For the above reasons, the obtained compounds can be considered a new group of promising anticancer agents, useful in the fight against p53-dependent drug resistance in cancers. They can also be treated as convenient, leading structures suitable for further optimization and searching for more active and selective molecules.Benzodiazepines that consist of one α- and one β-amino acid residues linked together in a seven-membered heterocyclic ring could be treated as small, rigid, cyclic dipeptides capable of exhibiting a wide range of biological activities. During our research on novel analogues of anthramycin, a tricyclic antibiotic benzodiazepine, we developed the synthesis of two benzodiazepine dimers, obtained through the cyclization of appropriate linear tripeptides. The synthesized compounds were tested on a panel of seven cancer and normal cell lines. The developed molecules exhibited promising cytotoxic activity against the lung cancer cell lines A549 and NCI-H1299 and the epidermoid carcinoma cell line A-431. Moreover, they showed significant selectivity compared to the reference cell lines . When tested on two isogenic cell lines, HCT116 and HCT116p53 The ratio of stereoisomers in the NMR spectrum was 54:46; 1H NMR  δ 11.06 , 8.86–8.76 , 8.55–8.47 , 8.40–8.29 , 7.30 , 4.09–3.98 , 3.75 , 3.09–2.99 , and 2.83–2.72 . 13C NMR  δ 170.65, 170.64, 165.1, 143.0, 142.1, 127.1, 127.0, 126.6, 126.2, 122.2, 51.93, 51.86, 35.1; 35.0, 28.4, and 28.3. HRMS (ESI): m/z [M + H]+ calculated for C21H19N6O8S2: 547.07003, found: 547.07069.3,3′-((methylenebis(sulfanediyl))bis(methylene))bis (11). The ratio of stereoisomers in the NMR spectrum was 58:42; 1H NMR  δ 10.81 , 8.89–8.76 , 8.05–7.90 , 4.05–3.95 , 3.73 , 3.08–2.98 , and 2.81–2.71 . 13C NMR  δ 170.85, 170.83, 166.2, 149.34, 149.33, 137.6, 132.62, 132.59, 131.35, 131.34, 118.1, 116.0, 115.9, 51.8, 35.2, 35.1, 28.5, and 28.4. HRMS (ESI): m/z [M + H]+ calculated for C21H19N6O8S2: 547.07003, found: 547.07034.3,3′-((methylenebis(sulfanediyl))bis(methylene))bis, lung carcinomas A549 (ATCC® CCL-185) and NCI-H1299 (ATCC® CRL-5803), colorectal carcinomas HCT116 (ATCC® CCL-247) and HCT116p53−/−, normal skin fibroblasts BJ (ATCC® CRL-2522), and normal lung fibroblasts MRC-5 (ATCC® CCL-171). The HCT116 p53−/− cell line was a kind gift from Professor Bert Vogelstein . The remaining cell lines were purchased from ATCC . The cells were cultured in minimal, Dulbecco’s modified Eagle’s, or Ham’s F-12K medium supplemented with 10% fetal bovine serum  and grown under standard culture conditions  in a humidified incubator with an oxygen concentration of 21%. All of the experiments were performed with mycoplasma-free cells.The following cell lines were used in this study: epidermoid carcinoma A-431  were performed for 72 h. The MTT stock solution  was added at a final concentration of 0.5 mg/mL. After 4 h of incubation at 37 °C, the water-insoluble formazan was dissolved in a lysis buffer containing 20% SDS, 50% DMF, 2.5% hydrochloric acid, and 2.5% acetic acid. The optical densities were measured at 570 nm using a scanning multi-well spectrophotometer . The ratio of the absorbance value of each tested group to the absorbance value of the untreated control was calculated, and each tested group’s cell viability was expressed as a percentage of the control. The cell viability assays were performed at least in triplicate using independent cell cultures.The viability of the cells was assayed by measuring the conversion of MTT -2,5-diphenyltetrazolium bromide) to formazan . The cells were seeded in 96-well culture plates at a density of 3500–5000 cells per well 24 h before the treatment. Six wells were considered for each experimental point . Treatments with increasing concentrations of https://www.sartorius.com/en/products/live-cell-imaging-analysis/live-cell-analysis-resources/live-cell-imaging-and-analsis-handbook (accessed on 21 October 2022)). The HCT116 and HCT116 p53−/− cells were seeded in 96-well culture plates at a density of 5000 cells per well 24 h before the treatment. Four wells were considered for each experimental point. Treatments with 10, 11, cisplatin (40 μM), or dimethyl sulfoxide  were carried out for up to 72 h, and images were recorded every 2 h during this time. The experiment was performed twice.The cell proliferation and death were estimated in the IncuCyte S3 live-cell imaging system using the SYTOX™ Green Dead Cell Stain  and analyzed using the IncuCyte 2019B Rev2 software , according to the manufacturer’s instructions , followed by a post-hoc Tukey’s test. The differences were considered significant if the p-value was ≤ 0.05. All of the statistical analyses were performed using the Statistica software package .The data are presented as means ± SDs. The differences among the variables were analyzed using a Student’s 10 and 11 utilizes a commercially available, naturally occurring, and non-proteogenic amino acid, known as djenkolic acid (12), which is isolated from legume seeds. In the first step, the djenkolic acid (12) was reacted with 5-nitroisatoic anhydride (13) or 4-nitroisatoic anhydride (14) in the presence of potassium carbonate, which led to the formation of the linear tripeptides 15 and 16 , and the following two non-cancer derived cell lines: normal skin fibroblasts BJ (a reference line to A-431) and lung fibroblasts MRC-5 (a reference line to A549 and NCI-H1299). A549 cells are used as models for studying lung cancer and developing drug therapies against it. HCT116 is also a cell line used in therapeutic research and drug screenings. The viability results and obtained half-maximal viability inhibitory concentrations (IC50) are shown in 10 in the lung cancer cell lines. The IC50 values varied from 1.07 ± 0.25 μM (NCI-H1299) to 1.6 ± 0.21 μM (A549) and were much lower than in the case of cisplatin . Thus, compound 10 was 5.33 times more cytotoxic for the NCI-H1299 cell line than cisplatin and 3.72 times more cytotoxic for the A549 cell line than cisplatin. The least cytotoxic effect was observed for the normal cell lines, BJ and MRC-5, where the IC50 values were, respectively, 16.72 ± 2.77 μM and 16.85 ± 2.96 μM for 10 and 23.1 ± 2.34 and 28.7 ± 7.6 μM for 11. Interestingly, the IC50 values of the BJ and MRC-5 cells exposed to cisplatin were significantly higher compared to the treatments with 10 or 11 .Compounds 10 showed very high selectivity indices, which are as follows: 10.54 ± 1.54, p < 0.001 for A549 and 16.08 ± 3.43, p < 0.001 for NCI-H1299, respectively . Contrary to cisplatin, the cytotoxic effects of 10 and 11 were not dependent on p53 and were comparable for both the HCT116 and HCT116p53−/− cell lines , was more sensitive to 10 than to cisplatin , which was used as a negative control, the HCT116 and HCT116p53−/− cells grew to near full confluence, reaching an average of 90% or 84% and 88% or 83% confluence, respectively, on the third day. When cultured in the presence of 40 µM of drugs, a drastic reduction in the confluence occurred in both cell lines, reaching only up to 16% confluence  than cisplatin; (ii) the calculated selectivity indices for compounds 10 and 11, obtained by comparing the values for the tumor and normal lines, were, in most cases, higher than those for cisplatin; (iii) contrary to cisplatin, the observed cytotoxic effect was not dependent on the p53 protein, which is one of the factors responsible for the drug resistance of tumors. We would emphasize that the synthesized products are low-molecular-weight compounds with lead-like properties. The applied molecular framework could serve as a convenient privileged structure for designing and developing novel bioactive molecules suitable for drug design, development, and optimization. The results suggest that this class of compounds could be applied to the treatment of lung cancers.During our studies, we identified compounds based on a unique, dimeric benzodiazepine scaffold that exhibited exceptional anticancer effects against various cell lines and were superior to the reference cancer drug cisplatin in the following three ways: (i) compound


Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties–minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects. Ganglioside GD2 is a validated clinical target overexpressed in multiple cancers such as neuroblastoma, glioma, breast cancer, sarcomas, small cell lung cancer, and melanoma. Its postnatal expression on healthy body cells is generally limited to the central nervous system, peripheral neurons, and skin melanocytes. Ganglioside GD2 performs complex biological functions in normal cells, including participation in cellular communication and adhesion, formation of lipid rafts, and modulation of signaling pathways. Being a glycosphingolipid, GD2 is directly synthesized from its precursor ganglioside GD3 by the enzyme GM2/GD2 synthase; however, its content in the cell is determined by the combined action of the various enzymes involved in ganglioside biosynthesis . The mosThe monoclonal antibodies dinutuximab and naxitamab, chimeric and humanized antibodies from the 14.18/14G2a and 3F8 families, respectively, are the only GD2-directed therapeutics that have been granted regulatory approval. Extensive research is underway to develop alternative GD2-specific strategies, including bispecific antibodies, immunocytokines, CAR T and NK cell therapies, and radioimmunotherapy .I1 drug, which was conjugated to the modified lysines of the antibody, and the conjugate was shown to significantly suppress liver metastases in a disseminated mouse model of neuroblastoma. In our recent work [Only two works regarding GD2-targeting conjugates with small cytotoxic molecules are present in the open literature. Lode et al.  developeent work , ADCs weChanging the antibody format within ADCs from full-length IgG molecules to smaller antibody fragments that results in antibody fragment-drug conjugates (FDCs) represents an important strategy for improving the efficacy of ADC therapy. All major antibody fragment formats such as Fab fragments, minibodies, diabodies, scFvs, and nanobodies, as well as smaller antigen-binding peptides, are being actively explored in preclinical studies as targeted molecules within drug conjugates; however, it is yet to be evaluated whether they represent a viable alternative to full-length mAbs in drug conjugates ,9. The pNo data exists on FDCs directed to GD2-expressing tumors. In this work, such FDCs were developed for the first time based on two antibody fragment formats: the scFv fragment from the clinically approved antibody ch14.18, and the corresponding minibody, which is the bivalent dimer of the same scFv fragment fused to the hinge region and the CH3 domain of the heavy chain of IgG1 antibody. MMAE and MMAF were site-directly conjugated to cysteines of the minibody hinge region and the terminal cysteine introduced into the scFv structure via a cleavable maleimide linker. We generated the FDCs with specific drug-to-antibody ratios (DAR) and evaluated their stability and binding to GD2 by the enzyme-linked immunosorbent assay (ELISA) and to cells with or without GD2 expression by flow cytometry and confocal microscopy. Furthermore, the cytotoxic effects of the conjugates were analyzed in GD2-positive and GD2-negative tumor cell lines by the MTT and propidium iodide (PI) assays.3S)4 linker for construct flexibility and protease resistance, the FLAG octapeptide tag (the sequence DYKDDDDK) for detection, and a C-terminal unpaired cysteine for site-directed thiol-maleimide conjugation were introduced into the sequence (3S)4 linker identical to and in the same orientation as those in the scFv fragment 14.18, with the addition of the IgG1 hinge region, a second GS linker (GGGSSGGGSG), and the IgG1 CH3 constant domain (Both the scFv antibody fragment and the minibody were designed based on the sequence of the light (VL) and heavy (VH) chain variable domains of the GD2-specific antibody 14.18 reported by Bolesta et al. . The scFsequence A. The mit domain A.Following transient gene expression in Expi293F cells and purification by protein L chromatography, both the minibody B and theWe also analyzed the scFv fragment and the minibody in 10% polyacrylamide gel electrophoresis (SDS-PAGE) C,E. The Several methods were used to confirm the specific binding of the obtained antibody fragments to the antigen. First, conjugates of the scFv 14.18 and minibody 14.18 with fluorescein 6-maleimide (FAM) were generated, and with these conjugates we directly demonstrated in flow cytometry and in confocal microscopy that both the scFv fragment and the minibody did not bind the GD2-negative B16 melanoma cell line, but manifested high binding to the B78-D14 melanoma cell line overexpressing GD2 A,B. The Additionally, the specificity of binding of the obtained antibody fragments was studied in direct ELISA using individual purified gangliosides GD2, GD3, GD1b, and GM2 C. When gWe generated the FDCs by conjugating MMAE or MMAF microtubule inhibitors to the C-terminal cysteine of the scFv fragment 14.18 or the reduced interchain cysteines of the minibody 14.18 by a maleimide linker that contains the cathepsin-cleavable valine-citrulline site. The reaction scheme and the FDC structures are presented in n = 4), while the average DAR for minibody-MMAE and minibody-MMAF in optimized conditions constituted 2.0 ± 0.1 and 2.1 ± 0.1, respectively. The absorption spectra of the representative scFv-based and minibody-based FDCs normalized at 280 nm together with the corresponding naked antibody fragment are shown in Based on the ultraviolet-visible (UV-VIS) spectroscopy analysis, the average drug-to-antibody ratios for both the scFv-MMAE and scFv-MMAF generated in optimized reaction conditions constituted 0.9 and were highly consistent between batches  and GD2-negative (B16 and NGP-127) cell lines were incubated for 48 h with a constant concentration of scFv fragment-based FDCs (scFv-MMAE and scFv -MMAF) and minibody-based FDCs (Mb-MMAE and Mb-MMAF) A.In the PI assay, all FDCs induced cell death in GD2-positive B78-D14 and IMR-32 cells, while none of them increased the level of cells with fragmented DNA in GD2-negative B16 and NGP-127 cell lines under the selected FDC concentration (100 nM) A. MiniboThe MMAE and MMAF drugs used to generate the FDCs belong to the family of dolastins, the mechanism of activity of which comprises inhibition of microtubule polymerization that leads to arrest in the G2 phase of the cell cycle and subsequent cell death ,21. In oChanging the format of the vector molecule in the ADCs from full-length antibodies to antibody fragments is an important strategy used to improve their efficacy ,22. VariResearchers are primarily guided by several criteria when choosing a small vector molecule format for developing FDCs. These include a balance between the circulation time of the vector molecule in the blood and its ability to penetrate into solid tumors. In addition, in the case of antibody fragments, it is necessary that they retain high affinity for the antigen and a significant rate of internalization, criteria which are also crucial for full-length antibodies . An addiIt is often difficult to predict in advance which antibody fragment format will be optimal in a particular case, even for tumor markers of protein nature. In the context of the ganglioside GD2, a relatively small glycosphingolipid molecule located only on the outer leaflet of the plasma membrane, this aspect demands even more consideration. In addition, the number of research papers and experimental data regarding anti-GD2 ADCs is minimal. Therefore, we formulated two main goals in the present work: 1) to analyze the fundamental possibility of GD2-directed FDCs to induce cytotoxic antigen-specific effects in tumor cells, and 2) given that the aforementioned effects are present, to compare the efficacy of FDCs created on the basis of different variants of GD2-binding antibody fragments. Results from our previous studies served as prerequisites for the following work.In our earlier paper, an scFv antibody fragment was generated based on the sequence of the ganglioside GD2-targeting murine antibody 14.18 which retained specific binding of the original antibody to GD2 . We haveFDCs based on both GD2-binding moieties, minibodies and scFv fragments, were successfully generated by site-direct conjugation to MMAE or MMAF via a cathepsin-B cleavable maleimide linker and with specific drug-to-antibody ratios . We confirmed specific binding of the FDCs to ganglioside GD2 in direct ELISA and to GD2-positive cells in flow cytometry and confocal microscopy analyses. Both formats induced selective cytotoxic and cytostatic effects in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. The IC50 values for both formats were in the nanomolar range in GD2-positive tumor cell lines.When analyzed in the MTT assay, the FDCs induced significant inhibitory effects in the B78-D14 mouse melanoma cell line that overexpresses GD2, in contrast to the GD2-negative B16 mouse melanoma cell line. Moreover, the FDCs based on both scFv fragments and minibodies conjugated to MMAF manifested stronger cytotoxic effects in this cell line than those conjugated to MMAE. The inhibitory effects of the FDCs were also significant in the GD2-positive human neuroblastoma cell line IMR-32. However, the FDCs based both on scFv fragments and minibodies were more effective in eliminating IMR-32 cells when they contained MMAE, in contrast to the FDCs with MMAF, which demonstrated a more prominent activity in B78-D14 cells. As in the case of GD2-negative mouse melanoma B16, cells of the GD2-negative human neuroblastoma NGP-127 were insensitive to our fragment-drug conjugates even at high concentrations.In order to differentiate the mechanisms of action of the FDCs, we used the PI assay which enabled us to study the contribution of cytotoxic and cytostatic effects to the overall activity of the anti-GD2 FDCs on the tumor cells. All FDCs induced cell death in GD2-positive B78-D14 and IMR-32 cells, while none of them increased the level of cells with fragmented DNA in GD2-negative B16 and NGP-127 cell lines. Minibody and scFv conjugates with MMAF showed the strongest effects in B78-D14 cells; FDCs containing MMAE manifested a less strong yet a pronounced effect. The FDCs also demonstrated a marked increase in cell death in a different GD2-positive cell line, IMR-32, characterized by a lower level of GD2 expression; however, the effects were expectedly weaker than in the B78-D14 cell line overexpressing ganglioside GD2. The results from the PI assay indicated a greater efficiency of the conjugates with MMAF for the B78-D14 cell line, and of those with MMAE for the IMR-32 cell line, which is in agreement with the results obtained in the MTT assay. The results also confirmed the absence of activity towards the GD2-negative B16 and NGP-127 lines at selected concentrations.We next set out to evaluate the effects of our FDCs on the cell cycle, for which the GD2-positive IMR-32 cells treated with the conjugates were analyzed in the PI assay. The distribution of cells with unfragmented DNA in the control samples and in the samples incubated with the FDCs containing MMAE clearly showed that most control intact cells were at the G0/G1 stage of the cell cycle, while the FDCs induced the transition of cells with unfragmented DNA that had not yet entered the late stages of apoptosis and had unfragmented DNA to the G2/M stage of the cell cycle. Moreover, the minibody-MMAE which had the strongest effect on the viability of these cells caused the highest accumulation in the G2/M stage. The data from the PI assay indicate that the mechanism of activity of the FDCs is directly related to the arrest of cells in the G2 phase of the cell cycle and that the total inhibitory effect of the conjugates observed in the MTT assay consists of both the cytotoxic and the cytostatic effects of MMAE or MMAF.The minibody-based FDCs with both MMAE and MMAF demonstrated more pronounced cytotoxic effects compared to scFv-based FDCs in all GD2-positive tumor cell lines within the analysis. This can be explained both by the higher DAR in minibody-based FDCs and by their stronger antigen-binding properties, resulting from the bivalent interaction of the minibodies with the antigen in contrast to monovalent interaction for scFv fragments. The valency of the interaction is an important criterion for the high binding affinity of an antibody or antibody fragment when interacting with ganglioside GD2, a carbohydrate antigen . In thisScFv fragments of GD2-specific antibodies 14.18 were produced by transient gene expression in modified HEK293 cells , as described previously [Minibodies were produced in the same expression system. To this end, the minibody monomer sequence was cloned between the XbaI and AgeI restriction sites of the eukaryotic expression vector pcDNA3.4, followed by verification of the construct identity by analytical restriction digests. The vector was then purified by QIAGEN Plasmid Maxi Kit , and transfection was performed in 50 mL cell culture volume following the protocol for the expression system. The cell culture was routinely checked for optical density and the percentage of viable cells, while the target protein content in the medium was assessed by polyacrylamide gel electrophoresis. The Antibody-Expressing Positive Control Vector from the expression system kit was employed for additional monitoring of protein expression level under recommended conditions.ScFv fragments and minibodies were isolated from the cell culture supernatants by protein L chromatography  as described previously , followeConjugation of the scFv fragments and the minibodies to MC-VC-PABC-MMAE or MC-VC-PABC-MMAF , further referred to as MMAE or MMAF, was carried out by thiol-maleimide chemistry. First, reduction of the C-terminal cysteine introduced intro the scFv fragment and the cysteines forming disulfide bonds between minibody chains was performed by 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP) in PBS buffer, for 1.5 h at room temperature (RT) and with agitation. The reducing agent was then removed by a Zeba Spin Desalting Column, 7K MWCO , followed by an immediate reaction of 1–2 mg/mL proteins with 4:1 molar excess of the corresponding auristatin drug over each antibody fragment (for FDCs with optimized DAR) in PBS buffer at pH 6.0 with addition of 0.2 g/l EDTA, for 3 h at RT and with agitation. The percent of DMSO from the drug stock solutions was kept below 3% in the reaction mix. The FDCs were then transferred to PBS in Zeba columns. Both scFv fragments and minibodies were additionally conjugated to fluorescein 6-maleimide , further referred to as FAM, in reaction conditions identical to those described above.Product purity and stability were analyzed by size-exclusion chromatography, as described above, and by gel electrophoresis. Visualization of the naked antibody fragments and the FDCs by SDS-PAGE was performed either in reducing or in non-reducing conditions, with modifications to . For redAverage drug-to-antibody ratio (DAR) for antibody fragments conjugated with MMAE or MMAF, as well as degree of labeling (DOL) for conjugates with FAM-maleimide, were calculated by UV-VIS spectroscopy  on a Bio450 in GM2-, GD1b-, or GD3-coated wells to OD450 in GD2-coated wells.Nunc MaxiSorp high protein-binding capacity 96-well ELISA plates  were coated with gangliosides GD2, GM2, GD1b, and GD3 at a concentration of 0.25 μg in 100 μL of 96% ethanol per well. GD2, GD1b, and GD3 were purchased from Sigma-Aldrich, and GM2 was obtained according to our previous work . FollowiL-glutamine, 100 μg/mL penicillin, and 100 U/mL of streptomycin .B16 and B78-D14 mouse melanoma cell lines were cultured in RPMI-1640, IMR-32 human neuroblastoma in EMEM, and NGP-127 human neuroblastoma in DMEM medium. All culture media were supplemented with 10% heat-inactivated fetal bovine serum, 2 mM IMR-32 and B16 cell lines were obtained from ATCC, and the NGP-127 cell line was kindly provided by Anton Buzdin . The GD2-positive B78-D14 mouse melanoma cell line generated by transfection of the GD2-negative B16 line with genes coding for GD3 and GD2 synthases  was a kiStaining of B16, B78-D14, IMR-32, and NGP-127 cells with FAM-labeled scFv fragments and minibodies was performed as described previously . In brieStaining of GD2-positive and GD2-negative cell lines for immunofluorescence analysis was performed by a modified protocol from our previous work . In brie4 cells/well, Greiner, Kremsmünster, Austria) with serial dilutions of FDCs for 72 h under standard culture conditions. Following incubation, the MTT solution  was added to each sample for 3 h. Reaction OD was assessed by Multiscan FC microplate reader at a wavelength of 540 nm. Cell viability was calculated using the formula (ODtreated cells − ODblank)/(ODcontrol cells − ODblank) × 100%, where ODblank represents OD in control wells containing no cells. SigmaPlot  was used to generate dose–response curves. All MTT experiments were reproduced at least three times.FDC-induced decrease in cell viability was analyzed by colorimetric MTT  assay previously described by Denizot and Lang , with mo6 cells per sample) were incubated with FDCs at concentration 100 nm for 48 h under standard culture conditions. Cells were subsequently fixed and permeabilized with ice-cold ethanol for 60 min at 4°C, and washed twice with PBS by centrifugation for 10 min at 300× g. Cell pellets were resuspended in DNA staining buffer , 20 μg/mL RNase A ) and further incubated for 30 min at 4 °C. For all samples, cell death and cell cycle were performed in triplicate. In each sample, at least 10,000 events were collected. The quantitation of DNA content was evaluated with BD FACSCalibur flow cytometer .Analysis of cell death or cell cycle was performed using propidium iodide (PI) staining in accordance with modifications  to the pp < 0.05 were considered statistically reliable.Graphs were created using SigmaPlot, GraphPad Prism, and MS Excel software. The data are represented as mean ± SEM of at least three independent experiments, or as one representative experiment from three. Statistical analysis was performed by unpaired Student’s t-test. Significance levels of


CRTC1-MAML2 chimeric gene was determined. Furthermore, the susceptibility of head and neck mucoepidermoid carcinoma to standard treatment drugs such as cisplatin, 5-fluorouracil, and cetuximab was investigated. We successfully established unique MEC cell lines, AMU-MEC1, from an initial surgical specimen and AMU-MEC1-R1 and AMU-MEC1-R2 from the recurrent biopsy specimen in the same patient. These cell lines exhibited epithelial morphology and developed in vitro-like cobblestones. They shared eight chromosomal abnormalities, including der(19)ins, which resulted in a chimeric CRTC1-MAML2 gene, indicating the same origin of the cell lines. The susceptibility of all cell lines to cisplatin and 5-fluorouracil was low. Interestingly, EGFR dependency for cell growth decreased in AMU-MEC-R1 and AMU-MEC-R2 but was retained in AMU-MEC1. These cytogenetic and biochemical findings suggest that the established cell lines can be used to investigate the disease progression mechanisms and develop novel therapeutics for MEC.Patients with advanced/recurrent mucoepidermoid carcinoma (MEC) have a poor prognosis. This study aimed to establish and characterize human mucoepidermoid carcinoma cell lines from the initial surgical specimen and biopsy specimen upon recurrence from the same patient to provide a resource for MEC research. MEC specimens from the initial surgical procedure and biopsy upon recurrence were used to establish cell lines. The established cell lines were cytogenetically characterized using multi-color fluorescence in situ hybridization and detection, and the sequence of the  CRTC1  gene at 19p13 or CRTC3 gene at 15q26 in-frame to exons 2–5 of the Notch coactivator mastermind like gene 2 (MAML2) gene at 11q21 resulting in the expression of a novel CRTC1/3-MAML2 fusion gene [CRTC1/3-MAML2 fusion genes are more prevalent in low- and intermediate-grade tumors than in high-grade tumors [Mucoepidermoid carcinoma (MEC) accounts for 5–15% of all salivary gland malignancies but less than 1% of all head and neck cancers . MEC is ion gene ,9,10,11.e tumors ,13. The e tumors ,14. CRTCe tumors .Only surgery is the standard treatment for MEC, though radiation is sometimes used as postoperative adjuvant therapy for advanced cases. Chemotherapy and radiation therapy can be combined, although the overall survival is not improved compared to radiation alone ,18,19,20Phase contrast microscopy images of the MEC cell lines are depicted . All thrCRTC1-MAML2 chimeric gene (detailed in the next section). Each of the three cell lines, AMU-MEC1, AMU-MEC-R1, and AMU-MEC-R2, shared seven distinct chromosomal abnormalities, including der(19)ins (The karyotypes of eight randomly selected cells from each cell line are listed in ) , suggest) . The uni) . CRTC1-MAML1 chimeric gene [CRTC1-MAML1 chimeric gene corresponded to the previously reported sequence (GenBank: AY040324.1) [CRTC1-MAML2 chimeric gene. All MEC cell lines displayed RT-PCR amplification of a 194bp gene product, which was consistent with the predicted length of the forward and reverse primers A,B. The ric gene , but not40324.1)  . Thus, MEC cell lines exhibited extremely limited growth . OSCC cell lines showed a drug concentration-dependent reduction in viability. IC50 of CDDP in HSC-2, HSC-3, and HSC-4 was approximately 6.0 µM, 2.0 µM, and 4.8 µM, respectively. The IC50 of 5-FU in HSC-2, HSC-3, and HSC-4 was approximately 2.1 µM, <0.3 µM, and <0.3 µM, respectively. However, MEC cell lines maintained 60–100% viability even at high CDDP and 5-FU concentrations . Thus, tCetuximab blocked EGF-EGFR signaling but not proliferation in AMU-MEC1-R1 and AMU-MEC1-R2 cells in all MEC cell lines treated with exogenous EGF; cetuximab inhibited EGFR phosphorylation. Cetuximab also suppressed the EGFR downstream protein, STAT1 phosphorylation A. These PD-L1 expression was not shown in all MEC cell lines. IFN-γ induced PD-L1 expression in AMU-MEC1 and AMU-MEC1-R1 but not in AMU-MEC1-R2 .CRTC1-MAML2 chimera is typically caused by the t translocation [CRTC1-MAML2, their chromosomal structural abnormality was der(19)ins instead of t. This is the first report of MEC with der(19)ins-generated CRTC1-MAML1. However, this investigation did not compare the biological and clinical aspects of translocation and insertion cases. These investigations need to be approached in the future. Low- and intermediate-grade patients have more CRTC1-MAML2 chimeric gene-positive cases than high-grade cases [CRTC1-MAML2 positive. Comparisons of these different histological-grade cell lines could be beneficial in the future. We established the AMU-MEC1 cell line from the surgical specimen of a MEC patient and the AMU-MEC1-R1 and AMU-MEC1-R2 cell lines from biopsy specimens at the time of recurrence of the same patient. The MEC-specific location ,14. Alththe t11;1 has been extensively used for various tumors. However, few clinical trials and basic research have focused on MEC immunotherapy, and its effects are unknown . RecentlCRTC1-MAML2 chimeric gene as a result of der(19)ins instead of t, and 3) the response to EGF or IFN-γ was heterogeneous among these three cell lines. MEC is a rare cancer with a favorable prognosis. However, there are few treatment options for advanced/recurrent cases, necessitating the development of novel targeted therapies. These cells could be used to study the mechanisms of MEC tumorigenesis and progression and to develop novel therapeutics. The three MEC cell lines established in this study are distinct in three ways, as described below. 1) The cell lines were derived from both surgical and biopsy specimens upon recurrence in the same MEC patient, 2) all cell lines generate the The patient is a 69-year-old male with a history of hypertension, hyperlipidemia, pneumonia, and herpes zoster. His father, sister, and aunt had a history of malignant lymphoma. Two months ago, he presented to the otorhinolaryngology clinic with a chief complaint of pain on swallowing and bilateral cervical lymphadenopathy. A laryngeal fiberoptic scan revealed a neoplastic lesion in close proximity to the epiglottis . After bMEC cell line, AMU-MEC1 was derived from surgically-resected salivary MEC tissue on 21 May 2019, while AMU-MEC1-R1 and AMU-MEC1-R2 were derived from recurrent carcinoma biopsy specimens on 15 October 2019 . The fol5 and 2 × 104 cells per well, respectively, serial concentrations of cisplatin (CDDP)  or 5-fluorouracil (5-FU)  were added in triplicate and cultured for five days. The supernatant in each well was completely aspirated, and the cells were incubated with 100 μL of WST-1 solution  diluted with DMEM (10% FBS) at 37 °C for 2 h. Absorbance was measured at 450 and 620 nm using a microplate reader . Cell viability was calculated using the following formula: % viability = 100 × (E − S)/(M − S), where E, M, and S denote the absorbances of the experimental well, well without drugs , and well with medium alone (without cells). After MEC and OSCC cells suspended in DMEM (10% FBS) were seeded in 96-well plates at 105 MEC and OSCC cells were seeded in 24-well plates and cultured for four days in DMEM (10% FBS). After seeding (days 1–4), the cells were treated with 10 μM BrdU (Fuji Film) and incubated at 37 °C for 1 h. After fixing cells with 70% ethanol at −20 °C for 30 min, double-stranded DNA was denatured with 2N HCl at room temperature for 30 min. After washing three times with buffer (PBS containing 0.2% HSA and 2mM EDTA), the cells were incubated with FITC-conjugated anti-BrdU monoclonal antibody  at 4 °C for 30 min. The cells were washed and resuspended in 1 μg/mL propidium iodide-containing washing buffer. For detection of PD-L1, 2 × 105 MEC and OSCC cells were seeded to 24-well plates, cultured for two days in DMEM (10% FBS), and treated with 25 ng/mL of interferon (IFN)-γ for two days. The cells were collected and incubated with PE-conjugated anti-PD-L1 monoclonal antibody  at 4 °C for 20 min. Then, flow cytometry was performed using BD LSRFortessa flow cytometer (BD Biosciences), and the data were analyzed using the FlowJo software v10.8.1 . Two × 105 cells/lane) and transferred to polyvinylidene fluoride membranes using an iBlot 2 Dry Blotting System . After blocking in tris-buffered saline (TBS) with 1% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies: EGFR , p-EGFR , p-STAT1 , and β-actin . β-actin was used as the internal control. After four TBS washes, the membranes were incubated with peroxidase polymer anti-mouse IgG or anti-rabbit IgG  at a 1:100 dilution for 30 min at room temperature. The chemiluminescence signal was detected using ECL Prime Western blotting Detection Reagent  and captured using an Amersham Imager 600 . The cell lysates in protein extraction buffer , and 2 mM 2-mercaptoethanol (pH 7.4)) were resolved on SDS-PAGE gels  was approached to conduct the staining and analysis. Cells were sequentially treated with 0.2 µg/mL colcemid for 3 h and 75 mM KCl for 10 min, then fixed using Carnoy’s solution. The fixed cells were denatured with 2 × SSC at 72 °C for 30 min and hybridized with the 24XCyte Human multicolor FISH probe  at 37 °C for 42 h. Images were captured using a Zeiss Axio Imager. Z2 microscope . 3 dishes, and 5 × 106 cells were collected per 1.5 mL tube. RNA was extracted using the Nucleo Spin RNA Kit , and Reverse transcription (RT)-PCR was performed using the Prime Script One-Step RT-PCR Kit ver.2  according to the manufacturer’s instructions. A 194-base-pair amplicon was generated to detect the CRCT1-MAML2 chimera using the primer sequences 5′-ATG GCG ACT TCG AAC AAT CCG CGG AA-3′(forward) and 5′-CCA TTG GGT CGC TTG CTG TTG GCA GGA G-3′(reverse). The amplified RT-PCR products of the CRCT1-MAML2 chimera were detected using 2% agarose gel electrophoresis. PCR products were sequenced using an ABI Prism 377 Genetic Analyzer .MEC cells were plated in 100 mmt-test. p < 0.05 was considered statistically significant.Differences between the two groups were examined with the Student


In Huang et alThe correct Figure 1 is given below:The correct Figure 7 legend is given below:Figure 7. RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real‐time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87 and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The western blot analysis of RGS16, TCF8, N‐cadherin and β‐catenin protein expression in LN229, U87 and U251 cell lines after applying RGS16 siRNA or negative control.  Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.  Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control.  Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.It earlier read as,RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real‐time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87, and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The Western blot analysis of RGS16, TCF8, N‐cadherin, and β‐catenin protein expression in LN229, U87, and U251 cell lines after applying RGS16 siRNA or negative control.  Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. (D) Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control. (E) Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.The authors apologize for these errors.


We screened a focused array of compounds based on the 4-anilinoquin(az)oline scaffold against both U-CH1 and the epidermal growth factor receptor (EGFR) inhibitor resistant U-CH2. To prioritize the hit compounds for further development, we screened the compound set in a multiparameter cell health toxicity assay. The de-risked compounds were then screened against a wider panel of patient derived cell lines and demonstrated low micromolar efficacy in cells. We also investigated the properties that gave rise to the toxophore markers, including the structural and electronic features, while optimizing for EGFR in-cell target engagement. These de-risked leads present a potential new therapeutic avenue for treatment of chordomas and new chemical tools and probe compound  Protein kinases have presented promising drug targets, with more than 70 inhibitors targeting the ATP binding site of kinases approved for use in the clinic3. This promising treatment avenue is currently being explored to treat chordomas using afatinib4.Cancer is the leading cause of death worldwide, responsible for nearly 10 million deaths in the last year5. Treatment of chordoma is challenging, with initial treatment focused on radical resection combined with proton beam therapy. Surgical and chemotherapeutic options are limited, meaning recurrent or metastatic disease is often fatal6.Chordoma is an osseous cancer, contained within the spine and central nervous system, with invasive and metastatic potential which is driven by the notochord transcription factor brachyury8. Several quinazoline-based clinical kinase inhibitors whose main target is EGFR have been shown to inhibit chordoma cell lines, including gefitinib, erlotinib, afatinib and lapatinib  is highly expressed in 40% of chordomas, and targeting EGFR has been shown to be both effective in chordoma cell lines and murine xenograft modelsnib Fig. 4,8–12.Fi17. They are also not effective at targeting some of the more resistant patient derived cell lines12. While exploring cyclin G associated kinase (GAK) as a potential collateral target to treat chordoma, we identified a series of potent EGFR inhibitor starting points based on the 4-anilinoquinazoline scaffold, exemplified by 112.While these drugs were designed to target EGFR, they show similar or higher affinity for several other kinases, making them limited tools for direct interrogation of EGFR and other kinase biology23. We initially synthesized and tested a panel of compounds based around compound 1. We synthesized compounds 1–45 through nucleophilic aromatic displacement of 4-chloroquin(az)olines (Scheme 10 (23%) and 16 (21%) both containing the trifluoromethyl quinazoline, and the 6,7-difluoroquinazoline analogue 6 (20%), all of which were consistent with previous reports22.Our objectives are to simultaneously improve the chordoma cellular potency and compound preclinical safety profile. Our strategy was to interrogate the structure activity relationships (SAR) of these literature starting points, using our in-house GAK inhibitor knowledge and library, along with a safety risk estimation screen, to optimize the 4-anilinoquin(az)oline scaffold1 with a series of  modifications, on both U-CH1 and U-CH2 chordoma cell lines. N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine (1) was previously identified as a narrow spectrum inhibitor of EGFR with only two other off-targets (GAK and Receptor-interacting serine/threonine-protein kinase 2 (RIPK2)) in a kinome wide screen18. Compound 1 was shown to be a low micromolar inhibitor of the U-CH1 cell line, consistent with our previous screen , significantly reduced activity against U-CH1 compared with 11, with no inhibition activity at all (IC50 = > 100 μM) on U-CH2. The 7-trifluoromethyl 16 showed some improvement against U-CH1 with an IC50 = 12 μM and some inhibition of U-CH2 (IC50 = 15 μM). The 6-cyano 17 and 7-cyano 18 substitutions demonstrated a sixfold reduction in inhibition of U-CH1 compared with 16. The 6-methylsulfone 19 showed little improvement. However, reverting to an electron donating substituent cyclizing the catechol first with a methyl spacer 20 and then with an ethyl spacer 21 moved potency back toward single digit micromolar IC50 values against U-CH1. Compound 21 had a similar inhibition profile to erlotinib on U-CH1, but while erlotinib showed activity against U-CH2 (IC50 = 18 μM), 20 and 21 did not (IC50 = > 100 μM).Switching the 7-position back to the methoxy 10. The initial compound 4-((3-ethynylphenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile (22) with the 6,7-dimethoxy substitution showed good inhibition against U-CH1 with an IC50 = 4.1 μM, and a weaker inhibition of U-CH2 , so did the potency of the compound in both U-CH1 and U-CH2 cell lines. The 6-iodo substitution 28 was the most potent with U-CH1 IC50 = 8.7 μM and U-CH2 IC50 = 14 μM. The 6-methylsulfone 29 had only limited potency against both cell lines, while the 7-position halogens (30–32) showed no affect at all on either cell line. However, switching back to the 7-methoxy 33 recovered activity to levels observed in 22.We then switched to the 3-cyanoquinoline scaffold, to evaluate if it was possible to increase the potency on U-CH1 and U-CH2, as had been demonstrated previously24. In addition, we ran an EGFR in-cell target engagement assay, measuring in-cell EGFR phosphorylation, using the human epidermoid carcinoma cell line A431. This cell line endogenously expresses a high level of EGFR, and stimulation of these cells with human epidermal growth factor (EGF) results in receptor tyrosine autophosphorylation25.We then selected a subset of the most potent compounds on U-CH1 and U-CH2, along with some structurally diverse compounds Table , to screN-(3-ethynylphenyl)-6,7-difluoroquinazolin-4-amine (6) displayed weaker activity against EGFR in A431 cells (IC50 = 12 μM), and no potency in the four additional patient-derived chordoma cell lines or WS1 control (IC50 = > 100 μM). The 6-iodo 9 was more potent against EGFR (IC50 = 0.83 μM), but was still weak in the four additional chordoma cell lines (IC50 = > 100 μM) and WS1 (IC50 = 98 μM). Similar results were observed for 7-methoxy 11 and 7-iodo 15. The 6-trifluoromethyl 16, while showing limited EGFR activity (IC50 = > 20 μM), displayed moderate activity against all four patient-derived chordoma cell lines, but this appeared to be driven by non-specific toxicity . The 7-cyano 17 was selected for structural diversity but was inactive at the top concentration in all chordoma cell line assays (IC50 = > 100 μM). The closing of the 6,7-dimethoxy to form a 6-member ring 21 showed some activity against EGFR (IC50 = 1.9 μM), but only weak activity on UM-Chor1 (IC50 = 64 μM). Erlotinib performed consistently as previously described12, with good potency against EGFR (IC50 = 0.030 μM), weak activity in four of the patient-derived chordoma cell lines, but also with similar non-specific toxicity observed with compound 16 (IC50 = 19 μM). Switching to the 3-cyanoquinoline scaffold, 4-((3-ethynylphenyl)amino)-6-iodoquinoline-3-carbonitrile (28) showed an improvement in potency in three of the four chordoma cell lines: CH22 (IC50 = 8.4 μM), UM-Chor1 (IC50 = 6.9 μM), and U-CH12 (IC50 = 7.7 μM), with only moderate non-specific toxicity in the WS1 control cells (IC50 = 34 μM) despite an eightfold reduction in EGFR activity compared to erlotinib. The observed potency could be related to a target other than EGFR, as the corresponding 7-iodo 32 has similar EGFR activity to 28, but was not potent in the patient-derived chordoma lines or the WS1 control (IC50 = > 100 μM).26. The screen works by estimating the risk using a supervised machine learning (ML) classifier to assess a multiparametric cellular stress phenotype, produced by the test compound, relative to a training set of known compounds  screen was developed to assess human safety risk for candidate pharmaceutical small molecules at the early hit-to-lead stage of developmentds Table . This sc26. This external curation informed an outcome of “high-risk” or “low-risk”. All 300 compounds were also processed through the automated FC screen, populating each of the two classes with frequency distributions of empirical cell-based phenotypes.The ML classifier was trained with 300 historically known compounds , which were divided into two outcome classes based upon literature, clinical trials, and market histories where applicableTo estimate human safety risk for an unknown compound, the ML classifier assesses the similarity of the test compound’s cell-based phenotype to the distributions of phenotypes in the two outcome classes. The final quantitative score, or Cell Health Index (CHI), is the probability (0–1) that the test compound’s phenotype belongs in the distribution observed for the high-risk outcome class in the training set Table . PhenotyTo produce the Cell Health Index, the ML classifier uses all twelve FC parameters simultaneously in a multidimensional logistic regression model, so the CHI contains the most predictive power. In addition to the Cell Health Index, Table N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine (1) produced an intermediate risk estimate, consistent with the WS1 toxicity counter screening12. The toxicity is potentially driven by cell morphology changes, but this is currently unclear. Compounds 6, 9, 11, 17 and erlotinib were consistent and found to be non-toxic in both assays; in contrast liabilities were consistently found in 16 and 21. The 7-iodo derivatives 15, 28 and 32 all performed poorly in the cell health secondary screening but showed no toxicity in the initial WS1 (IC50 = > 100 μM) counter screen. Interestingly, the 6-iodo derivative 9, appears to be more tolerated in this assay (for the complete data set see Tables S2–S3).The starting point 9 meant the first set of analogues focused on small changes of the aniline on 9. Shielding the anilino N–H group with a fluoro at the distal ortho-position 34, maintained EGFR activity, but decreased U-CH1 inhibition by fourfold compared with 9. Moving the fluoro to the proximal ortho-position 35 between the acetylene and quinazoline recovered the activity on U-CH1. The para-fluoro 36 was a threefold improvement in U-CH1 inhibition with EGFR activity maintained. Interestingly, switching to a para-chloro 37 reduced activity on U-CH1 by 11-fold, however, demonstrated relatively high activity on the difficult to treat U-CH2 cell line12, with an IC50 = 5.0 μM compared with no activity from the rest of the mini-series .In order to optimize the scaffold further, the 6- and 7-positions on the quinazoline ring were fixed, and the aniline portion was modulated with different halogens Table . The enc15. A fluoro at the distal ortho-position 38 showed no overall improvement, whereas in the proximal ortho-position 39 the fluoro improved potency on U-CH1 by sixfold (IC50 = 2.4 μM). Compound 39 also showed activity on the difficult to treat U-CH2 cell line (IC50 = 15 μM)12. The para-fluoro analog 40 showed no activity on U-CH1 or U-CH2, whereas the para-chloro derivative 41 demonstrated moderate inhibition on both U-CH1 (IC50 = 9.1 μM) and U-CH2 (IC50 = 16 μM) with low single digit micromolar EGFR inhibition (IC50 = 3.7 μM).The encouraging results with the 6-iodoquinazoline led to the exploration of the 7-iodo based around 1 has demonstrated potent inhibition on U-CH1 below 1 µM, suggesting that this substitution pattern may yield a more potent compound across multiple cell lines. The distal ortho-fluoro 42 reduced activity on the U-CH1 by tenfold compared to 1, whereas there was a threefold increase on U-CH2. This was despite 42 showing a tenfold decrease in in-cell EGFR activity. The proximal ortho-fluoro between the meta-acetylene and quinazoline ring system 43 demonstrated a threefold increase across U-CH1 and U-CH2 with EGFR activity increased by fivefold. The para-substituted analogues 44 and 45 both lost activity on U-CH1, but maintained some activity on U-CH2, and EGFR inhibition was maintained. The loss of inhibition on U-CH1 for 41, 44 and 45 is not easily explained and highlights the degree to which target-ligand interactions in cells may be governed by energetic phenomena that classical models inadequately address27.The 6,7-dimethoxy substitution present in 34–45, we screened them against the four additional patient-derived chordoma cell lines  and WS1 as a nonspecific cytotoxicity control . However, the 6-iodoquinazoline, para-chloro aniline analogue 41 showed some of the most potent inhibition to date on CH22 (IC50 = 0.48 μM) and U-CH12 (IC50 = 0.96 μM), along with good inhibition on the other two cell lines UM-Chor1 (IC50 = 25 μM) and U-CH7 (IC50 = 8.0 μM). There was some observed toxicity on WS1 (IC50 = 36 μM), but the ratios of inhibition to toxicity are 75-fold in the best case (CH-22/WS1). The 6,7-dimethoxy substitution analogues 42–45 showed good potential against the four cell lines but also showed some nonspecific toxicity in some cases. Compound 42 demonstrated good inhibition of CH22 (IC50 = 4.2 μM) but showed evidence of some non-specific toxicity . The same was observed for 43, with good inhibition of CH22 (IC50 = 5.8 μM) but some toxicity . The para-fluoro analogue 44 demonstrated excellent potency, but this was unfortunately likely driven by nonspecific toxicity . However, this nonspecific toxicity liability was able to be modulated with the para-chloro analogue 45 that showed potent inhibition on CH22 (IC50 = 1.2 μM) and U-CH12 (IC50 = 3.0 μM) with no observed toxicity .In order to better characterize these optimized compounds ol Table . The 6-i34–45 on the toxicity profiling platform and compared the results to some existing clinical compounds that have activity against chordoma (Table N-(3-ethynylphenyl)-6-iodoquinazolin-4-amine (9) performed well in the toxicity profile platform, while a fluoro substitution in the distal ortho-position 34 and a para-position chloro 37 were not well tolerated. However, other substitutions, such as an ortho-position fluoro 35 and a para-position fluoro on the same core, were tolerated. Interestingly, moving the iodo from the 6-position 9 to the 7-postion 15 led to an increase in toxicity risk estimation in the assay. However, the modifications to the pendant aniline 38–41 were well tolerated, demonstrating that trends cannot always be easily defined. The original starting point 1 was also tolerated but near the middle of the Cell Health Index at 0.41. The modifications 42–45 were consistent with the profile of 1 showing an acceptable toxicity window consistent with the WS1 data. Erlotinib demonstrates low-to-intermediate risk for toxicity, but other relevant literature inhibitors show high toxicity risk, including gefitinib and tesevatinib30.To further understand the toxicity liabilities in this scaffold we screened the optimized analogues ma Table . N- to measure the kinase activity of the 320 protein kinases at two concentrations (10 μM and 1 μM) (Table S6). Compound 41 only inhibited EGFR at 1 μM (> 50%), while at 10 μM (> 50%), several other kinases in addition to EGFR were moderately inhibited including Aurora-B, ERBB4, MAP4K4, PRKG2 and SLK. Compound 45 had a similar profile with only EGFR potently inhibited at 1 μM (> 50%), with some moderate inhibition of ERBB2 and ERBB4. The higher concentration of 10 μM (> 50%) affected several additional kinases in addition to EGFR including Aurora-C, DDR2, EPHA6, EPHB2, LCK, MAP4K4, MAP4K5, MINK1, MKNK1, MKNK2, NIK, PIM1, RIPK2, SLK, and VEGFR2. Beyond the kinases that can be screened in this assay format, GAK20, NLK20, STK10 also known as LOK22 and ADCK3 also known as COQ8A32 could also be potentially inhibited by this scaffold.To understand the selectivity profile across the kinome we screened 12.Chordoma tumors are challenging to effectively treat. The integrated nature of the tumor, combined with slow growth, creates a uniquely complex situation where standard treatment options are not well suited. These factors, combined with highly variable presentation and susceptibility to treatment, prevent a readily available and generalizable therapeutic approach. There have been a number of clinical trials that have evaluated EGFR inhibitors to treat chordomas, but treatment outcomes are highly variable, and molecular mechanisms underlying this observed variability are not well understood12. This was supported by the observation that 6, 11, 21, 28 and erlotinib all had single digit micromolar inhibition on U-CH1 and good EGFR activity . However, in later optimized derivatives (34–45), more correspondence between EGFR and cell line inhibition was observed  but no activity against U-CH1 (IC50 = > 100 μM), again highlighting the complexity of chordoma biology. This was despite 45 showing exciting results across all other cell lines with limited toxicity on WS1 and potent in-cell activity against EGFR, with IC50 = 0.018 μM. U-CH2 proved difficult to inhibit with the most potent compound 11 already known10, and even the lead compounds presented here, 41 and 45, only moderately inhibited U-CH2 . Compounds 41 and 45 both have good inhibition across the remaining chordoma cell lines screened. This activity, combined with limited toxicity in WS1 , support from the flow cytometry toxicity assay and a very narrow kinome spectrum at 1 μM, are all desirable characteristics for a lead compound. Compounds 41 and 45 not only have potent inhibition of EGFR, and good in-cell target engagement , they also appear to have stronger retention in the EGFR binding site than erlotinib via MD simulation  is a probe quality compound for EGFR, fulfilling several of the following criteria: (1) in vitro biochemical IC50 = < 50 nM, (2) ≥ 30-fold selectivity relative to other kinases in a large assay panel, and (3) cellular activity or target engagement IC50 = < 1 μM17.The two lead compounds 41 and 45 (UNC-CA359) both show very narrow kinome profiles and excellent activities across an array of patient derived chordoma cell lines. This is a potentially exciting step forward for a disease with a poor clinical prognosis.This screening and optimization effort has produced a series of useful tools for further interrogation of EGFR biology, and more specifically, chordoma inhibitor development. The lead compounds 34. Briefly, U-CH1 and U-CH2 cell lines were cultured in 4:1 IMDM:RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in gel-coated flasks. The WS1 cell line (ATCC:CRL-1502) was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. CH22, UM-Chor1, U-CH12, and U-CH7 cell lines were cultured in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cell lines all undergo regular mycoplasma screening9.Chordoma cell lines were cultured as described previously50 values were determined by four-parameter nonlinear regression analyses using GraphPad Prism™ version 8 software .Cells were seeded in 384 well plates and were treated with test compound in quadruplicate 24 h after plating. Cell viability was assessed at 72 h using alamarBlue . Fluorescence was measured using a Tecan Infinite 200 PRO plate reader with excitation at 535 nm and emission at 590 nm. ICComputational modelling was performed using Schrödinger Maestro software.31 was downloaded from RCSB, pre-processed and minimized using the protein preparation wizard tool of Schrödinger Suite 2020-4 . Structures of small molecule ligands were parametrized and minimized using LigPrep module  using OPLS3e force field35.Prior to docking studies, the selected x-ray structure of EGFR complexed with 4-anilinoquinazoline inhibitor erlotinib (PDB:1M17)Docking was computed using Induced Fit Docking workflow of Schrödinger employing SP-setting for Glide docking  and side chains 5 Å from initiative ligand poses were consider for conformational refinement with Prime module. At both stages of docking hydrogen bond was required to hinge amine of Met769. Graphical illustrations are made using PyMOL Molecular Graphics System, Version 2.4.1. Schrödinger, LLC.37. WaterMap links molecular dynamics (MD) simulations with statistical thermodynamic analysis of water molecules within a protein structure. Water molecules were analyzed within 6 Å from the docked ligand, and the 2 ns simulation was conducted with OPLS3e force field39.Schrödinger suite 2020-4 WaterMap  was used to evaluate the hydrations sites replaced upon ligand bindingsimulations were carried out using Schrödinger Desmond . Selected favorable Induced fit docking poses of corresponding EGFR inhibitors were selected as template structures. Prior to simulation set up each kinase-inhibitor complex structure was first minimized using Protein preparation wizard of Schrodinger with default heavy atom rmsd constraint of 0.3 Å and OPLS3e force field. In Desmond system builder the orthorhombic periodic the system was created, and it was solvated using TIP3 waters and 0.1 M NaCl– salt buffer. At the beginning of the simulation, the system was subjected to default relaxation protocol of Desmond and heated up to simulation temperature of 300 K. Unconstrained 1 s simulations were run using NPT protocol at temperature of 300 K, pressure of 1.01325 bar, Noe–Hoover thermostat and timestep of 2 fs. Trajectories were visually examined to see whether docking poses are stable, and interactions remain along the 1 s. The numerical analyzes were calculated in assistance of simulation quality analysis and simulation event analysis tools.Physical execution summaryAsedaSciences SYSTEMETRIC Cell Health Screen.2. Each dilution series was screened in duplicate, occupying a total of 20 wells, allowing 16 test compounds to be assayed per plate. Each row contained one positive and one negative control well, for a total of 16 replicate positive/negative control pairs on each assay plate. Compound formatting, cell deposition, and dye application were performed robotically so that final assay conditions comprised 100,000 cells in a 40 µL volume. After the 4 h compound exposure, cells were immediately stained with a panel of fluorescent dyes that report physiological signatures of both mitochondrial dysfunction and gross cell stress. Fluorescence and forward/side-scatter data were collected using automated FC with no gating. FC data are processed by an automated algorithm for producing quality control measures and ML classification of compound phenotypes.2.HL60 cell culture productionIn a 384-well platform, HL60 cells were exposed to a 10-step, 3 × dilution series of each test compound (5 nM–100 µM) for 4 h at 37 °C with 5% CO2 roller bottles with vented caps, at 1 RPM, 5% CO2, and 37 °C. Culture medium was RPMI 1640 without glucose, supplemented with 10 mM galactose and 10% dialyzed heat-inactivated FBS . Culture density was maintained at or below 1 × 106 cells/mL. Standard protocol for the Cell Health Screen is that a new production lineage of HL60 cells is started each month, and a crossover screen is performed in which the old and new production lineages are compared by using a set of 16 reference compounds to produce a known set of stress phenotypes (see supporting information). In this way, variation of screen performance is minimized by producing all screening cell populations within a narrow range of passage numbers, each checked for consistency of phenotypic performance with reference compounds. This process was performed prior to using cells to produce data for this study.3.Test compound format, cell exposure, and stainingHL60 cells were produced as suspension cultures in glass 850 cmP (Beckman Coulter) was used to deposit cells in all wells, at a density of 2.5 × 106 cells/mL, in a final assay volume of 40 µL per well . After cell deposition, each assay plate was sealed with breathable plate sealer, shaken at 2200 RPM for 10 s (Illumina® High-speed microplate shaker), and incubated for 4 h at 37 °C with 5% CO2.First fluorescent dye mix staining conditionsAll compound formatting, cell exposure, and staining with reporter dyes was performed in this study according to a standard protocol for the Cell Health Screen, which is described as follows. Compounds were formatted in groups of 16, with DMSO blanks loaded in unused screen positions for any smaller compound groups required to finish the complete study set. Each set of 16 test compounds was formatted in two replicate 384-well plates  for assays with two subsets of fluorescent dyes . Compounds in these replicate plates were identical except for the positive controls, which were chosen to produce an optimal response within each subset of fluorescent reporter dyes. Test compound dilution series and controls were formatted on a Biomek® 4000. Each compound was formatted as a 10-step, 3 × dilution series, in duplicate, on each of the two plates. Negative control wells contained the diluent used for both the test compound dilution series and positive controls. The diluent was RPMI 1640 (supplemented as above) with final working concentration of DMSO normalized to 1% in all wells. The positive and negative controls were distributed to plate wells from a single initial reservoir of each control mixture. Final assay concentration range for test compounds was 5 nM–100 µM. Prior to cell deposition, assay plates containing formatted compounds were sealed and stored at room temperature, protected from light, for 2 h, to allow binding equilibrium between serum components and test compounds. A Biomek NXP (Beckman Coulter) was then used to aspirate 20 µL of each well volume, after which 20 µL of dye mix was deposited in all wells. After dye deposition, the plate was re-sealed with its breathable plate sealer, shaken 2 × at 2200 RPM for 5 s each time , and incubated for 10 min at 37 °C with 5% CO2. The plate was then rapidly cooled to room temperature for 1 min in a shallow water bath, after which acquisition of flow cytometry data was started immediately.3b.Second fluorescent dye mix staining conditionsThe following protocol was applied to the first plate in each assay plate pair, inclusive of all compounds in this study. Dye mix buffer was 1 × PBS with 4% FBS, filter sterilized. The dye set consisted of Calcein AM, SYTOX™ Red, MitoSOX™ Red, and Monobromobimane . Dye concentrations were previously optimized, during the screen prototyping phase, to produce maximum dynamic range between positive and negative control wells. Prior to deposition of dye mix, the assay plate was removed from its 4-h incubation, and cells were gently pelleted at 300xg for 2 min. A Biomek NX2. The plate was then allowed to sit at room temperature for 15 min, protected from light. Acquisition of flow cytometry data was started immediately after this 15-min period.4.Acquisition of flow cytometry dataThe following protocol was applied to the second plate in each assay plate pair, inclusive of all compounds in this study. Dye mix buffer was 1 × PBS with 4% FBS, filter sterilized. The dye set consisted of JC-9, propidium iodide, and Vybrant® DyeCycle™ Violet . Dye concentrations were previously optimized, during the screen prototyping phase, to produce maximum dynamic range between positive and negative control wells. Cell pelleting and dye deposition were performed as above, in 3a. After dye deposition, the plate was re-sealed with its breathable plate sealer, shaken 2 × at 2200 RPM for 5 s each time , and incubated for 30 min at 37 °C with 5% CO26.5.Data processing and analysisFC data were acquired with a CyAn™ ADP flow cytometer (Beckman Coulter) with automated sampling performed by a HyperCyt® autosampler (Intellicyt). Autosampler settings were optimized to aspirate ≥ 10,000 cells per well. As described in section “Feature reductionAll well-specific FC data and matching plate map files were transferred to an EC2 server instance on Amazon Web Services (AWS). An automated algorithm converts the raw data to risk scores for each compound in two stages:26. This effectively quantitates the amount of change, in test cells relative to the negative control cells, that each concentration of the test compound caused in one of the 12 FC detection channels. For each FC detection parameter, the amount of change between each test well and the negative control was thus converted to a dose–response curve of QF distance values. The same process was executed for all 12 FC detection parameters, after which each of the 12 QF distance value curves was further reduced to two values: the point of the maximum rate of change and the range within which change occurs26. These two feature values for each FC parameter were then assembled into a vector representing all 12 FC parameters. This vector serves as the quantitative digital phenotype for the test compound, to be used in subsequent ML classification26.5b.Machine learning classificationFor each test each compound, all 12 ungated FC detection parameters were converted to a feature vector as follows. For each of the 10 concentration steps in a test compound dilution series, quadratic form (QF) distance was calculated between the empirical distribution of an FC detection parameter and that same parameter in the negative controlRisk scores were produced for test compounds with an ML classifier employing supervised learning, with a multidimensional logistic model. The classifier was trained on a set of 300 known compounds drawn from on-market pharmaceuticals, withdrawn drugs, research compounds, and a few industrial/agricultural compounds (representative set shown in Table 1–45) were prepared as previously reported and were consistent with previous reports22.4-Chloroquin(az)oline derivative (1.0 eq.), aniline (1.1 eq.) were suspended in ethanol (10 mL) and refluxed for 18 h. The crude mixture was purified by flash chromatography using EtOAc:hexane followed by 1–5% methanol in EtOAc. After solvent removal under reduced pressure, the product was obtained as a free following solid or recrystallized from ethanol/water. Compounds (Supplementary Information.


P. koraiensis SEs was heavily dependent on genotype (p = 0.001). There were high levels of PE III (pro-embryo III) number, ESE number, and soluble protein content, in the response cell lines (R cell lines), which were 1.6-, 3-, and 1.1-fold those of the obstructive cell lines (B cell lines), respectively. The B cell line had high levels of starch, auxin (IAA), Put, Spd, and putrescine: spermine (Put: Spm) compared to the R cell line. In addition, the numbers of PE III, ESEs, and soluble protein content were significantly positively correlated with SE yield. In contrast, the contents of starch, abscisic acid (ABA), Put, Spm, and Spd were significantly negatively correlated with SE yield. To ensure the accuracy of the results, we used nine cell lines to test the results. The PE III and ESE numbers and the Spm and Spd contents were positively correlated with SE yield, while the levels of starch, ABA, IAA, Put: Spd, and Put: Spm were negatively correlated with SE yield. Thus, we recommend using high PE III and ESEs as morphological indicators and low levels of starch, IAA, ABA, and Put: Spm as physiological markers to screen cell lines with a high somatic embryogenesis potential. In addition, we also found that the relationship between Spd, Spm, and SE yield was opposite in the two experimental results. Therefore, we speculate that the differences in Spd and Spm content are mainly affected by genotype. In conclusion, this study obtained the morphological and physiological markers of some high-somatic embryogenic cell lines by comparing the differences between nine somatic embryogenic cell lines. Our results can guide the improvement of conifer somatic embryogenesis technology and can provide a theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.Many cell lines in the embryogenic callus cannot produce somatic embryos (SEs) even if they meet the optimal SE maturation culture conditions during conifer somatic embryogenesis. This phenomenon hinders the progress of the industrial-scale reproduction of conifers. Therefore, there is an urgent need to obtain morphological and physiological markers to screen embryogenic calli in response to SE maturation conditions. To detect cell lines with high somatic embryogenesis potential during the proliferation process, we counted the number of pro-embryos and early SEs (ESEs) in different cell lines and storage substances, endogenous hormones, and polyamine contents. The results showed that the yield of  Pinus koraiensis somatic embryogenesis, we discovered that numerous cell lines in the embryonic tissue were unable to generate SEs even if they met the ideal SE maturation culture conditions . In the current study, we discovered that B cell lines had higher levels of Put than R cell lines did. Similarly, stifolia  and P. n, 25. In P. abies . This deic calli . HoweverP. nigra . TherefoP. koraiensis. The embryonic tissue induction medium uses mLV [−1 of 2,4-dichlorophenoxyacetic acid , 8.88 μmol·L−1 of 6-benzyladenine (6-BA), 30 g·L−1 of sucrose, 0.5 g·L−1 of casein hydrolysate (CH), 0.5 g·L−1 of L-glutamine, and 4 g·L−1 of gellan gum . To promote embryonic tissue proliferation, we transferred the obtained embryonic cell lines  to proliferation medium for propagation . The obtained embryonic tissue was subcultured at intervals of 2 weeks to maintain and proliferate. In this study, the embryogenic callus was used for subsequent experiments after 6 subcultures.Immature zygotic embryos were used to initiate embryonic tissue in uses mLV  as the b−1 of ABA, 68 g·L−1 of sucrose, 0.5 g·L−1 of CH, 0.5 L−1 of L-glutamine, and 12 g·L−1 of gellan gum. The cell lines were grown in darkness at 23 ± 1 °C for 10–12 weeks. Subculture was not required during SE maturation.In the next phase of our experiment, we collected 0.35 g of embryonic tissue from the proliferation medium and resuspended it in 25 mL of liquid mLV medium (a medium without PGR and L-glutamine). The suspended tissue was then collected by pouring 5 mL of aliquots onto five filter paper discs placed in a Büchner funnel. A vacuum pulse was applied to drain the fluid, and the filter paper with cells attached was placed in maturation medium. The maturation medium contained, supplemented with 80 μM·LThe 1-1, 1-19, 1-23, 1-100, 1-102, 8-7, 8-11, 8-16, 8-25, 8-30, and 8-18 cell lines were selected. After 14 days of subculture, 30 mg of the culture was randomly taken and stained with 2% acetocarmine, and the ESE morphology was observed under a Zeiss Ax microscope . The numbers of PE I, PE II, PE III, and ESEs were recorded under a 10× magnification. This procedure was repeated 10 times and the number of ESEs was counted within 20 fields of view each time.A total of 0.15 g of embryonic tissue was taken and transferred to fresh culture medium. The change in fresh weight (FW) was monitored every day, and the proliferation rate was calculated according to the formula below. Each measurement was repeated six times.−1) overnight at 4 °C. The supernatant was collected after centrifugation at 1000× g (4 °C) for 10 min, passed through a C18 Sep-Pak cartridge, and dried with nitrogen. The residue was dissolved in phosphate-buffered saline  and the content of IAA and ABA was determined. According to Dutra et al. [4, and 10 mL of the culture filtrate was freeze-dried and dissolved in 5% (w/v) HClO4 to analyze free and soluble conjugated polyamine from the supernatant of the centrifuged extracts. The polyamine dansyl derivatives were analyzed by high-performance liquid chromatography (HPLC). Each measurement was repeated three times.After 14 days of proliferation, embryogenic tissue was collected for physiological index determination. The contents of soluble sugar, soluble protein, and starch were determined according to the method described by Peng et al. . The meaa et al. , the polTo further validate the morphophysiological indicators that are closely related to SE yield in experiment 2.3, we used some genetically unrelated cell lines  to repeat the experiments on these morphological and physiological indicators.Data analysis was performed using Microsoft Excel 2019. SPSS  software was used for the one-way analysis of variance (ANOVA), Duncan’s multiple comparisons , and Pearson bivariate correlation analysis. SigmaPlot  as well as Microsoft Visio 2016 software was used for graph generation.In conclusion, this study compares the morphological and physiological characteristics of cell lines with various somatic embryogenesis capabilities. We discovered that the numbers of PE III and ESEs, the soluble protein content, and the Put: Spd ratio in R cell lines were significantly higher than those in B cell lines, which were 1.6-, 3-, and 1.1-fold those of B cell lines, respectively. The starch, IAA, Put, and Spd contents and the Put: Spm ratio in B cell lines were significantly higher than those in R cell lines, which were 1.2-fold those of R cell lines. In addition, the PE III and ESE numbers and soluble protein content were significantly positively correlated with SE yield, while the starch, ABA, Put, Spm, and Spd contents were significantly negatively correlated with SE yield. Through further verification of the above indicators, it was found that the numbers of PE III and ESE were significantly positively correlated with SE yield, whereas the quantities of starch, IAA, and ABA, and the Put: Spm ratio were significantly negatively correlated with SE yield. Therefore, we recommend using high levels of PE III and ESEs as morphological indicators, and low levels of starch, IAA, ABA, and Put: Spm as physiological markers for screening cell lines with a high somatic embryogenesis potential at an early stage. In addition, we also found that the relationship between Spd and Spm and somatic embryo yield was opposite in the two experimental results. Therefore, we speculate that the content differences of Spd and Spm are mainly affected by genotype. In general, our results provide a new perspective for the early screening of cell lines with high SE maturation potential and provide a theoretical basis for subsequent studies based on differences in somatic embryogenesis. Additionally, the found markers were used as indicators to develop the treatments along the SE propagation to find ones that result in the majority of the genotypes showing indicators favorable for high embryo production. As a result, the widespread application of somatic embryogenesis in forest tree breeding was promoted.


Osteosarcoma (OSA) is the most common bone tumor in both humans and dogs and has a nearly ten-fold higher incidence in dogs than humans. Despite advances in the treatment of other cancers, the overall survival rates for OSA have stagnated for the past four decades. Therefore, there is a great need to identify novel and effective treatments. We screened a series of tyrosine kinase inhibitors and selected sorafenib, a multi-kinase inhibitor, for further evaluation alone and in combination with cisplatin, carboplatin, and doxorubicin on canine and human OSA cell lines. Our data point to synergistic effects when sorafenib is combined with doxorubicin, but not when combined with cisplatin or carboplatin, in both human and canine OSA. Based on current findings, clinical trials using a combination of doxorubicin and sorafenib in proof-of-concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high and, in turn, provide useful data for the initiation of clinical trials in humans.  Osteosarcoma (OSA) is the most common primary bone tumor of both humans and dogs with about 1000 OSA cases reported in humans and 10,000 cases in dogs every year in the United States ,3,4. WitCDKN2A/B, AKT2, and BCL2 [SETD2 were found in 21% of examined tumor samples in three breeds tested  [Many studies have evaluated the genomic complexity of OSA and reported somatic copy-number and epigenetic changes ,6,7,8. Aand BCL2 . Alterattweiler) . Anothertweiler) . With th®, Whippany, NJ, USA), an oral small molecular inhibitor, inhibits a variety of signal transduction pathways. Sorafenib was primarily developed as a RAF inhibitor but was found to inhibit other targets, including VEGFR-2 , VEGFR-3, PDGFR (platelet-derived growth factor receptor), RAF-1, B-RAF, and c-KIT [Sorafenib  on three OSA cell lines and selected three TKIs  for further studies. We identified sorafenib as the potential therapeutic candidate based on the ICAs seen in 50 values were calculated using GraphPad Prism software  and three hOSA cell lines  was examined via MTS assay. After 72 h of incubation, dose–response curves were generated based on cell viability at a range of concentrations, and ICsoftware B,C. Two 50 values for the conventional chemotherapeutics cisplatin, carboplatin, and doxorubicin were also determined in our cell lines  and the MAPK/ERK  pathways ,38. In FWe next assessed the possibility of improving anti-tumor effects through combinations of drugs with different modes of action. We examined three drugs  in combination with sorafenib. Two OSA cell lines (D17 and SAOS2) were exposed to various concentration of sorafenib, one of the other three drugs, or their combination for 72 h. Later, cell viability was examined using MTS assay and the effects were calculated using CompuSyn software . As seen in We studied the effects of cell cycle changes with sorafenib and doxorubicin use on three OSA cell lines and found cell arrest at G2/M phase in all cell lines with their combined use. The distributions are very similar after treatment with 5 μM sorafenib alone A,B, wherIn bone cancers, targeting the MAPK/ERK pathway as a therapeutic strategy has been studied in vitro ,39,40, iIn the cell cycle assay, we compared three different concentrations  of sorafenib. Sorafenib alone from 1.25 μM to 5 μM did not have an effect on the phases of the cell cycle, which is consistent with a previous study . In addiIn humans, sorafenib has been used in a few clinical trials for osteosarcoma. Two clinical trials in osteosarcoma patients were reported in recent years. A phase II non-randomized trial explored sorafenib treatment in 35 patients with relapsed and unresectable OSA and reported that the median survival was 7 months . In anotIn dogs, there are very limited reports on sorafenib use. One report published pharmacokinetic studies on sorafenib in healthy dogs, and showed that 60 mg/kg per day was well tolerated in a 4-week study . To date50 values for dasatinib and sorafenib were 0.15 and 9.5 μM, respectively, suggesting that dasatinib was the most promising therapeutic agent. The patient survived for more than 730 days after the initial diagnosis after amputation and five cycles of carboplatin, followed by dasatinib treatments [The use of other TKIs such as dasatinib ,58 and teatments . Anothereatments . The fireatments . Later oeatments  and 22 [eatments  cOSA pateatments  and 95 [eatments  days in eatments ,60, otheeatments , which weatments . Anothereatments . In the eatments . These featments . Additioeatments . The expeatments , which sThe combination of sorafenib and doxorubicin has been studied and applied in clinical trials on hepatocellular carcinoma (HCC) ,70,71. A50s on OSA cell lines ranged from 3–9 μM, which is considerably lower than that of a human fibroblast cell line, MRC-5, 19.7 μM, as indicated by a recent report [In this present study, we demonstrated that sorafenib, a multi-kinase inhibitor, acts as an effective drug against both human and canine osteosarcoma cells. The ICt report . We provCanine OSA cell line D17 and human OSA cell line  were purchased from ATCC. The Abrams canine OSA cell line was provided by Dr. Elizabeth McNeil and originally established and shared by Dr. Doug Thamm. The BZ canine OSA cell line was established by our laboratory from a German shepherd dog. D17 was derived from a lung metastasis of a poodle; the canine Abrams cell line was derived from a metastatic nodule and the canine Gracie cell line was derived from a primary tumor sample. As indicated in the ATCC repository, the SAOS2 cell line was derived from a primary tumor in a Caucasian female, U2OS was from a tibia biopsy sample from a Caucasian female, and MG63 from a Caucasian male. 2. For cell culture maintenance, human OSA cell lines were incubated with DMEM medium and canine OSA cell lines were maintained with a-MEM medium, and all cells were supplemented with 10% fetal bovine serum and incubated in a humidified incubator at 37 °C with 5% COSorafenib was purchased from LC laboratories . Sorafenib was dissolved in DMSO while cisplatin was dissolved in PBS, and carboplatin was dissolved in water. Other TKIs, including cladribine, dasatinib, erlotinib, gefitinib, masitinib, nilotinib, sorafenib, sunitinib, toceranib, and tozasertib were all purchased from Sigma-Aldrich and dissolved in DMSO.50 values of sorafenib and other compounds on OSA cells. Cells were seeded in 96-well plates at a density of 2500–3500/well. After 24 h, cell culture medium was replaced by complete medium with each compound at the designated concentrations. Cells were treated for 72 h. Cell viability was analyzed using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) and determined by the amount of colored formazan dye produced by live cells. The absorbance of the formazan dye produced was measured at wavelength 490 nm, and IC50 values were calculated using PRISM Statistical Software. Each concentration for each drug was assayed in triplicate for the IC50 calculations.The MTS assay  was used to determine the ICIn this study, we treated three OSA cell lines  with a small panel of compounds including ten tyrosine kinase inhibitors  for 48 h. Percent growth inhibition was calculated for each treatment with the MTS assay with the vehicle control treatment containing 1% DMSO. For all TKIs, we used a high drug dose of 100 μM.The wound healing assay was used to examine the migration capacity of cells in a monolayer. Briefly, 100,000 cells/well were plated overnight and allowed to reach 70–80% confluence in 6-well plates. Then, scrapes were made on the plates using a 1-mL pipette tip. The cells were then incubated with cell culture medium with or without sorafenib . Each scrape was photographed after being made and at each specific time point thereafter.Cells were simultaneously incubated with two compounds at a fixed ratio  for 72 h. The synergistic effects of each pair of drugs were determined via isobologram and combination index (CI) analysis using CompuSyn software . The analysis was adapted from the median-principle methods of Chou and Talalay : resultsCells were treated with 1% DMSO (control), sorafenib , doxorubicin , or combination (ration 50:1) for 24 h, then collected by centrifugation and fixed with 70% ethanol at 4 °C overnight. The ethanol was removed via centrifugation, and cellular DNA was stained with propidium iodide (50 μg/mL) containing RNase (1 mg/mL). After cells were stained for at least 4 h, the PI fluorescence of individual nuclei were recorded with FACScan. The quantitative assessment of cell cycle phase and apoptosis were then determined using Modfit LtTM software after correction for debris and aggregate cell populations.The Abrams OSA cells  were treated with either vehicle (0.1% DMSO) or sorafenib for 24 h. Cells were lysed with 250 μL of CelLytic M lysis buffer , 2 μL of protease inhibitor , and 2 μL of phosphatase cocktail inhibitor B  according to manufacturer protocol. Protein concentrations were quantified with the QubitTM Protein Assay Kit. A total of 60 μg of protein per well was loaded on Bolt Bis-Tris 4–12% polyacrylamide gels (Thermo Fisher Scientific Inc.) and transferred to polyvinylidene difluoride membranes. The membranes were incubated with 5% bovine serum albumin (BSA) for 2 h at room temperature, then incubated with the following primary antibodies at 4 °C overnight to detect antigen: ERK (1:500), p-ERK (1:250), STAT3 (1:500), p-STAT3 (1:500), β-tubulin (1:4000) . After three washes in tris-buffered saline with 0.05% Tween 20, the membranes were incubated with appropriate secondary antibody  or goat anti-rabbit ) for 1 h at room temperature. The membranes were visualized using the Odyssey Infrared Imaging System  and analyzed using Image Studio™ Lite software .p-values of <0.05 were considered to be significant.Our results represent at least three separate experiments. Statistical analysis was performed with GraphPad Prism  and differences between categories were analyzed with one-way ANOVA; In conclusion, the findings from our studies suggest that the tyrosine kinase inhibitor sorafenib exhibits anti-tumor activity on its own through the inhibition of proliferation and the induction of apoptosis via inhibition of the ERK/MAPK and STAT3 pathways in OSA cells. Sorafenib is a comparatively novel drug in osteosarcoma; therefore, information on large-scale clinical data and other drug combinations is limited. In addition, combining sorafenib with doxorubicin was found to be synergistic in clinically relevant doses, inducing cell cycle arrest in the G2/M phase. Based on current findings, clinical trials using the combination of doxorubicin and sorafenib in proof-of-concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high and, in turn, provide useful information for the initiation of clinical trials in humans.