The aim of the present study was to examine the cytostatic effects of cold atmospheric plasma (CAP) on different head and neck squamous carcinoma (HNSCC) cell lines either in isolation or in combination with low dose cisplatin. The effect of CAP treatment was investigated by using three different HNSCC cell lines .Cell lines were exposed to CAP treatment for 30, 60, 90, 120 and 180 s (s). Cisplatin was added concurrently (cc) or 24 h after CAP application (cs). Cell viability, DNA damage and apoptosis was evaluated by dye exclusion, MTT, alkaline microgel electrophoresis assay and Annexin V-Fit-C/PI respectively.In all cell lines, 120 s of CAP exposure resulted in a significant reduction of cell viability. DNA damage significantly increased after 60 s. Combined treatment of cells with CAP and low dose cisplatin showed additive effects. A possible sensitivity to cisplatin could be restored in Cal 27 cells by CAP application.CAP shows strong cytostatic effects in HNSCC cell lines that can be increased by concurrent cisplatin treatment, suggesting that CAP may enhance the therapeutic efficacy of low dose cisplatin. Primarypression . Numeroupression . Severe pression . Novel rpression –9.202 and NO2 are the main reagents associated with CAP exposure of cells. The physical effects include thermal ultraviolet irradiation, and electromagnetic effect and have been shown to be minor in CAP in-vitro application in previous studies [Cold atmospheric plasma (CAP) is a promising alternative and additional treatment to the current cancer therapies , 11. CAP studies , 13. Stu studies –16. CAP  studies , 14. The studies , 18.in-vivo on the tumor would theoretically be possible. Due to the previously mentioned toxicity of cisplatin, an increase of the systemically applied dose is not possible in extensive or recurrent HNSCC. Therefore, the combination of CAP and cisplatin application could be of clinical relevance. In this study the common cellular responses were detected and analysed by comet assay (DNA damage), MTT assay  and trypan blue staining (cytoplasmic membrane damage). Physical factors of CAP exposure have been shown to be minor in CAP in-vitro application in previous studies. The culture temperature of the cell medium in our study was set at 37 °C. Studies have shown that even close CAP exposure will not increase the temperature significantly. Furthermore, the ultraviolet light exposure of cancer cells fails to show significant antiproliferative effects on cancer cells in-vitro and therefore physical factors were not specifically addressed in this study [Unlike many other tumors, HNSCC can be accessed directly via the oral orifice. Thus, a direct CAP application is study , 20.in-vitro.The purpose of the present study was to investigate the effect of CAP treatment  on common HNSCC cell lines (FaDu and OSC-19) in comparison to a chemo-resistant HNSCC cell line . A further aim was to examine the therapeutic efficacy of low dose cisplatin in combination with CAP for cisplatin sensitive and resistant HNSCC cell lines Welz et al. were used [cis-platinum.Three different HNSCC cell lines were used for this study. The same two cell lines (OSC 19 (JCBR Cell BANK) and FaDu ) as in the study of ere used , 21 and 2 at a temperature of 37 °C. All cells were cultured in 25 ml culture flasks (Nunc EasYFlasks) and medium was changed every 2–3 days. For this study, 5 × 105 cells of Cal 27, FaDu or OSC 19 were seeded onto a 6 well plate, and were left to attach for 16 h. For the experiments the cells seeded were splitted at least 3 times.OSC 19 cells were grown in DMEM/Ham´s F-12  and FaDu cells in DMEM (Biochrom). Both cell lines were also supplemented with 10% fetal bovine serum  and 10 U/ml Penicillin–Streptomycin (Biochrom). FaDu cells were additionally supplemented with 1% non-essential amino acids  and each 1% of L-Glutamine and Sodium Pyruvate (Biochrom). Cal 27 cells were cultured in DMEM also supplemented with 10% fetal bovine serum  and 10 U/ml Penicillin–Streptomycin (Biochrom). All cells were preserved under humidified conditions with 5% COA CAP device using the surface microdischarge (SMD) technology for plasma production was used for this study . An SMD A glass epoxy board, which is sandwiched by a stainless-steel mesh grid and copper foil layer creates the SMD electrode. The plasma is produced homogenously on the mesh grid side in the air by applying a high pulse-like voltage of 7 kV with a repetition frequency of 6.75 kHz. Via the mechanism of diffusion, the generated reactive species are transferred to the cells. The MiniFlatPlaSter® produces the plasma indirectly.One well of a 6-well plate has a rim diameter of 28 mm, fitting the CAP electrode exactly. The medium was then completely removed and the cells were exposed to CAP treatment. The CAP device was placed exactly onto the well rim, causing a closed volume condition (distance between the electrode and cells was 17.5 ± 0.5 mm). The respective controls were treated equally as the plasma treatments except for the CAP exposure. The treatment times used were 30 s, 60 s, 90 s, 120 s and 180 s. Immediately after the appropriate treatment medium was added.Ten µM cisplatin have been shown to induce sufficient DNA cross-links without inducing apoptosis in cell cultures . The cisPrior to cisplatin application, all cells were seeded and exposed to CAP treatment as stated above in the method and materials section. After the described CAP exposure each cell type was divided into another two treatment groups. For both groups the cells were incubated with 1 ml trypsin/EDTA solution for 8–10 min for trypsination and seeded at 8000 cells/well (100 μl) in a 96 well plate. The first group, cisplatin concurrent (Cis + cc) underwent a 2.5 µM cisplatin application directly after the different CAP treatments. In the second group, cisplatin consecutive (Cis + cs), the cells were incubated (37 °C with 5% CO2), in the 96 well plates in their according medium after CAP treatment and underwent a 2.5 µM cisplatin application after 24 h of incubation (37 °C with 5% CO2). For both groups 2.5 µM of cisplatin was added to each 96 well plate at the stated time point and the well plates were incubated under the same conditions as stated before for another 24 h. Following this, cisplatin was removed and each well plate was washed with PBS. PBS was then discarded and replaced with 10 μl labeling medium containing 0,5 mg/ml MTT and an MTT assay as described in detail below was performed.Trypan blue is used to distinguish between viable and dead cells. The method is based on the principle that the dye is not absorbed by living cells with an intact cell membrane. Dead cells take up the dye due to their damaged membrane and are stained blue . To anal2), 100 μl MTT-staining solution was added to each well followed by an incubation overnight. A VERSAmax™ ELISA- Reader  at a wavelength of 550 nm was used to quantify the purple formazan dye. The reference wavelength corresponds to 690 nm [n = 15). The mean cell viability reduction curves were standardized to the percentage of living cells, whereas the control cells were set at 100%.The MTT Assay is used to assess cytotoxicity, proliferation and cell viability by measuring the cellular metabolic activity . In thiso 690 nm , 18. EveFor the detection of DNA damages, the alkaline microgel electrophoresis (Comet assay) was performed after the different CAP treatments. Depending on the extent of damage/fragmentation, the DNA varies in its migration behaviour in an electrical field. Undamaged DNA shows no migration in comparison to fragmented DNA. The higher the fragmentation (damage) the further and faster the migration , 27.The alkaline microgel electrophoresis was carried out as published in the methods and materials section of Welz et. al. .2 EDTA and 1% N -lauroylsarcosine sodium salt). After the lysis process, the slides were placed in the gel electrophoresis chamber . Before applying an electric field, they were left with alkaline buffer solution containing 300 mMNaOH and 1 mM Na2EDTA at pH 13.2 for 20 min for the DNA double helix to denature. The electrophoresis was started at 0.8 V cm − 1 and 300 mA and continued for 20 min. The slides were then neutralised with Trisma base, 400 mM, pH 7.5 . After this, they were stained with 75 μl ethidium bromide  and analysed with a DMLB microscope . By random pattern, 80 cell nuclei per slide (2 slides per CAP treatment time) were selected and digitized with the attached monochrome CCD camera . Using the image analysis software Komet +  +  DNA-migration was measured. Cells containing damaged DNA have the appearance of a comet under the microscope, where the undamaged DNA is the „head “ and the damaged DNA is the „tail “ of the comet , cell counting and cell viability screen with a trypan blue exclusion test were carried out. A two layer agarose was used to ensure stability. The cells were resuspended in 75 µl of 0.7% low-melting agarose , applied to slides  which were covered with normal melting agarose (Biozym) to ensure the stability. The slides were then immersed with alkali solution for 1 h . Phosphatidylserin (PS) residues are found on the inner surface of the membrane in normal cells, hence being inaccessible to Annexin V. An early step of apoptosis is the translocation of phosphatidylserin (PS) from the internal to the external face of the plasma membrane as the cell membrane becomes permeable. Annexin-V is a Ca 2+ phospholipid binding protein with a high affinity to PS and can therefore be used as a sensitive marker for the apoptotic cells. Combining Annexin-V labelling with PI staining allows discriminating between (early) apoptotic and late apoptotic/necrotic cells, as PI will be able to stain the DNA in the nucleus. One hundred cells per CAP treatment were counted with the help of a hemocytometer and early apoptotic cells were identified with fluorescence microscopy, and indexes calculated out of 100 cells . CAP treatment and trypsinisation were carried out as described above. HNSCC cell lines were stained with Annexin V Fit-C and propidium iodide (PI) according to the manufacturer’s instructions. In this case, 1 × 10lls Fig. .Fig. 6.Figure p < 0.001). Both FaDu and OSC 19 had a similar cell survival after 60 s and 90 s (75.2% vs 69.0% and 52.1% vs. 48.2% respectively). OSC 19 showed the strongest reduction of cell viability reduction at 120 s and 180 s of CAP treatment compared to the other two cell lines (p ≤ 0.0207). Cal 27 cells showed the weakest reaction to CAP. After 180 s the mean number of viable cells was 53.1%. The half maximal inhibitory dose (IC50) was reached after 90 s of exposure in OSC 19 and after 120 s in FaDu cells.Figure In order to evaluate the DNA damage in the three different cell types after CAP treatment the alkaline gel electrophoresis was performed. Table p ≤ 0.0366), (Tables (p ≤ 0.0171).Figure , Tables . Only th, Tables . HoweverThe first significant difference of DNA damage between all three cell lines was seen after 90 s of CAP treatment time Fig. . The FaDAnnexin V Fit-C/PI staining was performed 24 h after CAP treatment in order to clarify if the time dependent increase of DNA damage induces apoptosis and if reduction in cell viability can be explained by programmed cell death. Figure p < 0.001). OSC 19 showed a significant percentage of apoptotic cells after 180 s of CAP treatment (p < 0.0001). OSC 19 showed the highest mean percentage of apoptosis (11.25% at 180 s) in comparison to the FaDu (8.50% at 120 s) and Cal 27 (8.20% at 180 s).Cal 27 and FaDu showed a significant increase in apoptosis after 120 s and 90 s CAP exposure respectively (p ≤ 0.0063). The mean cell viability was 83,87%, 83.0% and 93.03% for Cal 27, FaDu and OSC 19 respectively. The OSC 19 cell line showed a signifcantly higher cell viability after cisplatin monoapplication compared to the other two cells (p < 0.0001).Figure p < 0,0001). The Cal 27 cell line showed a significant difference between mono and consecutive cisplatin treatment a prior 180 s CAP exposure . FaDu cells already showed a significantly lower cell survival at 90 s CAP + Cis cc compared to a cisplatin monotreatment. The OSC 19 cell line presented similar results in cell viability in a combination therapy compared to a mono cisplatin treatment. CAP + Cis cc showed a significant cell viability reduction after 90 s compared to mono chemotherapy and CAP + Cis cs already after 60 s (p < 0.0001).All cell types showed a significant further reduction of cell viability with a combination treatment compared to mono treatment with cisplatin. Cal 27 and FaDu already showed a significant cell viability reduction compared to a mono treatment after 30 s CAP + cis cc . When comparing the different treatment groups, the significantly lowest cell survival was measured at all time points for the treatment with CAP + Cis cc application for the Cal 27 group except at 180 s (p ≤ 0.0237). An additional cisplatin treatment (concurrent and consecutive) after CAP treatment showed an additive effect on cell viability reduction to solely cisplatin or CAP therapy after only 30 s of CAP exposure time. The Cal 27 cell line only showed a significant difference between a mere CAP treatment and cisplatin treatment after 180 s CAP exposure. In both treatment groups the cell viability was > 80% at all other treatment times.The cell line Cal 27 showed significant difference in cell viability at almost all treatment time points. At treatment times of 30 s and 60 s there was a significant difference for all examined treatments . At 90 s no difference in cell survival could be seen between CAP and CAP + Cis cs. However, CAP + Cis cc showed a significantly lower cell viability at 90 s compared to the other treatments p ≤ 0.0015). There was no significant difference in viability to be seen for solely CAP treatment vs CAP + Cis cs application except at 180 s (p > 0.05).The FaDu cells showed a difference in cell survival at all treatment times between only CAP treatment and CAP + Cis cc treatment except after 90 s. A significantly higher cell reduction was to be seen by the additional Cis cc application for treatment times at 30 s and 60 s (p ≤ 0.0027). Here an additive effect was seen with the addition of low dose cisplatin cc and cs. There was no difference at these time point in cell viability between CAP + Cis cc and CAP + Cis cs. However, all three treatments showed lower cell viability at all treatment times compared to the cisplatin reference value.The OSC 19 cells had a significant difference in the amount of cell survival between solely a CAP treatment, CAP + Cis cc application and CAP treatment + Cis cs application at 90 s and 120 s , 60 s (59.0% and 81.3% respectively), and 90 s (61.7% and 76.8% respectively). The FaDu cells also showed a significantly higher cell reduction with an additional cisplatin cc treatment compared to OSC 19 at treatment times 30 s (60.2%), 60 s (57.2%), 90 s (42.1%) and 120 s (25.1%). FaDu cells measured a significantly lower cell viability compared to the Cal 27 cell line after CAP exposure times 90 s, 120 s and 180 s and Cis cc treatment.The adjusted The treatment with CAP treatment + Cis cs application showed a significantly lower cell survival after 90 s in the FaDu cell line (55.4%) compared to the Cal 27 (72.8%) and OSC 19 (68.4%) group. The duration of 120 s CAP exposure + cisplatin cs application presented significantly less viable cells in the FaDu cell group compared to Cal 27 (73.5%) and OSC 19 (50%), whereas Cal 27 had significantly higher survival compared to both other cell lines. After the CAP exposure time of 180 s + Cis cs application all cell lines show an average of less than 50% cell viability. The FaDu and OSC 19 cells lines measure a significantly higher cell reduction at this treatment time than the Cal 27 .In literature, CAP is one of the most promising new approaches to cancer therapy. Several studies have shown a successful application as an alternative therapy for malignant tumors , 31, 32.The aim of the present study was to analyze the effects of CAP on cell viability and toxicity of HNSCC cells in vitro either in isolation or in combination with low-dose cisplatin. Further aims were to test these effects on cells with respect to different exposure intervals to CAP and application times of low dose cisplatin.This study found that CAP has a cytotoxic effect on all three different HNSCC cells with increasing impact at longer exposure times. It is interesting to note that the inhibitory effects of CAP  varied with respect to the cell line. Faster proliferating cells (OSC 19) showed a better response to CAP mono treatment than the other two cell lines, FaDu and Cal 27 respectively. This effect can be enhanced by combining CAP application with low – dose cisplatinum treatment, especially on slower doubling times . A combined treatment CAP + cis cc showed a significantly lower cell viability in the chemo-resistant Cal 27 compared to any of the mono treatments for this cell line.The results are of clinical importance because the combination of topical CAP treatment in combination with systemic cisplatin administration could reduce the standard chemotherapy drug dosage currently administered and hereby reduce toxicity, side effects and improve prognosis. Additionally, a combination therapy could also improve the response of chemo-resistant HNSCC.Generally, as discussed by Keidar et al., the cellular response to CAP exposure is dependent on the individual cell line . DiffereWe carried out AnnexinFit-C/PI staining in order to evaluate if dose dependent DNA damage induces apoptosis. All three HNSCC cell types showed a significant increased percentage of apoptotic cells after 180 s of CAP treatment compared to their control. Again, the higher proliferating cells (OSC 19) showed a higher amount of apoptotic cells after 180 s than the slower proliferation cells . The different vulnerability of the cell lines towards CAP treatment may be explained by their cell cycle and doubling time. The most significant effect of CAP was observed in the fast-doubling OSC-19 (approx. < 25 h) and FaDu (approx. 30 h) cell lines, while Cal 27 (approx. 45 h) were affected the least –38. The Volotskova et al. showed in their study that cells are more likely to be damaged by CAP treatment during S-phase of the cell cycle . This isIn addition to the primary cytotoxic effect of CAP, we evaluated the response of malignant epithelial tumor cells with an additional cisplatin application. All epithelial cell lines showed a significant cell viability reduction after a mono treatment with cisplatin. Even Cal 27 cells which are supposedly resistant to chemotherapy with cisplatin , 37. In Cal 27 also showed a very low cell viability reduction after CAP treatment for all treatment times. Even after 180 s of CAP exposure mean cell viability was still 53.1%, showing a possible CAP-resistance. CAP exposure can lead to a reduction in redox regulators. This could be an explanation for chemo-sensitivity induction in the Cal 27 cell line. Furthermore, the theory of cell activation/sensitization, a unique feature of direct plasma application in comparison to indirect CAP treatment, could also be an explanation. Prior studies have shown that cancer cells show a different resistance towards reactive species, resulting in these cells remaining in an activation or sensitization state without viability reduction for approx. 5 h . Cal 27 Novel results imply that low intracellular ROS levels may be the decisive step in cisplatin resistance of cells . This stOnly Cal 27 cells showed a difference in cell reduction between a concurrent and consecutive cisplatin application after CAP exposure. An explanation could be that there was enough time for DNA reparation after minimal CAP damage and therefore no additive effect when applying cisplatin 24 h after CAP exposure. Only direct application showed an additive effect. The other cells (FaDu and OSC-19) showed no significant difference between cisplatin cc or cs applications, most likely because of maximum DNA damage already caused by only CAP exposure. These results could again be related and explained by the different doubling times of the cell lines. The faster doubling cell lines showed no difference in the cell viability results between the application times of cisplatin after CAP treatment, whereas the slower doubling Cal 27 cells showed a different sensitivity towards the time of cisplatin application after CAP therapy.In this study, only 30 s of CAP exposure combined with a single cisplatin cc application showed a mean cell viability of 64.5% in the otherwise chemo-resistant Cal 27 cell line. Studies have implied that agents with antioxidant activities could have a chemoprotective effect on healthy tissue against chemotherapy , 45 and Additionally, the chemo-sensitive cells FaDu and OSC 19 also showed lower cell viability when exposed to CAP and concurrently treated with cisplatin. In both cases 60 s of CAP treatment + Cis cc application showed a noticeable increased cell viability reduction when comparing to the cisplatin reference and only CAP exposure (FaDu > OSC 19). Again, a CAP treatment time reduction in combination with a single cisplatin application should be taken into consideration. This way, one could achieve a cisplatin dose reduction even in known chemo-sensitive tumor cells.The following limitation of the study have to be discussed. The chemoresistance to cisplatin of Cal 27 cells used was not significant which may be a result of an altered microenvironment. Exposure of these cells to increasing concentrations of cisplatin prior to CAP treatment and more precise control of the microenvironment (e.g. pH) to generate more reliable drug-resistant cells should be considered in further studies to re-evaluate the chemo sensitivity induction through CAP. However, other preclinical studies have shown that Cal 27 cells are not totally resistant to cisplatin and that the resistance is dose-dependent . A SMD pIn conclusion, this study demonstrates a decrease in cell viability in all three HNSCC cells types solely to CAP exposure, especially for treatment times 90 s and more. However, the supposedly cisplatin resistant cell line Cal 27 showed the least response to a CAP mono therapy. All HNSCC cell lines showed a CAP-dose dependent DNA damage as well as a significant increase in apoptotic cells with longer CAP exposure times. We observed an additive effect within all cell types, in particular for short CAP treatment times and an immediate subsequent cisplatin application. Especially Cal 27 cell line showed an increased vulnerability to treatment with cisplatin by the prior application of CAP for all exposure times, suggesting a possible chemo-sensitivity induction by a primary CAP treatment of only 30 s. Finally, we demonstrated the successful application of a direct plasma source for the treatment for HNSCC chemo-sensitive and –resistant carcinoma cells. Additionally, the study presents the potential of CAP and cisplatin combination therapy as a possible addition to adjuvant therapy options with low dose chemotherapy for the treatment of HNSCC.


G Protein Subunit Alpha I1 (GNAI1), which belong to GPRs, were stimulated in HHT-resistant cell strains in vitro and vivo, indicating that they may play a critical role in HHT resistance. In addition, these two genes have prognostic significance for AML patients. Taken together, we successfully constructed HHT-resistant cell lines with dynamic RIs and explored the resistance mechanisms, which will help identify new drugs for HHT-resistant AML patients.Homoharringtonine- (HHT-) based HHT, aclarubicin, and cytarabine (HAA) induction regimen is the first-line therapy for nonelder acute myeloid leukemia (AML) patients in China. However, drug resistance is a new challenge, and little attention has been devoted to excavating resistant mechanisms. This study used the classic method to construct six HHT-resistant cell lines with a gradually increasing resistance index (RI) to discover HHT drug resistance mechanisms dynamically. After HHT resistance, the cell growth rate decreased, cell cycle delayed, and P-glycoprotein  expression levels increased. Furthermore, we explored the changes in transcriptomics between HHT-sensitive and HHT-resistant cells using RNA-sequence. Through Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and hub gene analyses, we found that immune activity, especially G-protein coupled receptor (GPR) and related molecules, may mediate HHT resistance. Moreover, Calcitonin Receptor-Like (CALCRL) and  Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and is characterized by stem cell transcriptional dysregulation and leads to a block in the differentiation . NowadayCephalotaxus hainanensis, and it has significant antileukemic effects in AML, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) [p=0.00108) and the 3-year event-free survival (EFS)  in the HAA (HHT plus aclarubicin and cytarabine) treatment group were better than those in the DA (daunorubicin plus cytarabine) group [Homoharringtonine (HHT) is a cytotoxic alkaloid that was initially extracted from me (MDS) . One phae) group . The rese) group . HoweverStudies on the mechanisms of cytotoxicity and resistance to chemotherapy have been based on the development and analysis of resistant cancer cell lines . For hemBased on the above-mentioned background, we aimed to establish HHT-resistance AML cell lines with different RIs via exposure to gradually increasing HHT concentrations. After 15 months cultivated with HHT, 6 HHT-resistant cell lines were stabilization-established. Then cell characteristics including cell growth, apoptosis, cell cycle, and antigen p-gp were investigated to explore the biological behaviors difference between resistant cells and sensitive cells. From RNA-sequence data, we found that immune activity, especially G-protein coupled receptor and related molecules, such as CALCRL and GNAI1, may mediate HHT resistance. However, we have not further explored how CALCRL and GNAI1 mediate HHT resistance and the mechanism of resistance, and further exploration is needed in the future. The role of HHT in AML drug resistance is also explored from metabolomics or proteomics in the future.α, and U937 were cultured in RPMI-1640 medium ; MOLM-13 and MV4-11 were cultured in IMDM medium , and supplemented with 10% fetal bovine  at 37°C with 5% CO2. All cells were passaged less than 20 times and had cell identification certificates.AML cell lines THP-1 (CVCL_0006), HL-60 (CVCL_A794), NB4 (CVCL-0005), Kasumi-1 (CVCL_0589), MOLM-13 (CVCL_2119), MV4-11 (CVCL_0064), KG-1 (CVCL_0374), and U937 (CVCL_0007) were purchased from American Type Culture Collection (ATCC), and OCI-AML3 (CVCL_1844) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). THP-1, HL-60, OCI-AML3, NB4, Kasumi-1, KG-15 ml and treated with the indicated concentrations of HHT for 48 hours. Then 100 μL cell supernatant was transferred into 96-well plates and 10 μL CellTiter 96® Aqueous One Solution Cell Proliferation Assay was added per well. Absorbance was measured by a microplate reader  at 490 nm to determine the OD value after 4 hours. The GraphPad software (version 6) calculated the cell proliferation rate and half-maximal inhibitory concentration (IC50) value according to the OD.Proliferation and viability of AML cells were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay . Briefly, THP-1, HL-60, OCI-AML3, NB4, Kasumi-1, KG-1, U937, MOLM-13, MV4-11, and resistant strains were inoculated into 24 wells at a final concentration of 1 × 1050 value of those cells was calculated and the RI of those cells was calculated with the following formula: IC50 (resistance)/IC50 (sensitive).MV4-11 and MOLM-13 cells were treated with gradually increasing HHT concentrations (1 nmol/L to 50 nmol/L). The concentration of HHT was increased at approximately 2-week to 4-week intervals and maintained until cells grew normally in a complete growth medium in the presence of different concentrations of HHT for two weeks. Cells that grew normally in medium with 10% fetal bovine containing 10 nmol/L, 30 nmol/L, and 50 nmol/L HHT were preserved. These cells were designated as MV4-11 R10, MV4-11 R30, MV4-11 R50, MOLM-13 R10, MOLM-13 R30, and MOLM-13 R50. Then, the IC5/ml. Then cells were collected and washed twice with 1× phosphate-buffered saline (1× PBS) and inoculated into 300 μL 1× binding buffer with 10 μL Propidium (PI) and 5 μL Annexin-V for 30 minutes . For cell cycle analysis, cells were seeded in 24 wells at a final concentration of 3 × 105/ml cells per well and collected and fixed with precooled 75% ethanol at 4°C overnight. The next day, cells were washed twice with 1× PBS. Flow cytometry was utilized to detect the DNA content after PI  staining for about 30 minutes. For p-gp expression analysis, cells were inoculated into 300 μL of 1× PBS with 10 μL of p-gp antibody  labeled with APC for 30 minutes. Flow cytometry was performed and analyzed on ACEA NovoCyte .To examine the cell apoptosis, MV4-11 and MOLM-13 sensitive and resistant cells were inoculated into 24 wells at a final density of 1 × 106 MV4-11 and MV4-11 R50 cells (suspended in 100 μl 1× PBS), respectively. Tumor volumes were evaluated using the following formula: 1/2 × length × width2. After 7∼11 days, tumor volume reached 100–200 mm3, mice were randomly divided into MV4-11 group (n = 4) and MV4-11R group (n = 5), and HHT was administered by intraperitoneal injection every day at 0.5 mg/kg body weight for 7 days. Tumor volumes were measured twice daily using the caliper. All animal experiments were reviewed and approved by the Ethics Committee for Laboratory Animals of the First Affiliated Hospital, College of Medicine, Zhejiang University  (reference number: 20201522).Male BALB/c nude mice (5 weeks old) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Sciences . After one week of acclimatization, mice were subcutaneously injected with 1 × 10178 AML patients between March 2010 and June 2014 with detailed diagnosis and treatment information were included in this study. Patients with acute promyelocytic leukemia (APL) and patients undergoing bone marrow transplantation were excluded. The clinical characteristics of the patients are summarized in μl containing 1 μl of 100 ng/μl sample cDNA, 12.5 μl 2× PCR mix, 1 μl 0.5 μM of each primer, and 10.5 μl ddH2O. mRNA levels were normalized to GAPDH. The primers used for quantitative PCR are presented in Total RNA was isolated from cells using the TRIzol reagent . cDNA was prepared using the reverse transcription kit following the instructions . Real-time quantitative PCR (RT-qPCR) was performed in duplicate with the fluorescent DNA binding dye SYBR green . PCR reactions were performed in a total volume of 25 μg protein was loaded per well on 4–12% SDS-PAGE gel and transferred onto a PVDF membrane  preactivated with methanol. Membranes were blocked with 5% milk and incubated with antibodies against specific proteins. After overnight, membranes were washed with 1× TBST and incubated with horseradish peroxidase labeled secondary antibodies. The protein bands were visualized using enhanced chemiluminescence reagent . The antibody of GNAI1 12617-1-AP was purchased from Proteintech , CALCRL (ab84467) was purchased from Abcam , and GAPDH #5174S and the Rabbit (DA1E) mAb IgG secondary antibody were purchased from CST  (#3900).Total protein was extracted using RIPA buffer  supplemented with protease inhibitor and phosphatase inhibitor cocktail . The protein content was quantified using the BCA protein assay kit . An estimated 40 The mRNA expression profiles of MV4-11 sensitive cells and MV4-11 resistant strains (MV4-11 R10/R30/R50) were obtained using high-throughput sequencing (RNA-sequence). The sequenced data were obtained from Illumina HiSeq X Ten platforms, and the data were uploaded to NCBI's SRA and access to cite for these SRA data: PRJNA664675. For RNA-seq data, mRNA expression levels were calculated as RPKM (Reads Per Kilo-base per Million reads). We used DE Seq (1.18.0) R package to analyze the differentially expressed mRNAs.Overall survival (OS) was defined as the time from diagnosis to death due to any cause. Event-free survival (EFS) was defined as the time from diagnosis to removal from the study because of the absence of CR, relapse, or death. In the association between CALCRL expression and OS, EFS was estimated by the Kaplan-Meier method and log-rank test. In prognostic analysis of other genes like GNAI1, CALCRL was calculated via online database “GEPIA.”t-test, or ANOVA and Chi-square test were performed using IBM SPSS Statistics 20 software or GraphPad Prism 5. P < 0.05 was considered statistically significant. P < 0.05∗, P < 0.01∗∗, and P < 0.001∗∗∗.Experimental analysis, unpaired two-tailed Student's Protein-protein interaction (PPI) network was created by online database “STRING”  and visu50 value measurement. The IC50 values of MOLM-13 and MV4-11 were 6.858 nM and 7.207 nM, respectively, while other cell lines exceeded 10 nM  were utterly suppressed  were 7.13 nM, 23.75 nM, and 109.9 nM, respectively, which were 1.82-fold, 6.07-fold, and 28.11-fold higher than those of the sensitive cells. Contemporarily, the RIs of MV4-11 resistance cell lines  were 18.31 nM, 40.67 nM, and 58.82 nM, respectively, which were 4.92-fold, 13.08-fold, and 15.81-fold higher than those of sensitive cells . Furtherppressed . In addippressed . Then weppressed . Additioppressed .in vivo. Subcutaneous tumor mass in mice is mainly infiltrated by leukemia cells , which belong to the G-protein family. First, the mRNA expression levels of these genes were detected in the HHT innate resistant cell lines THP-1 and KG-1 and sensitive cell lines MV4-11 and MOLM-13. Only CALCRL and GNAI1 had higher expression levels in innate resistant cells THP-1and KG-1 . ContempWith the widespread use of HHT in AML treatment, drug resistance has become a great challenge, and few attempts have been made to explore the resistance mechanisms. In this study, we successfully constructed six HHT-resistant cell lines in two HHT-sensitive AML cell lines with different RIs. The growth rate of HHT-resistant cell lines was decreased and accompanied by a delay in the cell cycle and an increased p-gp (CD243) expression. GPRs mediate immune activity, for molecular mechanisms may play an essential role in HHT resistance. The GPR signaling, especially two GPRs, CALCRL and GNAI1, may mediate HHT resistance and have prognostic significance in AML.G1 phase was delayed and S phase was decreased in HHT-resistant cells.Drug-resistant cell lines can be used to study biological resistance changes. In this study, we constructed different RIs of cell lines to observe the resistance characteristics and mechanism dynamically. Recent studies have shown that the cell cycle plays a critical role in chemotherapeutic resistance in cancer. When cancer cells exhibit Palbociclib resistance, cells are delayed in the G1 phase . FurtherThe increase of p-gp expression in resistant cells was another crucial difference between resistant and sensitive cells. Overexpression of p-gp is considered the primary cause of multidrug resistance (MDR) in AML . p-gp functions as an ATP-dependent membrane protein and is involved in the absorption, distribution, and excretion of drugs, xenobiotics, and endogenous compounds . These rAfter KEGG and hub gene analyses, we found that immune activity may play an important role in HHT resistance, and top 10 upregulated hub genes were focused on GPRs. GPRs can regulate many fundamental biological processes, such as cardiovascular functions, immune responses, and hormones . DysreguIn summary, we successfully constructed six HHT-resistant AML cell lines with different RIs; these will be specific cell tools to study HHT resistance. The cell cycle delay and increase in p-gp expression may be the biological reasons for HHT resistance. At the transcriptional level, we found that GPRs, especially CALCRL and GNAI1, may mediate HHT resistance. There are some limitations in this study; although we found that GPRs may mediate HHT, we did not study how to mediate HHT resistance, and we need to further explore how CALCRL and GNAI1 regulate HHT resistance in AML and even chemotherapy resistance in AML. The interrelationship and role of CALCRL and GNAI1 also require further research in the future. We only explored the possible mechanism of HHT resistance in AML at the transcriptional level. Further, we need to use the constructed HHT-resistant cell lines to comprehensively study the HHT resistance mechanism at the metabolic and proteomic levels and to explore whether each omics level there is a cross.


Recent studies suggest that opioids have a role in the progression of HNSCC mediated by mu opioid receptors (MOR), however, the effects of their activation or blockage remains unclear. Expression of MOR-1 was explored in seven HNSCC cell lines using Western blotting (WB). XTT cell proliferation and cell migration assays were performed on four selected cell lines , treated with opiate receptor agonist (morphine), antagonist , alone and combined with cisplatin. All four selected cell lines display an increased cell proliferation and upregulation of MOR-1 when exposed to morphine. Furthermore, morphine promotes cell migration, while naloxone inhibits it. The effects on cell signaling pathways were analyzed using WB, demonstrating morphine activation of AKT and S6, key proteins in the PI3K/AKT/mTOR axis. A significant synergistic cytotoxic effect between cisplatin and naloxone in all cell lines is observed. In vivo studies of nude mice harboring HSC3 tumor treated with naloxone demonstrate a decrease in tumor volume. The synergistic cytotoxic effect between cisplatin and naloxone is observed in the in vivo studies as well. Our findings suggest that opioids may increase HNSCC cell proliferation via the activation of the PI3K/Akt/mTOR signaling pathway. Moreover, MOR blockage may chemo-sensitize HNSCC to cisplatin. Pain management is a major concern in cancer patients. Compared with other malignancies, head and neck squamous cell carcinoma (HNSCC) has the highest prevalence of pain, attributed to the rich innervation and function . Over 50The proposed mechanisms for these epidemiological findings associated opioids with tumor cell proliferation, invasion, angiogenesis, and anti-tumor immune response. Notably, mu opioid receptors (MOR), mediators for opioid effects, were found to be upregulated in several types of cancer cells ,7,8. SpePI3K/AKT/mTOR signaling axis, a frequently activated signaling route in HNSCC , promoteThis study aims to investigate, by means of in vitro and in vivo studies, the effects of MOR activation and blockage on HNSCC, with relation to disease progression, response to therapy, and activation of the PI3K/AKT/mTOR signaling axis. We hypothesize that these effects can be measured in vitro by parameters such as cell proliferation and migration, as well as in vivo by tumor volume. The findings of this study might lay grounds for determining the impact of opioid use in HNSCC patients. t-test. The study was performed at the Center for Translational Research in Head and Neck Cancer, Felsenstein Medical Research Center, Rabin Medical Center. The study investigated the in vitro and in vivo behavior of commercial human HNSCC cell lines when opioid agonists or antagonists are added, with or without conditions that correspond with therapy for HNSCC. Each in vitro assay was repeated at least three times and analyzed statistically by Student’s 2 in air at 37 °C incubator in the appropriate cell culture media that was supplemented with 10% heat-inactivated fetal calf serum, 2 mML-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL).Human HNSCC cell lines utilized in this study included: Cal-33 , FaDu , HSC2 , HSC3 , KYSE 70 , KYSE 180 , and LB771 . All cell lines were maintained in a humidified atmosphere of 5% COFour selected cells were treated with cisplatin , naloxone , and morphine  in a concentration gradient to determine the cytotoxicity and sensitivity doses to each agent. After establishing the concentrations required for treatment, cells were incubated with cisplatin  with or without 10 µM naloxone or 10 nM morphine and compared with controls .For immunoblotting, cellular and tissue homogenates were incubated with lysis buffer run on SDS-PAGE in polyacrylamide gel, transferred onto a nitrocellulose membrane, and developed with specific primary and secondary antibodies. The primary antibodies used in this study were anti MOR-1 and anti-phosphorylated (activated) form of AKT (p-AKT) and S6 (p-S6). Visualization of immunoreactive bands was achieved using fluorescent detection. Quantification of immunoblotting was obtained by densitometric analysis using ImageJ Software.For measuring in vitro cell growth, cells (5000 cells/well) were seeded into 96-well plates to a final volume of 100 μL and were allowed to grow for 24 h with the appropriate treatments of morphine or naloxone with and without combinations of cisplatin versus control. Subsequently, the activated XTT reaction solution was added to each well for additional 2–4 h incubation at 37 °C. Finally, the reaction was measured at a wavelength of 450 nm against a background control by a microplate reader.Measurement of in vitro cell migration was performed in a twenty-four transwell units with 8 μm pore size. Cells pretreated with morphine or naloxone treatments were incubated with 0.2 mL serum-free media in the upper chamber and allowed to migrate to media with 20% serum to the lower chamber through the pores for 18 h. Cells attached to both sides of the membrane were washed twice with PBS. Cells on the upper side of the membrane were removed by cotton swabs, and the migrating cells at the bottom of the membrane were visualized using a microscope, photographed, and counted.6 HSC-3 HNSCC cells  were subcutaneously injected to the flank of the mouse. Tumor volume was measured twice a week with a caliper. The tumor volume was calculated using the formula: 1/2 × length (mm) × [width (mm)]2.Male athymic nude mice were used for this study (8 weeks old and 27–38 g). Animals used in this study were humanely cared for, and all experiments were performed under the aegis of a protocol approved by the Tel Aviv University and were in compliance with the Guide for the Care and Use of Laboratory Animals, National Research Council. A total of 100 µL of 2 × 10In order to test the effect of MOR blockage on tumor’s growth, structure, and surrounding, the studied mice were administered with MOR antagonists in addition to or without an oncological intervention.3, the relevant subgroups received treatment as follows. Thirty animals were randomly divided into 4 groups: (1) control, (2) naloxone (1 mg/kg once a day), (3) cisplatin (3 mg/Kg every two days), (4) cisplatin (3 mg/Kg every two days) and naloxone (1 mg/kg once a day); different drugs were administered by separate injections. The selected doses for naloxone and cisplatin administration were based on data and findings of previous research [3. Tumor change in size were compared between the different study groups.When the tumors reached an average volume of 100 mmresearch ,16,17,18t-test, whereas the analysis of variance (ANOVA) test was used to compare multiple groups. The Bonferroni correction was applied for multiple comparisons. Linear model fit significance was calculated using F-test. Differences in the proportions of observations of two categorical variables were tested with a G-test.SPSS version 23 software  and GraphPad Prism version 6.0  was used to analyze and plot the data. Data were presented as mean +/− standard error (SE). Differences between two groups were analyzed using the independent All seven HNSCC cell lines examined express MOR-1 in Western blotting analysis (WB), as shown in p-value < 0.05) and 24%  in the FaDu and HSC2 cell lines, respectively , while a mild non-significant increase is observed in Cal-33 and FaDu cell lines, by 13% and 4%, respectively . Levels of p-AKT are attenuated with the addition of naloxone in two out of four cell lines and significantly increase in the HSC-2 cell line  in HSC-2 cell lines.Densitometric analysis of WB demonstrates that the addition of either naloxone (10 µM) or morphine (10 nM) alters phosphorylation of AKT and P6 in all cell lines c,d. Levep-value < 0.01) for HSC-3 . Therefore, the isobole of additivity can be viewed as linear [p = 0.02, t-test). Therefore, we performed an isobolographic analysis using an effect level higher than 50% [Cisplatin induces a reduction in cell viability in a dose-dependent manner . HNSCC cor HSC-3 a–d, suggor HSC-3 . ED50% vor HSC-3 a and cisor HSC-3 b were ins linear . The comthan 50% , specialp-value < 0.01, p-value < 0.05, Cell migration assays were performed for all four cell lines . The addp-value < 0.01, We used a mice model injected with HSC-3 tumor cell lines to further establish the proliferative effects MOR agonist and antagonist that were observed in the in vitro assays. Administration of naloxone (1 mg/Kg) decreases tumor proliferation by 37% compared to control. Analyzing the outcome in HSC-3-injected mice treated with a combination of cisplatin and naloxone, it demonstrates a synergistic effect from the addition of naloxone results in increased tumor volume reduction by 64% compared to cisplatin alone , a MOR-selective agonist on HNSCC cell lines. MOR activation by DAMGO increases proliferation, invasion, and migration, compatible with our results, while methylnaltrexone, a DAMGO antagonist combined with DAMGO, has the opposite effect. Interestingly, our results show that naloxone, an opioid antagonist, has an independent negative effect on cell proliferation. Moreover, we were able to demonstrate a synergistic effect between this opioid antagonist and chemotherapy. These are all novel findings in HNSCC research that warrant further investigations.The role of opioids in cancer cell proliferation, invasion, metastasis, and angiogenesis promotion was previously described in non-small-cell lung cancer (NSCLC), breast cancer, nasopharyngeal carcinoma, renal cell carcinoma, and hepatocellular carcinoma ,22,23,24The opioid receptors are divided into three major subgroups, µ, κ, and δ, and are all members of the G-protein-coupled receptor superfamily. MOR (µ) is the main target for opiates ,32. All Another receptor that might take part in the opioid agonists and antagonists effect on cancer cell proliferation, migration, and invasion is the epidermal growth factor receptor  . EGFR isRegarding signaling pathways, our findings suggest that morphine may have a role in the activation of the PI3K/AKT/mTOR axis, a dominant signaling pathway in HNSCC as well as in other cancer types ,18,31,42Another downstream pathway, investigated in both HNSCC and NSCLC ,44, is SThe synergistic effect of opioid antagonists and targeted chemotherapy/anti-cancer therapies was previously discussed in other types of cancer. Singleton et al. investigated the synergistic effect of methylnaltrexone, a selective peripheral μ-type opioid receptor antagonist, with anti VEGF (Avastin) and 5FU in endothelial cells, inhibiting VEGF-induced angiogenesis ,45. ThisRapamycin, an mTOR inhibitor, induces apoptosis and inhibition of cell growth . The synThese findings suggest the importance of opioids in cancer cell resistance to chemotherapy, and the potential of opioid antagonists in sensitizing HNSCC to targeted therapy, such as chemotherapy or even radiation. Therefore, our findings support what was previously suggested by several authors in other cancers, that opioid antagonists could be utilized as adjuvant therapy in advanced stage cancers ,20,24,31One limitation of our study is that naloxone, the non-selective opioid antagonist used, although it has the highest affinity towards MOR, may also target other opioid receptors such as the delta (DOR) and kappa (KOR) receptors in addition to the mu (MOR) receptor. This means that the effects of naloxone on cancer cell progression may not be solely due to its interaction with the MOR receptor. To gain a more specific understanding of the role of the MOR receptor in cancer cell progression, future studies could utilize a selective antagonist specific to the MOR receptor. This would greatly improve the specificity of our findings and provide direct information regarding the specific type of opioid receptors involved in cancer cell progression.Another limitation of our study relates to the in vivo experiments focusing on MOR antagonists alone. The decision to focus the in vivo study on naloxone and its combination with cisplatin was based on the findings of our in vitro experiments. Furthermore, the effects of MOR agonists were already investigated by previous studies .Our findings might provide a biologic explanation to previous epidemiologic studies on the relation between opioid use and cancer. Several studies reported that reduced doses of opioids following surgery for breast, lung, prostate, and colon cancers were associated with improved survival ,27,28,29This preclinical study provides an important insight into the mechanism of MOR activation and inhibition in HNSCC cells. Upregulation and activation of MOR may promote tumor proliferation via the PI3K/Akt/mTOR pathway and blockage of MOR may sensitize HNSCC to chemotherapy. Since opioids are widely used in pain management of HNSCC patients, further understanding of the molecular events triggered by opioid agonists and antagonists is of paramount importance.


Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease.To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins.NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors.We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM. MM is considered a treatable, but generally incurable disease, with a heterogeneous clinical course as one of its hallmarks. Several types of molecular alterations, such as single-nucleotide variants and cytogenetic abnormalities, are responsible for disease initiation, maintenance, and progression . AdvanceKRAS) oncogene is the most commonly mutated gene (36%) in the disease, followed by the neuroblastoma RAS (NRAS) (20%), with frequent co-existence of one or more variants in both KRAS and NRAS cancer-driver genes , mitogen-activated protein kinase (MAPK), and PI3K/AKT pathways, which are known to control proliferation and survival of MM cells , 3. Sequer genes –6. Interer genes . This suStudies in MM have shown that molecular features, including mutations, translocations and copy-number abnormalities, have both prognostic and predictive value and may enable further improvement in patient outcomes if employed to define personalized treatment strategies . InhibitBesides the RAS signaling pathway, the PI3K/AKT/mTOR pathway is activated in a significant proportion of MM patients . InhibitMM mutational status is currently used to guide the use of targeted agents in precision medicine trials, including the MyDrug study (NCT03732703), the CAPTUR study (NCT03297606), and the MATCH screening trial (NCT02465060). Protein expression and phosphorylation levels also impact drug responsiveness, arguing for integrating both genetic and functional analyses in treatment selection strategies –16.Here, we used high-throughput DNA sequencing to characterize the genetic make-up of nine MM cell lines. Based on the spectrum of mutated targets and their druggability, we designed a library of 33 targeted drugs, which was used on the same MM cell lines. Associations between mutational status and drug sensitivity were investigated. Next, we used a dataset of signaling readouts (n=31) available on the same cell lines  to studyOur findings showed that mutant NRAS/KRAS MM cells were highly sensitive to MEK inhibition, as compared to wild-type (WT) MM cells. Furthermore, a systematic correlation analysis of drug sensitivities and signaling protein readouts revealed several drug dependencies. Overall, both mutational status and protein phosphorylation/expression status may help elucidate drug-specific sensitivities in a small panel of MM cell lines, which could next guide future studies to predict biomarkers of drug sensitivity in the context of MM.TP53, and was used as a positive control for testing of the MDM2-P53 interaction inhibitor nutlin-3A. The MM cell lines were cultured in RPMI 1640 medium  supplemented with 2mM L-glutamine, 1% Penicillin-Streptomycin, 1x Sodium Pyruvate (NaPur), and fetal bovine serum (FBS) at 10%  or 15% FBS (U266), or 10% human serum  . The culture medium was supplemented with 2 ng/mL IL6 (ThermoFisher Scientific) for culturing of INA6, KJON, IH1, OH2, URVIN, and VOLIN. The cells were expanded, aliquoted and cryopreserved until experimental assays were performed. See Myeloma cell lines used in this study were U266 , JJN3 1, CAG 1919, INA6 Drugs (n=33) were selected based on mutated targets in the cell lines see . Normalihttps://cellmass.cytobank.org/cytobank/). Raw data were transformed to an arcsinh ratio relative to the signal of an isotype control, which was set to zero.Phospho flow assays were performed on freshly thawed myeloma cells as described previously . Antibodhttps://www.r-project.org/) with the four parameter log-logistic model, LL.4, or the logistic model, L.4, where LL.4 failed to converge. To quantify drug responses, a modified drug sensitivity score (DSS) was calculated for each drug . Welch’s t test  was used to compare two means, as indicated in the respective figure legends. ClustVis  was usedPredictive models for drug response (as defined by the DSS) were constructed using either mutation or phospho flow data as covariates. In each case, a type of multi-response penalized linear regression model known as tree-lasso , 30 was tree.lasso from the R package IPFStructPenalty, available at https://github.com/zhizuio/IPFStructPenalty. In each model, the tuning parameter Λ was set to a value at which the cross-validated MSE becomes essentially flat and the smoothing parameter for Smoothing Proximal Gradient descent (SPG) optimization was set to μ = 10-4. For the mutation-dependent model, the tuning parameter was set to Λ = 25 , and for the protein-dependent model it was set to Λ = 24 . The tree-lasso regression coefficients of each model were then computed using the optimal values of the tuning parameter Λ  as a function of the tuning parameter. The tree-lasso cost function was optimized using the sub-function ameter Λ 1A, B.2 = 1 - RSS/TSS, where the Residual Sum of Squares (RSS) and Total Sum of Squares (TSS) were both defined using all 9 cell lines at once. The R squared value was R2 = 0.39 for the mutation-dependent model, and R2 = 0.58 for the protein-dependent model.The amount of variance explained by each model was also assessed by the R squared value, which was computed for each tuned model as RThe covariates for the protein-dependent model were defined as the standardized phospho flow data, i.e., for each protein the phospho flow arcsinh ratios were centralized at their mean value across samples and normalized by their standard deviation prior to the tree-lasso model calculations. As a consistency check, an overall matching between the signs of the regression coefficients and those of the protein-drug Pearson correlation coefficients was observed (i) exclude known germline variants, and ii) enrich for coding, cancer-associated variants. Specifically, we excluded all variants that overlapped with germline variants found in the 1000 Genomes Project phase 3 , and NHLBI Exome Sequencing Project  (http://genetics.bwh.harvard.edu/pph2/) (Targeted DNA sequencing was performed using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons and untranslated regions (UTRs) of all known kinases and selected cancer‐related genes (n = 612). Paired-end sequencing reads of 100-bp length were aligned to the human reference genome (hg19) with Novoalign (version 2.08.3), followed by filtering and realignment with GATK tools and Picard. Single point mutations were identified using MuTect (version 1.1.4). Variant consequence annotation was performed with ANNOVAR, using RefSeq as the underlying transcript model. Details of the complete variant calling pipeline that was applied on the MM cell lines have been described previously . The meaulation) , 33. In ulation)  that hadulation) . All varu/pph2/) .NRAS/KRAS, BRAF, RAF-1, TP53, PIK3CA, PIK3R3, MTOR, FLT1, FLT3, FLT4, EGFR, and SYK, which are known to be frequently mutated in MM and other related blood malignancies , a tool that provides computational predictions for the functional impact of amino acid changes. We identified 31 amino acid changes with predicted damaging effects which were druggable , includi p.W590C , 41 , taselisib (mean DSS ± SD = 41.82 ± 17.10), pictilisib (mean DSS ± SD = 32.50 ± 13.66), and copanlisib (mean DSS = 19.83 ± 18.65), while treatment with idelalisib and GSK2636771 resulted in markedly low DSS   or RAS gene mutations   Table 2 PIK3CA and NRAS genes were selected as predictors for drug sensitivity to PI3K and MEK inhibitors  proteins correlated with their drug sensitivity.In order to identify relevant signaling proteins whose observed basal expression or phosphorylation levels across different cell lines can explain corresponding DSS values for different drugs, our first approach was a correlation analysis using Pearson correlation coefficients. Correlation coefficients for all possible protein-drug pairs, including the 33 drugs in our library, and the 31 relevant signaling proteins  selectedNotably, using the tree-lasso regression model we found that high levels of phospho-TBK1 inversely correlated with low drug sensitivity to MEK inhibitors, including trametinib, binimetinib, and cobimetinib  compared to the corresponding frequency for NRAS mutations (4/9). Nevertheless, our data are in agreement with results from a meta-analysis case in breast cancer, where the predictive role of PIK3CA mutation status on the clinical efficacy of PI3K inhibitors remains controversial .Trametinib induced the highest sensitivity across the MM cell lines, and sensitivity to both binimetinib and trametinib correlated with the presence of RAS mutations and prediction of MEK inhibitor sensitivity, the cell line IH1 harboring a p.G12V mutation in NRAS had the highest sensitivity to trametinib, followed by the OH2 and KJON cell lines harboring NRAS p.Q61K, and KRAS p.Q61H, respectively. The presence of RAS mutations and RAS pathway activation in lung cancer was previously shown to confer the highest sensitivity to MEK inhibitors among a panel of 500 oncology drugs . This may indicate that patients with PIK3CA mutations may be more addicted to taselisib, and further studies on this are warranted.Of the  reports , 57. Nexmutation . A phaseMEK inhibitors trametinib and binimetinib observed in some MM cell lines, including CAG and U266 with RAF-1 (CRAF) and BRAF mutations  proteins to test how these markers inform on drug sensitivity of targeted treatments. However, the small sample size of the cell lines in this study limited our ability to draw clear conclusions on the predictive value of this approach. The next step will be to improve the prediction significance by including a larger cohort of samples, which will also accommodate for more extensive multiple correlations analyses. While our results suggest that MM cells are sensitive to MEK and PI3K single agents, MM patients are often treated with more than one drug at the same time. Hence it would be of interest to test the efficacy of combined MEK/PI3K inhibition in the future, and also to add conventional agents, including proteasome inhibitors.The datasets presented in this article are not readily available because the data are human identifiable and cannot be openly shared according to Norwegian legislation. Data can only be shared on a collaborative basis based on approval from the Regional Ethics Committee. Requests to access the datasets should be directed to the corresponding author.SS and MG designed the research together with KT. MG and SS designed methodology, performed the experiments, analyzed and interpreted data together with KT and LS. LS performed modeling of prediction analysis. TH, SN, EC, KM, AW, EH, DV, and LM-Z provided the MM cell lines with DNA sequencing data, and performed analyses of raw sequencing data. MG wrote the paper with input from KT and SS. All authors read and commented on draft versions of the manuscript and approved the final version.This work was supported by the Regional Health Authority for South-Eastern Norway (2015031), the Research Council of Norway Centre for Digital Life PINpOINT project (294916) and the Stiftelsen Kristian Gerhard Jebsen (grant 19). This project has also received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement (801133), and the Research Council of Norway under the frame of ERA PerMed (322898). MG was a PhD fellow funded by the UiO: Life Science convergence environment PerCaThe when this work was performed. The exome data were produced by the Genomics Core Facility (oslo.genomics.no) as part of the Norwegian Cancer Genomics Consortium cohort.https://github.com/zhizuio/IPFStructPenalty). We would also like to thank Arnoldo Frigessi, from the Oslo Center for Biostatistics and Epidemiology, University of Oslo, for fruitful suggestions that helped to significantly improve the presentation of our results.We thank Martine Schrøder for excellent technical assistance, Thea Kristin Våtsveen for helping with cell culturing and for providing the FOLE cell line, and Alexandra Gade at the Chemical Biology Screening Platform at Centre for Molecular Medicine Norway (NCMM), University of Oslo, for assistance with drug sensitivity screens. We also thank Zhi Zhao at the Oslo Center for Biostatistics and Epidemiology, University of Oslo, for useful discussions and for suggesting his own R implementation of tree-lasso regression (available online at The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells  and peripheral blood lymphocytes (PBL) for different doses and radiation qualities ( DNA double-strand breaks (DSBs) are the most hazardous DNA lesions induced by ionizing radiation, causing the loss of genetic integrity and formation of chromosome aberrations (CA). Knowledge of the induction of DSBs in healthy and cancer cells and the efficiency of repair mechanisms is crucial in understanding of the biological response to different radiation qualities and success of the cancer radiotherapeutic treatment plans.In our previous studies, we have applied the conventional metaphase assay to characterize the chromosome aberration (CA) yield induced in human peripheral blood lymphocytes (PBL) by protons and heavy ions  and homologous recombination (HR). While the first one, lasting up to several minutes, is predominant in the fast, initial component of the DSBs repair, the second one is more time-demanding and takes many hours  model:In this study, the experimentally determined distributions of CA and PCC breaks as well as the fitted parameters of the LQ model obtained for both PBL and Cal 51 cells after exposure to the therapeutic proton beams and γ-rays will be exploited to discuss advantages and limitations of the applied methods for investigation of cellular repair mechanisms. The LQ model will be also used to determine relative biological effectiveness (RBE) of protons in human lymphocytes and Cal 51, which is especially interesting for proton therapy, for which a constant value of 1.1 is usually assumed. Due to the applied PCC technique, data obtained at two different time points after irradiation can be presented.0 cell cycle stage) were irradiated in 0.5 ml Eppendorf tubes. For PCC analysis, the lymphocytes were isolated by gradient centrifugation and seeded with a density of 0.5 × 106 cell/ml in the in RPMI medium supplemented by 20% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% phytohaemagglutinin (PHA). After 48–60 h of culture, the asynchronously growing lymphocyte population was exposed in suspension to 60Co γ-rays and protons (150 MeV and SOBP) in the culture flasks.The whole blood used for the study was obtained by venipuncture into heparinized vacuum containers. The samples were collected from informed, healthy volunteers, in accordance with local ethical regulations. For the conventional metaphase assay, the whole blood samples  supplemented with 10% fetal calf serum, 2 mM L-glutamine and 1% penicillin/streptomycin . Cells were stored at 37 °C in a 5% CO2 atmosphere. Asynchronously growing Cal 51 cells were irradiated as a monolayer in the culture flasks for both metaphase and PCC analysis. All exposures were done at room temperature, and controls were sham-irradiated.60Co γ-radiation source of the radiation therapy unit ROKUS-M was used. Dose rate at irradiation point was 0.82 Gy/min. Doses ranged between 0.5 and 5 Gy for metaphase assay and 0.5–2 Gy for PCC.Proton exposure was performed at the clinical proton beam facility of the medico-technical complex of Dzhelepov Laboratory of Nuclear Problems, JINR, Dubna, Russia  for metaphase accumulation, and stained with 3% Giemsa.For metaphase analysis, after irradiation Cal 51 cells were allowed to grow in a complete medium at 37 ℃ and 5% CO60Co γ-rays the blood samples (resting PBL at G0 cell cycle stage) were diluted in 4.5 ml of RPMI medium supplemented by 20% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1.5% phytohaemagglutinin (PHA), incubated at 37° C and 5% CO2, fixed at 48 h after PHA stimulation, proceeded by a 3 h colcemid treatment (200 ng/ml) for metaphase accumulation, and stained with 3% Giemsa. Typically, 100–300 metaphases were analyzed for every data point. Chromosomal aberrations were classified according to Savage (1976). All aberrations of the chromosome and chromatid types visible without karyotyping were recorded. The chromosome-type aberrations comprise paired fragments, dicentrics, centric and acentric rings  and translocations visible without karyotyping. The chromatid-type aberrations include the chromatid-type breaks and chromatid-type exchanges. The gaps were not scored as aberrations.After exposure to proton beams and ® CPT™  and cultured in the same RPMI medium 48 h prior to irradiation. Exponentially growing lymphocytes and Cal 51 cells were allowed to repair for various times after irradiation (0–12 h) and then were forced to condense chromatin prematurely by addition of 50 nM calyculin A (Sigma) immediately after irradiation and left for 1 h in 37° C. Then, the cells were treated with 0.075 M KCl for 10–15 min at 37 ℃ and fixed with methanol:glacial acetic acid (3:1). Cells were dropped onto a clean wet slide, airdried and stained with 3% Giemsa. Typically, 100–200 G2-phase cells were analyzed for every data point. The scoring and recording criteria followed those given in IAEA Manual  in human lymphocytes, as well as in Cal 51 cell line, was described by Poisson distribution. This stochastic distribution is used in the case of low-LET radiations for which the mean number of aberrations induced by a single particle transversal is low  and structure of Cal 51 cell line were markedly stable (data not shown). The level of spontaneous aberrations was 0.5–1%.α and β coefficients are given from the least squares regression. The uncertainties of β/α ratios were calculated using the total differential method.The percentage of aberrant cells, total CA yield and CA spectra in Cal 51 cells produced by photons and proton beams are listed in Table 0 PBL resulted in > 98% chromosome-type aberrations with 60–80% of exchange-type aberrations  or after repair completeness t12) The spontaneous level of PCC breaks was slightly higher in Cal 51 and accounted to 0.8–1.4 breaks/cell . The initial breakage (t0) was similar in both cell types.60Co γ-exposure, the mean number of breaks/cell induced in both cell lines are very similar for each examined dose. The difference is, however, significantly higher for both proton beams: 1.5 Gy of 150 MeV and SOBP protons induced in Cal 51 on average 31.7 ± 0.6 and 31.9 ± 0.6 breaks/cell, respectively, and in PBL 18.5 ± 0.4 and 21.8 ± 0.5 breaks/cell, respectively  calculated for 60Co γ-rays do not differ significantly between PBL and Cal 51. In contrast, for both proton beams the residual mean numbers of breaks/cell were significantly higher for Cal 51 as compared to PBL, the maximal registered values amounted 18 breaks/cell for Cal 51 and 8 breaks/cell in PBL (at 1.5 Gy SOBP protons).Twelve hours after irradiation Fig. , the numIn order to assess the repair efficiency of normal vs. cancer cells, the ratio of breaks measured at t0 and t12 was calculated Fig. . For eac0, Fig. 12, Fig. In Fig. Comparison of both proton beam RBE values based on CA yields in metaphases of both cell lines Fig. a revealsIn Fig. β parameter for SOBP protons in human lymphocytes does not allow to assess RBE for more than 4.8 breaks per cell. The RBE values determined at the level of 4 breaks per cell are as follows: 1.21 ± 0.14 vs. 1.7 ± 0.2 (fast protons) and 1.50 ± 0.13 vs. 2.5 ± 0.4 (SOBP protons) for PBL and Cal 51, respectively. They are considerable larger than those obtained for the metaphase and t0 analyzes.Similar tendency is also observed for measurements performed at t12 see Fig. c althougRecently we reported on chromosome damage induced in PBL by different radiation types, as obtained by conventional metaphase assay  model. Whereas the statistical distributions confirm generally stochastic character of damages described by the Poisson statistics , and Cal 51 as asynchronously growing population. Thus, the direct comparison of CA induced in G0-irradiated lymphocytes and in asynchronously growing carcinoma cells is not fully correct due to different sensitivity of G0- and G1-S-G2-irradiated cells. G0 cells have more time for repair, and another repair mechanisms dominates at different cell cycle stages: more error prone NHEJ is predominant in G1/G0 and early S-phase, whereas HR in the S- and G2-phase of the cell cycle  at irradiation time. In addition, there is a large difference in the nucleus sizes and geometry of the studied cells: the spherical compact heterochromatin nuclei of G0 PBL are much smaller than the flat ellipsoidal euchromatin nuclei of Cal 51.In order to determine the repair efficiency, the β/α ratios should be estimated (see Tables In contrast, for PCC analysis, both cell lines were treated under the same conditions as asynchronously growing populations. Equal experimental conditions in the case of PCC allow the direct comparison of chromosome breakage and repair. However, the β/α ratios obtained for the PCC study have very large statistical uncertainties, and, therefore, any direct comparison between the two cell lines is not possible Tables –4. FortuThis finding can be also supported by the analysis of RBE functions Fig. a–c. The In summary, we have conducted—for the first time to our knowledge—an investigation of breast cancer cell vs. PBL chromosomal radiosensitivity following photon and proton exposures using metaphase and PCC analysis. Both cytogenetic assays confirmed the higher efficiency of proton beams in tumor cells compared to PBL: Cal 51 cells have more efficient repair after photon treatment than PBL cells, but were shown to be more sensitive to protons. The lower DNA repair capacity in Cal 51 cell line after proton irradiation may be caused by defects in the DNA repair pathways, particularly homologous recombination. Grosse and co-workers (Grosse et al.


Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from  The use of herbal- and traditional medicines dates back as far as 5000 years ago across numerous civilisations with ancient texts indicating the use of at least 800 plants, with the earliest texts the instructing use of herbal remedies found in China, dating back to 2800 BC. Roughly 121 pharmaceutical products in use today have been formulated based on traditional knowledge developed over the last century ,4,5,6,7.Papaver somniferum. However, morphine and codeine are the most common BIAs isolated from the same plant  specific ELISA kit and spectrophotometry ,43. AutoResults showed that no statistically significant changes were induced as a result of 48 h exposure to PPV . In addiA recent study indicated that PPV exerts antiproliferative activity accompanied by oxidative stress and cell cycle abnormalities in a time- and dose-dependent manner in several tumourigenic cell lines . HoweverThe current study also explored the effects of PPV on pFAK and indicated that PPV does not significantly affect pFAK expression. FAK is a significant contributing signalling molecule to migration and cellular motility and functions as a motility regulator. Therefore, the upregulation of FAK is often associated with the upregulation of cell migration . HoweverIn addition, the present study also indicated that PPV exhibits dose- and cell line-dependent effects on VEGF B and VEGF R1 expression in all three cell lines. Exposure to PPV in MDA-MB-231 cells resulted in a decrease in VEGF B expression and an increase in VEGF R1 expression. Additionally, exposure to PPV in A549 cells resulted in a decrease in VEGF B and VEGF R1 expression. Exposure to PPV in DU145 resulted in a decrease in VEGF B and an increase VEGF R1. VEGF B is one of five VEGF ligands and has been implicated in hypoxia-related angiogenesis and the promotion of vascular cell survival. Hypoxia is a reduction or the impairment of oxygen tension in tissues and is a common abnormality observed in tumour progression ,42. IncrHowever, the exact function of VEGF B is still a topic of debate with some studies suggesting it is a survival molecule while others suggest it is an angiogenic factor ,49,50,51The present study found no statistically significant effects on VEGF R2 expression. VEGF R2 has been shown to mediate growth and survival signalling through the PI3K/Akt pathway and has been implicated in the activation of several downstream proteins, such as FAK. However, previous research has shown that PPV downregulates PI3K/Akt pathway . TherefoTumourigenesis is a complex process with multiple different pathways including the cAMP, PI3K/Akt, FAK, and VEGF pathways . The PDEThe current study has aided in understanding the effects of non-narcotic benzylisoquinoline alkaloid PPV on tumourigenic cell lines. Data obtained in the current study will contribute to the understanding of the effects of PPV on tumourigenic cell lines in addition to their biochemical molecular targets, including VEGF receptors and ligands. Unravelling the signalling transduction induced by PPV will improve cancer researchers’ understanding of phytomedicinal compounds, contributing to the existing knowledge regarding the influence of naturally occurring compounds in tumourigenic cells.2 in a humified atmosphere in 75 cm2 tissue flasks  specific ELISA kit.2 flasks. The cells were incubated at 37 °C and 5% CO2 in a humified atmosphere for 24 h to allow for attachment. Subsequently, the cells were exposed to PPV (10–150 µM) for 48- or 72 h since previous research showed optimal antiproliferative activity in tumourigenic cell lines  (100 µL) was added to each well. The plate was then covered with the sealer sheet, and the samples in the plate were mixed gently before being incubated for 1 h at 37 °C, after which the solution from each well was aspirated. Wash buffer (350 µL) (provided by the supplier) was then added to each well and left to soak for 2 min before aspirating the solution from each well and patting the plate dry against clean absorbent paper. This was repeated three times before HRP conjugate working solution (100 µL) (provided by the supplier) was added to each well and the plate covered with the sealer sheet and incubated for 30 min at 37 °C. The solution was then aspirated from each well before a wash buffer (350 µL) was added to each well and left to soak for 2 min before aspirating the solution from each well and patting the plate dry against clean absorbent paper. This washing procedure was repeated five more times. Subsequently, substrate reagent (90 µL) (provided by the supplier) was added to each well before covering the plate with a new sealer sheet. The plate was then incubated for 15 min at 37 °C. The plate was then wrapped in foil to protect the plate from light. Subsequently, a stop solution (50 µL) was added to each well. After which, the absorbance was measured at 450 nm using an EPOCH Microplate Reader ). MDA-MB-231, A549, and DU145 cells were seeded at a density of 500,000 cells per T25 cmll lines ,7,9,11. ll lines . Cells wp-value < 0.05 was calculated using the Student t-test for statistical significance and was indicated by an asterisk (*) using Jamovi statistical software version 1.6 (The Jamovi project (2021) . Image J software developed by the National Institutes of Health  was used to quantify the scratch assay.This study set out to determine the influence of PPV on cell migration and the expression of VEGF B, VEGF R1, VEGF R2, and FAK in a triple negative breast cancer cell (MDA-MB-231), alveolar adenocarcinoma cells (A549), and prostate adenocarcinoma cells (DU145). Three independent experimental repeats were conducted for all the techniques conducted where the mean and the standard deviation were calculated. Means were illustrated by using bar charts, and standard deviations were shown with errors bars. A This study aided in understanding the influence of PPV on tumourigenic cell lines and is one of the first studies to explore the effects of PPV on cell migration, VEGF, and pFAK expression in MDA-MB-231, A549, and DU145 cells. Results indicated that cellular migration was slowed as a result of increasing concentrations of PPV, with greater effects observed in A549 cells. The effects of PPV on cellular migration, pFAK, and VEGF B, VEGF R1, and VEGF R2 were investigated to determine the influence of PPV on these downstream pathways. No significant effects were observed in expression of pFAK and VEGF R2. However, dose- and cell line-specific alterations of VEGF B and VEGF R1 expression were observed, with more prominent effects observed in A549 cells. Therefore, this study contributes to the understanding of a naturally occurring compound already in clinical use in tumourigenic cell lines and may improve the understanding and use of phytomedicinal compounds in cancer research. Understanding the mechanisms affected by PPV may aid in a better understanding of the known effects of the compound and may give rise to more effective derivatives that may be better alternatives to current compounds in use on cancer cell lines.


In vivo effects were assessed in DSRCT and ERMS models. Concurrent pFAK and pSrc expressions (H-score >50) were observed in DSRCT (67%), ES (6%), ARMS (35%), and ERMS (19%) samples. Defactinib treatment decreased pFAK expression and reduced cell viability in all subtypes. Dasatinib treatment decreased pSrc expression and cell viability in each subtype. Combination treatment led to a complete reduction in pFAK and pSrc in each cell line and showed enhanced cell viability reduction, drug synergy, DNA damage induction, and a trend toward higher apoptosis induction in DSRCT, ERMS, and ARMS but not in ES cells. These promising in vitro results unfortunately do not translate into promising in vivo results as we did not observe a significant effect on tumor volume in vivo, and the combination did not show superior effects compared to dasatinib single-agent treatment.Desmoplastic small round cell tumors (DSRCTs), Ewing sarcoma (ES), and alveolar and embryonal rhabdomyosarcoma (ARMS and ERMS) are malignant sarcomas typically occurring at young age, with a poor prognosis in the metastatic setting. New treatment options are necessary. Src family kinase inhibitor dasatinib single-agent treatment has been investigated in a phase 2 study in patients with advanced sarcomas including ES and RMS but failed as a single agent in these subtypes. Since previous studies demonstrated high FAK and Src activities in RMS and ES tissue and cell lines, and dasatinib treatment was shown to upregulate activated FAK, we hypothesized that FAK-Src combination treatment could potentially be an interesting treatment option for these tumor types. We examined the effects of targeting the FAK-Src complex by addressing (p)FAK and (p)Src expressions in tumor sections of DSRCT ( Sarcomas are rare tumors of the connective tissue with over 70 subtypes. Desmoplastic small round cell tumors (DSRCTs) are very rare (incidence of 0.2–0.5/million persons per year) and highly malignant soft tissue sarcomas (STS) mainly seen in adolescent and young adult (AYA) men , 2. Ewinin vitro and in vivo antitumor effects following single-agent FAK and Src inhibition were reported in ES and RMS [n = 13), ES (n = 68), and RMS  and by determining the antitumor effects of the combined treatment of the FAK inhibitor defactinib and the multikinase (Abl/SFK) inhibitor dasatinib in DSRCT, ES, ARMS, and ERMS cell lines. Effects of defactinib and dasatinib combination treatment were assessed in an in vivo DSRCT and ERMS model.Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (non-RTK) involved in a myriad of cellular processes. FAK acts as both a signaling and a scaffolding protein. The signaling capacities of FAK are dependent on the phosphorylation of several kinase domains  and often involve interaction with Src, a non-RTK known to be of importance in sarcoma . Upon ac and RMS , 15–18.  and RMS . Single- and RMS . In hepa and RMS . Therefon = 13), ES (n = 68), ARMS (n = 21), and ERMS (n = 39) were stained for baseline FAK, Src, phosphorylated FAK Tyr397 (pFAK), and phosphorylated Src Tyr416 (pSrc) expression. pFAK expression could be evaluated in 13/13 DSRCT, 68/68 ES, 20/21 ARMS, and 39/39 ERMS tissues sections. pSrc expression could be evaluated in 12/13 DSRCT, 64/68 ES, 21/21 ARMS, and 37/39 ERMS tissue sections. Patient characteristics are presented in Tissue microarrays (TMAs) containing primary and post-treatment resections, metastatic or locally recurrent tumor tissue of DSRCT (The JN-DSRCT-1 cell line (EWSR1-WT1) was generously provided by Dr. Janet Shipley . The EW8 (Ewing sarcoma EWSR1-FLI1), RD (ERMS), Rh18 (ERMS), Rh30 (ARMS PAX3-FOXO1A), and Rh41  cell lines were generously provided by Dr. Peter Houghton , and the TC32 cell line  was generously provided by Dr. Friederike Meyer-Wentrup .2/95% air at 37°C.The JN-DSRCT-1 was cultured in DMEM : F12 GlutaMAX™ medium . EW8, Rh41, and Rh30 were cultured in RPMI 1640 medium . RD and TC32 were cultured in DMEM (Lonza) and Rh18 in McCoy's 5 A medium (Lonza). All culture media were supplemented with 10% fetal bovine serum (Gibco) and 0.1% gentamycin or 1% penicillin/streptomycin (Lonza). Cells were cultured in a humidified atmosphere of 5%COin vitro experiments. Defactinib and dasatinib were diluted in 0.5% hydroxypropyl methylcellulose (HPMC)/0.2% Tween-80 in sterile water and 9.5% DMSO/5.1% PEG-300/5.1% Tween-80 in sterile water [in vivo experiments, respectively.The FAK inhibitor defactinib and Abl/SFK inhibitor dasatinib were purchased from Selleckchem  and diluted in DMSO for le water  for in vμl), and plates were incubated for 2 h at 37°C. Extinction was measured at 490 nm . IC50 values were calculated with GraphPad Prism version 5.03 software.Cell viability was assessed by the MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Cells were treated with increasing drug concentrations for 72 h , 120 h (JN-DSRCT-1 and Rh41), or 144 h (Rh18), based on the estimated cell division rate. MTS solution  was added  and dose reduction index (DRI) were calculated using CompuSyn software [1 + D2) necessary to reduce x% cell viability (DX)1/2 divided by the dose necessary as a single agent to reduce the same x% cell viability (D1/2). D1 = defactinib, and D2 = dasatinib.Cell viability following simultaneous combination treatment of defactinib and dasatinib was assessed, and drug synergy was calculated as previously described . All drusoftware . CI < 1.50-based single-agent or combination treatment. A detailed description of each antibody can be found in the Supplementary Material.Western blot analysis was performed as previously described . Cells w50-based single-agent and combination treatment was measured using the annexin V/propidium iodide (PI) double-staining apoptosis assay . Detached and adhering cells were collected and subsequently incubated with annexin V-FITC and PI in a CaCl2-enriched culture medium. The number of apoptotic cells (annexin V+) was measured on the CytoFLEX flow cytometer  and calculated using FlowJo version 10.0.The level of apoptotic cells following 24–48 h IC# 2015-0109).All applicable international, national, and institutional guidelines for the care and use of animals have been followed. All procedures performed were in accordance with the ethical standards of the animal ethical committee of Radboud University, Nijmegen, Netherlands  solution, in male (JN-DSRCT-1) and female (RD) SCID mice (6–8 weeks of age). The experiment was started at a tumor size of approximately 0.25–0.4 cm3, and the animals were randomly allocated to the treatment groups (5 mice per group). Defactinib and dasatinib were administered at 50 mg/kg/day for 21 (RD) or 28 (JN-DSRCT-1) days. Tumor growth was monitored by caliper measurements twice weekly and depicted as relative tumor volume (RTV) as previously described [ƴH2AX, (p)FAK, and (p)Src expressions were assessed by IHC in the remaining viable tumor tissue as previously described and in Supplementary Materials [A total of 5 × 10escribed . Tumor vaterials .n = 13), ES (n = 68), ARMS (n = 21), and ERMS (n = 39). The number of samples can vary between the stainings since not all samples were evaluable for each staining. An example per staining intensity is depicted in Supplementary Baseline FAK, Src, pFAK (Tyr397), and pSrc (Tyr416) expressions were assessed in the tumor tissue of primary and post-treatment resections, metastatic or locally recurrent DSRCT (Baseline FAK expression (H-score >0) was seen in 92% of DSRCT (12/13), 85% of ES (52/61), 70% of ARMS (14/20), and 61% (22/36) of ERMS tissues.The pFAK expression (H-score >0) was seen in 77% of DSRCT (10/13), 25% of ES (17/68), 70% of ARMS (14/20), and 26% of ERMS tissues (10/39). High pFAK expression (H-score >100) was seen in 38% of DSRCT (5/13), 3% of ES (2/68), 15% of ARMS (3/20), and 13% of ERMS (5/39) .Baseline Src expression was seen in 100% of DSRCT (13/13), 88% of ES (56/64), 95% of ARMS (19/20), and 86% of ERMS (32/37). pSrc expression (H-score >0) was seen in 83% of DSRCT (10/12), 39% of ES (25/64), 76% of ARMS (16/21), and 35% of ERMS tissues (13/37). High pSrc expression (H-score >100) was seen in 58% of DRSCT (7/12), 8% of ES (5/64), 38% of ARMS (8/21), and 19% of ERMS (7/37) .pos and pSrcpos expressions were seen in 8/12 (67%) DSRCT, 4/62 (6%) ES, 7/20 (35%) ARMS, and 7/36 (19%) ERMS tumor tissues (high and pSrchigh expressions (p=0.045), ES , ARMS , and ERMS tumor tissue  (p=0.006) were significantly associated with a lower overall survival in ARMS patients  . Positivpatients . pFAK, p50 values ranging from 2.5 to 6.4 μM and 0.6 to 7.7 μM, respectively (μM treatment.The baseline expression levels of pFAK (Tyr397) and pSrc (Tyr416) in DSRCT, ES, ARMS, and ERMS cell lines and the effects of defactinib and dasatinib treatment on cell viability were determined. Two cell lines per subtype were used, except for DSRCT, for which only one established cell line was available. All cell lines showed pFAK and pSrc expressions, with low pSrc expression in the Rh18 cell line Figure 2/. Defactiectively . Cell viectively /S2B. DasTo investigate whether targeting the FAK-Src complex could increase the observed therapeutic responses after single-agent treatment, we investigated the effects of simultaneous defactinib and dasatinib combination treatment on (p)FAK and (p)Src expressions in one representative cell line per sarcoma subtype: JN-DSRCT-1, TC32, Rh30, and RD. Western blot results are shown in 50 value in the DSRCT, ERMS, and ARMS cell lines. ES cell lines showed a more additive effect following combination treatment (CI∼1.0)  /S1.γH2AX) expression, indicative of DNA damage, following combination treatment was increased compared to the single-agent treatments, especially in the TC-32, RD, and Rh30 cell lines . Phosphorylated H2AX . In addition, JN-DSRCT-1 cells showed a significant increase in apoptosis following 24-h combination treatment (60%) compared to defactinib (26%) and dasatinib (25%) single-agent treatment. No significant increase in apoptosis could be observed in the RD, Rh30, and TC32 cells following 24 h single-agent defactinib treatment (Twenty-four-hour single-agent defactinib (26%) and dasatinib (25%) treatment significantly increased apoptosis compared to vehicle-treated cells (9%) in JN-DSRCT-1 cells  in the DSRCT and ERMS models   expression was predominantly seen in the RD model and was significantly higher in the dasatinib-treated tumors compared to the vehicle group (p=0.03)  cells [In line with previous findings, single-agent treatment with defactinib or dasatinib led to a dose-dependent decrease in cell viability in the ERMS, ARMS, and ES cell lines , 15, 16.C) cells , 27, 32.We showed a clear decrease in pFAK after defactinib treatment and in pSrc after dasatinib treatment and a clear reduction in both pFAK and pSrc after combination treatment as expected. Total Src expression appeared to increase after dasatinib and combination treatment, which was unexpected but also shown in previous studies –41.In vivo analysis of simultaneous combination treatment did not show an effect on relative tumor volume. For the DSRCT model, this could be due to its very modest growth impeding the visualization of effects on tumor volume. The RD model on the other hand showed an exponential growth, which may have complicated treatment. We did show a reduction in tumor viability post dasatinib and combination treatment. Based on these findings, it can be suggested that, similar to pazopanib efficacy in advanced STS, additional measurements of tumor metabolism using radiological markers could potentially lead to a better representation of dasatinib and defactinib treatment efficacy [in vivo results revealed that the combination treatment does not show superior effects compared to dasatinib single-agent treatment as shown in vitro. The superior in vivo effect of dasatinib could potentially be explained by the anti-angiogenic properties of dasatinib [efficacy . Tumor vefficacy . The in asatinib .However, dasatinib single-agent treatment was already investigated in a phase 2 study in patients with advanced sarcomas and failed as a single agent in most sarcoma subtypes, including Ewing sarcoma and rhabdomyosarcoma . Other cThe effects of FAK-Src targeting in this study may presumably be underestimated because of the two-dimensional culture of cells in the absence of the extracellular matrix.in vivo combination treatment focused on tumor migration and tumor outgrowth at the metastatic site could potentially be of interest for further research [In addition, the role of the FAK-Src complex in tumor cell invasion and cell-cell adhesion suggests that research .in vitro results unfortunately do not translate into promising in vivo results as we did not observe significant effects on tumor volume and did not find superior effects of the combination in vivo compared to dasatinib single-agent treatment. Therefore, these results do not yet encourage further clinical research into the therapeutic potential of this combination treatment in DSRCT, ARMS, and ERMS.In conclusion, concurrent pFAK and pSrc expressions are present in a subset of DSRCT, ES, ARMS, and ERMS tumor tissue. This, in combination with the reduction in cell viability, induction of DNA damage and increased apoptosis following defactinib and dasatinib combination treatment in DSRCT, ERMS, and ARMS cells showed that targeting of the FAK-Src complex could enhance the antitumor effect in these sarcoma subtypes. These promising