourable outcome15, Cancer Cell Line Encyclopedia (CCLE)16, and Genomics of Drug Sensitivity in Cancer (GDSC)17 with data from an ALL cell line panel derived from the Resistant Cancer Cell Line (RCCL) collection and patient data to investigate the mechanisms underlying the discrepancy in the nelarabine sensitivity between T-ALL and B-ALL.Here, we used an approach that combined data from the large pharmacogenomics screens Cancer Therapeutics Response Portal (CTRP)The results show that low expression of Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) in T-ALL cells is a key determinant of nelarabine sensitivity and that SAMHD1 is a potential biomarker and therapeutic target for the improvement of nelarabine-based therapies for both T-ALL and B-ALL patients.16, Cancer Therapeutics Response Portal (CTRP)15, and Genomics of Drug Sensitivity in Cancer (GDSC)17. The CCLE contained data derived from 34 leukaemia cell lines  and the GDSC from 38 leukaemia cell lines , with an overlap of 19 cell lines  cell lines in the CTRP (Supplementary Table P < 0.05) in their abundance levels between T-ALL and B-ALL cells in the GDSC , CCLE , and CTRP   between T-ALL and B-ALL cell lines. Substantial proportions of transcripts displayed significant differences (http://pantherdb.org)20 also did not reveal processes that may underlie the increased nelarabine sensitivity of T-ALL cells  levels were significantly lower in T-ALL than in B-ALL cell lines in all three databases  hydrolase that cleaves physiological dNTPs and triphosphorylated nucleoside analoguesses Fig. . Similar 28 Fig. . Further29. While statistically significant differences in the expression of some of the respective genes were noted between B-ALL and T-ALL cell lines in some of the three datasets, none was consistent across all three and none was as robust as in the expression of SAMHD1 , SLC29A2 (ENT2), NT5C, NT5C2, PNP, RRM1, RRM2 and SLC22A4 (OCTN1)HD1 Fig. . In patiHD1 Fig. . Moreove30 and SAMHD1 expression levels are lower in T-ALL than in AML cells  cellslls Fig. . Hence, 31   , the product of the prodrug nelarabinenes Fig. . Notablynes Fig. . In contTaken together, these results confirm the CTRP data in showing that cellular SAMHD1 levels determine ALL sensitivity against AraG, the product of nelarabine, and that low SAMHD1 levels in T-ALL cells are associated with specific nelarabine/ Ara-G activity in this lineage. In contrast to the CTRP and GDSC data, the additional experimental analyses in the RCCL ALL cell line panel suggest that SAMHD1 levels may also affect cytarabine activity in ALL, albeit to a lower degree than AraG activity.41. Vpx is a protein encoded by HIV-2 and certain SIV strains that mediates proteasomal SAMHD1 degradation43. Vpx virus-like particles resulted in the sensitisation of ALL cells to AraG and cytarabine but exerted much more pronounced effects on the activity of AraG . Further, we investigated the SAMHD1 low/ non- expressing cell lines HEL (AML) and Jurkat (T-ALL) and their respective sublines transduced either with wild-type (WT) SAMHD1 or the triphosphohydrolase-defective mutant SAMHD1-D311A. In the AML cell lines, absence of functional SAMHD1 was associated with a 60-fold (THP-1/ THP-1-KO) and 6583-fold (HEL-SAMHD1_WT/HEL-SAMHD1_D311A) sensitisation to cytarabine, but only a 5.6- and 6.0-fold sensitisation to AraG  corresponding to the promoter sequence after HpaII digestion. Results indicated that the SAMHD1 promoter was methylated in all T-ALL cell lines but one (MOLT-16)   Fig. , which wroduct 99-bp correroduct 99-bp corre-1) Fig. . In agreion Fig. . Notablyion Fig. , suggest12. Although it had been known for decades that nelarabine is more active in T-ALL than in B-ALL cells46, the underlying mechanisms had remained elusive.Similar chemotherapeutic agents are used to treat T-ALL and B-ALL. However, nelarabine is specifically used for relapsed T-ALL40. SAMHD1 is a deoxynucleotide triphosphate (dNTP) hydrolase that cleaves and inactivates triphosphorylated nucleoside analogues including triphosphorylated AraG45. Moreover, T-ALL cells were characterised by substantially lower SAMHD1 levels than B-ALL cells. Previous studies had demonstrated an association between AraG efficacy and AraG triphosphate levels in leukaemia cells, but the mechanism determining AraG triphosphate levels had remained unknown47. Hence, SAMHD1 is the missing link explaining the discrepancy in nelarabine sensitivity between T-ALL and B-ALL.Here, we used an approach combining data derived from large pharmacogenomics screens , an RCCL-derived ALL cell line panel, and patient data and found that cellular SAMHD1 levels critically determined ALL cell sensitivity to nelarabine and AraG. Nelarabine is metabolised into AraG, which is then triphosphorylated by cellular kinases into the active form48. Thus, SAMHD1-mediated repair of nelarabine/ AraG-induced DNA damage may potentially also contribute to the increased nelarabine/ AraG resistance associated with high SAMHD1 levels in ALL cells. However, SAMHD1 expression was not associated with generally increased resistance to DNA damaging agents in the CTRP , which contains data from 34 ALL cell lines (18 B-ALL and 16 T-ALL)16, GDSC 17, and CTRP  15.ALL cell line drug sensitivity data and RMA-normalised gene expression values were obtained from the CCLE  of 0.05 (BH = (rank/n) × FDR, where n = the total number of genes compared). Gene expression levels between B-ALL and T-ALL cell lines were visualised using the ggboxplot function in R. Heatmaps showing gene expression levels were generated using the ggplot2 package in R.Gene expression was compared using the Mann-Whitney U (Wilcoxon) test for independent groups. Multiple test correction of p-values was performed using the Benjamini–Hochberg (BH) procedureIn all, 36 of the ALL cell lines  in the GDSC and 22 ALL cell lines  in the CTRP were treated with cytarabine. 23 ALL cell lines  in the CTRP were treated with nelarabine. Scatter plots and their associated Pearson correlations for each drug AUCs against gene expression were calculated using the ggplot2 package in R.P-values for each correlation were ranked and multiple test correction was performed .Expression of 18,542 genes was correlated with the nelarabine AUC in ALL cell lines and SAMHD1 expression was correlated with the AUC values of 441 drugs tested in ALL cell lines using the CTRPv2 dataset. Pearson correlation coefficients were calculated using the cor.test function in R. 20] based on genes significantly differentially expressed in B-ALL and T-ALL cell lines after Benjamini–Hochberg p-value correction (FDR = 0.05). Fisher’s exact test was applied to calculate over- vs. underrepresentation of classes. Heatmaps were prepared using the ggplot2 package in R.Pathway analysis was performed using the PANTHER (version 14.1) Overrepresentation Test . Cells were lysed in Triton X-100 sample buffer and proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted on a nitrocellulose membrane (Thermo Scientific). The following primary antibodies were used at the indicated dilutions: SAMHD1 , β-actin , pSAMHD1 , and GAPDH . Visualisation and quantification were performed using IRDye-labeled secondary antibodies  according to the manufacturer’s instructions. Band volume analysis was conducted by Odyssey LICOR. Uncropped blots are presented in Supplementary Fig. Western blotting was performed as previously described [Schneider et al.RNA extraction and TaqMan-based mRNA quantification of SAMHD1 (assay no. Hs00210019_m1) and RNaseP (TaqMan® RNaseP Assay (A30065)) as endogenous reference control were performed according to the manufactures protocol (Applied Biosystems). Total RNA was extracted using the RNeasy Kit from Qiagen and stored at −80 °C until use. Relative quantitative PCR analyses were performed on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). SAMHD1 mRNA expression levels were quantified by using the ΔΔCt method with RNaseP mRNA as an endogenous reference control. All samples were run in triplicate. Data analysis was conducted using the QuantStudio System Software (Applied Biosystems).SAMHD1 promoter methylation was determined as previously described45. SAMHD1 promoter contains five HpaII sites surrounding the transcription start site44. Methylation of the HpaII sites in the SAMHD1 promoter would prevent digestion by the HpaII, and the intact sequence would serve as a template for PCR amplification using SAMHD1 promoter-specific primers that flank the HpaII sites. To measure methylation of the SAMHD1 promoter genomic DNA was treated with the methylation-sensitive HpaII endonuclease or left untreated as described previously with some modifications44. PM3.fwd: TTCCGCCTCATTCGTCCTTG and PM3.rev: GGTTCTCGGGCTGTCATCG were used as SAMHD1 promoter-specific primers. A single PCR product (993-bp) corresponding to the SAMHD1 promoter sequence was obtained from untreated genomic DNA and treated DNA from cells with methylated but not from cells with unmethylated SAMHD1 promoter. To serve as input control, a 0.25-kb fragment of the GAPDH gene lacking HpaII sites was PCR-amplified using the same template DNA44. Uncropped agarose gels are shown in Supplementary Fig. 44.Cells were spinoculated with VSV-G pseudotyped virus-like particles carrying either Vpx or Vpr from SIVmac251, produced by co-transfection of 293T cells with pSIV3 + gag pol expression plasmids and a plasmid encoding VSV-G as previously described t-tests. Data are presented as means ± standard deviation (S.D.). Specific information on the number and nature of replicates is provided in the figure legends. Correlation analyses were performed using linear regression in GraphPad Prism resulting in r2 as a measure for goodness-of-fit and the P value, which is calculated from an F test, indicating whether the slope is significantly different from zero.Statistical data analyses were performed in GraphPad Prism version 7. Population means were compared using unpaired two-tailed Student’s Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Description of Additional Supplementary FilesPeer Review FileReporting Summary