Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. In this study, we show that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM. Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, our study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway.Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from  Anotignaling . Deficieignaling .Drosophila, JAK/STAT signaling is initiated by three cytokines of the Unpaired (Upd) family . The binding of Upd induces Domeless (Dome) dimerization and activation of the receptor-associated JAK molecules (termed Hopscotch). Activated Hopscotch then phosphorylates Dome, creating a docking site for the single Drosophila STAT family transcription factor, Stat92E. Phosphorylated Stat92E migrates into the nucleus in dimers, promoting target genes transcription (virus-induced RNA 1 (vir-1) and stress response genes Turandot A and M (TotA and TotM)  . Althougallenges , 19.Drosophila and Pacific white shrimp (Litopenaeus vannamei), the production of antimicrobial peptides (AMPs) was downregulated by Ect4 and LvSarm  protein is evolutionarily conserved from arthropods to mammals . The mamd LvSarm , 24, sugfections . The pos22.11118w flies were used as wild-type control. The IRw; L811fsXdcr-2 mutant flies have been previously described . For the generation of Ect4 mutant lines, transgenic U6:3-gRNA-Ect4 flies were crossed with the nos-Cas9 flies to get male F0  that were crossed with 1118w; TM3, Sb/TM6B, Tb to obtain F1 progenies. Singular F1 flies were crossed with 1118w; TM3, Sb/TM6B, Tb. PCR products amplified from F1 flies before being cloned into the pMD19-T vector according to the manufacturer’s instructions (TAKARA) for mutation identification.escribed . ubi-Galescribed . Ect4-IR2.2pAC5.1-Ect4-Flag was made by cloning Ect4 cDNA into pAC5.1-Flag vectors. For pAC5.1-Ect4-GFP constructs, the EGFP fragment was amplified from pEGFP-C1 and assembled with the Ect4 fragment into pAC5.1-V5 vectors using ClonExpress MultiS One Step Cloning Kit (Vazyme). Stat92E cDNA was inserted in pAC5.1-HA to generate pAC5.1-Stat92E-HA. The hop (or Dome) cDNA was inserted in pAC5.1-V5 to generate pAC5.1-hop-V5 (or pAC5.1-Dome-V5). For the truncated Ect4 constructs, ARM domain (aa 318-701), SAM domain (aa 680-826), and TIR domain (aa 829-1360) were amplified from pAC5.1-Ect4-Flag before assembled into pAC5.1-Flag empty vector, respectively.2.3S2 cells were cultured at 25°C in Sf-900™ III SFM (Gibco). All S2 cell transfection experiments were carried out with the Effectene Transfection Reagent (QIAGEN). For a co-immunoprecipitation assay, S2 cells were transfected with different plasmids. After 48h, cells were collected and lysed in lysis buffer . Lysates were incubated overnight at 4°C with Anti-Flag M2 affinity gel (Sigma) or EZview Red Anti-HA Affinity Gel (Sigma). After centrifugation, pellets were washed with 1ml lysis buffer three times before resuspension in 2X Laemmli SDS-PAGE buffer and detection by Western blot. Western blot was performed according to standard procedures.Primary antibodies: Mouse anti-V5 ; mouse anti-HA ; mouse anti-α-Tubulin ; goat anti-Stat ; mouse anti-FLAG ; rabbit anti-DCV ; mouse anti-PY20 . Secondary antibodies: HRP-linked anti-mouse IgG ; HRP-linked anti-rabbit IgG ; HRP-linked anti-goat IgG (1:5000 Millipore AP106P); Alexa Fluor 555 goat anti-mouse IgG .2.4RpL32 , vir-1 , DCV , TotA , TotM , Ect4 , Vago .According to the manufacturer’s instructions, total RNA was extracted from infected flies using RNAiso Plus (TAKARA), and cDNA was synthesized with the HiScript II Q RT SuperMix (Vazyme). The ChamQ SYBR qPCR Master Mix (Vazyme) was used for quantitative. Expression of the gene of interest was normalized to the Rpl32 RNA level. The following primers were used for qPCR: 2.52 and injected with PBS (Gibco) or virus suspension intra-thoracically using the Nanoject II injector (Drummond). Infected flies were monitored daily for survival rate or frozen for RNA analysis at the indicated time points.Virus stocks were prepared as described previously . All fly2.6pAC5.1-Ect4-GFP and pAC5.1-Stat92E-HA plasmids, and approximately 1×106 cells were transferred to 24 well plates containing coverslips 48 h after transfection. Twelve h later, cells were washed in 0.5ml PBS and fixed with 4% formaldehyde in PBS for 15 min, then washed twice in 0.5 PBT (PBS containing 0.1% Tween-20) before blocking with 5% BSA in TBST for 1 h. Cells were then incubated with primary antibody (anti-HA 1:1000) overnight at 4°C before 2×5 min TBST washes. The secondary antibody was incubated for 4 h at room temperature. Nuclei were stained with PBS with 10 μg/ml DAPI for 5 min. Immunostaining samples were photographed with a Zeiss confocal microscope.S2 cells were transfected with d old, raised at 25°C) of each indicated genotype were homogenized in methanol . After centrifugation, the supernatants were measured for absorbance at 480 nm.For eye pigment assay, the heads of 50 female flies  pre-incubated for 15 min was added to S2 cells to induce tyrosine phosphorylated Stat92E for 30 min. Cell lysates were prepared with the lysis buffer before immune precipitation with an anti-Stat92E antibody at 4°C and incubated with Pierce Protein A/G Plus Agarose (Thermo Scientific) beads. Co-immuno-precipitated proteins were detected with an anti-Stat92E antibody or anti-PY20 antibody.dsRNA targeting 2.8t-test. Grey value analysis was performed by the ZEN 2012 (blue edition) system. P-values below 0.05 were considered significantly different.Survival data were analyzed by the Kaplan-Meier method using GraphPad Prism. Quantification of immunoblots was performed with ImageJ 1.51p. Altered protein levels were presented as normalized fold change compared to the control value. Statistical analysis was performed using the Student’s 33.1Ect4 in Drosophila antiviral defense, an Ect4 mutant line was generated with the CRISPR/Cas9 system. The mutation, 17Ect4, covers a genomic deletion of 17 bp in the coding region of Ect4  position 598, located in the last exon, has the strongest effect on DCV susceptibility , thus limiting the effect of discordance in the SNP profile between different fly lines to the difference in DCV resistance.Variation in the pastrel gene is associated with natural resistance to DCV infection in tibility . SequencEct4 transcription in 17/+Ect4 heterozygotes was reduced by 45% compared with the wild-type flies . Therefore eye pigmentation of a Dcr-2 null mutant (L811fsX/dcr-2L811fsXdcr-2) in GMR>UAS-wIR background is red, whereas Dcr-2 heterozygous mutants (L811fsX/+dcr-2) display pale orange  acts as the first line of defense against viruses in osophila . There wosophila , 34. Sine orange . Our res Dicer-2 , did not3.4Drosophila (via the JAK/STAT pathway including virus-induced RNA-1 (vir-1), the stress-induced genes Turandot A and M (TotA and TotM) . As previously reported, DCV infection induced a strong up-regulation of vir-1, TotA, and TotM in wild-type flies , which encodes a hyperactive JAK kinase due to a G341E substitution  . To exampe flies . Howevertitution . Reducin hopTum-l, which e hopTum-l, which e hopTum-l, which e3.5Ect4 and JAK/STAT pathway, we examined whether Ect4 interacted with any known components of the JAK/STAT pathway. Differentially tagged forms of JAK/STAT pathway components and Ect4 were expressed in S2 cells, and co-immunoprecipitation studies were performed. As shown in To unravel the molecular mechanism underlying the relationship between Ect4 protein harbor three different domains: ARM (Armadillo motif) domains followed by two SAM (Sterile Alpha motif) domains and TIR (Toll -Interleukin-1 receptor) domain  was not detected by immunostaining, likely due to the transient activity of p-Stat92E dimers. Therefore, is Ect4 required for the nuclear translocation of Stat92E? As expected, reduced nuclear translocation of Stat92E in response to the pervanadate stimulus was detected in cells treated with Ect4 RNAi  and L. vannamei (LvSarm) were required to express antimicrobial peptides. Depletion of both led to decreased survival of the animals upon bacterial infections , and t stress , 46. TheEct4 reduced phosphorylated Stat92E upon pervanadate treatment in S2 cells, which suggests a role of Ect4 in regulating cell death through the modulation of JAK/STAT via the interaction with Stat92E.Ect4 interacts with Stat92E in S2 cells through the highly conserved TIR domain of SARM family origin, which have roles in cell death and neuronal destruction in mammals . As an ain vivo study revealed that Ect4 mutants showed enhanced mortality and increased viral load upon DCV challenge. We seek to further elucidate the unknown mechanisms of antiviral response in Drosophila by assessing whether Ect4 affects host resistance to viral infection by regulating cell death.In addition to antiviral immune defense, apoptosis is a conserved mechanism of programmed cell death that can prevent the infection before viral replication is completed , 51. OurDrosophila Ect4 in regulation of JAK/STAT signaling pathway and protection against DCV infection. It is still unclear if Ect4 also participated in the control of other virus infection. The contribution of JAK/STAT signaling to Drosophila antiviral protection is virus-specific. Although JAK/STAT pathway can be activated by RNA viruses, including DCV, CrPV, FHV, and DXV, it is only required for resistance against two Dicistroviridae family members, DCV and CrPV  polymerase PARP9 was recently reported as a noncanonical sensor for RNA viruses that depends on the PI3K/AKT3 pathway to produce antiviral type I interferon . PARP9 iThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ZH and PX carried out experiments. SG contributed to data collection. YH analyzed confocal images. YL, SY, and FS conducted statistical analysis. KA and W-MD revised the manuscript. JL and RJ contributed to the conception and design of the experiments. WW wrote the first draft of the manuscript. JC directed the studies. All authors contributed to the article and approved the submitted version.


Ultimately, the cellular base for all of these in vitro model systems is derived from human cell lines or patient samples, to investigate mechanisms of cancer and screen novel therapeutics, all of which aim to maximize successful outcomes in clinical trials. This in itself offers an opportunity to potentiate effective treatments for many groups of people, when diverse variables like genetic ancestry are consciously included into study design. This Perspective highlights the need for conscious inclusion of genetic ancestry in preclinical cancer tissue engineering, especially when it pertains to determining therapeutic outcomes.Diverse factors contribute to significant and dire disparities in cancer risk and treatment outcomes. To address this, there was a call for inclusion of sex as a biological variable, which resulted in more instances of careful inclusion of sex in preclinical studies of cancer. Another variable in cancer treatment is genetic ancestry. Although this is considered explicitly in clinical research, it is considerably neglected in preclinical studies. Preclinical research can use several 3D  Summary: Genetic determinants, like ancestry, impact cancer risk and therapeutic outcomes. Hence, this is an important variable to consider at the very initial stages of biomedical research at the bench. Several types of in vitro models are used widely in cancer research, including spheroids/organoids therapy and mortality. Even in cancers that are not related to sex organs, there are significant disparities in chemotherapy outcomes. For example, response to chemotherapy is more favorable in female compared to male melanoma patients . Furthern cancer . ImportaBRAF V600E mutation benefit from improved survival with vemurafenib treatment . Whether in monolayer cultures or 3D in vitro settings, these cell lines allow testing of targeted anti-cancer therapies (Cancer Cell Line Encyclopedia (CCLE) and the Catalogue of Somatic Mutations in Cancer (COSMIC) cell lines project, expanded the number of cell line models available for preclinical cancer research. However, the opportunity to include self-reported and verified genetic ancestry data was missed. Of the 1018 cancer cell lines in the COSMIC, 701 lines had unreported ancestry, of which 453 were inferred to be European , should compulsorily use diverse cell lines? The answer is likely no, because mandating this will exclude many scientists who simply lack the resources to do so, despite the awareness of conscious inclusion.”The Cancer Genome Atlas (TCGA), a multi ‘omics database, initially propelled the identification of oncogenes and driver mutations in cancer , but onlpatients . A comprTable S1 and in vitro models will enable the identification of the root cause of disparity in treatment, pharmacological variables and even tumor biology. If we continue to ignore this variable as an inconvenience, we are willfully contributing to a system of perpetuating inequity.To assess how much of this inclusion problem permeated into cancer spheroid models, I reviewed the literature published from 2017 to present (the past 5 years) using colorectal cancer spheroids. From a random cohort of 30 reviewed papers, 73.4% used established cell lines like HCT-116 and HT-29 in their studies, both of which cluster under white/European ancestry profiles, while only 26.7% used patient-derived cells , and there is occasional mis-reporting of this information in commonly used cell lines . SignifiOne obvious strategy to incentivize the inclusion of diverse samples in studies is recognizing and acknowledging the gap and adding it to the ‘Rigor’ criteria of research proposal evaluations. This lies in the hands of large funding agencies, and perhaps even journal publishers. This approach, while essentially forcing researchers' hands, has been widely successful in incorporating sex as a biological variable, and has thus set a precedent for including ancestry information as an equally relevant variable.A cautionary aspect of forcing inclusion, however, is the lack of formalized definitions for ancestral breakdown. The United States Census-based definition lacks inclusivity  and cons“If universities can extend their educational reach through global partnerships, they certainly can extend cooperative hands to stock biorepositories that capture ethnic diversity globally.”Does this mean that every study, at every point , should compulsorily use diverse cell lines? The answer is likely no, because mandating this will exclude many scientists who simply lack the resources to do so, despite the awareness of conscious inclusion. However, researchers who enjoy the privilege of using patient-derived cells/lines should report the known ethnicity of their samples when disseminating scientific data through peer-reviewed publications. The charge and onus to change this lies with bigger institutions, universities and funding agencies like the NCI. Universities that house precision medicine centers and biorepositories could curate ethnically diverse cell lines for broader use by their researchers. If universities can extend their educational reach through global partnerships, they certainly can extend cooperative hands to stock biorepositories that capture ethnic diversity globally. Federal agencies like the National Institutes of Health (NIH) and its institutes leading this charge will have implications not just on cancer research, but on all disease models that benefit from preclinical research.Any time a change in our fundamental way of doing things is asked of academics, there is resistance. However, combating health disparity is a collective responsibility borne by everyone at every training level in academic research, and not just medical and health professionals. Being conscious of inclusion during our development of disease models and using the weight of bioengineering to power preclinical studies are sure to make a difference. The aspirational goal of cancer research should be to make a difference for all people. Talking about disparity and inequity is simply not enough anymore, if it does not permeate into action at the academic research level. The time for that action is now.


Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide with 840,000 new cases and 420,000 deaths in 2020. Anticoagulants are widely prescribed medications routinely administered to help prevent blood clots. Despite the great relevance of these two topics, there is complete lack of knowledge regarding the potential effects that these drugs could exert on oral cancer patients. In this in vitro study, we comprehensively investigated the effect of anticoagulants on OSCC activity. This includes the effect of these drugs on cancer cell ability to survive, migrate to colonise distant sites, and resist treatment with conventional chemotherapy. We have demonstrated for the first time that various anticoagulants have anticancer effects on OSCC. Moreover, some of the anticoagulants tested were able to reduce the migratory ability of cancer cells. Finally, the great majority of anticoagulants studied reduced the effectiveness of the tested chemotherapeutic agent, allowing an increase in cancer cell proliferation. Our results highlight the need for urgent further research in the field, to improve the anticoagulant strategies in patients with oral cancer, and in turn their prognosis.Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. With anticoagulant usage on the rise, it is important to elucidate their potential effects on tumour biology and interactions with chemotherapeutics. The aim of the present study was to investigate the effects of anticoagulants on OSCC cell lines and their interactions with the drug 5-fluorouracil (5-FU). Cell proliferation was assessed using an MTS in vitro assay in two human OSCC cell lines (H357/H400) and in normal oral keratinocytes (OKF6) treated with the 5-FU (0.2/1/5/10 μg/mL), conventional anticoagulants warfarin (1/5/10/20 μM) and heparin (5/20/80 U), as well as four new oral anticoagulants, dabigatran (5/10/20 μM), rivaroxaban (5/10/20 μM), apixaban (0.1/1/5 μg/mL), and edoxaban (5/10/20 μM). Cell migration was assessed at 3 h intervals up to18 h using a wound healing assay. Our results clearly demonstrate, for the first time, that commonly prescribed anticoagulants exert in vitro antiproliferative effects on OSCC cells. Furthermore, treatment with some anticoagulants reduced the migration of OSCC cell lines. Nevertheless, most of the anticoagulants tested reduced the effectiveness of the chemotherapeutic agent tested, 5-FU, highlighting potential flaws in the current pharmacological management of these patients. Our findings showed the need for the immediate translation of this research to preclinical animal models. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, accounting for 90–96% incidence of all head and neck cancers . CurrentInterest has grown in the potential of anticoagulants for treating/preventing cancer, due to links between coagulation and cancer biology and prognosis. Certain angiogenic processes are favoured in carcinogenesis, due to cell-to-cell interactions, localised hypoxia, and the expression of particular cytokines and growth factors ,5. Anticoagulants are widely prescribed medications, routinely administered to help prevent blood clots. They are administered to people at high risk of blood clots, to reduce their chances of developing serious conditions such as stroke and heart attack. This may include people with atrial fibrillation or an irregular heartbeat. Anticoagulants may also be prescribed to people who have had major surgery, such as aortic valve replacement, or those with certain blood disorders. The most prescribed anticoagulant is warfarin, and for short-term reversible use, heparin. Overall, Vitamin K antagonists have been successfully used over the last five decades; however, more recently, newer types of anticoagulants have become available and are becoming increasingly common. These new oral anticoagulants (NOACs) include rivaroxaban, dabigatran, apixaban, and edoxaban. Warfarin and NOACs are taken as tablets or capsules, while heparin can be given by injection. The new types of anticoagulants have some advantages compared to vitamin K antagonist, e.g., they target a specific single factor of the coagulation cascade rather than inhibiting the whole vitamin K synthesis. Nevertheless, even if NOACs are becoming more popular, they have not fully replaced the use of warfarin and heparin presently.Recently, a study by Khan et al. systematically reviewed the use of new oral anticoagulants and direct oral anticoagulants in malignant patients . A totalWith anticoagulant usage on the rise, it is important to elucidate their potential effects on tumour biology and interactions with chemotherapeutics. However, little is known about these drugs’ effect on OSCC behaviour. Recently, we systematically reviewed the literature about this crucial topic, and have shown that there is a significant dearth of information surrounding interactions between anticoagulant therapy and OSCC . The stuThe aim of the present study was to investigate the in vitro effects of anticoagulants on OSCC cell lines and their interactions with the drug 5-FU. Our results have clearly demonstrated, for the first time, that commonly prescribed anticoagulants exert in vitro antiproliferative effects on OSCC cells. Furthermore, treatment with some anticoagulants reduced the migration of OSCC cell lines. Nevertheless, most of the anticoagulants tested reduced the effectiveness of the chemotherapeutic agent tested, 5-FU, unveiling a whole new dimension of chemotherapy resistance with dramatic potential clinical implications. Our findings showed the need for the immediate translation of these experimental conditions from bench to preclinical animal models.Two human OSCC cell lines derived from different intra-oral sites, H357/H400, and one normal human oral keratinocyte cell line, OKF6, were selected for this study. The OSCC adherent cell lines were established at Bristol Dental School, University of Bristol, UK, from primary explants of tongue (H357) and alveolar process (H400) squamous cell carcinoma . All OSC2, 37 °C). The normal human oral mucosal epithelial cell line, OKF6, was instead cultured in 100 mm Petri plastic dishes  and grown to 60–80% confluence before being further subcultured. OKF6 cells were cultured using keratinocyte serum-free medium (K-SFM)  containing 25 μg/mL bovine pituitary extract and 0.2 ng/mL human recombinant epidermal growth factor (as per manufacturer’s instructions), 0.4 mM CaCl2, 1% penicillin streptomycin mixture , and supplemented with 1% Newborn Calf Serum (NCS) . OKF6 cells were incubated at 37 °C, 5% CO2 for 5 to 7 days to reach 80% confluency. Epithelial cells grown to 80% confluency were subsequently detached via a pretreatment of 10 mM EDTA for 10 min, followed subsequently with incubation with a 0.25% trypsin in 1 mM EDTA solution  for 5 min. The viability of the keratinocytes was confirmed by trypan blue exclusion while passaging cells and during the seeding phase of each cell culture experiment .The OSCC cell lines were cultured as previously described . Cell liWarfarin , Enoxaparin sodium , Edoxaban , Apixaban , Dabigatran , Rivaroxaban R-enantiomer , and 5-FU , were reconstituted in acetone, filtered water, methanol, Dimethyl Sulfoxide (DMSO), HCl, DMSO, and DMSO, respectively. ® 3596, Corning, NY, USA) under standard conditions and treated with a combination of anticoagulant and/or chemotherapeutic agents at therapeutic concentrations at T = 0 [All the cell lines were tested with the chemotherapeutic agent 5-FU (0.2/1/5/10 μg/mL), as well as the two conventional anticoagulants warfarin (1/5/10/20 μM) and heparin 5/20/80 U), and four new oral anticoagulants, dabigatran (5/10/20 μM), rivaroxaban (5/10/20 μM), apixaban (0.1/1/5 μg/mL), and edoxaban (5/10/20 μM). H357, H400, and OKF6 cells were seeded in 96-well plates  and analysed with ImageJ Software . Due to the overall number of drugs to be tested within these experiments, pairs of anticoagulants were tested simultaneously with a shared control sample. All the wound healing assay experiments were performed in quadruplicate.Statistical analyses were performed using GraphPad Prism 8.0.1 . Differences between groups were analysed using the two-way ANOVA test.The conventional anticoagulants tested showed antiproliferative effects in the two cancer cell lines tested. Warfarin, at concentrations of 1 μM and 5 μM, significantly reduced cell growth at 72 h in H357 cell line . TreatmeTo assess if the anticoagulant effects observed were specific to oral malignant cells, the normal human keratinocyte cell line OKF6 was used as normal control in this study. Similarly, all the anticoagulants except heparin exerted antiproliferative effects in OKF6 cell line. This was seen with warfarin at 5 μM (time 48 and 72 h); 5 μM and 20 μM dabigatran at time points 72 and 48 h, respectively; 10 μM rivaroxaban at time 72 h; 0.1 μg/mL apixaban at time 72 h. Interestingly, edoxaban appeared to have no effects on OKF6 cell growth, even though it has to be noted that the drug solvent for edoxaban, 0.04% methanol, showed cytotoxic effects on OKF6 as early as 24 h.p ≤ 0.0001), but with the addition of 5 μg/mL and 20 μg/mL in H357; and with 20 μg/mL in H400, the growth was comparable with controls again, masking the antiproliferative effect of 5-FU. On the other hand, warfarin, edoxaban, and apixaban appear to not interact with 5-FU in H400 cell lines, and heparin, dabigatran, and rivaroxaban did not interfere with 5-FU effectiveness in H357 cell lines. Comparing the cancer cell lines with OKF6 control, all concentrations of warfarin and 10 and 20 μM rivaroxaban negated the chemocytotoxicity of 0.2 μg/mL 5-FU at time 72. All concentrations of heparin, 10 and 20 μM rivaroxaban, 0.1 μg/mL apixaban, and 10 μg/mL edoxaban reduced the effectiveness of 1 μM 5-FU at 72 h. Conversely, 10 μM dabigatran was able to act synergistically with 0.2 μg/mL 5-FU at 72 h to reduce OKF6 cell proliferation.The effect of anticoagulants on chemotherapy-induced cytotoxicity was assessed overtime in OSCC cells. The majority of anticoagulants either combated the chemocytotoxicity of 5-FU or appeared to not interact with 5-FU at all. In total, 5 and 10 μM concentrations of warfarin tested reduced the chemocytotoxicity of 5-FU in H357 cells at time 24 h. All concentrations of apixaban in H357 cells reduced the cytotoxicity of 0.2 μg/mL 5-FU at 72 h. With H400 cells, 20 U, 80 U heparin, and 5 μM dabigatran were able to decrease the effectiveness of 0.2 μg/mL 5-FU at 72 h. Edoxaban also showed potential to decrease cytotoxicity of 5-FU at 72 h. Across both cell lines, 0.2 μg/mL 5-FU alone caused significant inhibition of proliferation compared to its control (Conventional anticoagulants significantly impaired cancer cell migration. Addition of all concentrations of warfarin attenuated the ability of H400 cells to migrate . HoweverA comprehensive summary of the main findings from our in vitro experiments can be found in The present study has demonstrated for the first time that various anticoagulants exert antiproliferative effects on OSCC cell lines. In our experimental conditions, warfarin reduced proliferation of the H357 cancer cell line. Similarly, a study by Kirane et al. observed the antiproliferative effects of warfarin in pancreatic cancer cell lines . Bai et In vitro studies have shown that heparin and its derivatives are able to reduce tumour proliferation, migration, and invasion in epithelial cancers ,24,25,26The present study did not observe interaction between 5-FU and heparin in H357 cell lines. Interestingly, some concentrations of heparin attenuated the effects of low-concentration 5-FU on H400 proliferation; this effect has not been previously observed and warrants further investigation. Conversely, heparin and 5-FU worked synergistically to inhibit wound closure and thus migratory capacity of both cancer cell lines. While this drug combination has not been studied in human OSCC cell lines, the combination of cisplatin and heparin has been observed to result in reduced cell viability and cell migration capacity, as well as increased apoptosis of human OSCC cells ,33.Dabigatran, a direct thrombin inhibitor, has very limited research in the field of OSCC. Our study is the first to identify antiproliferative effect in OSCC cell lines. However, dabigatran failed to interfere with OSCC cells’ migratory phenotype. Decreasing cell viability is a finding supported by various studies testing dabigatran in numerous breast cancer cell lines ,37. De FLiterature regarding the pharmacodynamic interactions between anticoagulants, with the exception of heparin, and chemotherapeutic agents is sparse. The majority of anticoagulants studied reduced the chemocytotoxicity of 5-FU, allowing an increase in both normal and cancer cell proliferation. This novel finding warrants further urgent research. Additional in vitro research involving the use of further chemotherapeutic agents, such as cisplatin, and translation of this research line to preclinical animal models would add breadth to the present study and consolidate its findings. Our study demonstrated that there is real space for imminent and invaluable discoveries regarding the effect of anticoagulants in OSCC, with great impact on the future pharmacological management of oral cancer patients.


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.