DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic, MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the  BCR-ABL1-positive leukemia cells, which are clinically sensitive to the GVL effect [MLL (KMT2A)-rearranged (MLLr) acute lymphoblastic leukemia (ALL) cells, which are clinically resistant to the GVL effect [Two recently established immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have dramatically improved the therapeutic outcome in poor prognostic BCP-ALL patients. One is chimeric antigen receptor (CAR) T-cell therapy targeting at CD19 . Both thL effect  frequentL effect . In contL effect , generalL effect . We alsoL effect . MoreoveL effect . AccordiDR4 [DR4 and/or DR5 genes may be associated with the DR4 and DR5 expression and consequently with the TRAIL sensitivity in BCP-ALL cells. However, to date, little is known about the relevance of the epigenetic modification of the DR4 and DR5 genes in BCP-ALL.Hypermethylation of the CpG island in the gene promoter is an epigenetic modification of gene expression. We previously reported that cell lines and patients’ samples of T-cell ALL (T-ALL) showed a TRAIL resistance in association with low cell-surface expression levels of DR4 and DR5 . Semi-quDR4 . These oDR4 and DR5 gene promoter in BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer (NGS). We found that hypermethylation of the DR4 and DR5 CpG islands is highly associated with a lack of cell-surface expression of DR4 and DR5, and TRAIL resistance in BCP-ALL cell lines. We further investigated the significance of the methylation status of the DR4 and DR5 genes in clinical samples of BCP-ALL patients.In the present study, we quantified the methylation status of the CpG islands of the MLLr-ALL cell lines  [BCR-ABL1-positive ALL cell lines  [TCF3-PBX1-positive ALL cell lines ; four TCF3-HLF-positive ALL cell lines  [ETV6-RUNX1-positive ALL cell lines (KOPN79 and Reh); two MEF2Dr-ALL cell lines (KOPN61 and KOPN70); and two DUX4r-ALL cell lines  were used in this study (2 at 37 °C. Forty-nine cryopreserved samples of childhood BCP-ALL (35 samples at diagnosis and 14 samples at relapse after chemotherapy) (Nine  KOCL69) ; six BCR YAMN91) ; seven T HAL-O1) ; and twois study . All celtherapy)  were ana4 cells/well) were cultured in the absence or presence of 100 ng/mL of rhsTRAIL in triplicate in 200 μL of RPMI1640 medium supplemented with 10% FCS in a flat-bottomed 96-well plate. The plates were incubated for 42 h, pulsed for the last 6 hours of the incubation with 3H-thymidine (1 μCi/well) and harvested onto glass-fiber filters. The level of radioactivity incorporated into DNA was measured by liquid scintillation counting. The percent inhibition by rhsTRAIL was calculated as the ratio of the radioactivity to that in the absence of rhsTRAIL.Sensitivities to TRAIL were determined as previously reported ,15,18,191 or mAb on ice for 30 min. After washing, the cells were incubated with phycoerythrin-conjugated streptavidin  on ice for 30 min, and then analyzed by flow cytometry. The relative florescence intensity (RFI) was calculated as the ratio of the mean fluorescence intensity of specific staining to that of control staining.Cell-surface expression of DR4 and DR5 was determined as previously reported using the monoclonal antibodies (mAbs) specific to DR4 and DR5 ,15,18,19DR4 and DR5 were quantified as previously reported by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis [DR4 and DR5 transcripts, triplicated samples containing 9 μL of cDNA with 10 μL of Taqman Universal PCR Master Mix  and 1 μL of 20 × Assays-on-demand Gene Expression Product  were pre-incubated at 50 °C for 2 min and subsequently at 95 °C for 10 min. Amplification was obtained by 40 cycles of reaction at 95 °C for 15 sec and 60 °C for 1 min. Fluorescence data were quantitatively analyzed on ABI Prism 7500 Sequence Detection System (Applied Biosystems). Nalm1, a TRAIL sensitive chronic myelogenous leukemia blast crisis -derived cell line that expresses DR4 and DR5 [GAPDH; Hs 99999905_m1, Applied Biosystems) was performed.Gene expression levels of analysis ,19. Tota and DR5 , was useDR4 gene with forward primer (5′-GGAAGGAAGTTTAGGGTTAGTTAATAG-3′) and reverse primer (5′-TACCAAATCAATCCAAAAAACAAC-3′) and DR5 gene with forward primer (5′-AAGTGTTTTTTTTAATTTATTTTTTTTAAG-3′) and reverse primer (5′-CAACTACAAATTCCACCACAAATTA-3′) using one cycle of 95 °C for 4 min, 40 cycles of 95 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min, with a final cycle of 72 °C for 7 min. For conventional Sanger sequencing, PCR products were cloned into a pTAC-2 vector using a TA PCR Cloning Kit  and sequenced. For next-generation sequencing, amplicon libraries were generated by Ion Plus Fragment Library Kit  and Ion Xpress Barcode Adaptors Kit (Thermo Fisher Scientific). Briefly, 50 ng amplicon was end-repaired, nick-repaired, and Ion Torrent adapters P1 and Barcode were ligated with DNA ligase. Following Agencourt AMPure XP purification , the individual libraries were quantified by quantitative real-time PCR then diluted, and finally pooled in equimolar ratios. The libraries were processed with the Ion OneTouch™ 2 System using Ion PGM™ Template OT2 400 Kit (Thermo Fisher Scientific) to produce 400 base-read libraries. Sequencing was performed by an Ion PGM™ Hi-Q Sequencing Kit (Thermo Fisher Scientific) using 850 flows on the Ion 318 Chip Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After sequencing, single processing and base-calling were performed using Torrent Suite 5.0.2 (Thermo Fisher Scientific). Methylation analysis was performed using MethylationAnalysis_Amplicon plug-in v1.3 (Thermo Fisher Scientific).Bisulfite PCR was performed as previously reported . GenomicETV6-RUNX1, TCF3-PBX1, iAMP21, dic, BCR-ABL1, MLLr, hypodiploid (<45 chromosomes), and polyploid (>67 chromosomes). The data of the paired samples at diagnosis and at relapse were also analyzed in 24 relapsed BCP-ALL cases. Percent methylation of DR4 and DR5 was evaluated by mean methylation level of CG dinucleotides that were annotated as both 0–200 bases upstream of the transcriptional start site (TSS200) and CpG island using Illumina HumanMethylation450 BeadChip  [Gene methylation of childhood BCP-ALL clinical samples were investigated using 450 k DNA methylation array database of Nordic Pediatric Hematology and Oncology (NOPHO)  . Among 7Mann–Whitney U test, Fisher’s exact test, Chi-square test, and Spearman’s correlation analysis were performed using R software version 3.5.2 (R Core Team 2018).DR4  was observed between the mean percent methylation of the DR4 gene and that of the DR5 gene in 32 cell lines  in all of nine MLLr-ALL and two MEF2Dr-ALL cell lines, while it was unmethylated (<1%) in all of four TCF3-HLF-positive ALL cell lines (DR5 gene (DR5 gene was methylated (>10%) in six of nine MLLr-ALL cell lines and all of two MEF2Dr-ALL cell lines, while it was unmethylated in all of four TCF3-HLF-positive ALL and two ETV6-RUNX1-positive ALL cell lines. Despite being limited to a small number of cell lines, these observations suggest that methylation status of the DR4 and DR5 genes may be associated with karyotypes of BCP-ALL cell lines.We next analyzed an association of the methylation status of the ll lines G. A simiDR5 gene H. The DRDR4 and DR5 genes and the gene and cell-surface expression levels of DR4 and DR5 in 32 BCP-ALL cell lines. Mean percent methylation of DR4 gene showed a significant negative correlation, both with the gene expression level of DR4 (R2 = 0.37) and with the cell-surface expression level of DR4 (R2 = 0.39)  and the cell-surface expression level of DR5 (R2 = 0.38)  A. Simila = 0.38) B.DR4 and DR5 genes with the sensitivity to anti-leukemic activity of rhsTRAIL [DR4 gene showed a significant negative correlation with the percent inhibition by rhsTRAIL (R2 = 0.58)  between mean percent methylation of the DR5 gene and percent inhibition by rhsTRAIL was 0.23   as well as six unmethylated cell lines (DR4 and DR5 <1%)  were highly sensitive to rhsTRAIL (percent inhibition ≥80%). In contrast, three of five DR5-preferentially unmethylated cell lines  as well as 13 of 18 methylated cell lines (DR4 and DR5 ≥1%) were resistant to rhsTRAIL (percent inhibition <25%). Among them, all of the 11 highly methylated cell lines (DR4 and DR5 ≥10%) were resistant to rhsTRAIL (percent inhibition <25%). These observations indicate that the methylation status of DR4 and DR5 (particularly DR4) is tightly associated with rhsTRAIL sensitivity in BCP-ALL cell lines  C. To comwas 0.23 C. Since sitivity D. Of notll lines E.DR4 and DR5 genes in 49 childhood BCP-ALL samples by sequencing bisulfite PCR-product using the NGS. In contrast to cell lines, mean percent methylation levels of the DR4 and DR5 genes were less than 1% in the majority of the samples  was observed between the mean percent methylation of the DR4 gene and that of the DR5 gene in 49 samples  [R2 = 0.35, p < 0.001) was observed between the percent methylation of the DR4 gene and that of the DR5 gene in 459 BCP-ALL samples -positive; 2 of 28 MLLr-positive; 1 of 5 hypodiploid) ALL samples  ALL samples  in all of 187 high hyperdiploid ALL, 164 ETV6-RUNX1-positive ALL, 10 iAMP21-positive ALL, and 19 BCR-ABL1-positive ALL samples. In the bone marrow samples in complete remission and normal T-cells and B-cells, % methylation of the DR4 and DR5 genes were approximately 2–3%. In the majority of samples from patient at relapse, the percent methylation of DR4 and DR5 genes was almost unchanged compared to that of diagnosis. However, in the paired samples at diagnosis and at relapse, methylation levels of DR4 and DR5 genes were upregulated in several cases at relapse  . A signi samples C. In com samples D and 16  samples E showed  relapse F. These DR4 and DR5 genes by using a BCP-ALL cell line as a model system. In BCP-ALL cell lines, the methylation status of the DR4 and DR5 genes was associated with their mRNA and cell-surface expression levels and their rhsTRAIL sensitivities, indicating that epigenetic modification of the DR4 and DR5 genes due to hypermethylation is a mechanism for TRAIL resistance in BCP-ALL. However, we observed discrepancies in the methylation status of the DR4 and DR5 genes between the cell lines and the clinical samples. The DR4 and the DR5 genes were not methylated in the majority of clinical samples. This difference in methylation status may be partly attributed to the different distribution of karyotypes between the cell lines and clinical samples. In BCP-ALL cell lines, the DR4 and DR5 genes were unmethylated in ETV6-RUNX1-positive ALL cell lines, while being frequently methylated in MLLr-ALL cell lines. Similarly, in the genome-wide DNA methylation data of BCP-ALL samples in the NOPHO cohort, the DR4 and DR5 genes were exclusively unmethylated in the samples with ETV6-RUNX1 but relatively frequently methylated in the samples with MLLr. In our series of BCP-ALL cell lines, only 6% (2/32) of cell lines had ETV6-RUNX1, while 31% (10/32) of cell lines showed the MLLr karyotype. In contrast, in the NOPHO cohort, over one-third (164/459) of the samples had ETV6-RUNX1, while only 6% (28/459) of the samples showed the MLLr karyotype. We also observed upregulation of the DR4/DR5 methylation status in several relapsed cases  cell lines and is moderately sensitive to rhsTRAIL , suggesting that the DR5 gene, but not the DR4 gene, plays an essential role in CAR T-cell cytotoxicity against Nalm6 due to unmethylated status of the DR5 gene. Meanwhile, we previously reported that cell-surface expression of TRAIL on anti-CD19 CAR T-cells is upregulated by the co-culture with targeted BCP-ALL cells [As a new therapeutic modality, immunotherapy using anti-CD19 CAR T-cells and blinatumomab is promising for refractory BCP-ALL cases. In a recent genome-wide CRISPR-Cas9 screen of Nalm6, a BCP-ALL cell line, toxicity . In our LL cells . These oDR4 and DR5 genes is associated with rhsTRAIL resistance of BCP-ALL cell lines. However, gene silencing of the DR4 and DR5 genes due to hypermethylation was uncommon in the majority of BCP-ALL cases at diagnosis. Accordingly, resistance to immunotherapy due to TRAIL resistance as a result of hypermethylation of the DR4 and DR5 genes is unlikely in the majority of BCP-ALL cases, at least at disease onset. In several relapsed cases of the NOPHO cohort study, upregulation was observed in the methylation status of the DR4 and DR5 genes. Although the precise treatment in these cases was unknown, this observation suggests the possibility that acquired hypermethylation of the DR4 and/or DR5 genes may be one of mechanisms for relapse particularly after immunotherapy.Blinatumomab is a bispecific T-cell engager antibody simultaneously binding CTLs and CD19-positive BCP-ALL cells , suggestDR4 and/or DR5 genes was observed in certain karyotypes such as dic, MLLr, and hypodiploidy. Thus, when the BCP-ALL patients with these karyotypes are treated by immunotherapy, methylation status and/or gene/cell-surface expression levels of the DR4 and DR5 genes might be useful biomarkers to predict therapeutic responses. In this context, we previously reported that TCF3-HLF-positive ALL cells are highly sensitive to TRAIL, since TCF3-HLF fusion transcription factor effectively transactivates the DR4 and DR5 gene expression [DR4 and DR5 genes are unmethylated in all of four TCF3-HLF-positive ALL cell lines. TCF3-HLF-positive ALL is the most unfavorable type of childhood BCP-ALL due to resistance to conventional chemotherapy [TCF3-HLF-positive ALL patients resistant to a conventional chemotherapy [TCF3-HLF-positive ALL patients.Of importance, in the analysis of clinical samples, hypermethylation of the pression . Indeed,otherapy ,26. Of notherapy . Our finDR4 and DR5 genes is unlikely in the majority of BCP-ALL cases, particularly in the cases with favorable karyotypes such as hyperdiploidy and ETV6-RUNX1. Since TRAIL/death receptor system plays an essential role in the anti-leukemic activities of immunotherapy using anti-CD19 CAR T-cells, our findings provide an epigenetic rationale for clinical efficacy of immunotherapy in BCP-ALL patients. Moreover, in certain BCP-ALL cases with unfavorable karyotypes such as dic , MLLr, and hypodiploidy, evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict the efficacy of immunotherapy.The present study revealed that TRAIL-resistance due to hypermethylation of the


Metastases formation involves the formation, circulation and seeding of cohesive group of tumor cells called circulating tumors cells clusters at distant organs from the primary tumor. These clusters have a much higher metastatic potential than individual circulating tumor cell, it is therefore important to understand the molecular mechanisms involved in their formation. To this aim, in this study, from the analysis of the relationship between in vitro aggregation quantitative characterization of 25 cancer cell lines and their expression data, we identified genes significantly associated with aggregation. Interestingly, we found that these genes were strongly correlated with the transcriptional signature induced by HDAC inhibitors treatment and we finally showed experimentally that two HDAC inhibitors inhibits tumor cells cluster formation in vitro. These results open new therapeutic perspectives to prevent metastasis formation.Characterization of the molecular mechanisms involved in tumor cell clustering could open the way to new therapeutic strategies. Towards this aim, we used an in vitro quantitative procedure to monitor the anchorage-independent cell aggregation kinetics in a panel of 25 cancer cell lines. The analysis of the relationship between selected aggregation dynamic parameters and the gene expression data for these cell lines from the CCLE database allowed identifying genes with expression significantly associated with aggregation parameter variations. Comparison of these transcripts with the perturbagen signatures from the Connectivity Map resource highlighted that they were strongly correlated with the transcriptional signature of most histone deacetylase (HDAC) inhibitors. Experimental evaluation of two HDAC inhibitors (SAHA and ISOX) showed that they inhibited the initial step of in vitro tumor cell aggregation. This validates our findings and reinforces the potential interest of HDCA inhibitors to prevent metastasis spreading. The detection and monitoring of circulating tumor cells (CTC) have been the subject of major interest for several years to study and understand cancer pathology and to monitor the therapeutic response ,2. IndeeCell–cell adhesion involves multiprotein complexes located in intercellular junctions. In vertebrates, these cellular structures mostly comprise adherens junctions, gap junctions, desmosomes, and tight junctions. Proteins of the cadherin family are involved in adherens junctions (E-cadherin) and desmosomes (desmoglein and desmocolin); occludin, claudin, and ZO proteins are the most important components of tight junctions and Gap junctions connect two adjacent cells by establishing the continuity of connexons formed by connexins.Several years ago, it was shown that increased expression of E-cadherin, a major component of cell adherens junctions, enhances the formation of cell aggregates . This isTo identify additional molecular actors involved in the formation and/or stability of cancer cell clusters, study of the modalities and regulation of clustering also relies on in vitro assays to monitor intercellular interactions. We previously developed such an assay that we used to demonstrate the key positive role of E-cadherin and of two desmosomal proteins (DSG2 and DSC2), which interact with plakoglobin at this junction, in cancer cell aggregation . We alsoThe characterization of the molecular mechanisms involved in tumor cell clustering could open the way to new therapeutic perspectives. Therefore, it is essential to identify the regulators of this process and their specific roles. With this aim in mind, we chose an approach based on a basic observation. Specifically, among tumor cell lines of epithelial origin, some display very high anchorage-independent clustering capacities in vitro and aggregate very rapidly, while others have very limited, if any, clustering capacity. Therefore, we analyzed the anchorage-independent aggregation capacity of 25 tumor epithelial cell lines. Then, we selected three quantitative aggregation dynamic parameters to evaluate their relationship with gene expression data for these cell lines extracted from the CCLE database . This apAltogether, our observations support and strengthen the idea that deciphering the mechanism of tumor cell clustering and identifying the underlying regulatory pathways and interfering compounds might lead to new therapeutic opportunities.2 at 37 °C in DMEM , without or with 20 IU/L insulin , or in RPMI1640  medium according to the supplier’s recommendations, both supplemented with 10% fetal calf serum  and 1% penicillin/streptomycin . To study the impact of SAHA and ISOX, cells were incubated in culture medium containing 5 µM ISOX for 8 h or 10 µM SAHA for 24 h, then trypsinized and seeded for aggregation assay in culture medium containing the same concentration of ISOX and SAHA.The 25 cancer cell lines  were cul®, Corning, New York, NY, USA), except in the 36 peripheral wells to avoid edge effects. Plates were centrifuged at 400 g for 4 min, and then cell aggregation in each well was followed by time-lapse video-microscopy.The cell clustering assay used in this study was adapted with slight modifications from the one described in our previous reports ,13. SpecBased on our previous study ,13, imagFor each position/well/time, the whole stack of 2D images is fused into one single in-focus image. This process is based on a Laplacian of Gaussian filter to detect the in-focus image regions, followed by a Gaussian blending of these regions taken from the different focal planes.The background signal of the resulting in-focus image is estimated using a morphological opening and then subtracted.A Gaussian smoothing filter is then applied and the image intensities adjusted , followed by a simple automated thresholding, resulting in a binary mask that corresponds to all cell aggregates.2, because the XY resolution is 0.645 µm at 10× magnification). Detected objects with an area smaller than this threshold are excluded from the analysis. Holes inside the binary objects are excluded in the two object categories to measure standard parameters: number of aggregates, normalized area to the initial time point, and circularity. This image analysis pipeline was developed within a parallelized processing architecture using multi-core processors.Cell aggregates are individually detected in the binary image as connected components that match several criteria, and are divided in two groups according to their area using a threshold of 10,000 pixels ; a circularity of 1 indicates a perfect circle . [The gene expression data for the 25 cell lines used in this study were downloaded from the CCLE website e 2019). ,17. A spe 2019).  was used. ,17. A e 2019). .https://clue.io/connectopedia (accessed on: 31 August 2020).The Connectivity Map (CMap) is a resource that uses cell responses to perturbation to find relationships between diseases, genes, and therapeutics. The CMap database contains over one million gene expression signatures upon treatment of a variety of cell types with many different perturbagens . This resource has been successfully used in various studies in the field of cancer research to identify new candidates based on original signatures and to propose new drug repurposing opportunities  behaved very similarly in this assay.Our aim was to analyze the relationship between this variability and the gene expression profiles of the tested cell lines to identify new regulators of tumor cell clustering. Thus, to compare all 25 cell lines, we extracted three quantitative parameters that precisely described the aggregation kinetics and characteristics: the area-2h (early aggregation dynamics), the AUC (whole aggregation process), and circularity. These values are presented in www.broadinstitute.org/ccle accessed on: 4 June 2019), a collaboration between the Broad Institute and Novartis [To identify genes the expression of which was associated with the aggregation quantitative parameters, we extracted the expression data of 19,851 genes for the 25 cell lines used in our study from the Cancer Cell Line Encyclopedia (CCLE) project . The complete list of the median tau scores for the perturbagens is provided as As the genes associated with area-2h and AUC were different from those associated with circularity, we could not identify putative regulators of tumor cell aggregation directly with the sparse PLS method. Therefore, we used the CMap resource  to compaThese results suggest that HDAC inhibitors could regulate the capacity of cancer cells to form clusters.These results and a recent publication showing that HDAC inhibitors reduce CTC cluster size  led us tp < 0.0001) and the AUC  were significantly higher in SAHA-treated cells than controls. We obtained similar results in cells incubated with ISOX , which confirms the reliability of the methodology used.We focused on three quantitative parameters: area-2h, AUC, and circularity. First, we identified genes that showed expression variations in the 25 cell lines associated with these aggregation parameters using data from the CCLE transcriptional database ,17. ThenA recent comparative analysis  demonstr


Further in vitro and in vivo analyses in tumor-bearing mice are ongoing in our lab to support this claim and to unravel the full therapeutic efficacies of NSC777205 and NSC777207 in CRC.Genetic and environmental factors play important roles in cancer progression, metastasis, and drug resistance. Herein, we used a multiomics data analysis to evaluate the predictive and prognostic roles of genetic and epigenetic modulation of c-MET (hepatocyte growth factor receptor)/epidermal growth factor receptor (EGFR) in colorectal cancer (CRC). First, we found that overexpressions of c-MET/EGFR were associated with the infiltration of tumor immune cells and cancer-associated fibroblasts, and were of prognostic relevance in CRC cohorts. We also observed that genetic alterations of c-MET/EGFR in CRC co-occurred with other gene alterations and were associated with overexpression of messenger (m)RNA of some cancer hallmark proteins. More specifically, DNA-methylation and somatic copy number alterations of c-MET/EGFR were associated with immune infiltration, dysfunctional T-cell phenotypes, and poor prognoses of the cohorts. Moreover, we describe two novel gefitinib-inspired small molecules derivatives of 3-phenyl-2H-benzo[e] -oxazine-2,4(3H)-dione, NSC777205 and NSC777207, which exhibited wide-spectrum antiproliferative activities and selective cytotoxic preference for drug-sensitive and multidrug-resistant melanoma, renal, central nervous system, colon, and non-small cell lung cancer cell lines. We further provided in silico mechanistic evidence implicating c-MET/EGFR/phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibition in anticancer activities of those compounds. Our overall structure-activity relationship study revealed that the addition of an –OCH According to 2020 global cancer statistics, with more than 1.9 million new cases and 935,000 deaths, colorectal cancer (CRC) ranked third in terms of incidence, but second in terms of mortality in 2020 . CRC is Both genetic and environmental factors play important roles in the etiology of CRC. To date, several molecular hallmarks have been associated with CRC . These hMET gene, while the EGFR belongs to the ErbB family of RTKs, and is a trans-membrane protein. The binding of ligands to the extracellular domain of these receptors induces the formation of receptor homo- or heterodimers and subsequent activation of the intrinsic tyrosine kinase domain , is a tyrosine kinase receptor (RTK) that in humans is encoded by the e domain , which fe domain .in silico mechanistic evidence implicating c-MET/EGFR/phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition in anticancer activities of these compounds.Several treatment modalities exist, including immunotherapy, radiotherapy, neoadjuvant and palliative chemotherapies, laparoscopic surgery for primary disease, and more-aggressive resection of metastatic disease, and these provide alternatives for patients with primary and metastatic CRC . Howeverhttp://www.swissadme.ch) (http://way2drug.com/dr) (https://www.cbligand.org/BBB/) which operates based on support vector machine (SVM) and LiCABEDS algorithms on four types of fingerprints of 1,593 reported compounds (http://www.way2drug.com/PASSonline) (https://www.ebi.ac.uk/chembldb/), to predict the cytotoxic activities of NSC777205 and NSC777207 against various cancer cell lines.We analyzed the drug-likeness, PKs, medicinal chemistry, and toxicity of NSC777205 and NSC777207 using SwissADME software (adme.ch) , and com.com/dr) . We usedompounds  to analyompounds  to furthSonline)  created c-MET/EGFR in CRC tissues from The Cancer Genome Atlas (TCGA) database were compared with expression levels in normal tissues using the TNMplot module of the Kaplan-Meier Plotter (https://www.tnmplot.com/). Furthermore, we used the Human Protein Atlas (HPA) database (www.proteinatlas.org) to assess the immunohistochemical (IHC) profile of c-MET/EGFR in tumor samples from CRC patients. We used the MEXPRESS algorithm (https://mexpress.be/index.html) to depict the mechanisms of c-MET/EGFR dysregulation in colorectal cancer (https://precog.stanford.edu/precog_metaZ.datatable.php) , 95% confidence intervals (CIs), and log-rank test p values.Expression levels of l cancer . To analble.php)  to splithttp://www.cbioportal.org/) to mine the Colorectal Adenocarcinoma  dataset for c-MET/EGFR genetic alterations including mutations, copy number alterations, gene mutation co-occurrences, and microbiome signatures and evaluated the prognostic relevance of the alterations in 594 CRC patients using survival analyses of the cohorts (https://www.string-db.org) with the adjusted threshold confidence set to 0.400 (http://timer.cistrome.org/) to analyze correlations of c-MET/EGFR expression with somatic copy number alterations and infiltration of six tumor-infiltrating immune cell subsets  T cells, CD8 T cells, macrophages, neutrophils, and dendritic cells) in CRC. We also used the TIMER server to analyze correlations of c-MET/EGFR expressions and cancer-associated fibroblast (CAF) infiltration. In addition, we analyzed the prognostic relevance of these associations by employing a protocol described in a previous study (http://tide.dfci.harvard.edu)   to analyin vitro anticancer activities against 60 panels of human tumor cancer cell lines representing leukemia (six cell lines), NSCLC (eight cell lines), colon cancer (seven cell lines), CNS cancers (six cell lines), melanomas (nine cell lines), ovarian cancer (seven cell lines), renal cancer (eight cell lines), prostate cancer (two cell lines), and breast cancer (five cell lines) through the National Cancer Institute (NCI). Ranges of 5,000–40,000 viable cancer cells were seeded into each well of 96-well plates and incubated at 37 °C with 5% CO2, 95% air, and 100% relative humidity for 24 h. After incubation, cells were treated with either NSC777205 or NSC777207 at a single dose of 10 μM and further incubated for 48 h : [(Ti−Tz)/(C−Tz)] × 100 for concentrations for which Ti>/ = Tz or [(Ti−Tz)/Tz] x 100 for concentrations for which Ti < Tz.50) (μM) = [(Ti−Tz)/(C−Tz)] × 100 = 50.50% GI  = Ti = Tz.50; μM) = [(Ti−Tz)/Tz] × 100 = −50.50% Lethal concentration  did not meet the required effect on a particular cell line, the value for that parameter was expressed as greater than the maximum concentration tested (>100 μM).In these equations, Tz is the absorbance at time 0; C is the absorbance of the control after 48 h without treatment; Ti is the absorbance of drug-treated cells after 48 h; GIhttps://dtp.cancer.gov/databases_tools/compare.htm) to correlate anticancer activity fingerprints of NSC777205 and NSC777207 with NCI synthetic compounds, standard agents, and molecular targets based on established relationships between cell responses to therapy and gene expression profiles of the cell lines  (http://www.swisstargetprediction.ch/), which operates on the basis of similarity of the queried molecule with known active drugs (http://way2drug.com/dr)  , the Swive drugs , and the.com/dr) .http://avogadro.cc/) (https://pubchem.ncbi.nlm.nih.gov/). All mol2 and sdf files were converted to pdb files using the PyMOL Molecular Graphics System, vers. 1.2r3pre , while pdbqt files of ligands were generated from pdb files using AutoDock Vina  (https://www.rcsb.org/) in protein data bank (PDB) file format and subsequently converted into the Auto Dock Pdbqt format using AutoDock Vina tools. All receptors were charged, hydrogen atoms were added, and water (H20) molecules were removed prior to docking (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index)  molecular ball-and-stick models in mol2 format of NSC777205 and NSC777207 were obtained using the Avogadro molecular builder and visualization tool vers. 1.XX (dro.cc/) , while 3 States) . Crystal docking . Docking docking . Docking docking  and the p/index) .p value of <0.05. The KEGG and gene ontologies (GOs) were visualized using GraphPad prism software. DTP-COMPARE analyses were conducted using Pearson’s correlations. All survival analyses are presented using Kaplan-Meier plots. GI was calculated relative to cells without drug treatment and the time-zero control. Statistical significance was denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.All statistical analyses were conducted according to each server's instructions. We used the Wilcoxon test to compare statistically significant differences in c-MET/EGFR expressions between CRC cohorts and healthy tissues. Purity adjustment and partial Spearman’s correlations were used to analyze c-MET/EGFR expression correlations with infiltration of CAFs and various immune cells. Gene alteration co-occurrences on the cbioportal were considered significant only at an adjusted p = 1.24e-07) while EGFR expression is lower (p = 4.67e-04) in colorectal cancer cohorts compared to adjacent normal cohorts.  and READ  (p = 0.028) and primary tumors p = 0.04029) but less associated with metastasis or recurrent tumor. Furthermore, both c-MET and EGFR expressions show significant correlation with copy number alterations in colorectal cancer  . Further.01e-14) . We foun.01e-14) . Mean ag.01e-14) . Only 8..01e-14) . Our anal cancer . To assel cancer  and resi+ T cells, B cells, CD8+ T cells, neutrophils, dendritic cells, and macrophages) in CRC tissues  and READ , while MET exhibited no significant correlations with tumor purity in COAD  and READ . Collectively, the above results indicate that c-MET/EGFR expressions were correlated with immune infiltration in COAD and READ  = 0.008, p = 0.015) were risk factors for COAD  and READ . Similarly, c-Met expressions were positively correlated with CAF infiltration in COAD  and READ   . To evalval rate .We mined TCGA PanCancer Atlas dataset for c-MET/EGFR genetic alteration data and evaluated the prognostic relevance of alterations in CRC patients. We found that 3.0 and 4.0% of the 594 CRC patients ) in the dataset respectively harbored genetically altered EGFR and c-MET . Alteratp = 1.82E-05), Actinosynnema (p = 5.63E-05), Castellaniella (p = 8.47E-05), Moorella (p = 9.18E-05), Betaretrovirus (p = 9.45E-05), Pimelobacter (p = 1.25E-04), Arcanobacterium (p = 1.33E-04), and Cryobacterium (p = 1.36E-04) in EGFR non-altered cohorts compared to altered cohorts  enriched genetic alteration co-occurrences with EGFR alterations included SMARCD2, TRIM7, MCMDC2, HNRNPUL1, BEST3, GRB10, PDE6C, and SNX19, while SLC22A2, DST, AKT3, and TAB2 were the only significantly  enriched gene alterations that co-occurred with c-MET alterations  mRNAs in EGFR altered cohorts compared to non-altered cohorts were TRIM7, BARX2, CFAP43, TPBG, HPSE, GNB5, MARK3, FUT8-AS1, SS18, and KCNK1, while SMAD4, MBP, IER3IP1, OXCT1, ALKBH5, C18ORF25, WDR76, TYMS, LINC00909, and GPR3 were overexpressed in c-MET altered CRC cohorts  included cell adherent junctions, EGFR tyrosine kinase inhibitor resistance, central carbon metabolism, CRC, pancreatic cancer, hepatocellular carcinoma, gastric cancer, and melanomas (p < 0.05) included the c-MET, ERBB2, and EGFR signaling pathways, MAPKK activation, and negative regulation of cellular responses to drugs , CD8+ T cell (p < 0.001), neutrophil (p < 0.005), and dendritic cell infiltration in COAD, and macrophage (p < 0.005) infiltration in READ. Arm-level gains of c-MET were positively correlated with CD8+ T cell, B cell, neutrophil, and dendritic cell infiltration in colon cancer and dendritic cells infiltration in READ, while deep deletions of c-MET were associated with CD8+ T cell and neutrophil infiltration of tumors in READ  of a good drug candidate. The compounds were both lead-like and satisfied Lipinski's rules for drug likeness candidates. Furthermore, our in silico analysis suggested that both compounds can be easily absorbed in the gastrointestinal tract and are well permeant to the blood-brain barrier (BBB) especially NSC777205 which demonstrated 2-fold higher permeation of the BBB than NSC777207 based on in silico estimation  and other markers of eco-toxicity also identified both compounds as having low toxicities , brain (Hs683), lung (A549), and bone (SJSA-1) cancer cell lines, with NSC777205 demonstrating higher activities (Pa = 0.31–0.658) than NSC777207 (Pa = 0.303–0.565) against all cell lines (NSC777205 (3-(4-chloro-2-fluorophenyl)-2H-benzo[e]oxazine-2,4(3H)-dione) and NSC777207 (3-(4-chloro-2-fluorophenyl)-7-methoxy-2H-benzo[e]-oxazine-2,4(3H)-dione) were obtained at high purity (>95%) via condensation reactions between salicylic acid and the 2,4-disubstituted aniline moieties of gefitinib to produce a salicylanilide which was further cyclized to the 3-phenyl-2H-benzo[e]-oxazine-2,4(3H)-dione derivatives, NSC777205 and NSC777207, as summarized in timation ; Table 2ll lines .We initially screened NSC777205 and NSC777207 for anticancer activities against the full NCI-60 panel of human tumor cell lines. Interestingly, we found that with single-dose (10 μM) treatment, both NSC777205 and NSC777207 demonstrated antiproliferative activities against all of the NCI-60 cell line panels of breast, prostate, renal, ovarian, colon, melanoma, CNS, leukemia, and non-small cell lung cancers, with NSC777205 demonstrating higher activities than NSC777207. In addition, most of the leukemia (cell death (CD) = 0.77–10.28%), NSCLC (CD = 4.2–50.81%), CNS (CD = 013.71%), melanoma (CD = 0.57–46.24%) renal (CD = 7.01–30.91%), breast (CD = 10.48%), prostate, and ovarian cancer cell lines demonstrated some cytotoxic response to NSC777205 and NSC777207 single-dose treatment, while panels of colon cancer cell lines were completely insensitive to the cytotoxic activity of the compounds at 10 μM. Collectively, these studies demonstrated that both NSC777205 and NSC777207 exhibited a wide spectrum of anticancer activities but demonstrated selective and exclusive antiproliferative activities against all panels of colon cancer cell lines .50 = 11.90–20 µM), both NSC777205 and NSC777207 exhibited high anticancer activities with GI50 concentrations of <6 µM against all cell line panels , A498 (LC50 = 29.60 µM), and UO-31 (LC50 = 51.10 µM) renal cancer cell lines; the SK-MEL-5 (LC50 = 11.30 µM) and UACC-62 (LC50 = 54.10 µM) melanoma cell lines; the SF-295 (LC50 = 57.40 µM) CNS cell line; the COLO 205 (LC50 = 78.40 µM) colon cancer cell line; and the NCI-H522 (LC50 = 95.70 µM) NSCLC cell line  = 47–58) with fingerprints of some NCI synthetic compounds (p = 0.3–0.61) with a number of c-MET/EGFR/PI3K/AKT/mTOR inhibitors, including JNJ38877618, MTX211, SGX-523, altiratinib, TAS-6417, voxtalisib, PI-103 HCL, AMG-458 (c-MET), CC115, and others  docked well into the binding cavities of the receptors with respective binding affinities (ΔG) of −6.90, −6.80 and −6.80 Kcal/mol for EGFR; −8.95, 8.96 and −9.56 Kcal/mol for c-MET; −8.60, −7.90, and −9.0 Kcal/mol for PI3K; and −7.1, −7.6 and −9.2 Kcal/mol for mTOR . The intDespite the availability of various treatment strategies, CRC remains among the top ranked most diagnosed and leading causes of cancer deaths globally . In ordeSMARCD2, TRIM7, MCMDC2, HNRNPUL1, BEST3, GRB10, PDE6C, and SNX19, while alterations of SLC22A2, DST, AKT3, and TAB2 co-occurred with c-MET alterations in CRC. Genetic alterations and concurrent alterations of c-MET/EGFR with other genetic alterations have been well reported in lung cancer  . Tumor-iCAFs are apoptotic-resistant fibroblasts that promote tumorigenic and metastatic properties by secreting cytokines and remodeling the ECM . We founin silico drug-likeness and ADME profiles, and found that both NSC777205 and NSC777207 possessed good drug-likeness properties and obeyed Lipinski’s rule-of-five, a physicochemical-based rule set to evaluate drug candidates for suitability as oral remedy  than NSC777207 (Pa = 0.303–0.565). BBB penetration is an important limiting factor in the use of chemotherapy for treating glioblastomas and other CNS-associated diseases  brain (Hs683), lung (A549) and bone cancer (SJSA-1) cell lines with NSC777205 demonstrating higher activity (Pa = 0.31–0.658) than NSC777207 (Pa = 0.303–0.565). Of importance, among all cell lines, colon cancer cell lines stood out uniquely with all seven cell lines demonstrating strict antiproliferative sensitivity and no cytotoxic response to single-dose (10 μM) treatment of either NSC777205 or NSC777207. This suggests that NSC777205 and NSC777207 would be very useful in growth inhibition and treatment of CRC. Tumors often develop resistance to a wide range of anticancer drugs. Therefore, drug resistance represents an important problem in cancer chemotherapy  was 3-(2-fluoro-4-iodophenyl)-7-methoxy-2H-benzo[e]. In compliance with this recommendation, we used four computational tools including the COMPARE algorithm developed by the USA NCI to identify the most probable targets for NSC7772015 and NSC777207. In the COMPARE analysis, the anticancer fingerprints of NSC777205 and NSC777207 corresponded to anticancer fingerprints of known inhibitors of c-MET/EGFR/mTOR/PI3K pathways. Furthermore, the most highly correlated NCI synthetic compound (-benzo[e] oxazine--benzo[e]. In addi-benzo[e]-oxazin-3-benzo[e]. c-MET p-benzo[e]. It is tin vitro or in vivo validation. Non-covalent interactions such as hydrogen bonds, hydrophobic, ionic, and π-interactions, and van der Waals forces occurring between ligands and receptors are important for stabilization of a ligand within the binding cavity of a receptor (Molecular docking aids the identification of drug lead molecules by unraveling possible interactions between a drug candidate and protein targets, providing structure-activity relationships and therapeutic mechanisms of drug candidates prior to receptor . Our docreceptor . In addireceptor . Both NSBoth NSC777205 and NSC777207 exhibited high similarity with gefitinib and crizotinib, known clinical EGFR and c-MET inhibitors, respectively, in terms of common interacting residues with EGFR and c-MET and similar binding affinities with EGFR, suggesting that both NSC777205 and NSC777207 could be as promising as gefitinib and crizotinib at inhibiting EGFR and c-MET, serving as potential dual targets and thus superseding the strength of individual clinical drugs in arresting cancer proliferation and aggressiveness for which EGFR and c-MET are implicated. This multi-target potency of NSC777205 and NSC777207 could be attributed to the observed activities of NSC777205 and NSC777207 against NCI cell lines that are known to be multi-drug resistant including those that are resistant to more than 10 panels of TKIs .3 group to the salicylic core of NSC777207 was not favorable, as the added moiety did not contribute significant interactions with the receptor-binding residues and also led to overall less-favorable ADMET, physicochemical, and drug-likeness properties and weaker anticancer activities against the panel of NCI cell lines compared to the properties and activities demonstrated by NSC777205 that has no –OCH3 substituent group. However, further in vitro and in vivo analyses in tumor-bearing mice are ongoing in our lab to support this claim and to unravel the full therapeutic efficacies of NSC777205 and 777207 in CRC.Collectively, our study revealed that structural hybridization strategies yielded novel and potential c-MET/EGFR/PI3K/mTOR multi-target compounds, NSC777205 and NSC777205, with promising prospects for treating melanoma, renal, CNS, colon, and NSCLC cell lines. Although NSC777205 and NSC777207 share common interacting residues with the receptors, the overall structure-activity relationship study revealed that the addition of an –OCH3 substituent group from the salicylic core of NSC777205 was responsible for more highly favorable drug-likeness properties and anticancer activities, and more-robust interactions with c-MET/EGFR/PI3K/mTOR than those of NSC777207.In conclusion, our results indicated that, c-Met/EGFR are important biomarker signatures of cancer-associated fibroblasts and tumor immune infiltration, and are of clinical relevance in colorectal cancer. Genetic and epigenetic alterations of c-MET/EGFR were associated with dysfunctional T-cell phenotypes, and poor prognoses of the cohorts. c-MET/EGFR/PI3K/mTOR are therapeutic targets for NSC777205 and NSC777207 with consequent selective cytotoxic preferences for melanoma, renal, CNS, colon, and NSCLC cell lines. However, the removal of an –OCH


Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels. Liver cancer, mainly represented by hepatocellular carcinoma (HCC), is the second and sixth most common cause of cancer death among men and women, respectively . PrimaryDirect intercellular signaling is mainly mediated by gap junctions . Gap junLiver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis , such asIn order to assess connexin expression in liver cancer in vitro, a panel of seven cancer cell lines was selected . The panThe cell lines all varied in differentiation status and metastatic potential. In this respect, SK-HEP-1 cells are highly metastatic and display mesenchymal stem cell characteristics , while CMorphology pictures were taken during the exponential growth phase  of the cCx26 mRNA is more restricted to the periportal areas  at a concentration of 0.25 mg/mL. Maintenance medium was replaced 2 h later, after gelatinization of the Matrigel® layer at 37 °C, and again 2 days after addition of the Matrigel® layer. PHH sandwich cultures were maintained for 4 days after seeding.PHH were purchased from KaLy-cell  and cultured in a sandwich culture for scrape loading/dye transfer analysis and immunocytochemistry analysis. PHH were thawed and transferred to a tube with prewarmed cryopreserved hepatocyte recovery medium (CHRM) . After gentle mixing, PHH were pelleted by centrifugation at 100× ® TCP-1011™) was purchased from the American Type Culture Collection  and contained 7 human liver cancer cell lines, namely SK-HEP-1, C3A, SNU-449, SNU-423, SNU-387, SNU-475 and PLC/PRF/5  or Eagle’s Minimum Essential Medium (EMEM)  with addition of 1% MEM Non-Essential Amino Acids Solution  and 1% Sodium Pyruvate  supplemented with 10% fetal bovine serum (FBS)   and 100 units/mL penicillin and 100 µg/mL streptomycin .The Liver Cancer Panel  using a 10× objective. PHH sandwich cultures were imaged daily on the same microscope with a 20× objective. Scale bars were added with ImageJ software (version 1.52p) .TM Mammalian Total RNA Miniprep Kit  combined with an On-Column DNase I Digestion Set  according to the manufacturer’s instructions. RNA yield and purity were assessed using a NanoDrop 2000 spectrophotometer . Next, cDNA was generated with an iScript™ cDNA Synthesis Kit  on a MiniAmp Plus Thermal Cycler  followed by cDNA purification using a GenElute™ PCR Clean-Up Kit . RT-qPCR analysis was performed on an Applied Biosystems QuantStudio 3 Real-Time PCR system . Samples were tested in triplicate and amplified with TaqMan® Gene Expression Assays  targeted towards 5 housekeeping genes, namely actin beta (ACTB), beta-2-microglobulin (B2M), GAPDH, hydroxymethylbilane synthase (HMBS) and ubiquitin C (UBC), as well as towards Cx26, Cx32 and Cx43 . Efficiency was estimated based on a serial dilution of pooled cDNA mix from all samples and 3 no-template controls. Relative alterations compared to PHH were calculated according to the Pfaffl method [+ .The reaction mix contained 2 µL cDNA, 1 µL primer, 7 µL nuclease-free water and 10 µL of TaqManl method  in qbaseg, the resulting pellet was resuspended in the same protein lysis buffer as the cell lines. Next, lysates were transferred to a pre-cooled tube and put on ice for 30 min, during which the lysate was vortexed every 5 min. Supernatants were collected by centrifugation for 20 min at 4 °C and 14,000× g and stored at −80 °C. Protein concentrations were determined with the PierceTM BCA protein assay kit . For immunoblotting, proteins were separated on 10% (Cx43) or 12% (Cx26 and Cx32) Mini-PROTEAN TGX Stain-Free™ precast gels . Samples for detection of Cx26 were heated for 5 min at 95 °C. Gels were blotted onto a nitrocellulose membrane (Cx43 and Cx32)  or a polyvinylidene difluoride membrane (Cx26) . Blocking of the membranes was performed in 5% non-fatty milk in Tris-buffered saline solution (20 mM Tris and 135 mM sodium chloride) with 0.1% Tween-20 for 1 h at room temperature on an orbital shaker. Membranes were incubated overnight at 4 °C with primary antibodies raised against Cx26, Cx32 or Cx43 . Densiometric analysis was performed with Image Lab 6.0.1 software . Signals were normalized against total protein loading and by sum of all data points in a replicate [Membranes were washed 3 times for 10 min and incubated with a secondary antibody  diluted in blocking buffer for 1 h at room temperature. Membranes were washed and visualized by using the Pierce™ ECL Western Blotting Substrate kit  on a ChemiDoceplicate . Data arCell lines and PHH cultures were washed 3 times with ice-cold PBS for 5 min and fixed at −20 °C for 10 min with an acetone–ethanol (1:1) mixture that permeabilized simultaneously. Cells were then washed 3 times for 5 min with ice-cold PBS. Cells were blocked at room temperature with 1% bovine serum albumin (BSA)/5% donkey serum for 45 min and incubated overnight at 4 °C with primary antibodies. Primary antibodies targeted against Cx26, Cx32 and Cx43  were dil2, 5.55 mM glucose and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.4) or CBX buffer (100 µM CBX in SL/DT buffer). Next, a scratch was made with a fine needle in the presence of LY (1 mg/mL) dissolved in SL/DT or CBX buffer. This was incubated for 10 min at room temperature. Then, cells were washed 4 times with Hank’s Balanced Salt Solution (HBSS)/ HEPES buffer  and fixed for 10 min in 4% paraformaldehyde solution. Cells were washed again 4 times with HBSS/HEPES buffer and visualized on a Nikon Eclipse Ti-S microscope  with a 10× or 20× objective. The fluorescent area of the dye spread was measured via ImageJ software (version 1.52p) . Data were normalized to the area of the inhibited cells and expressed as a ratio to the non-inhibited signals in PHH.Cell lines and PHH were washed 3 times with scrape loading/dye transfer (SL/DT) buffer . The number of technical replicates (N) is specified in the figure legends. Statistical analysis was performed in GraphPad Prism v9.0 software , and data are presented as mean ± standard deviation. Normality was assessed by means of a Shapiro–Wilk normality test. Normally distributed RT-qPCR and immunoblot data were analyzed via an ordinary 1-way analysis of variance, followed by a Dunnett’s multiple comparisons test. Scrape loading/dye transfer data were analyzed in the same way when comparing the dye transfer of the cell lines to the dye transfer of the PHH. For comparing the inhibited to the non-inhibited fluorescent area within each cell line, multiple unpaired t-tests were performed with a Holm–Šidák correction for multiple comparisons. Significance levels are indicated by to the following symbols: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.One batch was used for PHH and all cell lines (


TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line . The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations , with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with  TP53 mutations, irrespective of histology [TP53 status of GCTs, combined with a pluripotent phenotype, high levels of PUMA and NOXA, and, often, low expression levels of CDKN1A (P21), result in a cellular disbalance and a favor towards apoptosis over DNA repair [TP53 mutations, or whether these mutations simply occur more in these tumors as these tumors harbor different intrinsic resistance mechanisms. To this end, we wanted to elucidate whether the removal of TP53 in a testicular GCT cell line can convey cisplatin resistance, and, thus, (partly) explain mediastinal GCT aggressiveness and treatment resistance. Firstly, we used the online cBioPortal tool to analyze an extensive GCT patient data set containing detailed clinical information, including treatment, tumor resistance, tumor stage, anatomical location, histology, and genetic mutations [TP53 background (TP53 mutant/loss in NCCIT from the mediastinum and TP53 wild-type in 2102Ep from the testis), were modified using a CRISPR/Cas9-mediated TP53 knock-out model system. We subsequently investigated the difference in cisplatin resistance in these testicular and mediastinal GCT cell lines. Using both GCT patient data characteristics and functional mechanistic cell line investigations, we show a role of P53 in GCT cisplatin resistance related to the anatomical location of the tumor.Germ cell tumors (GCTs) are the most common solid malignancies in young men ,2. Despiistology ,10. MoreA repair ,13,14,15A repair ,17,18,19A repair ,17. On tA repair ,17,20,21A repair ,7. WhileA repair . Apart fA repair ,17,19,23TP53 (and MDM2) alterations in cisplatin-sensitive and -resistant GCTs. Strikingly, while GCTs rarely harbor TP53 mutations, in line with their embryonic phenotype [TP53 are detected in cisplatin-resistant patients. Furthermore, we observe that MDM2 amplifications become increasingly abundant in patients with cisplatin-resistant GCTs. Note that, as expected, alterations regarding TP53 are often missense mutations or deep deletions. When comparing the disease-free survival of patients with alterations in the TP53 gene to patients with wild-type TP53 , we observed a highly significant  decrease in disease-free survival in patients harboring TP53 alterations  GCTs, we initially used the cBioPortal online tool. We investigated the MSKCC data set on refractory GCTs previously reported by Bagrodia and colleagues in 2016 . The rathenotype , alteraterations . As preve testis . Interest tumors A,B.TP53 status, whereas NCCIT carries a hemizygous one-base-pair deletion at nucleotide 949 (codon 272), resulting in a frameshift and a premature STOP codon at codon 347 .To study the difference between mediastinal and testicular GCTs, we used the well-established and -characterized NCCIT and 2102Ep GCT (EC) cell lines. While 2102Ep originates from the testis, NCCIT originates from the mediastinum, with a similar differentiation state . Furtherodon 347 A. This oMDM2 expression than both the 2102Ep cell lines, with 2102Ep resistance displaying the highest levels of MDM2 . RNA sequencing (RNA-seq) analysis showed that both the parental and resistant NCCIT cell lines had lower normalized  of MDM2 B, suppor of MDM2 C. In conll lines . Principll lines D,E. Thisterature ,16,23,27TP53 status [TP53 mutant status, the NCCIT cell line is considered to be inherently sensitive to cisplatin. Thus, we further compared the mutational status of the NCCIT cell line to the mutations found in refractory GCT patients , suggests that TP53 mutations could add additively to inherent cisplatin resistance mechanisms [TP53 in cisplatin resistance in the approach described. Therefore, we generated isogenic CRISPR/Cas9-mediated TP53 knock-out clones of both 2102Ep and NCCIT, as well as their resistant counterparts. Sanger sequencing, after mono-clonal picking and expansion, revealed mono-clonal sequence traces and a one-base-pair insertion at amino acid 48, resulting in a premature STOP codon at amino acid 51  profiling (TP53 and CDKN1A (P21) expression in both 2102Ep parental TP53 knock-out lines  and 2102Ep-resistant TP53 knock-out lines , which was also confirmed by Western blot (TP53 expression had no effect on the differentiation status. The miR371a-3p expression levels were checked because of its many implications in GCTs (mostly as a biomarker and marker of pluripotency in these tumors), together with the implications of P53 pathway regulation [TP53 knock-out clones, with cisplatin, we identified a clear significant  shift in cisplatin resistance when comparing the TP53 knock-out clone to its wild-type counterpart, with the 2102Ep-resistant TP53 knock-out clone demonstrating the highest cisplatin resistance  in the NCCIT-resistant TP53 knock-out clone compared to its NCCIT-resistant counterpart  shift in cisplatin resistance in the NCCIT-resistant TP53 knock-out clone compared to its NCCIT-resistant counterpart  [MDM4 alterations is not fully unexpected, as MDM4 is not able to directly ubiquitinate P53 and target it for proteasomal degradation; this is in stark contrast to MDM2, which is able to directly downregulate P53 through proteasomal degradation [MDM2 amplifications were mostly testicular in origin, whereas TP53-mutated tumors were primarily mediastinal [TP53 mutations in mediastinal GCTs could be due to a less favorable niche and more strict selection for these tumors [TP53 signaling pathway [TP53 mutations, and, thereby, a more treatment-resistant phenotype. In this study, we tried to elucidate if there was a causal link between the poor prognosis for mediastinal GCTs and their bias towards TP53 mutations. In line with cBioPortal analysis, both the Western blot and RNA-seq data indicated higher MDM2 protein levels and a higher MDM2 expression in the testicular 2102Ep GCT cell line compared to the mediastinal NCCIT GCT cell line, despite the lack of MDM2 amplifications in 2102Ep. This suggests a mechanism for treatment resistance within wild-type TP53 GCTs via MDM2 [MDM4 between the mediastinal and testicular cell lines. Finally, as P53 is known to regulate MDM2 and MDM4, and could, therefore, interfere with DNA-damage sensing, we studied the upstream activity of the DNA-damage signaling pathway in the TP53-mutated NCCIT cell line [TP53 mutation present in the NCCIT cell line to the mutational profile of TP53 mutations in refractory GCT patients, we noticed that the NCCIT mutant was located more distally within the protein and was unlikely to fully disrupt the function of the DNA-binding domain [TP53 in GCT cisplatin resistance and to interrogate the functionality of the mutant TP53 in the NCCIT cell line, we generated CRISPR/Cas9-mediated TP53 knock-out cell lines. As the loss of TP53 can contribute to chromosomal instability and, therefore, cisplatin resistance, we used GSA analysis to verify the genomic changes between the original and knock-out clones. No major copy number changes were identified related to the TP53 knock-out procedure and subsequent selection. However, small copy number alterations could be observed, possibly related to clonal selection and expansion. Interestingly, although rarely observed in testicular GCT patients, full knock-out of TP53 in the testicular EC cell line 2102Ep resulted in a significant increase in cisplatin resistance in both a parental and resistant background. This coincided with both the reduced expression levels of TP53 and CDKN1A (P21), as well as the reduced protein levels of P53 and P21, suggesting a strong dependency on intact TP53 for CDKN1A expression in this cell line. Strikingly, when we knocked out TP53 in the TP53 mutant mediastinal EC cell line NCCIT, we also observed an increase in cisplatin resistance in the resistant cell line only. The knock-out of TP53 resulted in a full loss of P53 at the protein level and a minor reduction at the mRNA level. Interestingly, the parental TP53 knock-out cell line showed reduced expression of microRNA371a-3p, while the resistant TP53 knock-out line showed an increase. It remains to be elucidated whether this NCCIT observation is a passenger effect or is possibly related to previous findings regarding the negative regulatory effect of this microRNA cluster on TP53 expression [TP53 knock-out is relevant in the context of the informativity of this molecular biomarker for GCTs, as recently reviewed by Leão and colleagues, as well as in the case of refractory disease [TP53 does not induce differentiation, excluding the possibility of increased resistance due to a differentiated phenotype [TP53 status of these cell lines, being wild-type, hemizygous mutant, or full knock-out, it did not interfere with the detection of the microRNA cluster 371a-3. In other words, the miR371a-3 cluster appears to retain its suitability as a GCT biomarker irrespective of the tumor’s TP53 status and, thus, also partially the level of cisplatin resistance [TP53 in the NCCIT-resistant (but not 2102Ep) cell line resulted in increased protein levels of P21, an effect not readily observed at the mRNA level, suggesting a negative effect on the P21 levels of the mutant TP53 present in the resistant NCCIT subclone. This is interesting in light of previous studies indicating increased tumor resistance, malignancy, and aggressiveness caused by the P53-independent upregulation of P21 in TP53 mutant tumors [Here, we present a study on the effect of the loss of t tumors . Of noteficance) . This loradation ,21,39. Siastinal . One coue tumors . As ment pathway . It coulvia MDM2 . This isvia MDM2 ,21,42. Well line ,27. Cellell line ,44,45. Oell line ,44,45. Wpression . The abs disease . In conthenotype . It is isistance ,47,48,49t tumors ,52,53,542. The resistant isogenic clones were generated by the group of Christoph Oing and Friedemann Honecker, University of Hamburg, Germany. These resistant cell lines were obtained by exposing the parental cell lines to cisplatin over a period of 6–9 months to increase the sublethal concentrations of these cells to cisplatin. Cisplatin dose was kept the same for two subsequent treatments and stepwise increased by 30–50%. Each cisplatin treatment was applied for 24 h in 80% confluent cells, followed by a medium change. Cells were allowed to rest with regular medium change over a period of 5–7 days until re-growth was detectable, and cells were re-plated afterwards. After one passage to regenerate, the next treatment was applied as mentioned above. Cells were passaged for a maximum of 7 cycles, followed by intermittent cryopreservation to prevent differentiation. For experimental use, obtained resistant subclones were cultured similarly to their sensitive parental lines without continuous cisplatin supplementation. The resistant subclones were maintained similarly to the parental cells .The parental (T)GCT cell lines used were previously reported and further characterized by us . NCCIT  and selecting the germ cell tumors  data set under testis tumors [TP53 and MDM2 in the gene query section. Subsequent analysis was performed using the tools provided by cBioPortal.Patient data were analyzed by using cBioPortal ,25 by vis tumors . We querSTAR (v2.5.3a) was used as aligner for RNA-seq data, using 2-pass mapping for each sample separately. Mapping quality plots were generated and checked based on sambamba flagstat (v0.6.7) statistics. Count files, with the number of RNA-seq reads for each gene were created with subread FeatureCounts (v1.5.2) and normalized for library size to counts per million (CPM).Cell lysates were made using RIPA buffer, followed by measuring protein concentrations according to Pierce BCA Protein Assay Kit . A total of 25 µg protein was loaded onto a 4–15% Mini-Protean TGX Stain-Free Protein Gel . After separation, the proteins were transferred to a 0.2 µm PVDF membrane with the Turbo Trans-Blot system . The following primary antibodies were added to the membranes: mouse anti-MDM2 (IF2) , mouse anti-MDMX , anti-β-actin antibody , mouse anti-human p53 , rabbit anti-human p21 Waf1/Cip1  and mouse anti-vinculin , as a loading control. After incubating the membranes overnight at 4 °C, either goat anti-mouse IgG(H+L) cross-absorbed, HRP  or goat anti-rabbit IgG (H+L) cross-absorbed, HRP  were added as secondary antibodies and the membranes were incubated for 2 h at RT. To detect the proteins, Clarity Western ECL substrate  was added to the membranes.TP53 , using the Neon Transfection System . To confirm a successful TP53 knock-out and to determine introduced mutations, of all the different cell lines, DNA was isolated using QuickExtract according to manufacturer’s protocol, followed by PCR with the following primers: forward: CAGTCAGATCCTAGCGTCGA and reverse: CACTGACAGGAAGCCAAAGG. Sequencing was performed by Macrogen Europe and knock-out efficiency was determined with the online ICE analysis tool by Synthego ICE analysis. Available online: https://ice.synthego.com/#/ (accessed on 15 September 2021).Both parental and resistant cells of NCCIT and 2102Ep were exposed to a ribonucleoprotein complex made of resuspension buffer R, 61 µM Cas9 protein and 100 µM gRNA duplex consisting of cRNA and tracrRNA, which was designed to target the gene of interest, High-quality total RNA was extracted from the above-mentioned cell lines using TRIzol reagent  according to the manufacturer’s instructions. Quantity and quality were assessed on Nanodrop One  and with Qubit 4 fluorometer .Targeted miRNA profiling was performed on diluted RNA (5 ng) using TaqMan MicroRNA Reverse Transcription Kit  and TaqMan Assays RNU48 (001006), hsa-miR371-3p (002124), and hsa-miR885-5p (002296) as described before . RNU48 wHPRT (hs02800695_m1), TP53 (hs01034249_m1), TP73 (hs01056231_m1), CDKN1A/P21 (hs99999142_m1), BBC3/PUMA (hs00248075_m1), PMAIP1/NOXA (hs00560402_m1), SOX2 (hs01053049_s1), POU5F1 (hs00999632_g1), POU5F1 (hs04195369_s1), POU5F1 (hs03005111_g1). The 2× TaqMan Advanced PCR Master Mix  was used and reactions were run in 96-well plates on QuantStudio 12K Flex System . HPRT was used as a housekeeping gene for normalization purposes and relative gene expression levels were computed as 2−ΔΔCt. Fold change between parental and knock-out clones was plotted.Diluted RNA (50 ng) was reverse transcribed using SuperScript IV reverse transcriptase . RT-QPCR was run using the following TaqMan gene expression assays: 2 overnight. Then, the following concentrations of cisplatin were made from 1 mg/mL cisplatin stock  diluted in fresh medium enriched with 0.9% NaCl: 32 µM, 16 µM, 10 µM, 8 µM, 6 µM, 4 µM, 2 µM, 1 µM and 0.33 µM, or, 32 µM, 25 µM, 20 µM, 17.5 µM, 15 µM, 12.5 µM, 10 µM, 5 µM and 2.5 µM, dependent on expected IC50. The medium in the wells was replaced with 100 µL of the corresponding cisplatin medium. The plate was incubated at 37 °C and 5% CO2 for 72 h.The sensitivity of the cells (both NCCIT and 2102Ep) to cisplatin was determined using a viability assay. To test the sensitivity of the cells to cisplatin, 10.000 NCCIT and 2102Ep parental cells and 4.000 2102Ep-resistant cells  were seeded in 100 µL RPMI or DMEM (respectively) medium per well in a 96-well plate. The plate was incubated at 37 °C and 5% CO® 2.0 Luminescent Cell Viability Assay kit  was used to lyse the cells to be able to measure the viability. Afterwards, 100 µL of the lysate was transferred to a Pierce white opaque 96-well plate (Thermo Fisher). The luminescence was measured on an ID3 Spectramax  or a FLUOstar Omega .After incubation of either 2102Ep cells or NCCIT cells with cisplatin concentrations, CellTiter-Glot-test or one-way ANOVA with a Tukey’s multiple comparisons post hoc test.The viability assays were performed with three technical replicates and at least on three separate occasions . Data were visualized and interpreted using GraphPad Prism 9. To extract IC50 values, concentrations were transformed to logarithms, non-linear S-curves were fit through the data set using a GraphPad algorithm to extract absolute IC50s and the S-curves were interpolated with y = 0.5 to derive the absolute IC50s. Statistic differences were calculated using either an unpaired Student’s Genomic changes between original and knock-out subclones were identified after mono-clonal expansion and a minimum of ~3 months of separate cell culture using Infinium Global Screening Array-24 v3.0 BeadChipGSA (GSA) profiling. Array data were obtained from the HUGE-F as a Genome Studio vs. 2.0.4  project using the hg38 reference genome.® 5.3.2 and GraphPad Prism 9.Data were visualized using cBioPortal (v3.7.12), Adobe Illustrator (2020), SnapGeneTP53 status in cisplatin resistance in GCTs, related to the anatomical location associated with molecular constitution. The results obtained show that the investigated cell lines, independent of intrinsic resistance, demonstrate a beneficial effect of the loss of TP53 regarding cisplatin resistance, as schematically represented in TP53, originally present in the commonly used NCCIT, is functional in the context of cisplatin sensitivity, as the knock-out of this mutant resulted in increased cisplatin resistance. The isogenic generated cell lines provide a novel informative model system to study the involvement of TP53 in the original cellular background (of NCCIT ad 2102Ep), and provide insight into the clinical behavior of GCTs. Moreover, we provide, to our knowledge, for the first time, insights into the functionality of the hemizygous TP53 mutant present in NCCIT, and, additionally, we developed a cell line harboring a bona fide TP53 null status. More GCT cell lines originating from both tumor sites , or even tumor-derived organoids from these sites, could provide more insights into the role of TP53 in the clinical behavior and chemotherapy response of these tumors. These data can aid in patient stratification for optimal clinical decision making, especially for mediastinal tumors in which TP53 mutations are more common. Patients could benefit from screening for intrinsic TP53 mutations in the primary tumor or acquired TP53 mutations in the refractory malignancies. This study illustrates the contribution of TP53 not only to known cisplatin sensitivity, but also as a potential target for acquired cisplatin resistance.In conclusion, this study, combining GCT patient data characteristics and functional mechanistic cell line investigations, illustrates the role of


Currently, no clinically useful biomarkers for radioresistance are available in head and neck squamous cell carcinoma (HNSCC). This study assesses the usefulness of Cell Line Microarray (CMA) method to enhance immunohistochemical screening of potential immunohistochemical biomarkers for radioresistance in HNSCC cell lines.Twenty-nine HNSCC cell lines were cultured, cell pellets formalin-fixed, paraffin-embedded, and arrayed. Radioresistance features of the cell lines were combined to immunohistochemical stains for p53, NDFIP1, EGFR, stem cell marker Oct4, and PP2A inhibitor CIP2A.Expression of p53, EGFR or CIP2A did not indicate intrinsic radioresistance in vitro. Stem cell marker Oct4 nuclear positivity and NDFIP1 nuclear positivity was correlated with increased intrinsic radioresistance.The usefulness of CMA in analysis of HNSCC cell lines and discovery of biomarkers is demonstrated. CMA is very well adapted to both testing of antibodies in a large panel of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC.The online version contains supplementary material available at 10.1186/s12885-021-08618-6. Radiotherapy has a major role in the treatment of head and neck squamous cell carcinoma (HNSCC). Albeit the general radiosensitivity of HNSCC, radioresistance of a subset of the tumors is a major clinical problem requiring further study both for an enhanced mechanistic understanding of radioresistance, and to overcome clinical radioresistance in patient therapy. Putative mechanisms responsible for clinical radioresistance include hypoxic environment, EGFR-pathway alterations, epithelial-mesenchymal transition, deregulation of p53, angiogenesis, and cancer stem cells . DespiteThe inability to correctly identify clinically significant molecular events in cell line studies remains a significant problem, since virtually all in vivo cancer studies are preceded by in vitro cell line investigation into cancer behavior and characteristics. The genomic and molecular diversity of different cell lines complicates the selection of cell lines for in vitro studies. Behavior of different cell lines may vary according to the planned experiment e.g. due to differences in protein expression or genomic features. Thus, selection from the multitude of available cell lines happens often by trial and error. Thus, there is a high demand for high-throughput screening method for cell line characteristics.Tissue microarray (TMA) methods have proven to be an excellent tool for profiling and screening tumor samples , 5. ReceIn this study, we aimed to assess a carefully selected panel of five immunohistochemical stains of putative radiotherapy biomarkers – p53, EGFR, Oct4, NDFIP1 and CIP2A – in head and neck cancer cell lines. Mutations of the tumor suppressor p53 occur frequently in HNSCC tumors and cell lines , 9. Whil2 in Dulbecco’s Modified Eagle’s Medium (DMEM), with 10% FCS, glutamine and antibiotics (penicillin and streptomycin). The intrinsic radioresistance of UT-SCC cell lines was previously determined using the 96-well clonogenic assay for radioresistance [The included 26 UT-SCC cell lines are summarized in Table sistance , 9, 22. Three previously established stable shRNA-transfected UT-SCC cell lines were included, two shCIP2A-silenced variants of UT-SCC-24A and one shCIP2A-silenced variant of UT-SCC-14A. shRNA cell lines were generated using pGIPZ lentiviral vectors consisting of a GFP tag and puromycin resistance (Open Biosystems). Cells transfected with lentiviral vector pGIPZ.NS shRNA containing non-silencing shRNA served as control cells expressing high CIP2A. Two stable cell lines for low CIP2A expression were generated using pGIPZ.shRNA (#556) and pGIPZ.shRNA (#557) containing targeting CIP2A antisense sequences TACATCAGCAGCAAGTTTG and TACTCAATGTCTTTATGTG, respectively. Lentivirus production followed the standard protocol. At the time of transduction, cell line confluency was approximately 40–50%. After infection, the cells were selected for puromycin resistance, and if the number of GFP-positive cells was low, the cells were sorted with FACS for further experiments. Finally, all cell lines were tested negative for replication competent viruses (RCV test) as well as for Mycoplasma, Acholeplasma, Entomoplasma and Spiroplasma . Western blot using anti-CIP2A antibody  was used to confirm the successful silencing of CIP2A expression  the supernatant was removed and 10 ml of buffered formalin was added for 48 h, after which the pellets were stored in PBS (+ 4 °C). Microfuge tubes were cut open, the pellet transferred into a tissue cassette, and submitted for paraffin embedding  with OptiView DAB kit and with 64-min CC1 preparation and 32-min antibody incubation. Mouse monoclonal anti-CIP2A antibody  was used. For PME-1, SET, LIMA1, NDFIP1, and Oct4 stainings, immunohistochemical stainings were done as previously described , 24. TheAll stainings were analyzed independently by at least two authors , and contradictory cases were discussed until consensus was reached. For microscopic photographs, slides were scanned using Pannoramic 250 Flash slide scanner. Using the CaseViewer software, photographs of either 10-fold or 20-fold magnification were exported in 300 dpi quality. Images were cropped in image editing software, while no color adjustments were performed.p53 staining was analyzed using a previously established 3-tier system consisting of deletion-like absence of p53 staining, wild-type pattern of staining, and mutation/amplification-associated aberrant-type overexpression. EGFR staining was scored in a 4-tier system  for cytoplasic/membraneous staining pattern. For CIP2A, the intensity of the cytoplasmic/membraneous staining was scored on a scale of 1 to 3 as weak/negative, intermediate or strong, taking into account the number of positive cells. In patient samples, CIP2A was uniformly present and was scored similarly based on cytoplasmic/membraneous staining intensity. Oct4 immunostaining was analyzed for the presence of positive nuclei. Oct4 positivity was present only in a subpopulation of cells, and thus the presence of strong nuclear immunoreactivity of individual cells was regarded positive. NDFIP1 staining was scored positive, when strong, uniform nuclear staining pattern was present, whereas cytoplasmic staining was not taken into account. A three-tier scoring system was used for uniform nuclear PME-1 and SET staining .p value < 0.05 was deemed significant.Cell line data, patient data, and results of immunohistochemistry were entered in SPSS 25 software. Dependence of cell line radioresistance to the staining results was analyzed using General Linear Model statistics. Both main effects and interactions were observed. Estimated marginal means were calculated and 95% confidence intervals determined using bootstrapping. Correlation was determined by Spearman’s method. Throughout, For the CMA construction, UT-SCC cell lines with previous data on radiosensitivity were preferentially selected. Altogether 26 UT-SCC cell lines were included, the majority of which were derived from male patients with oral cavity cancer Table . Six celp = 0.020), but no correlation between the expression of other biomarkers. Patient age and gender or tumor site, T class or nodal positivity were not directly linked with the expression of any investigated biomarkers.Immunohistochemical stains for p53, EGFR, Oct4, CIP2A, and NDFIP1 were analyzed (Fig. The associations between staining results and intrinsic radioresistance of the cell lines were analyzed. Expression of p53, EGFR, and CIP2A were not associated with radioresistance (Fig. To test the functionality of CMA in the validation of antibody specificity, and to study protein interactions in genetically modified cancer cell lines, three CIP2A shRNA-silenced cell lines were included in the CMA. CIP2A shRNA-silencing was confirmed by Western blot Fig. A and is Since CIP2A failed to predict radioresistance in the CMA and since CIP2A silencing did not affect other putative biomarkers expression, we were interested, whether CIP2A would have an association with other PP2A inhibitors and performed immunohistochemistry of two well-established endogenous PP2A inhibitors, PME-1 and SET, which are not pronouncedly identified as radiotherapy biomarkers (Supplemental Figure HNSCC consists of a genetically and behaviorally heterogeneous group of malignancies. The observed genomic instability is due to mutagenic insults on the mucosal lining of the upper aerodigestive tract such as tobacco and alcohol . AccordiPrior studies presenting similar methods of cell line array construction have mostly included relatively small amounts of cell lines, and have been constructed with other than head and neck cancers , 31, 32.In addition to efficiency, CMA provides other notable benefits as well. Use of exclusively cellular material allows strictly focusing the evaluation of IHC staining positivity on cancer cells and removing the majority of unspecific sources of unwanted IHC signaling positivity. This also provides a fixed, paraffin-embedded biobank of cell lines. It is possible to manipulate and modify the cell lines before constructing CMA, allowing e.g. molecular profiling of cell lines by inhibiting or enhancing the expression or effect of certain genes. This may also be applied to drug development and discovery studies, since cell lines may be exposed to drug of interest during culture. Moreover, in a CMA, all types of analysis available for FFPE patient samples can be used. As the CMA paraffin block is handled similarly to FFPE samples, cell line results can directly be compared with patient staining results. Our UT-SCC CMA included three CIP2A-shRNA-silenced cell lines, which demonstrate a highly successful silencing after lengthy cell line culture. CMA offers a powerful tool for the characterization of cellular effects of targeted silencing.A major weakness of cell line experiments in general and thus also of this study, is that cell line models are unable to take into account patient-related factors such as immunological responses, that may decisively affect the success of radiotherapy in HNSCC patients. However, the intrinsic radioresistance of the cancer cells is an important phenomenon in clinical radioresistance , 9, 22. Oct4 is a transcription factor essential for pluripotency in embryonic stem cells and testicular maturation. Oct4-related stemness has been found to have a role in radiotherapy resistance of various cancers, including HNSCC , 33–35. This study, in addition to confirming the feasibility of CMA methodology to screen for new predictive factors in head and neck cancer, revealed potential immunohistochemical biomarkers associated with radioresistance in head and neck cancer cell lines. Especially interesting for its potential clinical implications is the stem cell marker Oct4 which has been demonstrated to have a significant predictive role in previous studies , 14. In Additional file 1: Figure S1. Representative examples of low and moderate PME-1 and moderate and strong SET stainings.