Helleborus species have been found to be useful in various chronic diseases. This has brought Helleborus to the attention of medical researchers. H. purpurascens is a plant characteristic of the Carpathian area, known since ancient times for its beneficial effects. The aim of the study was to evaluate the flavonoids composition of a hydroalcoholic extract of H. purpurascens, as well as to assess its antioxidant activity and antitumor potential at the level of two healthy cell lines and four tumor cell lines. In addition, the expression of the genes involved in the apoptotic process  were evaluated. The results indicated that the extract has a high concentration of flavonoids, such as epicatechin, quercetin, and kaempferol. The extract has an increased antioxidant activity, very similar to that of the standard, ascorbic acid and cytotoxic effects predominantly in the breast cancer cell line, being free of cytotoxic effects in healthy cell lines. Underlying the cytotoxic effect is the induction of the process of apoptosis, which in the present study was highlighted by decreasing the expression of anti-apoptotic genes (Bcl-2) and increasing the expression of pro-apoptotic genes (Bad and Bax). In conclusion, the hydroalcoholic extract of H. purpurascens can be considered an important source for future medical applications in cancer therapy.In the field of oncology, the plant kingdom has an inexhaustible supply of bioactive compounds. Phytochemical compounds isolated from  The threat of cancer continues despite scientific advancements in the medical field. According to data provided by the World Health Organization, cancer was the leading cause of death globally in 2020. The main types of cancer diagnosed, except skin cancer, are breast, lung, and colorectal cancers . Skin caIn recent years, remarkable progress has been made in the field of oncology. Thus, the neoplastic process has been more closely studied and understood and, implicitly, the antitumor treatment has shown considerable improvements. Conventional cancer therapy consists of chemotherapy, radiation therapy, and surgery. However, the main disadvantage of classical antineoplastic therapy is the non-selective mode of action which results in the appearance of toxic reactions at the systemic level, as well as the low response rate to treatment . For thiHelleborus species represent a new treatment opportunity. The genus Helleborus is part of the Ranunculaceae family and includes over 20 different species with a predominant distribution in Europe and West Asia [Helleborus, namely, H. purpurascens and H. odorus. Although H. purpurascens has a high toxic potential, it has been used since ancient times to treat various diseases such as psychiatric and cardiovascular disorders or various pains [H. purpurascens include muscle relaxation and analgesic properties. However, the exact phytocomponents responsible for these biological activities are not known [H. purpurascens root extract caused an immunostimulatory effect in vivo by stimulating lymphocytes and neutrophils, [H. purpurascens, it was determined that this plant has a rich composition of natural compounds with biological effects, such as polyphenols, tannins, and glycosides [H. purpurascens extracts [Recent research in the field of phytocompounds has shown that est Asia . Numerouest Asia . In the us pains . Multifaot known . Additiorophils, . Followiycosides ,10. In lextracts . Regardiextracts . One of the modern directions of antitumor therapy is based on the elimination of cancer cells through the process of apoptosis . ApoptosH. purpurascens (HPex) in terms of the composition of polyphenols. In addition, the antioxidant activity of the extract was determined and the potential cytotoxic effect were assessed on four different cancer cell lines: squamous carcinoma—A431; murine melanoma—B164A5; and breast cancer—MCF-7 and MDA-MB-231. For a complete evaluation of the cytotoxic effect, two healthy cells line were selected: human keratinocyte cell lines (HaCaT) and murine epidermal cells (JB6). Finally, the expression of the main genes involved in the apoptosis process was determined, thus providing a possible explanation for the cytotoxic effect of H. purpurascens extract.For this reason, the present study focused primarily on evaluating the composition of a hydroalcoholic extract of The extract obtained was subjected to LC-MS quantification and in The  TAOxA of the four different extract concentrations was evaluated by DPPH radical scavenging assay. Ascorbic acid, positive control, exerts a TAOxA of 97.79% as can be observed in Cell viability was determined by the Alamar blue method and expressed as a percentage of viable cells compared to control cells.According to To determine the in vitro cytotoxicity effect, four tumor cell lines were chosen: MCF-7, MDA-MB-231, B164A5 and A43, at which the five concentrations previously tested on healthy cell lines were evaluated.In At the first four concentrations tested, the viability of the murine melanoma cells, B164A5, showed a slight decrease, being around 89%. However, at the highest concentration, cell viability dropped to about 68%. A plateau value of cell viability was also recorded in the case of squamous cell carcinoma, A431, where at the lowest concentration tested cell viability was similar to that of control cells, and at concentrations of 250, 500, and 1000 µg/mL, the values of cell viability were similar, approximately 85%, 84%, and 83%, respectively .Following the results obtained in the cell viability test, it was observed that HPex causes a marked decrease in the cell viability of breast cancer cells—MCF-7. In order to provide a more detailed picture of the mode of action of HPex at the level of this cell line, it was decided to determine the expression of the genes involved in the apoptosis process, namely: Bax and Bad—pro-apoptotic genes; Bcl-2—anti-apoptotic gene. H. purpurascens is a widespread plant in Eastern Europe, especially in the Carpathian Mountains. Although the plant has a high toxic potential, it is used in traditional medicine for a wide range of pathologies [Helleborus species are currently being considered a promising antitumor therapy [Rannuculaceae family, including the species of H. purpurascens, they were studied for their content rich in active compounds and for antitumor activity [H. purpurascens has been studied for its antitumor effect in vitro, but the biological mechanisms associated with this therapeutic activity have not been fully elucidated [hologies . Due to  therapy . Regardiactivity . In addiucidated .H. purpurascens. This therapeutic effect has been studied in conjunction with the evaluation of phytocomposite content, as well as with the evaluation of the beneficial antioxidant effect in antitumor therapy.Based on existing scientific considerations, the present study aimed to provide innovative information on the potential antitumor effect of a hydroalcoholic extract of To elucidate the cytotoxic effect of HPex, four tumor cell lines were chosen, two of breast cancer (MCF-7 and MDA-MB-231) and two of skin cancer (B164A5 and A431). Additionally, the effects exerted by HPex were studied in two healthy cell lines (HaCaT and JB6), to provide a more accurate picture of the cytotoxic effect. Cell viability was determined using the Alamar blue method, after 24 h of stimulation. Results of the study indicated that HPex has a selective cytotoxic effect, meaning healthy cells are not affected by its use. In contrast, in tumor cells there was a decrease in cell viability depending on the concentration tested. The most visible cytotoxic effects were observed in the breast cancer cell line (MCF-7), where cell viability decreased by up to 24%. The cytotoxic effects were also observed in the other cells studied, but the effect on cell viability was moderate. Helleborus species have been tested for their antitumor potential. In the preliminary study by Lindholm et al. [H. cyclophyllus extract showed antitumor effects, but the mechanism of action has not been fully elucidated. Another example is represented by H. caucasicus which has proven its cytotoxic effects on various tumor cell lines such as lung cancer (A549) or colorectal cancer (DLD-1) [H. niger on several tumor cell lines, including melanoma and breast cancer. The results revealed that H. niger has a concentration-dependent cytotoxic and antiproliferative effect on all cell lines used [H. niger extract on the melanoma cell line due to its ability to induce cell apoptosis [H. odorus, H. multifidus, and H. hercegovinus have been tested in vitro on Burkitt’s lymphoma B cells (BJAB). The results revealed that the strongest antiproliferative effect was observed in H. multifidus [H. purpurascens species, previous studies have shown that the alcoholic extract has an antiproliferative effect on the tumor cell line of cervical cancer (HeLa), causing a decrease in the number of mitoses [H. purpurascens, an aqueous extract and a hydroalcoholic extract. They determined that both extracts have cytotoxic effects on the HeLa tumor cell line, but the hydroalcoholic extract has a significantly more intense activity than the aqueous extract [H. purpurascens. Many m et al. , more th (DLD-1) . A similnes used . In addipoptosis . Other sltifidus . Regardi mitoses . Voichit extract . Thus, tH. niger on the effect on genes involved in apoptosis, mainly the Bcl-2 gene, noting that it induces Bcl-2-dependent cellular apoptosis [H. cyclophyllus, Yfanti and collaborators determined that it has pronounced cytotoxic effects in the lung adenocarcinoma cell line, A549. In addition, the group of researchers observed that H. cyclophyllus extract induces morphological changes characteristic of the apoptosis process, and in addition, causes a decrease in procaspase-3 levels and cleavage of PARP1 [The B-cell CLL/lymphoma 2 (Bcl-2) family plays a major role in the process of mitochondrial-mediated apoptosis in breast cancer. This family of proteins is divided into two categories: anti-apoptotic members such as Bcl-2 and pro-apoptotic members such as Bax and Bad . A featupoptosis . Regardiof PARP1 .H. purpurascens, the studies showed a correlation between its polyphenol content and the antitumor effect exerted at the level of different tumor cell lines [H. purpurascens extract, namely epicatechin, rosmarinic acid, quercetin, and kaempferol, have also been studied for their potentially beneficial role in the treatment of cancer. Epicatechin has previously been studied for its effect in the treatment of triple negative breast cancer using a murine model. It has been found to have strong antitumor activity in combination with doxycycline, inhibiting cell proliferation by modulating adenosine monophosphate-activated protein kinase-mediated pathways [p53 gene, known as a tumor suppressor protein [p53 gene, quercetin has found its utility in the treatment of skin cancer. In addition to this mechanism, quercetin also increases the activity of the tyrosine kinase enzyme, thus causing the antioxidant effect [As a means of better understanding the cytotoxic effect, this study used the LC-MS method to quantitatively determine the phenolic composition of the HP extract. Thus, it was found that the hydroalcoholic extract has a high content of epicatechin, rosmarinic acid, quercetin, and kaempferol, the latter having the highest concentration (approximately 68 µg/g). A wide range of evidence suggested that polyphenolic compounds could be therapeutic in diseases such as cardiovascular disease ,29,30 anpathways . Similarpathways . An in-dpathways . Rosmaripathways . The ben protein . Additiot effect . Kaempfet effect . H. purpurascens has many phytoconstants that underlie its antitumor activity. Thus, Franz et al. discussed the composition of aqueous and organic extracts of H. purpurascens. Thus, they identified the fact that in terms of aqueous extract, it has a high amount of amino acids, especially asparagine [H. purpurascens extract in more detail, providing information on its rich composition in carbohydrates, glycosides, saponins, and tannins [Helleborus species is hellebrin, which is found in the largest amount in the species of H. purpurascens [H. caucasicus, studies have shown that these natural compounds are involved in reducing the viability of tumor cells by inducing apoptosis due to decreased expression of the GRP78 gene [H. niger, it was determined that due to the composition of bufadienolide and ecdysteroid, the extract has antitumor capacity by inducing apoptosis [However, in addition to its rich flavonoid composition, studies have shown that paragine . Similar tannins . Probablurascens . Studiesurascens , pancreaurascens , breast urascens , and othurascens . In addiurascens , but alsurascens . RegardiP78 gene . Similarpoptosis .As for the MCF-7 breast cell line, it differs from the other MDA-MB-231 breast cancer cell line, mainly due to the fact that MCF-7 is hormone dependent, having positive estrogen and progesterone receptors, while MDA-MB-231 is triple negative. For this reason, MDA-MB-231 does not respond to antiestrogen therapy . Over tiH. purpurascens. A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was applied to evaluate the antioxidant activity of four extract concentrations . The results obtained were related to the antioxidant activity exerted by ascorbic acid and considered 100%. Consequently, the antioxidant activity was higher than 50% in all concentrations, while the most intense antioxidant activity was recorded at the concentration of 100 µg/mL, approximately 78%. Its remarkable antioxidant properties are due to the compounds that were found in H. purpurascens extract, which have been associated with the neutralization of oxygen-free species [H. purpurascens extracts, one aqueous and one alcoholic, were tested. The obtained results showed that the alcoholic extract has a higher antioxidant activity than the aqueous one, with a value of 78% [Helleborus species. Thus, Öztürk et al. evaluated the flavonoid content and antioxidant activity of an H. orientalis extract. They determined that the extract has a significant antioxidant activity, observing an increase in the antioxidant activity and the protective effect against oxidative alterations with increasing concentration of the tested extract [H. vesicarius. In this study it was determined that this species has an intense antioxidant activity as well as an increased oxidizing potential [Helleborus, H. odorus, H. multifidus, and H. hercegovinus. In this study, it was determined that the extract made from the leaves has a more intense antioxidant activity than that obtained from the root [In light of the fact that flavonoids are antioxidant compounds  and that species . The ant species . Similare of 78% . These r extract . Mohammeotential . Čakar ethe root .Helleborus purpurescens) used in this study, was harvested from Timis County, Western Romania, and certified at Pharmaceutical Botany Department (voucher herbarium specimen no. HP/S12/2020), Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy. The plant was prepared for extraction according to European Pharmacopoeia (Ph. Eur.) 10th Edition.The plant material (Helleborus purpurescens extract (HPex) the maceration method was used. Plant material (5 g), crushed and homogenized, was placed in the flask and 25 mL of ethanol (EtOH) 70% were used to sequentially extraction for seven days. The final extract was filtered through filter paper, the solvent was removed by a rotary evaporator (Heidolph Hei-VAP Advantage Rotary Evaporator package) under vacuum, the pellet being lyophilized and stored in a dark glass tube at 2–8 °C until further analysis.To obtain The hydro-alcoholic hellebore extract, was subjected to a LC-MS analysis, and the following steps were accomplished: (i) extract homogenization  and centrifugation for 120 s at 10,000 rpm ; (ii) LC-MS analysis on a Shimadzu chromatograph ; (iii) chromatographic conditions, extraction time 60 min, room temperature, EC 150/2 NUCLEODUR C18 Gravity SB 150 mm × 2.0 mm × 5 μm column , flow rate 0.2 mL/min, two mobile phases , wavelengths 280 and 320 nm; (iv) gradient elution—0–20 min 5% B, 20–50 min 40% B, and 50–60 min 95% B; (v) calibration curves between 10 and 50 µg/mL and limit of quantification 0.3 µg/mL.−4 M) was prepared in ethanol and 103 µL was added to 3 × 103 µL of hydro-alcoholic HP extracts, at four different concentrations: 10, 25, 50, and 100 μg/mL; (ii) the absorbance was measured continuously for 15 min at 516 nm T70 UV/VIS Spectrophotometer, PG Instruments Ltd., UK); (iii) ascorbic acid was used as positive control, while distilled water as negative control. The final TAOxA was presented as percentage obtained after applying the formula presented in our previous studies [DPPH  free-radical scavenging assay was selected to evaluate the total antioxidant activity (TAOxA) of hydro-alcoholic HP extract, according to the method described in the literature ,77. Brie studies ,77.®, Manassas, VA, USA, CRL-1555™); murine melanoma cell line—B164A5 ; breast cancer cell lines—MCF-7  and MDA-MB-231 . Healthy cell lines were represented by human keratinocyte cell lines—HaCaT ; murine epidermal cells—JB6Cl41-5a . HaCaT, B164A5, A431, and MDA-MB-231 were cultured in the specific culture medium represented by Dulbecco’s Modified Eagle’s Medium high glucose in which were added 10% fetal bovine serum  and 1% penicillin/streptomycin solution . JB6Cl41-5a and MCF-7 were cultured in Eagle’s Minimum Essential Medium, which was supplemented with 5% fetal bovine serum  and 0.1% non-essential amino acids for JB6Cl41-5a and 10% fetal bovine serum  for MCF-7. To prevent microbial contamination, 1% penicillin/streptomycin solution mixture  was added. During the experiments, the cells were maintained in constant conditions of temperature and humidity (5% CO2 and 37 °C). Countess II Automated Cell Counter , was used to determine the number of cells in the presence of Trypan blue. The tumor cell lines selected in the present study were squamous cell carcinoma cell line—A431 . The treated cells were incubated for 24 h. After this interval, cell viability was determined using the Alamar Blue method using the protocol described above [In order to determine the cell viability following the treatment with the hydroalcoholic extract of ed above . Thus, aed above .6 cells/well. After reaching a confluence of approximately 90%, the cells were stimulated with the sub-cytotoxic concentration of HPex (100 µg/mL) for a period of 24 h. After this time, RNA was isolated using Trizol reagent and the Quick-RNA ™ purification kit and its amount was determined using a DS-11 spectrophotometer . Finally, RNA transcription was completed using Maxima® First Strand cDNA Synthesis Kit , and quantitative real-time PCR analysis was performed using Quant Studio 5 real-time PCR system  in the presence of Power SYBR-Green PCR Master Mix.Given that, following the cell viability test, the most affected cell line was breast cancer MCF-7, it was decided that the influence of HPex on gene expression should be established by applying the reverse transcription–polymerase chain reaction (RT-PCR) method to this cell line. To determine the expression of the Bax, Bcl-2  and Bad , the cells were cultured in 6-well plates in a number of 1 × 10H. purpurascens, a medicinal plant with therapeutic effects applied in traditional medicine since ancient times. LC-MS analysis revealed that the extract contains high levels of flavonoids, especially quercetin, kaempferol, and epicatechin. First of all, the hydroalcoholic extract exerted a strong antioxidant effect, close to that of ascorbic acid, mainly at a concentration of 100 µg/mL. Further, starting from the beneficial role played by antioxidants in antitumor therapy, the hydroalcoholic extract proved its selective cytotoxic effects in the four tumor cell lines used, the proliferation of healthy cells not being affected by stimulation with H. purpurascens extract. Finally, the influence of HPex on increasing the expression of pro-apoptotic genes (Bad and Bax) and decreasing the expression of anti-apoptotic genes (Bcl-2) was highlighted in the breast cancer cell line—MCF-7. In conclusion, the extract has an important therapeutic value for the future of oncological therapy, but, nevertheless, additional studies are needed to unravel the mechanism of action and identify the key phytoconstituents which explain these biological effects.The present study was designed to determine the chemical composition and biological properties of a hydroalcoholic extract of


Stable cell lines derived from primary tissues and tumors are widely used in medical research. This study presents interesting findings from evaluation of publicly available gene expression profiles (GEPs) of primary tissues derived from the normal esophagus, Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), as well as available esophageal cell lines. We observed that the GEPs of currently available BE cell lines deviate from the GEPs of primary BE tissues (columnar) and EAC tumors (glandular), and are unexpectedly similar to the GEPs of normal esophageal mucosa and ESCC tumors. In vitro exposure to an acid and bile environment was not sufficient to reverse this “squamous like” GEP adopted by a BE cell line, BAR-T. This incomprehensible change in the GEP may result in ambiguous changes in the phenotype of the BE cell lines, and needs careful consideration during experimental design.hi) had considerably lower expression in EAC cell lines than EAC tumors. However, more surprisingly, unlike various normal cell lines  which were very similar to various tissues from normal esophagus, BE cell lines  were extremely different from primary BE tissues, as BE cell lines had substantially lower levels of EAChi and substantially higher levels of ESCChi gene expression. This ESCC-like profile of the BAR-T remained unaltered even after prolonged exposure to an acidic bile mixture in vitro resulting in malignant transformation of this cell line. However, primary BE tissues had EAC-like gene expression profiles as expected. Only one EAC case from the Cancer Genome Atlas resembled BE cell lines, and while it had the clinical profile and some mutational features of EAC, it had some mutational features, the copy number alteration profile, and the gene expression profile of ESCC instead. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrants careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.Esophageal adenocarcinoma (EAC) is strongly associated with Barrett’s esophagus (BE), a pre-malignant condition resulting from gastric reflux. Esophageal squamous cell carcinoma (ESCC), the other major subtype of esophageal cancer, shows strong association with smoking and alcohol intake and no association with gastric reflux. In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors using publicly available datasets, and subsequently assessed the enrichment levels of these signatures in commonly used EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors but still more similar to EAC tumors than ESCC tumors, as the genes up in EAC vs. ESCC (EAC Esophageal cancer has two distinct histologic subtypes  with difDue to asymptomatic disease progression in esophageal cancers, the diagnosis is delayed until later stages of disease, leading to increased mortality and only 20% 5-year survival after diagnosis . Therefohi) show substantially lower expression in BE cell lines than primary BE tissues, but also the genes up in ESCC vs. EAC (ESCChi) had substantially higher expression in BE cell lines than primary BE tissues. We validated this “squamous like” GEP of BE cell lines in a previously published in vitro model of BE carcinogenesis (BEC) [In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors, and subsequently quantified the enrichment of these signatures in the established EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors. The normal cell lines  were very similar to various tissues from normal esophagus, but the BE cell lines  were extremely different from primary BE tissues. This was because not only did the genes up in EAC vs. ESCC (EACis (BEC) ,21,22,23is (BEC) , chromosis (BEC) , and malis (BEC)  of a benis (BEC)  but did is (BEC)  resemblehttps://gdc.cancer.gov/about-data/publications/pancanatlas) (accessed on 1 March 2020). The list of EAC (n = 72) and ESCC (n = 91) tumors and the contributions of COSMIC17 and APOBEC mutation signatures [Gene expression data of tumor and adjacent normal samples were obtained from the TCGA Pan-Cancer Atlas website , a list of ESCC cell lines was obtained from https://depmap.org/portal/context/esophagus_squamous (accessed on 4 April 2021), and gene expression data of these cell lines were obtained from https://www.cbioportal.org/study/summary?id=ccle_broad_2019 (accessed on 17 June 2021). For the Genotype-Tissue Expression (GTEx) dataset [https://www.gtexportal.org/home/datasets (accessed on 17 June 2021). Gene expression data of 13 datasets were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=) (accessed on 17 June 2021): GSE2144 [https://www.ebi.ac.uk/gxa/experiments/E-MTAB-2706/Downloads) (accessed on 17 June 2021), E-MTAB-3983 (https://www.ebi.ac.uk/gxa/experiments/E-MTAB-3983/Downloads) (accessed on 17 June 2021), and E-MTAB-4054 [https://www.ebi.ac.uk/gxa/experiments/E-MTAB-4054/Downloads) (accessed on 17 June 2021).For the Cancer Cell Line Encyclopedia (CCLE) dataset , a list  dataset , gene ex GSE2144 , GSE9768 GSE2144 , GSE1337 GSE2144 , GSE3720 GSE2144 ,33,34,35 GSE2144 , GSE5896 GSE2144 , GSE1300 GSE2144 , GSE1652 GSE2144 , GSE1731 GSE2144 , and GSE GSE2144 . Gene ex GSE2144   tables from GEO, and subsequently matched with Entrez IDs from the TCGA pan-cancer atlas. For genes with multiple probes, the probe with highest mean of log2 transformed expression was chosen.https://www.ensembl.org/biomart/martview/) (accessed on 17 June 2021). In datasets where Ensembl IDs were available, they were converted to Entrez IDs using Ensembl BioMart, and subsequently matched with Entrez IDs from the TCGA pan-cancer atlas. In a dataset where RefSeq IDs were available instead, they were first converted to Ensembl IDs using Ensembl BioMart, and then processed as described above. In datasets where only gene symbols were available, they were first matched with gene symbols from the TCGA pan-cancer atlas, and then corresponding Entrez IDs were used as input in Ensembl BioMart to retrieve gene length (if necessary).For RNA-sequencing datasets, if normalized data was unavailable, then read count data was converted to RPKM using gene lengths from Ensembl BioMart (hi) were included in the EAC signature (hi) were included in the ESCC signature (hi) were included in a longer EAC signature (hi) were included in a longer ESCC signature (hi and ESCChi genes (100 and 500) in each sample of each dataset. A two-sided Wilcoxon ranksum test was used to compare the enrichment levels in different types of samples, and statistical significance was assessed at p < 0.05.A total of 9485 genes were common between the 22 datasets analyzed in this study; therefore, all datasets were restricted to these 9485 genes. To create gene expression signatures of EAC and ESCC, differential expression analysis was performed on the rank-normalized TCGA dataset. A two-sided Wilcoxon ranksum test was used for pairwise comparison, and the Benjamini–Hochberg method was used for multiple hypothesis testing correction. The genes that differed a full quartile  between EAC and ESCC were sorted by false discovery rate, and the top 100 genes up in EAC  and ESCC (n = 91) cases from TCGA (hi) (shown in red in hi)  and ESCC cases  from TCGA, and samples from other datasets .By comparing EAC  from the ESCC cases (green) in TCGA , except for one EAC case (see result section below). Thus, the gene expression signatures of EAC and ESCC passed the consistency check.Enrichment levels of the 100 EACn = 23) from GSE130078 [n = 17) [n = 15) [n = 51) [n = 77) [To validate these gene expression signatures, we tested ESCC cases    ,33,34,35[n = 51)     (accessed on 29 July 2021). As a representative example, the ESCC cell line OE21 from GSE57130 [hi genes compared with ESCC cases from TCGA, they are quite similar to primary ESCC tumors and therefore ESCC-like.First, we investigated ESCC cell lines A–D and EGSE57130   from GTEx  show tisWhile the normal esophageal cell line EPC2 from GSE173169  Figure G was ESChi and 500 ESCChi genes, respectively).Similar results were observed  for longhi genes and low expression of EAChi genes (or vice versa).It should be noted that the classification of normal mucosa and EPC2 as ESCC-like does not mean they are tumor-like, it means they are “squamous like” instead of “columnar/glandular like”. Since we are plotting the enrichment levels of genes differentially expressed between EAC and ESCC , normal samples can cluster with ESCC (or EAC) tumors from TCGA if they have high expression of ESCCPrimary BE tissues with specialized intestinal metaplasia from GSE58963 A, non-dyhi genes than primary BE tissues , but also a substantially higher enrichment level of the 100 ESCChi genes than primary BE tissues (which is not true for EAC cell lines vs. EAC tumors).Unlike primary BE tissues, the BE cell line CP-A from GSE62909  Figure G and GSEhi and 500 ESCChi genes, respectively).Similar results were observed  for longThe notable observation that (unlike primary BE tissues) BE cell lines are ESCC-like (instead of EAC-like) was subsequently validated in a previously published in vitro BEC model ,21,22,23hi genes.We sequenced 10 samples with various levels of ABS exposure obtained from the BEC model  and 8 ABS unexposed samples in total at 3 different centers, and the three RNA-sequencing datasets  confirmehi and 500 ESCChi genes  but were low in expression in the BE cell lines CP-A and BAR-T , while most of the ESCChi genes showed the opposite trend (hi genes and low expression of ESCChi genes (except for SK-GT-4 which had high expression of some ESCChi genes) and may be considered EAC-like. The majority of EAChi and ESCChi genes were low in expression in normal muscularis, GEJ, and Het-1A, which are neither EAC-like nor ESCC-like  clustered with/near BE cell lines B–D, so w2 transformed gene expression data clustered this case with ESCC cases instead of other EAC cases  had a high contribution from COSMIC17 mutation signature , even hiThe mechanism of CDKN2A loss in this case—oncogenic mutation accompanied by heterozygous deletion—is consistent with the EAC diagnosis, as the mechanism of CDKN2A loss in ESCC is homozygous deletion D. HoweveThus, the only EAC case in TCGA that had a GEP similar to BE cell lines was an unusual case that had features of both EAC and ESCC: the clinical profile of EAC, and some mutational features typical of EAC, but the copy number alteration profile and the gene expression profile of ESCC, and some mutational feature typical of ESCC.hi and ESCChi genes that could reliably discriminate between primary EAC and ESCC tumors, this study found that unlike the normal cell line EPC2 that had a GEP similar to normal mucosa, the GEPs of representative BE cell lines (BAR-T and CP-A) showed notable deviation from the expected primary BE/EAC-like profile to ESCC-like profile , ESCC-like (low enrichment of EAChi genes and high enrichment of ESCChi genes), or neither (low enrichment of both EAChi and ESCChi genes) based on the top 100 genes selected from each histological type. Similar results were observed for the top 500 genes which confirmed that the similarities or differences observed are global with respect to gene expression and may be associated with the tissue/cells of origin (squamous or glandular epithelium), particularly in case of the primary tumors, and normal and primary BE tissues. However, this was not true for BE cell lines: while EAC cell lines had considerably lower enrichment of EAChi genes compared with EAC tumors  if RNA-sequencing data of those samples are available. While a set of 100 EAChi genes  were usehi genes B. Howevehi and ESCChi genes may be useful in selecting appropriate cell lines for an experiment. Our results suggest that EPC2 may be suitable for studying normal mucosa, while Het-1A may be more suitable for experiments studying normal muscularis or GEJ. Similarly, while some EAC cell lines such as KYAE1 and ESO26 are very similar to EAC tumors, certain EAC cell lines such as OACM51, FLO1, and SK-GT-4 may not be suitable for studying EAC tumors. While most ESCC cell lines are quite similar to ESCC tumors, certain ESCC cell lines such as OE19 and TE-4 may not be suitable for studying ESCC tumors. Commonly used BE cell lines  were extremely different from primary BE tissues and the only EAC case from TCGA (TCGA-IN-A7NT) that was comparable to BE cell lines was an unusual case that had features of both EAC and ESCC  (accessed on 4 April 2021), clustered with EAC tumors from TCGA. This cell line is indeed labeled as EAC cell line in Cellosaurus (https://web.expasy.org/cellosaurus/) (accessed on 29 July 2021), suggesting the possibility that it may be an EAC cell line accidentally misclassified as an ESCC cell line, and warrants further investigation.Another potential utility of our method could be identification of misclassified EAC and ESCC cases, which may have clinical relevance. While validating the gene expression signatures of EAC and ESCC, we observed that a small number of EAC and ESCC cases clustered with ESCC and EAC tumors from TCGA, respectively. While some of these may be rare cases of EAC that resemble ESCC  or vice-versa, most of these are likely ESCC and EAC cases, respectively, that were accidentally misclassified. A similar example was observed in case of cell lines, where OE19, a cell line labeled as ESCC cell line in DepMap (This study points to notable changes in the gene expression profiles of established Barrett’s esophagus cell lines that deviate significantly from primary Barrett’s esophagus tissues thus making them more “squamous like”. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrant careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.


Photobacterium phosphereum T3) and ryegrass (Lolium perenne L.). Compared with Fe-EDTA, the transfer factors of nZVI and B-nZVI were 65.0% and 66.4% lower, respectively. This indicated that although iron nanoparticles accumulated in the roots of ryegrass, they were difficult to be transported to the shoots. The results of this study indicate that B-nZVI has a strong application potential in in situ environmental remediation.The aggregation of nanoscale zero-valent iron (nZVI) particles and their limited transport ability in environmental media hinder their application in environmental remediation. In this study, the Cr(VI) removal efficiency, transport performance, and toxicity of nZVI and bentonite-modified nZVI (B-nZVI) were investigated. Compared with nZVI, B-nZVI improved the removal efficiency of Cr(VI) by 10%, and also significantly increased the transport in quartz sand and soil. Increasing the flow rate can enhance the transport of nZVI and B-nZVI in the quartz sand columns. The transport of the two materials in different soils was negatively correlated with the clay composition. Besides, modification of nZVI by bentonite could reduce toxicity to luminous bacteria ( Nanoscale zerovalent iron (nZVI) is an engineered nanoparticle that is increasingly used in environmental remediation ,2. Due t−1) of nZVI inhibited the rice seedlings growth and inhibited active iron transportation in rice plants [2) to form reactive oxygen species (ROS) [In addition to agglomerate, the potential hazards of nZVI have also raised great concern . As an ee plants . In the e plants ,34,35. Ae plants . Iron ioe plants . Moreovees (ROS) . If excees (ROS) . TherefoThe main aims of this study are: 1) To synthesize nZVI and bentonite modified nZVI (B-nZVI) and investigate the Cr(VI) removal efficiency of these two materials; 2) to investigate the mobility of nZVI and B-nZVI in quartz sand and soil columns; 3) to determine their toxicity toward bacteria and plants. Results of this study could provide a theoretical basis for the application of iron-based nanomaterials in environmental remediation.3∙6H2O), bentonite (Al2O3·4(SiO2)·H2O), anhydrous ethanol, potassium borohydride (KBH4), polyvinylpyrrolidone , and potassium dichromate (K2Cr2O7) were purchased from Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China.Ferric chloride hexahydrate  were dissolved in 100 mL ethanol–water solution (30% V/V) in a three-necked flask. After mechanical stirring under N2 environment for 10 min, 100 mL 0.2 M KBH4 (dissolved in 1% NaOH) was added dropwise by a constant pressure separation funnel. Ferric ions were reduced to zero-valent iron by the following reaction:Nanoscale zero-valent iron (nZVI) and bentonite-supported nZVI (B-nZVI) were prepared through a modified chemical reduction ,39. Brie2 environment for 30 min, the products were washed with anhydrous ethanol for three times. The products were dried under vacuum and sealed storage. The preparation of nZVI used the same procedure, but without bentonite.After mechanical stirring under NThe morphology and elementary composition of nZVI and B-nZVI was characterized by scanning electron microscope  and transmission electron microscopy . Gas absorption operation was carried out for the determination of particles’ specific surface area in a surface area analyzer . X-ray diffraction (XRD) was performed in a X-ray diffractometer  with Cu Kα radiation (λ = 0.154 nm). The scan range was set from 10° to 90° at 40 kV/40 mA. X-ray photoelectron spectroscopy was operated on a X-ray photoelectron spectrometer  with Al Kα radiation (hv = 1486.6 eV) at 12 kV/6 mA.−1 Cr(VI) was prepared by dissolving desired amount of K2Cr2O7, and the initial pH of the solution was adjusted by 0.2 M NaOH or HCl. Cr(VI) removal experiments were performed using conical flasks (500 mL) with an oscillation frequency of 200 rpm. The removal efficiency of Cr(VI)  by nZVI and B-nZVI  was investigated at different initial pH . After 2, 5, 10, 20, 30, 60, 120, 240, 360, 960, and 1440 min of reaction, 2 mL solution was sampled and filtered through a 0.22 μm membrane filter. The concentrations of Cr(VI) in the solutions were determined using the diphenylcarbohydrazide method [A solution with 300 mg L−1, and the background solution was NaCl solution prepared with anaerobic water . The column was first injected with four pore volumes (PVs) background solution in up-flow mode by a peristaltic pump to ensure that the column reaches water saturation and a stable flow state. Subsequently, 4 PV nZVI or B-nZVI suspension and 4 PV background solution were pumped into the column. The suspensions were continuously sonicated to avoid the sedimentation of nZVI and B-nZVI. The effluent was mixed with hydrochloric acid in a ratio of 1:1 with a water bath at 90 °C for 1 h. The total Fe content in the solution was determined by flame atomic absorption .The column used for column transport experiments was a cylindrical plexiglass column with a length of 10 cm and an inner diameter of 1.3 cm. The column transport experiments were carried out in two media: quartz sand (20–30 mesh) and soils . The conPhotobacterium phosphereum T3) was purchased from Institute of Soil Science, Chinese Academy of Sciences . The freeze-dried luminous bacteria were mixed with 1 mL 2% NaCl solution in an ice bath for 2 min. Subsequently, 10 μL of the luminous bacteria solution was added into 2 mL sample solution (prepared with 3% NaCl). After being incubated at 20 °C for 15 min, the bioluminescence intensity was measured by DXY-2 microtox test instrument . Each sample was determined at least three times. HgCl2 was used as a reference in this test, and the relative fluorescence intensity had a linear relationship with the concentration of HgCl2  [−1) was evaluated in addition to the nZVI and B-nZVI treatments.The luminous bacteria (ctively) . The toxLolium perenne L.) seeds were surface sterilized in 3% hydrogen peroxide solution for 30 min and thoroughly washed with deionized water. Then the ryegrass seeds were transferred to moist gauze and incubated in darkness at 28 °C for 6 days. The 6-day-old seedlings were transferred to a hydroponic solution with 1/3 strength heavy elements (1 mM Ca(NO3)2, 0.5 mM Ca(H2PO4)2, 0.5 mM K2SO4, 1 mM MgSO4, and 1.5 mM NH4NO3) and full strength trace elements . The hydroponic solution was adjusted to pH 5.8. Different doses of nZVI and B-nZVI  were added into the hydroponic solution for the treatment groups. Control  and ionic treatments using Fe-EDTA  were prepared in addition to the nZVI and B-nZVI treatments; three parallels for each treatment, and 24 seedlings for each parallel. The plant culture was performed in a growth chamber at 25 °C for 16 h in the day and at 20 °C for 8 h in the night with 50–60% relative humidity. The hydroponic solution was replaced every 3 days.Selected uniform perennial ryegrass . Fresh plant samples were mixed with 4 °C phosphate buffer (PBS), homogenized in a glass homogenizer. The homogenate was centrifuged at 4 °C, and the supernatant was separated for the determination. SOD activity assay kit  and catalase assay kit  were used for enzyme activity assay. Lipid peroxidation in plants was determined by MDA content by 2-thiobarbituric acid (TBA) reaction .After 21 days of growth, ryegrass was harvested and washed with deionized water. Then shoots and roots were separated, and their dry weights were measured. The contents of Fe in plants were determined by ICP-MS  after being digested with HNO2/g) to 26.5 m2/g, and the adsorption/desorption average pore diameters of nZVI and B-nZVI were 16.6/6.1 nm and 16.2/8.4 nm, respectively. As observed from the XRD patterns of nZVI and B-nZVI  facet of bcc iron (JCPDS no. 06-0696) [θ of 19.8°, 35.8°, and 62.8° can correspond to montmorillonite (JCPDS no. 29-1498) [θ of 27.8° was observed in the pattern of B-nZVI, which is consistent with the characteristic peak of silicon oxide (JCPDS no. 82-1566), one of the main components of bentonite.The morphology and size of the newly prepared nZVI and B-nZVI were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) . The SEMd B-nZVI , a diffr06-0696) . No othe0 and Cr(VI) is a process of consuming hydrogen ions, and thus the increased concentration of hydrogen ions in the solution can promote the reaction to proceed to the positive direction [In order to investigate the Cr(VI) removal efficiency of nZVI and B-nZVI, the removal experiments of Cr(VI) were studied under different initial pH conditions . The results showed that for both nZVI and B-nZVI, the removal process of Cr(VI) was very fast . The remirection . In addiirection ,47.3/2 and Cr 2p1/2 photoelectron peaks at 576.8 eV and 586.5 eV represent that Cr(OH)3, Cr(OH)O, and Cr2O3 are the main species of Cr [2Cr2O7 [3/2 and Fe2p1/2 are 710.9 eV and 724.5 eV, respectively. This indicates that Fe in the product basically exists in the form of Fe(III), and the possible chemical structure is iron oxide hydrate (FeOH), magnetite (Fe3O4), and hematite (Fe2O3) [0 after the reaction, and no such photoelectron peak was found in the spectra of B-nZVI. Li et al. also found that Fe0 can be still detected in the nZVI after reacting with Cr(VI) for 24 h [0 more sufficient, thus Fe0 was completely oxidized to Fe(III) after the reaction.Further analysis of the reaction products by XPS showed that the reaction products were mainly composed of Fe, Cr, O, and C elements . The Cr es of Cr . The spies of Cr ,49. In a [2Cr2O7 . This ma (Fe2O3) . In addifor 24 h . This ca−1 to 3.0 cm min−1, the plateaus increase from 0.02 to 0.04. Previous studies have also found that increasing the flow rate can improve the mobility of nanoparticles, mainly because increasing the flow rate can generate stronger hydrodynamic forces to keep the colloids in the mobile phase [The transport behaviors of nZVI and B-nZVI were studied by column transport experiments. The breakthrough curves of two materials begin at 1 PV and reach their plateaus at about 1.5 PV . For nZVle phase ,55.−1, the breakthrough plateaus can reach 0.35, which is about eight times that of nZVI. The attachment of nZVI to bentonite decreased the magnetic attraction and created steric repulsion between nanoparticles, thus facilitating the transport of nZVI through quartz sand column [After modification with bentonite, the mobility of nZVI in quartz sand column has been greatly improved b. At thed column . Moreoved column . Similard column .The mobility of nZVI and B-nZVI in soil columns packing with three different soils was studied . Similar−1 of nZVI can reduce the relative bioluminescence intensity by 93.7%, which indicates that high concentration of nZVI is highly toxic to the luminous bacteria. Some studies reported that nZVI had no cytotoxicity toward microorganisms. For example, 1000–10,000 mg/L of nZVI had no effect on the survival and activity of Klebsiella planticola, and did not cause significant cell damage [Escherichia coli were inactivated with the treatment of 70 mg/L nZVI [Escherichia coli, Bacillus subtilis showed a stronger tolerance to nZVI, but the survival rate of Bacillus subtilis also decreased with the extension of nZVI treatment time [−1 treatments. However, the relative bioluminescence increased from 6.3% to 14.6% with the treatment of 500 mg L−1, indicating that the toxicity of B-nZVI was lower than nZVI. In the study reported by Qiu et al. [−1 nZVI can decrease the relative bioluminescence intensity of luminous bacteria by 49.3%, and the generated iron ions were considered to be the main source of toxicity. In order to figure out the effect of iron ions to luminous bacteria, Fe-EDTA treatments were prepared as ionic controls. Compared with nZVI and B-nZVI, the treatment of 50 mg L−1 Fe-EDTA had no significant effect on the relative bioluminescence intensity. The toxicity of Fe-EDTA to luminous bacteria was produced as the treated concentration was increased. However, compared with the same concentration of nanomaterials, its toxicity was significantly reduced. This suggests that the toxicity of nZVI and B-nZVI to luminous bacteria may not only come from the dissolved iron ions, but also from the nanoparticles themselves. Metal-based NPs have been found to induce perturbations on microbes not only by the dissolved ions, but also by the particles themselves [2−, and H2O2 [The toxicity of nZVI and B-nZVI was evaluated by luminous bacteria toxicity test . The rell damage . Howeverg/L nZVI . Compareent time . Surfaceent time . After tu et al. , the preemselves . Due to and H2O2 . The excand H2O2 .−1 of nanomaterials had no significant effects on the biomass of the plants  after 21 days of hydroponic culture. Although iron is one of the essential nutrients for plants, excessive iron concentration can inhibit plant growth [The phytotoxicity of nZVI and B-nZVI was investigated by hydroponic experiment of ryegrass. The results indicated that the addition of 50 and 200 mg Le plants . Howevert growth .−1 nanomaterials was 10.5 times and 5.6 times that of the control group, respectively. The Fe accumulation in the shoots also increased by 2–3 times compared with the control group, while the Fe content in the group treated with B-nZVI was relatively independent of the dosage of B-nZVI. Similarly, the total Fe content in the plants with the treatment of Fe-EDTA was higher than in the control group. However, compared with the same concentration of nanomaterials treatment groups, the Fe-EDTA treatment group had a lower Fe content in the roots. As a result, the translocation factor (Cshoot/Croot) of the Fe-EDTA treatment group was higher than that of the nanomaterials-treated groups  of nZVI has also been found to up-regulate the CAT activity and MDA content in rice plants [Overall, the treatments of iron-based NPs had greater effects on the roots of plants rather than the shoots. The results of antioxidative enzymes and lipid peroxidation correlated well with the results of biomass and Fe contents. Excessive iron accumulation can inhibit plants growth, cause oxidative stress, and stimulate plants to up-regulate antioxidative enzymes activity. Oxidative stress caused by iron-based NPs has been reported in other research. The exposure of 100 mg Ln plants . High doe plants .Photobacterium phosphereum T3) and perennial ryegrass (Lolium perenne L.). The transfer factors in nZVI and B-nZVI treatments were much lower than those in the Fe-EDTA treatments. This indicated that although iron nanoparticles accumulated in the roots of ryegrass, they were difficult to be transported to the shoots. The results show that bentonite is an ideal carrier, which can not only enhance the removal ability of nZVI to Cr(VI) and improve its migration performance in porous media, but also reduce its ecological toxicity. B-nZVI has a strong potential in the remediation of groundwater and soil polluted by Cr(VI).In this study, it was found that modification of nZVI by bentonite could improve the removal efficiency of Cr(VI) by 10%. The transport of nZVI was limited both in quartz sand and soil columns, and the transport performance can be improved by increasing the flow rate. Compared with nZVI, the transport of B-nZVI in quartz sand and different types of soil was increased by 8 times and 2–10 times, respectively. The transport of the two materials in different soils was positively correlated with the sand composition. Modification of nZVI by bentonite could reduce the toxicity to luminous bacteria (


Bothrops jararaca snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 μg/mL (low) or 2.5 μg/mL (high) of B. jararaca venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of B. jararaca venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with  Bothrops genus [Bothrops jararaca (B. jararaca) snake is a complex mixture composed of proteins, peptides, amino acids, nucleotides, lipids, and carbohydrates that present a range of different actions when they are isolated or together [Bothrops venoms [Bothrops venoms are composed of various classes of toxin, including metalloproteinases, serine proteinases, phospholipases A2, and C-type lectins, the most abundant components participating in the local and systemic envenomation effects.Ophidian accidents constitute a serious public health problem in Brazil, with an average of 29,000 cases and 125 deaths reported every year  [together ,4,5,6,7,together ,9. Severs venoms ,14,15,16B. jararaca engenders three main activities: proteolytic, coagulant, and hemolytic. The proteolytic activity causes degradation of extracellular matrix proteins, plasma, and cell surface [B. jararaca venom is composed of a complex mixture of proteins, mainly metalloproteinases, serine proteinases, phospholipases (PLA2), and L-amino acid oxidases. The metalloproteinases comprise most of the venom composition [The venom of  surface , which r surface ,18. In a surface . About 9position ,21. Theyposition ,23. In tposition . The thiposition . Finallyposition , are flaposition ,28. Howeposition .Echis carinatus snake venom and produced by Millennium Pharmaceuticals and Schering-Plow. It is used as an antiplatelet drug [B. jararaca venom. It is used for the treatment against hypertension and renal insufficiency [2+, inducing the release of cytochrome C, decreasing or increasing the expression of proteins that control the cell cycle, and causing damage to cell membranes [Snake venom constituents have been isolated and studied for their therapeutic potential in the treatment of various diseases. One example is Eptifibatide, marketed as Integrilin, derived from the let drug . Anotherficiency ,32. In aficiency , severalembranes ,34,35,36embranes .With the rapid advances of nano-scale liquid chromatography (nLC) and mass spectrometry (MS) technologies in the last two decades, nLC-MS/MS-based proteomics analysis has been widely applied as a powerful tool for biomarker discovery to improve cancer therapy ,39. A nuB. jararaca snake venom treatment on breast cancer-related cell proteome. In the present study, nLC-MS/MS was used to characterize the effects of sub-toxic doses of B. jararaca snake venom on two different breast cancer cell lines MCF7 and MDA-MB-231. MCF7 and MDA-MB-231 are non-metastatic and metastatic tumor cell lines, respectively. They are characterized by a high degree of glycolytic efficiency that promotes the interaction between the tumor cell and the extracellular matrix [B. jararaca snake venom in these cell lines highlight proteins and cell pathways that could be targeted in cancer therapy.However, to the best of our knowledge, there has been no report that describes the effects of r matrix . Althougr matrix ,48. ProtB. jararaca venom cytotoxicity assay on MCF7 and MDA-MB-231 cell lines was monitored using the WST-1 reagent. This analysis showed that although cell viability was similar between both MCF7 and MDA-MB-231 cell lines, they had different venom resistance profiles where MDA-MB-231 cells showed to be more resistant to the venom when compared to the MCF7 cells. Although both cell lines started to die at doses higher than 2.5 μg/mL, at the 5.0 μg/mL of venom, only about half of the MDA-MB-231 cells have died while most of the MCF7 have died at this venom concentration , 725 proteins were identified in cells treated with 0.63 µg/mL of venom, and 713 proteins were identified in the cells treated with 2.5 µg/mL of venom. Comparative analysis of the identified proteins showed that among all of the 789 proteins identified, 637 proteins were in common to all conditions, 657 proteins were in common between the control and the 0.63 µg/mL venom treatment, 656 proteins were in common between control and the 2.5 µg/mL venom treatment, and 677 proteins were in common between the 0.63 µg/mL and 2.5 µg/mL venom treatments  of proteins whose abundance increased more than 1.5× in the MCF7 cell line treated with 2.5 μg/mL of venom showed a high interconnection of proteins related to metabolic process (in red) and metabolism pathways (in blue) a. InterePPI analysis of the MCF7 cell lines with FC ≥ 1.5 treated with 0.63 μg/mL and FC ≤ 0.67 when cells were treated with 0.63 μg/mL and 2.5 μg/mL of venom are shown in We further analyzed proteins exclusively identified in one or two of the three conditions in both MCF7 cell line  and MDA-Of the proteins identified exclusively when cells were treated with 0.63 μg/mL of venom there were 28 proteins from MCF7 cell line, from which we highlight Cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1), Latexin (LXN), Vesicle-associated membrane protein-associated protein B/C (VAPB), Cysteine and glycine-rich protein 1 (CSRP1), and DDB1- and CUL4-associated factor 7 (DCAF7). From MDA-MB-231 cell line, 20 proteins were identified from which we highlight Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), low molecular weight phosphotyrosine protein phosphatase (ACP1), Transcription factor A, mitochondrial (TFAM), and Rae1 protein homolog (RAE1) .Proteins identified exclusively at the high dose treatment of 2.5 μg/mL of venom included 17 proteins detected in the MCF7 cell line, from which we highlight 26S proteasome non-ATPase regulatory subunit 6 (PSMD6) and 60S ribosomal protein L36 (RPL36). Similarly, we identified 44 proteins in MDA-MB-231 cell line from which we highlight protein Ubiquitin carboxyl-terminal hydrolase (HEL-117) .B. jararaca venom. From the MCF7 cell line, we identified 40 proteins from which we highlight: Serine/threonine-protein kinase ATR (ATR) and Jupiter microtubule-associated homolog 1 (JPT1). From the MDA-MB-231 cell line, we identified 22 proteins from which we highlight Angiomotin (AMOT) and Small nuclear ribonucleoprotein G (SNRPG)  . Among the proteins that were identified in the control group and at the 2.5 μg/mL venom treatment, we highlight: Small nuclear ribonucleoprotein Sm D1 (SNRPD1) in MCF7, and Epididymis secretory protein Li 71 (Hel-S-71) and Isoform Far upstream element-binding protein 3 (FUBP3) in MDA-MB-231 cell line . Of protB. jararaca venom treatment of MCF7 and MDA-MB-231 cells, including SNX3, HEL-S-156an, UQCRC1, RPL36, and ATR identified in MCF7 cell line and H3C15/HIST2H3, HEL-S-132P, MTCH2, TFAM, KCTD12, RPL34, and RPL37A identified in MDA-MB-231 cell line, and histone H3F3B and LAP3 in both cell lines.The present study successfully identified cancer-related proteins that undergo significant changes upon B. jararaca snake venom [With the discovery of rattlesnake venom’s antitumor activity in 1931 by Essex and Priestley  and lateke venom ,117,118,ke venom ,120.2+ influx. Several studies have revealed that these enzymes are critical regulators of cancer pathologies in several types of cancers including breast cancer [B. jararaca venom may have an important antitumor effect on Ehrilich ascites tumor cells in vivo and in vitro [Snake venoms have a complex mixture of proteins that can account for 95% of the total dried weight. Metalloproteases, serine proteases, LAAOs, and PLA2s are some of the most abundant proteins present in bothropic venoms, and the toxic action of these proteins are responsible for myotoxic effects, disruption in the coagulation cascade through thrombin-like proteins (serine proteases), imbalance in the blood homeostasis by the action of some metalloproteinases, apoptosis induction, and changes in the protein expression in oxidative stress and energy metabolism by the increase of Cat cancer ,126,127.in vitro .Aiming at the drug development, several groups have used proteomics approach to observe up- or down-regulated effects of proteins resulting from disease activity or side effects of the treatments ,130,131.Calloselasma rhodostoma, Macrovipera lebetina and Bothrops mattogrossensis significantly inhibit cell viability, either with crude or fractionated venoms and in different tumor cell lines such as LS174T , HCT116 , HT29 , HEL92.1.7 (erythroleukemia), and SK-BR-3 (breast adenocarcinoma) cell lines [Proteomics studies using tumor cell lines has been performed to systematically characterize protein complexes that may be important in disease or drug development, comparing the protein levels of cells in normal versus pathological situations . Studiesll lines ,134.B. jararaca venom, we tested two different invasive breast cancer cell lines with different phenotypic and genotypic differences, MCF7 and MDA-MB-231 cell lines. They were treated with different venom concentrations ranging from 0.32 µg/mL to 20 µg/mL. When treated with over 10 µg/mL of venom both MCF7 and MDA-MB-231 cell lines showed rounded and irregular morphologies and the MCF7 cell line detached from the plate. These results are similar to the results observed by Bernardes-Oliveira et al. (2016) when testing 50 µg/mL of venom from B. jararaca and Bothrops erythromelas for 48 h on SiHa HPV-16 and HeLa cells, both derived from cervical tumors. They observed that the cells became more rounded, with gradual size reduction and detachment from the cell monolayer [B. jararaca venom treatment. Based on both morphological and cell viability analyses, we selected the concentrations of 0.63 µg/mL (low sub-toxic dose) and 2.5 µg/mL (high sub-toxic dose) for further proteomic analyses.To evaluate the cytotoxicity of onolayer . In addiB. jararaca venom presented less proteins with FC ≥ 1.5 when compared to the MCF7 cell line. We identified 137 proteins with FC ≥ 1.5 at 2.5 μg/mL venom treatment in MCF7 while 34 proteins in MDA-MB-231 at 2.5 μg/mL venom treatment. At the low 0.63 μg/mL venom treatment, 25 proteins in MCF7 presented FC ≥ 1.5 and 16 proteins in MDA-MB-231. However, MDA-MB-231 showed more proteins with FC ≤ 0.67 compared to MCF7 cell lines. MDA-MB-231 showed 28 proteins presenting FC ≤ 0.67 at 0.63 μg/mL venom treatment and 19 proteins in MCF7; and 41 proteins with FC ≤ 0.5 at 2.5 μg/mL venom treatment in MDA-MB-231 cell lines compared to 23 proteins in MCF7 cell line. Among these proteins, our data showed that B. jararaca venom was able to modulate, up or down, the abundance of several proteins playing essential roles in tumorigenicity. For example, the venom treatment increased the abundance of SNX3, HEL-S-156, MCCC2, and GSTM3 in the MCF7 cell line, and H3C15/HIST2H3 and MTCH2 in the MDA-MB-231 cell line whose overexpression has been related to increase in tumorigenicity  increased more than 1.5×, and proteins that abundance decreased less than 0.67×, after 24 h treatment with 2 in RPMI 1640 culture medium  supplemented with 10% inactivated fetal bovine serum  and 25 mg/mL ampicillin and 100 mg/mL streptomycin antibiotics. Cells were split when the confluence reached 80%.MCF7 and MDA-MB-231 breast cancer cell lines were previously acquired from ATCC  and maintained at our laboratory cell bank. Cells were thawed and cultured at 37 °C in 5% COB. jararaca (lot 01/09-2) used in this study was a pool made from the extraction of 697 snakes collected at various Brazilian locations . Extracted venom were pooled and lyophilized at the department of herpetology at Butantan Institute under the coordination of Dr. Marisa Maria Teixeira da Rocha, and assigned for use and testing at the Laboratory of Applied Toxinology at Butantan Institute. The methods and use of venom in this work were approved by Butantan Institute Ethics Committee under the certification CEUAIB #9766150719 (2019).The venom of 4 cells were plated in each well of a flat bottom 96-well microtiter plate. Cells were cultured at 37 °C in 5% CO2 and on the day prior to analysis (70–80% confluency), culture medium from each well was exchanged to 100 μL of medium in the absence or presence of venom in different concentrations. After 24 h, 10 μL of the WST-1 reagent previously dissolved into an Electro Coupling Solution (ECS) were added to each well and incubated for 4 h at 37 °C in 5% CO2. The absorbance at 450 nm of each well was measured using a microplate reader FlexStation 3 spectrophotometer . The determination of the absorbance and the quantification of viable cells were calculated using SoftMax Pro  and Microsoft Excel software.The cytotoxicity assays were carried out by treating the cells with different concentrations of venom in the range from 0.1 μM to 20 μM. The cytotoxicity tests were performed using the colorimetric method of cell viability analysis using the WST-1 cell proliferation reagent kit  according to manufacturer’s instructions. Briefly, 105 cells were plated and after the cells had reached a confluence of 70–80% (2–3 days), different concentrations of B. jararaca venom were added to the culture medium, starting from the cell cytotoxicity threshold up to a 1000-fold dilution for 24 h. For mass spectrometry analysis, cells were washed with ice-cold PBS and lysed with 1 mL ice-cold 8 M urea supplemented with cOmpleteTM protease and phosphatase inhibitors cocktail . Fifty microliters of the cell lysate were separated for protein quantification using BCA  and the lysate were stocked at −80 °C until further preparation of the sample for mass spectrometry analysis. Both cytotoxicity and proteomics experiments were performed in three biological replicates.In a six-well plate, 2 × 10To the cell lysates, four microliters of 10 mM dithiothreitol (DTT) were added to reduce the disulfide bonds and incubated at 56 °C for 1 h. After incubation with DTT, 40 μL of iodoacetamide (IAA) was added for the alkylation of the cysteines for 1 h in the dark at room temperature. After incubation, 2.5 mL of ammonium acetate and 20 μL of trypsin  were added enough to have an enzyme:protein ratio of 1:50 and incubated at 37 °C overnight. The reaction was stopped by adding 30 μL of 100% acetic acid. Samples volume were reduced in a SpeedVac  until the volume has lowered to 50 μL and were further desalted on in-house manufactured stop-and-go extraction tips (Stage-Tip) with three SDB-XC  membranes as previously described . The stam/z, and a dynamic exclusion of 70 s. Protein identification was performed using Mascot , MaxQuant , and Peaks Studio  against the Homo sapiens database downloaded from the Uniprot in March 2019 (www.uniprot.org accessed on 1 October 2019). As search parameters, oxidation of methionine was set as a variable modification and carbamidomethylation of cysteine was set as a fixed modification. Searches were performed with mass error tolerance of 10 ppm for MS and 0.3 Da for MS/MS, and trypsin was selected as the proteolytic enzyme used in the digestion of proteins, and up to two miscleavages were allowed. Mass spectrometry-based proteomic analysis of MCF7 and MDA-MB-231 cell lysates, treated or not treated with low or high doses of B. jararaca venom were performed in triplicate. All raw data files for these analyses were uploaded and are available at: http://massive.ucsd.edu/MSV000084138/ accessed on 5 July 2021.Dried samples were resuspended in 0.1% formic acid and analyzed on LTQ-Orbitrap Velos mass spectrometer  coupled to an EASY II nano liquid chromatographer (Thermo Scientific) using the shotgun approach. The spectrometer was equipped with a nanospray source connected to an in-house prepared analytical column (10 cm × ID 75 μm × OD 360 μm) packaged with 7 cm of 5 μm C18 resin . Precolumn (7 cm × ID 75 μm × OD 360 μm) was also prepared in-house packed with 5 cm of 10 μm C18 resin . The LC-MS/MS analyses were carried out by injecting 5 μg of the peptide extract and the peptides eluted from the column with a gradient of 5–40% in 100 min of solvent B  at a flow rate of 200 nL/min. The nanospray source was operated at 1.8 kV. The peptide mixture was analyzed by the acquisition of spectra in the full scan mode at a resolution of 30,000 for the determination of molecular masses (MS) of up to 10,000 Da. The 10 most intense peaks were automatically selected via data dependent acquisition (DDA) for the subsequent acquisition of spectra of the ions product to MS/MS for amino acid sequence determination at a resolution of 7500, a maximum injection time of 30 ms, a range of 200 to 2000 geneontology.org accessed on 1 October 2019) and PantherDB (pantherdb.org accessed on 1 October 2019). The proteins presenting different profiles in the venom conditions were also analyzed using KEGG (www.genome.jp/kegg accessed on 1 October 2019) canonical pathways to determine the protein group or pathways that have undergone changes with different concentrations of the venom treatment. The PANTHER Classification System  is a research tool that covers evolutionary and functional information on protein family genes and has been widely used to understand protein evolution and its functional classification [B. jararaca venom that presented FC ≥ 1.5 in order to identify an enrichment pattern of protein families and pathways affected by the venom in the cell lines.The entire proteome was analyzed to classify protein profiles by biological processes, molecular function, cellular components, and cellular pathways using Gene Ontology . The set of protein dissimilarities were computed using the “Euclidean” distance with the function “dist” to the hierarchical clustering based on the package and function “hclust”. There was employed the agglomerative method with “ward.D2”. The fold change and the protein–protein interactions provided by information from the String database [B. jararaca venom using the Homo sapiens reference genome.Semi-quantitative protein analysis was performed using the Label Free Quantification MaxLFQ algorithm from MaxQuant software with an FDR rate of ≤1% to compare relative abundance of proteins in each of the cell lines . Data gedatabase  was usedhttp://www.sthda.com/english/rpkgs/factoextra accessed on 1 November 2020) for graphical visualization.Principal component analysis (PCA) was applied to find which linear combinations of the differentially expressed proteins with ≥1.5 would explain most of the variability for the different cell lineage conditions. The PCA analysis was performed using the R-statistics packages FactoMineR (accessed on 1 November 2020)  and Fact


The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines  Summary: Generation and characterization of two phenotypically different and transplantable mouse model lung squamous cell carcinoma cell lines – UN-SCC679 and UN-SCC680 – to be used in different research scenarios, including immunotherapy. Lung cancer is the leading cause of cancer-related death worldwide . The incOf all lung cancers ∼20-30% are classified as lung squamous cell carcinoma . There are two types of lung cancer, i.e. small lung cell cancer (SCLC) and non-small cell lung cancer (NSCLC), and LUSC is more strongly associated with tobacco smoking than any other subtype of NSCLC. Age, exposure to second-hand smoke, asbestos, mineral and metal dust, and radon constitute other risk factors for LUSC.Basal stratified squamous lung epithelial cells are the most likely origin for LUSC . Within LUSC is a less-studied NSCLC subtype than LUAD. Different factors have contributed this fact. First LUSC biology is more challenging than that of LUAD and, on average, human LUSCs have more mutations per megabase than LUAD . Thus, LIn this present work, we performed exhaustive molecular and functional characterization, and comparison of two novel LUSC research cell line models. These models consist of two A/J mouse-derived syngeneic cell lines obtained by using NTCU-induced LUSCs. These cell lines might become robust tools for the study of squamous cell lung cancer in a reliable and reproducible manner, and when testing novel antimetastatic and immunotherapy agents. In fact, one of the two cell lines (UN-SCC680) has successfully been used by our group to evaluate a strategy of combined immunotherapy . Here, win situ carcinomas were observed in mouse bronchi upon NTCU treatment . Progressive de-differentiation of the typical squamous phenotype was observed with increasing passages, as previously reported for human lung LUSC cancer cell lines . We compared the differentially expressed genes identified in UN-SCC679 with those in UN-SCC680 cells. We observed that ∼80% of the differentially expressed genes are present in both cell lines. This indicated that, despite their differences in mutational patterns, UN-SCC679 and UN-SCC680 cells share many molecular similarities that reflect their identical squamous origin. Shared and non-shared differentially expressed genes are listed in Table S5. Compared to normal lung tissue, we found that both cell lines are enriched in p63  and that expression of genes related to the classic subtype described for human LUSC genomic subtypes, such as Gsto1, Aldh3a1, Bcl6, Atp5g3, Dld, Odc1, Trp63, Gsta4, Ndufb5, Ephx1 or Cox5b (data not shown) is increased . Finally, both cell lines overexpress Arnt2, which has been previously associated to lung cancer , UN-SCC679 cells showed an advantageous in vivo tumor growth in immune-competent mice compared to that of UN-SCC680 cells , i.e. anti-PD-1 therapy . Taking 80 cells A,B, sugg80 cells A. These 80 cells B, the reThe success of checkpoint blockade in NSCLC, both squamous cell carcinoma and adenocarcinoma, is strongly influenced by the nature of the immune infiltrate present in the tumor . To bettWe completed our immune population study by performing a multiplexed immunohistochemistry experiment using VECTRA technology D and E. 5 UN-SCC679 or UN-SCC680 cells that had been transfected with a triple modality GFP-luciferase-thymidine reporter gene . To explore the specific metastatic potential of the cell line models, we inoculated the left heart ventricle of A/J mice with 1×10ter gene A. Thus, ter gene B. At the has been shown to promote proliferation and cell autophagy via ERK1/2 signaling in human lung cancer cells (DNAH6) belongs to the dynein family, whose members encode large proteins that constitute the microtubule-associated motor protein complex; it has recently been related to smoking-associated lung cancer (CDH23) – a member of the cadherin superfamily, whose genes encode calcium-dependent cell-cell adhesion glycoproteins – is downregulated in lung cancer, working as a suppressor of cell migration (CSMD3) to be a frequently mutated gene in human LUSC, resulting in an increased proliferation of airway epithelial cells (FBN1), encoding an extracellular matrix glycoprotein that serves as a structural component of calcium-binding microfibrils, seems to play an important role in tumor-related immune infiltration and has been proposed as a prognostic and predictive biomarker for immune therapy against pancreatic ductal adenocarcinoma (PDAC)  . Finallya (PDAC) . Our stuRelevant LUSC studies coincide in emphasizing the molecular heterogeneity of this tumor type, which makes the development of targeted therapies very difficult . Thus, wThe National Comprehensive Cancer Network guidelines for LUSC treatment  do not rKRAS and P53 are partly responsible for the resistance of lung tumors to immunotherapy , which could be involved in its resistance to anti-PD-1 treatment. However, UN-SCC680 cells, which responded to immunotherapy to some extent, contained mutated Kras. Nevertheless, the specific Kras mutation found in UN-SCC680 cells was a rare alteration of the gene, which differed to the classic G12V/D mutation that characterizes lung cancer. This detail must be taken into account, since Kras is not a gene characteristically mutated in LUSC and because the mutation we found in our cell line might have other functional and biological implications regarding the tumoral context, which are different to those expected with the classic G12V/D mutation.It is well stablished how specific mutations drive immune evasion in cancer. Specifically, mutations in otherapy . UN-SCC6In the context of sensitivity to immunotherapy and based on the tumor immune landscape, six immune subtypes (ISs) have been identified in SCC. The most common for LUSC – comprising ∼41% of cases – is IS1, followed by IS5 (25%) and IS3 (14%). Both IS1 and IS3 are ‘immune-cold’, characterized by a low lymphocyte infiltration rate, a high immunosuppressive microenvironment and a poor prognosis. IS3 has the worst outcome among all subtypes . AccordiThe immune landscape and mutational burden have been shown to predict the response of LUSC to immunotherapy. In general, a tumor with a high lymphocytic infiltrate and an elevated mutational burden will have a greater response to immunotherapy treatments . UN-SCC6A well-known feature of human lung tumors is their selective metastatic tropism. Lung cancer most commonly spreads through bloodstream to the liver (34.3%) and adrenal glands (32.6%), followed by bones (14.9%) and other organs, such as the central nervous system (CNS) (12%), kidney (10.9%), myocardium (9.1%), pancreas (5.1%), spleen (4%), small or large intestines (3.4%), stomach (2.3%), thyroid gland (1.7%) and ovary (0.6%) . The twoIn conclusion, we have generated and thoroughly characterized two mouse LUSC cell models, i.e. cell lines UN-SCC679 and UN-SCC680 derived from the NTCU-induced mouse model in A/J mice. UN-SCC679 and UN-SCC680 cells carried different somatic mutations and gene expression profiles, with tumor mutation burdens comparable to those of LUSC patients. UN-SCC680 cells responded partially to treatment with anti-PD-1 antibody, whereas UN-SCC679 cells were refractory to the treatment. The immune landscape study revealed a cold IS of both cell lines, with a more-immunosuppressive tumor microenvironment in UN-SCC679 tumors. Finally, both cell lines demonstrated metastatic potential with the ability to grow metastatic lesions in the brain, bones, liver and adrenal glands of different degree of aggressiveness. In summary, we believe we have generated a very valuable tool for further LUSC research, comprising two cell lines with complementary traits that recapitulate the complexity of LUSC in humans.Seven-week-old A/JOlaHsd mice (hereafter referred to as A/J mice) were obtained from Harlan Laboratories (Harlan-Winkelmann). All animal experiments were conducted in accordance with the standards set by the Declaration of Helsinki. The protocols were approved by the institutional animal care committee of our institution , following the legal and ethical requirements demanded by the European Communities Council Directive 2010/63/UE and according to the regulations of the Spanish Royal Decree 53/2003, which were drawn up to ensure that pain and suffering would be limited to the lowest level.−/−IL2Rg−/− immunodeficient mice and engrafted into syngeneic 8-week-old A/J mice as previously described . Both cell lines were cultured in RPMI 1640 supplemented with 10% Fetalclone serum (Thermo Fisher Scientific) and 100 U/ml penicillin-100 µg/ml streptomycin (Thermo Fisher Scientific). All cells were grown in a humidified incubator under 5% CO2 at 37°C. Cell lines were routinely tested for mycoplasma.LUSC tumors were induced by N-nitroso-tris-chloroethylurea  treatment applying 0.04M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Lungs of euthanized mice were excised, and tumor cell lines UN-SCC679 and UN-SCC680 were derived and cultured for 25 passes to ensure they were immortalized. Cells were then subcutaneously injected into the flanks of 6-week-old female Rag2escribed  , catalog no.: AM071-10M, 1:50, TE pH 9.0, 20 min at 95°C; TTF1 (Dako) catalog no.: M357529-2, 1:40, TE pH 9.0, 6 min at 98°C; P40  catalog no.: 3066, 1:200, TE pH 9.0, 20 min at 95°C; P63 (Abcam) cataloge no.: ab735, 1:100, EDTA pH 8.0, 20 min at 95°C. All primary antibody incubations were carried out overnight at 4°C. After detection of primary antibodies was performed with EnVision System (Dako), slides were counterstained with hematoxylin, dehydrated, and covered with DPX mounting medium.Whole-exome sequencing was performed on extracted DNA from the tumor-derived cell lines. Enrichment for sequencing was performed using the SureSelectXT Mouse All Exon kit (Agilent) following the manufacturer's protocol. Exon-enriched libraries were subjected to paired-end sequencing, 2×100 base pairs read length and 60× minimum coverage.https://uni-koeln.sciebo.de/s/JJdOLcanGNN7U92.Raw sequencing reads were aligned to the respective mouse reference genome (NCBI37/mm9). Alignment was performed with the BWA aligner. Concordant read-pairs were identified as potential PCR duplicates and subsequently masked in the alignment file. Somatic mutations and copy number alterations were determined using our in-house analysis pipeline . Data arGSE185054.Samples were prepared with the Illumina TruSeq Stranded mRNA kit following the manufacturer's instructions and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. For RNA sequencing analysis, raw fastq files were trimmed with Trimmomatic/0.36. Pseudoalignment was carried out to the mm10 reference genome and gene level counts were determined with Kallisto . Differein vivo tumor growth studies, 2×106 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in both flanks of 8-week-old female Rag−/− IL2Rg−/− mice (Harlan-Winkelmann). Tumor volume (weight2×length×0.52) was measured with a digital caliper twice a week until sacrifice.Cell proliferation in 2D was assessed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, MTS (Promega). Experiments were carried out on the days indicated, according to the manufacturer's instructions and normalized to day 1 post seeding. For 6 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice (Harlan-Winkelmann). When tumors reached a volume of 75 mm3, mice were randomized (n=6 per group) and treated intraperitoneally with three doses of 100 µg of the following antibodies: anti-PD-1 , anti-CTLA4  or PBS every 3 days from day 7 post inoculation. Tumor volume (weight2×length×0.52) was measured with a digital caliper twice a week until euthanasia.2×106 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in one flank of 6-8-week-old female A/J mice (Harlan-Winkelmann). Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis as previously described  by using the murine-specific kit (NEL840001KT) from Akoya Biosciences  following the manufacturer's instructions. Extra Alexa Fluor tyramides Opal 540 (FP1494001KT), 620 (FP1495001KT) and 650 (FP1496001KT) from Akoya Biosciences were used as well. Citrate buffer pH6 was used for all antigen retrievals and removal of primary-secondary complexes formed with previous antibodies. Primary antibodies and its corresponding Opal reagents were as follows: FOXP3  at 1:600 dilution and combined with Opal 540 fluorophore; CD4 (Abcam ab183685), 1:400 dilution, Opal 520; CD8 , 1:500 dilution, Opal 620; and F4/80 , 1:400 dilution, Opal 650. Acquisition of multispectral images and unmixing were conducted with the Vectra Polaris Automated Quantitative Pathology Imaging System, using the Phenochart and InForm softwares .Multispectral analysis was performed using QuPath 0.3.0 software . Tumor r5 cells as detailed by in vivo system . For imaging analysis, M3 Vision software (v 1.1) was used. Photon flux was calculated for each mouse by using a circular region of interest for the whole body and extracted as photons/s/cm2/sr. All in vivo experiments were normalized to the luciferase signal at day 0.Cells had previously been transfected with a triple modality construct expressing GFP, luciferase and thymidine kinase . Eight-wt-test. Non-normal samples were analyzed using the Mann–Whitney test  or the Median test .Sample size was chosen based on similar experiments previously published by the authors. For comparison of two groups, sample normality and variance were explored . Groups with normal distribution followed a two-tailed


O-propargylated mollugin with aryl azides. All the compounds were evaluated for their cytotoxicity on five human cancer cell lines  using MTS assays. Among the synthesized series, most of them showed cytotoxicity and most of all, compounds 14 and 17 exhibited significant cytotoxicity of all five cancer cell lines.A new series of mollugin-1,2,3-triazole derivatives were synthesized using a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of corresponding  Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) . Briefly, cells were seeded into each well of a 96-well cell culture plate. After 12 h of incubation at 37 °C, the test compound (40 μM) was added. After incubated for 48 h, cells were subjected to the MTS assay. Compounds with a growth inhibition rate of 50% were further evaluated at concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicate, with cisplatin and paclitaxel (MeilunBio) as positive controls. After the incubation, MTS (20 μL) was added to each well and the incubation continued for 4 h at 37 °C. After sufficient reaction, the light absorption value of each well was read by Multiskan FC at 492 nm. The IC50 value of each compound was calculated with Reed and Muench’s method.The following human cancer cell lines were used: HL-60, A-549, SMMC-7721, MCF-7, and SW-480. These cells were obtained from American type culture collection (ATCC) . All the cells were cultured in RPMI-1640 or Dulbecco’s modified Eagle medium (DMEM) medium , supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO14 and 17 exhibited cytotoxicity of all five cancer cell lines significantly and compound 36 could enhance the cytotoxicity of lung cancer cells (A549) specifically. Structure and activity relationship (SAR) analysis reveals that electron-donating groups including hydroxyl, methoxy, and alcohol hydroxyl groups are essential for retaining the cytotoxicity to derivatives. In addition, for derivatives containing methoxy groups that the cytotoxicity may increase with the number of methoxy groups. Based on the SAR studies, we believe that the enhancement of cytotoxicity of the derivatives may be caused by the aromatic ring becoming electron-rich or the electron-donating atoms with lone pairs provided by electron-donating groups available to serve as hydrogen bond acceptors with the active site, which is worthy of further study.In conclusion, 40 1-substituted 1,2,3-triazole-mollugin derivatives were synthesized through Huisgen 1,3-dipolar cycloaddition reaction and evaluated for cytotoxicity against a series of five different human cancer cell lines  along with the parent molecule. Most of the derivatives showed better cytotoxicity than parent molecule. It is worth mentioning that our experiment results showed that compound