Pinus nigra cell lines with contrasting embryogenic capacity, accounting for variability between genotypes. Our results showed that cell wall-related and carbohydrate-acting proteins were the most differentially accumulated. Peroxidases, extensin, α-amylase, plant basic secretory family protein (BSP), and basic secretory protease (S) were more abundant in the medium from the lines with high embryogenic capacity. In contrast, the medium from the low embryogenic capacity cell lines contained a higher amount of polygalacturonases, hothead protein, and expansin, which are generally associated with cell wall loosening or softening. These results corroborated the microscopic findings in cell lines with low embryogenic capacity—long suspensor cells without proper assembly. Furthermore, proteomic data were subsequently validated by peroxidase and α-amylase activity assays, and hence, we conclude that both tested enzyme activities can be considered potential markers of high embryogenic capacity.Somatic embryogenesis is an efficient mean for rapid micropropagation and preservation of the germplasm of valuable coniferous trees. Little is known about how the composition of secretome tracks down the level of embryogenic capacity. Unlike embryogenic tissue on solid medium, suspension cell cultures enable the study of extracellular proteins secreted into a liquid cultivation medium, avoiding contamination from destructured cells. Here, we present proteomic data of the secretome of  The process has been initiated in many plant species such as herbal species, angiosperm, and conifer trees. The developmental pattern of SE differs from that of angiosperms (Somatic embryogenesis (SE) is a complex process by which somatic embryos develop from somatic cells under iosperms . Von Arniosperms . Cell suiosperms .Pinus caribaea var. hondurensis). Secreted proteins are involved in the regulation of many biological and physiological processes, such as cell expansion, cell division, cell-to-cell communication, cell adhesion, signal transduction, and response to stress or stimuli (reviewed by Pinus nigra) at the level of the cellular proteome. A comparison of three types of tissues obtained from two cell lines  revealed a higher impact of cell line/genotype on proteome than the type of investigated tissue. An altered accumulation of proteins active in the biosynthesis of cell wall components was also found, comparing the embryogenic and non-embryogenic tissue of both cell lines  in the SE . Previoull lines .P. nigra. We compared the population of proteins secreted into the suspension culture medium from cell lines with different embryogenic capacities by label-free mass spectrometry. In our comparison, we included two cell lines with high embryogenic capacity (HEC) and two with low embryogenic capacity (LEC) to weigh the influence of genotype. Our results contribute to a better understanding of complex regulation of early stages of SE, when bipolar structures are already formed. Finally, we proposed potential markers of early stages of SE in pine species.In the present study, we aimed to obtain comprehensive secretome profiles and to identify possible players in the SE of conifer 22.1P. nigra enclosed in megagametophytes , squashed, and covered by a coverslip . The pre2.3On the eighth to ninth day after the last subculture, the tissues were resuspended in a liquid basal DCR medium without growth regulators and organic additives. Aliquots of 100–120 mg of tissue in the suspension were pipetted on stacked filter paper discs, and after absorbing the liquid, the filter papers with cells on their surface were placed on a maturation medium. The maturation medium was based on basal DCR medium supplemented with ABA , enzymatic casein hydrolysate (500 mg/L), glutamine (50 mg/L), myo-inositol (200 mg/L), glycine (2 mg/L), nicotinic acid (0.5 mg/L), pyridoxine (1 mg/L), thiamine (1 mg/L), and maltose 6%. The maturation medium was solidified with 1% gelrite  and E492, E494 (HEC). On the eighth day after the last subculture, 2.5 g of well-growing embryogenic tissue (taken from different plates) was resuspended in 25 ml of liquid basal DCR proliferation medium. The suspension cultures were cultivated in 100-ml Erlenmeyer flasks on a rotary shaker at 80–90 rpm in the dark. After 1 week of cultivation, the suspension was poured into 25-ml glass cylinders, and the cells were allowed to settle for 30 min. We estimated the sedimented cell volume  as a non-destructive growth parameter . Subsequ2.5Extraction of secreted proteins was performed in duplicate for each cell line. The culture medium (50 ml) was concentrated and partially desalted in two steps by ultrafiltration at 4 °C using Amicon Ultra-15  and Amicon Ultra-0.5  to a final volume of 200 µl. According to the manufacturer’s instructions, concentrated secreted proteins were precipitated with 2-D Clean-Up Kit , and pellets were solubilized in 40 µl of solubilization buffer . Proteins were quantified using the Pierce BCA Protein Assay kit (Thermo Scientific).To estimate potential contamination by cytosolic proteins, the activity assay of cytosolic enzyme glucose-6-phosphate dehydrogenase  was performed using the G6PDH assay kit (Sigma-Aldrich) according to the manufacturer’s instructions.Secreted proteins were separated in 12% polyacrylamide separation gel with 4% upper stacking gel. Prior to loading, protein samples (25 µg) were mixed with loading buffer  in a 1:1 ratio and heated at 70 °C for 10 min. Electrophoresis was performed on the Protean II xi Cell unit (gel dimensions 16 × 20 cm) (Bio-Rad) as follows: constant voltage of 60 V (30 V per gel) until the dye front reached separation gel, then at a constant voltage of 180 V (90 V per gel) until the run was complete. The protein bands were visualized with colloidal Coomassie blue (G-250) and digitalized using the GS-800 Calibrated Densitometer (Bio-Rad).2.6Each gel lane was divided into nine sections of similar size and cut into small pieces. Gel pieces were washed with 50 mM ammonium bicarbonate containing 50% acetonitrile until complete removal of the dye. Subsequently, gel pieces were reduced with 10 mM dithiothreitol in 100 mM ammonium bicarbonate, alkylated with 50 mM iodoacetamide in 100 mM ammonium bicarbonate, and dehydrated with 100% acetonitrile. Finally, the gel pieces were saturated with 10 ng/µl trypsin (Promega) in 10 mM ammonium bicarbonate containing 10% acetonitrile for 2 h at 4 °C and digested at 37 °C overnight. The digestion was stopped by 1% trifluoroacetic acid in 70% acetonitrile and tryptic peptides were extracted by 70% acetonitrile with 1% trifluoroacetic acid. Extracted peptides were stored at −20 °C until analysis.2.7Pinus taeda protein database . The samples were loaded onto a trap column  and separated with a C18 column  on an Ultimate 3000 RSLCnano system (Dionex) in a linear 120-min gradient (3%–43% B) and a flow rate of 300 nl/min. The following two mobile phases were used: 0.1% FA (v/v) (A) and 80% ACN (v/v) with 0.1% FA (B). Eluted peptides were sprayed directly into an Orbitrap Elite mass spectrometer  and spectral datasets were collected in the data-dependent mode using the Top 15 strategy for the selection of precursor ions for the HCD fragmentation . Each ofdatabase . The masXD040247 .2.8Peroxidase activity was measured in the cell culture medium of cell lines E489, E490, and E483 (LEC) and E494, E482, and E486 (HEC) in triplicate. The secreted proteins for the peroxidase activity assay were concentrated as described above, followed by buffer exchange [phosphate-buffered saline ] after the ultrafiltration step. Peroxidase activity was determined spectrophotometrically at 420 nm using the Peroxidase (POD) Assay kit (Elabscience) according to the manufacturer’s instructions. Peroxidase activity was expressed in units per milligram of secreted protein (U/mg protein). One unit was defined as the enzyme activity that catalyzes 1 µg of substrate by the 1 mg of protein extract per minute at 37 °C.The α-amylase activity was measured in the culture medium of cell lines E489, E490, and E483 (LEC) and E456, E477, and E494 (HEC) in triplicate. Secreted proteins were concentrated using Amicon Ultra-15 . The activity was determined spectrophotometrically at 405 nm using the Amylase Activity Assay Kit (Sigma Aldrich) according to the manufacturer’s instructions. The α-amylase activity was expressed in nmol/min·ml. One unit is the amount of α-amylase that cleaves ethylidene-pNP-g7 to generate 1.0 μmol of p-nitrophenol per minute at 25 °C.2.9t-test) on proteomic data was done in Perseus (version 1.5.5.3). Two cell lines with similar embryogenic capacity were treated as replicates. Statistical evaluation of enzymatic activity essays was done in Prism  using ANOVA followed by post-hoc Tukey test. Each of the six cell lines was considered as a separate group.A powerful classification approach, partial least square discriminant analysis (PLS-DA) with pareto scaling, was carried out by script EZinfo 3 for Waters (Umetrics). In essence, PLS-DA discovers a combination of variables (specific proteins), which separate sample groups (cell lines with HEC versus cell lines with LEC). To assess the relative influence of a proteomic variable on the mathematical model, variable importance in the projection (VIP) scores were calculated. VIP score is a weighted sum of the squared correlations between elements of the model. Additionally, conventional statistics  and TargetP-2.0 server . Only proteins predicted to have a signal peptide by both programs were considered. Those proteins were further scanned against PS00014 motif using the ScanProsite server (https://prosite.expasy.org/scanprosite/) to determine if they are retained in the endoplasmic reticulum (ER). Proteins containing ER retention motif were excluded from the list of secreted proteins. The localization prediction was accepted when three out of four used prediction tools  concurred.To predict the subcellular localization of differentially accumulated proteins, we used a set of bioinformatic tools. The presence of signal peptides (SP) was predicted using SignalP 5.0 server (http://gpcr2.biocomp.unibo.it/gpipe/index.htm) was used for the prediction of GPI-anchored proteins (specificity: ≥99.5%). For the prediction of transmembrane helices in proteins, they were queried in TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), CCTOP (http://cctop.enzim.ttk.mta.hu/), and MEMSAT analysis tool on PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/). Transmembrane predictions were accepted in the case of the presence in at least two predictors. We assigned the most probable location for each identified protein by cross-referencing data obtained from the bioinformatics tools mentioned above.The PredGPI prediction tool  were classified as HEC cell lines ; moreover, they did not reach the cotyledonary developmental stage. These structures necrotized or were overgrown due to intensive tissue cell proliferation. Two types of cell lines also differed in the structural organization of early stages of bipolar somatic embryos. HEC cell lines contained bipolar somatic embryos with well-formed embryonal parts composed of tightly packed meristematic cells (“head”). The embryonal part was connected with a suspensor composed of a bundle of long vacuolated cells (3.2P. nigra with different regeneration capacity: two LEC (E489 and E490) and two HEC cell lines (E492 and E494) growing in the proliferation basal DCR medium. We analyzed secreted proteins by the label-free LC-MS/MS approach with quality control of protein extract by denaturing gel electrophoresis in two biological replicates per cell line  to the separation of biological groups in the mathematical model Figure 23.3P. nigra, we also included medium from additional cell lines into the analysis. Altogether, peroxidase activity was measured in the medium from six cell lines: E489, E490, and E483 (LEC), and E482, E486, and E494 (HEC). Detected peroxidase activity was in the range 19.7 ± 9.8 to 66.4 ± 11 U/mg protein in medium from LEC cell lines, and differentially higher activity (108.8 ± 4.1 to 144.8 ± 6.1 U/mg protein) was measured in the medium from HEC cell lines  acting on the different components of the cell wall, and their activity is often affected by post-translational modifications . Among t radiata . However radiata . Cell waCyclamen persicum, and it was proposed as a potential morphological marker of SE  activity. The β-glucuronidase indirectly participates in Hmodeling . Eudes emodeling . The autmodeling . Severalmodeling , cell–cemodeling , and celmodeling .Additionally, we identified two isoforms of xyloglucan endotransglycosylase/hydrolase  of the GH16 family, each accumulated in the medium from different types of analyzed cell lines Figure 42O2 and reactive oxygen species, thus causing a wide range of cell wall modifications. As a result of their action, cell wall loosening or cell wall stiffening may occur. Cell wall stiffening through the formation of suberin, lignin, or cross-linking of cell wall components, such as extensins, led to the inhibition of cell elongation growth , hydroxyproline-rich glycoprotein (HRGP) with homology to leucine-rich repeat extensin (EXT) proteins, and dirigent protein (DIR) in the medium from HEC cell lines Figure 4n growth . Overexpn growth , and celn growth . On the n growth  and contn growth . Our resQuercus (Three thaumatin-like proteins (TLPs), all more abundant in the medium from HEC cell lines Figure 4Quercus . TLPs afQuercus . IncreasQuercus , similarAraucaria angustifolia occurred at the later stages of development. Contrastingly, starch accumulated early in the proliferation phase in the blocked line, and starch degradation occurred during the maturation phase , more abundant in the medium from HEC cell lines Figure 4on phase . In accolomerata .Expansin-like A1 protein (EXLA1) was more abundant in the medium from LEC cell lines Figure 44.2We found that LysM-domain containing receptor-like kinase (LysM-RLK) accumulated in the medium from HEC cell lines Figure 4Ginkbilobin-2 and Gnk domain-containing protein both accumulated in the medium from HEC cell lines Figure 44.3Extracellular nuclease S1 and two cysteine proteinases (both papain-like C1 family) accumulated in the medium from LEC cell lines Figure 4Other interesting proteins, plant basic secretory family protein (BSP) and basic secretory protease, accumulated in the medium from HEC lines Figure 45Our study revealed differences in secretome comparing the cell lines with different embryogenic capacities. The majority of altered proteins are involved in changes of the cell wall composition and remodeling, although some proteins such as peroxidases can have, depending on the developmental context, both loosening and stiffening effects on the cell wall. Both proteomic and microscopy results suggest that cell walls of HEC cell lines, in which cotyledonary embryos are developing, undergo rather stiffening or rigidification and, in contrast, LEC cell lines in which cotyledonary somatic embryos are not developing undergo loosening, softening, or even cell separation. In such scenario, higher activity of peroxidase in HEC might indicate feruloylate cross-linking or lignin deposition, rather than radical-mediated polymer degradation. Although further biochemical and biomechanical experiments are needed to reconcile this, changes that we detected clearly point to the protein-mediated cell wall and starch dynamics likely regulating and defining embryogenic capacity in pine cell cultures. Both peroxidase and α-amylase activities in the culture medium can be used as a marker to distinguish cell lines with HEC and LEC.https://www.ebi.ac.uk/pride/archive/, PXD040247.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: KK and TS contributed to the conception and design of the study. MP, TS, JB, PB, MD, and KK performed the experiments. MD carried out the statistical analysis. KK wrote the manuscript. All authors contributed to the article and approved the submitted version.


Infertility affects 10.7–15.5% of couples worldwide, and it is defined as the inability to conceive after 6–12 months of unprotected sexual intercourse  and the Many research and clinical groups worldwide use various approaches to study, prevent, diagnose and treat reproductive disorders . These tAlso, further advancements in computer and information sciences have been contributing to providing more targeted treatments in reproductive medicine , which iJournal of Translational Medicine named “Reproductive Medicine & Clinical Reproductive Sciences”. This section welcomes new research focusing on the basic mechanisms or the translational and clinical aspects of human reproductive biology. Also, we encourage the submission of preclinical and clinical studies whose results have the potential to prevent, diagnose, and treat reproductive issues, as well as studies that describe and characterize the genes and the mechanisms regulating human reproductive biology in cell lines or animal models. Reviews on existing and novel technologies, and commentaries on scientific breakthroughs in the field are also welcome. We look forward to receiving your contributions.We are delighted to launch a new section in the


Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL. in vitro and/or in vivo.  In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells.  As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database.  Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients. As an aggressive hematological tumor, T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% and 25% of childhood and adult ALL, respectively, and presented poor sensitivity to chemotherapy drugs and prone to the drug resistance . About 1As a large cluster composed of several adjacent near-ordinary enhancers, super enhancer (SE) is commonly observed in high-density transcription factors, cofactors, and enhancer-related epigenetic modifications. By regulating lineage-specific genes and oncogens, SE is critical for cell identity and the pathogenesis of multiple cancers –10. InhiThe FYN binding protein 1 (FYB1) encodes the adhesion and degranulation-promoting adapter protein (ADAP) and is highly expressed in T cells, NK cells, bone marrow cells, and platelets, responsible for the signal transduction upon T cell receptor (TCR) stimulation with integrin activation .Studies have shown key roles of FYB1 gene in megakaryocyte-specific platelet production  and autoin vitro and in vivo through the downstream effector, IGLL1. Our results proposed FYB1 as a potential vulnerability of T-ALL cells, representing an interesting therapeutic target.In this study, we identified FYB1 as an SE-driven gene using public H3K27ac ChIP-seq data from T-ALL cell lines, acute myeloid leukemia (AML) cell lines and hematopoietic stem and progenitor cells (HSPCs). As a SE-driven gene, FYB1 gene was highly expressed in T-ALL cell lines, suggesting plausible dependance of T-ALL cells on the high expression of FYB1 gene. Downregulating FYB1 blunted the growth of T-ALL cells both 2 gas at 37°C and subjected to routine mycoplasma testing. In 2020 and 2021, all cell lines were confirmed by short tandem replicates.Human T-ALL cell lines, including HUT78, J-gamma1, CCRF-CEM, MOLT-4, Jurkat and 6T-CEM, human AML cell lines, including MV4-11 and NB4, and human CML cell line K562, were all cultured with Roswell Park Memorial Institute (RPMI)-1640 medium  supplemented with 10% FBS  and 1% penicillin–streptomycin . Human neuroblastoma cell lines SK-N-BE2 and IMR32, human gastric cancer cell lines OACP4C and HGC-27, and human osteosarcoma cell line HOS were all cultured with DMEM medium  supplemented with 10% FBS  and 1% penicillin–streptomycin . All cell lines mentioned above were purchased from the Chinese Academy of Sciences Cell Bank and were cultured in a humidified incubator with 5% CO3. Following the instructions of the manufacturer, Cell Counting Kit-8 (CCK8)  was applied to determine the cell viability. Community multiplication was computed as a percentage of cell growth in the control medium. Cell density was measured three times and repeated in at least three separate trials. Plots and statistic analysis were performed by Graph Prism software 8.4.3 .Cells  were plated in a 96-well plate with a density of 1 × 10ΔCT method. Sequences of primers used in this study were shown as below: GAPDH: forward: TGCACCACCAACTGCTTAG, reverse: GATGCAGGGATGATGTTC, FYB1 forward: GGATGTCTCAGTCAATAGCCG, and reverse: GGTTCCTTGTCAGGCTTTTCC.The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted as mentioned previously . TRIzol®μm filter. Leukemia cells were then incubated with lentivirus for 24 hr supplemented with polyurethane (Sigma–Aldrich) of 10 μg/ml. Puromycin (10 μg/ml) selection was used to make stable T-ALL cell lines was prepared with 10 μg/ml puromycin .shRNAs targeting FYB1 and IGLL1 (sequences were shown in P < 0.05 and Log2 (fold change) >0.5 or Log2 (fold change) <−0.5). The RNA sequencing data have been uploaded to the GEO database (https://www.ncbi.nlm.nih.gov/geo) under the session of GSE197450.In accordance with the protocol advised by Novogene , RNA sequencing was carried out as brief described as below. Total RNA was first retro-transcribed into cDNA for library construction, followed by next-generation sequencing. The original reads were then filtered and mapped against HISAT for clean reads. Gene expression levels were then computed (in fragments per kilobase exon model mapped per million reads). The differentially expressed genes (DEGs) were identified by DESeq2 analysis (μg/ml polyurethane (Sigma–Aldrich). Stable cell lines were selected with 10 μg/ml of puromycin . Leukemia cells obtained after 5 days of screening were washed with precooled 1x PBS and suspended in a 1x binding buffer and then treated using the FITC-Annexin V Apoptosis Kit  according to the manufacturer's instructions. Flow cytometry  was used to analyze the apoptosis.Apoptosis was analyzed as mentioned previously . T-ALL cμmol/l; Catalog no.: P4170; Sigma–Aldrich; Merck KGaA) and 25 μg/ml RNase A, and kept in dark for 1 hr. Beckman Gallios™ flow cytometry  was used to evaluate cell cycle distribution. The MultiCycle AV DNA analysis software  was used to analyze the proportion of cells at different stages of the cell cycle.The cell cycle was analyzed as mentioned previously . The celWestern blotting was analyzed as mentioned previously . The RIP6 Jurkat cells via tail vein injection. On the 18th day after cell injection, luciferase was injected into the abdominal cavity of each mouse and followed by immediate anesthesia with isoflurane gas. Then, the NightOWL in vivo imaging system  was used for the mouse of each group on the 25th, 30th, and 35th days. Mice weight and fur were monitored every 5 days. Mice were euthanized when they reached the humane endpoint. The liver, spleen, peripheral blood (PB), and bone marrow of mice in the sh-NC group and the sh-FYB1 group were collected; and the liver and spleen organs were subjected for imaging. The flow cytometry  was used to analyze the expression quantity of antihuman CD45+ after grinding the liver, spleen, PB, and bone marrow of mice and the differences between the sh-NC Group and the sh-FYB1 group were compared. Immunohistochemistry and HE (hematoxylin and eosin) staining were conducted for each organ specimen. Cleaved-Caspase 3  and Ki67  were used according to the manufacturer's instructions.All animal procedures in this study have been approved and licensed by the Animal Care and Use Committee of Children's Hospital of Soochow University (CAM-SU-AP #: JP-2018-1). Female NSG mice aged 4–8 weeks (NOD-Prkdc) Scid II2rgEm1/Smoc, NM-NSG-001) were purchased from Shanghai Model Organizations. Mice were maintained in a standard SPF room and used in all studies of the sh-NC Group and the sh-FYB1 Group with 12 for each. On Day 0, each mouse was injected with 2 × 10http://www.cistrome.org/). The obtained ChIP-Seq H3K27ac datasets  were aligned to the reference genome (UCSC hg38) with alignment parameters -p 4 -q -x [Public ChIP-Seq H3K27ac datasets of T-ALL cell lines, AML cell lines, and HSPCs were downloaded from the Cistrome database ( 4 -q -x . Peaks w 4 -q -x  for the  4 -q -x . Then wehttp://www.broadinstitute.org/ccle).The expression level of FYB1 and IGLL1 mRNA in different types of tumor cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE) . Supplementary 1. A total of 213 genes were selected which were commonly correlated with SEs in at least 6T-ALL cell lines, including CDK6, CCND3, ETV6, and FYB1 . In this study NB4, MV4-11, and THP-1 were also used as representative AML cell lines. Additionally, we included three HSPC) samples, to compare the H3K27ac signals among those T-ALL, AML, and HSPC cell samples. The principal component analysis (PCA) result and clustering result based on the peak signals clearly distinguished T-ALL samples from AML or HSPC Figures . Putativand FYB1 . As showhttps://portals.broadinstitute.org/ccle) (FYB1 was identified as one of the SE-driven genes in T-ALL cells . SE was rg/ccle) . Furtherrg/ccle) . CollectP < 0.001) . In contrast, overexpression of FYB1 resulted in enhanced proliferation of J-gamma1 and Jurkat cells  with high expression of FYB1 by shRNAs . Knockin Figures . In addi Figures . In cons Figures , which c Figures . Taken tin vitro, we therefore proceeded to evaluate this in vivo. To this end, Jurkat cells with or without knocked-down FYB1 were transplanted into Nod–Scid mice   . No obvi 0.0026) . All theP < 0.05 and Log2 (fold change) >0.5 or Log 2(fold change) <−0.5, Supplementary 3. IGLL1 was among the top 20 downregulated genes (Supplementary 2). All these results point to IGLL1 as a potential downstream target of FYB1 signaling.To reveal the potential targets of FYB1 responsible for T-ALL cell proliferation, we performed RNA-seq in J-gamma1 cells with or without knocking down FYB1 (GEO ID: GSE197450). About 1,051 DEGs were identified after knocking down FYB1 gene, including 757 downregulated genes and 294 upregulated genes  and found that the expression of IGLL1 was highly expressed in T-ALL (Supplementary 2). Besides, we also determined the effects of knocking down IGLL1 on the apoptosis and found enhanced apoptosis in J-gamma1 and 6T-CEM cells as a result of knocking down IGLL1 (To further explore the role of IGLL1 in T-ALL, we analyzed the CCLE dataset (in T-ALL . To evalin T-ALL  were usein T-ALL . In agrein T-ALL , at leasin T-ALL . Moreovewn IGLL1 . In addiwn IGLL1 . Taken tAs an invasive hematological malignancy, T-ALL was characterized of heterogenous phenotypes and genetics. The past decades have seen improved treatment outcome, thanks to intensified combination therapy. However, around 20% of T-ALL patients would experience relapse and relapsed/refractory T-ALL patients have even worse prognosis –26. TherOver the past decade, mounting evidence has shown that SE, associated with the pathogenesis of various solid tumors and hematological malignant tumors, plays a significant role in the regulation of key oncogenes –31. In tActing as an essential adapter of the FYN and LCP2 signaling network, FYB1 is critical in bridging T-cell signaling to remodeling of the actin cytoskeleton and important for the fitness of normal T cell. However, whether FYB1 is essential for the fitness of T-ALL cells is largely unknown , 38.in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis (Figures Crucial for TCR signaling transduction, FYB1 has been reported for its involvement in the pathogenesis, invasion, and metastasis of various solid tumors –17. Howe Figures  and 4. IAmong the top genes that were downregulated due to knocking down FYB1 and IGLL1 was also driven by T-ALL specific SE . In consGenerally, our results indicate that FYB1-IGLL1 axis might play important roles in the pathogenesis of T-ALL, providing new insights for the biology of T-ALL. Given its role in controlling cell proliferation and apoptosis, FYB1-IGLL1 represents an interesting target for T-ALL therapy.


There is a clear need to expand the toolkit of adequate mouse models and cell lines available for preclinical studies of high-grade neuroendocrine lung carcinoma  and large cell neuroendocrine carcinoma (LCNEC)). SCLC and LCNEC are two highly aggressive tumor types with dismal prognoses and few therapeutic options. Currently, there is an extreme paucity of material, particularly in the case of LCNEC. Given the lack of murine cell lines and transplant models of LCNEC, the need is imperative. In this study, we generated and examined new models of LCNEC and SCLC transplantable cell lines derived from our previously developed primary mouse LCNEC and SCLC tumors. RNA-seq analysis demonstrated that our cell lines and syngeneic tumors maintained the transcriptome program from the original transgenic primary tumor and displayed strong similarities to human SCLC or LCNEC. Importantly, the SCLC transplanted cell lines showed the ability to metastasize and mimic this characteristic of the human condition. In summary, we generated mouse cell line tools that allow further basic and translational research as well as preclinical testing of new treatment strategies for SCLC and LCNEC. These tools retain important features of their human counterparts and address the lack of LCNEC disease models. High-grade neuroendocrine lung carcinomas are among the most malignant lung tumors. They consist of small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) and account for >15% and 2–3% of all lung tumors, respectively ,5 and twThe prognosis of patients is dismal. Most SCLC or LCNEC patients are diagnosed at advanced stages, when surgery is not a therapeutic option and survival rates at 5 years are as low as 7–8%  or even Rb1, Rbl1, Pten, and Trp53 in basal epithelial airway keratin K5 expressing cells led to SCLC with a remarkable resemblance to its human counterpart [Rb1, Rbl1, Pten, and Trp53 in a wide variety of lung epithelial cells (using an adeno-CMVcre virus). Therefore, there is an urgent need to expand the availability of experimental preclinical in vitro and in vivo models for this lung cancer tumor type.Researchers have developed and analyzed several genetically engineered mouse models (GEMMs) of SCLC ,4,13,14,nterpart ,21. MeanIn this study, we developed novel in vitro and in vivo models of high-grade neuroendocrine carcinoma of the lung. We established, from the abovementioned spontaneous tumors, new SCLC and LCNEC cell lines that can be successfully transplanted into immunodeficient or immunocompetent mice and develop the corresponding SCLC or LCNEC tumor allografts. The derived cells and tumors maintained neuroendocrine properties and transcriptomic characteristics of the tumors from which they originated and displayed strong similarities to their human counterparts. Moreover, the SCLC syngeneic model showed the ability to develop metastases, mimicking this characteristic of the human disease. These cell lines might become robust tools for basic and translational research of high-grade neuroendocrine lung cancers as well as preclinical studies of novel therapeutic strategies. In addition, the syngeneic immunocompetent mice may be particularly suitable for testing novel immunotherapeutic and anti-metastatic approaches. The intratracheal administration of adenovirus Ad-CMVcre to adult QKO mice produced LCNEC in lungs . An examRb1, Rbl1, Pten, and Trp53 loci  or immunocompetent (QKO) backgrounds and observed that both the 46LCNEC and 54SCLC subcutaneous tumors developed from the same cell passage reached a similar size irrespective of the recipient mice  and showed the ability of these cell-derived tumors to colonize distant organs, thus mimicking the primary tumors arisen in the lungs and the human condition. As tumors injected with 54SCLC grew very rapidly in the syngeneic mouse . Immunohistochemical detection of the neuroendocrine markers chromogranin A or CGRP as well as the expression of TTF-1 or the absence of p63 that were observed in subcutaneous tumors (Given the high metastatic rates reported in human SCLC patients (about 70% at diagnosis) , we sougnd Trp53 ,21, the ouse see , we surgs tumors  corrobors tumors .These data show the ability to form metastases of both the 54 and 57SCLC cell lines in the syngeneic model, which reproduces the pattern observed in human SCLC and makes these cell lines particularly suitable to address anti-metastatic therapeutic approaches in defined molecular settings.To further characterize our models, we performed RNA sequencing from LCNEC and SCLC cell lines , their corresponding derived syngeneic allograft tumors , and normal lung tissue from littermates used for 57SCLC and 46LCNEC syngeneic allografts (lung). As expected, principal component analysis (PCA) showed that triplicates from each sample type grouped together in the bi-dimensional space A. To anaWe evaluated whether our cell and allograft syngeneic models resembled the spontaneous tumors from which they originated. In a previous work , we charThis study presents new mouse LCNEC and SCLC cell lines, which were generated from spontaneous tumors arisen in our previously generated GEMMs ,21. SubcGenetically engineered mouse models (GEMMs) faithfully reproduce human tumors in a physiological and immunocompetent environment . HoweverThe relevance of mouse cell lines for basic, translational, and preclinical research depends on how closely they resemble their human tumors’ counterparts. The comparative transcriptomic analyses performed here shows that LCNEC and SCLC cells and subcutaneous tumors retain the transcriptomic profile of the original spontaneous tumors and share strong similarities with those of human LCNEC and SCLC ,21,28. TSCLC is a highly metastatic and recalcitrant carcinoma, with metastasis as a major cause of mortality. While human SCLC cells fail to metastasize when transplanted into mice, few murine SCLC cell lines have been reported to generate metastasis after subcutaneous engraftment ,29 to seIn summary, we report the establishment and precise characterization of high-grade neuroendocrine lung carcinoma (LCNEC and SCLC) cell lines and transplanted tumors in different backgrounds. To our knowledge, these are the first murine LCNEC cell lines that have been generated. Our data support the neuroendocrine properties of these novel cell lines and their transcriptomic similarities among cells, allograft, and spontaneous and human tumors of the same cancer type (LCNEC and SCLC). Importantly, the SCLC cell lines showed metastatic potential with the ability to grow metastatic lesions. In summary, we showed that these cell lines might serve as reliable model systems for further neuroendocrine lung cancer research and identification of novel molecular targets and immunotherapies for the treatment against highly aggressive SCLC and LCNEC.All the animal work was approved by the Animal Ethical Committee (CEEA) and conducted in compliance with Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) guidelines.Rbl1, Pten, and Trp53 alleles along with a null Rbl1 gene and have been described elsewhere [nu/nu immunodeficient 6–8-week-old mice were obtained from Janvier (Saint-Berthevin). QKO mice bear floxed lsewhere . PrimaryFor the isolation and culture of tumor cells, CMV-QKO and K5-QKO mice  were sacCells grew either in suspension (SCLC cells from K5-QKO tumors) or in adherent monolayer (LCNEC cells from CMV-QKO tumors). To split the cell cultures, suspending aggregates were harvested and mechanically disaggregated by pipetting with micropipette. Adherent cultures were washed (PBS) and detached by incubating the cells in trypsin-EDTA solution (MilliporeSigma) for 3 min at 37 °C.2 flasks and cultured for 5 days. Every day, the cell number was counted in a Countess 3 Automated Cell Counter . To assess cell growth kinetics, 100,000 LCNEC or SCLC cells were seeded into 75 cmAll experiments were performed with mycoplasma-free cells.2) × (short side/2))/3. Tumors were allowed to grow until a maximum volume of 1500 mm3; then, mice were euthanized and processed for histology. Complete necropsies were performed in search of metastases. For mice injected with 54SCLC, subcutaneous tumors were surgically excised and allowed to regrow. Organs were processed for histology, as described below  and injected subcutaneously into the flanks of recipient (immunodeficient NMRI nude or immunocompetent QKO) mice. Tumors were measured twice a week with a caliper, and tumor volume was calculated with the formula (4 π ((long side/2)elow see .F/FRb1, -/-Rbl1, F/FTrp53, and F/FPten control lungs, cells, and tumors using DNeasy Blood & Tissue Kit . Primer sequences, amplified fragments, and PCR amplification product sizes appear in Genomic DNA was isolated from Adherent cell lines (LCNEC) were seeded in chamber slides and grew there for 48 h. Floating cell lines (SCLC) were spun in Shandon CytoSpin 2 centrifuge at 400 rpm for 5 min. For the immunofluorescence analyses, the cells were (1) fixed with MetOH-acetone (1:1) at −20 °C for 10 min; (2) incubated with horse serum 10% for 1 h at room temperature; and (3) washed three times with sterile phosphate-buffered saline (PBS) (pH 7.5) prior to incubation with the appropriate primary antibodies diluted in horse serum 10%. Primary antibodies were used as follows: 1/200 dilution of anti-NCAM CD56 ; 1/100 dilution of anti-SSTR2 ; and 1/2000 dilution of anti-CGRP . Secondary antibodies were used as follows: 1/1000 anti-rabbit AlexaFluor488  and 1/1000 anti-mouse AlexaFluor488 (A11001Molecular Probes). Diamidinophenylindole (DAPI) was used to counterstain the nuclei or chromosomes.Necropsies were performed at the end of the experiments. Upon necropsy, tumors and tissues were fixed in formalin for 24 h. Fixed tissues were dehydrated and embedded in paraffin wax. Sections (5 μm) were stained with hematoxylin and eosin (H&E) for histological analysis or processed for immunohistochemistry. Immunohistochemical analyses were performed as follows: the sections were (1) deparaffinized; (2) incubated with 10% horse serum for 30 min at 37 °C to block non-specific binding; and (3) washed three times with sterile phosphate-buffered saline (PBS) (pH 7.5) prior to incubation with the appropriate primary antibodies diluted in horse serum 10%. Primary antibodies were used as follows: 1/100 dilution of anti-chromogranin A ; 1/2000 dilution of anti-CGRP ; 1/200 dilution of anti-TTF1 ; 1/50 dilution of anti-p63 ; and 1/100 dilution of anti-CC10 . Secondary antibodies were used as follows: 1/1000 biotin anti-mouse  and 1/1000 biotin anti-rabbit .Cells and tissues were embedded in RNALater  according to the manufacturer’s instructions. RNA yield and quality were determined using an Agilent 2100 Bioanalyzer.n = 3 57SCLC cell line samples, passage 11, from 3 different cell flasks; n = 3 46LCNEC cell line samples, passage 17, from 3 different cell dishes; n = 3 57SCLC syngeneic allograft tumors (from transplanted cell passage 11); n = 3 46LCNEC syngeneic allograft tumors (from transplanted cell passage 17); and n = 3 normal lung tissue from littermates of mice used for 57SCLC and 46LCNEC syngeneic allografts. RNA sequencing was performed after poly-A selection using Illumina NovaSeq 2 × 150 bp sequencing. Reads were aligned to GRCm39 mouse genome with “Rbowtie2”, counts extracted with featureCounts function from “Rsubread” and gencode annotation release 27 (gencode.vM27.annotation.gtf), and differential expression with DESeq2. Principal component analysis graph was plotted after variance stabilizing transformation (vst function in DESeq2). Transcriptome sequencing was performed from www.broadinstitute.org/gsea (accessed on 5 May 2023)) [We developed signatures of genes specifically up-regulated in tumors from our CMV-QKO LCNEC (moLCNEC) and K5-QKO SCLC (moSCLC) models . Brieflyy 2023))  was usedp-values for each SA. The SubMap module uses permutations to compute p-values. Number of permutations for nominal-p of Fisher’s statistics: 1000. The p-value correction method: FDR and Bonferroni.We analyzed the association between our cells and allograft RNAseq expression data using the SubMap module of Gene Pattern ,25. SubMThe 300-gene LCNEC/SCLC classifier from George et al. (2018)  was convp-value threshold of ≤0.01 and an expression fold change (FC) ≥2 were selected. DAVID [https://david.ncifcrf.gov/home.jsp (accessed on 5 May 2023)) from the NIAID/NIH was used to identify Gene Ontology Biological Process (GOBP) functional categories (GOTERM_BP_DIRECT). GOBP terms were listed by p-value based on EASE score [DESeq2  was usedd. DAVID  web servSE score  and manuData were analyzed with GraphPad Prism 9 software . Statistical analysis was performed using U Mann–Whitney test.Patients with SCLC or LCNEC have very poor prognoses, low survival rates, and few effective treatments. Surgery is not usually an option and, as a consequence, human material for research is scarce. In an attempt to address these issues, this study described newly generated mouse cell lines and in vivo models of SCLC and LCNEC that closely resemble their human counterparts. These constitute relevant tools for further basic and translational research as well as preclinical testing of potential antitumor compounds, including immunotherapies. It is of note that no mouse LCNEC cell lines have been previously described.


Calendula officinalis flower, and Aloe vera on breast cancer vs. epithelial cell lines. Various extraction methods were used, and the proliferative effect of the resulting extracts was assessed by proliferation assay on breast cancer and epithelial cell lines. Carrot, Aloe leaf, and Calendula flower extracts were extracted by hexane and methanol methods, and their semi-purified extracts were able to specifically inhibit the proliferation of breast cancer cell lines. The extract composition was investigated by colorimetric assays, UHPLC-HRMS, and MS/MS analysis. All the extracts contained monogalactosyl-monoacylglycerol (MGMG), while digalactosyl-monoacylglycerol (DGMG) and aloe-emodin were found in Aloe, and glycerophosphocholine (GPC) derivatives were identified in Calendula, except for the isomer 2 detected in carrot, suggesting that their observed different anti-proliferative properties may be associated with the different lipid compounds. Interestingly, Calendula extract was able to strongly inhibit the triple negative breast cancer MDA-MB-231 cell line proliferation , supporting MGMG and GPC derivatives as potential drugs for this BC subtype treatment.Breast cancer (BC) remains the leading cause of mortality in women, despite significant advancements in diagnosis. Thus, the identification of new compounds for its treatment is critical. Phytochemicals are known to exhibit anti-cancer properties. Here, we investigated the anti-proliferation potential of extracts from carrot,  The first fragmentation, detected in ToF-MS/MS spectra, arose from the neutral loss of methyl formate (60 Da). This behavior has been described as pivotal for the identification of the GPC lipid class and seems to involve a gas-phase SN2-like reaction, in which the formate ion acts as the nucleophile and the methyl group of the quaternary ammonium polar head as the electrophile [m/z 224.069 , while the one at m/z 295 could be ascribed to its hydroxy-derivative, already described in plants belonging to Calendula genus [m/z 279), oleic acid , and saturated palmitic acid .Calendula sample was mainly composed of glycerophosphocholine (GPC) derivatives, differently substituted. Their molecular ions shared even trophile . Therefo 224.069 . In partla genus . The lat® Structure Database (LMSD) [Lipid Mapse (LMSD)  was consm/z 699.3803) was found only in Aloe semi-purified extract. Additionally, these metabolites appeared as formate adducts, which in MS/MS experiments immediately gave rise to deprotonated structures. Then, ions at m/z 415.1470, assigned to the digalactosylglycerol moiety, and the corresponding dehydrated fragment at m/z 397.1363, were pivotal for their characterization.Apart from GPCs, one monogalactosyl-monoacylglycerol (MGMG) was tentatively identified in all the samples, whereas a DGMG  was identified as aloe-emodin according to the literature [Finally, in terature .Calendula, Aloe, and carrot were dried, weighted, solubilized in DMSO at a final concentration of 1 mg/mL, and tested at the final concentration of 0.5 µg/mL (assay concentration of DMSO 1.0% v/v). These semi-purified extracts were tested on four different cell lines: MCF10A as breast cell line of control and MCF7, SKBR3 and MDA-MB-231 breast cancer cell lines, representing oestrogen receptor positive, and Her-2 positive and triple negative breast cancer subtypes. All cell lines were treated upon 48 h with all the semi-purified extracts, and cell survival was evaluated by CYQUANT® cell proliferation assay.Semi-purified extracts from v/v of DMSO; the carrot semi-purified extract  isomer 2 as well as the hydroxy-octadecadienoic acid compound also detected in carrot. Instead, all the extracts contained one monogalactosyl-monoacylglycerol , while a digalactosyl-monoacylglycerol  and aloe-emodin were only identified in Aloe leaves semi-purified extract  derivatives, differently substituted in m/z 277.2174) detected in the extracts from all vegetables. Previously, it has been reported that the monogalactosyldiacylglycerol (MGDG), another type of glycoglycerolipid found in the plant cell membrane, showed therapeutic anti-cancer activity [Calendula flowers was higher, compared to the other extracts from carrot and Aloe, on the MDA-MB231 breast cancer cell line. This effect may be due to the presence of the different glycerophosphocholine (GPC) derivatives detected only in Calendula flowers, except for the GPC (16:0) isomer 2 which was also found in carrots.All together, these data suggest that the anti-proliferative effect may be associated with the presence of the glycoglycerolipid compound . BC includes several distinct subtypes based only on genetic, morphologic, and clinical features [Lipid compounds are getting attention for their anti-tumoral, anti-inflammatory, and anti-viral activities. It has been reported that they are able to induce greater apoptosis in various cancer cells  than in normal cells, both in vitro and in vivo, by generating reactive oxygen species, suppressing angiogenesis via the Akt cell signalling pathway, and inducing apoptosis ,40,41,42features . Among a


Globally cells adapted by decreasing expression of genes involved in metabolism, proliferation, and protein maturation, and increasing alternative splicing. They accumulated mutations, especially frameshift insertions, and harbored increased copy number alterations, indicating increased genomic instability. Cells showing highest TMB simultaneously experienced a significant downregulation of DNA replication and repair and chromosomal maintenance pathways. A sixteen-gene common response to chronic hypoxia was put forth, including genes regulating angiogenesis and proliferation. Our findings show that chronic hypoxia enables survival of tumor cells by metabolic reprogramming, modulating proliferation, and increasing genomic instability. They additionally highlight key adaptive pathways that can potentially be targeted to prevent cancer cells residing in chronically hypoxic tumor areas from thriving.Hypoxia afflicts the microenvironment of solid tumors fueling malignancy. We investigated the impact of long hypoxia exposure on transcriptional remodeling, tumor mutational burden (TMB), and genomic instability of cancer cells that were grouped based on their inherent sensitivity or resistance to hypoxia. A hypoxia score was used as a metric to distinguish between the most hypoxia-sensitive (hypoxia high (HH)), and most resistant (hypoxia low (HL)) cancer cells. By applying whole exome sequencing and microarray analysis, we showed that the HH group was indeed more sensitive to hypoxia, having significantly higher TMB ( Hypoxia is common to solid tumors manifesting upon the drop in oxygen below physiological levels. Moving away from the functional vasculature within the growing tumor mass, different levels of oxygenation levels exist creating sites of intermittent, or cyclic, hypoxia experiencing fluctuations in perfusion leading to episodes of hypoxia and reoxygenation; sites of acute hypoxia due to transient disruption of perfusion lasting minutes to hours; and areas of chronic hypoxia where there is continuously limited oxygen diffusion . HypoxiaIn vitro investigations have shown that hypoxia could lead to genomic instability through its downregulation of DNA repair genes and their respective pathways of MMR . Hypoxis of MMR  (reviewes of MMR ). In a ss of MMR . How proin vitro and in vivo; an effect that was not observed in cells proficient for MMR  cells not only resulted in increased TMB, but also dynamic mutational profiles that led to neoantigens renewal  for MMR . This in for MMR . A longigen load . Xenogragen load . We havegen load . While tgen load . In thatgen load .in vitro by applying different oxygenation levels and exposure times, but a comprehensive study on the prolonged exposure of tumor cells to hypoxia is yet to be conducted. This is highly relevant, since the actual time tumor cells can remain in a hypoxic state within the tumor can range from hours to even weeks  values for the eight signature genes corresponding to three interdependent experiments carried out in duplicate were obtained from our previously published work . There whttps://www.cbioportal.org/) to download RNA sequencing data  for the 8 signature genes for the following datasets: Breast Invasive Carcinoma ; Colorectal Adenocarcinoma ; Ovarian Serous Cystadenocarcinoma  and Pancreatic Adenocarcinoma . Subsequently the patients in each dataset were given a hypoxia score as previously described . They were cultured in appropriate Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F-12), GlutaMAX Supplement  or in Roswell Park Memorial Institute Medium 1640 (RPMI 1640) Medium, GlutaMax supplement , supplemented with 10% Fetal Bovine Serum (FBS) , 1% Sodium pyruvate  and 1% Penicillin - Streptomycin , as previously described . Cells were passaged twenty times in the two conditions and DNA and RNA was isolated and processed as described below.The three highest scoring cell lines, H226, MCF-7 and HT-29, as well as the three lowest scoring cell lines, Capan-1, SKOV-3, MIA PaCa-2 were maintained in a 5% COescribed . Two ind® Hybridization oven 640 (Affymetrix). Arrays were subsequently washed and stained using appropriate reagents from the GeneChip™ WT PLUS Reagent Kit (Applied Biosystems™) as well as the GeneChip Fluidics Station 450 (Affymetrix/Thermo Scientific inc) following manufacturer’s recommendations. The arrays were immediately scanned with the GeneChip™ Scanner 3000 7G (Affymetrix/Thermo Scientific inc).Total RNA was first isolated using EasyBlue  following manufacturer’s instructions, and subsequently purified with repeated washes and in 75% ethanol. After drying, isolated pellets were resuspended in nuclease free DEPC treated water and concentration and purity were evaluated using the Nanodrop. The integrity of RNA was determined by running 300 ng on 1% agarose gel and the 28S/18S bands were observed. Only RNA with acceptable integrity (28S twice the intensity of 18S and without smearing), as well as purity ratios  were processed for transcriptome analysis with the Human Clariom™ D Assay . Aliquots of 50 ng/μL were prepared and 100 ng of total RNA was used to prepare fragmented and labeled single stranded cDNA. The steps were conducted with the GeneChip™ WT PLUS Reagent Kit (Applied Biosystems™) following manufacturer’s instructions. Each sample was hybridized to one array respectively by placing the arrays in specialized trays and incubating them for 17 h at 45°C and 60 rotations per minute using the Genechipp-value ≤0.05 were considered significant. The lists of significant differentially expressed TCs per comparison per cell line can be found in The images and raw data files from scanned arrays were first checked to ensure correct alignment and that there were no bubbles or debris affecting the image’s clarity. Subsequently, cell probe defined files (.CEL) files were processed using the Transcriptome Analysis Console (TAC) 4.2 software (Applied Biosystem). Gene + Exon summarization using the Signal Space Transformation-Robust Multi-Array Average (SST-RMA) algorithm was first applied to summarize, background-subtract, and normalize the CEL files. Further QC checks were applied to ensure that all samples were properly labeled and hybridized and had acceptable positive signal intensity. The resulting CHP files were imported back to TAC to carry out differential gene expression analysis using ANOVA method for statistical testing with ebayes  correction for small sample size. The hypoxia samples in each cell line were compared to their respective normoxia samples from passage zero and passage twenty. Only transcript clusters (TCs) that showed a fold change (FC) of ≤ −2 or ≥2 with a false discovery rate (FDR) Pathway analysis on the differentially expressed genes per cell line was conducted using GSEA v4.2.2. Default settings were used , except that the permutation type was gene_set and not phenotype, given that the sample numbers per group were less than 7. The metric for ranking genes was the ratio of classes, which is equivalent to the fold change. Curated gene sets from HALLMARK , KEGG , REACTOME , BIOCARTA  and WIKIPATHWAYS (//pub/gsea/gene_sets/c2. cp.wikipathways.v7.5.1. symbols.gmt) were used for enrichment analysis. Enriched pathways are given with a normalized enrichment score (NES) that is an enrichment score which has been normalized across the analyzed gene-sets. The enrichment score itself, represents the degree to which a particular gene-set is over-represented at the top or bottom ranked list of genes present in the expression data. Significantly enriched pathways were considered as those having a NES with an FDR q-value of at least 0.05.p-value of less than 0.05 are considered differentially expressed. In addition, based on the signals of the PSRs and JUCs of a gene, the event of alternative splicing is determined as being an exon cassette (splicing out of exon from primary transcript), intron retention , alternative 5′ donor site , alternative 3′ acceptor site , mutually exclusive exons (2 back to back exons excluded from the mature mRNA transcript), alternative last exon and alternative first exon, as well as complex events that do not fit in these categories. How well the event explains the splicing pattern visualized is given by an event score with values from 0 – 1. The event with the highest value is considered as the most possible mode of alternative splicing. Alternatively spliced TC without any reported event score were excluded from the analysis.Potential alternative splicing was assessed due to the presence of probe selection regions (PSRs) that represent gene exons and junctions (JUCs), which are used to probe the inter-exon boundary. Eventpointer algorithm is applied to predict the presence of alternative splicing events considering the signals of the JUCs and PSRs upon their normalization to the expression levels of the gene itself. The summary expression signals of JUCs and PSRs are then used to determine the splicing index in condition 2 (hypoxia) compared to condition 1 (normoxia). TC with exon splicing index of greater than 2 or less than −2 and an exon FDR ® Genomic DNA Kit  following manufacturer’s recommendation. In brief, DNA pellets were lysed by vortexing in 200 μL PureLink® Genomic Lysis/Binding Buffer following treatment with Proteinase K and RNase A. DNA was subsequently bound on the PureLink® Spin Columns upon addition of 200 μL absolute ethanol. The DNA was washed with appropriate buffers, which were removed by centrifugation and finally eluted using 40–60 μL low binding TE buffer . The purity of gDNA was confirmed with the Nanodrop and the integrity was checked by running 500 ng on agarose gel electrophoresis. The Ion S5 System  was used for whole exome sequencing. Before starting, gDNA was accurately quantified by fluorometry using the Qubit dsDNA HS Assay Kit  and the Qubit 4 Fluorometer (Invitrogen). Subsequently 100 ng of DNA was used for exome library preparation with Ion AmpliSeq™ Exome RDY Kit and Ion Xpress™ Barcode Adapters 17–32 Kit  or Ion Xpress™ Barcode Adapters 33–48 Kit  following manufacturer’s protocols. For every sample, target regions were amplified by PCR and the resulting amplicons were pooled, partially digested then ligated with suitable adaptors to achieve barcoded libraries. Libraries were purified using the CleanNGS system  then finally quantified by qPCR using the Ion Library TaqMan Quantification Kit  and diluted to 100pM. The Ion 540™ Kit–Chef  was used to implement the sequencing reactions following recommended protocols. The Ion template preparation on 540 Ion Chip™ was done using the automated Ion Chef™ System (Ion Torrent™) and the sequencing reaction was carried out in the Ion S5™ sequencer (Ion Torrent™). Two differentially barcoded libraries could be sequenced simultaneously at 25 pM per library.Genomic DNA was isolated using the PureLinkvcf2maf, as described in (n = 2 across both the files) were removed as a part of the data cleaning process from further analysis. Furthermore, two MIA PaCa-2 files were removed from the downstream analyses as they generated TMB levels that were 300 orders of magnitude higher than all values achieved for this cell line, as well as the other cell lines. The TMB values were also higher than those previously reported for the cell line as per the cancer cell line encyclopedia on cbioportal . Therefore, there were 44 files that were analyzed further. The full information of these samples can be found in the Raw sequencing data was first aligned against hg19 with Ion Torrent Suite software to generate Binary Alignment Map (BAM) files. Low stringency somatic variant and indel calling was then conducted with the Torrent Variant Caller by applying default settings to obtain Variant Call Format (VCF) files. VCF files were converted to MAF files using ribed in . Two Capmaftoolsv2.6.05 was used to analyze MAF files. Mutational landscape across the samples HL was analyzed after normalizing the variants called in the hypoxia-treated cancer cells against the normoxia cancer cells used as the control. In this way only variants that emerged in the cancer cells after exposure to long term hypoxic conditions could be identified. The mutational landscape was then reported as follows: 1. Tumor Mutational Burden, 2. Gene mutation summary and 3. Sample mutational summaries. Next, Copy Number Variations (CNVs) were calculated using the command line version of Control-FREEC. The analysis was run on. bam files. Control-FREEC requires an additional custom config file with all the parameter values to be run according to the data input and the desired output. CNVs were computed, normalized, and segmented for each hypoxia tumor sample against a corresponding normoxia sample as a control dataset using a window size of 50kb. breakPointThreshold was set to 0.8, contamination parameter was set to 0, minMappabilityPerWindow was set to 0.85 and ploidy was set to multiple values of 2, 3, 4 and the ploidy was allowed to be chosen according to the value that explains most observed CNVs across all samples. All the supporting files including input, output, config and codes written to carry out the analysis can be found at the following github link: https://github.com/Mohak91/hypoxia_pan_cancer_types_genomics_study.R package −ΔΔCq method  following manufacturer’s instructions. Twenty nanograms of cDNA were subjected to 10 μL qPCR reactions run in triplicate with the Maxima SYBR Green/ROX qPCR Master Mix (2X) . Select genes were quantified by RT-qPCR using specifically designed primers . Quantitq method . Beta acp-value ≤0.05. Heatmaps of expression were generated using Rstudio version 1.3.1073  by applying the gplots package. Hierarchical clustering was performed using Euclidean distance and ward. D2 method.Statistical analysis was conducted using GraphPad Prism 9.3.1. Unpaired two-sided t-test was applied for comparisons between only two groups, otherwise 2-way ANOVA with the recommended correction for multiple comparisons was used. Statistical significance was considered as in vitro 8-gene hypoxia signature   identified on the chip, 14′799 unique transcript clusters were differentially expressed in at least 1 cell line. The bulk of response in all cell lines, except for HT-29, was cell-line specific. Indeed, the biggest contributing factor to the variation seen among samples was their cell line-dependent characteristics . The celAK4, the pro-angiogenic gene VEGFA and the cell adhesion gene DSG2 were upregulated in all 3 cell lines, while the translation regulation gene, EIF4G2 and the mitochondrial gene PGAM5 were downregulated. Among the six HL specific genes, two showed variable expression , while one was consistently downregulated (PSAT1P1) and three upregulated  in all 3 cell lines. The upregulation of the cell adhesion gene CEC2B and the glycoprotein hormone STC1 were validated by RT-qPCR , were downregulated in hypoxia. The expression of five of these genes was validated by RT-qPCR . In part RT-qPCR .PGK1, NDRG1 and P4HA1, maintained significant differential expression in all cell lines. Significant VEGFA increased expression was also maintained in the HH group.In terms of the common response to hypoxia that was achieved in all cell lines, three genes were downregulated, two of which are involved in metabolism, while the remaining thirteen genes were consistently upregulated across all 6 cell lines. Those included long non-coding RNAs, genes involved in glycolysis, apoptosis, extracellular matrix (ECM) remodeling, proliferation, and angiogenesis . Of intePDK1, NDRG1, LDHA, and SLC3A2 were alternatively spliced in all the cell lines. In addition, HIF-1α was significantly spliced in all cell lines . There were 175 pathways in H226 (51 specific), 183 pathways in MCF-7 (63 specific), 26 pathways HT-29 (2 specific). With respect to the HL group, there were 58 pathways in Capan-1 (6 specific), 34 pathways in SKOV-3 (16 specific) and 47 pathways in MIA PaCa-2 (3 specific) (Supplementary File 3). Taken together, the HH group observed a higher number of enriched pathways compared to the HL group.The enriched pathways could be grouped into six major subsets: cell signaling and response; metabolism; DNA/RNA/protein processing; cell cycle; DNA replication and repair; and immune response . The onlvia NFκB, among others. On the other hand, MCF-7 demonstrated hypoxia-specific enrichment of the P53 transcriptional network and pathway, while HT-29 was enriched for glycolysis. MIA PaCa-2 and Capan-1 did not show any specific increased enrichment in hypoxia, while SKOV-3 showed the highest number of increased pathways in hypoxia, including pathways of EMT, Extracellular matrix degradation and organization, apical junction, and focal adhesion among others. Indeed, this cell line had quite a unique response to hypoxia, while most of the cell-cycle, and cell-signaling pathways were downregulated in hypoxia for the cell lines, in SKOV-3, eukaryotic translation initiation, Dectin1-mediated non-canonical NFκB signaling, and RHO GTPase related signaling were upregulated; so were pathways of the cell-cycle and cell-cycle checkpoints and control.Other pathways positively enriched in hypoxia were seen in a cell line dependent manner . H226 deBased on these results, it can be concluded that long passaging in hypoxia leads to cell cycle inhibition, decreased metabolic activity as well as nucleic acid and protein processes. Our data also indicate that cells seem to switch to an energy conserving and indolent state.2. Mutational landscape across the samples was analyzed after normalizing the variants called in the hypoxia-treated cancer cells against the normoxia cancer cells used as the control.To determine the relationship between the sensitivity of cancer cell lines to hypoxia with their ability to accumulate mutations, the three highest scoring cell lines, H226, MCF-7 and HT-29, and three lowest scoring cell lines, Capan-1, SKOV-3, and MIA PaCa-2 were cultured in chronic hypoxic conditions, indicated as twenty passages at 1% OTMB achieved across cell lines was variable, ranging from a mean of 1.040 mutations/Mb in SKOV-3 (95%CI: 0.3649–2.394) to 5.37 mutation/Mb (95%CI: 3.801–6.939) in MCF-7 . All celp = 0.03), suggesting that cells that are more sensitive to hypoxia are also more mutable. Looking into the classification of identified variants, the majority were frame-shift insertions, followed closely by missense mutations in both hypoxia groups with the most common form being C>T and T>C transitions  to the hypoxia low (HL) groups, HH displayed a mean of 3.007 mutations per megabase of genome (95%CI: 1.805–4.209), while the HL group accumulated 1.588 mutations (95%CI: 0.9353–2.241) . Indeed,nsitions . Among t samples .FLG, GPRIN2 and PCDHB5, no effects could be reported for most of the genes, however, in the case of MCF-7, seven genes were affected. One was differentially spliced (BRIP1), two were only differentially expressed  and four were both differentially expressed and spliced  (ZNF431) was differentially expressed in H226, and one (ZNF273) differentially spliced in MIA PaCa-2  . With reA PaCa-2 . These rin vitro were also altered in patient samples, we applied our signature to stratify tumors of TCGA datasets based on their hypoxia score into high hypoxia and low hypoxia. For each cancer type, the percent alteration of the top ten in vitro mutated genes were compared within the two groups. Results showed a good concordance level between the genes that were reported as affected in vitro and their mutation in more hypoxic patient tumors . Therefore, while the effect of long passaging in hypoxia on chromosomal instability is cell-line dependent, cells that are more sensitive to hypoxia are more prone to experiencing copy number losses. These changes did not give rise to any discernable phenotypic changes, as cells presented with similar morphology when comparing early and late passages in hypoxia , number of gains (CNG) and losses (CNL) for each cell line . RegardiL groups . Upon ex hypoxia .via ATR as well as DNA damage response were downregulated in H226; REACTOME pathways of G1-S DNA damage checkpoints and transcription coupled NER were downregulated in MCF-7; and REACTOME gene sets of HDR, activation of ATR in response to replication stress and DNA strand elongation, as well as KEGG DNA replication were downregulated in Capan-1. No effects on such pathways could be reported for HT-29 , transcription or translation regulation , and biogenesis of ribosomes  and purines (ATIC), it can be deduced that cells in the HH group mounted a bigger attenuation of key cellular processes when compared to those in the HL group. When considering results of pathway analysis at the cell-line level, the only commonly enriched pathway was that curated for hypoxia. This gene-set includes 200 genes reported to be upregulated in hypoxia and including HIF-1α targets. In addition, all the cell lines, except SKOV-3, showed significant downregulation of DNA/RNA/protein processing-, and cell cycle-related pathways. It seems like SKOV-3 showed bigger resistance to hypoxia than estimated based on the hypoxia score. Nonetheless, all the cell lines experienced a downregulation of pathways of metabolism. Interestingly, a common response to hypoxia could be determined that included sixteen genes. Three of these genes were downregulated, and among them two IDH3A and COA7 are involved in metabolism. While IDH3A has been associated with stabilization and transactivation of HIF-1α  (TP53). It is important to note that through our experiment, we cannot distinguish between the effect of passaging in hypoxia on accumulation of mutations and hypoxia alone as a genotoxic stress. This is relevant given the findings of Ben-David et al., which eloquently highlight the genetic heterogeneity and extensive clonal diversity that exists in cancer cell lines, wherein selection pressure could lead to genetic instability and genetic diversification  , which cnt (HRD)  and can fication . Regardification . Nonethe hypoxia . How theGenomic instability could be due to replication stress and deregulation of DNA repair pathways, both of which have been reported by us  and otheFLG, GPRIN2 and PCDHB5. Based on literature findings  cells, while promoting the inhibitory role of regulatory T-cells . Furtherolecules . Of inteversus hypoxia alone. One limitation of this work is that we did not directly compare the response of these cells in the given condition with other hypoxic conditions, such as acute or cycling hypoxia. This could have firmly underlined chronic hypoxia-specific processes. In addition, the issue faced with the sequencing of two MIA PaCa-2 samples meant we only had two hypoxia samples for this cell line instead of four, as with the other cell lines. Another limitation is that by not carrying out RNA sequencing, we are only capturing a fraction of the altered transcriptome in hypoxia. Functional assays implicating the roles of the reported genes in the adaptation to hypoxia would be required to confirm their relevance to chronic hypoxia. Finally, without neoantigen load data it is difficult to make a complete conclusion on the effect of hypoxia on immunogenicity across the investigated cells.This study provides a global view on the reactivity to hypoxia of different tumor cells with varying inherent sensitivity to the same condition. The choice of focusing on the three highest and three lowest scoring cell lines was to ensure the presence of more than 2 cell lines per group to carry out proper comparative analysis. It would be interesting to see how the findings vary if all the scored cell lines are analyzed. This step would be necessary to determine the robustness of the signature in classifying the cells as HH and HL, and validate the association seen for higher TMB in HH compared to HL. Furthermore, clonality analysis would be necessary for distinguishing between the consequence of high passaging in hypoxia on clonal selection in vitro in hypoxia were frequently mutated in patients with more hypoxic tumors, compared to those with less hypoxic ones, shedding light on their potential relevance to this condition. A global response to chronic hypoxia could also be observed, wherein there was an increase in alternative splicing of both HIF-1-target and non-target genes across cells, and we could report a common signature composed of 16 genes, suggesting the presence of a core response to chronic hypoxia. This core response included well-acknowledged hypoxia-response genes, such as PGK1 and NDRG1, as well as much less investigated genes, including COA7 and the oxygen sensing gene, PAM, whose roles in chronic hypoxia deserve further attention. Finally, our results also hint at the role of chronic hypoxia in depressing tumor cells’ inherent immunogenicity through the downregulation of antigen processing and presentation related pathways. Therefore, chronic hypoxia could influence the adaptation of tumor cells, enabling their survival by promoting metabolic reprogramming, modulating proliferation, and increasing genomic instability.In conclusion we have shown that in chronic hypoxia cancer cells are reprogrammed to an indolent state, and yet they incur genomic instability in the form of high tumor mutational burden, mainly in the form of frameshift insertions, and copy number alterations. This could be due to the downregulation of pathways related to DNA damage response and repair, replication stress, as well as chromosomal maintenance. Of interest, cells that were ranked as being more sensitive to hypoxic stress accumulated a mean TMB that is significantly higher than the more resistant cells. They further experienced a higher rate of copy number loss. We also found that most genes altered


New chemotherapy agents are warranted for head and neck squamous cell carcinoma (HNSCC), particularly for incidence-rising HPV-positive tumors. Based on the evidence of Notch pathway involvement in cancer promotion and progression, we aimed to gain insights into the in vitro antineoplastic effects of gamma-secretase inhibition in HPV-positive and -negative HNSCC models.All in vitro experiments were conducted in two HPV-negative  and one HPV-associated HNSCC cell line (SCC154). The influence of the gamma-secretase inhibitor PF03084014 (PF) on proliferation, migration, colony forming, and apoptosis was assessed.We observed significant anti-proliferative, anti-migratory, anti-clonogenic, and pro-apoptotic effects in all three HNSCC cell lines. Furthermore, synergistic effects with concomitant radiation were observable in the proliferation assay. Interestingly, effects were slightly more potent in the HPV-positive cells.We provided novel insights into the potential therapeutic relevance of gamma-secretase inhibition in HNSCC cell lines in vitro. Therefore, PF may become a viable treatment option for patients with HNSCC, particularly for patients with HPV-induced malignancy. Indeed, further in vitro and in vivo experiments should be conducted to validate our results and decipher the mechanism behind the observed anti-neoplastic effects. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is associated with low survival rates . Due to HPV-related oncogenesis revolves primarily around the HPV-associated oncoproteins E6 and E7, which promote degradation of the tumor-suppressor genes TP53 and RB1 and modulate important cell cycle processes, resulting in tumor promotion and progression  resultinHPV-positive tumors differ from HPV-negative disease with regards to genetic alternations. For example, FGFR2 and FGFR3 mutations are uniquely found in HPV-positive tumors . Due to Cisplatin is the most commonly used systemic agent treatment option for advanced HNSCC. However, the response to therapy remains highly variable among patients. One important limitation is the cisplatin resistance, which often associates with termination of treatment in HNSCC patients . FurtherThe Notch pathway is involved in cell processes such as cell proliferation, differentiation, and survival. It operates by transmitting extracellular information, depending on the cell–cell contact interactions, and plays a role in cell cycle regulation. It is often upregulated in cancer, and many Notch signaling alterations have been linked to oncogenesis , 15. PreAs no substantial improvements in chemotherapy options for HNSCC in recent years, efforts need to be given to the identification of new therapeutic agents –13. TherAll experiments were conducted in three HNSCC cell lines, two HPV-negative  and one HPV-positive cell line  , which wIn order to determine the cytototoxic effects of PF and calculate its half-maximal inhibitory concentration (IC50) value of PF, we performed a dose–response assay in 96-well plates  for each cell line. In particular, 5000 cells/well were seeded in 100 μl DMEM. After 24 h (h) of incubation, cells were exposed to gradually increasing concentrations of the inhibitor. The inhibitor was reconstituted in dimethyl sulfoxide (DMSO)  and diluted in cell culture medium . Vehicle control was conducted using 0.1% DMSO. PF treatment was followed by irradiation with 2, 4, and 8 Gray (Gy) with the YXLON device . After 72 h, the medium containing the inhibitor/vehicle control was removed and replaced with 100 μl of 56 μM resazurin  solution. FaDu and Cal27 were then incubated for 90 min (min), while SCC154 were incubated for 180 min. Measurements were performed using the TECAN Spark reader .In order to assess the anti-migratory potential of the PF, we used 24-well plates  with Ibidi inserts  to create the initial gap. For this, 70,000 Cal27 and FaDu cells were seeded in Ibidi chambers  and 250,000 SCC154 cells/ibidi chamber in starvation medium (DMEM with 1% P/S and 1% FBS). Ibidi inserts were removed after reaching 100% cell confluency. Upon removal of the Ibidi inserts, wells were washed with DPBS and introduced with starving medium containing two different inhibitor concentrations , as well as the vehicle control (0.1% DMSO). Visualizations using the TECAN Spark plate reader were utilized after 0, 24, and 48 h. Calculations and analysis of the obtained images were performed with ImageJ.Furthemore, to determine a single-cell’s ability to form colonies after treatment, we performed a clonogenic assay. In particular, 1000 SCC154 cells/well and 350 cells/well for Cal27 and FaDu were seeded in 12-well plates  containing 1 mL of medium. Treatment was performed 24 h after seeding . After 72 h of treatment, cells were incubated for 10  and 14 days (FaDu). Subsequently, wells were washed with DPBS to remove cell debris, and the visualization was performed with the TECAN Spark plate reader.Caspase 3/7 Glo Assay  was conducted to assess the pro-apoptotic effects of the inhibitor. For Cal27 and FaDu, 7.500 cells/well and 15,000 SCC154 cells/well in 100 μL DMEM were seeded into 96-well plates . After 24 h, the medium was cells were treated. After 72 h, the medium was collected and mixed in a 1:1 ratio with Caspase 3/7 Glo reagent in three conditions: blank , tested sample , and negative control (media containing vehicle control + reagent and treated cells). We prepared the reagent according to the manufacturer’s instructions (Promega). Cells were incubated for 30 min and the luminescence was measured with the TECAN Spark plate reader.In order to compare differences in cell viability between different inhibitor concentrations, we conducted a two-way ANOVA analyses. On the other hand, differences in the migration, the colony formation, and the apoptosis assays were analyzed with a one-way ANOVA. For both, multiple comparison testings were performed on all possible pairwise means. We used GraphPad Prism  for graphical representation. All figures show mean values ± standard deviation (SD). Synergistic interactions between the inhibitor and radiation therapy were evaluated using the online tool Synergy Finder using a zero-interaction potency (ZIP) model.In order to assess the cytotoxicity of the PF in HNSCC, we performed the dose–response assay. For these, 5000 cells/well were seeded into 96-well plates with in 100 μl DMEM for each cell line. After 24 h of incubation, we treated cells with increasing concentrations of the inhibitor. We observed significant anti-proliferation effects of PF in all three cell lines (Fig. We utilized a wound healing assay to assess the effects of PF on the migratory potential of three HNSCC cell lines and graphically depicted these results in Fig. Next, in order to assess the colony forming abilities, we performed the clonogenic assay with 1000 SCC154 cells/well and 350 cells/well for Cal27 and FaDu seeded into 12-well plates. Cal27 and SCC154 cells were treated with three PF concentrations and a DMSO vehicle control. In total, strong anti-clonogenic properties of PF were shown in both cell lines. These results are shown in Fig. Finally, we assessed the pro-apoptotic potential of PF with the Caspase 3/7 Glo assay. For this, 7500 Cal27 and FaDu cells/well and 15,000 SCC154 cells/well were seeded into 96-well plates in 100 μL DMEM. All three cell lines were treated the corresponding IC50 PF concentration. Notably, the treatment induced apoptosis in all three cell lines. In particular, a significant increase was observed in FaDu and SCC154 cells. These results are depicted in Fig. The prognosis in patients with advanced HNSCC remained poor in recent years. Moreover, no major advancements regarding chemotherapy were made in the last decades. Furthermore, widely used cisplatin is associated with various adverse effects, often causing a transition to another agent or treatment termination. Finally, particularly treatment of HPV-positive HNSCC is set to become a major challenge in the coming decades, due to the aforementioned global rising incidence rates , 11–13. The data on targeting the Notch pathway via GSI is sparse in HNSCC. To the best of our knowledge, only one study reported on the effects of PF in HNSCC. In particular, Zhang et al. showed that PF in combination with erlotinib suppressed cancer growth . No furtGenerally, HPV-associated HNSCC patients have a more favorable prognosis than HPV-unrelated HNSCC. In particular, 5-year survival rates are reported as high as 80% in HPV-related HNSCC. Meanwhile, the 5-year survival rate is reported around only 50% in HPV-negative HNSCC . Still, Another broadly used treatment option in HNSCC is radiation therapy . TherefoBesides cytotoxic effects, the anti-neoplastic potential of PF was further demonstrated in a migration assay. In particular, treatment with PF significantly decreased the gap closure in all cell lines. These are, to the best of our knowledge, first insights into the anti-migratory potential of PF in HNSCC. With regards to the anti-clonogenic effects of PF, its IC50 concentration seemed sufficient to disrupt colony formation in SCC154. Correspondingly, treatment with PF had a similar anti-clonogenic potential in the Cal27 cell line. Disrupting the clonogenic abilities via Notch pathway inhibition has been observed in other studies as well. For example, Lan et al. showed adverse effects on cell growth and migration by Notch inhibition in embryonic microenvironment . FurtherLastly, we provided evidence on the pro-apoptotic properties of PF in HNSCC in vitro. In particular, slightly stronger pro-apoptotic effects were shown in the HPV-positive cell line. Certainly, the mechanistic background underlining our findings remains unclear and warrants further investigations. Interestingly, HPV infection has shown to have anti-apoptotic properties  and therIn total, our study provided novel results and we gained insights into the therapeutic potential of GSI in HNSCC. However, certain limitations need to be addressed. First, we conducted all experiments solely in 2D cell cultures and these do not completely reflect the native tumor microenvironment of HNSCC patient tumors. Furthermore, the mechanistic background behind the antitumor effects of PF was not analyzed and these could be deciphered with molecular biology experiments such as Western blotting. In particular, the direct downregulation of the Notch pathway by the GSI was not assessed. Most importantly, we assessed PF antitumor properties only in one HPV-positive cell line. Therefore, in order to be able to make definitive statements on PF effects in HPV-positive HNSCC, our findings need to be validated in further HPV-dependent cell lines.