Ten of twelve cell lines harbored alterations in common HRR-related genes, and five cell lines were HRD-positive, although this parameter did not correlate well with Olaparib sensitivity. Moreover, functionally relevant APC and β-catenin mutations were registered, which were also detected in 4/176 (2.3%) samples on a CCA microarray. Although rare, these alterations were exclusive to large duct type CCA with associated intraductal papillary neoplasms of the bile duct (IPNB) in 3 cases, pointing at a distinct form of cholangiocarcinogenesis with potential specific vulnerabilities.Cholangiocarcinoma (CCA) features a dismal prognosis with limited treatment options. Genomic studies have unveiled several promising targets in this disease, including fibroblast growth factor receptor (FGFR) fusions and isocitrate dehydrogenase (IDH) mutations. To fully harness the potential of genomically informed therapies in CCA, it is necessary to thoroughly characterize the available model organisms, including cell lines. One parameter to investigate in CCA is homologous recombination deficiency (HRD). While mutations in homologous recombinational repair (HRR)-related genes have been detected, their predictive value remains undetermined. Using a targeted next-generation sequencing approach, we analyzed 12 human CCA cell lines and compared them to 62 CCA samples of the molecular tumor board cohort. The AmoyDx FGFR2 fusion-positive pretreated patients by the Food and Drug Administration (FDA) in 2020  , Ivosidenib (IDH1-inhibitor) [range 3.13–200 µM] (MedChemExpress), Foretinib (MET-inhibitor) [range 0.13–8 µM] (MedChemExpress), Trametinib (MEK-inhibitor) [range 3.13–200 µM]  or Mirdametinib (MEK-inhibitor) [range 3.13–200 µM]  were applied. The measurements were obtained in triplicates for a total of 72 h.Cell viability assays were performed using the xCELLigence50 values were computed in the RTCA software using the formula for sigmoidal dose-response (variable slope) across the observational period.Raw data were analyzed with the RTCA software . The data were normalized to the timepoint of inhibitor addition. Moreover, ICTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) at a ratio of 100:1. The Pierce BCA Protein Assay Kit (ThermoFisher Scientific) was used to determine the protein concentration. Protein denaturation was done in sample buffer (Laemmli Sample buffer: β-ME = 19:1) at 95 °C for 5 min. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was placed onto a nitrocellulose membrane . The membrane was blocked in EveryBlot Blocking Buffer (Bio-Rad Laboratories) at room temperature for 15 min and subsequently incubated with primary antibodies , membranes were developed with the Clarity Western ECL Substrate (Bio-Rad Laboratories). Image acquisition was performed on a ChemiDocTM MP Imaging System (Bio-Rad Laboratories).Cell pellets were homogenized and lysed in T-PER Mammalian Protein Extraction Reagent  with the Halttibodies  at 4 °C tibodies  at room ®  for 10 min. The primary antibody was diluted in Dako Antibody Diluent® (S2022). Sections were incubated herein in a humidity chamber at room temperature overnight (see ® (S3006), the secondary antibody Histofine Simple Stain MAX PO® anti-rabbit or anti-goat was administered for 60 min at room temperature. Following two additional washes in Dako washing solution® (S3006), the chromogenic reaction was performed with the Dako Liquid DAB + Substrate Chromogen System®. Slides were stained in Mayer’s hemalum for 10 s. Coverslips were automatically placed on top with the Ventana BenchMark Ultra® . β-catenin antibodies used with respective dilutions are listed in For immunohistochemical analyses, two µm thick histological sections were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Sections were incubated for 30 min at a 70 °C temperature. Afterward, slides were deparaffinized through a series of xylene and gradient alcohols to water. For antigen retrieval, slides were cooked for 8 min at 110 °C in citrate pH 6.0 and then placed in iced water. Next, slides were incubated in 1× Dako Peroxidase-Blocking Solution®  was used for image acquisition. The 20× magnification objective was selected. Stitched images were visualized using the SlideViewer® (Sysmex) software. Screenshots of relevant regions were generated with a 300 ppi resolution.The slide scanner Pannoramic 1000 Flash RX® RSC RNA FFPE Kit  and Maxwell® RSC (Promega) device. For RNA concentration, the Agilent TapeStation®  and a fluorometer  were used.Nucleic acids from formalin-fixed, paraffin-embedded patient (FFPE) tissue were isolated using the Maxwell® DNA Mini Kit  was used for DNA extraction from CCA cell lines. Total RNA extraction from CCA cell lines was performed with the NucleoSpin® RNA Plus Kit . RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). qRT-PCR was performed on the CFX96TM Real-Time System . The following protocol was applied: 1.0 µL primer mix, 5 µL TaqMan Universal PCR Master Mix (ThermoFisher Scientific), 2.5 µL cDNA (100 ng), and 2.1 µL d2H2O. Cycling conditions: polymerase activation at 95 °C for 2 min, 40 cycles: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Primers for qRT-PCR were ordered from Thermo Fisher Scientific and listed in The AllPrepTM 550Dx (Illumina). Variant annotation was performed using Qiagen Clinical Insight (QCI) Interpret . In addition, the software vcf2maf converter  was employed to generate maf files, which were uploaded to a locally installed version of cBioportal [The hybrid capture-based TruSight Oncology 500 Library Preparation Kit  was employed for library preparation according to the manufacturer’s protocol. Unique molecular identifier (UMI)-containing adaptors were ligated to the DNA fragments. Next, a pool of oligos specific to the 523 genes included in TSO500 were hybridized to the DNA libraries. Streptavidin magnetic beads captured the probes hybridized to the targeted regions. Primers were used to amplify the enriched libraries before purification with sample purification beads. Afterward, the libraries were pooled, denatured, and diluted. TSO500 libraries were sequenced on a NextSeqioportal ,33 for a® HRD-focus panel  to screen 24,000 single nucleotide polymorphisms for HRD scoring [TM550Dx device (Illumina). The calculation of the HRD score (GScore) was done by summing loss of heterozygosity, large-scale state transition, and telomeric allelic imbalance by means of the ANDAS software (Amoy Diagnostics). HRD-positivity was defined as a GScore ≥ 50 following the manufacturer’s instructions.Genomic DNA from CCA cell line samples was extracted for library construction and analyzed with the AmoyDx scoring . 100 ng ® PanCancer Pathways panel (NanoString Technologies), including 770 genes from thirteen canonical pathways, was chosen.The comparison of differential mRNA expression was carried out using the NanoString nCounter . This technology relies on direct molecular barcoding of defined target mRNA sequences. Specifically, 100 ng of total RNA (OD260/280 ratio 1.7–2.2) was hybridized overnight with reporter and capture code set at 65 °C. Excess probes were washed off using a two-step magnetic bead-based purification on the nCounter Prep station (NanoString Technologies). The purified complexes were eluted off the beads, immobilized on a cartridge, and subsequently aligned. Data were collected using the nCounter Analyzer (NanoString Technologies) at 555 fields of view through epifluorescence microscopy and CCD capturing. The nCounterTM analysis software Version 4.0 (NanoString Technologies). All samples passed quality control in this software. Additional analyses were conducted using the nCounter Advanced Analysis 2.0 plug-in (NanoString Technologies). Automated normalization was chosen by selecting those genes that minimize the pairwise variation statistic. For visualization, unsupervised clustering was selected to generate a heatmap based on the normalized data counts of individual mRNAs. Differential expression was plotted as a volcano plot with individual genes −log10.  and log2 fold change compared to the control group. The Pathview module was used to assess gene expression changes within the WNT pathway visually. The undirected global significance score of the included Kyoto Encyclopedia of Genes and Genomes (Kegg) gene sets was calculated and displayed as a heatmap to determine group pathway changes. Details can be obtained from the nCounter Advanced Analysis  documentation. For differential expression analyses, p-value adjustments with the Benjamini-Yekutieli method were selected.Data were analyzed using the nSolver®-targeted sequencing panel. Tissue microarray samples were obtained from the primary hepatic tumors. The tissue samples of our molecular tumor board registry were derived either from the primary tumor (39/62) or from metastatic sites (23/62).All patients provided written informed consent under our institutional review board-approved protocol, and the study was conducted following the Declaration of Helsinki. FFPE tissue specimens of 62 CCA samples of the molecular tumor board cohort (34 intrahepatic cholangiocarcinomas (iCCA), 10 perihilar cholangiocarcinomas (pCCA), 9 distal cholangiocarcinomas (dCCA), 1 mixed hepatocellular cholangiocarcinoma (HCC/CCA), and 8 gallbladder carcinomas) and 4 CCA samples of a tissue microarray  displaying positive nuclear β-catenin staining were analyzed using the TSO500®-based targeted sequencing of 523 cancer-related genes. These analyses met the prespecified quality control parameters of coverage in exonic nucleotides of a minimum of 50 UMI-reads of 90%. In addition, the variant call cut-off was set to a 5% allele fraction. Panel-based microsatellite analysis was possible for all samples, since a sufficient sequencing depth was achieved for more than 40 monomorphic microsatellite loci in each case. Moreover, an evaluation of the tumor mutational burden (TMB) was performed, as the sequenced region was more than 1 MB with a sufficient sequencing depth. RNA-based analysis for genetic rearrangements and a splice variant analysis were also possible for all studied cell lines. The same analyses were carried out on 62 CCA patient samples to compare the frequencies of the detected genetic events and assess the available CCA cell lines as a suitable disease model.RNA and DNA extracts of the twelve iCCA cell lines under investigation were subjected to TSO500KRAS mutations occurred in 58% of cell lines as opposed to 16% of patients. In patients, KRAS mutations were detected more frequently in pCCA  and dCCA  than in iCCA , while none was found in the gallbladder carcinoma specimens . TP53 tumor suppressor gene mutations were observed in cell lines and CCA tissue samples with 50 and 23% occurrence frequencies, respectively. Furthermore, tumor suppressor genes with a mutational frequency of >10% in cell lines involved SMAD4, SMARCA4, ARID1A, ARID2, and RBM10. Notably, the latter 4 genes are either implicated in chromatin remodeling or heterochromatin assembly in the case of RBM10 [IDH1 mutations were found in 2 cell lines  and 4 CCA samples . A TERT-promoter mutation was exclusive to one cell line. Individual mutations in cell lines were registered in the histone methyl transferases KMT2A and DNMT3A, the former also in one CCA sample. NSD1, a ligand-regulated transcription factor activated by steroid hormones, while also possessing activity as a histone methyl transferase [MAP2K1 gene could be found in a CCA cell line  and in a case of gallbladder adenocarcinoma . The former mutation has been described to confer sensitivity to a combination of trametinib and panitumumab in a case of colorectal cancer before [TSC1, FBXW7, and SPEN were each mutated in one cell line and one CCA sample. Especially the role of the ubiquitin ligase FBXW7 has been implicated in cholangiocarcinogenesis via activated AKT signaling and the induction of c-Myc [APC and CTNNB1 in CCA cell lines further suggest an involvement of the WNT pathway, which will be extensively discussed below. Also, considering variants of unknown significance, mutational frequencies of genes defined in various Kegg pathways were analyzed  and MAP2K2 (25%) with a high frequency. MAP2K2 mutations could, however, not be found in the CCA patient cohort. Here also, various alterations could be detected in the RTK-RAS pathway, including ERBB2 (8.2%), ERBB3 (8.2%), FGFR2 (16.4%), MET (9.8%), NRAS (4.9%), and MAP2K1 (3.3%) as potentially druggable targets. Furthermore, mutations occurred in relevant genes of several more pathways both in CCA cell lines and tissue samples: the PI3K pathway , the Notch pathway (NOTCH1-4), which has been proven to be a prerequisite for cholangiocarcinogenesis [RB1, FBXW7, TP53).Based on these results, we first evaluated the occurrence and frequencies of putative driver mutations as annotated by either OncoKB  or QCI . The dis 5.9%, 2/, while nof RBM10 . IDH1 munsferase , was mutr before , while tr before . TSC1, Fof c-Myc . Of noteanalyzed . Mutatio in pCCA %, 4/10 aogenesis , and theNext, the GScore was evaluated in CCA cell lines and correlated with mutations in HRR-related genes ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK2, CHEK1, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, MRE11, NBN, PALB2, RAD50, RAD51, RAD51B, and RAD51D . Among tApart from HRD, 2/12 (16%) of CCA cell lines had a TMB > 10 mut/MB (YSCCC cells with 34.6 mut/MB and KKU-M213 cells with 46.1 mut/MB), while a high TMB was only detected in 1 iCCA among 43 evaluable tissue samples (2.3%). Microsatellite instability (MSI), as inferred from panel diagnostics and defined as >10% unstable MSI sites, was not present in the cell lines. One of 25 MSI (4%) evaluable patients among the CCA tissue samples reached the criteria for MSI, as evaluated by the targeted panel diagnostics.MET breakpoint in the former fusion was located in exon1 so that no functional kinase domain would result. The functional significance of the detected EWSR1-ADRBK2 fusion is unclear. EWSR1 fusions have rarely been described in adenocarcinomas such as colorectal cancer. The underlying mechanism is usually an enhanced activation of a C-terminal transcription factor [MET-splice analysis, several different splice variants were detected in 5/12 (42%) of CCA cell lines. In part, multiple splice variants were detectable in a single cell line, such as OZ and KKU-M156  in TKKK cells and two cases of EGFR CNVs > 2. Both alterations can represent important oncogenic drivers and are potentially amenable to targeted therapies. Likewise, the CCA samples also included one EGFR-CNV positive iCCA , which was confirmed by immunohistochemistry (H-Score 270), and one ERBB2-CNV positive extrahepatic CCA (CNV 11.896). In addition, CCA cell lines also displayed RICTOR copy number gains , which were recapitulated in human CCA samples, where 3/62 samples (4.8%) were positive for RICTOR copy number gains .Finally, we analyzed copy number variations (CNV) of the 59 genes included in the TSO500relevant . PrimariKRAS driver mutations, an elevated ratio of phosphorylated ERK/ total ERK was evident in HUCC-T1, KKU-M055, KKU-100, KKU-M213, OZ, and SG231 cell lines. In contrast, the cell lines KKU-M156 and RBE only displayed a minor elevation in the ratio compared to KRAS WT cell lines CC-LP-1 and HuCC-A1, even though they harbored classical activating KRAS mutations at positions G12 and G13. Particularly noteworthy is that the KKU-M156 cell line, derived initially from KKU-M213 cells, is not defined by strong ERK activation, although the mutational background is highly congruent, and the same KRAS G13C mutation is present in both cases. KKU-M055 containing a MAP2K1-mutation showed a slightly elevated ERK ratio. The cell line TKKK also showed a high ratio of phosphorylated ERK despite being KRAS WT, presumably because of ERBB2 amplification The described EGFR copy number gains were confirmed to result in an elevated expression of EGFR in the case of HuCC-A1 and YSCCC cells, while elevated protein levels were not observable in TKKK. In addition, the ERBB2-copy number gain in TKKK translated into a high protein expression. The diverse reported MET splice variants were not clearly associated with a change in c-Met protein levels. Regarding RICTOR copy number gains, low to medium Rictor protein levels were observed by Western Blotting. The effectors of mTORC2 signaling p-EPHA2 and p-GSK3β were in part upregulated (in CC-LP-1 and HuCC-A1). In addition, p-RPS6 and p-4EBP1, canonical effectors of mTORC1, were positive in most cell lines.To determine the biological relevance of the detected genetic alterations in CCA cell lines, Western Blot analyses were performed . In the 50 values were calculated for a total of 10 cell lines, which were suitable for xCelligence® gradient concentration experiments (OZ and TKKK were omitted because of very slow growth characteristics). The two cell lines with the highest GScore (>80) were found to be most sensitive to Olaparib treatment and had IC50 values of 59 and 91 µM, respectively (KKU-M156 and HuCC-T1). However, two other cell lines evaluated as HRD-positive demonstrated IC50 values that were either in the range of HRD-negative cell lines  or even much higher . When comparing the IC50 values for the MEK-inhibitors Mirdametinib and Trametinib with regard to KRAS mutational status, no difference could be observed. Using the MET-inhibitor Foretinib, the CC-LP-1 cell line, which contained a MET-CAV1 fusion and several MET splice site variants of unknown significance, had the lowest IC50 value (1.05 µM) compared to cell lines without MET splice variants. Surprisingly, the two IDH1 mutated cell lines RBE and YSCCC did not show a decreased IC50 compared to IDH1 WT cell lines when using the IDH1 inhibitor Ivosidenib. Possible confounders could be the cooccurrence of a KRAS mutation in RBE cells, an unusual event that has only been described in one case of biliary cancer to date [EGFR in YSCCC cells. Both of these concomitant alterations could inhibit the antiproliferative effects of Ivosidenib.Next, we investigated the predictive value of variants detected in the CCA cell lines , Table 4 to date , and theCTNNB1 mutation (p.G34V) in CC-LP-1 cells and a likely pathogenic APC mutation in the KKU-M055 cell line (p.D1636fs*2) (CTNNB1 (Y30*/pCCA and I610M/ iCCA). Furthermore, besides the CTNNB1-mutation, CC-LP-1 also harbored an APC variant of unknown significance (A1670V). Hence, the frequency of APC alterations in CCA cell lines was 16.7% (2/12) compared to 6.6% of CCA samples . Furthermore, alterations could be found in Ring finger protein 43 (RNF43) in 1 CCA cell line  and 2 CCA tissue samples . RNF43 is a negative regulator of WNT/β-catenin signaling by reducing the membrane level of Frizzled [AMER1 displayed alterations both in CCA cell lines and CCA tissue samples, while mutations in AXIN1, AXIN2, and TCF7L2 were exclusive to patients’ samples.Given the surprising occurrence of a pathogenic 636fs*2) , we broa636fs*2) a. SpecifFrizzled . FinallyCTNNB1 and APC in the two CCA cell lines CC-LP-1 and KKU-M055, Nanostring® gene expression analyses using the PanCancer Pathway Panel were conducted in comparison with the two WNT pathway WT cell lines HuCC-A1 and TKKK  pathogenic mutations in and TKKK b,c. HereTcf axis . Simulta reduced . In cont® software  change across samples were generated using the nSolversoftware c. Here,  to TKKK . The relKKU-M055 b,c. AltoCTNNB1 mutations. Whole slides were also stained with equivalent results. By targeted sequencing using the TSO500®-panel, three patients were identified to harbor likely pathogenic APC mutations (patients (P)1–3), and one patient had a pathogenic activating β-catenin mutation  displayed marked nuclear positivity of β-catenin tested with three different β-catenin antibodies , which ction P4; , explains before . Another®-analysis was conducted. For comparison, 3 additional samples from our CCA sequencing cohort without APC/CTNNB1 mutation were randomly selected (P5–7), and membranous β-catenin staining was verified by immunohistochemistry  and overall survival (OS) has been observed in the case of fluorescent in situ hybridization positive extrahepatic cholangiocarcinomas (eCCA), comprising both pCCA and dCCA, which was, however, not statistically significant. The association was instead not found for iCCA and appeared even reversed in gallbladder carcinoma [MAP2K1 mutation in the KKU-M055 cell line. This mutation also resulted in an increase in the p-ERK/ERK ratio. Nonetheless, this increase was lower than most KRAS mutations, which overall showed the most substantial effect on this parameter. In the CCA cohort, another driver MAP2K1 mutation occurred in a case of gallbladder adenocarcinoma (p.G128D). MAP2K1 activating mutations are known in the literature to play a critical role in histiocytic neoplasms, where in individual cases, responses to a cobimetinib therapy have been described [KRAS mutations as a positive predictive factor, which is contrary to retrospectively analyzed findings of a study of binimetinib plus capecitabine in CCA patients, where RAS/RAF/MEK/ERK pathway mutations showed significantly better tumor response, PFS and OS [This study aimed at comprehensive molecular profiling of CCA cell lines and comparison with actual patients’ tumor samples. Altogether, a frequency of major genetic alterations in accordance with the available literature could be determined . Recurrearcinoma , promptiescribed . FurtherS and OS .Altogether, cell lines had a higher proportion of oncogenes and tumor suppressor genes, while maintaining similar mutational characteristics to patients’ samples. The increased occurrence of oncogenes could favor the growth characteristics of CCA cell lines, increasing their aptitude for research. Thereby, a slight bias is incorporated into experiments involving CCA cell lines. This should be accounted for when choosing in vitro models to mirror the human disease.ARID1A mutations were found not to be a good discriminator for HRD positivity, as they were found both in HRD positive and negative CCA cell lines. In contrast, a driver mutation observed in RAD50 in HuCCT-1 occurred together with a high GScore, presumably indicating an association. When extending the analysis to VUS of DDR genes, 10/12 cell lines harbored alterations in DDR genes, and overall the affected genes were similar between cell lines and patients’ samples. Of note, 38% (24/62) of patients’ samples harbored pathogenic mutations in HRR-related genes. Although the association to HRD might not be clear, the frequency should warrant further experimental elucidation to determine the extent of HRD positivity in CCA and specify the relevant HRR-related genes and mutations that result in a clinically actionable phenotype. Undoubtedly, the high frequency in CCA cell lines is an encouraging finding in this respect. Since individual studies have purported the predictive value of BRCA1/2 mutations in CCA [IDH1 mutations did not result in an increased GScore, although IDH1 plays a critical role in DNA repair  were HRD positive was unexpected, especially since no clear association to known driver mutations in DNA-damage related (DDR) genes could be established. s in CCA , it woulme ALKBH ). The clme ALKBH . It willAPC and a β-catenin mutation in cell lines. In CCA, the activation of the β-catenin pathway has been evaluated by immunohistochemistry before. In particular, one study reported a reduction in membranous expression in 82% (of 71 samples) and an erroneous nuclear expression in 15% of samples. However, the reason for the nuclear localization of β-catenin had not been determined in this particular study, potentially because only CTNNB1 mutations were analyzed, which could not be found there [CTNNB1 or APC mutations. Of particular interest is the finding that the respective CCAs with aberrant nuclear expression were all CCA of the large duct type. On closer examination, an association with IPNB could be determined in three of four cases, clearly delineating a special mode of cholangiocarcinogenesis occurring in the large bile ducts and involving the classical canonical WNT pathway. In contrast, the non-canonical WNT pathway has been postulated to play a critical role in intrahepatic cholangiocarcinogenesis [AXIN2. The occurrence of β-catenin in IPNB has been described before, albeit without the association with invasive carcinoma [CTNNB1 mutation detected here. This finding hints at the ability to harness WNT pathway alterations therapeutically, and large duct type CCA originating preferentially from IPNBs could be potential candidates for such treatments.Finally, we set out to study the importance of the WNT/β-catenin pathway prompted by the occurrence of an nd there . In contogenesis . Of notearcinoma . The gaparcinoma  determinOverall, the current study provides a detailed overview of the mutation landscape of human CCA cell lines. Noticeably, the data presented here indicate that the genetic alterations occurring in CCA cells are also present in human CCA specimens, implying the potential use of these cell lines as a platform for drug testing. Nonetheless, we found that the response to targeted treatments of CCA cell lines harboring actionable mutations is heterogeneous, suggesting that the effectiveness of tailored therapies might depend on the intricated crosstalk between driver mutations and additional genetic and epigenetic events.


Since T-VEC is already approved for treatment of melanoma, its promising efficacy shown here also for NUT carcinoma (NC) cell lines may create a rapid transition to individual treatments as well as clinical trials in NC patients. The idea of combining T-VEC immunotherapy with BET inhibitors is strengthened by the assumption that the initial rapid response of NC to BET inhibitor therapy and the additional direct tumor cell lysis triggered by virotherapeutics may be able to effectively stabilize or even shrink the tumor cell mass to bridge the time gap until the durable immune response, induced by immunovirotherapy, can lead to complete tumor remission. This would signify a real breakthrough for patients suffering from this extremely aggressive tumor, whose average survival time is currently in the range of only six months.talimogene laherparepvec (T-VEC) and the iBET compounds BI894999 and GSK525762 were assessed in vitro in monotherapeutic and combinatorial approaches. Viral replication, marker gene expression, cell proliferation, and IFN-β dependence of T-VEC efficiency were monitored. T-VEC efficiently infected and replicated in NC cell lines and showed strong cytotoxic effects. This implication could be enhanced by iBET treatment following viral infection. Viral replication was not impaired by iBET treatment. In addition, it was shown that pretreatment of NC cells with IFN-β does impede the replication as well as the cytotoxicity of T-VEC. T-VEC was found to show great potential for patients suffering from NC. Of note, when applied in combination with iBETs, a reinforcing influence was observed, leading to an even stronger anti-tumor effect. These findings suggest combining virotherapy with diverse molecular therapeutics for the treatment of NC.NUT carcinoma (NC) is an extremely aggressive tumor and current treatment regimens offer patients a median survival of six months only. This article reports on the first in vitro studies using immunovirotherapy as a promising therapy option for NC and its feasible combination with BET inhibitors (iBET). Using NC cell lines harboring the BRD4-NUT fusion protein, the cytotoxicity of oncolytic virus  NUTM1 (formerly known as NUT (nuclear protein in testis)) gene [NUTM1 is a rather unstructured protein with an unknown function that can be linked to various fusion partners, including BRD4 (70%), but also BRD3 [NSD3 [ZNF532 [NUTM1 then results in the expression of fusion oncoproteins that interact with chromatin and induce abnormal histone acetylation, which in turn leads to an epigenetic blockade of cellular differentiation and uncontrolled cell growth [NUTM1 rearrangement [NUT carcinoma (NC), formerly known as NUT midline carcinoma (NMC), is a rare but very aggressive tumor, genetically defined by a reciprocal translocation involving the lso BRD3 , NSD3 [3l growth . The incing BRD4 0%, but aThe current treatment approach is surgical resection before or after chemoradiation, resulting in a median survival of only about six months ,9,10. ThEven application of highly specific inhibitors of BET proteins (iBETs) ,12 that Moreover, results of recent preclinical and clinical studies  indicate that this tumor type requires a combination of different treatment approaches, also including novel ones, to counteract the highly aggressive growth of NUT carcinoma.Oncolytic immunotherapy has already demonstrated its potential for cancer treatment in other tumor types, but has not been investigated in NUT carcinoma so far. Oncolytic viruses (OVs) are replication-competent viral vectors, which are capable of selectively infecting tumor cells, replicating massively within and destroying infected tumor cells, concomitantly releasing thousands of progeny virus particles . Furthertalimogene laherparepvec (T-VEC), already approved for the treatment of melanoma [ICP34.5/ICP47-deleted version of strain JS1, which may further enhance the virus-induced systemic anti-tumor immune response [In this preclinical study, the oncolytic herpes simplex virus type 1 (HSV-1) melanoma , was useresponse . The iBEresponse ,23, wereresponse .Here, the purpose was to investigate for the first time whether immunovirotherapy with T-VEC could become a new treatment option for NC patients. Moreover, combinatorial treatment with well-characterized iBET compounds was performed, in order to exclude any possible mutual negative effect of both therapeutic modalities as a basic prerequisite for clinical application, but also to investigate a possible additive anti-tumor effect. In this line, parameters such as cytotoxicity, tumor cell proliferation, viral replication, and interferon dependence of the oncolytic effect were investigated in six human tumor cell lines harboring the BRD4-NUT fusion oncoprotein.BRD4-NUTM1 fusion gene, were employed in this study. NC cell lines 143100 and HCC2429 were kindly provided by Dr. Xin Zhang from University Hospital Essen, Germany. NC cell lines 14169, 10-15, and JCM were a kind gift from Dr. Chris French, Boston, MD, USA. NC cell line Ty-82 was purchased from JRCB Cell Bank (No.: JCRB1330). Cell line authentication was performed on all NC cell lines by STR profiling at the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Germany. Vero cells (African green monkey kidney cells) were obtained from the DSMZ (No.: ACC 33) and were used for virus titration only. All cell lines were cultured in DMEM supplemented with 10% FCS. All cell lines were tested negative for mycoplasma contamination prior to utilization .A panel of six human NUT carcinoma (NC) cell lines, all harboring the BI894999 (Amredobresib) was kindly provided by Boehringer-Ingelheim RCV ; GSK525762 (Molibresib) was obtained from Seleckchem . NC cells were seeded in 24-well plates and treated 24 h later with the respective iBET compound. For this purpose, medium was replaced with cell culture medium containing iBETs at desired concentrations, and cells were incubated until their respective readout.talimogene laherparepvec) was kindly provided by Amgen Inc. . NC cell lines were infected with T-VEC as described previously [The oncolytic herpes simplex virus type-1 (HSV-1) construct T-VEC  cell viability assay as described previously [NC cells were seeded in E-96-well-plates and viral infection and/or iBET treatment started 24 h later. Real-time dynamic cell proliferation was monitored in 30 min intervals during the complete observation period of 120 h using the xCELLigence RTCA SP system . Cell index values were calculated using the RTCA Software .NC cells were seeded in 6-well plates and infected with T-VEC at suitable MOIs with and without additional iBET treatment. Viral replication was quantified by performing plaque assays at 1, 24, 48, 72, and 96 hpi, as described previously [NC cells were seeded in 24-well plates and pretreated with 2 ng/mL IFN-β for 16 h before infection with T-VEC at cell line-adjusted MOIs. At 16, 24, 72, and 96 hpi, supernatants were harvested for quantification of T-VEC-mediated GM-CSF expression by using MAX™ Deluxe Human GM-CSF ELISA Kit  according to manufacturer’s instructions.HCC2429 NC cells were infected with T-VEC at MOI 0.0001 or 0.001, trypsinized and fixed in Karnovsky fixative at 48 hpi and stored at 4 °C. Cell pellets were embedded as described earlier [The 143100 NC cells were seeded in tissue culture dishes and pretreated with 2 ng/mL IFN-β for 16 h or left untreated as control, before they were infected with T-VEC at MOI 0.1 or left uninfected (MOCK). At 8 and 16 hpi, cells were harvested and frozen for cryosectioning. Fresh frozen 5 µm sections were fixed with periodate–lysine–paraformaldehyde. Sections were blocked using donkey serum and stained with a primary rabbit-anti-HSV1 antibody . Bound antibody was visualized using donkey-anti-rabbit-Cy3 . For nuclear staining, Yopro  was used. Sections were analyzed using an LSM 800 confocal laser scanning microscope  and processed with the software ZEN 2.3 .p values were determined: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).Statistical analysis was performed with GraphPad Prism Version 9 . Reduction in cell mass between two treatment groups was analyzed by Brown-Forsythe and Welch ANOVA and Dunnett’s multiple comparison test. In experiments regarding IFN-β pretreatment, significance between two treatment groups was analyzed by one-way ANOVA and Šidák’s multiple comparison test. Four different The clinically licensed virotherapeutic compound T-VEC was employed to analyze for the first time the general susceptibility of human NC cell lines to virus-mediated oncolysis and to find out whether the replication cycle of T-VEC in NC cells is comparable to other already characterized tumor entities.In a first approach, transmission electron microscopy (TEM) images were taken exemplarily from T-VEC-infected HCC2429 NC tumor cells at 48 h post infection (hpi) . Steps oBRD4-NUTM1 cell lines were infected with T-VEC and the remaining NC tumor cells were measured at 96 hpi using SRB cell viability assay . However, when NC tumor cells were pretreated with IFN-β for 16 h, SRB assays at 96 hpi revealed that the potent oncolytic activity of T-VEC was almost completely impaired when compared to the IFN-β-untreated control , albeit Moreover, quantification of T-VEC-mediated GM-CSF expression in 143100 NC cells at different time points during the course of infection by ELISA indirectly provided insights into the timeline of impairment of T-VEC-mediated gene expression and thus viral replication of T-VEC B. While Similar results were observed for the other five NC cell lines . In NC cFurthermore, early infection processes were visualized by immunofluorescence staining in both IFN-pretreated and -untreated 143100 cells after infection with a high T-VEC MOI of 0.1 . In IFN-In order to develop a potent and interacting therapeutic approach for highly aggressive NC tumors, a combination of oncolytic immunovirotherapy with iBETs was investigated next. First, all six NC tumor cell lines were treated with the iBET compounds BI894999 or GSK525762 in monotherapy. Here, the anti-tumoral effects of both compounds that are already under clinical investigation for NC treatment could be confirmed for all investigated NC cell lines, using SRB cell viability assay . An iBETp < 0.01 to p < 0.0001), respectively. While for the NC cell line 10-15, neither T-VEC alone nor the combinatorial approach was able to outperform the iBET monotherapy /mL for all tested NC cell lines, indicating a high susceptibility of all six NC cell lines to T-VEC infection. Thereby, a correlation between the reduction in viable tumor cells in the SRB assay and the viral titers could be observed: in the more susceptible NC cell lines 143100, HCC2429, and 14169, viral titers increased up to 107 PFU/mL (6 PFU/mL) without a decrease at 96 hpi  surNotably, oncolytic viruses are able to indirectly target these small tumor remnants or metastases, as they can activate the body’s immune system to provide an in situ anti-tumoral immune response , and areIn this study, six human NC cell lines harboring the BRD4-NUT fusion oncoprotein were evaluated for their response to immunotherapy with T-VEC, either alone or in combination with one of two different BET inhibitors .As a major result, it was shown that in all six NC cell lines, monotherapeutic treatment with T-VEC resulted in a significant reduction in tumor cells when applied at sufficiently high MOIs. However, in order to better classify the virus concentrations used, a systematic review of the efficacy of this immunotherapy in a variety of different tumor cell lines, such as those included in the NCI-60 tumor cell panel, would be beneficial. Such systematic NCI-60 screens have already been performed with other virotherapeutics, for example, with an oncolytic measles vaccine virus  or with Type I interferons, including IFN-β, play an essential role in the immune response of healthy cells to viral infections as they can diminish the replication of viral genomes, protein translation, and virus egress . In cancIn order to demonstrate the general sensitivity of T-VEC to IFN-β and conversely show that NC tumor cells may be particularly amenable to immunovirotherapy with T-VEC due to possible defects in IFN signaling pathways, NC tumor cells were pretreated with IFN-β, before infection with T-VEC, and subsequently, anti-tumor efficacy was investigated. As a result, it was demonstrated that in all examined NC cell lines, pretreatment with IFN-β resulted in massively reduced oncolytic efficiencies, which was conversely confirmed by quantification of T-VEC-mediated GM-CSF expression. Of note, the sensitivity of T-VEC to IFN-β was found to vary depending on the NC cell line studied and the MOI used, indicating that there may be an NC cell line-specific critical virus concentration at which IFN-triggered inhibition can be overcome.The observation that pretreatment with IFN-β suppresses T-VEC-mediated gene expression at very early time points after T-VEC infection (at 16 hpi) suggests that not only viral replication but also possibly initial infection of NC tumor cells may be suppressed. However, early infection processes visualized by immunofluorescence staining in both IFN-pretreated and untreated 143100 NC cells provide evidence that IFN, at least in this NC cell line, is unable to completely prevent T-VEC infection induced by a quite high viral concentration (MOI 0.1), but nevertheless slows down the replication process significantly. Both the results obtained here, and the observation that none of the six NC cell lines intrinsically produce IFN-β or are able to react to T-VEC infection with an IFN-β response (data not shown), suggest that NC tumors indeed are particularly well suited for immunotherapy with this generally IFN-sensitive virotherapeutic agent and that a possible explanation for this may lie in distinct defects in IFN signaling pathways in this hitherto rather uncharacterized tumor entity. Whether this assumption can be confirmed must be clarified in further investigations.In many clinical studies, carried out with different iBET agents, NC tumors initially responded well, but progression often started again only after weeks or months ,32,33. TA combination of T-VEC-mediated immunotherapy with iBET compounds provides the opportunity to initially shrink the NC tumor cell mass of this fast-growing and aggressive tumor through a direct reinforcing attack consisting of the antiproliferative iBET therapy and the oncolytic effect of T-VEC, thus keeping the tumor under control until the long-lasting anti-tumor immune response triggered by T-VEC becomes effective and can lead—in the best case—to a complete tumor remission.In our preclinical study presented here, the groundwork for a combination therapy was laid for the first time by ensuring in vitro that the two therapeutic approaches (OV/iBET) do not generally affect each other negatively. Combinatorial treatment of the six NC cell lines with T-VEC and iBET compounds BI894999 or GSK525762 showed no evidence of mutual negative interference, either in terms of cell mass reduction or viral replication. Furthermore, in five out of six cell lines a significantly enhanced NC tumor cell mass decrease could be observed compared to the corresponding most effective monotherapy. A recent study using several iBETs, including NEO2734, GNE-781, and OTX015, to treat NC cell lines demonstrated that these iBETs are able to suppress the IFN response in NC cells ,37, whicIn our combinatorial approaches, it was quite challenging to choose NC cell line-specific suitable MOIs and concentrations due to the high susceptibility of the NC cell lines to T-VEC as well as to the iBET compounds. The initial goal of achieving a viable NC tumor cell mass of approximately 75% with monotherapeutic treatment was met only with difficulty, as T-VEC proved to be exceedingly effective in terms of its virus-induced oncolysis of NC cells. Considering the possible biological variations at these low concentrations of T-VEC, this may have led to the observed non-significant reduction in 10-15 NC tumor cells after combination therapy. While this is a problem for in vitro tests, it could be an advantage for clinical applications. Thus, in initial clinical trials using GSK525762 for the treatment of NC, a dose of 80 mg/day was found to be well tolerated and resulted in plasma concentrations of 2.5 µM at baseline . TherefoIn 2006, it was shown that BRD4 represses gene translation of the human papillomavirus . It was BRD4-NUTM1 translocation were investigated. Stirnweiss et al. reported on a study comparing the general efficiency of iBET therapy in different NC cell lines. It was shown that the type of NUTM1 fusion expressed may be one of the factors that contribute to iBET sensitivity, with NC tumor cells expressing the BRD4-NUTM1 ex11:ex2 fusion being on average more than tenfold more sensitive to iBET treatment than cells with BRD4-NUTM1 ex15:ex2 or ex14:ex2 fusions [BRD3, NSD3, or other gene fusion characteristics are needed.In this study, only NC cell lines harboring the most often seen  fusions . UnfortuOur data demonstrate that immunotherapy with T-VEC, even as monotherapy, constitutes a highly efficient tool for the treatment of NUT carcinoma cells. As a next step, it would be beneficial to test the efficacy of this therapeutic regimen in NC organoid models and further in immunocompetent mouse models. To date, only immunodeficient mouse models could have been developed ,36,40,41In a recent phase Ib clinical trial undertaken in advanced melanoma patients, it was shown that T-VEC combined with anti-PD-1 heightens the treatment efficiency compared to treatment with anti-PD-1 alone, due to changes in the tumor microenvironment . However


This poor responder profile was preserved in recurrences compared to matched resections (p = 0.0007). PI3K pathway activity, inversely linked with FOXO activity, was higher in CRT poor responder cell lines compared to CRT good responders. Poor CRT responder cell lines could be sensitized to CRT using PI3K inhibitors. To conclude, by using a novel method to measure signaling pathway activity on clinically available material, we identified an association of low FOXO transcriptional activity with poor response to nCRT. Targeting this pathway sensitized cells for nCRT, underlining its feasibility to select appropriate targeted therapies.In this study we aimed to investigate signaling pathways that drive therapy resistance in esophageal adenocarcinoma (EAC). Paraffin-embedded material was analyzed in two patient cohorts: (i) 236 EAC patients with a primary tumor biopsy and corresponding post neoadjuvant chemoradiotherapy (nCRT) resection; (ii) 66 EAC patients with resection and corresponding recurrence. Activity of six key cancer-related signaling pathways was inferred using the Bayesian inference method. When assessing pre- and post-nCRT samples, lower FOXO transcriptional activity was observed in poor nCRT responders compared to good nCRT responders (The online version contains supplementary material available at 10.1186/s12967-022-03376-w. Over the past three decades, incidence rates of esophageal adenocarcinoma (EAC) have been progressively increasing . This inEAC is characterized by a complex network of aberrant signal transduction pathways, making therapeutic targeting challenging , 5. In oThe aim of this study was to identify aberrant signaling pathways that drive therapy resistance in EAC. To reveal candidate signaling pathways as targets for targeted therapy, we investigated key signal transduction pathways in material available from daily clinical routine. Tissue specimens before and after nCRT, and for primary tumor and recurrent disease were assessed and compared to clinicopathological outcome data. Using patient-derived cell lines, candidate signaling pathways were targeted to study their potential therapeutic relevance.2 body-surface area), carboplatin (AUC 2 ml/min), 41.4 Gy radiotherapy in 23 fractions of 1.8 Gy, followed by resection. As part of a randomized phase II study [Medical records of a national referral center for esophagogastric cancer, the Amsterdam UMC, location Academic Medical Center, were systematically searched for patients with histologically proven EAC, including gastric esophageal junction (GEJ), treated between June 2004 and May 2013. (i) Resectable disease: patients treated with curative intent by an esophagectomy as single treatment or by the CROSS regimen: nCRT with paclitaxel . qPCR data from extracted RNA were subjected to stringent quality checks (QC) prior to determining signaling pathway activity scores of the androgen receptor (AR), estrogen receptor (ER), Phosphatidylinositol 3 Kinase (PI3K)-Forkhead Box O (FOXO), Hedgehog (HH), Transforming growth factor receptor Beta (TGF-β) and Wingless-Integrated (WNT) pathways by applying the computational model described below [Using hematoxylin and eosin (H&E) stained slides of included biopsies and resection specimens, FFPE tumor blocks containing the highest tumor percentage were selected by a trained pathologist. Selected H&E slides were scanned using Philips Ultra-Fast Scanner . Tumor areas were marked by a trained pathologist using the Digital Pathology Images portal . A minimal area of 2 mmed below , 10–12.Measuring signal transduction pathway activity on Affymetrix U133 Plus2.0 data has been described extensively before. Pathway activity was inferred from target gene mRNA levels of six key oncogenic signaling pathways that play a role in tumor growth and metastasis  using a Primary EAC cell lines EAC007B, EAC031M, EAC058M, EAC081R and EAC289B were established from patient EAC material as previously described . PrimaryPre-treatment cells were lysed in RIPA buffer  containing phosphatase and protease inhibitor cocktail . Protein levels were determined by BCA (Pierce). Samples were heated for 5 min at 95 ºC loaded on 4–20% polyacrylamide precast gels (Bio-Rad) and transferred to PVDF membranes. Samples were blocked with 5% BSA (Lonza) in Tris buffered saline with 0.1% Tween-20 (TBS-T), and incubated overnight at 4 °C with primary antibodies . Cells were seeded into 96-well plates in triplicates. After cell adhesion overnight, cells were treated. After one week, cell viability was measured by adding 20 μL of Cell Titer-Blue reagent to each well followed by three hours incubation. Plates were read at 560/590 nm in a cytofluormeter (BioTek Instruments). Viability was calculated from values from CRT cells with or without PI3K inhibitors, minus baseline cell viability.Proliferation was determined using IncuCyte™ live cell imaging system (Essen BioScience), quantitatively detecting live cells. Cells were imaged after one week of treatment.Apoptosis was similarly assessed using the IncuCyte system, by incubating cells in 0.33 mg/mL annexin V-FITC, administered simultaneously with drugs. Cells were imaged after one week of treatment. Apoptotic fraction was calculated by the ratio of FITC-positive cell area, divided by confluence .PI3K_AKT_mTor_signaling gene set was correlated with the Broad Hallmark Epithelial_Mesenchymal_transition gene set in two publicly available datasets: Esophageal Adenocarcinoma Fitzgerald (GSE96669)[https://gdc-portal.nci.nih.gov/projects/TCGA-ESCA) [http://r2.amc.nl).Gene expression of Broad Hallmark GSE96669) and EsopGA-ESCA) . Analysip-value of p < 0.05 was considered statistically significant.Pathway activity between normal and EAC tissue were calculated using the Mann–Whitney test to compare ranks. In case of paired samples—between matched pre-treatment biopsies and resection specimen or resection specimen and recurrence, the Wilcoxon matched-pairs signed ranked test was used. Correlation of pathway activities was assessed using Pearson’s correlations. Sensitivity analyses were performed for patients receiving panitumumab . Surviva2 and 38.4 mm2 for resection specimens. In total, 67 biopsies and 73 resection specimens were excluded due to reasons of availability or insufficient tissue and could therefore not be processed for pathway analysis. After stringent quality control, 242 specimens of 181 patients were included, comprising 92 biopsies and 150 resections. Biopsy and resection could be matched for 67 patients, rendering 134 matched and 108 unique samples. Of the 92 biopsies, 79 later received nCRT, of which 77 a mandard score could be determined after surgery.In order to identify aberrant signaling pathways that drive therapy resistance in EAC, clinically obtained material from the Amsterdam UMC that was stored in FFPE was retrospectively screened for eligibility. Two EAC patient cohorts were assembled: a resectable disease cohort (i), and a recurrent disease cohort (ii). For the resectable disease cohort (i), 236 EAC cases with matched biopsies of the primary tumor site and post-nCRT resection specimens were identified Fig. A. The ma2 and 20.9 mm2 for recurrences.Matched material of resected primary tumor site and metachronous recurrence for the recurrent disease cohort (ii), was available for 66 patients Fig. B. Seven p = 0.002, respectively, Fig. p = 0.038, TGF-β: p = 0.066). These data show that patients with a worse pathological response to nCRT have persistently lower FOXO activity in both biopsy and resection specimens compared to patients with a good response to nCRT. Unfortunately, EAC pre-treatment biopsies showed no significant differences in activity of the assessed pathways , TGF-β (p < 1∙10–3) and HH (p = 0.003) activities were increased after nCRT, compared to no difference or a small decrease in patients who received surgery only . Pathway signal transduction activity of six key oncogenic signal transduction pathways  was measFig. p < ∙10–3, TGp < 3.3∙10–6; Fig. p = 2∙10–7, p = 0.0023, and p = 0.011, respectively).Although tumor tissue is characterized by high activation of oncogenic signal transduction pathways, which pathways is cancer type specific. To assess if any pathway activity scores from EAC tumor tissue are differentially expressed from the healthy tissue from which it is derived, non-treated EAC tissue (N = 84) was compared to adjacent normal healthy esophageal tissue N = 20). EAC tumor tissue was characterized by lower FOXO activity   patients, while this was not the case in Mandard low 1–2) patients, supporting the hypothesis that combined low activity of both FOXO and TGF-β pathway after nCRT is related to poor prognosis . In patients who had recurrent disease after nCRT and esophagectomy, reduced combined FOXO and TGF-β activity was seen in recurrent sites compared to matched resection specimens . High pS6K cell lines were relatively resistant to CRT , whereas low pS6K cell lines were more sensitive . We take these data to indicate that in vitro, pre-treatment PI3K pathway activity as determined by pS6K, associates with, and potentially drives poor response to CRT.FOXO transcription factors are known to be under negative control by the PI3K pathway, by activity of kinases AKT and S6 –23 and aAiming to improve anti-tumor efficacy of CRT in poor responder cell lines, the additive effect of PI3K inhibition to the CRT regimen was assessed. Four PI3K inhibitors with positive Phase I clinical results were selected; LY3023414, Alpelisib, Pictilisib and Idelalisib , characterized by a mesenchymal cellular morphology and therapy resistance . Indeed,GSE96669 , and R =CGA-ESCA ; AdditioCGA-ESCA , most noUsing clinically available FFPE material we were able to determine activity of key cancer driving pathways before and after nCRT using small amounts of EAC tissue. Persistent low FOXO transcriptional activity was associated with poor response to nCRT in EAC patient samples. This poor nCRT responder profile was also seen in recurrences of nCRT-pretreated patients. In addition, heterogeneity in high PI3K activity, inversely linked with FOXO, was seen in patient-derived cell lines, providing a valuable tool to experimentally target candidate signaling pathways, such as PI3K-FOXO. Our exploratory data demonstrate that PI3K inhibition can indeed sensitize nCRT-resistant cell lines.As EAC is known to be a heterogeneous tumor, the full characteristics of the tumor might not be represented by the analysis of single tumor sites. This could be problematic, as large intratumoral heterogeneity might hamper patient selection for targeted therapy. In addition, not merely tumor cells but also stromal and immune cells influence EAC tumor biology . These eHere we described that low FOXO transcriptional activity is associated with worse clinical outcome, sometimes combined with low TGF-β pathway activity. TGF-β signaling has a complex dual role in human cancer , 33. ViaThe phosphatidylinositol 3-kinase (PI3K) pathway is one of the main cellular growth factor signaling pathways frequently hyperactivated in cancer . BecauseTo experimentally test novel therapeutic strategies as PI3K pathway inhibition, preclinical cancer models representative of patients’ tumor biology are essential. We found that patient-derived cell lines show heterogeneity in PI3K pathway activity, allowing their use as a tool to test predictive signals for response to PI3K pathway inhibition. In line with our patient data, both good and poor nCRT responder cell lines were identified with corresponding PI3K pathway activity on phosphorylated protein level. In the relatively small number of cell lines we could not find and association of nCRT on FOXO transcriptional activity level. However, as phosphorylation plays an essential role in activation of the comprehensive PI3K pathway, we were able to detect a relation on protein phosphorylation level. Although these experimental data are encouraging, caution must be taken drawing conclusions as additional experiments should be performed in 3D models.Nonetheless, we identified a PI3K inhibitor that could give a beneficial additional effect to nCRT; LY3023414, a novel PI3K/mTOR inhibitor, with potent activity in EAC in rat models . InteresTo conclude, we were able to determine the activity of key cancer driving pathways before and after nCRT in clinically attainable amounts of EAC tissue. The poor nCRT responder profile was detected in tissue of the primary site, recurrences of nCRT-pretreated patients and patient derived cell lines. Hence, the pathway activity model described here may be used to identify patients irresponsive to nCRT and select for appropriate targeted therapies.By using a novel Bayesian inference method to measure signaling pathway activity on clinically available material, we identified an association of low FOXO transcriptional activity with poor response to nCRT. Targeting this pathway sensitized cells for nCRT, underlining its feasibility to select appropriate targeted therapies.Additional file 1: Figure S1. Poor responder phenotype in patient samples. A Pathway signal transduction activity of six key signal transduction pathways was measured in the resectable disease cohort (i). Pre-treatment biopsy pathway activity scores were subtracted from all matched resections that both passed QC, i.e. delta activity score (N = 69). Two-sided Wilcoxon signed-rank statistical tests were performed between all post-nCRT (N = 56) and surgery only (N = 13) patients. p-values are indicated in the figures. Boxplots represent median with interquartile range. B All post-nCRT resection specimens from the resectable disease cohort were assessed for correlations between FOXO and TGF-β activity. Spearman correlations were performed, N = 138, separated for low , middle  and high  Mandard score. C Pre-treatment biopsies were assessed for correlations between FOXO and TGF-β activity. Spearman correlations were performed, N = 77, separated for low , middle  and high  Mandard score obtained after nCRT. D Disease free survival of patients with combined low FOXO and TGF-β pathway activity versus combined high pathway activities in post-nCRT resection specimens (N = 83). Cut-off by median pathway activity score.Additional file 2: Figure S2. Validation of PI3K pathway inhibition and sensitization of poor CRT responder cells. A Correlation of baseline FOXO transcriptional activity with PI3K pathway activity based on P-S6, P-AKT and P-ERK PI3K in all eight EAC cell lines. B Poor CRT responder cell line 031 M and good CRT responder cell line 289B were exposed for 7 days to the CRT regimen in combination with 500 nM LY3023414, Alpelisib, Pictilisib or Idelalisib. Cells were lysed on day 8. Western blot analysis of PI3K-FOXO pathway by P-AKT and P-S6K as proteins of interest, β-actin as loading control. C Quantification of Western blot in A corrected for α-tubulin. D Cells were treated for 7 days with the CRT regimen, including a concentration range of 0, 62.5, 125, 250, 500 and 1000 nM Alpelisib, Idelalisib, Pictilisib or LY3023414. Percentage viable cells were measured on day 8 and plotted normalized to CRT. Data represents two biological replicates with SEM.Additional file 3: Figure S3. PI3K inhibitors can revert CRT-induced EMT. A Correlation of gene expression of Broad Hallmark Pi3K_AKT_mTor_signalling gene set with the Broad Hallmark Epithelial_Mesenchymal_transition gene set in two publicly available datasets, GSE96669 and Esophageal Carcinoma Tumor Cancer Genome Atlas (TCGA-ESCA), respectively. B FACS analyses of mesenchymal marker CXCR4 after 7 days of treatment with CRT with or without 500 nM LY3023414. 031 M poor CRT responder, 289B good CRT responder. gMFI = geometric mean fluorescent intensity.Additional file 4: Figure S4. Apoptosis induced by PI3K pathway inhibitors in poor responder CRT cell lines. Poor CRT responder cell lines 007B, 058 M and 081R and good responder cell lines Flo1, OE19 and OE33 were treated for 7 days with the CRT regimen in addition to 500 nM of PI3K pathway inhibitors (based on average IC50 of four compounds). Apoptosis measured by percentage of green fluorescent Annexin V-FITC. Data points represent biological replicates, mean with SD.Additional file 5: Table S1. Characteristics of the five established patient derived cell lines. Table S2. FDA approved PI3K inhibitors tested in this study. Table S3 Western blot antibodies used in this study.


Triple negative breast cancer (TNBC) is currently associated with a lack of treatment options. Arsenic derivatives have shown antitumoral activity both in vitro and in vivo; however, their mode of action is not completely understood. In this work we evaluate the response to arsenate of the double positive MCF-7 breast cancer cell line as well as of two different TNBC cell lines, Hs578T and MDA-MB-231. Multimodal experiments were conducted to this end, using functional assays and microarrays. Arsenate was found to induce cytoskeletal alteration, autophagy and apoptosis in TNBC cells, and moderate effects in MCF-7 cells. Gene expression analysis showed that the TNBC cell lines’ response to arsenate was more prominent in the G2M checkpoint, autophagy and apoptosis compared to the Human Mammary Epithelial Cells (HMEC) and MCF-7 cell lines. We confirmed the downregulation of anti-apoptotic genes  by qRT-PCR, and on the protein level, for TGFβ2, by ELISA. Insight into the mode of action of arsenate in TNBC cell lines it is provided, and we concluded that TNBC and non-TNBC cell lines reacted differently to arsenate treatment in this particular experimental setup. We suggest the future research of arsenate as a treatment strategy against TNBC. Breast cancer is a common female malignancy, representing 11.6% of all cancer cases worldwide, Ref. ,3,4. AmoStandard therapy across breast cancer subtypes includes surgery, chemotherapy, and radiation. Non-selective chemotherapies are especially important for TNBC patients, because no targeted therapy is available. Also, for those patients who have developed metastases, surgery alone is not sufficient, and additional general chemotherapy is necessary ,9,10,11.Among non-specific cancer treatments, arsenic-derived compounds are commonly used ,13,14. IArsenate is known to cause direct and indirect DNA damage through reactive oxygen species,  and can The purpose of this study was to investigate whether arsenate can be a selective chemotherapeutic agent against TNBC cell lines. For this, we examined the response to arsenate treatment in autophagy, apoptosis and cell proliferation via phenotypic and transcriptomics readouts. Firstly, we evaluated the effect of arsenate treatment on the cellular level. We performed functional tests, including colony assay formation, autophagy and apoptosis investigation using fluorescence microscopy, and measured cytoskeletal response by immunofluorescence staining followed by confocal microscopy evaluation and dark field microscopy for morphologic alteration. Then, after seeing that arsenate causes apoptosis specifically in the Hs578T and MDA-MB-231 cell lines, we investigated whether there is any mechanistic change in the transcriptome of the cell lines responding to the low arsenate concentration using cDNA microarrays and regularized discriminant analysis (RDA). Finally, findings from the gene expression analysis were validated by qRT-PCR, and at the protein level by ELISA and immunofluorescence confocal microscopy.We first assessed the effect of arsenate exposure on colony formation capacity, which is a widely used test to assess the sensitivity of tumor cell lines to therapeutic agents . The tesp = 0.0004). However, the increase is not observed for arsenate treated vs. untreated HMEC cells (p > 0.05 in a t-test). The number of autophagic cells in the Hs578T and MDA-MB-231 cell lines increased significantly .Given the decrease of colony formation ability described above, we next investigated its mechanistic background, focusing on apoptosis and autophagy. We stained the cells with monodansylcadaverine (MDC), a fluorescent marker for the acidic endosomes, lysosomes, and late-stage autophagosomes . An incrp = 0.026 in a t-test), but not in the HMEC cell line (p > 0.05).The cells were then simultaneously stained with Annexin-V and FITC/PI to visualize apoptosis. Only Annexin-V FITC staining visualizes early apoptosis, meanwhile both Annexin-V and FITC PI are markers for late apoptosis. An increased apoptotic rate was observed in the triple negative breast cancer cell lines as follows. In the Hs578T cell line, both the early and late apoptotic state was visible, while in case of the MDA-MB-231 cells, the majority of cells appeared in the late apoptotic phase 70–90%; C. The nuThese data suggest the involvement of apoptosis and likely autophagy as a response to arsenate exposure, which, based on the data shown here, is more pronounced in the TNBC cancer cell lines. Consistent with that, arsenic in oxidation state +3 has previously been found to interfere with intrinsic and extrinsic apoptosis and autophagy mechanisms in the breast cancer cell lines MCF-7 and MDA-MB-231 . ArsenicAutophagy has been presented in previous literature as a protective mechanism against arsenite induced oxidative stress, which causes genome damage . MeanwhiWe next used dark field microscopy to assess cellular morphology, which allows for the assessment of cytoskeleton alterations and hence the actin and tubulin status of a cell. It can also resolve more discrete features such as membrane disorganization, blebbing and apoptotic bodies. In the case of HMEC cells, only a slight modification of morphology was observed . The celCellular stress becomes visible through abnormal elongated cells for MCF-7 and Hs578T cells and irregular nuclei surrounded by sparse apoptotic bodies D,F. NumeTo assess whether the morphological response of the cellular cytoskeleton to arsenate treatment is in accordance with the dark field microscopy images, we next stained the actin cytoskeleton with Phalloidin-FITC dye and the cell nuclei with DAPI staining, visualized in Alteration of the cytoskeletal organization is overall more pronounced in breast cancer cells. Hs578T cells treated with arsenate do not present significant alterations to the cytoskeleton CII; howeWe next investigated the mode-of-action of arsenate treatment in the four different cell lines based on gene expression data see B showed q-value < 0.1 in a Benjamini-Hochberg False Discovery Rate-corrected t-test and |log2 FC| > 1; see We next evaluated gene expression on an individual gene and pathway level. First, we found the number of differentially expressed genes in the four arsenate-treated cell lines, which were 81 for HMEC, zero for MCF-7, 1231 for Hs578T and 275 for the MDA-MB-231 cell line  to differentiate the response to arsenate between the cell lines as follows. For this kind of comparison, we used the fold change values as input. We treated the two TNBC cell lines as one and the normal and DPBC cell line together as a second set of cell lines. This way we intended to investigate whether the RDA analysis will show transcriptomic changes according to the morphological results obtained. Indeed, we found enrichment in the apoptosis, the mTORC and the cell cycle hallmarks using the RDA value as input data . This suThe mTORC signalling was differentiated between the TNBC and the DPBC/normal cell lines. The mTORC signalling was generally downregulated in the TNBC cell lines. This downregulation included various metabolic enzymes such as glucose-6-phosphate dehydrogenase (G6PD) or sorbitol dehydrogenase (SORD), amino acid transporters such as cystine/glutamate transporter (SLC7A11) and large neutral amino acid transporter small subunit 1 (SLC7A5). The mTORC is the master regulator of autophagy, inhibiting it in the case of adequate metabolic flux . These rHowever, all these responses on the transcriptome are weak, possibly due to the low concentration of the arsenate treatment used. Next we selected the key regulators of the various processes  for further validation experiments to validate our results. The activation of apoptosis, autophagy and cell cycle arrest are the key outcomes of arsenate treatment as observable from the genes shown in Given that apoptosis regulation is a complex process, we next used network visualization to gain further insights into the mode of action of arsenate treatment . The higThe genes were selected based on their function in apoptosis according to the network figure  and theiFollowing microarray-based gene expression analysis, we selected four genes for qRT-PCR in order to validate our results, which have a central role in apoptosis  and whicBCL2 is an apoptosis regulator which blocks BAX from releasing Cytochrome C out of the mitochondria. This represents the initiation step of the intrinsic apoptotic process and activates the caspase cascade . MCL1 haIt can be seen  that in The expression of antiapoptotic regulators (BCL2 and MCL-1) hence significantly decreased after arsenate exposure, which provides a mechanistic rationale for in apoptosis facilitation via the intrinsic apoptosis pathway . CCND1 iWe next quantified alteration at the protein level as a validation step for the alteration on the transcriptome level. The results from BCL2 protein quantification by confocal immunofluorescence are presented in p < 0.05 t-test, Finally, we quantified TGFβ2 and IL6 by an ELISA assay (at the protein level) after 24 h and 48 h from cultures of HMEC, MCF-7, Hs578T and MDA-MB-231 cells for treatment vs. control, the results of which are shown in Arsenic derivatives showed antitumoral activity in the case of many types of cancer such as arsenic trioxide on head and neck tumors  or on epArsenate derivatives have been researched extensively regarding their medical applicability as well as biological effects. Arsenate affects cancer progression through coding and non-coding genes related to a wide range of biological processes . A partiMost of the studies that presented the biological effect of arsenic are related to the oxidation state +3 (arsenite); meanwhile for the oxidation state +5 (arsenate) there is much less information about its known mode of action. Despite the fact that arsenate efficacy in the treatment of breast cancer was demonstrated, Ref.  its antiIn a wide range of cellular models ,71,72,73Arsenite showed the ability to induce S-phase arrest, autophagy and apoptosis on various tumors by modulating genes such as Forkhead box O3 (FOXO3a) and Cyclin D1 (CCND1) , or sustIt was demonstrated previously that the apoptotic and autophagic responses have very specific cross-talk . EvidencThe analysis described in our paper shows that arsenate reduced cell proliferation as well as activation of autophagy and apoptosis in breast cancer cells. In this study the cytotoxic effect of arsenate was found to be largely cell type specific, as observed previously also in hepatocellular carcinoma cells . Our stuEach cell line in this study responded to arsenate treatment differently . In FigIn the case of Hs578T cells, we observed an alteration of chromatin pathways, DNA replication and telomere signaling pathways, and chromatin modification  correlating with microscopy data from previous studies ,83. ChroArsenate target genes involved epigenetic reprogramming , which lOur data suggest the involvement of apoptosis and autophagy in the effects of arsenate exposure, which furthermore appears to be specific to cancer cell lines, based on the data generated here. In our in vitro experiments we have not distinguished whether arsenate or arsenite had the biological effect in the intracellular milieu. Nevertheless, the outcome of the apoptosis and autophagy assay suggest a cell line specific cytotoxic effect.Arsenic in oxidation state +3 has previously been found to interfere with intrinsic and extrinsic apoptosis and autophagy mechanisms in the breast cancer cell lines MCF-7 and MDA-MB-231 . Arsenic2.HMEC  were maintained in HMEC basal serum free medium  and HMEC supplement kit . The DPBC cell line MCF-7  was cultured in MEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% nonessential amino acids. The Hs578T cell line (ATCC collection) was maintained in MEM  high glucose (4500 mg/mL glucose) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% nonessential amino acids  and 0.01 mg/mL insulin. The MDA-MB-231 cell line (ATCC collection) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. All cells were maintained in a humidified incubator at 37 °C with 5% CO5+ in solution directly because arsenate is internalized easily by phosphate carriers. [5+) presenting less direct cell toxicity than (As3+). [3 → H3AsO4 + 5NO2 + H2O)  and diluted to the required concentration.We used the Asarriers.  Hence, t (As3+).  Arsenaten = 3). A graphical representation and t-test results were shown.Treated and untreated cells were seeded in six-well plates at a density of 250 cells/well/2 mL in triplicate. After 14 days, the cells were washed with PBS 1×, fixed with 1 mL of methanol 80% for 15 min, stained with 300 µL of Trypan Blue 0.2% and then washed with PBS 1×. The colonies were counted by a visual observer without the use of visual augmentation devices. Images of the plates have been taken with a c300 machine  using the white light and then they were counted directly from the plate  using a 20× objective for magnification.For autophagy detection, the cells were treated with an Autophagy/Cytotoxicity Dual Staining Kit  that contains monodansylcadaverine (MDC) for autophagic vacuole detection in cultured cells and propidium iodide (PI) for necrotic cell detection. Staining was applied after 24 h of arsenate treatment. Apoptosis was detected by the Annexin V-FITC/PI Apoptosis Detection Kit . The kit contains Annexin V-FITC that stains in green the apoptotic cells that translocated membrane phospholipid phosphatidylserine to the outer leaflet of the cellular membrane, while the PI part of the composition stains the nuclei. Late apoptotic cells are hence double stained with both PI and Annexin-V-FITC. The staining was performed according to the manufacturer’s protocol followed by fluorescence microscopy evaluation (20× magnification). On four different images the apoptotic and autophagic cells were counted in both, treated and untreated conditions. The average cell count of the untreated condition was set to 100% and changes in cell number were reported as multiples of this number.Dark-field microscopy was performed using an Olympus B × 43 microscope  equipped with a CytoViva Enhanced Dark-Field Condenser , an UPlanFLN60×, NA = 1.2 oil immersion objective  and a 6.4 µm/pixel CCD camera . Images were calibrated for scale and annotated in ImageJ2.0  and conv2. Thereafter, 100 µL of 200 nM Phalloidin-FITC was added and the samples were incubated at room temperature under no illumination for 30 min. 200 µL of 100 nM DAPI was added over the coverslip for 30 s and washed with a phosphate saline buffer. The coverslips were mounted with 90% glycerol. Images were captured using a UPLSAPO40 × 2, NA:0.95 objective  and excitation wavelengths/emission windows were automatically selected according to the fluorescence dye spectral information database inside the acquisition software .The fluorescent staining protocol used DAPI  for the labelling of the nucleus and Phalloidin-FITC  for the cytoskeleton. After treatment, the cells were fixed in 4% paraformaldehyde followed by 0.5% Triton × permeabilization for 1 h. The cells were incubated at 37 °C and 5% COFor microarray experiments, cells from three serial passages were seeded in a six-well plate, using 0.3 million cells/well for each triplicate of control and arsenate treated cells. RNA extraction was performed using TriReagent, which was then purified using the RNeasy Mini kit .The microarray samples were prepared according to the Agilent Low Input Quick Amp Labeling (5190–2305) protocol to synthesize equal quantities of 100 ng of total RNA, followed by purification of the hybridization products using the RNeasy Mini kit (Qiagen). A NanoDrop2000 spectrophotometer  was used to perform probe quality control, with results showing that all the probes had a specific activity higher than 6 pmol/µL Cy3/µg cRNA (specific activity > 8 pmol Cy3/µg cRNA). Fragmentation and hybridization were performed based on the Agilent One-Color protocol . ,95 The sGene expression values were determined using the Agilent G4851C microarray slides for the three arsenate treated samples and three controls across four cell lines .normexp” method and then quantile normalization [avereps” function in R. The probe sets were translated using the annotation file of the microarray chip [prcomp” function in R [Resulting data were analyzed using the limma  package lization  was perflization  Probes tray chip . This reray chip . From thion in R  for visup-value < 0.1 and |log2 FC| > 1) were tested in the Gorilla [Based on the experimentally determined gene expression profiles, we calculated the average log2 fold change value per gene for each cell line responding to arsenate in R, using the functions lmFit and eBayes . Signifi Gorilla  tool for Gorilla ,102. For Gorilla .The calculation of log2 fold changes between each treatment and each non-treated sample resulted in nine fold-change values per cell line (three treated and three untreated replicates in each distinct cell lines). Next, Robust Regularized Discriminant Analysis (RDA) was performed on these fold changes using the R package RDA . After aAfter optimizing parameters, we calculated a centroid value per gene, which indicates the extent to which the given gene is able to differentiate the two TNBC cell lines from the double positive and normal cell line. This centroid was the subject of the subsequent Gene Set Enrichment Analysis (GSEA)  using thWe next generated an apoptosis reference network by using the genes from the MolSigDB apoptosis pathway  and mappTotal RNA extraction was performed using TriReagent  according to the manufacturer’s protocol. A NanoDrop-1100  was used to evaluate RNA concentration and quality by measuring the absorbance of UV light. For gene expression evaluation, total RNA (1000 ng) was reversely transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit . We used the Assay Design Center from Roche for the primer design . The primers for each gene are listed in The expression level of TGFβ2 released in the cell culture medium was detected by ELISA using the Human TGF-beta 2 DuoSet ELISA , DuoSet Ancillary Reagent Kit 2  and Sample Activation Kit 1 . For IL6 quantification from cell culture, ELISA was performed using the IL6 DuoSet ELISA Kit  along with the DuoSet Ancillary Reagent Kit 2 .® 488 . Laser scanning confocal microscopy was performed on an Olympus FV1200MPE microscope equipped with UPLSAPO40×2, NA:0.95 objective.For immunofluorescence staining, a Human/Mouse BCL2 Antibody  was used. Incubation was done with 5 µg/mL overnight followed by washing steps and incubation for 2 h with secondary antibody Goat Anti-Rabbit Alexa Fluort-tests, our considered significance level was p < 0.05, and the statistical analyses were performed using GraphPad Prism software version 6 for Windows . For microarray differential gene analysis, we used moderate t-statistics with a Benjamini-Hochberg correction and p < 0.05. For Gene-set enrichment analysis we used the Kolmogorov Smirnov test for p < 0.05 and FDR < 0.1.For pairwise comparisons we used two-sided In this study we were able to demonstrate that arsenate induces a cell line specific morphological and transcriptomic alteration at low concentration. Arsenate induces the cytoskeletal alteration and cell death in TNBC cell lines through activating autophagy and apoptosis and reduces the clonogenic capacity.The novelty of this study stands in the fact that arsenate therapeutic stress produces pro-apoptosis signals largely selectively in TNBC cells . In addition, arsenate showed no effects in HMEC cells and only moderate effects in MCF-7 cells.Regularized discriminant analysis showed that the low concentration of arsenate affected the G2M checkpoint, autophagy and apoptosis cell line specifically. The downregulation of anti-apoptotic genes  was confirmed by qRT-PCR, and on the protein level, for TGFβ2, by ELISA, concluding that TNBC and non-TNBC cell lines in this particular experimental setup reacted differently to treatment.The alteration of gene expression levels demonstrates a crosstalk among autophagy, cell cycle and apoptosis as a potential mechanism of action of arsenate which must be investigated in future pharmacological interventions.This study is a step toward understanding arsenate TNBC-type specific effects which potentially correlates with active DNA repair pathways. However, further studies are necessitated to demonstrate arsenate metabolism and mechanism of action, considering the importance of intracellular reduction of the metalloids for biological effects. Nevertheless, this study makes the use of arsenate to be a potential selective chemotherapeutic drug treating triple negative breast cancer one step closer to reality.