In this study, we evaluated for the first time Trastuzumab emtansine (T-DM1), a novel antibody–drug conjugate, against multiple epidermal growth factor receptor-2 (HER2)-positive USC cells in vitro followed by developing a supportive in vivo model. Fifteen primary USC cell lines were assessed by immunohistochemistry (IHC) and flow cytometry for HER2 protein expression. C-erbB2 gene amplification was evaluated using fluorescent in situ hybridization. Sensitivity to T-DM1 and trastuzumab (T)-induced antibody-dependent cell-mediated cytotoxicity was evaluated in 5-h chromium release assays. T-DM1 and T cytostatic and apoptotic activities were evaluated using flow-cytometry-based proliferation assays. In vivo activity of T-DM1 versus T in USC xenografts in SCID mice was also evaluated. High levels of HER2 protein overexpression and HER2 gene amplification were detected in 33% of USC cell lines. T-DM1 was considerably more effective than trastuzumab in inhibiting cell proliferation and in causing apoptosis (P = 0.004) of USC showing HER2 overexpression. Importantly, T-DM1 was highly active at reducing tumor formation in vivo in USC xenografts overexpressing HER2 (P = 0.04) and mice treated with TDM-1 had significantly longer survival when compared to T-treated mice and control mice (P ≤ 0.0001). T-DM1 shows promising antitumor effect in HER2-positive USC cell lines and USC xenografts and its activity is significantly higher when compared to T. T-DM1 may represent a novel treatment option for HER2-positive USC patients with disease refractory to trastuzumab and traditional chemotherapy.Amplification of  Trastuzumab emtansine  is a novel antibody–drug conjugate that comprised trastuzumab covalently linked to the antimicrotubule agent DM1 Uterine serous carcinoma (USC) accounts for ∼10% of endometrial cancer. This subtype of endometrial cancer is highly biologically aggressive and accounts for a disproportionately large number of endometrial cancer deaths Although the mechanisms underlying trastuzumab's clinical benefit have not been completely elucidated, its therapeutic benefit is attributed to disruption of signaling through the PI3K/AKT/mTOR pathway in HER2 overexpressed malignant cells as well as through the recruitment of NK cells and initiation of antibody-dependent cell-mediated cytotoxicity (ADCC) c-erbB-2 gene amplification by fluorescent in situ hybridization (FISH).Study approval was obtained from the Institutional Review Board, and all patients signed consent prior to tissue collection according to the institutional guidelines. A total of 15 USC cell lines were established after the sterile processing of fresh tumor biopsy samples, as described previously μm sections of cell blocks after deparaffinization and rehydration, using the c-erbB-2 antibody  at 1:800 dilution. HER2 staining intensity was graded per the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) 2007 breast scoring criteria.Cell blocks were obtained from all 15 USC cell lines and reviewed by a gynecologic surgical pathologist to confirm the presence of serous carcinoma cells. The level of HER2 expression was then evaluated as described previously μm were deparaffinized and rehydrated, followed by acid pretreatment and proteinase K digestion. A probe mix containing an orange probe directed against the c-erbB-2 gene  and a green probe directed against the pericentromeric region of chromosome 17 (Vysis CEP 17) were added and specimens were denatured for 5 min at 73°C. Slides were then incubated overnight in a humidity chamber at 37°C and washed the day after when a fluorescence mounting medium, containing 4, 6-diamidino-2-phenylindole (DAPI), was applied. Fluorescent signals in at least 30 nonoverlapping interphase nuclei with intact morphology were scored using a Zeiss Axioplan 2 microscope  with a 100× planar objective, using a triple band-pass filter that permits simultaneous blue, green, and red colors. A case was scored as amplified when the ratio of the number of fluorescent signals of c-erbB-2 gene to chromosome 17 was ≥2.Fluorescent in situ hybridization analysis was performed using the PathVysion HER2 DNA FISH Kit  according to the manufacturer's instructions. Cell block sections of 5 T) method was used for the calculation of amplification fold as specified by the manufacturer.RNA isolation from all 15 USC cell lines and from normal endometrium cell controls used in these experiments was performed using TRIzol Reagent  according to the manufacturer's instructions. Quantitative PCR was carried out to evaluate the expression level of HER2 in all samples with a 7500 real-time PCR system using the recommended protocol by the manufacturer . Each reaction was run in duplicate. The internal control, glyceraldehyde-3-phosphate dehydrogenase Assay-on-Demand Hs99999905_ml (Applied Biosystems), was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle  5 μg/mL was used as a negative control. For staining, a fluorescein isothiocyanate-conjugated goat anti-human F(ab1)2 immunoglobulin was used as a secondary reagent . Analysis was conducted with a FACScalibur, using Cell Quest software . Cell cycle position was identified by the DNA content of cells as determined by propidium iodide (PI) staining. For each analysis 10,000 gated events were collected to permit cell cycle analysis. Data analysis was performed using Cell Quest (BD Biosciences) and FlowJo v8.8.2 Trastuzumab  is a humanized mAb of the IgG1 isotype that binds with high affinity to the extracellular domain of the HER2 receptor. The USC cell lines were incubated with 2.5 51Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-Hypaque-separated peripheral blood lymphocytes (PBLs) from several healthy donors in combination with trastuzumab or T-DM1 against the 15 USC target cell lines at effector to target ratios (E:T) of 15:1 and 30:1. The release of 51Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to 2.5 μg/mL of trastuzumab or 2.5 μg/mL of T-DM1. Dose–response experiments were performed in order to determine the optimal antibody dosing for ADCC experiments. Chimeric anti-CD20 mAb rituximab 2.5 μg/mL was used as the negative control for trastuzumab and T-DM1 in all bioassays. The percentage cytotoxicity of trastuzumab or T-DM1 was calculated by the following formula: % cytotoxicity = 100 × (E − S)/(T − S), where E is the experimental release, S is the spontaneous release by target cells, T is the maximum release by target cells lysed with 0.1% SDS.Standard 5-h chromium  were given a single intraperitoneal (i.p.) injection of 7.5 × 10 USC ARK-2 cells (high HER2 expressor that does not harbor a PIK3CA mutation) in ∼400 2 transforming them to ΔCT values. Group means with 95% confidence intervals (CI) were then calculated for the relative copy numbers. Differences in HER2 expression by flow cytometry were analyzed by the unpaired t-test, and a P < 0.05 among the samples was considered to be significant. The Wilcoxon rank-sum test was used to compare USC cell lines to normal endometrium for differences in IHC staining intensity. The unpaired t-test was used to evaluate the differences in ADCC levels by 5-h chromium release assays and the inhibition of proliferation in the USC cell lines after exposure to trastuzumab versus T-DM1. Overall survival data were analyzed and plotted using the Kaplan–Meier method. Survival curves were compared using the log-rank test. Statistical analysis was performed using GraphPad Prism  version 6. A P < 0.05 was considered as the level of statistical significance.For quantitative real-time PCR (qRT-PCR) data, right skewing was removed by taking copy number ratios relative to the lowest expressing normal endometrium (relative copy number), and then logHigh levels of HER2 protein expression by immunohistochemistry (+3 staining) were detected in 33% of the USC cell lines . Three cell lines had no HER2 expression on IHC, whereas the other seven cell lines had +1/+2 expression . The mRNA copy number for USC ARK-1, USC ARK-2, USC ARK-3, USC ARK-9, and USC ARK-21 (high expressors) were 2749.6, 4478.9, 4993.8, 4323.6, and 750.4, respectively, with a group mean mRNA copy number of 3459 and 95% CI; 1310–5606. This was compared with mRNA copy numbers of between 0.02 and 117.2 for USC ARK-4, USC ARK-5, USC ARK-6, USC ARK-7, USC ARK-8, USC ARK-11, USC ARK-12, USC ARK-13, USC ARK-14, USC ARK-19 (low expressors) with a group mean and 95% CI of 45.34; 15.44–75.24; c-erbB-2 gene amplification was detected in USC ARK-1, USC ARK-2, USC ARK-3, USC ARK-9, and USC ARK-21, thus supporting the premise that strong receptor expression and high HER2 mRNA levels were likely as a result of c-erbB-2 gene amplification. In contrast, USC ARK-4, USC ARK-5, USC ARK-6, USC ARK-7, USC ARK-8, USC ARK-11, USC ARK-12, USC ARK-13, USC ARK-14, and USC ARK-19 were found to be negative for c-erbB-2 gene amplification  at E:T ratios of 15:1 and 30:1 .All 15 primary USC cell lines were tested for their sensitivity to PBL-mediated cytotoxicity when challenged with heterologous PBLs collected from several healthy donors in a standard 5-h μg/mL and T-DM1 at 2.5 μg/mL. T-DM1 and trastuzumab (T) were similarly effective in inducing strong ADCC against all primary USC cell lines expressing high levels of HER2  with mean cytotoxicity ± SEM, 61.6 ± 5.3% versus 58.4 ± 5.74%, TDM-1 versus T, P = 0.686  after 48–72 h of exposure to 2 μg/mL of T-DM1. T-DM1 showed activity  against all USC cell lines tested including cell lines endowed with primary resistance to the cytostatic effects of T. In contrast, no significant G2/M phase arrest was noted in low expressor HER2 cell lines after exposure to T-DM1  had to be euthanized or died from tumor burden between days 56 and 66 after tumor injection, whereas three of five mice in the trastuzumab group required euthanasia or died from tumor burden between days 98 and 116 after tumor suspension injection , trastuzumab, or T-DM1 intraperitoneally (i.p.) once per week was initiated 7 days after USC suspension inoculation in mice in each treatment group. All drug treatments were given weekly for 5 weeks after which mice were placed into observation until death or meeting criteria for euthanasia according to the IACUC guidelines. Palpable tumors and/or ascites were noted after approximately 3 weeks after USC tumor cell injection in the rituximab and trastuzumab groups. Specifically, all mice in the rituximab group .Overall T-DM1 appears to be well tolerated as a single agent given at 3.6 mg/kg every 3 weeks. This dosing differs from the 6 mg/kg every 3-week trastuzumab dosing. TDM-1 also has a shorter half-life than trastuzumab (∼4 days for conjugated T-DM1 vs. 3–4 weeks for trastuzumab), which may also result in a decrease in adverse effects including cardiotoxicity associated with trastuzumab c-erbB2 amplified primary USC cell lines evaluated in this study were indeed found resistant to the cytostatic effect of T in vitro. While the mechanisms resulting in T resistance are not completely understood, recent studies have identified multiple potential molecular alterations to explain in vivo lack of clinical activity. Potential mechanisms include significant intratumoral heterogeneity in HER2 protein expression in USC c-erbB2 amplification also harbored PIK3CA mutations.Despite the overexpression of HER2 in up to 61% of USC, 38% of clear cell, and 11% of grade 3 endometrioid adenocarcinoma of the uterus found in large cooperative Gynecologic Oncology Group studies  Regardless of the mechanism of resistance to T active in USC, our experimental data clearly demonstrate that T-resistant USC may be highly sensitive to T-DM1 effects in vitro and in vivo. These results strongly support the working hypothesis that T-DM1 may represent a significantly more effective therapeutic tool when compared to T for the treatment of chemotherapy resistant/recurrent endometrial cancer overexpressing HER2.c-erbB-2 amplified USC cell lines in the presence of PBL. In vitro data also suggest that T-DM1 is dramatically more effective than T in inhibiting cell proliferation and in inducing apoptosis after G2/M phase cell cycle arrest. Our In vitro findings were confirmed in an in vivo model, where T-DM1 was significantly more effective at inhibiting HER2-positive USC xenograft growth and establishment than T. Due to the significant fraction of USC patients overexpressing HER2 In conclusion, we have shown that T-DM1 and T are similarly effective in inducing strong ADCC against primary The authors declare no conflict of interest or previous publication and fulfill all conditions required for authorship.


Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC Inhibitors of BRAFV600E have changed the clinical management of patients with BRAF-mutant advanced melanoma, since significant improvements in progression-free survival (PFS) and in overall survival (OS) have been reported in Phase 3 trials –2. TheseIn the attempt to address all these limitations associated with targeting of a single (MAPK) pathway, several pre-clinical studies have suggested that combinatorial targeting of MAPK and PI3K/mTOR intracellular pathways may be a potential approach to improve target therapy of melanoma and to overcome resistance and cross-resistance mechanisms –17. Howe50 > 1 μM, n = 21), 81% (seventeen) showed strong or intermediate cross-resistance to the MEK1/2- and the PI3K/mTOR-specific inhibitors. Extensive drug interaction analysis on all 49 cell lines and mechanistic studies in cross-resistant cell lines indicated that co-targeting of MEK1/2 and PI3K/mTOR, in-vitro and in-vivo, was a more effective combinatorial treatment, compared to co-targeting of BRAF and PI3K/mTOR, to counteract the primary cross-resistant phenotype.Recently, we observed instances of primary cross-resistance to a MEK1/2 (AZD6244) and to a dual PI3K/mTOR (BEZ235) inhibitor in some melanoma cell lines (18). Starting from this initial evidence, in this study we assessed responsiveness to BRAFV600E-, MEK1/2-, dual PI3K/mTOR- and dual mTORC1/2-specific inhibitors in 49 BRAF-mutant melanoma cell lines and in an independent set of 23 BRAF-mutant short-term melanoma cell cultures, all isolated from patients never treated with target therapy. Among cell lines with strong intrinsic resistance to the BRAF inhibitor  was found in 21/49 cell lines , intermediate resistance (IC50 = 0.1 to 1 μM) and susceptibility (IC50 < 0.1 μM) to PLX4720 were observed in 16/49  and 12/49 cell lines , respectively , MEK1/2 (AZD6244), dual PI3K/mTOR (BEZ235) and dual mTORC1/2 (AZD8055) inhibitors Figure . Three sy Figure  classifiy Figure . Hierarcm Figure . Spearmad Figure .in-vitro passage), obtained from 23 BRAF-mutant metastatic specimens of patients not previously treated with target-specific inhibitors, was used to test responsiveness to the same set of inhibitors. The same classification into three subsets based on ranking of PLX4720 IC50 values was applied. We found that 6/6 PLX4720-resistant melanoma cell cultures (group 1) showed strong (i.e. IC50 > 1 μM) or intermediate (i.e. IC50 > 0.1 μM) cross-resistance to MEK1/2 and PI3K/mTOR inhibitors, and 11/13 cultures in group 2 (intermediate resistance to PLX4720) showed also strong or intermediate cross-resistance to PI3K/mTOR inhibitors  and short-term melanoma cell cultures (Me_cc117 and Me_cc128) from the cross-susceptible group 3 , indicatell line . Taken thi and MITFlo cell lines and short term cultures were found in each of the three susceptibility groups (data not shown).The panel of 49 melanoma cell lines shown in Figure Taken together, these results indicated that intrinsic resistance to BRAFV600E inhibition can be frequently associated with cross-resistance to MEK1/2 and/or PI3K/mTOR inhibitors in BRAF-mutant melanoma cells.arrows). By clonogenic assays  against the whole panel of 49 melanoma cell lines compared to PLX4720-BEZ235, in three out of nine experimental conditions where both AZD6244 and PLX4720 were present at equivalent doses  matrix  where AZin-vivo of AZD6244-BEZ235 and PLX4720-BEZ235 combinatorial treatments. To this end, we established a SCID mouse model based on s.c. xenograft of a cell line from group 1 (Me13). This cell line was characterized by strong primary resistance to PLX4720 (IC50 > 1 μM) and by intermediate resistance to AZD6244 and BEZ235 (IC50 > 0.1 μM). Both treatments exerted a significant inhibitory effect on tumor growth, compared to control animals treated with vehicle, but the AZD6244-BEZ235 association was significantly more effective than PLX4720-BEZ235 treatment . Me13 and Me6 have a higher ICTaken together, these results indicated that the association of MEK1/2 and PI3K/mTOR inhibitors induces a more effective inhibition of p-ERK at both early and late time points compared to BRAF and PI3K/mTOR dual blockade, in PLX4720-resistant melanoma cells and even in cell lines with a cross-resistant phenotype.50 values , in tumors from mice treated with AZD6244-BEZ235 compared to PLX4720-BEZ25 . Significantly modulated genes by each combination, identified by class comparison through BRB Array Tools, were subjected to downstream effect analysis through IPA software. “Cell death and survival” was the top biological function affected by the three combinatorial treatments, but the most significant P value for association with such function was observed for the AZD6244-BEZ235 treatment (data not shown). Based on Z scores > 2 or < −2, the function “cell death” was predicted to be increased, while the function “proliferation” was predicted to be decreased by the three combinatorial treatments  in addition to gene expression changes shared by two or even three different combinatorial treatments . Thus, AZD6244-BEZ235 upregulated a set of 79 genes and downregulated a different set of 83 genes, not significantly affected by the other two combinatorial treatments. By canonical pathway analysis by IPA  and, b) the differences, among the three combinatorial treatments, in the fraction of significantly modulated genes belonging to each pathway  specific differences in the modulatory effects induced by the three combinatorial treatments were identified  and results of published trials .Female SCID mice, 8–10 weeks old (Charles River Laboratories) were provided with food and water Melanoma apoptosis was assessed after staining with APC-conjugated Annexin V (BD Pharmingen) and propidium iodide  as described –38. DNA ™ Cell Analyzer (Merck Millipore). For apoptosis determination, cells were collected and resuspended according to manufacturer's instructions with the working solution of the Muse™ Annexin V & Dead Cell Kit (Merck Millipore) for 20 minutes at room temperature. For caspase 3/7 activity cells were collected, resuspended in 1X Assay Buffer BA, and incubated with Muse™ Caspase-3/7 Assay Kit (Merck Millipore) reagent working solution for 30 minutes in the 37°C incubator with 5% CO2. After incubation, Muse™ Caspase 7-AAD working solution was added for 5 minutes at room temperature. Data were analyzed by Muse 1.4 Analysis Software.In some experiments, apoptosis and caspase 3/7 activation were assessed by the MuseMelanoma cells were treated with MAPK and PI3K/mTOR inhibitors for 48 h. The Human Apoptosis Array Kit (R & D Systems) was used, as described  accordinTransient silencing experiments were carried out with ON-TARGET Plus Human BAX siRNA smart-pool  containing four oligos specific for Bax  or corresponding negative control . Oligos were used at 10 nm final concentration according to Lipofectamine RNAiMAX guidelines (Thermo Fisher Scientific). Silencing was checked by Western blot analysis at 48 h and at 96 h. At 48 h after transfection with Bax siRNA smart pool, or with negative control oligos, cells were treated with AZD6244 plus BEZ235 (0.1 μM + 0.1 μM), or with PLX4720 plus BEZ235 (0.1 μM + 0.5 μM) and analyzed after 48 h by staining for Annexin-V/Propidium Iodide or for active caspase-3.Immunohistochemistry was performed with formalin-fixed, paraffin-embedded tissues as described . SCID miwww.ingenuity.com) as described [Melanoma cells from Me13 cell line were treated with AZD6244 (0.1 μM), BEZ235 (0.1 μM), AZD8055 (0.3 μM) or PLX4720 (0.5 μM), or with the AZD6244-BEZ235, PLX4720-BEZ235, AZD6244-AZD8055 combinations for 8 hr. Three biological replicates for each treatment were set up. Total RNA isolation, clean-up, DNase treatment and assessment of RNA integrity and purity were performed as described , 38. Sinescribed .Ct slope method to ensure that the quality of each complementary DNA and the dynamic range of amplifications were comparable [Ct−Δ, reflecting target gene expression normalized to endogenous control levels [c-Fos (Hs99999140-m1), CTNNB1/β catenin (Hs003550489-m1) and, as endogenous control, GAPDH (Hs00266705-g1) TaqMan Gene Expression Assays  were used. Total RNA (1 μg) was reverse transcribed with oligo d (t) using a Transcriptor First Strand cDNA Synthesis Kit . Preliminary experiments were conducted for the endogenous control using the mparable . Real-til levels .50 values were clustered by Cluster 3.0 software and clustering was visualized by Java TreeView. Correlation of susceptibility profiles to different inhibitors was tested by Spearman correlation analysis. Comparison of IC50 values, of combination index (CI) values and of fraction affected (FA) data, in different groups of melanoma cell lines, was done by Kruskal-Wallis and Dunn multiple comparison test. Analysis of FA values by combinations of inhibitors in the same group of melanoma cell lines, or in all cell lines, when equivalent doses of different inhibitors were used, was carried out by Wilcoxon matched pair test. Significance of different treatments on melanoma apoptosis, caspase activation, modulation of apoptosis-related molecules was assessed by ANOVA, followed by Student-Newman-Keul (SNK) multiple comparison test. Analysis of the antitumor activity of different treatments in-vivo was carried out by mixed effects model ANOVA [ICel ANOVA  by XLSTA


We evaluated the expression levels of EpCAM and the 51Cr release-assays. The proliferative activity, activation, cytokine secretion  and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the pleural fluid of a CS patient were also evaluated by CFSE and flow-cytometry assays.EpCAM expression was evaluated by flow cytometry in a total of 5 primary CS cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against the panel of primary CS cell lines expressing different levels of EpCAM in standard 4 h P < 0.0001). Ex vivo incubation of autologous TAL with EpCAM expressing malignant cells in pleural effusion with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers , and a reduction in number of viable CS cells in the exudate (P < 0.001).Surface expression of EpCAM was found in 80.0 % (4 out of 5) of the CS cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-h chromium-release assays . In contrast, after incubation with solitomab, EpCAM positive CS cells became highly sensitive to T-cell-cytotoxicity (mean killing ± SEM of 19.7 ± 6.3 %; range 10.0-32.0 %; Solitomab may represent an effective treatment for patients with recurrent/metastatic and/or chemo-resistant CS overexpressing EpCAM. Gynecological carcinosarcomas (CS), also known as Malignant Mixed Mullerian Tumors (MMMT), are rare biphasic tumors that most commonly arise in the uterus and ovaries . HistoloOverexpression of epithelial cell adhesion molecule-1 (EpCAM), also known as TROP-1 or TACSTD1, is a known poor prognostic biomarker across a large number of carcinomas and carcinosarcomas , 5. EpCAin vivo resistance to multiple chemotherapy agents was confirmed in vitro by MTT chemotherapy resistance assays against multiple cytotoxic agents (data not shown). Primary carcinosarcoma cell lines were tested for presence of EpCAM by Quantitative Real-time PCR and by flow cytometry as described below. An additional tumor sample was collected from a CS patient with recurrent disease and a large pleural effusion. The fluid sample was cytologically confirmed to contain a large number of EpCAM + carcinosarcoma cells at the time of a therapeutic thoracentesis. The fresh sample of pleural fluid was plated into 6-well microtiter plate for treatment using solitomab and a nonspecific BiTE® control antibody construct without prior processing. Cell numbers and viability were determined by flow cytometry as described below. Patient characteristics of all carcinosarcoma cell lines and the pleural fluid exudate are described in Table All patients signed an informed consent form according to institutional guidelines and approval for this in vitro study was obtained from the institutional review board. A total of five primary carcinoma cell lines  were established after sterile processing of surgical biopsy specimens, as described previously  x 100. Similarly, using the same equation the number of CD8+/HLA-DR+, CD4+/CD25+ and CD4+/HLA-DR+ expression was calculated.Solitomab induced T cell activation was measured by detecting CD25 protein surface expression and HLA-DR expression on CD8+/IFN gamma containing cells = [number of CD8+/IFN gamma cells/total number of CD8+ cells] × 100. Similar calculations were performed for CD4+ T cells .The level of solitomab dependent cytokine induction was compared to the corresponding value of percentage of cytokine released in the non-specific antibody control wells. This was performed by treating the solitomab and control non-specific antibody construct wells with phorbol myristate acetate and ionomycin followed by a 3 h incubation period to allow for lymphocyte stimulation. Brefeldin A was added and a further incubation for 3 h occurred in order to enhance intracellular cytokine staining signals. Cytokine analysis of the supernatants was performed by FACS analysis after adding anti-CD8-FITC antibody for surface staining followed by fixation, permeabilization and intracellular staining with anti-IL-4-PE antibody and anti-IFN gamma-PE antibody. Solitomab mediated release of each of these cytokines was calculated according to the following exemplary formula: Percentage of CD851Cr) release assay was used to measure the cytotoxic reactivity of Ficoll-Hypaque-separated peripheral blood lymphocytes (PBLs) from several healthy donors against five carcinosarcoma cell lines at effector to target ratios (E:T) of 10:1 and 20:1. The release of 51Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to a concentration of 1 μg/ml of solitomab. The negative control conditions were the incubation of target cells alone or with PBL without BiTE® antibody construct. As a positive control condition, 1 % sodium dodecyl sulfate (SDS) was used to achieve complete lysis of target cells. The control BiTE® huMEC14 at 1 μg/ml was used as the negative control for solitomab in this bioassay. The control BiTE® antibody construct shared the CD3 binding arm with solitomab, but otherwise recognizes an herbicide as an irrelevant antigen instead of recognizing EpCAM. The percentage cytotoxicity of solitomab was calculated by the following formula: % cytotoxicity = 100 X (E-S)/T-S), where E is the experimental release, S is the spontaneous release by effector cells, T is the maximum release by target cells lysed with 1 % SDS.The standard 4-h chromium  and three primary ovarian carcinosarcoma cell lines . Table 51Cr) release assay cytotoxicity using solitomab (EpCAM BiTE®) at a concentration of 1 μg/ml, as described previously [+ T cells and 10–20 % NK cells (CD56+/CD16+). In previous titration experiments we found ADCC against tumor cells to plateau at 1 μg/ml concentration (10). This concentration was therefore used as optimal concentration in the cytotoxicity experiments described below. We found CS cell lines to be highly resistant to NK and T-cell-mediated killing when challenged with PBL at target: effector ratios of 1:10 and 1:20  . As expected, the low EpCAM expressing cell line (SARARK-1) demonstrated low to negligible levels of cytotoxicity when challenged with PBL in the presence of solitomab . Anti-EpCAM antibodies were used to label tumor cells to be counted by flow cytometry and human recombinant IgG1 anti-EpCAM monoclonal antibody (mAb) MT201 combined with propidium iodide (PI) for exclusion of dead cells. As shown in Fig. + T and CD4+ T cells at baseline and after 7-days co-cultures with solitomab were analyzed by flow cytometry for the expression of the T-cell activation markers CD25 and HLA-DR. As demonstrated in the Fig. + and CD4+ T cells in the solitomab treated wells when compared to the wells treated with the control BiTE®. These data are consistent with our previous results with solitomab in the ascitic fluid of ovarian cancer patients [Next, we explored whether solitomab is capable of inducing T cell activation in the presence of carcinosarcoma cells in minimally manipulated pleural fluid samples . CD8patients .Fig. 3UpNext we investigated the level of the solitomab dependent cytokine induction in PBL when compared to the cytokine release of PBL stimulated with non-specific BiTE® control wells in the presence of EpCAM positive CS. The flow analysis was performed after 7 days of co-incubation of solitomab with tumor cells and PBLs before lymphocyte stimulation in the presence of brefeldin A as described in the Methods section. The analysis by FACS revealed a significant increase in IFN-gamma production in EpCAM + pleuric fluid exposed to solitomab when compared to control BiTE® wells in both CD8+ T and CD4+ T cells, , as a new therapeutic strategy against chemotherapy-resistant EpCAM expressing CS.Cell lines established from patients harboring advanced and/or chemotherapy resistant carcinosarcoma were assessed for EpCAM expression as were free tumor cells and spheroids collected via thoracocentesis from a patient. EpCAM was overexpressed in the majority of the primary CS cell lines. These data are similar to data we previously reported for EpCAM in ovarian cancer patients , 14 as wNK cells are known to play a critical role in the elimination and surveillance of cancer. Interestingly, EpCAM-positive CS cell lines were highly resistant to natural killer cytotoxic activity. While in the present study we did not investigate the mechanisms of resistance to NK cells, efficacy of cancer immunotherapy with antibodies activating cellular and complement-mediated cytotoxicity is known to be limited by the overexpression of multiple membrane-bound complement-regulatory protein (mCRPs). Consistent with this view, recent data from our group in uterine serous carcinomas, a subset of biologically aggressive tumors histologically similar to CS, demonstrate overexpression of mCRPs CD46, CD55 and CD59 in the majority of the tumors .51Cr release assays. Data also show that ex vivo CS single cell suspensions and tumor spheroids isolated from the pleural fluid of a CS patient harboring chemotherapy resistant disease may also be highly sensitive to solitomab-mediated cytotoxicity. These results were obtained by culturing freshly collected tumor and tumor associated lymphocytes ex vivo. After adding solitomab to the culture, without immune-stimulatory cytokines, tumor killing was observed. These data suggest that solitomab may be an efficacious treatment for recurrent disease in vivo.Importantly, primary CS cell lines in our study were highly sensitive to solitomab-mediated T cell cytotoxicity in 4-h Our data suggests that solitomab is a potent stimulator of T cell cytotoxicity even at low effector to target ratios while simultaneously inducing significant CD8+ and CD4+ T lymphocyte proliferation. TAL isolated from the pleural fluid of a heavily pretreated CS patient, and subsequently exposed to solitomab, increase their production of type I cytokine production. The integrity and size of CS tumor spheroids, found in ex vivo cultures containing tumor cells expressing EpCAM and autologous tumor associated lymphocytes, were diminished after exposure to solitomab. Previous studies have suggested that TIL and TAL associated with tumors or ascitic fluids are frequently anergic and/or tolerogenic towards autologous tumor cells –21. The Solitomab, and other antibody/BiTE®-based therapies, may be effective through either IV or intraperitoneal/intrapleural administration because of the frequency of expression of EpCAM on the surface of CS combined with its minimal expression on mesothelial type cells in the abdominal or pleural cavity. EpCAM/CD3-bispecific antibody construct (MT110) is currently being explored in phase 1 studies of patients harboring advanced stage tumors . A trifuOvarian and uterine carcinosarcoma are aggressive tumors that are difficult to treat initially and particularly in the recurrent state. The data presented here demonstrate that EpCAM is expressed in the majority of carcinosarcomas evaluated (80 %) and that solitomab effectively induced ADCC and significant tumor death in carcinosarcoma cell lines expressing EpCAM that were incubated with PBL. Solitomab, compared to control BiTE®, caused an increase in the production of type 1 cytokines and cytotoxic activity of tumor associated lymphocytes isolated from pleural fluid in CS harboring patients and may increase recruitment as well as activation of CD4 and CD8+ T cells. Clinical trials should be designed to assess the efficacy of solitomab as a novel therapeutic approach in patients with chemotherapy refractory carcinosarcoma.


The purpose of this study was to assess the possibility of predicting Cellular uptake of NBTXR3 nanoparticles was assessed in a panel of human cancer cell lines (radioresistant and radiosensitive) by transmission electron microscopy. The radioenhancement of NBTXR3 nanoparticles was measured by the clonogenic survival assay.NBTXR3 nanoparticles were taken up by cells in a concentration dependent manner, forming clusters in the cytoplasm. Differential nanoparticle uptake was observed between epithelial and mesenchymal or glioblastoma cell lines. The dose enhancement factor increased with increase NBTXR3 nanoparticle concentration and radiation dose. Beyond a minimum number of clusters per cell, the radioenhancement of NBTXR3 nanoparticles could be estimated from the radiation dose delivered and the radiosensitivity of the cancer cell lines.in vitro biological effect of NBTXR3 nanoparticles exposed to ionizing radiation.Our preliminary results suggest a predictable  Ionizing radiation is a universal killer with ability to generate double strand breaks in the DNA; as such it is the principle target for cancer cell killing. Ionizing radiation-induced damage correlates with the energy dose deposition in each biological structure . As it iRecently, nanotechnologies have paved the way to new approaches in local cancer therapy. Nanoparticles with high electron density offer the possibility to deposit high amounts of energy within the cancer cells when activated by ionizing radiation, providing appropriate bioavailability and persistence ,3.Nanocrystals of hafnium oxide, NBTXR3 nanoparticles, were designed for efficient cancer cell uptake and interaction with different types of ionizing radiation. The high density material at the nanoscale and ionizing radiation interactions render feasible the physical mode of action of radiotherapy directly from within the cancer cells.in vitro research program to investigate if we could predict the biological response of cells treated with NBTXR3 nanoparticles exposed to ionizing radiation. First, we estimated the nanoparticle uptake in a panel of human cancer cell lines with increased nanoparticle concentration. Subsequently the NBTXR3 nanoparticle’s effect on cell survival was evaluated using the clonogenic survival response curve. Our results suggest that above a minimum number of nanoparticles per cell, the radioenhancement of NBTXR3 nanoparticles could be predicted from the radiation dose delivered and the intrinsic radiosensitivity of the cancer cell line.Using Monte Carlo calculation, activation of NBTXR3 nanoparticles with high energy photons (1 or 6 MeV) already demonstrated a local energy deposit in specific subcellular structures containing the nanoparticles . Advanci2) crystallites, bearing a marked negative surface charge in aqueous solution at pH 6-8. Specifically, the studies reported in the present article were performed with spherical nanoparticles with size and surface charge of approximately 50 nm and -50 mV respectively  and Hs913T (fibrosarcoma) were purchased from the American Type Culture Collection  and the Interlab Cell Line Collection  respectively.The radioresistant cell lines HT-29 , FaDu (pharinx squamous cell carcinoma), HT-1080 (fibrosarcoma) and PANC-1 , were purchased from the American Type Culture Collection (LGC). The radioresistant cell lines NCI-H460-luc2  and CAL-33 (human head and neck squamous cell carcinoma) were purchased from Caliper life science . The radioresistant cell line 42-MG-BA (glioblastoma multiforme), was purchased from the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH German Collection of Microorganism and Cell Cultures .2 humidified atmosphere.Cells were grown according to each manufacturer’s recommendations  to the cells. Cells were then pre-fixed with 2.5% glutaraldehyde in cacodylate 0.1 M at pH 7.4  for 2 hours. Cells were subsequently post-fixed with 1% osmium tetroxide (Sigma Aldrich) for 45 minutes, washed and dehydrated in graded concentrations of ethanol  (Sigma Aldrich). Cell samples were embedded with EPON . The resin sample block was trimmed, 90 μm thin-sectioned to thickness of 70 nm, , and collected on grids (Euromedex) for observation under transmission electron microscopy . For each cell line, a minimum of 20 cells were observed to estimate NBTXR3 nanoparticles cell uptake. Parameters evaluated included cluster number per cell and the cluster size. Size was split between “small cluster” with a medium of size around 0.45 μm of diameter and “large cluster” with a diameter around 1.8 μm.Cells were seeded into 6-well plates (in duplicate) at the density of 20 000 cells/cm2 humidified atmosphere, allowing cells to form colonies. The colonies were fixed and stained with crystal violet solution (Sigma Aldrich) and individual colonies were counted using the standard procedure [n = 3)) and mean Dose Enhancement Factor (DEF mean ± SD (n = 3)).Cells were seeded into 6-well plates (in triplicate) within the range of 200 to 8000 cells/well. NBTXR3 nanoparticles concentrations from typically 50 μM up to 800 μM were tested. When cells were attached to the plate, NBTXR3 was added overnight (12 h-16 h) to the cells before delivering the radiation dose. The cells were cultured with antibiotics  from 10 to 15 days at 37°C under a 5% COrocedure . The DosCell survival curves were obtained by analysing the mean surviving fractions (SF mean) with the linear quadratic (LQ) model (Equation 1) using the GraphPad Prism® V5.0 Software [S represents the survival after radiation dose (D). α (Gy−1) and β (Gy−2) are the linear and the quadratic parameters respectively.−1 using X-Ray generator . The 6-well plates were irradiated with a single dose (0 Gy (sham control) up to 4 Gy or 6 Gy) after incubation with NBTXR3, and then replaced into the incubator until colonies formed.X-ray irradiations were delivered at dose rate of 1.26 Gy minThe cellular uptake of NBTXR3 nanoparticle at 400 μM by epithelial , mesenchymal  and glioblastoma 42-MG-BA cell lines was investigated by TEM .Epithelial, mesenchymal and glioblastoma cancer cells, (radiosensitive and radioresistant), were selected to evaluate the surviving fraction and DEF of cells treated with NBTXR3 at increasing concentration and exposed to ionizing radiation Figure . The pla50, we determined the DEF as a ratio of surviving fractions for a given radiation dose, i.e. for a given radiation dose (D) we determined a survival fraction in the presence of NBTXR3 nanoparticles, SF+NP(D). We then determined for the same given radiation dose (D) the survival fraction in the controls (i.e. without nanoparticles), i.e. SF-NP(D). For a given radiation dose, the DEF value was obtained by taking the ratio of the two survival fractions, i.e. DEF = SF-NP(D)/SF+NP(D).In the epithelial models , the measured mean DEF values (over the triplicate measurements) ranged between 1.05 ≤ DEF ≤ 1.64 and 1.25 ≤ DEF ≤ 2.63 for NBTXR3 (400 μM) activated by 2 or 4 Gy respectively. NBTXR3 at 800 μM showed 1.21 ≤ DEF ≤ 2.24 and 1.57 ≤ DEF ≤ 5.09, activated by 2 or 4 Gy respectively. For the mesenchymal and glioblastoma cell lines , the DEF values for NBTXR3 (400 μM) activated by 2 or 4 Gy ranged between 1.57 ≤ DEF ≤ 2.75 and 3.44 ≤ DEF ≤ 5.36 respectively. The DEF values increased according to the NBTXR3 concentrations and radiation dose  for NBTXR3 concentration ≥ 800 μM in the epithelial cancer cells lines. Meanwhile, this equation was applicable at lower nanoparticle concentrations, typically between 100 μM and 400 μM, in the mesenchymal and glioblastoma cancer cell lines (Table 2) of the cell line , whereas cells treated with NBTXR3 at 400 μM and exposed to ionizing radiation, alpha ranged between 0.43 Gy−1 and 0.74 Gy−1. The quadratic coefficient β, for both controls and NBTXR3 treated cell lines, ranged between 0.01 Gy−2 and 0.09 Gy−2.The parameters associated with the survival curves are summarized in Table 9 HT-1080 and Hs913T cells respectively  with presence of NBTXR3 nanoparticles, whereas a total dose of approximately 45 Gy and 30.5 Gy respectively was necessary for the same outcome in these cell lines with radiotherapy alone.We established an effective survival curve model in the context of fractionated radiotherapy (2 Gy) once daily, for 25 days, for the fibrosarcoma cell lines exposed to 400 μM of NBTXR3, and compared the results of this model to corresponding predictions for radiotherapy alone. We assumed that the window between successive fractions has allowed the physiological repair  in cells. The α and β parameters using the LQ model for the HT-1080 and Hs913T cell lines with NBTXR3 concentration of 400 μM and using the X-ray generator 200 kVp, were used in this simulation . The exact mechanism for this differential uptake deserves more studies and is beyond the objectives of this paper.via secondary electron emission (photoelectric and/or Compton effect), photon emission and also possibly via characteristic x-ray photon, Auger cascade and subsequent radical production. In addition, significant in vitro radioenhancement has already been reported to be related to the nanoparticles location at the subcellular level [Radiation dose deposit within any tissue sample is linked to its ability to absorb/interact with x-rays. Introduction of material with a high electron density into the x-ray pathway can increase absorption as compared with water. Only the use of such material at the nanoscale can bring the physical mode of action of radiotherapy from within the cancer cells. Local dosimetry at the cellular level has yielded evidence of increased deposit of energy in specific subcellular structures containing the high electron density nanoparticles, ar level ,3,19.The Linear Quadratic model assumes that lethal radiation damage, as a result of double-strand breaks in the DNA, is created either as a consequence of a single ionizing event or as a consequence of two separate sublethal ionizing events that interact pairwise to create lethal damage . α and βin vitro NBTXR3 cellular uptake was observed to be lower in CAL-33 and FaDu cell lines at NBTXR3 concentration 800 μM (data not shown).Radioenhancement of NBTXR3 nanoparticles in all tested human cancer cell lines was found to increase with both NBTXR3 nanoparticle concentration and radiation dose. For the following cancer cell lines, HCT 116, NCI-H460-luc2, PANC-1, Hs913T, HT-1080, and 42-MG-BA, above a certain nanoparticle concentration and 2 Gy, equation 2 predicted an observable effect. According to equation 2, for a radiation dose (D) of 2 Gy and more, the DEF values were found to vary linearly with the dose D . The nanoparticles concentration was dependent on the cell line. For the epithelial cancer cell lines, a minimum NBTXR3 concentration of 800 μM was necessary, whereas in the mesenchymal and glioblastoma cancer cell lines a lower nanoparticle concentrations, typically between 100 μM and 400 μM, was needed. The NBTXR3 nanoparticles cell uptake was related to some extent to the biological behavior of each cancer cell line. For instance, in the 42-MG-BA model, a marked uptake was seen in all the observed cells at the low NBTXR3 concentration of 100 μM, whereas for HCT 116 cells it was observed at 800 μM or above  were determined by dynamic light scattering technique . The surface charge of the nanoparticles in aqueous solution at pH values between 6 and 8 (about -50 mV) was estimated by zeta potential analysis (Zetasizer NanoZS). Spherical nanoparticle shape was determined using transmission electron microscopy technique .Click here for fileHuman cancer cell lines and culture conditions. The HCT 116 and HT-29 cells were grown in McCoy’s 5a medium , supplemented with 10% (v/v) of heat-inactivated fetal calf serum (FCS) (Invitrogen). The NCI-H460-luc2 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX™ medium (Invitrogen), supplemented with 10% of FCS . The PANC-1, HT-1080, CAL-33 and Hs913T cells were grown in DMEM with GlutaMAXTM (Invitrogen), supplemented with 10% of inactivated FCS (Invitrogen). The 42-MG-BA cells were grown in DMEM with GlutaMAXTM (Invitrogen) and Minimum Essential Media (MEM) (1:1) (Invitrogen), supplemented with 10% FCS (Invitrogen). The FaDu cells were grown in MEM with GlutaMAXTM (Invitrogen), supplemented with 10% FCS (Invitrogen). All cells lines were kept in an incubator at 37°C under 5% CO2 humidified atmosphere.Click here for file


Methods: We processed native tumor material obtained intra-operatively within 24 h for cell culture. Epithelial membrane antigen (EMA) immunohistochemistry was performed after the first passage to confirm that cells were meningioma cells. For 5-ALA-PDT treatment, about 5000 cells per well were seeded in 20 wells of a blank 96-well plate. Each block of 4 wells was inoculated with 150 µL of 0, 25, 50 and 100 µg/mL 5-ALA solutions; one block was used as negative control without 5-ALA and without PDT. Following incubation for 3 h PDT was performed using a laser . The therapeutic response was analyzed by the water soluble tetrazolium salt (WST-1) cell viability assay 90 min after PDT. Results: 5-ALA-PDT was performed in 14 primary meningioma cell lines. EMA expression was verified in 10 primary cell cultures. The remaining 4 were EMA negative and PDT was without any effect in these cultures. All 10 EMA-positive cell lines showed a significant and dose-dependent decrease in viability rate (p < 0.001). Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL was 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. For the negative controls (no 5-ALA/PDT and ALA/no PDT), the viability rates were 101.72% ± 3.5% and 100.17% ± 3.6%, respectively. The LD50 for 5-ALA was estimated between 25 and 50 µg/mL. Conclusion: This study reveals dose-dependent cytotoxic effects of 5-ALA-PDT on primary cell lines of meningiomas. Either 5-ALA or PDT alone did not affect cell survival. Further efforts are necessary to study the potential therapeutic effects of 5-ALA-PDT in vivo.Objective: Five-aminolevulinic acid (5-ALA)-induced porphyrins in malignant gliomas are potent photosensitizers. Promising results of ALA-PDT (photodynamic therapy) in recurrent glioblastomas have been published. Recently, 5-ALA-induced fluorescence was studied in meningioma surgery. Here, we present an experimental study of ALA-PDT in an  Meningiomas account for 20%–34% of intracranial tumors. With gliomas, they are the most common primary intracranial tumors with an annual incidence ranging from two to seven per 100,000 for women, and from one to five for men ,6,7. RecFive-aminolevulinic acid (5-ALA) has been under close scrutiny for fluorescence-guided resections because it elicits the accumulation of fluorescing porphyrins in malignant gliomas ,18,19,20An endogenous precursor molecule in the heme synthesis, 5-ALA is metabolized to protoporphyrin IX (PPIX). PPIX fluorescence under blue light has been used for intraoperative visualization (photodiagnosis) of malignant gliomas . In addiSystemic 5-ALA application causes fluorescence of gliomas and also of meningiomas, but not of adjacent brain tissue enhancing visual distinction of tumor tissue from brain tissue. This may help to improve the extent of resection (photodiagnosis) but may also be used for PDT ,32. ExpeTo assess whether 5-ALA-induced PDT is a useful tool in therapy of malignant and recurrent meningioma, a better knowledge of PDT susceptibility of different meningioma cell lines is necessary. Therefore, in this study, we evaluated the 5-ALA induced PDT susceptibility of 21 primary meningioma cell cultures.p = 0.002) difference of EMA expression depending on the latency of the first passage could be identified, with high EMA expression corresponding to an early passage in average after 7 days (range 5–10 days). Chi2-test did not reveal any correlation of EMA expression to WHO grade.Of the 14 cell lines that underwent immunohistochemical detection of epithelial membrane antigen (EMA), ten (71%) were EMA positive and four (29%) were EMA negative . A highlWater soluble tetrazolium salt (WST)-1 cell viability assay showed toxicity to the tested cell lines exclusively with the combination of 5-ALA application and laser irradiation. No toxicity (decreased cell viability) was detected in wells containing 50 μg/mL 5-ALA not subjected to irradiation, neither was toxicity detected in wells without 5-ALA subjected to irradiation .p < 0.001) at concentrations 25 μg/mL and above. The viability rates, documented by the WST-1 assay, are demonstrated in No toxicity was seen in EMA negative cell lines. Application of 5-ALA and subsequent PDT was found to be toxic in all 10 tested EMA positive cell lines, and toxicity was found to be dose-dependent in all cell lines. Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL were 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. Cell viability decreased significantly (p < 0.001). LD50 was estimated between 25 and 50 µg/mL. The four EMA negative populations showed no significant effect by 5-ALA-PDT.50 between 25 and 50 µg/mL. Hefti et al. recently reported a very interesting in vitro experiment on two immortalized meningioma cell lines (BEN-MEN 1 and HBL-52). Within the same cell lines the results on ALA-PDT were reproducible with a high reliability and validity. However, between the two cell lines, they detected a variable response to the irradiation therapy [in vitro. All primary cell lines responded positively to ALA-PDT with a significant decrease in viability with increasing concentrations of 5-ALA. The cell lines showed different sensitivities, reflected by the high variance and standard deviation at 25 µg/mL. The significance of this high variance compared to the other used concentrations remains unclear; however, it may reflect the borderline of the LD50 between 25 and 50 µg/mL ALA solutions.In the present study, we demonstrated that 5-ALA-PDT is effective in decreasing viability of primary meningioma cell lines in a dose-dependent manner, with an estimated LD therapy . We confin vivo. The intraoperative use of 5-ALA for the visualization of meningiomas showed different fluorescent intensity between and within the resected meningiomas [Nevertheless, our results confirm Coluccia’s observations ingiomas .et al. found dose-dependent toxicity of PDT in five meningioma cell lines and no toxicity in control cell lines. However, the sample size was small and hematoporphyrin derivate (HpD) was used as photosensitizer, which is associated with severe adverse effects and is not tumor specific. Rather, it transgresses the blood-brain barrier along with edema and is thus unspecific for intracerebral tumor tissue [The major determinant of recurrence and prognosis in therapy of meningiomas is the extent of surgical resection of the tumor. Complete resection may be hindered by infiltration or tight contact to dural sinuses, cranial nerves, vessels or brain parenchyma. PDT technically offers a possibility of selective destruction of infiltrating tumor tissue, thereby potentially reducing the rate of recurrence. The first investigations of PDT of meningiomas were carried out in the 1990s ,35. Markr tissue ,18.A subsequent study of 5-ALA-PDT of immortalized glioma and meningioma cell lines found increased fluorescence and PDT sensitivity of glioma cell lines compared to meningioma cell lines . Althouget al. revealed inhomogeneity of fluorescence within meningiomas [Several groups have demonstrated reliable fluorescence of meningiomas after 5-ALA application, which reflects substantial PPIX accumulation ,32,34,39ingiomas , a phenoingiomas . This iningiomas ,42, may ingiomas ,43. Mostingiomas ,44. In oTumor material used in this study was obtained from 14 patients undergoing surgery for resection of a meningioma. There were five (35.8%) males and nine (64.2%) females. Age at resection ranged from 30 to 82 years with an average of 55.3 years. Twelve were classified as benign (WHO grade I), one as atypical (WHO grade II) and one as anaplastic (WHO grade III) according to the WHO 2007 classification criteria . Informeet al. [n = 20), we had modified Hardy’s method continuously in order to establish the following procedure in our laboratory: After surgical resection, meningiomas were washed in Hank’s balanced salt solution (HBSS)  and separated from macroscopically visible blood vessels and dura mater, before being minced and centrifuged (500 rpm/400 G). Tissue was then washed again and incubated at 37 °C with 0.05% Trypsin  for 30 min. Medium  with 10% FCS , 1% glutamax, 1% penicillin, 1% streptomycin and 1% amphotericin B ) were added and the tissue centrifuged again before performing another two washing and centrifugation steps. The obtained cell pellet was re-suspended in medium in a 0.01% collagen type single-coated T25 cell culture flask and cultured as a monolayer. Cells were passaged when a confluence of 70% was reached.Culture of meningioma cells after resection of the tumor was performed by a method adapted from Hardy et al. . In prel® .As culture of primary cells often bears the risk of overgrowth of fibroblasts, we immunohistochemically stained for EMA, which is a specific marker expressed in meningothelial cells. For immunohistochemical detection of EMA, a portion of cells were transferred into a Petri dish containing a cover slip and cultured for several days until the cells had overgrown the cover slip. The cover was then fixed in acetone at −20 °C for 10 min before being left to air drying for 15 min. The dry cover slip was then glued onto a glass slide and was then ready for polymer-conjugated detection of EMA expression by the EnVision™ Detection System . This method involves detection of monoclonal mouse anti-EMA antibodies by binding of peroxidase–polysaccharidpolymer-conjugated anti-mouse secondary antibodies. Firstly, slides were washed in tris buffered saline with tween 20 , and then incubated in Dako REAL™ Peroxidase-Blocking Solution  for 10 min. Under subsequent washing, slides were incubated in primary antibody ) and then in secondary antibody ), each in a humidity chamber for 30 min. The slides were then washed again and incubated in substrate chromogen solution ) for 4–6 min. After washing in demineralized water, they were contrasted with hemalaun, dehydrated in ethanol and finally mounted with Eukitt2 incubator for 4 h (PDT and control wells were filled with 100 μL of cell solution containing 50 cells per 1 μL medium (counted using trypan blue with PBS 1:2 in a Neubauer counting chamber) corresponding to 5000 cells per well, whereas blank wells were filled with 100 μL of colorless medium. After incubation for 48 h 5-ALA powder  was diluted with aqua dest, in a sterile setup avoiding direct light, to the required concentration, before 50 μL were added to PDT and control wells. Fifty μL of colorless medium were added to blank wells before incubating the microplate in a CO for 4 h .2, which was executed for 625 s resulting in a radiation dose of 18.75 J/cm2. After irradiation, the microplate was returned to the incubator for 90 min before performing the cell viability assay . It includes a diode laser , which emits light at a wavelength of 635 nm through a fiberglass probe with a diffusor lens, producing a cone of light. This allows homogeneous irradiation of the wells of the 96-well microplate. Irradiation of the wells by backscatter rays from other wells was avoided by leaving at least two rows of distance between the experimental wells. The light source was arranged 9.3 cm from the microplate in order to achieve an intensity of 30 mW/cmty assay .Standard WST-1 assay , which is based on living cells’ capacity to convert red tetrazolium salt into dark red formazan, which can be photometrically quantified, was used to determine cell viability after PDT.2-test, student’s t-test, Kruskal–Wallis-Test and one-way ANOVA, in which p <0.05 and p < 0.01 were considered significant and highly significant, respectively. Data was expressed as average ± standard deviation. Graphs were created using SPSS.SPSS Statistics 21 , was used to perform Chiin vivo in meningiomas.Photodynamic therapy using the photosensitizer 5-ALA is effective in a dose-dependent manner in primary human meningioma cell lines. Further investigations are necessary to understand the transduction pathway of 5-ALA in tumor cells and to understand variation between individual meningiomas. This may open the possibility of clinical trials to test the therapeutic effect of ALA-PDT


Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus  Glioblastoma Multiforme (GBM) is the highest grade glioma (grade IV) tumor and the most malignant form of astrocytoma. Despite the wide range of treatments, including surgery, radiotherapy, and chemotherapy, a majority of the therapies for GBM have a limited improvement on patients’ survival, largely due to the highly proliferative, invasive, and often drug-resistant nature of the tumor. GBM’s median survival time is approximately 14.6 months  × 100% (where T is the treatment time and T0 is the time that the wound was induced).Wound healing assay was employed to assess cell migration, as described elsewhere . In brie3VO4, 1 mM NaF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin) and centrifuged at 13,000 rpm to pellet the insoluble material. The protein concentrations of cell lysates were determined using was determined with the bicinchoninic acid (BCA) assay method. Equivalent amounts of protein were separated in SDS-PAGE gel and transferred to nitrocellulose membrane  using a semi-dry transfer method . The membrane was then blocked with 5% milk in TBS with 0.1% Tween-20 at room temperature for 1 h, and immunoblotted with primary antibodies overnight at 4 °C as follows: anti-TRPM7 (1:1000), anti-p-Akt (1:1000), anti-Akt (1:1000), anti-p-ERK1/2 (1:1000), anti-ERK1/2 (1:1000), and anti-β-actin (1:1000) antibodies, followed by incubation with the corresponding HRP-labeled secondary antibody  for 1 h at room temperature in conjunction with a chemiluminescence reagent system . Densitometry was carried out using Image-Pro Plus software.Western blotting experiments were carried out as previously described ,57. Tota2, 20 HEPES, and 10 glucose (pH was adjusted to 7.4 with NaOH). The cell was perfused with bath solution before perfusing with xyloketal B (300 μM). When the effects of xyloketal B reached the maximum platform, the cell was reperfused with bath solution to wash out xyloketal B.Whole-cell patch-clamp recording was used to analyze TRPM7 currents using an Axopatch 700B , as previously described . Brieflyt-test. All reported p-values were two-sided and were considered to be statistically significant at p < 0.05.Data are presented as the mean ± SEM. One-way ANOVA with subsequent Newman–Keuls test was used to determine the statistical significance for multiple comparisons. Comparison between two groups was analyzed using Student’s in vivo study, is essential to explore its value as a potential therapeutic agent for treatment of glioblastoma.Marine compound xyloketal B reduces cell viability, proliferation, and migration through inhibition of TRPM7 and modulation of the PI3K/Akt and MEK/ERK signaling pathways. Thus, marine compound xyloketal B may be a potential target for drug development. Further research, especially


Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies.High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this  Neuroblastoma is a developmental tumor of early childhood arising from the neural crest , 2. NeurHere, we investigated the druggability of NRAS in neuroblastoma. We show that NRAS can be targeted by MEK inhibitors AZD6244 (Selumetinib) and MEK162 whereas wild-type cells are refractory to this treatment. We observed that single PI3K inhibitors were ineffective in blocking PI3K signaling and also cell growth. However, the use of single mTOR inhibitors Everolimus and AZD8055 resulted in a significant reduction of cell growth exclusively in NRAS mutant cell lines. More importantly, Everolimus alone was sufficient to induce apoptosis in NRAS mutant neuroblastoma cell lines but not in wild-type cell lines. Moreover, combination of mTOR and MEK inhibitors block cell growth synergistically. These findings open new perspectives in the treatment of high-risk and relapsed neuroblastoma.https://ocg.cancer.gov/programs/target). The Children's Hospital of Philadelphia Institutional Review Board approved this study and was responsible for oversight of this study. Flash-frozen tumor samples were analyzed for percent tumor content by histopathology, and samples with <75% tumor content were excluded.Patient samples are derived from high-risk neuroblastoma patients as described in  as part AZD6244, MEK162, BKM120 and PD0325901 were purchased from Selleck Chemical. Everolimus, AZD8055, GSK690693, MK2206 and BKM120 (as a control) were purchased from MedChemExpress. All inhibitors were solubilized in dimethyl sulfoxide (DMSO) at stock concentrations of 1mM.CHP-212 and SK-N-AS were purchased from ATCC. NGP and CHP-134 were purchased from DSMZ . SH-SY5Y was provided by Prof. A. Aguzzi, Institute of Neuropathology, Zurich. CHP-212 cells were cultured in DMEM and F12 medium (1:1) supplemented with 10% fetal calf serum (FCS) and 1mM L-glutamine. SK-N-AS cells were cultured in DMEM supplemented with 0.1mM non-essential amino acids and 1mM L-glutamine. NGP cells were cultured in high-glucose DMEM (4.5 g/L glucose) supplemented with 10% FCS. SH-SY5Y cells were cultured in DMEM supplemented with 10% FCS. All cell lines were purchased within less than one year and regularly tested for mycoplasma contamination.CHP-212, SK-N-AS, SH-SY5Y, CHP-134 and NGP were transfected with either control or two different siRNAs against NRAS . Accell siRNA was purchased from Dharmacon and used according to manufactor’s protocol. In brief, siRNAs were diluted in Accell siRNA Delivery Media from Dharmacon to a final concentration of 1μM. Knockdown efficiency was observed after 96 hours.6 cells were lysed for 30 minutes in ice-cold MPERM buffer supplemented with 25mM NaF, 1mM dithiothreitol, and complete protease inhibitor cocktail from Roche Diagnostics. Cell debris was removed by centrifugation and then, proteins were blotted onto a nitrocellulose membrane  followed by blocking with 5% bovine serum albumin in phosphate-buffered saline/Tween . The following antibodies were used: anti-phospho-ERK , anti-ERK , anti-phospho Akt , anti-panAKT , anti-phospho MEK , anti-MEK , anti-phospho mTOR , anti-mTOR , anti-phospho S6 , anti-S6 , anti-NRAS rabbit  and anti—tubulin (Sigma-Aldrich).Western Blots were performed as described previously . In brieIdentification of mutations was performed as described previously . Total cCell lines were treated with indicated concentrations of inhibitors and apoptosis was measured after 48 hours and 72 hours. Apoptosis was assessed by AnnexinV—APC (Enzo Lifescience) and propidium iodide (PI) (Sigma-Aldrich) by FACS. Both reagents were diluted to a final concentration of 1.5%. Specific cell death was calculated by the following equation: specific cell death % = /(100%—% spontaneous cell death) x 100 .Cell growth was measured with the Cell-Titer-Glo Reagent (Promega) according to manufacturer’s instructions. Cells were plated in 96-well plates at a density of 500–2500 cells per well. The next day, drugs were added at indicated concentrations and cell growth was measured 96 hours later. Proliferation was made with a 96-well plate luminometer/plate reader . Data shown are calculated as relative values for a given drug concentration compared to non-treated cells. All experimental points were set up in duplicate replicates and were conducted at least 3 independent times. IC50 were calculated with GraphPad Primsm. Combination index (CI) values with CalcuSyn Software (Biosoft) according to Chou . CI valuWithin the TARGET consortium whole genome or whole exome sequencing of newly diagnosed matched neuroblastoma tumor and blood-derived DNAs was performed , 19. We Analysis of the NRAS gene in our panel of neuroblastoma cell lines revealed two NRAS mutations at position Q61K in the CHP-212 and SK-N-AS cell line, which was also the most common mutation in the primary tumors , adenocarcinoma of the lung (1%), neuroblastoma (0.83%) and cutaneous T-cell lymphoma (4%). We and others have shown that NRAS mutations sensitize towards inhibition of MEK in cutaneous T-cell lymphoma, lung cancer and neuroblastoma cell lines , 13, 15.In our study, we demonstrate that inhibition of the mTOR pathway by Everolimus alone is sufficient to block cell growth in NRAS mutant neuroblastoma cells compared to wild type cells . More imIn conclusion, we show that mutant NRAS neuroblastoma can be targeted by single MEK and mTOR inhibitors or their combinations. Especially Everolimus as currently available drug could be an alternative option for NRAS mutated neuroblastoma patients being refractory to other treatments.S1 FigA) PCR obtained from cDNA of respective cell lines were sequenced by Sanger sequencing for the complete NRAS coding region. B) Cell lines were incubated with indicated concentrations of MEK162 and IC50 values were calculated with GraphPad Prism. C) Accell siRNA against NRAS (siNRAS1 and siNRAS2) was transfected in NRAS mutant cancer cell lines according to manufactor´s instruction. Then, 96h later cells were lysed and lysates subjected to Western blot. Efficiency of knock-down was measured by anti-NRAS antibodies. Equal loading was controlled by anti-tubulin antibodies. D) Accell siRNA against NRAS (siNRAS1 and siNRAS2) was transfected in NRAS mutant cancer cell lines according to manufactor´s instruction. 96 hours after transfection cell growth was assessed by Cell titer Glo. P values were calculated with t-test and * defined as p<0.05.(EPS)Click here for additional data file.S2 FigA) NRAS mutant and NRAS wild-type cell lines were incubated with indicated concentrations of MEK inhibitors MEK162 for 72 hours. Then, cell death was determined by Annexin V and PI staining.(EPS)Click here for additional data file.S3 FigA) CHP-212, SK-N-AS and BT-474 (used as planned positive control) cells were treated with 0.5μM and 1μM of BKM120 for 3 hours. Then, cells were lysed and analysed by Western blot. B) L-363 was used a positive control to investigate whether BKM120 might work in our hands. L-363 cells were treated with 0.5μM and 1μM of BKM120 or GDC0032 for 3 hours. Then, cells were lysed and analyzed by Western blot. C) L-363 was left untreated or treated with indicated concentrations of BKM120 for 96 hours. Next, cell growth was measured by Cell Titer Glo according to the manufacturer’s instructions.(EPS)Click here for additional data file.S4 FigA) CHP-212 cells were treated with MEK162 or Everolimus or combinations thereof as indicated for 48h. Then, cell growth was assessed by Cell titer Glo. B) Same as C) but the readout was done after 72h. C) Same as B) but the readout was done after 96h. Combination index (CI) values with CalcuSyn Software (Biosoft).(EPS)Click here for additional data file.


Metformin has been shown to inhibit cell proliferation and induce cell death in many cancer cell lines. However, some cell lines exhibit resistance to the cytotoxic effects of metformin in tissue culture medium with high glucose levels (25 mM). Recent studies suggest when glucose is lowered; metformin becomes increasingly cytotoxic to cancer cells. However, the precise mechanism for metformin’s cancer cytotoxicity remains unidentified.Cell death assay using Sytox Green Nucleic Acid Stain Sytox Green nucleic acid stain (10 μM) was added directly to cells in 96 well plates and read to obtain dead cells. Total cell number was obtained after adding 0.4% Triton X-100.ATP assay 10 μl lysate of equal numbers of cells was used following the manufacturer’s protocol for ATP assays from Invitrogen .Lactate Assay Medium was tested for lactate concentration using L-Lactate Assay Kit (Eton Biosciences Inc.). Relative lactate level was obtained after normalized by total cell number.Measurement of Extra Cellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) ECAR and OCR were measured using a Seahorse XF24 analyzer according to manufacturer’s instructions (Seahorse Bioscience). ECAR and OCR were plotted after normalizing to total cell number.Metformin became more cytotoxic in all of the tested breast cancer cell lines when glucose concentration was titrated to low levels in tissue culture medium. Metformin enhanced cytotoxicity in low glucose medium was correlated with significant reduction of ATP that was not observed in high glucose containing medium. Supplementing low glucose medium with fructose or galactose failed to prevent the reduction of ATP or to rescue the cells from metformin induced cell death. This suggested that metformin treated breast cancer cells cannot efficiently use fructose or galactose to maintain ATP. Metformin is known to inhibit complex I and prevent ATP production. However, in high glucose containing medium metformin treated cells maintained ATP levels via feedback activation of glycolysis. This enhanced glycolysis was either diminished or blocked when metformin treated cells were switched to low glucose, high fructose or high galactose medium. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis.Our data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production via diminishing or preventing metformin stimulated glycolysis.


PTEN tumor suppressor, but also a number of targets that are less frequently mutated in specific cancer types, including ABL1 oncoprotein in acute myeloid leukemia. An application to leukemia patient primary cell models demonstrated how the target deconvolution approach offers functional insights into patient-specific addiction patterns, such as those indicative of their receptor-type tyrosine-protein kinase FLT3 internal tandem duplication (FLT3-ITD) status and co-addiction partners, which may lead to clinically actionable, personalized drug treatment developments. To promote its application to the future drug testing studies, we have made available an open-source implementation of the TAS calculation in the form of a stand-alone R package.Deconvoluting the molecular target signals behind observed drug response phenotypes is an important part of phenotype-based drug discovery and repurposing efforts. We demonstrate here how our network-based deconvolution approach, named target addiction score (TAS), provides insights into the functional importance of druggable protein targets in cell-based drug sensitivity testing experiments. Using cancer cell line profiling data sets, we constructed a functional classification across 107 cancer cell models, based on their common and unique target addiction signatures. The pan-cancer addiction correlations could not be explained by the tissue of origin, and only correlated in part with molecular and genomic signatures of the heterogeneous cancer cells. The TAS-based cancer cell classification was also shown to be robust to drug response data resampling, as well as predictive of the transcriptomic patterns in an independent set of cancer cells that shared similar addiction signatures with the 107 cancers. The critical protein targets identified by the integrated approach were also shown to have clinically relevant mutation frequencies in patients with various cancer subtypes, including not only well-established pan-cancer genes, such as  Drug Discovery Collection: The authors take a pan-cancer approach that provides functional insights into molecular vulnerabilities across various cancer subtypes and patient-specific target addiction patterns that could lead to actionable treatment strategies. With the emerging challenges of translating the cancer genome sequencing information into clinically actionable, targeted drug treatment strategies, it has become evident that we also need complementary strategies for functional investigation of druggable vulnerabilities and cellular addictions of cancer cells. Advances in cell-based high-throughput compound screening have made it possible to profile the cellular activity of an extensive collection of bioactive small molecules, thereby enabling a functional phenotype-based approach to unbiased identification of candidate compounds for individual cancer patients  is an important aspect of the rational phenotype-based drug discovery approach . Knowledin vitro and to patient-derived samples ex vivo; for a given cancer cell model, it provides a functional importance scoring of the drug targets, according to an estimate of how much the cell sample is addicted to the particular target protein. In an application to >100 cell lines, we deconvoluted the target signal patterns across the heterogeneous cancer cell collection, hence revealing pan-cancer correlations in their addiction signatures between both shared and unique genetic and tissue backgrounds. A specific application to leukemia patient primary cancer cell models demonstrates how the target deconvolution approach can yield functional and actionable insights into patient-specific addiction patterns towards clinical translation.BackgroundCell-based drug sensitivity testing is becoming a popular approach to provide a functional profile not only for cancer cell lines but also for primary patient cells to map pathway dependencies in specific cancer subtypes and to select optimal personalized treatments for cancer patients. However, several critical challenges remain; in particular, how to deconvolute signals of addiction – a phenomenon describing the dependency of certain tumors on a single oncogene for growth and survival – behind the drug sensitivity and resistance phenotypes, which is a critical step in the phenotype-based drug development process and an important prerequisite for many translational applications.ResultsIn this study, the authors implemented a network-based experimental-computational approach that makes use of polypharmacological effects of drug compounds to identify pharmacologically actionable target signals in individual cancer cell subtypes and in patient-derived cell models. When applied to >100 cancer cell lines, this approach mapped target signal profiles across heterogeneous cancer cell models; these profiles revealed pan-cancer correlations in the addiction signatures of different cancer cell subtypes that have both shared and unique genetic and tissue backgrounds. Specific application to leukemia patient primary cell models demonstrated how the target deconvolution approach offers functional insights into patient-specific addiction patterns that might lead to clinically actionable treatment strategies.Implications and future directionsThis computational target deconvolution method represents an open-source and extendable approach that can be used to identify target addiction profiles across heterogeneous cancer cell lines and leukemia patient primary cell models. This approach should thus provide a valuable resource for the community to enable translation of drug sensitivity testing results into many exciting drug development and repurposing opportunities.In the present study, we applied an experimental-computational target deconvolution approach in a diverse collection of cancer cell models. Our computational target addiction scoring (TAS) algorithm integrates cell-based compound sensitivity profiling with global drug-target interaction networks that capture a broad spectrum of both on- and off-target effects of the compound panel, comprising not only kinase inhibitors but also other important drug target classes. The integrated approach is applicable both to cell line models We implemented an experimental-computational platform that identifies critical drug targets in individual cancer cell lines or patient-derived cell models . The appsupplementary material Table S1). As the primary drug response metric, we used the drug sensitivity score (DSS), which was recently shown to improve the prediction of drugs' mechanism of action, as well as the identification of drug-responsive cell lines and patient-derived cancer cells (50), as well as the area under the dose-response curve (AUC), as available in the GDSC resource. The pairwise addiction signatures between the cell lines were compared with those derived from the genomic signatures of the selected cell lines, using congruence analysis, and later the predictive power of the TAS signatures was assessed in an additional set of 20 cell lines from GDSC.We first systematically tested the TAS approach using the comprehensive cell line data from the Genomics of Drug Sensitivity in Cancer (GDSC) project, which is the largest public resource for information on drug sensitivity in cancer cells along with their comprehensive genomic and molecular characteristics . We calcer cells . We alsoP=0.0001, permutation test of the Kendall coefficient). In contrast, the cell line clustering based on the target addiction profiles was effectively independent of the tissue type . The same was also true when using the DSS alone, although not as prominent as with the DSS-TAS transformation . For instance, gastrointestinal (GI) tract cell lines formed a clear sub-cluster in the expression-based clustering, whereas these cells were scattered in various sub-clusters of the TAS-based functional classification. This indicates that the pan-cancer functional relationships in the addiction patterns carry information beyond the tissue origin alone, which may therefore be more closely connected to the underlying biology shared by multiple cancer subtypes.We first derived unsupervised molecular and functional classifications of the 107 cancer cell lines based on their transcriptional, drug response and target addiction profiles. As expected, the cancer cell line clustering based on the gene expression profiles was largely driven by the tissue origin of these cells . The novel GFA analysis resulted in both unique and common genes in the shared active components, including, for instance, well-known cancer oncogenes, such as MYCN and CDKN2A, which were common to all the three data sets, whereas TP53 and KRAS were unique to the MUT data set only , copy number (CN) and mutation status (MUT) across the genetically heterogeneous cancer cells, we used a sparse extension of the Bayesian group factor analysis GFA; , which eset only A.Fig. 3P=0.003, permutation test). Likewise, the AUC response metric showed slightly improved concordance after its TAS transformation, whereas the IC50 metric showed a totally opposite trend in concordance. Surprisingly, the combination of the CN and MUT components resulted in a lower concordance with the addiction signatures, compared with that when using the mutation status alone, perhaps owing to the relatively high dependency (anti-correlation) of the genomic information captured by the copy number alterations and somatic point mutations, as observed in recent pan-cancer analyses .When comparing the target addiction signatures and the combined genomic signatures from the GFA analyses, it was found that the TAS version calculated with DSS led to the overall best concordance . Although the average robustness values over the whole spectrum of sub-cluster structures were relatively similar between the three response metrics, the DSS-TAS combination resulted in the most consistent cluster solution over the bootstrap samples. Notably, each of the cluster solutions made without the TAS transformation had sub-clusters with 0% robustness also at the upper levels of hierarchy in the corresponding dendrograms , indicating that these larger sub-clusters at higher levels were in fact very sensitive even to small changes in the drug response input data. In contrast, the TAS-based cluster solutions resulted in notably higher robustness values for each sub-cluster, especially at the upper levels of the dendrogram , compared with their TAS-free counterparts .To verify that the TAS-based cell line cluster solutions were not obtained by chance, for instance, owing to the specific data sample only or a single run of the clustering algorithm, we repeated the cell line clustering on 10,000 bootstrap samples of the original 107 cell lines. Strikingly, the robustness of the cell line classifications based on each drug response metric could be improved by using the TAS transformation A, most ssupplementary material Table S1). Within each of the detected eight addiction sub-clusters, we compared the GE, CN and MUT profiles of the newly added cell lines with those of the original cell lines, often representing different tissue types . As expected, the gene expression profiles were highly correlated among all the cancer cell line pairs, leading to a shifted background distribution with a positive mean (Spearman correlation 0.8). However, the expression correlations of the new cell lines with those predicted to share similar addiction patterns were significantly higher . Likewise, the TAS-based clusters were fairly predictive of the CN profiles, whereas the mutation status of the new cell lines could not be predicted accurately (data not shown). This is likely to result from the relatively small number of genes whose mutation status was available .To evaluate whether the DSS-TAS profiles could also carry a predictive signal for the genomic characteristics of the cancer cell lines, we combined 20 additional cell lines from GDSC (tested with fewer than 138 compounds) with the original analyses done on the basis of the 107 cell lines  and CN  and none with the mutational profiles; although this surprisingly small overlap may be attributed in part to the rather sparse mutation panel, it also demonstrates that the functional drug/target profiling provides added value to the genomics-only-based analyses and that the targets deemed functionally critical by the addiction scoring are not necessarily overlapping with those detected by genomic analyses of the same cancer cells.Next, we combined the target addiction signatures of the cancer cell lines into GFA in order to study the similarities and differences in the active components revealed by the genomic and functional profiles. Overall, the shared active components had only a few common genes, indicating the complementary nature of these profiles A. In parABL1, ABL2, GPRC5A and CTNNB1). An interaction network was constructed by combining these genes with each other using direct, experimentally validated and manually curated interactions from the Ingenuity Pathway Analysis knowledgebase . In addition to novel links, the network analysis also identified missing players between these four genes, including RAS oncogene, EGFR and BCR, all of which are well established as having a role in the pathogenesis and treatment of many cancer types, including breast, colorectal, pancreatic and lung cancer; these molecules are either serving as biomarkers for cancer prognosis and/or prediction of treatment efficacy .As a specific example, we focused more closely on the four common genes that were included in the shared active components between DSS-TAS, GE and CN  and HOXA1 in lung adenocarcinoma  resource. In addition to pan-cancer recurrent genes, such as arcinoma B. In leuleukemia .in silico personalized medicine’ case study. Specifically, we applied the TAS approach to the cell-based drug response data obtained from a recent study where the sensitivity of primary leukemia patient cell samples was assessed to a panel of 66 kinase inhibitors ex vivo . Based on our previous analysis of ex vivo drug response patterns in AML patients, where receptor-type tyrosine-protein kinase FLT3 internal tandem duplication (FLT3-ITD) mutation status was found to be associated with functional classification of the AML subtypes . PCR analysis revealed one of the patient samples (ID 08024) to exhibit FLT3-ITD with loss of the wild-type FLT3 allele in the original study . The calculation was based on a number of compounds with both on- and off-targeting of FLT3-ITD, including sunitinib, sorafenib and ponatinib . Whereas VEGFR2 and RET shared a number of common inhibitors with FLT3-ITD, partly explaining their correlated addiction profiles, GAK displayed a more distinct inhibitor panel, with only one potent inhibitor common with FLT3-ITD  or gain of function . In practice, only proteins with activating mutations can be targeted directly with small-molecule inhibitors. In contrast, loss-of-function cancer genes, including ethality . Howeverhenotype ; hence, FLT3 (FLT3-ITD), which are detected in approximately 20% of AML patients associated with a poor prognosis  mutations in rognosis . Clinicaassembly . In addil growth , highly l growth , as welll growth . GAK is assembly , and patassembly . Given ssupplementary material Table S3), because the observed drug response (that we want to capture) is elicited not only through the direct binding targets, but also through secondary and downstream effects . Even though we made our best effort to include only the most reliable targets, through stringent filtering criteria and manual curation, it is likely that the target mappings from these databases include both false-positive and false-negative interactions , future improvements in both the compound and target coverage of the quantitative bioactivity mappings should lead to further improvements in the performance of computational target deconvolution methods.To model the polypharmacological effects and mechanism of action of multitargeting compounds, we wanted to consider here a wide spectrum of both direct and indirect compound-target interactions  response parameters. We had access also to the raw dose-response data , enabling us to compute our drug sensitivity score ; more specifically, we also collected secondary and downstream targets for the compounds using a number of additional resources. For the kinase targeting compounds, we extracted quantitative bioactivity data from the large-scale kinase inhibitor selectivity profiling assays by 50, Kd, Ki and potency ≤35 nM to focus on the most potent targets only. We also manually filtered out genes involved in cellular metabolism, such as cytochrome (CYP) and ATP-binding cassette (ABC) genes. This resulted in an interconnected network among 138 compounds and 749 protein targets, capturing both direct and indirect interactions, which was used for modelling the comprehensive spectrum of not only on-target but also off-target and downstream effects of the compounds .For the system-level modeling of drugs' mode of action and polypharmacological effects, we extended the compound-target mappings beyond the direct binding interactions and the primary targets listed at Genomics of Drug Sensitivity in Cancer (GDSC) database of the Sanger Institute  for a particular target t is calculated by averaging the observed drug response (DR) over all those tn compounds that are known to target protein t, as follows:The target addiction score gives an estimate of the sensitivity of a cell to the inhibition of a particular protein target, thereby extending the concept of kinase addiction maps introduced by https://bitbucket.org/BhagwanYadav/target-addiction-score-tas-calculation.2 scale. Supplementary material Fig. S3A shows the standard deviation of all genes in the expression data. The genes with high enough standard deviation (above the red line) were used in GFA. All the data sets were scaled to unit standard deviation, giving them equal weight. GFA was run with a varying number of components (K) to model the total variation in the three data sets. Supplementary material Fig. S3B shows the activity of the components in GFA. A component is present for a data set when shown in black, and otherwise absent. The run resulted in components absent in all the data sets, indicating that K=30 was large enough to model the data.Factor analysis is a statistical method, which allows description of the dependencies among many observed variables using a few factors. Group factor analysis (GFA) is a recent extension of the standard factor analysis to capture joint variability across multiple data sets . Here, GW) is a non-parametric statistic to assess the agreement between two rankings . The statistical significance of the congruence was assessed by performing 10,000 permutations. We also performed Mantel's randomization test using ‘ade4’ R package . We retrieved mutation information of the selected genes in various cancer patients from The Cancer Genome Atlas (TCGA) resource. The TCGA data were downloaded and extracted using the ‘cgdsr’ R package from cBioPortal . We investigated the cancer relevance of the selected genes using QIAGEN's IngenuityPathway Analysis . Within each sub-cluster, we performed Spearman correlation analysis between the newly added cell lines and the original cell lines within the sub-cluster to evaluate their average similarity in gene expression and copy number. To assess the statistical significance of the correlation coefficients, we computed the background null distribution as pairwise Spearman correlation coefficients of every pair of the cell lines.Beyond the 107 cell lines in our original analysis, we later selected 20 additional cell lines, which were tested with 134-137 compounds. We added the TAS profiles of the 20 new cell lines to the original clustering based on the TAS profile of the 107 cell lines, and used the same distance function and clustering algorithm to re-compute new clusters. The complete panel of cell lines was divided into eight sub-clusters (FLT3-ITD.For the leukemia case study, we extracted the drug screening and clinical data for 151 primary leukemia patient samples . In this