Omics-based tools were coupled with bioinformatics for a systeomics analysis of two biopharma cell types: Chinese hamster ovary (M-CHO and CHO-K1) and SP2/0. Exponential and stationary phase samples revealed more than 10,000 transcripts and 6000 proteins across these two manufacturing cell lines. A statistical comparison of transcriptomics and proteomics data identified downregulated genes involved in protein folding, protein synthesis and protein metabolism, including PPIA-cyclophilin A, HSPD1, and EIF3K, in M-CHO compared to SP2/0 while cell cycle and actin cytoskeleton genes were reduced in SP2/0. KEGG pathway comparisons revealed glycerolipids, glycosphingolipids, ABC transporters, calcium signaling, cell adhesion, and secretion pathways depleted in M-CHO while retinol metabolism was upregulated. KEGG and IPA also indicated apoptosis, RNA degradation, and proteosomes enriched in CHO stationary phase. Alternatively, gene ontology analysis revealed an underrepresentation in ion and potassium channel activities, membrane proteins, and secretory granules including Stxbpt2, Syt1, Syt9, and Cma1 proteins in M-CHO. Additional enrichment strategies involving ultracentrifugation, biotinylation, and hydrazide chemistry identified over 4000 potential CHO membrane and secretory proteins, yet many secretory and membrane proteins were still depleted. This systeomics pipeline has revealed bottlenecks and potential opportunities for cell line engineering in CHO and SP2/0 to improve their production capabilities. Enhanced production of biotherapeutic proteins is critical for pharmaceutical companies to offer efficacious and affordable drugs to patients. Chinese hamster ovary (CHO) cell lines are the most widely used production hosts compared to other cell lines, including mouse myeloma (SP2/0), baby hamster kidney (BHK-21), murine myeloma (NS0), and human embryonic kidney (HEK)2. The implementation of stable cell lines is integrated together with bioprocess optimization, medium development, and possibly additional cell line engineering in order to achieve efficient growth rates, along with high productivities, product yields and quality appropriate for scale up and production of clinical grade biologics3.Mammalian expression systems are the predominantly used platforms for FDA-approved drugs and biologics at pre-clinical and clinical stages6 have set the foundation of a systems biology era for understanding CHO cell line physiology for enhanced protein production7. Proteomic, transcriptomic and microRNAomic9 technologies have also been applied to quantitatively track the changes in protein, mRNA and microRNA levels between cell lines exhibiting different growth rates and productivities in order to increase our knowledge about production hosts. Coupling this information with cell line engineering and media development technologies not only enhances mammalian growth and productivity but also ushers in a new era for understanding the cellular properties that may make a particular host line appropriate for production for each molecule of interest.Recent advances in omics technologies have been one avenue to elucidate biological changes in mammalian expression systems that may be relevant to recombinant protein production. Genetic sequencing of the CHO cell line and the Chinese hamster10. Similar methods can be used for the systematic identification of depleted and enriched pathways in mammalian expression platforms12 to help identify the bottlenecks and limitations related to cell growth, production yields and product quality.Another emerging approach that can help improve our understanding of complex biological pathways and their roles in bioproduction is the application of systeomics. Systeomics, in biomedicine, is the integration of proteomics, transcriptomics and genomics data, often at the pathway level, for the discovery of novel gene targets and biomarkers for various diseases13. In order to better understand the overall cellular characteristics and the underlying reasons for the expression behavior in these widely used cell lines, we performed a comparative systeomics analysis on the exponential and stationary phases of an AstraZeneca production cell line called M-CHO and a SP2/0 cell line. Understanding the advantages and disadvantages of these two cell lines can provide us the gene or pathway targets that may be appropriate for cell engineering of superior host platforms. Transcriptomic and proteomic experiments were performed in which Illumina HiSeq was used for mRNA sequencing, and filter aided sample preparation (FASP) technique was coupled with two-dimensional LC–MS to reach deep sequencing at the protein level. This is, to our knowledge, the first comprehensive proteomic study done comparing and contrasting SP2/0 and suspension CHO cell lines.Although both the CHO and SP2/0 cell lines are commonly used host platforms in industry, they exhibit different cellular characteristics. Due to its natural secretory properties, SP2/0 can often generate high mAb titers. On the other hand, CHO cell lines are the primary expression systems in the $113 billion pharmaceutical market due to their manufacturability, high growth profiles, and glycosylation capability14. For the functional annotation, gene ontology (GO) analysis was used, whereas Kyoto Encyclopedia Genes and Genome (KEGG) and Ingenuity Pathway Analysis (IPA) were used to statistically compare the metabolic, signaling and cellular processes at a systems biology level, schematically illustrated in Fig. Furthermore, to elucidate the differences across CHO cell lines, a CHO-K1 adherent cell line was compared to an M-CHO (proprietary AstraZeneca CHO cell line) suspension cell line using RNAseq and proteomic dataIt will be helpful for future cell designers and bioproduction engineers to understand the capabilities of CHO versus SP2/0 by analyzing omics data. To our knowledge, this is the first large scale study that reveals the specific differences at the gene expression, protein, and pathway level of two of the most widely used mammalian expression systems. This analysis can then serve as the basis for understanding the advantages and disadvantages of these critical production hosts as well as suggesting opportunities to improve their capabilities and capacities in the future through genetic intervention or process design.16. In order to obtain even greater insights from these data sets, an emerging approach called comparative systeomics was used in this study to analyze whole cell proteomic and transcriptomics data of CHO and SP2/0. Firstly, a comprehensive omics study was performed on both exponential and stationary phases of two model cell lines, M-CHO and SP2/0, in order to evaluate differences in their proteome and transcriptome expression patterns, as well as the changes in each cell line between the exponential and stationary phases  and others11, and another analysis of two CHO cell lines (CHO-S and CHO DG44) identified 9359 unique proteins12. The protein and peptide information belonging to M-CHO exponential, M-CHO stationary, SP2/0 exponential, SP2/0 stationary and a control CHO-K1 ATCC stationary are compiled in Supplementary Tables RNAseq resulted in the identification and quantification of more than 10,500 transcripts for M-CHO, whereas the sequencing of SP2/0 identified and quantified more than 13,500 transcripts, likely due to the superior annotation of the mouse genome. The identified mRNA, along with their normalized values and triplicates belonging to M-CHO exponential, M-CHO stationary, and CHO-K1 stationary phases are tabulated in Supplementary Table 19. Firstly, the genes having both mRNA and protein expression were mapped for both cell lines under the two conditions, resulting in 5500–6000 genes exhibiting both mRNA and protein expression in the separate phases  for each cell line  was used to correlate and compare the mRNA values of the samples, whereas the abundance level across the proteins and between the samples was compared using the normalized spectral abundance factor (NSAF), accounting for the length of the identified proteins20. The growth curves of these two cell lines can be found in Figs. Next, pair-wise comparisons were performed; as indicated in Figs. 21.In addition to the cell cycle and protein folding pathways, apoptosis and actin cytoskeleton signaling pathways were found to be differentially expressed between the two cell lines. The actin cytoskeleton expression was found to be lower in the exponential phase SP2/0 proteome data relative to exponential phase in M-CHO. Interestingly, the actin cytoskeleton was also found to be a biological hub, providing crosstalk with PAK and RAC signaling  from the apoptosis pathway, EDEM1, CRYAB, and Mbtps1 from the protein processing pathway were shown to be expressed in the current M-CHO study. Other proteins, such as ERGL and S2P involved in protein processing pathways, were identified in SP2/0 cells in this study even though they were absent from the CHO proteome.In order to further explore differences between CHO and SP2/0 cell lines at the systems level, we applied pathway analytical tools, including KEGG and IPA, along with biological, molecular and cellular functional analysis tools such as GO Fig. . Both CHShown in Fig. 23, we were especially interested in looking for the differences between SP2/0 cells, coming from mouse spleen, and CHO cells. The KEGG pathway analysis of calcium signaling and pancreatic secretion in Fig. 24, and the importance of calcium signaling, both in endocrine and exocrine secretory cells has been previously demonstrated25. Pla2, a calcium-dependent lipase associated with phospholipid remodeling of bio-membranes in many cell types, and MaxiK , which plays a key role in regulating calcium-sensitive potassium channels for membrane potential and is important to exocytosis, mapped to the SP2/0 but were depleted in M-CHO and CHO-K1. This result is not unexpected since calcium signaling is important to the development and function of B cells26.A heat map for a group of proteins found to be more depleted in CHO cells compared to SP2/0 cells was generated in Fig. p-values for MF, BP and CC were found and are listed in Supplementary Tables 1128. The enrichment results of the M-CHO and SP2/0 cell lines are summarized in Fig. In order to gain a better understanding of the biological process (BP), molecular function (MF) and cellular component (CC) of the transcriptomic and proteomic profiles of M-CHO and SP2/0 cells, gene ontology (GO) analysis was implemented to identify over-represented (enriched) and under-represented (depleted) categories. The GO-CHO database was used, and enrichment and depletion Interestingly, the integral components of membrane, cell surface and plasma membrane terms, and secretory granules were found to be under-represented for the CC analysis in M-CHO cells and enriched in SP2/0 cells. Individual genes representing the secretory granule category were compared between the SP2/0 and CHO cells with the resulting overlap shown in Fig. 30. Although M-CHO cells are widely used both for secreted and membrane protein expression, poor expression of membrane proteins has been previously reported31. In order to further examine the presence of key membrane and vesicle proteins in M-CHO cells, we applied three different enrichment methods to explore the M-CHO membranome. Two step ultracentrifugation, cell surface biotinylation and hydrazide chemistry-based glycoproteome enrichment methods were coupled with LC/MS/MS as shown in Fig. 3 treatments allowed for the isolation of the vesicular proteome, exosome and plasma membranome.Both transcriptomics and proteomics data, gene ontology and KEGG pathway analysis revealed that membrane or secretion associated pathways were often depleted in M-CHO or CHO-K1 cells, whereas these pathways were enriched in SP2/0 cells. Membrane biogenesis is known to be enriched in murine cells, but these findings also suggest that this category of proteins may also be low in M-CHO cells32, SignalP28, TargetP33, Phobius34, and WolfPSort35 in order to identify those containing transmembrane domains and/or signal peptides27. Although glycoproteome enrichment provided the highest percentage of either membrane or secreted proteins, the ultracentrifugation-based membrane proteomics technique revealed the highest number (1483) of membrane and/or secreted proteins. For this reason, peptides from the ultracentrifugation enrichment were separated into 48 fractions using bRPLC followed by tandem mass spectrometry analysis. Coupling enrichment technology with the two-dimensional fractionation technique identified 86,646 peptides belonging to 8736 proteins, with an average of 10 peptides per protein. Of these proteins, 2478 were predicted to be on the membrane, based on WolfPsort, TMHMM and Phobius-TM, whereas 2804 were predicted to be secreted, based on WolfPsort, SignalP, TargetP and Phobius-SP  present on the synaptic vesicles serve as calcium sensors for exocytotic processes. Interestingly, in most secretory systems, exocytosis is often initiated by increases in the calcium concentration inside the cell40, and it is worth noting that calcium signaling mechanisms were depleted in CHO. These examples illustrate the capacity of systeomics to serve as a worthwhile starting point in order to discover and characterize genes and pathways that are deficient in particular production hosts such as CHO. We believe that systeomics serves primarily to elucidate why particular cell lines such as SP2/0 or CHO are efficient at certain tasks such as survival or secretion. Secondly, we believe efforts such as those described here can serve as a key launching point for subsequent synthetic biology and metabolic engineering interventions aimed at generating engineered cell lines with improved properties such as cell growth, protein folding, vesicular transport and secretion for biopharmaceutical applications.Since proteins residing on the membrane or functioning in the secretory pathway are often in low abundance when using conventional cell isolation methods, glycoproteomics, cell surface biotinylation and ultracentrifugation-coupled membrane enrichment methods were followed by LC/MS/MS analysis to isolate and identify them in CHO. Bioinformatics tools such as TMHMM, SignalP, TargetP, and Phobius and GO helped to further elucidate the secretory and vesicle compartmental microenvironment of CHO cells. Even after enrichment, depletion tests verified the absence of membrane and secretory granule proteins in CHO cells. For example, only 1 out of 16 secretory granules were identified by membrane enrichment experiments. Proteins representing genes such as Stxbpt2, Syt1, Syt9 and Cma1 were still not found by secondary isolation, although they were noted in SP2/0. The importance of Stxbpt2 in intracellular membrane trafficking and functioning of SNAREs (soluble NSF attachment protein receptor) for membrane fusion and vesicular transport is known11. Both cell lines were grown in shaking incubators at 37 °C/5% CO2/120 rpm. A Vi-cell TMXR cell Viability analyzer  was used for cell counting.A proprietary AstraZeneca CHO cell line was cultured in CD-CHO  medium and supplemented with 6 mM L-glutamine . SP2/0 cells were cultured in EX-CELL® Sp2/0 Serum-Free Medium with 8 mM L-glutamine . CHO-K1 cells used in this study are the adherent cells grown in serum containing mediaA total of three biological replicates were undertaken for the transcriptomics studies. Qiagen RNeasy kit was used to extract the mRNA from each cell line using the manufacturer’s protocol . After assessing the quality and quantity of mRNA with a Nanodrop spectrophotometer  and Agilent Bioanalyzer , Poly A was depleted. Illumina’s Truseq RNA preparation methodology was used to prepare RNAseq libraries according to the manufacturer’s suggested protocol. Agilent DNA High Sensitivity Bioanalyzer and Library quantitation qPCR kit  was used to assess the quality and quantity of the RNAseq libraries. Illumina HiSeq 2000 was used for library sequencing using a 100 bp paired end sequencing strategy. A TruSeq RNA Sample Preparation kit  was used to prepare RNA libraries, and the paired-end (2 × 75 bp) reads were sequenced on a HiSeq 2000.41. HTSeq (http://www-huber.embl.de/users/anders/HTSeq/) was used for counting the number of mapped reads per gene and eliminating ambiguous alignments. The count table was normalized with a DESeq package from Bioconductor.The reference sequences for CHO cell lines were obtained from the NCBI RefSeq database, which has a total of 109,151 contigs. The N50 size of these contigs was 502 bp, and the total size was 2,399,770,464 bp. Contigs <  = 5 kb in length were discarded for more accurate analysis. The retained 9,020 (9.0%) contigs, with a total length of 2,330,772,269 bp (97.1%), were used for further analysis. The Tophat2 program was used to map the RNA-seq reads to the CHO reference sequencesCells were washed with cold PBS at 4 °C prior to lysing in 2% SDS supplemented with 0.1 mM PMSF and 1 mM EDTA solution at pH 7.6. After sonication, a BCA assay was done to measure the protein concentration. The required protein amount was reduced with 4.5 mM TCEP at 60 °C and alkylated with 8.5 mM MMTS at room temperature for 30 min. All the samples were incubated with 9 M sequanol grade urea for 1 h at room temperature. To remove the SDS, 10 kDa FASP filters were used. The samples were then digested in FASP tubes with Trypsin/LysC  enzymes at 37 °C. The dried peptide samples were subjected to bRPLC (basic reversed phase liquid chromatography) fractionation.N-glycopeptide capture, the samples were reduced, alkylated and digested before oxidization with 10 mM sodium periodate  at room temperature in the dark for 1 h. The peptides were mixed with AffiPrep Hz Hydrazide beads  and 100 mM aniline  and left at room temperature gently shaking overnight. After washing away the non-glycosylated peptides, the glycopeptides attached to the beads were digested in 25 mM ammonium bicarbonate with 3 µL PNGase F  at 37 °C, as shown previously by Zhang et al.42. The released glycopeptides were analyzed by LC–MS/MS. M-CHO cells were also subjected to the ultracentrifugation technique for membrane proteome enrichment. The cells were lysed in a sucrose buffer  and centrifuged at 50,000 rpm for 96 min. Initial ultracentrifugation pellets were brought up in a resuspension buffer  before a second ultracentrifugation spin. Final membrane-enriched pellets were resuspended in sonication buffer in 2% SDS and subjected to the FASP method before LC/MS/MS analysis.For the biotinylation experiment, M-CHO cells were harvested, washed and biotinylated with a Pierce Cell surface biotinylation kit  according to the manufacturer’s protocol. After elution, the proteins were subjected to the FASP method and digested with Trypsin/C-Lys-C Mix  shaking at 37 °C overnight. For Digested peptides were fractionated on a basic reversed phase column . Fractions were merged into final 24 or 48 sample groups prior to LC/MS/MS analysis. Tandem mass spectrometry analysis of the peptides were carried out on the LTQ-Orbitrap Velos  attached to an Eksigent nanoflow 2D liquid chromatography system with an auto sampler . Peptides were enriched on a 2 cm trap column (YMC gel ODS-A S-10 µm), fractionated on a Magic C18 AQ, 5 µm, 100 Å , 75 µm × 15 cm column and electrosprayed through a 15 µm emitter . The reversed-phase solvent gradient consisted of solvent A (0.1% formic acid) with increasing levels of solvent B  over a period of 90 min. The LTQ Orbitrap Velos was set at 2.0 kV spray voltage, full MS survey scan range of 350–1800 m/z, data dependent HCD (higher energy collision dissociation) MS/MS analysis of top 10 precursors with minimum signal of 2,000, isolation width of 1.9, 30 s dynamic exclusion limit and normalized collision energy of 35. Precursor and the fragment ions were analyzed at 60,000 and 7500 resolutions, respectively. All the raw data has been deposited to NIST.Cricetulus griseus database for CHO cell lines and M. musculus database for SP2/0 cell lines , with oxidation on methionine (variable), deamidation NQ (variable), phosphoSTY (variable), and methylthiomethane on cysteine (fixed) as modifications, using Mascot software interfaced in the Proteome Discoverer  workflow. Mass tolerances on precursor and fragment masses were 15 ppm and 0.03 Da, respectively. Data was analyzed using Proteome Discoverer 1.4 software. In addition to this, CHO-K1 mass spectrometry raw data was compiled from the study used in Baycin et al. 2012. All the MS raw data was reannotated with the same strategy as M-CHO cells. 1% FDR  was used for both peptides and proteins identification.Peptide sequences were identified from isotopically-resolved masses in MS and MS/MS spectra extracted with and without deconvolution using a Thermo Scientific MS2 processor and Xtract software. Data was searched against all entries in the 44 compared to distributed normalized spectral abundance45, normalized spectral index46, and exponentially modified protein abundance index47.The NSAF and FPKM values were calculated for protein and mRNA values and were compared and plotted using TIBCO Spotfire 3.1. In this current study, NSAF method was applied due to its capability of providing high reproducible data on the quantification of proteinshttp://www.ingenuity.com/). The data from all the cell lines were annotated with the Gene Ontology (GO) molecular function, biological process and cellular component categories. For GO annotation of the CHO genes, GO Cross Homology was obtained using GOCHO platform version ’14-04,’ which is publicly available at http://ebdrup.biosustain.dtu.dk/gocho. The Mouse Genome Informatics database was accessed on 11 June 2014 to download corresponding GO terms of mouse genes for SP2/0 cell line (ftp://ftp.informatics.jax.org/pub/reports/index.html#go)48. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways were downloaded from the KEGG website (http://www.genome.jp/kegg/) on 11 June 2014 for mouse and Chinese hamster species50 All calculations and programming tasks were performed using MATLAB version 2010a and R software51. Enrichment and depletion p-values are the outcome of a hypergeometric distribution calculated using MATLAB’s hygecdf and hygepdf functions. Adjusted p-values, Bonferroni correction was used in this study. Genesis software (release 1.7.6) was used for making heatmaps51. KEGG pathway mapper was used for calcium signaling and pancreas secretion pathways coloring52.Fold changes (FC) were used as selection criteria to identify candidate individual proteins of interest and to explore enriched canonical pathways along with protein/gene networks in the Ingenuity Pathway Analysis Software (Supplementary Information 1.Supplementary Information 2.


Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer.An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15–20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed  Breast cancer is the most frequently diagnosed cancer in women, contributing 23% of total cancer diagnoses and 14% of total cancer deaths in vivo activity, increasing the life span of mice with melanoma and leukemia Unlike ER+, PR+, or HER2+ breast cancers, TNBCs cannot be treated with endocrine therapies or HER2-targeted agents, and chemotherapies are standard of care. Platinum-based compounds carboplatin and cisplatin are often first-line therapies for basal-like TNBC in vivo using human basal-like TNBC PDXs. Single-cell RNA sequencing (scRNAseq) and immunohistochemistry assessed the expression of drug targets in human basal-like TNBC cell lines and PDXs. As presented here, these pre-clinical studies suggest that co-targeting XPO1 and PI3K/mTOR is a promising approach for the treatment of basal-like breast cancer.Due to high rates of metastasis, basal-like disease is associated with relatively worse prognosis than other TNBC subtypes ,19 The pFour basal-like TNBC cell lines- MDA-468, HCC-1143, HCC-1187, and SUM-149- were used in this study. MDA-468 cells were provided by Dr. Youngman Oh (VCU). SUM-149 cells were purchased from Asterand. HCC-1143 and HCC-1187 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 GlutaMAX media (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were negative for mycoplasma infection (ATCC Mycoplasma Detection Kit).d-luciferin  . The IVIS Spectrum In vivo Imaging System (Xenogen IVIS-200) and living image software (PerkinElmer) were used to image cells and quantify luciferase activity Firefly luciferase-GFP lentiviral transduction was performed on all cell lines to induce luciferase expression. CMV-Luciferase (firefly)−2A-GFP was purchased from GenTarget Inc. Puromycin was used to select for labeled cells to establish stable, GFP-luciferase labeled cell lines. Cell lines were plated in 96-well plates at 1500 to 5000 cells (cell-line dependent) per well. Cells were incubated overnight to allow for adherence. Then, cells were treated with drugs for 72 h. At hour 72, cells were imaged to measure luciferase activity  two minutes after the addition of https://bioinfogp.cnb.csic.es/tools/venny/) was used for cytotoxicity visualization. 64 of these drugs displayed exceptional relative cytotoxicity across all four cell lines. From these 64 drugs, six drugs targeting different genes were selected for further study: MLN2238, crizotinib, afatinib, KPT-330, dovitinib, and GSK2126458. ABT-263 and dasatinib were also included, as they showed exceptional cytotoxicity in three of the four cell lines. Finally, sorafenib and cobimetinib were selected due to their unique gene targets, BRAF and MEK respectively. Sorafenib and cobimetinib were the most cytotoxic BRAF and MEK inhibitors studied. Heatmap depicting relative cytotoxicity of 68 drugs of interest on human basal-like TNBC cell lines and PDXs was created using Morpheus (https://software.broadinstitute.org/morpheus). Data were hierarchically clustered by both samples (cell lines/PDXs) and drugs using the one minus Pearson correlation metric and average linkage method.MDA-468, HCC-1143, HCC-1187, and SUM-149 cells were treated with 1363 drugs (ApexBio DiscoveryProbe FDA-approved Drug Library) at 10 µM for 72 h. Cell viability was quantified by normalizing treated wells to vehicle wells to produce a percent of vehicle value. The 1363 drugs were ranked by cytotoxicity in each cell line. Venny  was used to obtain single cell biomass measurements every ten minutes as described by Murray et al. (2018) UCD52 mammary gland tumors were excised from mice once they reached ∼10 mm x 10 mm in size. Tumors were prepped into a single-cell suspension using the protocol described previously Single-cell RNA sequencing (scRNAseq) was performed on four human basal-like TNBC cell lines- HCC-1143, HCC-1187, MDA-468 and SUM-149 and four human basal-like TNBC PDXs- HCI-001, WHIM2, WHIM30, and UCD52. ScRNAseq was performed using the Chromium Single Cell Gene Expression Kit (10X Genomics) per the manufacturer's protocol. Samples were aligned and gene expression calculated using the 10X Genomics CellRanger v3.1 software suite of tools, and dead/poor quality cell removal was done using an in-house R script utilizing the Seurat v3.1.5 package. PDX samples went through additional filtering and realignment to remove mouse cells prior to creating a final merged dataset containing only human cells using CellRanger. 10X Loupe Cell Browser v4.0.0 was used to visualize cell clusters and perform differential gene expression analyses across clusters ,25. ScRNImmunohistochemical staining was performed on formalin-fixed, paraffin-embedded tumors. Heat-induced antigen retrieval was performed in pH 9 Tris-EDTA using a Dakocytomatin Pascal Pressure Chamber. XPO1  antibody was diluted 1:200 in SignalStain Antibody Diluent  and was applied to sections from the aforementioned tumors. Detection was performed using the Rabbit Dako EnVision System (Agilent K406511–2). Slides were imaged using Zeiss Axio Observer.N = 140) were rank-ordered based on XPO1 expression and divided into quartiles. Kaplan-Meier curves were generated with GraphPad Prism V9.0.0.The PDX RNA-sequencing data was obtained from a previously published dataset (GEO Accession: GSE118942) in vitro assays, HSLCI, or serial passaging into mice.Four basal-like TNBC PDXs were used in this study: HCI-001, WHIM30, WHIM2, and UCD52. HCI-001 was obtained from the Huntsman Cancer Institute. WHIM2 and WHIM30 were obtained from Washington University, St. Louis. UCD52 was obtained from the University of Colorado. Tumor fragments were implanted in the mammary gland of female non-obese diabetic severe combined immunodeficient gamma (NSG) mice. Tumors were allowed to grow until they reached approximately 10 mm x 10 mm in size. Then, tumors were removed and prepped into a single-cell suspension according to the protocol described previously 2 asphyxiation followed by cervical dislocation. Tumors were then excised, weighed ex vivo, and photographed. Mice reaching maximum tumor burden prior to completion of the study were sacrificed, and their tumor measurements were not considered in endpoint analyses.All studies involving mice were in accordance with the VCU Institutional Animal Care and Use Committee (IACUC). Single cell suspensions were prepped from harvested PDX mammary gland tumors. Tumor cells were resuspended in Matrigel (Corning) and injected into the right mammary gland  of female non-obese diabetic severe combined immunodeficient gamma (NSG) mice. Drug treatment began when all tumors were ∼ 3 mm x 3 mm. Mice were randomized into treatment groups. All drugs were dissolved in a solution of 1% methylcellulose + 0.1% Tween-80. All drugs were administered via oral gavage. KPT-330 was administered thrice weekly at 5 mg/kg for 21 days. MLN9708 and GSK2126458 were administered twice weekly at the appropriate doses, 4 mg/kg and 2 mg/kg respectively, for 21 days. Mice receiving combination treatment received KPT-330 thrice weekly and the second drug twice weekly. Combination-treated mice never received both drugs on the same day. Tumor growth was monitored via biweekly caliper measurements. After 21 days, all mice were euthanized via COin vivo or relative chemoresistance in vitro . MSupplemental Table S1). This led to the exploration of 45 unique drug combinations targeting distinct molecular pathways (b). From this dataset, three drug combinations from each cell line that showed increased cytotoxicity in combination compared to as a single agent were identified .A single dose of each selected drug that killed approximately half of the cells compared to vehicle (IC50) was used in a combinatorial assay with every other drug at its pre-defined dose   [20], [2.Fig. 2Nu(a), and active cell death corresponds with decreasing mass (b). In this study, UCD52 PDX cells were treated with vehicle, KPT-330, MLN9708, GSK2126458, KPT-330 + MLN9708, or KPT-330 + GSK2126458, respectively. Treatment with KPT-330, MLN9708, and GSK2126458 as single agents decreased hourly growth rate relative to vehicle. However, combination treatment with either KPT-330 and MLN9708 or KPT-330 and GSK2126458 decreased hourly growth rate to a significantly greater extent than treatment with a single agent alone .Cell biomass measurements with high speed live cell interferometry (HSLCI) have been utilized to determine the effect of drugs on individual cells within a population, the proof-of-principle of which we have previously demonstrated with carboplatin and BRAF inhibitors ,31. In ta, and acing mass b. In thint alone c, d.Fig.in vivo, including HCI-001, UCD52, WHIM2, and WHIM30. Analysis of bulk RNA-sequencing (RNAseq) from these PDX samples revealed consistent, positive expression of XPO1 transcript across basal-like TNBC PDX samples , with WHIM2 showing significantly lower XPO1 expression than the other three PDXs. Immunohistochemistry found that XPO1 protein was expressed in all tested PDXs . These data correlate with XPO1 immunohistochemistry performed by The Protein Atlas on 11 patient tumor samples where homogeneous moderate to high protein expression was found in > 75% of epithelial tumor cells for each sample. . ScRNAseq revealed that XPO1 was abundantly expressed in the majority of the basal-like cells, with more heterogenous expression present within the PDX samples than the cell lines . In each PDX, a subset of cells demonstrated XPO1 overexpression compared to the bulk of the population . At the cellular level, there were significant positive correlations between XPO1 expression and MTOR expression as well as XPO1 expression and PIK3CA expression . Interestingly, there was also a significant positive correlation between expression of XPO1 and expression of MKI67, a known marker of proliferation Supplemental Fig. S4) Fig. 4h). High G2M pathway scores have been associated with high expression of other proliferation-related gene sets and worse clinico-pathologic features Four basal-like patient-derived xenografts were utilized for a set of experiments in vivo was evaluated on four basal-like TNBC PDXs: HCI-001, UCD52, WHIM2, and WHIM30 . The patients from whom the HCI-001 and WHIM2 PDXs were derived presented with distant metastases and subsequently died from metastatic disease; the patient from whom the WHIM30 PDX was derived presented with no metastases [in vivo treatments found that the combination of KPT-330 and GSK2126458, but not KPT-330 and MLN9708, resulted in significantly smaller tumors than treatment with either monotherapy in the WHIM2 PDX . Further studies found that combination treatment with KPT-330 and GSK2126458 produced antitumor activity in all four PDXs . In WHIM2 and HCI-001, the combination of KPT-330 and GSK2126458 demonstrated significantly greater antitumor activity than either single agent, as determined by final tumor surface area and tumor mass . Mice did not demonstrate any signs of acute toxicity, and on average, there were negligible changes in mouse mass (± 5%). Hematological analysis of drug treated mice did not find any appreciable differences in erythrocytes or thrombocytes between vehicle and drug combination treated mice.The efficacy of KPT-330, MLN9708, GSK2126458, or combination treatment with KPT-330 and MLN9708 or GSK2126458 for inhibiting tumor growth tastases ,38. HCI-tastases ,39. Thertastases . Initialp < 0.001) in tumors as compared to normal tissue (a). Across all TCGA samples, XPO1 was, on average, significantly overexpressed in TCGA breast cancer patient samples compared to adjacent normal tissue (b). When XPO1 expression was compared across TCGA breast cancer samples, basal-like tumors showed significantly greater XPO1 expression than other breast cancer subtypes . Similar trends in XPO1 expression were observed utilizing a separate public dataset .The Cancer Genome Atlas (TCGA) data for ductal breast carcinoma was used to evaluate XPO1 expression across different normal and cancerous breast sample groups. In patient-matched normal breast and breast tumor samples, XPO1 was significantly overexpressed , which is an FDA-approved therapeutic for multiple myeloma and lymphoma. Arango et al. (2017) demonstrated the promising preclinical antitumor activity of KPT-330 against TNBC in vivoKPT-330 has previously been shown to synergize with a variety of chemotherapies and targeted drugs in both TNBC and other models of cancer. Arango et al. (2017) demonstrated that KPT-330 synergized with select chemotherapies in TNBC PDXs We found that within basal-like patients, XPO1 overexpression was positively correlated with brain relapse and lung relapse. High cellular proliferation rates have long been known to be drivers of metastatic ability All data are available upon publication. The scRNAseq data is available at the NCBI Gene Expression Omnibus: GSE174391.Narmeen S. Rashid: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Visualization. Nicole S. Hairr: Investigation, Writing – review & editing. Graeme Murray: Methodology, Formal analysis, Investigation, Writing – review & editing. Amy L. Olex: Software, Formal analysis, Data curation, Writing – review & editing. Tess J. Leftwich: Investigation, Writing – review & editing. Jacqueline M. Grible: Investigation, Writing – review & editing. Jason Reed: Resources, Supervision, Writing – review & editing. Mikhail G. Dozmorov: Software, Formal analysis, Writing – review & editing, Visualization, Supervision. J. Chuck Harrell: Conceptualization, Methodology, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Background: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. Methods: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients. Malignant pleural mesothelioma (MPM) is a malignancy originating from the pleural mesothelium, a serosal membrane covering the thoracic cavity. This rare type of cancer is heavily associated with asbestos exposure and has an infaust prognosis with median survival ranging from 9–15 months after initial diagnosis  or carboMost human mesothelial cells affected by asbestos-related microfibers undergo apoptosis. Some escape the programmed cell death and accumulate pro-oncogenic mutations, resulting in malignant transformation and MPM development . These mNew therapeutic approaches are needed to overcome the high mortality of MPM, bearing in mind that the incidence peak of MPM is expected within this decade . TherefoThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.p = 0.036, MK12: p = 0.044)  .The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect could be observed using CM conditioned by IMR-90 than those conditioned by MRC-5, the two activated myofibroblast cell lines used for conditioning of the medium by secretion of (soluble) factors. In general, epithelioid NCI-H2052 cells treated with CM of FB showed a distinct phosphorylation pattern illustrated by unsupervised clustering, with many hyperphosphorylated phosphosites (blue shaded) . BiphasiThe viability of treated cell lines was analyzed using enzyme activity-based assays. Overall, enhanced phosphorylation of RON, MAPK3, CD3Z, EGFR, GSK3B, and JAK1 showed significant associations with increased viability rates in each treated cell line, compared to cell lines treated with control medium .Moreover, enhanced apoptotic rates are associated with the phosphorylation of multiple phosphosites . Contrarp = 0.034), resulting in increased migration behavior of MPM cells treated with FB CM.In addition, the invasive behavior of MPM cells under the influence of FB CM was assessed by the implementation of cell migration and invasion assays for each MPM cell line. Kinase activity analysis of these treated cells showed a significant association between decreased phosphorylation of CDK4  was performed. GSEA utilizes molecular interaction networks outlined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) to map out increased target phosphorylation in a specific molecular pathway, depending on a response variable.p ≤ 0.001, normalized enrichment score: 1.88), Oocyte meiosis , the Fc epsilon RI signaling pathway , the MAPK signaling pathway , the Neurotrophin signaling pathway , the signaling pathways regulating pluripotency of stem cells , Long-term potentiation  and Axon guidance . Furthermore, significant enrichment has been calculated for the Fc gamma R-mediated phagocytosis  and Melanogenesis . The mentioned pathways all show activation in MPM cell lines treated with FB CM , Prostate cancer , Glioma , and Tuberculosis  , Pancreatic cancer , Choline metabolism in cancer , Kaposi-sarcoma-associated herpesvirus infection , ErbB signaling pathway , and Fc epsilon RI signaling pathway . Activation of all of those pathways is significantly associated with increased levels of apoptosis  was enriched in association to elevated cell viability  . Furtherpoptosis . Moreoveiability .p-value of enrichment, exact targets included in the gene sets, and those differentially regulated, can be found in Details of the GSEA regarding cell state, including normalized enrichment score, the Until recently, the tumor microenvironment (TME) role and importance have not been in the focus of mesothelioma research. However, the complex interactions of extracellular signaling within TME itself and its influence on tumor cells gain more attention, especially regarding new potential treatment options. Interactions between CAFs, tumor cells, and the host’s immune system are of particular interest as they hinder an effective immune response and aid tumor progression, thus ultimately leading to failure of chemotherapy . RegardiThe tumor-stromal tissue comprises fibroblasts, endothelial/immune cells, and extracellular matrix (ECM) components, all different from their dormant counterparts in homeostatic tissues . In homeIn this study, we simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. Limits of this study are the miss of nontumoral cells and the use of already FAP-expressing myofibroblasts. This has to be considered by analyzing the gained results. The use of a nontumoral cell line as control is challenging. The cell line MET-5a is often used as a benign version of MPM cells. However, this cell line is SV40-immortalized and thus do show altered culture performance . The mesWe observed that the effect of treatment with FB derived CM in MPM varies for each cell line related to the overall kinase pattern. A much stronger effect was observed after the usage of CM from IMR-90 in comparison to those from MRC-5. In a previous study, we were able to identify that cell–cell interaction via cytokines seems to be of greater importance in patients with an epithelioid subtype of MPM . These rThe MAPK pathway is a complex, interconnected signaling cascade with frequent involvement in oncogenesis, tumor progression, and drug resistance . These hThe results of our approach are consistent with this line of thought, as both MAPK and RAF signaling pathways are affected in FB CM-treated cell lines, especially in the NCI-H2052 cell line. Several of these interlocking kinases showed altered activation levels, probably significantly contributing to measured changes in viability and increased proliferation rates.Concerning the induction of apoptosis, MAPK signaling pathway showed the highest enrichment score in Since targeted therapies have already been successfully used in several cancers to counteract the enormous survival advantage of cancer cells that establish a supportive TME , based oFibroblasts pass different states of activation in a so called “life cycle” . The norIn addition, MEK inhibition by trametinib is thought to reprogram CD8+ T lymphocytes into memory stem cells, further enhancing their antitumor properties . ThroughWe simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052 , MSTO-211H , and NCI-H2452  cell lines represented MPM. Embryonal myofibroblast cell lines, IMR-90 and MRC-5, were selected since they present a CAFs-like phenotype ,37. In aKinase activity of treated samples, as well as control cells (conditioned medium from the cell line itself), were prepared by creating a total of 17 experimental conditions (5 Control + 2 of each MPM + 3 of each FB) and subsequently analyzed using the PamStation12 .In short, all cells were cultivated in Rosswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (RPMI-mixture). Washing steps were performed twice in 5 mL of Dulbecco’s phosphate-buffered saline (DPBS) before media replacement. Trypsinization was performed with 2 mL of a 10% Trypsin-EDTA solution applied to the flask for 10 min at room temperature. CM was generated by applying two million cells of the desired cell type into T-175 flasks and a total volume of 24 mL of the RPMI-mixture mentioned above. After an incubation time of 48 h, the conditioned media was extracted and immediately used to treat awaiting cells. Treatment lasted for 24 h, after which cells were counted, thereby delivering proliferation rates. The ApoTox-Glo Triplex Assay kit  was used to measure viability, necrosis, and apoptosis. The evaluation was carried out using the Promega GloMax Multi + Detection System. For assessment of MPM cell-invasive behavior under the influence of FB CM, a 24-Well Cell migration and Invasion Assay  was used for all available MPM samples.All samples that did not serve as positive controls went through treatment in technical triplicates in all analyses mentioned above. The experiment has been set up independently twice, resulting in biological replicates. All technical triplicates were summarized as a mean for all further calculations. Biological replicates were treated as separate samples within the data analysis.Following treatment, protein isolation was performed according to the protocol 1160 from the PamGene platform. Cells were lysed using M-PER Mammalian Protein Extraction Reagent containing HALT phosphatase inhibitor cocktail and HALTprotease inhibitor cocktail EDTA-free . Lysed cells were harvested by using a cell scraper. Lysates were stored in 5–20 μL aliquots at −80 °C. The protein concentration was determined via fluorometric quantification  using the protein assay kit according to the manufacturers’ instructions.®-4 was by application of 30 µL of 2% BSA (PamGene) while preparing the master mix with the appertaining reagent kit for PTK or STK PamChip arrays (PamGene). 5 µg (PTK assay) or 0.5 µg (STK assay) of desired sample protein lysate were then applied.Subsequently, also following manufacturers’ instructions, the Protein Tyrosine Kinase— and the Serine/Threonine Kinase Assay  were performed in order to determine changes in kinase activity compared to generated controls. Blocking the PamChipImage analysis of both PTK and STK assay was performed using the Bionavigator software (PamGene). Determination of kinetics for each phosphosite, normalization between spots, and cutoff calculation for countable phosphorylation of individual phosphosites have been performed according to the manufacturers’ instructions.t-test (parametric). To compare ordinal variables and factors with more than two groups, either the Kruskal–Wallis test (nonparametric) or ANOVA (parametric) was used to detect group differences.Statistical and graphical analyses of specific phosphosite phosphorylation levels were carried out using the R statistical programming environment V 4.0.2 . The Shapiro–Wilks test was applied to test for the normal distribution of each data set for ordinal and metric variables before exploratory data analysis. The resulting dichotomous variables underwent either the Wilcoxon–Mann–Whitney rank-sum test (nonparametric) or two-sided student’s Double dichotomous contingency tables were analyzed using Fisher’s exact test. To test the dependency of ranked parameters with more than two groups, Pearson’s chi-squared test was used. Correlations between metrics were tested applying Spearman’s rank correlation test and Pearson’s product-moment correlation testing for linearity.2 fold changes between means (if parametric) or medians (if nonparametric) of compared groups. Significant pathway associations were identified by gene set enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit  . Each run was executed with 1000 permutations. Finally, all associations were ranked according to the false discovery rate (p < 0.05).Basic quality control of run data was performed by mean-vs.-variances plotting to find outliers in the target or sample level. True differences were calculated by correlation matrices analysis. Pathway analysis is based on the KEGG database and was performed using the “pathview” package of R. Differences were specified by −logp-values were adjusted using the false discovery rate (FDR). The level of statistical significance was defined as p ≤ 0.05 after adjustment.Due to the multiple statistical tests, the The factors secreted by fibroblasts into fibroblast CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in analyzed MPM cell lines. This pathway plays a decisive role in cancer progression since it affects cell proliferation, differentiation, migration, senescence, and apoptosis. Based on our results, MEK- or ERK- inhibitors could be potent agents for therapy. Additionally, the inhibitors might offer a positive synergistic effect in combination with cisplatin-based treatment, as the cancer-progressing factors would be inhibited and additionally cisplatin would enhance cells undergoing apoptosis. The synergistic effect hopefully enhances the therapeutic outcome and therefore the survival time of MPM patients.


Glioblastoma is one of the most devastating cancer worldwide based on its locally aggressive behavior and because it cannot be cured by current therapies. Defects in alternative splicing process are frequent in cancer. Recently, we demonstrated that dysregulation of the spliceosome is directly associated with glioma development, progression, and aggressiveness.SF3B1 expression was also explored at the single-cell level across all cell subpopulations and transcriptomic programs. The association of SF3B1 expression with relevant clinical data  in different human cohorts was also analyzed. Different functional (proliferation/migration/tumorspheres and colonies formation/VEGF secretion/apoptosis) and mechanistic  assays were performed in three different glioblastomas cell models (human primary cultures and cell lines) in response to SF3B1 blockade (using pladienolide B treatment). Moreover, tumor progression and formation were monitored in response to SF3B1 blockade in two preclinical xenograft glioblastoma mouse models.Different human cohorts and a dataset from different glioma mouse models were analyzed to determine the mutation frequency as well as the gene and protein expression levels between tumor and control samples of the splicing-factor-3B-subunit-1 (SF3B1), an essential and druggable spliceosome component. BCL2L1 splicing.Our data provide novel evidence demonstrating that the splicing-factor-3B-subunit-1  is low-frequency mutated in human gliomas (~ 1 %) but widely overexpressed in glioblastoma compared with control samples from the different human cohorts and mouse models included in the present study, wherein SF3B1 levels are associated with key molecular and clinical features . Remarkably, in vitro and in vivo blockade of SF3B1 activity with pladienolide B drastically altered multiple glioblastoma pathophysiological processes  likely by suppressing AKT/mTOR/ß-catenin pathways, and an imbalance of Together, we highlight SF3B1 as a potential diagnostic and prognostic biomarker and an efficient pharmacological target in glioblastoma, offering a clinically relevant opportunity worth to be explored in humans.The online version contains supplementary material available at 10.1186/s13046-022-02241-4. Gliomas are the most frequent (> 80%) primary malignant brain tumor in adults . They arGFAP , TCGA [WEseq data [n = 746] and MSKCC [DNAseq data [n = 923] were interrogated through the cBioportal website  and OncoPrinter tool   Table S.n = 22) were obtained by intracranial surgery and non-tumor samples from 4 healthy brain donors by autopsy  and CGGA  were interrogated through the GlioVis-Tools (http://gliovis.bioinfo.cnio.es) (Table Sn = 5528)  were used as previously described , 23.SF3B1 simultaneously in human samples and cell lines. Specific primers for human transcripts including SF3B1, key GBM tumor markers, selected signaling pathway endpoints genes and 3 housekeeping genes were specifically designed with the Primer3 4.0.0 software (Table SACTB), hypoxanthine-guanine phosphoribosyl-transferase (HPRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH); Table STotal RNA from fresh non-tumor and tumor human samples and from GBM cell lines was extracted and DNase-treated, the concentration quantified, and the RNA retro-transcribed for qPCR analyses as previously described , 23. As n = 13) and control/non-pathologic samples (n = 4) from our cohort  and cultured according to the supplier’s recommendations. These cell lines were previously checked for mycoplasma contamination by PCR as previously reported .IDH1wt.Fresh tissue samples were collected within 15 min after intracranial surgery and immediately transported to the cell culture room in sterile cold S-MEM medium  complemented with 0.1% BSA (#A2153), 0.01% L-glutamine (#G7513), 1% antibiotic-antimycotic solution  and 2.5% HEPES (#H3537). Fresh tissue samples were dispersed into single-cells within the following 30 min by a mechanic/enzymatic protocol as previously reported . The sin50 determination (at 48 h) of pladienolide B in GBM cell lines and primary-GBM cell cultures. Least-squares regression was used as a fitting method to IC50 determination. As previously described  andescribed , cell prescribed .Apoptosis induction in response to pladienolide B treatment in GBM cell lines  was performed by using Caspase-Glo® 3/7 Assay  as previously reported . In addiPreviously described assay  was carrThe VEGF Human ELISA Kit  was used to quantify VEGF secretion in response to pladienolide B in GBM cell lines, following the manufacturer’s instructions and previously described methods .Colony formation assay was performed in GBM cell lines. Briefly, 300 or 500 cells/well (6-well plate) of U-87 MG and U-118 MG were seeded, respectively. Cells were pre-treated with pladienolide B (for 24 h and 48 h) before seeding the experiment to evaluate its effect on tumor onset/formation. Then, cells were seeded, medium was replaced, cells washed with PBS 1x, and crystal violet 0.5% plus glutaraldehyde 6% was added and incubated 45 min at room temperature. Finally, cells were rinsed 3 times with distilled water and left to dry at room temperature. Colonies (particles per well) were measured by ChemiDoc-XRS+ System  and analyzed using ImageJ 1.8.0_172 software.To determine protein levels, cell pellets were resuspended using pre-warmed SDS-DTT sample buffer  followed by sonication for 10 s and boiling for 5 min at 95 °C. Proteins were separated by SDS-PAGE electrophoresis with different poly-acrylamide percentage and transferred to nitrocellulose-membranes . Membranes were blocked with 5% non-fat dry milk (#T145.3) in Tris-buffered saline/0.05%-Tween-20 (#93773) and incubated with the primary-antibody , and their appropriate secondary-antibodies [anti-rabbit  or anti-mouse ]. Proteins were detected using an enhanced chemiluminescence-detection system  with dyed molecular-weight markers. As previously reported , a densiFoxn1nu/nu mice  were injected subcutaneously with 3 × 106 of U-87 MG cells in both flanks [resuspended in 100 μl of basement membrane extract ]. Once the tumor was clearly measurable, each mouse received an intra-tumor injection (12 days after cell-inoculation) with 50 μl of pladienolide B (100 nM) into one flank and vehicle  into the other flank. Tumor growth was monitored every 2 days using a digital caliper. Eight days after injection, mice were sacrificed and each tumor was dissected, fixed, and sectioned for histopathologic examination after H&E-staining. Examination of mitosis number, vascular proliferation, and necrosis was performed by expert pathologists. Additional tumor pieces were placed in liquid nitrogen and then frozen at -80 °C until RNA or protein extraction using Trizol-reagent or SDS-DTT buffer, respectively, and as previously reported [A preclinical xenograft mouse model to test pladienolide B in vivo was developed. 5-week-old ATHYM-reported . Micro-CFoxn1nu/nu mice were injected subcutaneously with 3 × 106 of U-87 MG pre-treated cells [n = 6 mice/condition ] using similar approaches described above. In this case, Slicer 4.11 software was used for 2D analysis together with 3D imaging rendering in the Micro-CT imaging. These experiments were performed according to the European Regulations for Animal Care under the approval of the university/regional-government research ethics committees.In addition, a preclinical xenograft mouse model was developed by inoculating U-87 MG cells previously pre-treated with pladienolide B at 100 nM in vitro for 24 h and 48 h. Specifically, 5-week-old ATHYM-KLF6, CRK, MST1R, CASP2, RAC1, MCL1, BIRC5, SPP1, and BCL2L1 using specific primer pairs of each gene. Specifically, primer design for BCL2L1 was performed using primers with specific annealing in ExonIIa (forward-sequence) and ExonIII (reverse-sequence) neighboring the splicing event . Details of the end-point PCR to detect splicing events have been previously reported . Speciel Fig. Sf.KLF6/CRK/MST1R/CASP2/RAC1/MCL1/BIRC5/SPP1/BCL2L1). Specifically, we performed a screening of the SVs of these genes in U-87/U-118 MG cells using end-point PCR-methodology (data not shown). Among them, only BCL2L1 showed an alteration in the SV-profile  analysis, and/or their presence by RT-qPCR. This latter analysis revealed that Bcl-xS/Bcl-xL ratio was elevated after SF3B1 inhibition with pladienolide B in GBM in vivo  and GBM cells in vitro (U-87/U-118 MG cells and primary-GBM cell cultures)  splicing event in U-87/U-118 MG cells  Fig. h, but noes) Fig. i. Particlls Fig. j, primarlls Fig. k, and inlls Fig. l. AdditiBCL2L1 is a potential driver of pladienolide B-mediated antitumor effects, we designed, validated, and used different ASOs that might be able to revert the Bcl-xS/Bcl-xL splicing process observed in response to pladienolide B. First, we performed an initial screening using U-87 MG cells to optimize the ASOs transfection and to identify which of the 5 designed ASOs were efficient in inhibiting the pro-apoptotic Bcl-xS variant and in promoting the anti-apoptotic Bcl-xL variant in response to pladienolide B treatment. Among the 5 designed ASOs, only two (ASO1_BCL2L1 and ASO3_BCL2L1) were able to efficiently revert the splicing process of BCL2L1 after pladienolide B treatment  GBM cells  treated with pladienolide B  transfected with ASO1_BCL2L1 and ASO3_BCL2L1 and treated with pladienolide B  in response to pladienolide B treatment was significantly blunted compared with non-transfected cells treated with pladienolide B  compared to other cancer pathologies /PDGFRA)  and diffr group) , in humaSF3B1 expression is directly associated with a worse OS rate in GBM patients, certainly, the main clinical problem in this pathology. This finding was corroborated in two external patient cohorts with GBM (Rembrandt/CGGA-dataset) and further supported by similar observations found in other tumor pathologies . Most notably, SF3B1 blockade strikingly decreased also GBM-stem/progenitor cells in terms of tumorspheres number and area, both relevant functional results that may help to explore the GBM onset and how to overcome the well-known GBM -resistance to different/current drugs , 51. It SF3B1K700E mutation can modulate the expression of key components of the AKT pathway with resulting increases in the migration/invasion of breast cancer cells  and their association with SF3B1 expression of different GBM cell lines. (b) Heatmap generated by the number of drugs  and their association with SF3B1 expression of different GBM cell lines. (c) Pathway annotation of different drugs hit in (a) and (b). Figure S4. Determination and accuracy of in vitro models and pladienolide B dose. (a) Comparison of the mRNA levels of SF3B1 between non-tumor brain tissues, GBM tissues, and GBM cell lines U-87 MG and U-118 MG. (b) Dose-response of pladienolide B in GBM cell lines  and (c) in primary patient-derived GBM cells. (d) IC50 of pladienolide B in vitro in the two GBM cell lines and primary patient-derived GBM cells. (e) Stratification of the primary GBM cell cultures used in the present study (n = 6) based on the percentage of reduction in the proliferation rate in response to pladienolide B  and on the SF3B1 expression levels in the same primary GBM cell cultures (bottom-heatmap). (f) Correlation between SF3B1 expression levels and the effect of pladienolide B (48 h of incubation) in terms of proliferation rate in the primary GBM cell cultures used in the present study. Asterisks  indicate statistically significant differences across different conditions. Figure S5. Unveiling the potential drivers of pharmacological blockade of SF3B1. (a) Heatmap showing the phosphorylated-protein level and (b) total-protein levels of several components of AKT/mTOR and ß-catenin pathways in GBM cells (U-87 MG and U-118 MG) after pladienolide B administration. (c)SF3B1 mRNA levels after pladienolide B administration in GBM cell lines U-87 MG and U-118 MG, in primary patient-derived GBM cells and the preclinical-xenograft GBM model. (d) Protein levels of SF3B1 after pladienolide B administration in GBM cell lines U-87 MG and U-118 MG. (e) Correlation of SRSF1 with SF3B1, MTOR, and CCTNB1 using CGGA-dataset. (f)SRSF1 mRNA levels after pladienolide B administration in GBM cell lines U-87 MG and U-118 MG, primary patient-derived GBM cells, and in the preclinical-xenograft GBM model. (g)BCL2L1-xS and BCL2L1-xL determined by qPCR in response to pladienolide B treatment in the preclinical-xenograft GBM model, (h) in GBM cell lines (U-87 MG and U-118 MG), and (i) in primary patient-derived GBM cells. (j)BCL2L1-xS and BCL2L1-xL determined by qPCR after pladienolide B administration in a primary non-tumor brain cell culture. (k) Correlation of SF3B1 expression with BCL2L1 A5SS PSI in GBM cell lines (U-87 MG and U-118 MG) after pladienolide B administration. Asterisks  indicate statistically significant differences across different conditions. Table S1. Clinical characterization of patients with gliomas obtained from the CGGA-dataset, TCGA-dataset and MSKCC-dataset (WEseq) to detect SF3B1 somatic mutations. Table S2. Clinical characterization of patients with GBM from three different cohorts . Table S3. Top 10 cellular markers used to identify different clusters identified in single-cell RNA seq dataset. Table S4. Clinical characterization of patients with GBM obtained from the CPTAC dataset. Table S5. Results of enrichment analysis from CGGA-dataset using SF3B1 high correlated genes (r > ± 0.800). Enrichment analysis was performed based on KEGG Pathways Analysis. Table S6. Specific primers for human transcripts used in this study were specifically designed and used in qPCR-based microfluidic assays. The official name of the genes, NCBI accession number of the transcripts, primers sequences, and product sizes of the amplification products are included. Table S7. Antisense Oligonucleotide (ASO) sequences for human BCL2L1 were used in this study to specifically promote the splicing of Bcl-xL variant. The 3′-5′ ASO sequence [2′ O-methoxy-ethyl (MOE) residues are shown in bold)], 5′-3′ BCL2L1 Targeted Region, and oligo sizes are included. Table S8. Drug resistance hits together with its corresponding annotated targeted pathway in SF3B1 high-expression cell lines using the Genomics of Drug Sensitivity in Cancer dataset. Table S9. Drug sensitive hits together with its corresponding annotated targeted pathway in SF3B1 low-expression cell lines using the Genomics of Drug Sensitivity in Cancer dataset.


The lack of treatment options for high-grade brain tumors has led to searches for alternative therapeutic modalities. Electrical field therapy is one such area. The Optune™ system is an FDA-approved novel device that delivers continuous alternating electric fields (tumor treating fields—TTFields) to the patient for the treatment of primary and recurrent Glioblastoma multiforme (GBM). Various mechanisms have been proposed to explain the effects of TTFields and other electrical therapies. Here, we present the first study of genome-wide expression of electrotherapy (delivered via TTFields or Deep Brain Stimulation (DBS)) on brain tumor cell lines. The effects of electric fields were assessed through gene expression arrays and combinational effects with chemotherapies. We observed that both DBS and TTFields significantly affected brain tumor cell line viability, with DBS promoting G0-phase accumulation and TTFields promoting G2-phase accumulation. Both treatments may be used to augment the efficacy of chemotherapy in vitro. Genome-wide expression assessment demonstrated significant overlap between the different electrical treatments, suggesting novel interactions with mitochondrial functioning and promoting endoplasmic reticulum stress. We demonstrate the in vitro efficacy of electric fields against adult and pediatric high-grade brain tumors and elucidate potential mechanisms of action for future study. Glioblastoma multiforme (GBM) is the most common and aggressive adult primary brain tumor . The curRecently, exciting work has demonstrated that glioma cells may rely on functional synaptic connections with both neurons and other tumor cells ,10. PotaAt present, the only clinical electrotherapy approved for adult GBM is the Optune™ system . The system includes a portable electric field generator which delivers low intensity 1–3 V/cm), intermediate frequency (200 kHz) and alternating electric fields—termed Tumor Treating Fields (TTFields) ,6. The T V/cm, inDevelopment of internalized electrotherapies seek to exploit the electrosensitivity of tumor cells while overcoming limitations of delivery from an external system, as well as overcoming concerns of cost of the TTFields therapy . Xu and Electrical fields have been delivered clinically to the human brain for many years via Deep Brain Stimulating (DBS) electrodes. In contrast to TTFields, DBS are internalized wires implanted surgically into the brain parenchyma. They have shown considerable success in treating movement disorders such as Parkinson’s disease, through delivery of electrical fields to designated anatomical targets such as the subthalamic nucleus or globus pallidus. Many thousands of patients have benefitted from DBS, and the electrode system is well-tolerated, internalized and safe to implant. IMT electrodes, in contrast, have not yet been used in humans. The electrical field delivered by DBS also differs from TTFields in frequency, generally being in the hundreds of hertz range, as opposed to the hundreds of kilohertz for TTFields.Here, we present our investigations into the effects of TTFields on pediatric brain tumor cell lines as well as comparative studies into the use of repurposed DBS electrodes as delivery devices for low frequency electric fields. We demonstrate how electric field treatments of varying intensities and frequencies delivered by either TTFields or DBS electrodes significantly affect primary and commercial brain tumor cell line viability and cell cycling, while not affecting the viability of non-dividing astrocyte cells. We also show how efficacy of electric fields, including the clinically relevant TTFields (Optune™), may be significantly improved with the addition of TMZ and mitotic inhibitors. Finally, we present genome-wide expression analysis of electric field-treated GBM cells, examining the genetic effects of both TTFields and DBS treatments on tumor cells, giving further suggestions to possible mechanisms of action.p ≤ 0.0001; t-test). The medulloblastoma cell lines, DAOY and UW228-3, had an optimal frequency of 300 kHz and 100 kHz, with reduction of 30% and 47% in the cell counts . The ependymoma cell lines tested, DKFZ-EPN1 and BXD-1425EPN, had optimal frequencies of 100 kHz and 200 kHz, with reductions of cell counts of 60% and 40% . Interestingly, the pediatric GBM cell line SF188 was determined to have an optimal frequency of 400 kHz as opposed to the accepted 200 kHz for adult GBM in the clinic; however, KNS42 cells were most affected by TTFields at 200 kHz . We determined the optimum frequency of TTFields for our cell lines over a panel of frequencies. TTFields have been tested in various cancer cell lines within the 100–500 kHz range , and thu 200 kHz .p = 0.0018 and p = 0.0024; t-test) following treatment with 200 kHz and 400 kHz TTFields, respectively. There were no significant differences between the 200 kHz and 400 kHz treatments  neural stem cells and post-mitotic astrocytes. We treated both types of cell for 72 h at 200 kHz and 400 kHz TTFields, followed by a PrestoBlue assay for assessment of cell viability and a cell count relative to the control cells. Cell counts confirmed our hypothesis that TTFields would only negatively affect the actively dividing cells, with a reduction in neural stem cell numbers but no significant effect on astrocyte numbers. The reduction of human neural stem cell counts following TTFields treatment were 41% and 37%  for U87 cells, 69%  for KNS42 cells, 65%  for SF188 cells, 39%  for GIN-5 cells and 30%  for GIN-31 cells  and KNS42  cell lines. Relative to treatment with 130 Hz, these reductions translate to a further reduction in U87  and KNS42   B.p ≤ 0.0001; t-test) when treated with DBS electric fields. There were no significant differences between the 200 kHz and 400 kHz treatments . Secondly, there was significant G0-phase accumulation following DBS treatment for U87, KNS42 and SF188 cells, with an increase of 15% , 14%  and 17% , respectively. Thirdly, S-phase depletion following DBS treatment was apparent in our cell lines U87, KNS42 and SF188, with a decrease of 13% , 10%  and 18% , respectively. Finally, there was no significant effect on the G2-phase of the cell cycle following DBS treatment for any of our cell lines  staining and flow cytometry. Firstly, DBS treatment appeared to have minimal cytotoxicity activity as shown through the lack of cell accumulation in the subG0-phase; however, U87 cell accumulation did achieve significance  for KNS42 and SF188 cell lines, 11% and 12%  for DAOY and UW228-3 cell lines and 13% and 8%  for BXD-1425EPN and DKFZ-EPN1 cell lines, respectively. The cell lines DAOY, UW228-3 and DKFZ-EPN1 also had significant G0-phase depletion at 8%, 7% and 7% , respectively. These cell lines also experienced a slight S-phase accumulation as well: 6%, 4% and 10% . None of the cell lines had significant levels of subG0-phase accumulation, suggesting that TTFields has a predominantly cytostatic over a cytotoxic effect in the tested pediatric brain tumor cell lines  for U87 cells, 61% and 69%  for KNS42 cells and 39% and 65%  for SF188 cells, respectively , KNS42 cells at 58% and 17%  and SF188 cells at 67% and 43% , respectively  in U87 cells, 9%  in KNS42 cells and 10%  in SF188 cells and when compared to cell lines treated with high dose paclitaxel alone. This combination did also cause a significant depletion of G0-phase accumulation, with no significant differences in accumulation in the G2/M-phase for our cells when compared to DBS-treated cells alone. Finally, combining the lower dose of paclitaxel with DBS treatment has no significant effect on the cell cycle  for SF188 cells, 32% and 28%  for UW228-3 cells and 23% and 30%  for BXD-1425EPN cells.As our data suggests that TTFields target the G2/M-phase of the cell cycle, we decided to further target this phase with the tubulin-interacting mitotic inhibitors, paclitaxel and mebendazole. This strategy of targeting the same region of the cell cycle with different therapies seeks to overcome the axis-dependent nature of TTFields which may limit efficacy of the treatment. We established the IC50 of paclitaxel (0.4–4.6 nM) and mebendazole (0.02–1.2 µM)  on variop ≤ 0.0002 and p = 0.0006, two-way ANOVA), UW228-3 cells at 34% and 22%  and BXD-1425EPN cells at 27% and 34%  , UW228-3 cells at 25% and 29%  and BXD-1425EPN cells at 19% and 16%   and 33% (high dose plus TTF) (y ANOVA) A. We alsy ANOVA) C. Overalp ≤ 0.0001 and p = 0.003, two-way ANOVA), KNS42 cells at 42% and 9% , SF188 cells at 49% and 21%  and GIN-31 cells at 17% and 9% , respectively , GCE-77 cells at 22% and 29% , GIN-28 cells at 2% and 5% , and GIN-31 cells at 12% and 7% , respectively  and MGMT-unmethylated (GIN-28 and GIN-31) cell lines, to be more representative of the clinical challenges of GBM treatment. We first established an IC50 of TMZ (0.8–1.2 µM)  on GBM cectively D.Overall, brain tumor cell lines all showed additional reduction in viability from the addition of electric fields to chemotherapy. Interestingly, the MGMT-unmethylated cell lines (GIN-31 and GIN-28) both benefited from additional electric field treatment with the TMZ, though to a much more modest degree than with methylated cell lines.To further explore alternative potential mechanisms of electric fields as a whole, we ran TTFields or DBS-treated and control KNS42 and GIN-31 cell lines on a genome-wide mRNA expression array (Clariom™ S Human Assays) to produce genome-wide profile of expression for all genes. Hierarchical clustering analysis showed differential expression profiles of our cell lines. Firstly, we observed that DBS electric fields treatment caused differential expression compared to untreated controls of 4539 and 5406 genes for KNS42 and GIN-31 cells, respectively . Of thesGene ontology analysis implicated that the commonly differentially expressed genes were related to genes involved in mitochondrial and ER functioning, including, electron transport, metabolism, ion signaling and protein folding. It is worth noting that all of these associated genes are significantly downregulated, implying that these processes are also downregulated. PrestoBlue assay changes are significantly dependent on mitochondrial function and show substantial decreases as illustrated in previous results sections. Furthermore, KEGG pathway analysis demonstrated significant over-representation of genes known to be involved in the Parkinson, Alzheimer and Huntington disease pathways in our data . AlthougFinally, we chose MT-ND5, CTSB and BTNL9 to validate the gene expression findings at a protein level via Western blot; of note, MT-ND5 was the most downregulated gene (>−100-fold downregulation) and BTNL9 was the only upregulated gene of the top 50 target genes . Our WesWe have shown that both electrical treatments (TTFields and DBS) exhibit efficacy against adult and pediatric brain tumor cell lines in vitro, and this efficacy increased with optimization of frequency and other parameters. DBS electric field efficacy was increased through increasing the intensity of the field and through manipulation of frequency between 60 Hz and 190 Hz. We tested frequencies approaching the threshold that is clinically relevant for DBS purposes (~130 Hz)  to test TTFields treatment efficacy was increased through manipulation of the frequency between 100 kHz and 400 kHz; we did not explore the effects of manipulating the intensity of TTFields as this has already been established in vitro . We founPrevious reports have hypothesized that an actively dividing cell is the target of electric field treatment , and a fWe have demonstrated how both electrical treatments cause significant shifts in cell cycle phase accumulations, with DBS electric fields promoting G0-phase accumulation and TTFields treatments promoting G2/M-phase accumulation in brain tumor cell lines. The accumulation of TTFields treated cells in the G2/M phase has been observed in many different cell lines and tumor types and has been attributed to perturbation of key mitotic proteins, e.g., tubulin and septin proteins ,18. A nuWe have tested a variety of chemotherapies in combination with DBS electric fields, showing that the efficacy of these drugs may be increased with the addition of electric fields treatment. Of particular note, we tested a variety of MGMT-unmethylated and MGMT-methylated GBM cell lines and still observed an increase in drug efficacy in the MGMT-unmethylated cell lines when combining electric fields treatment. This may be of future clinical relevance given how MGMT-unmethylated tumors are typically more chemoresistant to chemotherapies, particularly alkylating agents . TTField+ to NADH (Nicotinamide Adenine Dinucleotide) [We examined how electric fields affect treated GBM cells at the genetic level to help guide future research and identify novel mechanisms of action. Firstly, we clearly observed striking similarities between the gene expression profiles of DBS electric fields and TTFields treated cells, as with the gene ontologies. A significant number of the affected genes and pathways were associated with mitochondrial and endoplasmic reticulum functioning synonymous with the unfolded protein and the ER stress response. The downregulation of mitochondrial and ER functioning could potentially be a result of the prolonged mitotic arrest from the electrical treatment, with free radical accumulation leading to cellular organelle damage and the corresponding alteration in the gene expression profile. Related observations have already been made in TTFields studies where it has been shown that prolonged TTFields exposure may promote ER stress , giving leotide) . This re2 incubator and have been STR genotyped to ensure correct identification and, in the case of the primary low-passage cell lines, that they maintain the same genetic profile as their parent tumor over multiple passages.GIN-28, GIN-31 and GCE-77 cells (patient-derived primary low-passage GBM lines) were grown in DMEM  supplemented with 15% FBS. U87-MG (adult GBM) cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). KNS42 and SF188 (Pediatric GBM) cells were grown in DMEM/F-12  supplemented with 10% FBS and 1% L-glutamine. DKFZ-EPN1 and BXD-1245-EPN (pediatric ependymoma) cells were grown in DMEM supplemented with 10% FBS. DAOY (SHH medulloblastoma) cells were grown in MEM  supplemented with 10% FBS, 1% L-glutamine, and 1% sodium pyruvate. UW228-3 (SHH medulloblastoma) cells were grown in DMEM/F-12 supplemented with 10% FBS. Neural stem cells and post-mitotic astrocytes were derived from human induced pluripotent stem cells and grown as described by Julia et al., 2017. All cells were maintained at 37 °C in a COTTFields were applied using the Inovitro™ system . The system consists of two pairs of perpendicular transducer arrays on the outer walls of a ceramic Petri dish. A sinusoidal waveform generator was attached to the transducer arrays to produce alternating electric fields at frequencies between 100–400 kHz and 1.75 V/cm intensity. The electric fields alternated orientation of 90° every second. Temperature was measured to be 37 °C inside the dishes by 2 thermistors attached to the ceramic walls. DBS electric field treatment was achieved through delivering electric fields from neurostimulators  via 1 × 8 low impedance compact leads  and programmed with the N’Vision™ programmer  with a bipolar configuration with one anode and multiple cathode contacts. The parameters manipulated for the electric field treatment were intensity (V), frequency (Hz) and pulse-width (µs). DBS leads were inserted into T25 flasks containing cells and were programmed to chronically deliver electric fields at the desired parameters.Cells were plated and treated for 7 days with DBS electric fields, with cell viability measurements taken at the Day 0, 1, 5 and 7 time-points. A range of intensities were tested: 0 V (sham), 0.5 V, 1.0 V, 2.5 V, 5.0 V and 10.0 V, and a range of frequencies were tested: 60 Hz, 90 Hz, 130 Hz, 160 Hz, 190 Hz, 500 Hz and 1000 Hz.PrestoBlue™ was added to each sample at a time-point at a 1:10 dilution. Samples were incubated at 37 °C for 30 min. 100 µL (triplicate) of the PrestoBlue solution was transferred to a black-bottomed 96-well plate, and fluorescence was analyzed via UV-Vis Spectroscopy at excitation/emission wavelengths at 560/590 nm with the FLUOstar Omega (BMG Labtech).Cells were treated with DBS electric fields for 5 days and then fixed in cold methanol and stored at 4 °C. Samples to be analyzed for cell cycle were incubated in a propidium iodide (PI) solution containing 0.1% Triton X-100, 10 µg/mL PI  and 100 µg/mL DNase-free RNase A in cold PBS. The samples were incubated at room temperature in the dark for 30 min. Cell cycle analysis was performed via flow cytometry using FC500 Flow Cytometer  and data were processed with Weasel V.3.1. software.Cells were treated with TMZ , paclitaxel  and mebendazole  as a monotherapy or in combination with DBS electric fields. Each of the drugs were administered at their IC50 (high dose) or 10% IC50 (low dose), with the IC50s being determined through drug-response curves. The drugs were administered with and without DBS electric fields for 5 days before cell viability and cell cycle were assessed.Human Clariom™ S Arrays  were used for an unbiased, whole transcriptome gene expression analysis of TTFields and DBS-treated GBM cell lines. Microarray processing was performed by Dr. Marcos Castellenos and Dr. Iqbal Khan of Nottingham Arabidopsis Stock Centre (NASC), University of Nottingham.®. The criteria of significant genes were a false discovery rate (Bonferroni Correction) and p-value of <0.05, and a fold-change of <−2.0 and >2.0. An unsupervised clustering method was used, and analyses were displayed as heatmaps on a genome-wide scale within each electrical treatment. Gene ontology and KEGG pathway analyses were performed using Enrichr . Enrichr was used at http://amp.pharm.mssm.edu/Enrichr/ (accessed on 1 September 2021).Hierarchical clustering analyses were performed using Partekg for 30 min. Protein lysates were then retrieved and quantified via Bradford assay. 20 µg of each protein sample was separated on a 10% acrylamide gel and transferred via electrophoresis to nitrocellulose membranes. The membranes were blocked and then incubated at 4 °C overnight with the primary antibodies: MT-ND5 , CTSB , BTNL9  and GAPDH 1:10,000 . Membranes were washed and then incubated at room temperature for 1 h with complimentary secondary antibodies. Membranes were washed and then incubated in enhanced chemiluminescence and Western blotting detection solutions before Western blot images were developed.Cell pellets were collected following electric field treatment and then treated with protease cocktail buffer on ice before centrifugation at 13,000×


Background: Hexokinase 2 not only plays a role in physiological function of human normal tissues and organs, but also plays a vital role in the process of glycolysis of tumor cells. However, there are few comprehensive studies on HK2 in esophageal carcinoma (ESCA) needs further study.Methods: Oncomine, Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were used to analyze the expression differences of HK2 in Pan-cancer and ESCA cohort, and to analyze the correlation between HK2 expression level and clinicopathological features of TCGA ESCA samples. GO/KEGG, GGI, and PPI analysis of HK2 was performed using R software, LinkedOmics, GeneMANIA and STRING online tools. The correlation between HK2 and ESCA immune infiltration was analyzed TIMER and TCGA ESCA cohort. The correlation between HK2 expression level and m6A modification of ESCA was analyzed by utilizing TCGA ESCA cohort.Results: HK2 is highly expressed in a variety of tumors, and its high expression level in ESCA is closely related to the weight, cancer stages, tumor histology and tumor grade of ESCA. The analysis results of GO/KEGG showed that HK2 was closely related to cell adhesion molecule binding, cell-cell junction, ameboidal-type cell migration, insulin signaling pathway, hif-1 signaling pathway, and insulin resistance. GGI showed that HK2 associated genes were mainly involved in the glycolytic pathway. PPI showed that HK2 was closely related to HK1, GPI, and HK3, all of which played an important role in tumor proliferation. The analysis results of TIMER and TCGA ESCA cohort indicated that the HK2 expression level was related to the infiltration of various immune cells. TCGA ESCA cohort analyze indicated that the HK2 expression level was correlated with m6A modification genes.Conclusion: HK2 is associated with tumor immune infiltration and m6A modification of ESCA, and can be used as a potential biological target for diagnosis and therapy of ESCA. Recent studies show that Esophageal carcinoma (ESCA) ranks seventh in terms of incidence and sixth in mortality overall . DespiteHexokinase (HK) is an enzyme capable of phosphorylating hexose and is a rate-limiting enzyme in the glycolytic pathway. Four subtypes of human HK have been discovered, which are encoded by the genes HK1, HK2, HK3, and HK4, respectively . HK1 is Tumor immunotherapy and N6-methyladenosine (m6A) modification are hot spots in tumor treatment, which are extensively used in the investigation and therapy of ESCA . HoweverOn this project, we processed The Cancer Genome Atlas (TCGA) ESCA cohort and performed bioinformatics analysis using the R software, online website and other databases. The differences of HK2 expression in different cancer tissues were studied, and the mRNA and protein expression of HK2 in ESCA were verified by cell assay and immunohistochemistry (IHC). The co-expression gene networks of HK2 in ESCA were analyzed, and the possible biological mechanisms and signal pathways involved in associated genes were analyzed. Eventually, the correlation between the expression difference of HK2 and the tumor immune cell infiltration and m6A modification of ESCA will be explored, which will help to study the potential pathogenesis of ESCA.The protocol of this study had been approved by the Ethics Committee of Taihe Hospital Affiliated of Hubei University of Medicine  (document NO.2021KS021) and conducted according to the principles stated in the Declaration of Helsinki, and the requirement to obtain informed consent was waived.1  cohort to analyze the difference of HK2 expression between ESCA and normal tissues. UALCAN database5  function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway that co-expressed genes may participate in, and the ggplot2 software package was used to visually analyze the data.The co-expression network of HK2 in TCGA ESCA cohort was analyzed using LinkedOmics7  module of the TIMER tool was used to associate the genetic copy number variation (CNV) of HK2 with the relative abundance of tumor infiltrating cells. The CIBERSORT . These results believe that HK2 plays a potential role in the occurrence and development of ESCA.The ESCA cohort of TCGA and GEO was utilized to analyze the differential expression of HK2 in ESCA tissue samples and normal tissue samples. Both TCGA and GEO cohort analysis indicated that the HK2 expression level in ESCA tissue samples was remarkable higher than that in normal tissue samples . To furtP < 0.05). The expression of HK2 in normal weight patients was higher than that in extreme weight patients (P < 0.05). The expression of HK2 in esophageal squamous cell carcinoma was higher than that in esophageal adenocarcinoma (P < 0.05). The expression of HK2 in tumor grade 3 was lower than that in tumor grade 2 (P < 0.01) and 1 (P < 0.05), respectively. However, the expression level of HK2 did not differ in different gender and age groups , 6 cell component (CC), 19 molecular function (MF) and 6 KEGG. The bubble graph shows the top 15 messages of GO and KEGG, including 5 messages of BP, CC, and MF. The results of GO functional annotations showed that HK2 co-expressed genes were mainly participated in the cell adhesion molecule binding, cell-cell junction, and ameboidal-type cell migration . KEGG paA GGI network composed of 21 genes was constructed, and the HK2 gene was surrounded by 20 nodes. Their functionality was analyzed using the GeneMANIA database . These nUse STRING to further analyze PPI network of HK2. The results show that HK2 is related to HK1 (hexokinase 1), GPI (glucose-6-phosphate isomerase), HK3 (hexokinase 3), G6PD (glucose-6-phosphate 1-dehydrogenase), PGM1 (phosphoglucomutase-1), FBP1 , GFPT1 (glutamine–fructose-6-phosphate aminotransferase 1), MPI (mannose-6-phosphate isomerase), GFPT2 (glutamine–fructose-6-phosphate aminotransferase 2), GNPNAT1 (glucosamine-phosphate N-acetyltransferase 1), PGM2 (phosphoglucomutase-2), H6PD (hexose-6-phosphate dehydrogenase), PFKM (ATP-dependent 6-phosphofructokinase), TALDO1 , PFKL (ATP-dependent 6-phosphofructokinase), PFKP (ATP-dependent 6-phosphofructokinase), ENO1 , TPI1 (triosephosphate isomerase 1) and PGD (6-phosphogluconate dehydrogenase). The combined scores were 0.986, 0.97, 0.967, 0.955, 0.944, 0.941, 0.937, 0.935, 0.933, 0.931, 0.929, 0.925, 0.918, 0.905, 0.866, 0.864, 0.704, 0.66, and 0.495, respectively .r = –0.157, P = 3.62E-2), CD4+ T cell  and macrophage  , CD4+ T cell , macrophage , neutrophil  and dendritic cell  , CD4+ T cell  and macrophage  , CD4+ T cell , macrophage  and dendritic cell  , CD4+ T cell  and macrophage  , CD4+ T cell , macrophage  and neutrophil  , CD4+ T cell (r = 0.209 P = 5.02E-3) and macrophage   . In addi2.59E-4) . The exp1.30E-4) . The exp2.28E-2) . LOC72872.33E-6) . RASSF4 6.16E-4) . PBXIP1 1.80E-6) . These rtic cell .P = 0.004), resting NK cells (P = 0.018), M0 macrophages (P = 0.002), activated mast cells (P = 0.042) and resting mast cells (P = 0.025). Overall, HK2 and its co-expression genes have correlation with tumor immune cell infiltration in ESCA , HNRNPA2B1 , YTHDC2  and YTHDF2  (n = 81) and low expression group (n = 81) according to the expression level of HK2. We further evaluated the expression abundance of m6A associated genes between high and low HK2 expression groups to study whether m6A modification was different between the two groups (P < 0.05). These analyses suggest that HK2 is strongly associated to m6A modification in ESCA.The TCGA ESCA cohort was analyzed to study the association between the expression of HK2 and the expression of 20 m6A related genes in ESCA. Analysis indicated that the expression of HK2 was remarkable positively associated with 4 m6A associated genes in ESCA, including IGF2BP2 (= 0.010) . The sca= 0.010) . In addio groups . AnalysiAs one of the isozyme subtypes of HK, HK2 is the first rate-limiting enzyme in the catalytic glycolysis pathway and mainly distributed in the cytoplasmic region . HK2 ovein vitro experiments. Bioinformatics is widely used in tumor research  can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Taihe Hospital Affiliated of Hubei University of Medicine. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.X-SL conceived the project and wrote the manuscript. X-SL, Y-JC, F-YL, and R-MW participated in data analysis. X-SL, J-ML, FT, D-BZ, WL, YG, and HZ participated in discussion and language editing. Z-JP reviewed the manuscript. All authors contributed to the article and approved the submitted version.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.