The expression of 84 key genes associated with unfolded protein response (UPR) was assessed in four OS cells  by RT2 Profiler PCR Arrays. Based on results, we performed both siRNA and inhibitor assays focusing on IRE1α-XBP1 and PERK pathways. All OS cell lines showed resistance to PERK inhibitors. Furthermore, ATF4 and EIF2A inhibition by siRNA did not affect the survival of OS cell lines. On the other hand, IRE1α-XBP1 inhibition by toyocamycin suppressed OS cell growth (IC50: < 0.075 μM) and cell viability was suppressed in all OS cell lines by silencing XBP1 expression. The expression of XBP1s and XBP1u in OS cell lines and OS surgical samples were confirmed using qPCR. In MG63 and U2OS, toyocamycin decreased the expression level of XBP1s induced by tunicamycin. On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by tunicamycin. However, morphological apoptotic changes and caspase activation were observed in these two cell lines. Inhibition of the IRE1α-XBP1s pathway is expected to be a promising new target for OS.Osteosarcoma (OS) is the most common primary malignant bone tumor. However, the therapeutic results of the advanced cases at the first visit were still extremely poor. Therefore, more effective therapeutic options based on molecular profiling of OS are needed. In this study, we investigated the functions of endoplasmic reticulum (ER) stress activities in OS and elucidated whether ER stress inhibitors could exert antitumor effectsThe online version contains supplementary material available at 10.1007/s12672-021-00453-2. Osteosarcoma (OS) is the most common primary malignant bone tumor; it peaks during childhood/adolescence and after the age of 50 years. The standard protocol for the treatment of patients with OS was established more than 30 years ago , and limited therapeutic progress has been made since then . The theRecently, studies have explored the therapeutic effects of targeting endoplasmic reticulum (ER) stress and unfolded protein response (UPR) using these inhibitors in several tumors , 3. Our The 143B and MG63 cell lines were obtained from the American Type Culture Collection (ATCC). The KHOS and U2OS cell lines were provided by Dr. Melinda Merchant . All cell lines were grown in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin.-∆∆Ct method.The overall expression of 84 key genes associated with the UPR was determined with the RT2 Profiler PCR Arrays , using an RT2 SYBR Green ROX qPCR Master Mix (Qiagen). Arrays were analyzed using mRNA from four OS cell lines. Thermal cycling was performed using ABI-7500Fast  with initial denaturation at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The signal was acquired at 60 °C for each cycle. The cycle threshold (Ct) values obtained in quantification were used to calculate fold changes in mRNA abundance using the 2EIF2A (Assay ID Hs00230684_m1), ATF4 (Assay ID Hs00909569_g1), and GAPDH (Assay ID Hs02758991_g1). Custom qPCR sets were designed for XBP1s, XBP1u, and TATA-box binding protein (TBP) for separate quantifications. These primer and probe sequences were as follows: XBP1s , XBP1u , TBP . Plasmids for standard curves were generated by cloning cDNA fragments of XBP1s, XBP1u, and TBP into the pCRII TOPO vector (Invitrogen). The amounts of XBP1s and XBP1u relative to the housekeeping gene, TBP, were determined using the standard curve method. The amounts of other genes relative to the housekeeping gene, GAPDH, were determined using the comparative Ct method.RNA was extracted using RNeasy Plus Mini kit . All quantitative real-time PCR (qPCR) was performed with TaqMan Fast Advanced Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus Real Time PCR System in accordance with standard protocols. qPCR was performed using predeveloped TaqMan assays  for XBP1 siRNA knockdown was also performed using pre-designed XBP1 siRNA , EIF2A siRNA , and ATF4 siRNA  using Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific). After 72 h, RNA from each cell line was isolated, and its expression was validated using quantitative real-time PCR.For the knockdown expression studies, we used four cell lines . XBP1, EIF2A, and ATF4, 2000 to 5000 OS cells were plated into 96-well plates on day 1. Transfection was performed on the same day with 25–50 nM of the siRNA reagents, as described above. After 72 h, the cell proliferation ability of OS cell lines was assessed using a Cell Counting Kit-8  and a microplate reader .For knockdown proliferation studies with respect to XBP1 pathway inhibitor. GSK2606414  and ISRIB  were used as PERK pathway inhibitors. OS cells were seeded into 96-well plates at a density of 3000–10000 cells/well. The next day, different concentrations of inhibitors or DMSO (as a vehicle control) were added to each well. After 72 h, the inhibitory effect of these inhibitors on the growth of OS cell lines was assessed using a Cell Counting Kit-8  and a microplate reader . The IC50 was calculated using GraphPad Prism software version 9.2.0 .Toyocamycin  was used as an IRE1α-–9 μM: minimum dose) for 12 h, and after DMSO (as a vehicle control) exposure for 18 h.Because two OS cell lines (143B and KHOS) kept unexpectedly high-level expression of XBP1s even after Toyocamycin treatment while showing morphological apoptotic change, apoptotic assays were performed for these cell lines. These cells were plated into 96-well plates at a density of 5000 cells/well, and the next day, tunicamycin (TM) or DMSO (as a vehicle control) were added to each well. After 3 h and 6 h, apoptosis (caspase-3/7 activity) was measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit . Furthermore, apoptosis assay (caspase-3/7 activities) was performed at the following time points: after TM stimulation for 18 h, after TM stimulation for 6 h and subsequent toyocamycin stimulation . ATF4 and EIF2A showed the highest and second highest expressions, respectively, among ER stress genes across all OS cell lines. In addition, XBP1 showed the third highest expression in three cell lines , and the fifth highest expression in KHOS. Furthermore, stimulation with tunicamycin led to enhanced expression of PERK pathway genes, including ATF4, EIF2A, DDIT3, PPP1R15A, and DNAJC3 . Furthermore, HSPA5, an upstream gene of the main UPR pathway, was also upregulated by tunicamycin stimulation. These findings indicated that tunicamycin stimulation enhanced the upstream UPR pathway gene and stimulated the PERK pathway among the three ER stress pathways. Based on these findings, we focused on the PERK and IRE1α pathways for further analysis.Three major signaling pathways in the ER stress response are inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6), all of which are involved in tumorigenesis –5. We peEIF2AK3 of the PERK pathway, while ISRIB is an inhibitor of EIF2A phosphorylation of the PERK pathway. The IC50 of GSK2606414 was shown to be 1.7 µM in ARPE-19  treated with GSK2606414 for 72 h [Recently, two PERK inhibitors have been developed: GSK2606414 and ISRIB. GSK2606414 is an inhibitor of ATF4 and EIF2A was performed via siRNA-mediated knockdown of ATF4 and EIF2A in the four OS cell lines. qPCR confirmed a significant decrease in ATF4 and EIF2A mRNA levels in all OS cell lines . In the cell proliferation assays, by silencing the expression of ATF4, cell viability was not significantly suppressed, except for U2OS . Additionally, silencing EIF2A expression did not significantly suppress cell viability in any of the OS cell lines .To investigate the association between the PERK pathway and viability of OS cells, the inhibition of XBP1 inhibition on OS cell lines. We had previously reported that toyocamycin showed the highest anti-tumor effect on Ewing’s sarcoma cells [Next, we examined the effect of IRE1α-ma cells . In Ewinma cells . ToyocamXBP1 pathway and malignant behaviors in Ewing sarcoma cells [XBP1s and XBP1u expressions were analyzed in the four OS cell lines and eight clinical surgical materials. All OS cell lines showed the mRNA expression of XBP1s and XBP1u .Our previous proteomic analyses demonstrated critical associations between the IRE1α-ma cells . XBP1s aP1u Fig. A. MG63 cXBP1 pathway and the survival of OS cell lines, inhibition of XBP1 by siRNA was performed in four OS cell lines. The knockdown of both XBP1s and XBP1u was confirmed using qPCR. In the cell proliferation assays, we also confirmed that cell viability was suppressed in all OS cell lines due to the silencing of XBP1 expression  induced the expression of XBP1s in a time-dependent manner and suppressed the expression of XBP1u in all OS cell lines . On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by TM. However, morphological apoptotic changes were observed in these two cell lines .Toyocamycin is a selective IRE1α inhibitor that shows antitumor effects and induces apoptosis in cancer cells. Tunicamycin (TM) generally induces ER stress and enables the processing of nes Fig. . We nextTo verify the different effects of toyocamycin after TM stimulation on the two OS cell lines (143B and KHOS), we evaluated apoptotic activity using the caspase-3/7 assay. After TM stimulation for up to 6 h, caspase-3/7 activity was not evident in the OS cells, and morphological apoptotic change was not evident . Toyocamycin treatment at a low dose after TM stimulation elevated caspase-3/7 activity in two OS cell lines (143B and KHOS), and morphological apoptosis changes were evident . On the other hand, in MG63 and U2OS cells, morphological apoptosis changes were not evident  after TM stimulation for 6 h and toyocamycin treatment at a low dose after TM stimulation. These findings were consistent with the morphological changes observed in the OS cell lines following stimulation with toyocamycin.The endoplasmic reticulum (ER) is a major intracellular compartment involved in protein folding and maintenance of cell homeostasis , 8. To m-EIF2A-ATF4 activation contributes to tumor progression and metastasis, and is ultimately associated with drug resistance [Tumor cells escape from ER stress by UPR, making the adjacent environment suitable for tumor survival and tumor growth , 10. IREsistance , whereassistance . In thisXPB1 is a downstream transcriptional factor of the IRE1α pathway and plays an important role in cancer progression. It has been shown that the loss of XBP1 induces a terminal UPR that blocks proliferation and differentiation during mammary gland development [XBP1 strongly inhibited cell proliferation in all OS cell lines, which is consistent with a previous study showing the antitumor effect of XBP1 knockdown in two OS cell lines [XBP1 mRNA splicing [Several studies have revealed an association between the IRE1α pathway and malignant tumors, including apoptosis, cell differentiation, invasion, metastasis, and drug resistance . XPB1 iselopment . In thisll lines . Functioll lines , 16, 17.splicing . These fXBP1 expression during TM/toyocamycin treatment, we found that TM stimulation induced XBP1s expression in all OS cell lines. Furthermore, we confirmed that XBP1s expression was decreased and XBP1u was increased after treatment with toyocamycin in two OS cell lines (U2OS and MG63). However, this switching of XBP1 expression after toyocamycin treatment was not clear in the other two OS cell lines (KHOS and 143B), and XBP1s expression remained at a high level. Interestingly, these two OS cell lines were not examined in a previous study showing anti-tumor effects on XBP1 blocking in OS [XBP1s overexpression and apoptosis, it has been known that activation of JNK (MAPK8) cooperates with p38 and induces apoptosis [MAPK8 expression was not enhanced . Furthermore, it has been reported that sustained activation of XBP1 splicing induces apoptosis in normal tissues [XBP1 splicing induces apoptosis in tumor cells, we observed caspase activation in 143B and KHOS cells, after toyocamycin treatment at a low dose after TM stimulation, and morphological apoptotic changes were evident. Interestingly, TM treatment for 6 h followed by toyocamycin treatment for 12 h induced morphological apoptotic changes in 143B and KHOS with caspase activation, while high levels of XBP1s expression were preserved in these two cells as well as under TM stimulation. The reason for this paradoxical change in OS cells was unclear because high levels of XBP1s were preserved while morphological apoptotic changes occurred.Regarding ng in OS . Notablypoptosis , 13. How tissues , 19. Alt, we investigated the functions and malignant activities of ER stress response in OS, and further elucidated whether inhibitors of ER stress response had antitumor effects. Our findings demonstrated critical associations between ER stress response and malignant behavior in OS. Furthermore, we found that IRE1α inhibitors exerted antitumor activity in OS. As XBP1s expression was consistently observed in OS clinical samples and XBP1s expression was not related to the chemotherapeutic state, inhibition of this pathway is expected to be a new promising target for OS patients.In ConclusionAdditional file 1: Table S1. List of the 84 key genes associated with the UPR (unfolded protein response) on RT2 Profiler PCR Arrays. (XLSX 15 KB)Additional file 2: Table S2. Clinicopathological information of eight OS clinical samples. (XLSX 9 KB)Additional file 3: Figure S1A-1. Gene expression data of the 84 key genes associated with the UPR without any stimulation (143B and MG63). Green: PERK pathway; Yellow: IRE1α pathway; Blue: ATF6 pathway. Figure S1A-2. Gene expression data of the 84 key genes associated with the UPR without any stimulation (KHOS and U2OS). Green: PERK pathway; Yellow: IRE1α pathway; Blue: ATF6 pathway. Figure S1B: ER stress response analysis using the RT2 Profiler PCR Arrays after TM stimulation shows a high expression of DDIT3 (CHOP) followed by EIL2A and ATF4. XBP1s and XBP1u were not included in this array , and were, therefore, separately examined using qPCR. Figure S2: Cell viability by ATF4 knockdown in OS cell lines. ATF4 siRNA knockdown in four OS cell lines  was performed to examine the associations between ATF4 expression and cell viability. A Suppression of ATF4 expression evaluated using qPCR. The expression of ATF4 in each OS cell line is suppressed by all ATF4 siRNAs. B The cell viability in OS cell lines is not affected by ATF4 siRNA knockdown. Figure S3 Cell viability following EIF2A knockdown in OS cell lines. EIF2A siRNA knockdown in four OS cell lines  was performed to examine the association between EIF2A expression and cell viability. A Suppression of EIF2A expression is evaluated using qPCR. The expression of EIF2A in each OS cell line is suppressed by all EIF2A siRNAs. B The cell viability in OS cell lines is not affected by EIF2A siRNA knockdown. Figure S4. Caspase-3/7 assay in OS cell liens. Caspase-3/7 activity is not evident in OS cells after TM stimulation for up to 6 h. However, it is activated by prolonged stimulation (18 h) in KHOS cell. Caspase-3/7 activity in 143B and KHOS cells is elevated by toyocamycin treatment after TM stimulation. Figure S5. Morphological apoptotic changes in OS cell liens. In MG63 and U2OS cells, morphological apoptosis changes are not evident after TM stimulation for up to 6 h and toyocamycin treatment after TM stimulation. On the other hand, in 143B and KHOS cells, morphological apoptosis changes are evident after low dose toyocamycin treatment after TM stimulation. (PPTX 4759 KB)


Epirubicin (EPI) is one of the potent breast cancer (BC) chemotherapeutic agents, but its adverse effects limit its efficacy. Herein, EPI was selected to be loaded in liposomal carrier, which has been targeted by a monoclonal antibody, Herceptin. The preparation process of liposomes was a modified ethanol injection method followed by Herceptin conjugation. The in vitro cell toxicity and cellular uptake of optimum formulation against HER2+ and HER2− cancer cell lines were evaluated. The results showed that the drug loading (DL%) and encapsulation efficiency (EE%) of liposome preparation method yielded 30.62% ± 0.49% and 62.39% ± 8.75%, respectively. The average size of naked liposomes (EPI‐Lipo) and immunoliposomes (EPI‐Lipo‐mAb) was 234 ± 9.86 and 257.26 ± 6.25 nm, with a relatively monodisperse distribution, which was confirmed by SEM micrographs. The release kinetic followed Higuchi model for both naked and immunoliposomes. In vitro cytotoxicity study on three different BC cell lines including BT‐20, MDA‐MB‐453 and MCF‐7 demonstrated higher toxicity of EPI in the Herceptin conjugated form (EPI‐Lipo‐mAb) in comparison with the free EPI and EPI‐Lipo in HER2 overexpressing cell line. In addition, the cellular uptake study showed a higher uptake of immunoliposomes by MCF‐7 cells in comparison with naked liposomes. In conclusion, these data show that the targeted delivery of EPI to breast cancer cells can be achieved by EPI‐Lipo‐mAb in vitro, and this strategy could be used for breast cancer therapy with further studies. Despite different therapeutic strategies, BC has the highest mortality rate after lung cancer . More thAnthracyclines exist in many cytotoxic regimens for the treatment of several malignancies such as sarcoma, BC and lymphoma as they diminish disease relapse and improve survival rate . EpirubiHerein, EPI was encapsulated in the novel immunoliposomes consisting of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE) and cholesterol (Chol), and was targeted for HER2 receptors by Herceptin functionalization. Liposomes were prepared by ethanol injection method followed by extrusion and mAb conjugation. Immunoliposomes were optimized according to physicochemical properties, cellular uptake was assessed and, finally, cytotoxicity was studied for the optimized formulation in three different BC cell lines to investigate the in vitro tumour cell cytotoxicity.22.1Mw 145.5 kDa) was supplied by LuyePharma. Epirubicin was purchased from Sigma‐Aldrich Co.DOPE was purchased from Lipoid AG. Chol, N‐hydroxycarbidiimide (NHS), 3‐‐2,5‐diphenyltetrazolium bromide (MTT), 1‐ethyl‐3‐(3‐dimethyl aminopropyl) carbodiimide (EDC), trypsin‐EDTA, were obtained from Merck. Potassium dihydrogen phosphate, dimethyl sulfoxide (DMSO), analytical grade ethanol and acetone were purchased from Sigma‐Aldrich Co. Trastuzumab  was obtained and stored at 4°C for further analysis  [Amide bond formation is the basis of trastuzumab conjugation. So, for the preparation of functionalized nanoliposomes, the mAb should be activated with EDC and NHS. NHS ester creates reactive acylating agents with nucleophiles such as primary amine and releases its leaving group to form a stable amide bond. The addition of aminated antibody to prepared liposomes was performed under NGermany) .Figure 2.2.3The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. For this aim, 180 μl of Coomassie dye plus reagent was added to an ambient amount of free dispersion of mAb, and after 10 min of incubation, the absorbance was measured at 595 nm using a UV‐Visible instrument (Schimadzu). The results were compared to a standard curve of mAb solution in the concentration range of 15–60 μg/ml [2.3Wa, Ws, and WDOPE,Ch were the weight of the added drug, the analysed weight of the drug in the dialysis medium, and the weight of DOPE and cholesterol added to prepare liposomes, respectively.The percentage of incorporated EPI was calculated indirectly and has been reported as drug loading (DL)  and enca2.42.4.1The samples (naked liposomes and immunoliposomes) were diluted with distilled water, and after drying for 12 h, they were placed on an aluminium pan and coated with a gold thin layer. The scan was conducted at an accelerating voltage of 10 kV by capability of point‐to‐point scanning by XL30 microscope  for scanning electron microscopy (SEM) images .The particle size, poly dispersity index (PDI) and zeta potential of the naked liposomes and immunoliposomes were measured by dynamic light scattering (DLS) . The vesicles were diluted with distilled water (ratio of 1:3) and measurements were conducted at room temperature.2.4.2−1.Fourier transform‐infrared spectroscopy (FT‐IR) spectra of samples were studied by Schimadzu IR‐prestige 21 FTIR spectrometer to find the sample interactions. Therefore, the evaluated samples were naked and immunoliposomes, free mAb, Chol, DOPE and EPI. Each sample tablet was prepared by mixing 2 mg of sample with 10 mg KBr followed by compression. Transition mode was applied for sample evaluation and the spectral region was 450–4000 cm2.4.3Thermal characteristics of the samples including naked and functionalized liposomes, free mAb, cholesterol, DOPE, EPI and physical mixture were characterized by differential scanning calorimeter  at the temperature range of 20–375°C and heating rate of 10°C/min. Reference was An empty aluminium pan was used as a reference, and dry nitrogen was used as effluent gas during the experiment .2.5Franz diffusion cell was selected to evaluate the in vitro drug release profile of naked liposomes and immunoliposomes . The relR2) of the models given in Table Different kinetic models were evaluated to find the best model fitted to in vitro release data. The analysis was done according to correlation coefficient R of the m2.64 cells/well followed by 24‐h incubation for sufficient adhesion of cells. The different formulations containing different concentrations of free EPI, phospholipid, naked EPI‐loaded liposomes and EPI‐loaded immunoliposomes were added to wells and incubated for 24 h; each sample was repeated six times. Then the samples were replaced with 200 μl fresh Dolbecco's modified eagle's medium followed by 20‐min incubation. The medium was removed, then 200 μl RPMI medium and 20 μl of MTT dye solution (10% in phosphate buffer pH = 7.4) were added to each well and incubated for 2.5 h (at 37°C and 5% CO2). Finally, the medium was removed, and formazan crystals were solubilized by 200 μl of DMSO; the reacted dye was dissolved and the absorbance was measured at 570 nm by a microplate reader .The cytotoxicity assay was studied in three different cell lines containing MCF‐7, MDA‐MB‐453, and BT‐20; the method was based on MTT assay, which shows the viable cells. For this purpose, the cells were cultured in 96‐well plates ; the cell density was 1.5 × 10A)test/(A)control × 100, where (A)test is the absorbance of each test sample and (A)control is the absorbance of control sample. The culture medium without cells was considered as control, and the IC50 of samples (the concentration which inhibits the growth of 50% of cells) was calculated by Sigma plot software [The cell viability (%) was calculated by . According to the results of Table One of the important factors for drug release is particle size by the way that smaller particles lead to higher surface area so the drug exposure for dissolution will increase and higher amount of drug will be dissolved in the medium . Figure 3.550 of each formulation was evaluated; the results are depicted in Figure MTT assay was selected for in vitro cytotoxicity investigation of formulations. MCF‐7, MDA‐MB‐453 and BT‐20 cell lines were exposed to different concentration of formulations. The viable cells were counted and ICIt can be concluded that the phospholipid (DOPE) did not show any cytotoxicity effect on none of the cell lines in the treated concentrations.50 of EPI in three formulations , as tabulated in Table 50 of EPI in MCF‐7 cells is higher for immunoliposomes compared to naked liposomes. As this cell line is HER2−, the immunoliposomes could not attach to cell surface as much as to cause lower IC50 and so higher potency. On the contrary, the IC50 of EPI in BT‐20 cell line is much lower for immunoliposomes than naked liposomes. The IC50 of EPI in MDA‐MB‐453 is much lower even than the difference of BT‐20 for immunoliposomes in comparison with naked liposomes. The reason can be the overexpression of HER2 for MDA‐MB‐453 cells that leads to higher efficacy of loaded EPI in mAb coupled liposomes in this cell line.Evaluation of the average IC50, which can be related to the ER+ and PR+ characteristic of MCF‐7 cell line. The overexpression of ER and PR on the cell surface can lead to higher affinity of immunoliposomes [50 has been reduced to about a third of the free drug.Comparison of the effect of immunoliposomes in three cell lines shows that EPI is more effective in MCF‐7 and BT‐20 than MDA‐MB‐453 and confirms by lower ICiposomes . The resCellular uptake of naked and immunoliposomes to MCF‐7 cells was studied by fluorescence microscopy. There was no need for addition of a fluorescent dye, due to EPI fluorescence property. The amount of cellular uptake of EPI increased along with higher exposure time of formulation to cells but the fluorescence intensity was high enough to be detected even after four hrs of exposure. As shown in Figure 4In the described experiments, EPI‐loaded liposomes and HER2‐targeted liposomes were prepared, and after the physicochemical characteristic evaluations, these formulations were assessed by in vitro cytotoxicity and cellular uptake study. As the liposomes were targeted for HER2 receptors by Herceptin, the drug efficacy was higher than free EPI in HER2 overexpressing cells. Therefore, this kind of EPI‐loaded drug delivery system can be a potent carrier for HER2+ BCs.Higher efficacy of immunoliposomes can be due to cytostasis/cytolysis which is caused by the carrier binding to HER2 receptors as was elucidated by Vaidya et al. They prepared dual functionalized immunoliposomes by trastuzumab (anti‐HER2) and OKT‐3 (anti‐CD3) antibodies to study the doxorubicin efficacy. The results showed high cytotoxicity for the functionalized formulations, especially when effector cells exist. They demonstrated that the mechanism can be cytostasis/cytolysis in addition to slow drug release from functionalized liposomes .The mechanism of EPI uptake in HER2+ cell lines can be through receptor‐mediated endocytosis as reported by Amin et al. in the study of mAb conjugated liposomes containing idarubicin in HER2+ BC cell lines. It has been observed that trastuzumab conjugated liposomes cause more efficient attachment of drug delivery to overexpressed HER2 cell lines and so higher cellular uptake will happen .Yan Wu et al. studied the EPI pharmacokinetic parameters in animal model after encapsulating in thermosensitive liposomes. The results showed that liposomes can increase the circulation time of EPI, and higher drug exposure will lead to higher antitumour activity .Rodallec et al. studied the efficacy and tumour cell uptake of targeted liposomes containing Docetaxel for HER2+ BC model in vitro and in vivo. They found that immunoliposomes caused higher efficacy than free drug and antibody. They related this effect to better drug delivery because despite the higher tumour internalization, the localization of immunoliposomes was not higher for immunoliposomes, even in HER2+ cells. The results show that the EPR effect is as potent as antibody conjugation in drug targeting .5Further in vivo analysis should be conducted to confirm the results.In the present study, Herceptin conjugated EPI‐loaded liposomes were prepared and the physicochemical properties were evaluated. EPI was successfully encapsulated which was confirmed by high EE%. The negative surface charge of particles leads to high stability of formulation and a good choice for intravenous injection. The Higuchi release kinetic shows that drug will be released continuously during 24 h. Different formulations based on various aqueous‐to‐organic phase ratio and components were prepared. The optimum formulation according to size and size distribution was selected and in vitro cellular uptake and cytotoxicity studies showed that Herceptin conjugated liposomes have higher cell toxicity in HER2+ cell lines compared to naked liposomes. Therefore, Herceptin decorated liposomes can improve EPI efficacy in HER2+ BCs. The authors declare no conflict of interest.


Salmonella enterica serovars are frequently associated with disease outbreaks in low-moisture foods (LMF) due to their ability to adapt efficiently to desiccation stress. These serovars are often persistent during food processing. Disruption of these resistance responses was accomplished previously using the membrane-active lipopeptide, paenibacterin. This study was initiated to determine how desiccation resistance mechanisms are overcome when Salmonella Tennessee, a known resistant serovar, is treated with the membrane-active food additives carvacrol and thymol. Knowing that the minimum inhibitory concentrations (MICs) of carvacrol and thymol against Salmonella Tennessee are 200 and 100 µg/mL, the concentrations tested were 100–400 and 50–200 µg/mL, respectively. Results show that desiccation-adapted Salmonella Tennessee, prepared by air drying at 40% relative humidity and 22–25 °C for 24 h, was not inactivated when exposed for 4.0 h to less than 2xMIC of the two additives. Additionally, treatment of desiccation-adapted Salmonella Tennessee for 120 min with carvacrol and thymol at the MIC-level sensitized the cells (1.4–1.5 log CFU/mL reduction) to further desiccation stress. Treating desiccation-adapted Salmonella Tennessee with carvacrol and thymol induced leakage of intracellular potassium ions, reduced the biosynthesis of the osmoprotectant trehalose, reduced respiratory activity, decreased ATP production, and caused leakage of intracellular proteins and nucleic acids. Carvacrol, at 200–400 µg/mL, significantly downregulated the transcription of desiccation-related genes  as determined by the reverse-transcription quantitative PCR. The current study revealed some of the mechanisms by which carvacrol and thymol combat desiccation-resistant Salmonella Tennessee, raising the feasibility of using these additives to control desiccation-adapted S. enterica in LMF.Some  Salmonella enterica serovars are highly adaptable to dry conditions, and this adaptation allows the pathogen to survive for extended periods in low-moisture foods (LMF), and cause frequent disease outbreaks [Salmonella Tennessee, Agona, Bredeney, Mbandaka, Montevideo, and Enteritidis, which caused salmonellosis linked to consumption of products such as peanut butter, breakfast cereal, tahini, and almonds. Serovars having this phenotype invoke several mechanisms upon exposure to desiccation conditions; these mechanisms include accumulation of potassium ions, biosynthesis of the osmoprotectant trehalose, increased fatty acid metabolism, and production of cellulose and fimbriae [Salmonella serovars to dry conditions was found to involve collateral adaptive responses, such as the ability to form biofilm and induction of viable but nonculturable state [Some nontyphoidal utbreaks . Dry-tolfimbriae . Adaptable state ,4. Collele state .Salmonella serovars on dry products such pistachios, nuts, or in-shell pecans; these methods include heat-spray, irradiation, and hot water treatment [S. enterica serovars; the lipopeptide rendered the pathogen sensitive to dry conditions [Salmonella, in similar fashion to paenibacterin, were investigated [Salmonella serovars.Researchers explored the suitability of different methods to combat desiccation-resistant reatment ,7,8. In nditions . Howevernditions . Alternastigated . Of thesSalmonella, in view of their activity against bacterial cytoplasmic membranes. In a previous study, the two additives sensitized desiccation-adapted Salmonella to heating at 55 °C for 15 min [Salmonella effect of carvacrol was apparent during spray- or freeze-drying of liquid milk, and this effect was noticeable during storage of the dried milk at room temperature [Salmonella. Unveiling such mechanisms will help in confirming the ability of the two compounds to overcome desiccation adaptation in Salmonella. This knowledge could facilitate developing membrane-active agents that de-adapt desiccation-resistant Salmonella, with the goal of improving the safety of LMF. Therefore, the present study was conducted to investigate the mode of action of membrane-active carvacrol and thymol against the desiccation resistance mechanisms employed by S. enterica.Carvacrol and thymol are monoterpene phenolic compounds that have been studied extensively for their antimicrobial, antitumor, and anti-inflammatory activities ,14,15,16r 15 min . Furtherperature . These fCarvacrol and thymol  were dissolved in sterile water containing dimethyl sulfoxide  at 5% level. Control experiments were completed using sterile aqueous DMSO (5%).Salmonella enterica serovar Tennessee E2007000304, a known desiccation-resistant strain [Salmonella Tennessee demonstrated stronger resistance to desiccation stress when compared to Salmonella Typhimurium [t strain , was obtt strain , and to himurium , and a gSalmonella Tennessee cells were prepared as described previously [S. enterica cells were grown overnight in Tryptic Soy broth  at 37 °C to a cell density of 109 CFU/mL, as determined by plating on Tryptic Soy agar . Cells of the incubated cultures were harvested by centrifugation at 4 °C and 8000× g for 5 min, and finally resuspended in fresh TSB. Aliquots  from cell suspensions were placed in empty plastic Petri dishes  with a final population of 109 CFU per plate, and air dried in a biosafety cabinet for 24 h at 22–25 °C under ca. 40% relative humidity; survivors are considered desiccation-adapted cells. The adapted cells were collected by dispensing 1 mL of the resuspension medium onto the dried cells. The resuspension medium was saline (0.85% NaCl), TSB, or M9 minimal medium supplemented with glucose, depending on the medium required in a given experiment, to obtain desiccation-adapted cell suspension  containing 5 mM glucose . Portions of these cell suspensions (90 µL) were added to wells of a black 96-well microplate (Corning) followed by the addition of the PBFI probe at a final concentration of 2 µM. Then, 10 µL of carvacrol or thymol was added to each well to achieve final concentrations of 80 to 800 µg/mL of each compound, and polymyxin (10 µg/mL) served as a positive control. Concentrations of released potassium ions were measured as changes in fluorescence using a microplate reader  at excitation and emission wavelengths of 346 and 505 nm, respectively. Fluorescence measurements were captured every 100 s and normalized by subtracting the background noise. The potassium-sensitive benzofuran isophthalate probe  was used to determine the release of potassium ions from desiccation-adapted eviously ,11. BrieSalmonella Tennessee desiccation-adapted cells in saline  at 280 and 260 nm, respectively, and results are expressed as µg/mL. n saline  were treSalmonella Tennessee desiccation-adapted cells in TSB , an electron acceptor that indicates dehydrogenase activity, was used as described previously [To measure respiratory activity in eviously . BrieflySalmonella Tennessee cells in saline  from additive-treated and untreated cells were distributed into 96-well, clear-bottom, black microtiter plates (Corning), and the ATP levels were determined using an assay kit  according to the manufacturer’s instructions. The ATP levels were determined by measurement of changes in luminescence using a microplate reader (PerkinElmer). The decrease in ATP levels in treated cells was calculated in reference to the untreated control.Desiccation-adapted n saline  were treSalmonella Tennessee desiccation-adapted cells in TSB  and the reference gene, gapA, which encodes glyceraldehyde-3-phosphate dehydrogenase [Salmonella Tennessee cells and compared to the untreated control cells using the 2−ΔΔCt method [s in TSB  were treus study  using rerogenase , were met method . Primer p value of <0.05.All experiments were performed in triplicates and each experiment was repeated twice, independently. Each data point was represented as mean ± SD of the two independent repeats. The statistical analysis was performed using a statistical software . Analysis of variance (ANOVA) with Tukey pairwise comparisons were used to determine significant differences between treatment groups or comparing pairs of treatment. Statistical significance was considered at Salmonella Tennessee was performed as reported in previous studies [The preparation of desiccation-adapted dry cells of  studies ,11, descSalmonella Tennessee E2007000304 were determined in a previous study to be 200 and 100 µg/mL, respectively [Salmonella Tennessee, suspended in saline  than those of the untreated control , when compared to the untreated control (p > 0.05) Salmonella populations when compared to the untreated control during the 4.0-h exposure period. These findings suggest that 2× MIC, at least, of carvacrol or thymol is required to inactivate desiccation-adapted Salmonella Tennessee. The minimum inhibitory concentrations (MICs) of the food additives carvacrol and thymol against non-desiccation-adapted cell suspensions of ectively . Based on saline , was tre control A. Simila control B. HoweveSalmonella Tennessee cells were treated with carvacrol or thymol at 1× MIC level for 120 min and then subjected to a second round of desiccation, the populations behaved differently, compared to the desiccation-adapted additive-untreated control , respectively, compared to the untreated populations, which decreased only by 0.3 log CFU/mL after the second cycle of desiccation. This indicates ability of carvacrol or thymol to sensitize Salmonella Tennessee to desiccation stress. When desiccation-adapted  control . Results+) is required for S. enterica to adapt to desiccation conditions [+ leakage could counteract desiccation resistance. Carvacrol or thymol at levels ranging from sublethal to lethal concentrations caused rapid leakage (p < 0.05) of potassium ions (after 100 s of exposure to the additive) from desiccation-adapted Salmonella Tennessee cells, compared to the untreated desiccation-adapted control (p < 0.05) when the concentration of carvacrol or thymol was >100 or >80 µg/mL, respectively.Accumulation of potassium ions  as shown in p < 0.05; p > 0.05) when the two additives were compared for their ability to induce leakage of the intracellular nucleic acids.The ability of carvacrol and thymol to induce potassium leakage prompted us to investigate the capability of these additives to invoke the leakage of intracellular macromolecules, such as proteins and nucleic acids. Results showed that carvacrol caused leakage of intracellular proteins in a concentration-dependent manner . Thymol caused similar effect at all tested concentrations but the decrease in trehalose levels was significant only when 100 and 200 µg/mL of the compound were tested (p < 0.05). The capability of carvacrol and thymol to inhibit trehalose biosynthesis in desiccation-adapted iccation . Figure e tested B. GeneraSalmonella Tennessee desiccation-adapted cells, suspended in M9 broth medium that was supplemented with glucose  among the various concentrations of each food additive or even between the inhibitory effects of the two additives.  glucose , were me glucose . SalmoneSalmonella cells, we explored their ability to affect the production of ATP, which is produced through cytoplasmic membrane- and respiration-related processes [Salmonella to adapt to desiccation [Salmonella cells suspended in saline (p < 0.05).Because carvacrol and thymol are membrane-active compounds, and both decreased the respiratory activity in rocesses . Producticcation ,19. Desin saline  were expn saline A, with tn saline B, but thproV, kdpA, otsB, and STM1494) are necessary for desiccation adaptation and are mainly involved in proline accumulation, K+ transport, trehalose synthesis, and osmoprotection, respectively [proV and STM1494, whereas a concentration of 400 µg/mL  was needed to significantly downregulate (>2-fold decrease) the expression of proV, STM1494, and kdpA (The genes investigated in this study (ectively ,26. Exprand kdpA . In contS. enterica in LMF, the bacterium continues to cause frequent outbreaks linked to these foods [S. enterica in LMF, such as almonds, pecans, pistachios, and walnuts, among others [Salmonella expresses thermal resistance [Despite the efforts of processors to control se foods ,28. The se foods  and causg others . Howeversistance , which csistance ,32. Salmonella and to sensitize the pathogen to drying and mild thermal processing. One of these strategies is utilizing cytoplasmic membrane-active agents to de-adapt desiccation-resistant Salmonella into a phenotype that is sensitive to drying and thermal processing. The incorporation of membrane-active agents, such as carvacrol and thymol, in liquid foods before drying could be an effective approach to sensitize Salmonella to the mild food processing [Several antimicrobials, including oxidizing agents, such as peroxyacetic acid, hydrogen peroxide, and propylene oxide, were used for nonthermal decontamination of LMF. Use of these agents may cause oxidation of food components, which could alter product quality . Althougocessing .Salmonella Tennessee and Eimsbuettel in response to desiccation [Salmonella Tennessee E2007000304. Carvacrol and thymol are membrane-active food additives that were found capable of sensitizing desiccation-adapted S. enterica to mild heat treatment, and spray- or freeze-drying [Salmonella Tennessee showed tolerance to 1× MIC carvacrol or thymol; these additives were needed at 2× MIC to cause significant reduction in Salmonella populations (S. enterica serovars in response to treatment with the membrane-active paenibacterin or other chemical antimicrobials [In a previous study, the microbial-produced lipopeptide paenibacterin disrupted the adaptation mechanisms employed by iccation . In the e-drying . In the ulations . Similarcrobials ,35.Salmonella Tennessee to accumulate K+, synthesize trehalose and ATP, retain respiratory activity, and maintain the intracellular contents, is indispensable for desiccation adaptation [+ in a concentration-dependent manner at levels ranging from sublethal to lethal concentrations (+ may be attributed to the increased cytoplasmic membrane permeability, which can be facilitated by carvacrol or thymol [Salmonella’s early responses to hyperosmotic shock or desiccation conditions [+ leakage results, the additives also caused the release of intracellular proteins and nucleic acid (The ability of aptation . Thus, ttrations ; this efr thymol . Potassinditions . Similareic acid ; this coSalmonella, since the sugar protects the bacterium against cellular collapse during water loss by forming hydrogen bonding with the membrane phospholipids [Salmonella Tennessee (Salmonella to desiccation. Evidence for the role of trehalose in desiccation resistance was demonstrated in the finding that desiccation-sensitive Salmonella Typhimurium LT2 accumulates inadequate amounts of trehalose [The biosynthesis of the trehalose is important for desiccation adaptation in holipids . Carvacrennessee . This efrehalose .Salmonella Tennessee by more than 70% (Based on the results of the current study, carvacrol and thymol reduced the respiratory activity in desiccated than 70% . A plausthan 70% , may be proV, STM1494, and kdpA (Salmonella. In contrast, the anti-desiccation effect of thymol could not be attributed to ability of this compound to alter the expression of desiccation-related genes (Salmonella Enteritidis with carvacrol decreased expression of a panel of cellular proteins that include few transporter proteins [Origanum vulgare, which include carvacrol and thymol, were capable of crossing the cytoplasmic membrane of Salmonella Enteritidis and acting directly on the bacterial DNA [It was observed that carvacrol at 200–400 µg/mL downregulated the expression of and kdpA ; these ged genes . The dirSalmonella is presented schematically in Salmonella Tennessee permeabilizes the cells, causing leakage of the intracellular small ions, such as K+, whose intracellular accumulation is needed as a primary event in resistance to desiccation. With the increase in cell-membrane permeability, efflux of other intracellular molecules, such as DNA and proteins, starts. Compromising the cytoplasmic membrane permeability could also alter the proton motive force and reduce respiration. Additionally, the phenol group of carvacrol or thymol may interact with ATPase, causing its inactivation. Several other direct or indirect cascades of events could affect the transcription of genes necessary for desiccation adaption; however, this inference needs future research.The proposed mechanism of action of carvacrol or thymol against desiccation-resistant S. enterica in low–moisture foods. The ability of carvacrol and thymol to act on the cytoplasmic membrane of Salmonella Tennessee impaired the desiccation resistance in this pathogen via altering the membrane permeability and inducing the leakage of intracellular osmoprotectants. Considering that Salmonella Tennessee has been linked to salmonellosis outbreaks in several low-moisture foods, the current findings imply that carvacrol, thymol, or similar membrane-active agents can protect these foods against this pathogen and potentially against other desiccation-resistant serovars. However, further research is needed to confirm the efficacy of these additives under different desiccation conditions and in various low-moisture foods.This study provided molecular evidence that the additives carvacrol and thymol are feasibly usable in mitigating desiccation-resistant


Transcriptional and phosphoproteomic profiling reveals that the combination converges to downregulate the MAPK-ERK signaling pathway, a result not predicted by single-agent analysis alone. Correspondingly, the addition of a MEK inhibitor synergizes with either dasatinib or cabozantinib to increase its efficacy. This study, by using approved, clinically relevant drugs, provides the rationale for the design of effective combination treatments in NCCRCC that can be rapidly translated to the clinic.The lack of effective treatment options for advanced non-clear cell renal cell carcinoma (NCCRCC) is a critical unmet clinical need. Applying a high-throughput drug screen to multiple human kidney cancer cells, we identify the combination of the VEGFR-MET inhibitor cabozantinib and the SRC inhibitor dasatinib acts synergistically in cells to markedly reduce cell viability. Importantly, the combination is well tolerated and causes tumor regression   Non-clear cell renal cell carcinomas have poor prognoses and limited treatment optionsDrug screens identify cabozantinib and dasatinib combination as synergistic in NCCRCCCombining cabozantinib with dasatinib induces regression of NCCRCC tumors in miceIntegrated multi-omic analyses links therapeutic efficacy to suppression of MAPK activity One important and challenging question in kidney cancer is how do we treat treat non-clear cell kidney cancers? Lue et al. tackle this question by performing drug screens in non-clear kidney cancer cells and mouse models and identify the cabozantinib-dasatinib combination as a promising clinically relevant therapeutic option. VHL tumor suppressor gene. Consequently, clear cell RCC (CCRCC) is responsive to drugs that directly or indirectly inhibit angiogenesis.VHL gene, and are broadly classified as non-clear cell RCC (NCCRCC) or variant histology RCC. NCCRCC show less responsiveness to antiangiogenics and have no effective treatment options.Renal cell carcinoma (RCC) accounts for >400,000 new cancer cases and 175,000 deaths per year worldwide.,We and others identified SRC, an intracytoplasmic tyrosine kinase, as a novel therapeutic target in RCC.VHL intact (wild type [WT]) and null, resulting in single-agent and dasatinib combination responses. These studies revealed cabozantinib as a promising drug combination with dasatinib. Cabozantinib inhibits several tyrosine kinases that are biologically relevant in RCC, including VEGFRs, MET, and AXL,,in vivo testing in representative VHL intact RCC models. Strikingly, while single-agent dasatinib and cabozantinib recapitulated the clinical responses to restrain tumor growth, the combination caused marked tumor regression. Comprehensive integration of transcriptome and phosphoproteomic analysis of the combination therapy revealed rewiring of the kinome, with inhibition of mitogen-activated protein kinase (MAPK) signaling required for cytotoxic synergy. Our studies have identified promising drug combinations that transcend lineage and genetic landscape to induce cytotoxicity, suggesting broad utility across different kidney cancer subgroups.Here, we took a systematic approach toward identifying co-targeting strategies for dasatinib by performing a combination drug screen using a chemogenomic library of mechanistically annotated, clinically relevant approved and investigational drugs that inhibit pathways involved in growth, metabolism, and apoptosis in human RCC cell lines that were VHL WT: ACHN, SN12C, TK10, UO31, CAKI-1, and VHL Null: 786-0, 769-P, A498; 50 (the drug concentration necessary to inhibit growth by 50% compared to the untreated condition), the minimum viability, and the AUC (area under the dose-response curve) between drug alone and drug+dasatinib. Subsequently, we determined the leads for secondary screening with the following rationale: a drug should have an effect in multiple cell lines, but an effect across all cell lines is not necessary. Therefore, for a specific measurement such as GI50 or AUC, we considered drugs that were in the top 50% of the measurement for all drugs in more than half of the cell lines  and drugs that were in the top 25% of the measurement for all drugs in >1 cell line . We applied these criteria to 3 calculations  and noted the drugs that passed the criteria for multiple measurements , generating a further 6,720 dose-response signatures. The growth inhibition values from this secondary screen across different drug doses and combinations were analyzed for synergy using the Bliss independence model.25  of dasatinib and observed a leftward shift with corresponding decreases in cell viability  to the 5 human VHL WT RCCs in our panel, cabozantinib remained as one of the top combination partners for dasatinib . In agreement with the CellTiter-Glo data, combined treatment with dasatinib and cabozantinib increased apoptosis and decreased proliferation in RCC cells in the panel compared to single-agent treatment B and S4CThese experiments suggested several potential therapeutic partners for dasatinib. We selected cabozantinib for further evaluation because it has been approved for both first- and second-line treatment in CCRCC,in vivo in 2 xenograft tumor models. While dasatinib and cabozantinib exhibited antitumor effects on ACHN and CAKI-1 human RCC xenografts, the combination potently inhibited tumor growth and caused tumor regression  of ACHN human NCCRCC cells treated with dasatinib, cabozantinib, and the combination for 24 h. Based on prior work and reports, robust transcriptional signatures emerge ∼24 h after treatment with drugs that affect cell viability such as cell-cycle- and cell death-related signals. These can be captured by RNA-seq profiling, in addition to more specific treatment-related changes, and inform on mechanisms of action.To home in on the genes and phosphopeptides changes specifically induced by the combination treatment, we developed a statistical and network approach to compare across these treatments B. We ana,,Within the transcriptomic data, 324 genes were significantly better explained after the inclusion of an interaction term, indicating that these genes are preferentially activated after combination treatment (likelihood ratio test: q < 0.01). To identify molecular nodes that may mediate the response to the combination treatment, we performed a master regulator (MR) analysis to identify transcription factors or signaling molecules that serve as signal integration hubs. Specifically, we used the MARINa  and VIPER  algorithms and a gene regulatory network derived from The Cancer Genome Atlas cervical kidney renal papillary cell carcinoma (TCGA KIRP) cohort to infer MR activity from genes ranked by the effect of interaction of expressionWe used a similar analytical approach to analyze the proteomic data and identified 959 proteins that could be explained by an interaction between dasatinib and cabozantinib. Kinase activity driving the effect of drug interaction was inferred from the phosphoproteomic data using KSEA (q < 0.01), and demonstrated the concordant downregulation of cell-cycle-associated kinases CDK1/CCD2. Notably, KSEA also suggested the downregulation of multiple nodes of the MAPK pathway  with the dasatinib-cabozantinib combinationTo integrate across transcriptional and phosphoproteomic datasets, we leveraged the TieDIE algorithm with the Multinet interaction network to combine inferred transcriptional MRs, inferred kinases, and kinases into an integrated network associated with response to the combination drug treatment.To better define the effect of MAPK pathway inhibition, we examined 9 drugs with known MEK inhibitor activity  in combination with dasatinib against the 5 VHL WT RCC cells  in our screen. We generated drug dose-response curves for these MEK inhibitors alone and with dasatinib to examine the drug effect. All 9 structurally unrelated MEK inhibitors have limited single-agent activity, but they were more potent in combination with dasatinib, as seen by the decreased cell viability and leftward shift in dose-response curves F and 7G.−20). Conversely, antimetabolite chemotherapy agents were significantly de-enriched by the same analysis—for example, gemcitabine (p = 4.52 ×10−22)  resource, L1000FWD, which provides interactive visualization of 16,000 drug and small-molecule-induced gene expression signatures in 68 cell lines. ×10−22) B. These MET oncogene, chromophobe tumors exhibit alterations in mitochondrial DNA, RCCs that occur due to TFE translocation) and some that are not. For advanced NCCRCC, the only treatment options have been largely extrapolated from agents studied in CCRCC, including anti-angiogenics and mTOR inhibitors. In general, studies have demonstrated that patients with NCCRCC have a worse prognosis, with lower response rates to these therapies.11,,,,NCCRCC (or variant histology RCC) accounts for ∼25% of kidney cancer, with papillary, chromophobe, clear cell papillary, collecting duct, medullary, and sarcomatoid variants accounting for the majority of subtypes.The lack of treatment options for NCCRCC patients represents a critical unmet clinical need. Here, we sought to identify preclinical synergistic drug combinations that would transcend lineage and genetic landscape to induce cytotoxicity in NCCRCC and ultimately lead to deep and durable responses in patients. Accordingly, we present a model to predict and develop novel combination therapies through joint analysis of high-throughput datasets that interrogate multiple aspects of the kidney cancer cell . We posit that our phosphoproteomics signaling output likely reflects the direct results of treatment , whereas the transcriptional signatures likely represent the consequences that come from signaling pathway modulation . Consequently, integrating the phosphoproteome and transcriptome is a strength because we gained insight into several previously undescribed combinations in NCCRCC—for example, dasatinib with cabozantinib, dasatinib with MEK inhibitors, and cabozantinib with MEK.in vivo, confirming the robustness of our experimental screening approach. We observed that multiple NCCRCC cells appear to respond similarly to the combination, suggesting broad utility across this histologically diverse group of cancers. Mechanistically, the dasatinib-cabozantinib combination converges to suppress MAPK signaling to induce cytotoxicity. Critically, this suggests that combining dasatinib or cabozantinib with MEK inhibitors could be an alternate combination. In support of this, we observed decreased cell viability with these previously untested combinations. Taken together, our preclinical findings provide rationale for novel combination clinical trial designs.We demonstrate that the dasatinib-cabozantinib combination has potent synergy in 2-dimensional (2D) cell culture models. Importantly, this combination induces tumor regression ,What then is the most straightforward path to the clinic? We speculate that these findings have direct clinical implications for cabozantinib, providing rationale for further clinical study of cabozantinib in NCCRCC and supporting future combination treatments. Retrospective studies suggest that cabozantinib has activity in patients with NCCRCCin vitro, we and others,in vivo. This suggests that the cabozantinib may not directly induce apoptosis or may stimulate other types of programmed cell death mechanisms in vitro.The caveats here are limitations of cell culture and mouse xenograft studies and how these can be extrapolated to clinical trials. While we noted that increasing doses of cabozantinib alone suppressed proliferation in human RCC cells Although the dasatinib-cabozantinib combination was well tolerated in mice, the potential clinical toxicities in the setting of patients who have exhausted multiple lines of therapies and with comorbidities are part of the real-world study considerations. Accordingly, appropriate dose-finding studies will be needed as a first step to explore safety and feasibility, either stand-alone or incorporated into larger efficacy trials.thomasge@ohsu.edu)Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, George V. Thomas  supplemented with 10% fetal bovine serum at 37°C in a 5% COWe did screening using a library of 292 small molecules  GI50 fold change: Drugs that have at least 5-fold change in GI50 ratio between drug alone and drug+dasatinib (ratio GI50 drug/ GI50 combination) in 50% of cell lines (n = 38). 2) AUC difference: Drugs that have AUC difference of more than 0.9 (average is 0.9) between drug alone and drug+dasatinib in at least 50% of cell lines (n = 31). 3) AUC % change: Drugs that have AUC % change of more than 25% (average is 25%) between drug alone and drug+dasatinib in 66% (4 of 6) cell lines (n = 31). 4) GI50 of combination: Combinations that are in top 50%  in 50% (3 of 6) cell lines and pass through the minimum viability cutoff of 15% across cell lines that  (n = 18). 5) AUC of combination: Combinations that are in the top 50%  and in 50% (3 of 6) cell lines and pass through the minimum viability cutoff of 15% across cell lines that  (n = 13). We shortlisted the drugs based on the above criteria and picked 28 drugs that were identified by at least 4 of the 5 parameters. These 28 drugs were further subjected to the secondary screening, which involved a dose-matrix of 6X8 (6 doses of dasatinib and 8 doses of the drug). The growth inhibition values from the secondary screening were subjected to the estimation of synergy using Calcusyn. Calcusyn calculates Combination Index (CI) for drug combinations: CI < 1 is synergistic; CI = 1 additive and CI > 1 is antagonistic effects.6 cells were diluted in 50 μl of PBS and 50 μl of Matrigel (BD Biosciences) and were injected subcutaneously into the right and left flank of each mouse (8 mice per treatment arm). Tumors were monitored until they reached an average size of 50-80mm3 (approximately 2 weeks), at which point treatments were begun. dasatinib  was administered by oral gavage 5 days/week. Cabozantinib  were administered by oral gavage 3 days/week. The in vivo dosing for dasatinib and cabozantinib were chosen based on prior work from our group and others: Dasatinib has been administered as a single agent in the 25-50mg/kg range,,,in vivo, we examined our in vitro 6x8 dose matrix dasatinib-cabozantinib combination results, which demonstrated higher doses of cabozantinib were required to decrease ACHN cell viability and so we chose 25mg/kg dasatinib+30mg/kg cabozantinib in vivo dose. Conversely, the in vitro dasatinib-cabozantinib combination indicated that lower doses of cabozantinib when combined with higher doses of dasatinib elicited decreased CAKI-1 cell viability. Dasatinib and cabozantinib were dissolved in NMP/PEG. Tumors and mouse weights were measured twice weekly. At least 6-8 mice per treatment group were included in the analyses , and the investigators were not blinded to the identity of the study groups. All mice were euthanized using CO2 inhalation followed by cervical dislocation per institutional guidelines at Oregon Health and Science University.All animal experiments were approved by the Institutional Animal Care and Use Committee at OHSU (IACUC protocol: TR02_IP00000022). The studies are compliant with all relevant ethical regulations regarding animal research. All mice were housed in the OHSU animal facility approved by AAALAC in accordance with NIH guidelines. The animal facility provides care for a variety of animals and provides services of feeding and watering, cage cleaning and room sanitation, plus observation and health assessment of laboratory animals. All nude mice were housed in sterile conditions and handled in a sterile laminar airflow room by trained staff, supervised by several veterinarians. Six-week old female mice  were utilized for human renal cell carcinoma xenografts. For both ACHN and CAKI-1 cell lines 2x10,Cell viability assays were performed by plating cells/well in 96-well plates in triplicate and treating the following day with the indicated agent: dasatinib (dose range of 0–400 nM) and cabozantinib (dose range of 0–10 μM). These doses were based on prior work from our group and others.Cells were plated in 6 well dishes and treated the following day with the indicated agents. Treatments were for 24 hours, after which cells were washed with ice cold PBS and lysed with RIPA buffer (Sigma). Phosphatase inhibitor cocktail set II and protease inhibitor cocktail set III (EMD Millipore) were added at the time of lysis. Lysates were centrifuged at 15,000 g x 10 min at 4°C. Protein concentrations were calculated based on a BCA assay (Thermo Scientific) generated standard curve. Proteins were resolved using the NuPAGE Novex Mini Gel system on 4% to 12% Bis-Tris Gels (Invitrogen). For western blotting, equal amounts of cell lysates (15-20 μg of protein) were resolved with SDS-PAGE, and transferred to membranes. The membrane was probed with primary antibodies, washed, and then incubated with corresponding fluorescent secondary antibodies and washed. The fluorescent signal was captured using LI-COR  Odyssey Imaging System, and fluorescent intensity was quantified using the Odyssey software where indicated. The following antibodies were used for western blots: p-SRC (Y416), SRC, p-MET (Y1234/1235), p-p44/42 MAPK (Erk1/2) (T202/Y204), ERK and β-actin (AC15). Ki67 and cleaved caspase 3 were used for immunohistochemistry. Dasatinib, cabozantinib and cobimetinib were purchased from Selleck chemicals.Immunostaining was performed following deparaffinization and rehydration of slides. Antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Nonspecific binding was blocked using Vector mouse IgG blocking serum 30 min at room temperature. Samples were incubated at room temperature with rabbit monoclonal antibodies cleaved caspase 3, and Ki67. Slides were developed with Vector Immpress rabbit IgG and Vector Immpress mouse IgG (Vector Laboratories) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (Vector Laboratories) for 3 min. Slides were counterstained with hematoxylin. A 3DHistech MIDI Scanner (Perkin Elmer) was used to capture whole slide digital images with a 20x objective. Images were converted to into MRXS files and computer graphic analysis was completed using inForm 1.4.0 Advanced Image Analysis Software (Perkin Elmer).H&E slides of formalin fixed, paraffin embedded tissue was used to assess morphological integrity of tumor samples. Once integrity was confirmed, immunofluorescent analysis was performed for p-SRC (Y416), p-MET (Y1234/1235). Four μ sections were cut, de-paraffinized and rehydrated. Antigen retrieval was performed using citrate for 4 min in a pressure cooker. Slides were blocked using 2.5% normal goat serum for 30 min then incubated in primary antibody for 1hr followed by secondary antibody mouse anti-rabbit Alexa 488  for 30 min. Slides were rinsed in PBS, air-dried, and coverslipped using Dako mounting media with Dapi.RNA was extracted from triplicate 24-hour treated cell lines using the RNeasy extraction kit (QIAGEN), and the quality if the extracted RNA was verified using an Agilent 2100 Bioanalyzer. Unstranded, poly-A-selected RNA-seq libraries were prepared using a TruSeq RNA Library Prep kit (Illumina). Read depth and splice junction details are in Differentially expressed genes in drug-treated samples were identified with DESeq2 (v1.18.1)qij is proportional to the predicted true concentration of transcripts in sample j, xjr are the design matrix components for sample j, and βir are the coefficients (log2 fold changes). The full modelxjdβid), the cabozantinib effect (xjcβic), and an interaction effect (xjdcβidc). The reduced modelxjdβid) and the cabozantinib effect (xjcβic).Significance of the cabozantinib-dasatinib interaction effect on gene expression was assessed using a likelihood ratio test to compare two generalized linear models. Both models feature logarithmic link functionsWe considered genes to be affected by the interaction of the drugs if the full model explained the observed effects significantly better than the reduced model .Enrichment of specific pathways and ontological terms in the gene most affected by the drug interaction was assessed using the Broad Gene Set Enrichment Analysis (GSEA) (v3.0) Java applicationβidc), using a network generated from gene expression profiles of the TCGA kidney renal papillary cell carcinoma cohort.Master Regulator analysis was used to infer transcription factors responsible for the observed drug interaction-induced changes in gene expression. The R package VIPER (v1.12)Interaction effects in the phosphoproteomic data were analyzed in R using the limma (3.36.3)βidc. A heat diffusion algorithm was then run to model diffusion of “heat” from the input nodes to nearby nodes in the network, and a subnetwork of agreement between the data types was identified by identifying nodes that receive heat from both data sources.An integrated network of inferred drug interaction effects on transcription and phosphoprotein levels was created using TieDie (v1.0)Similar and opposite drug-induced expression signatures were identified from lists of differentially expressed genes using the L1000 Fireworks Display (L1000FWD) web service. Differentially expressed genes were identified by selecting genes significantly better explained using a full model containing an interaction term, compared to a reduced model containing only terms for single agents (as described above in https://www.uniprot.org).Phosphopeptides were enriched and analyzed by mass spectrometry as previously described in detail.Quantitative phosphopeptide data were log10 transformed and missing data were imputed before applying quantile normalization as previously described.,Kinase substrate enrichment analysis (KSEA) was performed as previously described.P value less than 0.05 was considered statistically significant. Further statistical details, including values of n and definitions of what n represents, can be found in the figure legends. Additional statistical analysis specific to phosphoproteomics and RNA Seq are listed above. Immunohistochemistry: P-values were calculated using one-way ANOVA, with Bonferroni’s multiple comparison test. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01, and ∗∗∗ denotes p < 0.001. The plots here were created using R version 3.6.1. Packages needed include ggplot2 and dplyr.Statistical significance between two groups was assessed by unpaired Student’s t test. Mouse tumor size was analyzed by 2-way ANOVA with time and drug as factors, using GraphPad Prism. Mouse weight during treatment was analyzed by repeated-measures 2-way ANOVA, with time and drug as factors. All cell culture experiments were per-formed at least two times. Data are presented as mean ± s.e.m., and a


G12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid–Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-Ras Editors' choice: We developed a new conditional genetically engineered mouse model of rhabdomyosarcoma (RMS) with homologous molecular signature to human RMS that provides valuable pre-clinical models for evaluating novel therapies. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite improvements in patient survival rates over the past few decades, over one-third of RMS patients still succumb to the disease. Therefore, identification of additional effective therapeutic regimens is crucial towards improving the outcome for these patients. RMS can be categorized histologically into alveolar RMS (ARMS) and embryonal RMS (ERMS) . The alvKRAS oncogene has been implicated in various cancers, with up to one-third of RMS cases involving the activation of one of the three RAS isoforms, including K-RAS , rendering ARMS to be grouped as fusion positive and ERMS as fusion negative. Although ARMS rarely involves the inactivation or mutations of p53 (TP53), ERMS has a relatively high frequency of mutations or aberrations in the p53/MDM2 axis . In adding K-RAS .Although rapid progress has been made in the field of RMS, most research has been based on xenotransplantation using human cell lines into immunodeficient models. These xenograft models do not recapitulate the complete tumor biology, including the tumor immune microenvironment. Thus, there is a significant need to develop murine RMS models that can develop and progress in immunocompetent model systems.In this study, we developed and characterized a conditional p53 and/or K-Ras fusion-negative RMS (FN-RMS) genetically engineered mouse model (GEMM). We also performed histologic and genetic analysis of GEMM tumors, and found that these tumors had similar phenotypic and molecular features to human RMS. Based on the homologous histological and molecular traits to RMS, we believe that this mouse model, and the derived cell lines, could significantly contribute to our understanding of the molecular pathogenesis of RMS and provide valuable immunocompetent models for investigating novel therapeutic strategies for treating RMS.R172H or K-RasG12D alleles. Four distinct MyoD-Cre transgenic genotypes were monitored for tumor incidence: K-Ras-wild type (WT) mice with floxed p53 allele/WT p53 allele or LSL-p53R172H allele/WT p53 allele (K-RasWTp53F/+ and K-RasWTp53R172H/+) and K-Ras-mutated mice with floxed p53 allele/WT p53 allele or LSL-p53R172H allele/WT p53 allele (K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+) -Cre mice with germline floxed p53, Lox-Stop-Lox (LSL) p53R172H/+) A.Fig. 1During necropsy, we assessed for metastatic lesions in all of the major organs, noting lungs to be the most frequent site of metastasis, which we noted ranged between 10% and 20% of the animals, while there seemed to be no significant difference in incidence of tumor generation between each group B. The siWTp53F/+ and K-RasWTp53R172H/+) lived significantly longer than K-Ras-mutated mice (K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+) (P<0.0001). In addition, there was a significant survival difference between K-RasWTp53F/+ and K-RasWTp53R172H/+ (P=0.0269) mice, but there were no statistically significant differences between the K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+ cohorts (P=0.58).We examined the overall survival of these genetically engineered mice, comparing the survival rates of K-Ras-mutated and K-Ras-WT mice D. We notFig. S1). Most tumors had identifiable elongated ‘strap’ cells or cells with visible cross striations that are found in RMS.Following the evaluation of tumor incidence, primary tumor development and metastatic potential in this murine model, we next sought to characterize the tumors histologically. Tumor morphology was evaluated by a veterinary pathologist with experience in mouse sarcoma models, and skeletal muscle differentiation was evaluated using phosphotungstic acid–Hematoxylin (PTAH) histochemical staining and immunohistochemistry for desmin and myogenin, markers of muscle and skeletal muscle differentiation A-D. PrimG12D tumors (p53R172H/+), three K-RasWT tumors (two p53R172H/+and one p53F/+) and normal gastrocnemius muscle tissue from three non-tumor bearing mice as controls. We generated gene signatures of at least 1.5-fold change and a false discovery rate (FDR)≤0.05 for K-RasG12D or K-RasWT compared to normal murine gastrocnemius muscle tissue, as well as human RMS (either fusion negative or fusion positive) to normal muscle tissue. To determine the extent of overlap between the differentially expressed genes (DEGs) from the K-RasG12D/+ and K-RasWT tumors with human RMS, we compared the murine DEGs to the human fusion-positive RMS (FP-RMS) and FN-RMS gene signatures. Of the 7900 FN-RMS DEGs, 30% (2391 genes) and 45% (3585 genes) overlapped with our K-RasWT and K-RasG12D, respectively (WT and 43% (3547 genes) overlap with K-RasG12D (We performed RNA sequencing (RNA-seq) from primary GEMM tumors to examine whether gene expression in tumors from our model overlapped with gene expression changes seen in human RMS. For our studies, we used three K-Rasectively A. Simila-RasG12D A.Fig. 3GSE108022 and murine Kras gene signatures in other human RMS cohorts. Although both the K-RasG12D and K-RasWT signatures were significantly correlated with our human RMS signature in all four cohorts analyzed, the K-RasG12D signature consistently had much higher Pearson correlation coefficients, similar to those of FN-RMS and FP-RMS, compared to K-RasWT (R172H/+ resulted in the rapid development of UPS in the setting of an activating K-Ras mutation (WT signature had a higher correlation coefficient (0.97) with the UPS gene signature compared to K-RasG12D (0.89), which had comparable correlation coefficients to UPS with human FN-RMS and FP-RMS. Taken together, these data indicate that the K-RasG12D GEMM more closely mimics the gene expression changes seen in human RMS across multiple datasets compared to the K-RasWT model, which has gene expression changes more closely related to those seen in human UPS.We further asked whether the overlapping gene changes were consistent across multiple human RMS datasets. We performed signature correlation between our human RMS gene signature derived from the Gene Expression Omnibus dataset  K-RasWT B. A prev K-RasWT , and Doymutation . We therG12D mouse model captures human RMS pathway changes, we ran gene set enrichment analysis (GSEA) using the mouse and human DEG signatures. Interestingly, based on unbiased hierarchical clustering using the resulting normalized enrichment scores (NESs), the K-RasG12D clustered closer to human RMS than the K-RasWT . Not unexpectedly, immune-related pathways were not consistent between our K-RasG12D tumors and cell lines. We further assessed for the ability of these cell lines to generate tumors in vivo by injecting 1 million cells for each K-RasG12D cell line, KMR19 (p53R172H/+), KMR46 (p53F/+) and KMR72 (p53R172H/+), into the gastrocnemius of C57BL/6J mice  and p53F/+ (MF21) in C57BL/6 mice (Table S1), but, after several months (2-4 months), no tumors were evident upon necropsy of the animals.After isolation of GEMM tumors, as outlined in our methods, we developed primary cell lines from several GEMM tumors. We performed RNA-seq analysis on four cell lines  and subsequently performed GSEA analysis on the DEGs between our cell lines and normal gastrocnemius muscle tissue. RNA splicing, mRNA processing and signaling pathways were consistent with the results from our K-Ras/6J mice A, with pM tumors B,C. In aM tumors B,C. The G12D cell lines were injected via the tail vein into three C57BL/6J mice. One to two weeks following injection, mice were sacrificed and examined for metastatic disease. We observed that all mice showed evidence of macroscopic lung metastasis, which was further analyzed by Hematoxylin and Eosin (H&E) staining  values between our murine RMS lines and the human RMS cell lines  were purchased from American Type Culture Collection . The cells were grown and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cultured in a humidified 5% COR172H mice or K-RasG12D mice. Both male and female mice were used. Genotyping was performed using PCR. All research was performed in compliance with the Baylor College of Medicine IACUC  and AAALAC recommendations as published in The Guide for the Care and Use of Laboratory Animals (NRC1996). All animals were monitored closely and underwent comprehensive necropsies following humane euthanasia at an endpoint. All major organs and tumors were evaluated, and tissues suspected of metastatic disease were submitted for histological analysis.Transgenic mouse lines were generated through MyoD promoter-driven Cre recombinase with core enhancer, which directs the early activation of MyoD, as reported by 2 incubator at 37°C. All murine RMS cell lines are available upon request from the corresponding author (J.T.Y.).Murine RMS cell lines  were isolated from primary K-Ras-mutated RMS tumors. Resected tumors were diced and dissociated using a Tumor Dissociation Kit, mouse  and a gentleMACS Dissociator (Miltenyi Biotec). The dissociated cells were resuspended and filtered through a 70 μm cell strainer (Corning), then seeded, established and subcultured. The established cell lines were cultured in RPMI-1640 medium  supplemented with 1% penicillin/streptomycin and 10% FBS, and cultured in a humidified 5% CO6 cells per 25 μl PBS and then mixed with 25 μl Matrigel matrix . The cell–Matrigel mixture was subsequently injected into the gastrocnemius of C57BL/6J mice (n=3/cell line), and the tumors were resected once palpable.KMR19, KMR46, KMR72, KMR78, RD, Rh30 and PDX40 cells were harvested and resuspended at a concentration of 1×106 cells per 200 μl PBS. The cells were injected into the tail vein of the mice (n=3/cell line), which were subsequently sacrificed a week after injection. Cell lines were authenticated using short tandem repeat analysis and frequently checked for mycoplasma contamination.KMR19, KMR46, KMR72 and KMR78 cell lines were harvested and resuspended at a concentration of 1×103 cells per 96-well plate (Corning) for 24 h and then incubated with various concentrations of vincristine, irinotecan, SN38, MK1775 and trametinib  for 72 h. Subsequently, the degree of cell viability was evaluated using the Cell Counting Kit-8  according to the manufacturer's protocol.To assess cell viability, cells were seeded at 5×10GEMM and orthotopic model-derived xenograft tumors, as well as clinical specimens, were embedded in paraffin, fixed, and analyzed by Hematoxylin, Eosin, myogenin  and desmin  staining. For IHC analysis, a VECTASTAIN ABC Kit, Rabbit IgG  was used, and the staining was visualized using a Vector NovaRED Peroxidase Substrate Kit (Vector Laboratories).For PTAH staining, we used a Rapid PTAH Stain Kit . Briefly, tumor sections slides were placed in Langeron's iodine after deparaffinization and rehydration, and then transferred to 5% sodium thiosulfate. After rinsing in water, slides were stained in pre-heated PTAH.Images of H&E and IHC staining were obtained using a BX53 Biological Microscope . The whole pictures of lung sections were obtained by tile scanning, which merged 20× images and allowed the acquisition of overview images of the specimen, and Leica DMi8 and LAS X software .RNA samples underwent quality control assessment using the RNA tape on Tapestation 4200 (Agilent Technologies) and were quantified using a Qubit Fluorometer (Thermo Fisher Scientific). The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with QIAseq Stranded Total RNA Library Kit (Qiagen) using 500 ng input RNA. mRNA was enriched with Oligo-dT probes attached to Pure mRNA beads (Qiagen). RNA was fragmented, reverse transcribed into cDNA, and ligated with Illumina sequencing adaptors. The size selection for libraries was analyzed using the DNA 1000 tape Tapestation 4200 (Agilent Technologies). The prepared libraries were pooled and sequenced using NextSeq 500 (Illumina), generating ∼17million 2×76 bp paired-end reads per samples.GSE108022, which includes 101 RMS (35 FP-RMS and 66 FN-RMS) samples and five normal muscle samples. Differential expression analysis was performed using DESeq2 R package (1.28.1)  software package; significance was achieved for adjusted q-value<0.25.Paired-end sequencing reads were trimmed using trimGalore software. The trimmed reads were mapped using STAR against the murine genome build UCSC mm10 and quantified using featureCounts  against (1.28.1) . P-valuen=30; n=147; n=58; GSE66533) datasets in R2:Genomics Analysis and Visualization Platform (http://r2.amc.nl). The UPS cohort RNA-seq counts data were kindly provided by Dr Nischalan Pillay .