Leontopodium alpinum Cass. (edelweiss) is recognized as a frequent constituent of anti-aging skin care products, providing increased antioxidant and anti-inflammatory defense. Considering the growing demand and the protected status of edelweiss in many countries, alternative methods of production have been developed, one of them being callus culturing. This study reports the phytochemical composition of a methanolic extract of L. alpinum callus cultures, characterized by liquid chromatography coupled to ion-mobility high resolution mass spectrometry (UPLC/IM-HRMS). The methanolic extract exhibited strong free radical scavenging activity (122.19 ± 7.28 mg AAE/g dw), while the quantitative evaluation revealed that four major constituents (phenylpropanoid derivatives) represent 57.13% (m/m) of the extract. Consequently, a screening of antiproliferative effects was performed on ten cancer cell lines, representative of prostate, colon, lung and breast cancer, showing inhibition of colony formation in all cases. These results provide a comprehensive phytochemical characterization of L. alpinum callus cultures using advanced IM-HRMS, while the in vitro explorations confirmed the potent antioxidant properties of edelweiss which are worth exploring further in cancer prevention. Leontopodium alpinum Cass. (edelweiss) is one of the most well-known members of the Asteraceae family. It grows on limestone surfaces, prefers the alpine climate and it is a protected species in many countries. In traditional medicine, edelweiss was used as remedy in digestive and respiratory illnesses as powder, infusion and tincture [tincture  or in thtincture . NowadayCallus culture  is a developing technique for the production of useful compounds, including pharmaceuticals . The mosLeontopodium alpinum phytochemical composition highlighted a number of compound classes, such as phenylpropanoid, sesquiterpene, benzofuran and lignan derivatives. However, data regarding the composition of callus cultures is rather limited [Previous investigations ,10,11,12 limited ,14. So f limited , showingL. alpinum extracts effects has been focused on anti-inflammatory effect evaluation [During the last decade, the preclinical research on aluation , leukotraluation , anti-agaluation  and memoaluation . Howeveraluation , due to The aim of this study was to perform a comprehensive phytochemical characterization of edelweiss callus cultures. Following the assessment of the antioxidant capacity evaluation, two major cancer hallmarks were also investigated (proliferation and resisting cell death) using a panel of ten cancer cell lines representative of breast, prostate, lung and colon cancers.L. alpinum extract was obtained by microwave assisted extraction (MAE). In comparison to ultrasound assisted extraction (UAE), this technique was found to offer similar yield in a much shorter time (5 min for MAE compared to 45 min by UAE). The total extraction yield was calculated to be 26.4% .The Further, the methanolic extract of edelweiss callus cultures was subjected to phytochemical profiling using a reversed phase LC/IM-HRMS method, achieving a good separation of the detected compounds . ConsideL. alpinum, whereas for the untargeted approach, a diagnostic product ion (DPI) strategy was implemented [L. alpinum extract: glucaric acid derivatives and quinic acid derivatives. Therefore, glucaric acid (m/z 191.0192), peaks 2 and 3 , and quinic acid (m/z 191.0556), peak 1  were used as DPI  based on the fragmentation pattern and previous reports [Both targeted and untargeted strategies have been implemented to identify most of the compounds present in the extract. The targeted approach was based on literature reports of major phytochemical constituents in lemented . Using t DPI see . Peak 4  reports . Out of  reports  and a maL. alpinum callus cultures. These derivatives are formed by the esterification of D-glucaric acid hydroxyl groups with caffeoyl and 3-hydroxybutanyl moieties. In total, 11 glucaric derivatives have been identified. Among these, peak 17 leontopodic acid A  and peak 13 leontopodic acid B  were the most abundant and their identification was validated using standards. Isomers of both leontopodic A and B were detected as peak 18 and 15, respectively.Glucaric acid derivatives are the most abundant class of secondary metabolites identified in 24H22O14, 1.2 mDa) isomers were identified as peaks 6, 7 and 8, while peak 24 represents tetra-caffeoyl glucaric acid , a tetra-substituted derivative [26, it is most likely to be a penta-substituted glucaric acid derivative , with two 3-hydroxybutanyl and three caffeoyl moieties.A series of di-caffeoyl glucaric (Cm/z 191.0556) was used as DPI. Peaks 5 and 14 were identified by comparison with standards and annotated as chlorogenic acid  and 3,5-dicaffeoyl quinic acid , respectively. Two other di-caffeoyl quinic isomers  were identified as peaks 12 and 16.For the identification of quinic acid derivatives, quinic acid , while the in vitro antioxidant activity of the extracts expressed in ascorbic acid equivalent antioxidant capacity (AEAC) was 122.19 ± 7.28 mg AAE/g dw, as determined using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay.The total polyphenol determination revealed that the The phytochemical characterization of the LA extract highlighted high concentrations of polyphenolic derivatives with strong antioxidant activities. Consequently, we exposed a panel of diverse cancer cell lines  to LA extract and performed several in vitro screenings. Particularly, we focused of two hallmarks of cancer, namely sustaining proliferative signaling (cellular proliferation and colony formation capabilities) and resisting cell death .L. alpinum extract on cancer cell panel viability was studied by 3--2,5-diphenyltetrazolium bromide (MTT) assay. The assay involves conversion of 3--2,5-diphenyltetrazolium bromide by NADPH-dependent oxidoreductases to a purple-colored formazan product whose VIS absorbance is proportional to the number of metabolic active cells. Cells were grown in 96-well plates and treated with complete medium containing serial dilutions of extract (6.25–400 µg/mL). A blank control (complete medium only) and a positive control (100% DMSO) were included in every plate. The extract showed significant effect on cell viability at the highest tested concentration (400 µg/mL) on most cell lines : this is the case for SK-MES-1 (67.2 ± 23.8%) lung cancer cell line and MDA-MB-231 (77.21 ± 4.9%) and HS578T (67.4 ± 13.3%) TNBC representatives.Effect of ll lines , exceptiL. alpinum extract had the greatest impact on cell viability and was used in the in vitro functional assays conducted further.The 400 µg/mL dose of L. alpinum callus cultures’ methanolic extract on cellular morphology and cell distribution was evaluated by 4′,6-diamidino-2-phenylindole (DAPI) and Fluorescein phalloidin  staining, used for nuclei and actin filaments labeling. The levels of toxicity and the antiproliferative effects were assessed by comparing control and treated cells groups. A summary of observed effects is found in The effect of the A substantial reduction of cell population was seen in the human non-cancer fibroblast (BJ cell line) and MDA-MB-231 TNBC in accordance with the viability assay conducted. Reduction of cell population was also observed in the case of both HCT-116 and DLD-1 colon cell lines. Particular to BJ cell line was the absence of signs of nuclear and cytoskeletal damage.L. alpinum extract induced nuclear fragmentation on most cell lines, excepting BJ, HS578T and SK-MES-1. Furthermore, multilobed nuclei were observed for HS578T, SK-MES-1, 22RV1, and HCT116, associated with rounding of cells. This last phenomenon was found also for H1792 and LNCAP in a less extensive manner. A marked cytoskeleton fragmentation was observed in the case of prostate cancer compared to the other cell lines.The L. alpinum extract, few cells presenting nuclear fragmentation and cytoskeleton damage, with most of the cells having similar shape to the control.Compared to TNBC cell lines, MCF-7 was less sensitive to Another cancer feature is the capacity to develop secondary cancer sites, also known as metastatic sites. Colony assay was chosen to analyze this ability in vitro, by measuring the colony diameter of both control and treated cells .L. alpinum extract generated small and hardly visible colonies in the captures, the colony formation capacity of 22RV1 being half of control, while for LNCAP cells the capacity was even lower.After treatment, 22RV1 and LNCAP prostate cells colonies were considerably reduced and both cell lines exhibited a low ability to create new colonies . CompareHCT116 colon cancer cells showed higher capacity to form colonies compared to DLD-1. The extract considerably inhibited the colony formation of both cell lines, with a pronounced effect on HCT116. Lung cancer cell lines (H1792 and SK-MES-1) showed high colony formation capacity, but the treatment reduced the colony number and size. The inhibitory effect of the treatment was confirmed on all three cell lines, the colony diameter being decreased by more than half compared to that of control. Similar to lung, colon and prostate cell lines, the extract inhibited colony formation was seen in all breast cancer cell lines. Breast cancer cell lines exhibited different colony formation capacities, the most potent being MCF-7, followed by MDA-MB-468 and the other two TNBC cell lines.L. alpinum was used mostly for its anti-inflammatory properties. Today, its current use is limited due to the protected species status that the plant has in many countries. However, the growing demand coming from the cosmetic industry, due to the antioxidant properties of L. alpinum, required the development of alternative means to obtain the specific constituents, such as callus cultures of edelweiss, now a world-wide substitute.In traditional medicine, In medicine, many plant antioxidants have been shown to possess antitumoral properties . AlthougMicrowave assisted extraction (MAE) and ultrasound assisted extraction (UAE) were assessed for the targeted extraction of polyphenolic compounds from edelweiss callus cultures, using the same extraction solvent (70% methanol). MAE was found to offer a faster extraction time (5 min), compared to UAE (45 min), both having comparable yield (data not shown). The dried extract was easily soluble in aqueous solutions, even at high concentrations (4 mg/mL stock solutions).L. alpinum extract was performed using LC/IM-HRMS. Compared to conventional mass spectrometry, ion-mobility mass spectrometry offers a supplementary separation dimension. Being a gas phase separation technique, ions are resolved based on their size and shape, depending on their collisional cross section (CCS). Ion-mobility mass spectrometry proved to be useful for differentiating between isomeric species [L. alpinum extract, no co-eluting isomeric species could be identified, all of them being resolved chromatographically before detection. The CCS values of the molecular ions of the constituents detected in the extract are presented in 6, 7 and 8) and di-caffeoyl quinic acid isomers . However, their separation by ion-mobility alone would probably not be possible considering the low resolution of today’s commercially available technology.The polyphenol profiling of the  species . Still, L. alpinum) such as isomers of leontopodic acids A and B, disubstituted derivatives of both glucaric (di-caffeoyl glucaric acids) and quinic acids (di-caffeoyl quinic acids), together with two highly substituted derivatives of glucaric acids (tetra-caffeoyl glucaric acid and a penta-substituted glucaric acid derivative).The analysis of the extract revealed 27 major constituents. The two detected classes of polyphenols were derivatives of glucaric and quinic acids, the most abundant being leontopodic acids A and B, chlorogenic acid and 3,5-dicaffeoyl quinic acid. Besides these already known constituents, several other derivatives were observed  with other compounds reported in the composition of terpenes ,10,11, lterpenes , or flavterpenes .L. alpinum    revealedConsidering the high quantity of polyphenols determined in the edelweiss callus cultures, an in vitro screening procedure was implemented which allowed the evaluation of the extract on three cancer hallmarks. Its effects were studied on ten cancer cell lines, representative of four different cancer types. A summary of the obtained results is presented in L. alpinum callus cultures extract was evaluated in vitro using the MTT assay. Decreased cell viability was observed at the highest concentration tested (400 µg/mL) for TNBC breast cell lines MDA-MB-231 and HS578T, both colon , and in lung cancer cell lines (SK-MES-1 and H1792). In the specific case of MCF-7, an estrogen dependent cell line, pro-proliferative effects have been observed. It has already been documented that polyphenols, due to their chemical structure, can interact with estrogen receptors with different affinity towards estrogen receptors α and β. A deeper insight into the mechanisms involving polyphenols’ effects on hormone dependent cancer cell lines can be found in a comprehensive review published by Cipolletti et al. [The cytotoxic effect of i et al. .Next, other cancer hallmarks were studied in order to observe potential induced morphological changes and colony formation capabilities. Colony formation was inhibited for all cell lines, presenting substantially reduced diameters , with prThese results could be attributed to the high quantity of polyphenols which are known to modulate pathways relevant to proliferation, migration or colony formation and in some particular cases cell death could be triggered ,28,29,30Using in vitro models, polyphenols were previously described to possess anticarcinogenic properties, capable of inhibiting tumor growth, metastasis and inflammatory processes ,32,33. TL. alpinum extract, 3-5-dicaffeoylquinic acid, acts as antioxidant agent with neuroprotective effects [50 below 300 µM  [The second quinic acid derivative found in high quantities in  effects . On top, effects . The anteatment) . The autL.alpinum callus cultures, and remains a subject open to research.Unfortunately, less is known about the potential anticancer mechanisms of actions of the leontopodic acids A and B, the other two major constituents of The reference standards of chlorogenic acid, 3,5-dicaffeoylquinic acid and leontopodic acids A and B which were used in this work were acquired from Extrasynthese . The organic solvents, methanol and acetonitrile, used for mobile phases and extractions were of LC-MS purity and were purchased from Merck . Ultrapure water was produced with a Purelab water filtration system , while MS grade formic acid was produced by Carlo Erba . 2,2-Diphenyl-1-picrylhydrazyl and Folin-Ciocâlteu’s phenol reagent were purchased from Sigma , ascorbic acid was produced by International Laboratory , and sodium carbonate by Lachner .Leontopodium alpinum Cass. (www.semintelegumeflori.ro (accessed on 10 December 2021), batch Pl730/12/210/1274A, SC XTREME ANG MARKETING SRL, Bucharest, Romania), aseptically germinated, and seedlings were used to initiate the callus culture on Gamborg medium [Commercial seeds of g medium , supplemL. alpinum callus cultures from three batches were freeze-dried  using a LyoQuest -55 Plus instrument , then all samples were mixed to create a representative sample which was further used for this study. Ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) were assessed in order to find the most suitable extraction conditions.Prior to extraction, v/v), then sonicated for 45 min at 37 kHz  [v/v) [v/v) was performed 3 times , the organic phase being discarded [For UAE, 20 mg of freeze-dried sample was suspended in 1 mL of 70% methanol (Germany) . MAE (byy) [v/v) . The expy) [v/v) ,42,43. Tiscarded . The metm/z, using 2 functions. The collision energies were set to 4 eV (trap) and 2 eV (transfer) for the first function and a ramp between 20–45 eV (trap) for the second function. Leucine-encephalin was used as single point reference (m/z 554.2615).All analyses performed for the phytochemical characterization were carried out on a Waters I-Class UPLC instrument coupled with a Synapt G2-Si high resolution mass spectrometer . The separations were achieved by reversed phase using a BEH C18 (100 × 2.1 mm) stationary phase  and mobile phases formed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), being pumped at a flow rate of 0.3 mL/min at a temperature of 45 °C. Gradient elution was implemented as follows: 0 min 3%B–10 min 40%B–15 min 98%B. The mass spectrometer was set in resolution mode using negative electrospray ionization (2 kV capillary voltage), with source temperature of 130 °C. Data independent acquisition was achieved in two modes: (i) HDMSE (ion-mobility enabled) for qualitative runs and identification purposes and (ii) MSE (Q-ToF only) for quantification. Data was acquired in the range of 50 to 1200 Data processing  was carried out using Progenesis QI software version 3.0.76 . A first identification was performed on the most abundant compounds by using an in-house natural compounds library. Major compounds were putatively annotated based on their accurate mass, fragmentation pattern, available reference standards and on other previous studies.Quantitative determinations of leontopodic acids A and B, chlorogenic acid and 3-5-dicaffeoylquinic acid were made by constructing a 5-point calibration curve (0.1–40 µg/mL) for each analyte. Each standard solution was injected 2 times and the quantitative analysis was performed using the TargetLynx module of MassLynx software , by measuring the molecular peak areas of the analytes. Injection repeatability was assessed by injecting 5 times the previously mentioned standards (RSD less than 3.85%). Two replicates of the extract were prepared, then analyzed twice, after a 40-fold dilution.v/v) and shaken for 60 s. The mixtures were kept for 40 min in darkness at room temperature, then absorbance at λ = 515 nm was measured using a Clariostar Plus microplate reader . The blank sample consisted of 20 μL of water with 180 μL of methanol:water , while the control sample was 20 μL of water with 180 μL of DPPH solution. Each determination was performed in triplicate and the percentage of quenched DPPH was determined using Equation (1).Antioxidant assay was performed using a microplate protocol described by Navarro et al. . Two serBy calculating the concentrations of sample and extracts equivalent to 50% quenched DPPH, the ascorbic acid equivalent antioxidant capacity was determined.2CO3 was added, followed by incubation at room temperature and darkness for 2 h. The solution absorbance was measured at λ = 765 nm . The determination was made in triplicate. The gallic acid calibration curve was built, showing a linear range for concentrations between 10–200 μg/mL gallic acid. The total polyphenol content of extracts was expressed as mg of gallic acid equivalents (GAE)/g dry weight (dw), using the Equation (2) based on the calibration curve [T = the total phenolic content in mg/mL as GAE; C = the concentration of gallic acid from the calibration curve in mg/mL; V = the volume of the extract solution in mL; M = the weight of the extract in g.The total polyphenols determination was made according to the method described by Singleton et al.  and modion curve .(2)T=C L. alpinum extract. Non-cancer human epithelial fibroblast (BJ—ATCC CRL-2522) and double positive breast cancer cell line (MCF7—ATCC HTB-22) were cultivated in MEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. Triple negative breast cancer cell lines , lung cancer cell lines (H1792—ATCC CRL-5895 and SK-MES-1—ATCC HTB-58), colon cancer cell line DLD-1 (ATCC CCL-221) and prostate cancer cell lines (LNCAP—ATCC CRL-1740 and 22RV1 ATCC CRL-2505) were maintained in RPMI supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamine, while HCT116 colon cancer cell line (ATCC CCL-247) was cultivated in McCoy’s supplemented with 10% FBS and 1% Penicillin/Streptomycin. All cell lines were maintained in a humidified chamber, at 37 °C and 5% CO2. All the reagents needed for cell culturing were purchased from Gibco .A panel consisting of twelve human cell lines was chosen for the in vitro evaluation of L. alpinum extract stock solution was prepared at a concentration of 4 mg/mL by dissolving the necessary amount of freeze-dried extract in the appropriate culture medium for each cell line. Subsequent dilutions were prepared at specific concentrations needed for the different assays.The L. alpinum callus cultures’ methanolic extract was investigated using the MTT assay. Cells were grown at a density of 1 × 104 cells/well in a 96-well flat-bottom plate, each treatment having 3 replicates. After 24 h incubation, cells were treated with different concentrations of extract (6.25–400 μg/mL) for 48 h. To remove possible extract residues that might interfere with MTT assay, a wash step with Phosphate Buffer Saline 1× (PBS) was performed, then cell viability was determined after incubation for 3 h with 100 μL 1 mg/mL MTT. Afterwards, the solution was removed and 150 μL of DMSO were added to dissolve the crystals. The absorbance corresponding to the viable cells was measured at λ = 570 nm on a SPARK10M multiplate reader . A blank control (medium only), and a positive control (DMSO 100%), were included on every plate. The experiment was repeated three times. Statistical analysis was performed using Graphpad Prism (version 8) software . Cell viabilities were expressed as percentage of control (set as 100%) and represented as mean ± SEM. Data was analyzed by one-way ANOVA followed by Dunnett’s multiple-comparison test.The effect of L. alpinum extract (400 µg/mL) and incubated for 48 h, then the cell culture medium was replaced with treatment free medium. Cells were observed daily up to 14 days and when the colonies reached the proper size (more than 50 cells/colony) or when the colonies were too close to another, the experiment was stopped and the cells were fixed with methanol and stained with crystal violet  [t-test was applied to determine significant differences in colony numbers. *An equal number of cells (500 cells/well) were seeded in a 6-well plate, both for control and treatment. After 24 h the cells were treated with MO, USA) ,50. The L. alpinum extract (400 µg/mL), cells were analyzed in bright field with an inverted microscope. The same instrument was used in combination with a fluorescent source  for details regarding the cytoskeleton and nucleus, after staining these cell compartments with Phalloidin-FITC (for the cytoskeleton)  and DAPI (for the nucleus)  using a previously published protocol [To investigate the morphological changes induced by the 48 h treatment with L. alpinum callus cultures revealed high quantities of polyphenols, most of them being derivatives of glucaric and quinic acids. The four most abundant represent more than 57% (m/m) of the dry-frozen extract and around 15% (m/m) of the dry-frozen callus culture, demonstrating that in vitro culturing can be a useful alternative to produce natural compounds.The phytochemical profiling of L. alpinum callus cultures extract demonstrated interesting effects regarding its capacity to inhibit proliferation and colony formation. L. alpinum callus cultures extract showed considerable inhibitory effect on SK-MES-1, MDA-MB-231 and HCT116 cell lines, most probably being related to the high content in polyphenolic compounds which exhibit strong antioxidant capacity.The screening of cancer cell lines revealed that the L. alpinum as chemosensitizer might represent a perspective. To better understand the possible effects of leontopodic acids A and B, further research could also focus on evaluating the proliferation, migration or colony formation assays on cancer cells treated with these two natural products.Considering its abilities to inhibit cancer proliferation and colony formation, the use of Leontopodium alpinum Cass. (Edelweiss) and the crude extract, OSIM Romania Patent Application A/00740/17.11.2020.Cogălniceanu G. C., Mitoi E. M., Ciocan A. G., Holobiuc M. I., Maximilian R. C., Helepciuc F. E., Morosanu A. M. Biotechnological procedure for initiating and obtaining high proliferative cell mass producing bioactive compounds in


Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking—in part due to the difficulty in assessing migration using high‐throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the ‘identification of Migration control genes by differential Expression’. MigExpress exploits the combination of in‐depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration‐associated genes by their differential expression in slow‐ versus fast‐migrating cells. We applied MigExpress to investigate non‐small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration. Cell migration is an essential process in development and disease—prominently in cancer metastasis. We present MigExpress, a high‐throughput approach to systematically identify factors governing cell migration by combining migration quantification with molecular profiling. MigExpress on a panel of 54 NSCLC cell lines discovered differentially expressed mRNAs, circRNAs, and proteins between fast‐ and slow‐migrating cells, which controlled migration. BICC1bicaudal C homolog 1CDH1E‐CadherinCDH2N‐CadherincDNAcomplementary DNAcircRNAcircular RNACPA4carboxypeptidase 4DSEdermatan sulfate epimeraseECMextracellular matrixFLNAfilamin AFLNBfilamin BFLNCfilamin CFPKMfragments per kilobase per millionGOGene OntologyLOXL2lysyl oxidase‐like 2LUADlung adenocarcinomaMitCmitomycin CNSCLCnon‐small cell lung cancerp53tumor protein 53RNA‐seqRNA sequencingRT‐qPCRreverse transcription quantitative PCRshRNAshort hairpin RNAsiRNAsmall interfering RNATCGAThe Cancer Genome AtlasTUBB6tubulin beta 6VIMvimentin1Cell migration plays a central role in a large number of developmental and physiological processes. During embryogenesis, cell migration is essential for gastrulation or development of the nervous system . During Metastasis is a complex process in which cancer cells remodel the extracellular matrix (ECM), migrate into the vascular system, and move to distant sites where they establish metastatic nodules. Within a cell population, fast‐migrating cancer cells are likely to be more effective at giving rise to metastases than slower‐migrating cells . Lung caWhile it is thus important to understand the molecular mechanisms that govern cancer cell migration, the determination of cell migration activity in higher throughput is a challenging task. Hence, while thousands of screens have been carried out for cancer cell viability and proliferation, only a handful of screens have identified genes controlling cell migration. For preselected genes, migration screens have been carried out by introducing cDNAs , 16, 17 In the present study, we present MigExpress  as an effective approach to uncover genes governing cell migration by simultaneously quantifying cell migration and gene expression. Application of MigExpress to a broad panel of NSCLC cell lines discovers and functionally validates a novel set of genes essential for lung cancer cell migration.22.12 in a humidified incubator. Cells were obtained from ATCC  and / or fingerprint, and cells were regularly tested for mycoplasma.All 54 NSCLC cell lines were cultured in RPMI medium  supplemented with 10% FBS at 37 °C and 5% CO2.2m DiR . 1 million cells were incubated in 1 mL for 15 min at 37 °C followed by a washing step.Cells were incubated prior to seeding in medium with 2.5 µ2 in a humidified incubator to allow cell attachment before the stopper was removed. The subsequent steps were performed according to the manufacturer’s instructions. Directly after removing the stopper, the 0‐h time point was recorded for all DiR‐stained cell lines . After 24 and 48 h, this measurement was repeated. All plates were fixed after 48 h with 4% formaldehyde in PBS for 15 min at RT followed by DAPI staining with 300 nm DAPI  in PBS for 5 min. The mask was placed under the plate, and the stopper area was imaged using an Olympus Scan^R High Content Screening Station. The area was measured using imagej  and compared to a cohort of control wells determining the 0‐h time point (area of possible migration). Each cell line was tested at least in biological duplicates up to biological quintuplicates and technical duplicates up to technical quadruplicates.For all 54 cell lines, cell migration was investigated using the Oris™ Cell Migration Assay TriCoated (Platypus Technologies). Cells were stained prior to seeding with a live dye (DiR)  and stai2.3RNA was extracted using either the RNeasy Mini Kit , the Direct‐zol™ RNA MiniPrep Kit , or the Quick‐RNA™ MiniPrep Kit (Zymo Research). Whole‐cell RNA that was used for RNA‐seq experiments was isolated using RNeasy Mini columns (Qiagen).2.4The method has been described in detail before , 28. In 2.5m Urea, 40 mm Tris/HCl pH 7.6, 1X protease inhibitor complete mini‐EDTA‐free , 1× phosphatase inhibitors 1, 2, and 3 ). The protein concentration was determined by the bicinchoninic acid assay. The sample preparation for mass spectrometry included in‐solution tryptic digestion followed by solid‐phase extraction (SPE) peptide purification (SepPAK). Samples were tagged with TMT10plex™ reagent (Thermo Fisher Scientific™) [Cells at 80% confluency were scraped and lysed on ice in Tris/Urea lysis buffer (8 ntific™) , fractiontific™) , and subntific™)  on a TheData analysis was performed using MaxQuant (version 1.5.5.1) . To normFor the comparison of both RNA‐seq and mass spectrometry data between fast versus slow cell lines, the fold change was calculated for each coating  separately and the average fold change was used for candidate selection. For the determination of the statistical significance of differentially expressed genes, the fast versus slow cell lines were compared on the basis of their average migration capacity on the three coating surfaces.2.6https://david.ncifcrf.gov) [The pathway analysis was carried out using DAVID analysis online tool (crf.gov) , 37. The2.7m  of nontargeting control (siNT) or siPOOL of all targets (siTOOLs Biotech) using 6 µL Lipofectamine™ RNAiMAX  in 6‐well plates, or 0.2 µL/well in 96‐well plates. For the scratch assays, cells were seeded at ~ 50% confluency in collagen I precoated 96‐well cell culture plates  and reverse‐transfected. Cells from 6‐well plates were used for RNA extraction. siPOOL sequences are listed in Table For knockdown of the six targets using siPOOLs, cells were reverse‐transfected with 10 n2.8® S3 Live‐Cell Analysis System according to the manufacturer’s instructions. Every 1 h, pictures of each well were taken. For the analysis of different cell lines in the correlation graph, the analysis was performed manually. The size of the wound was measured at three positions over each scratched area between 0 h and up to 24 h. The migrated area was calculated by the subtraction of the average size of the gap (measured at the three positions) after 24 h from the average size of the gap (measured at the three positions) at 0 h. Each cell line was tested in biological duplicates and technical triplicates/quadruplicates.Scratch assays were performed using the WoundMaker™  and measured in the IncuCyte−1). Cells were reverse‐transfected with siPOOLs and plated at 50% confluency. After 24 h, the scratch wound was made with WoundMaker™ (Essen Bioscience) and imaged in the IncuCyte® S3 Live‐Cell Analysis System every 1 h for 24 h. For scratch assays in the presence of mitomycin C (Sigma‐Aldrich F1141), cells were reverse‐transfected in medium containing 10 µm MitC. After 24 h, the scratch was made and the wells were replenished with medium containing 7.5 µm MitC and images were recorded for 24 h at 1‐h intervals. All experiments were done in technical triplicates and biological triplicates to quintuplicates.For scratch assays after siPOOL‐mediated knockdown, the plate was initially coated with collagen I rat tail , hole fill (0.01), and adjust size (3). After analysis, information about wound confluence was exported for each well to every measured time point.After recording the wound closure within the IncuCyte2.9For cDNA conversion, 2 µg of total RNA was subjected to reverse transcription using Thermo Scientific™ RevertAid RNA Transcriptase using random primers. For real‐time qPCR, equal amounts of cDNA were used with Applied Biosystems™ SYBR™ Green PCR Master Mix and Applied Biosystems™ StepOnePlus™. The sequences of all primers are provided in Table 2.10Ten thousand cells were plated in duplicates for each condition and each time point (48 and 72 h) in 24‐well plates. After 48 and 72 h, cells were trypsinized and resuspended in complete medium. An equal volume of trypan blue was mixed with the cells, and the cells were counted in a Bio‐Rad TC20™ cell counter. The initial cells plated were taken as cell count at 0 h.2.11Circular RNA expression had been previously analyzed in the RNA‐seq dataset of the 54 cell lines , 38. Dif33.1A panel of 54 NSCLC cell lines enriched for LUAD was screened for their migration capacities on different matrix surfaces  using the ORIS™ migration assay to quantitatively and robustly assess the migration capacity of each cell line. The cell lines were classified into ‘fast’, ‘medium’, and ‘slow’ cell lines depending on their migration properties. In parallel, the cell lines were subjected to RNA sequencing and mass spectrometric analyses for transcriptomic and proteomic profiling, respectively. Gene expression differences were determined from the RNA‐seq and mass spectrometry data to identify genes that were significantly up‐ or downregulated between the cells classified as fast or slow. The overall workflow of MigExpress is summarized in Fig. As an example, representative images of slow (NCI‐H1623) versus fast (NCI‐H2009) cell lines from the ORIS™ migration assay for 0, 24, and 48 h time points show an apparent difference in their migration capacities Fig. . In thisR = −0.10; Fig. Next, we further validated the platform for the quantitative analysis of cell migration. Cell proliferation is considered as a potentially confounding factor for the analysis of cell migration as cells proliferating faster could also enter uncovered areas faster. Hence, we also determined the proliferation rate of all 54 cell lines in terms of their cell doublings within 72 h. A correlation analysis of the proliferation rate and the migration capacity verified that there was no relevant correlation of cell proliferation with cell migration  was observed between the migration capacities of the NSCLC cells determined on different matrix surfaces  and found a very high correlation (R = 0.86) for the fold change between fast versus slow cell lines between the transcript and the protein level  displayed an overlap with the proteomic hits with 14 upregulated and five downregulated genes Fig. . ImportaIn summary, MigExpress identified candidates linked to cell migration by combining differential migratory capacities of the broad panel of cell lines to their transcriptomic and proteomic profiles.3.3CDH2 (N‐Cadherin) [CPA4 (carboxypeptidase 4 [FLNC (filamin C [TUBB6 (tubulin beta 6 [DSE [CPA4) and was not detected by mass spectrometry. BICC1  , which htidase 4 , 44), FLilamin C , 46), ann beta 6 ) as canda 6 [DSE , 49) hadomolog 1 , 51) wasAll six selected genes were upregulated in the fast compared with the slow cell lines in our RNA‐seq, RT‐qPCR, and mass spectrometry data Fig. . From Th3.4® migration assays uncovered a significant impact of each of the knockdown of the selected genes with a significant decrease in the cell migration of NCI‐H1792 cells , the overall expression of FLNC was found lower compared with the higher expressed FLNA and FLNB , we identified at the gene level  based on the analysis of back‐splicing reads , 38. Wheel Table  28 genesel Table . At the el Table , we founel Table . We seleel Table E,G.4Cell migration is a fundamentally important process in health and disease. Therefore, the systematic evaluation of cell migration and identification of genes controlling this process in a high‐throughput fashion is required, but has so far been a difficult task. In our study, we designed and implemented an effective method called MigExpress to successfully tackle this challenge. MigExpress  combines two quantitative analyses: cell migration and molecular profiling. The cell migration analysis is performed in parallel for a large number of cell lines using the robust and quantitative ORIS™ assay. The cell lines can also be grown on different matrix surfaces to monitor their migratory behavior based on extracellular signaling and attachment. The molecular profiling is performed by subjecting the same panel of cell lines to transcriptomics  and / or proteomics . Genes differentially expressed between fast and slowly migrating cell lines can then be identified as candidate genes controlling cell migration and can then be individually scrutinized for validation.Metastasis is the leading cause of death in most cancers, and it occurs when cancer cells migrate from the primary site and form new tumor nodules at a secondary site . Yet, ouThe established standard assays for analyzing cell migration are the scratch / wound‐healing assay or the Boyden chamber assay. However, both methods come with limitations—the scratch assay has limited reproducibility due to the difficulties in producing even scratches and the Boyden chamber assay is time‐consuming and cannot be used for high‐throughput studies . We findRegarding the surface coating, we observe that the migration capacities of the NSCLC cell lines are largely independent of the matrix surface, which suggests that cancer cells have inherent molecular properties that render them more migratory, which may not be influenced by ECM signaling. For example, a p53 mutation increases the migration capacity of cells by regulating Rho GTPase family proteins , 61.For the molecular profiling, we evaluated the expression levels of 58 096 transcripts, 148 811 circRNAs, and 9304 proteins using replicates of RNA‐seq and mass spectrometry, respectively, in all 54 cell lines. The observed expression levels between transcriptomics and proteomics correlated very well, while the overlap in candidate hits was lower due to the stringent expression cutoff in both analyses. Thereby, we identified genes differentially regulated between fast‐ and slowly migrating cells. This reflects two important advantages of MigExpress: (a) It covers the entire transcriptome and / or proteome allowing the identification of a broad spectrum of genes at the RNA (both protein‐coding and noncoding) and protein levels, and (b) it employs a large number of cell lines allowing the identification of generally or broadly relevant factors not restricted to single‐cell lines. Indeed, this comprehensiveness is unique among the previously described screens, which were either restricted by a small number of cell lines and / or the limited number of genes queried , 24, 25.The statistical analysis identified 84 and 90 differentially regulated candidate molecules between fast‐ and slow‐migrating NSCLC cell lines at the RNA and protein level, respectively. Hence, transcriptomics and proteomics identified comparable numbers of candidates, although the transcriptomic analysis quantified a larger number of genes due to its higher sensitivity but many of these were lowly expressed and hence filtered out in our stringent analysis. GO analysis of the genes upregulated in fast cell lines revealed the significant enrichment of several terms such as ECM organization, signal transduction, cell migration, and cell adhesion , 63, 64.Beyond this state of the art, MigExpress importantly also identifies genes not previously linked to cell migration. To further validate these novel players in lung cancer cell migration, we selected five candidates—DSE, CPA4, FLNC, TUBB6, and BICC1—and the positive control CDH2, which were upregulated in fast cells and also associated with poor overall survival in NSCLC patients. Indeed, all of these genes possess strong pro‐migratory properties. The siPOOL‐mediated knockdown of these genes in five different NSCLC cell lines reduced the cell migration capacities to varying degrees. Notably, this effect was independent of cell proliferation, which was not significantly affected by the knockdown, nor did mitomycin C treatment affect the observed migration phenotypes.Filamin C (FLNC) is one migration factor from our MigExpress study with a striking impact. To further evaluate the specificity of our findings, we carefully looked at the filamins—a family of actin‐binding proteins that enhance the stability of the cytoskeleton and are involved in mechanotransduction, cell adhesion, and migration . They coAs a future outlook, similar approaches combining the molecular profiling and functional screening of larger sets of cell lines can also be used to discover new factors governing other cellular properties such as proliferation, apoptosis, or drug response or the impact of molecular aberrations such as canonical or noncanonical mutations on the expression of cancer genes , 76, 77.5in vivo. As a comprehensive resource, we provide a deep transcriptomic and proteomic profile of a NSCLC cell line panel and a quantitative assessment of their proliferation and migration properties. For the future, MigExpress can be broadly employed to uncover migration control genes across all fields of life science research.In summary, we developed, applied, and validated MigExpress as a method to systematically identify genes impacting the highly relevant process of cell migration by combining quantitative molecular profiling with robust quantification of cell migration in a broad panel of cell lines. As a proof of principle, we successfully discovered and validated multiple genes essential in lung cancer cell migration. Future studies will need to unravel whether these factors differentially expressed and regulating migration in cell culture correlate with the metastatic potential S.Di. is co‐owner of siTOOLs Biotech GmbH, Martinsried, Germany. All other authors disclose no competing interests.JP analyzed, validated, and extended the migration screen; ACB performed and analyzed the migration screen; SDh, JS, and MA conducted and analyzed experiments; YS analyzed the transcriptome data; JB, CM, CL, and BK performed and analyzed the proteome data; SDi conceived, coordinated, and supervised the project; and JP and SDi wrote the manuscript and all authors read, edited, and approved the manuscript.Not applicable.Not applicable.Fig. S1. Migration assay validation. A. Standard error of mean (SEM) plotted for replicate experiments of each cell line grown on three different coated matrices. The mean is represented by red solid line. The red dashed line represents SEM of 5. B. Lack of correlation between proliferation of the 54 NSCLC cell lines at 72 h and the percentage migration from ORIS™ assay (uncoated). R represents Pearson’s correlation coefficient. C. Representative images of 18 further fast and slow cell lines used for validation in the IncuCyte® scratch assay at 0 h, 24 h and 48 h.Fig. S2. Gene expression validation in fast versus slow NSCLC cell lines. A‐C. Genes downregulated in fast cell lines compared to slow cell lines from RNA‐seq (n = 54) and RT‐qPCR (n = 16) data – A. CEACAM6, B. PRR15L, C. AGR2. D‐F. Genes previously reported as regulators of NSCLC cell migration – D. VIM, E. LOXL2, F. CDH1. Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001.Fig. S3. Gene Ontology (GO) analysis for pathway enrichment of candidate genes. A. GO analysis of upregulated genes from RNA‐seq and mass spectrometry data (n = 98). B. GO analysis of downregulated genes from RNA‐seq and mass spectrometry data (n = 76). All pathways selected are statistically significant  and have a minimum of four genes enriched in the pathway.Fig. S4. Effect of knockdown of candidate genes on NCI‐H2009 cell migration. A. Knockdown verification by real‐time qPCR (n = 3). Expression in cells treated with the nontargeting (NT) siPOOL was normalized to 100%. All genes show efficient knockdown. –Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. B‐G. Scratch assay results for knockdown of candidate genes (n = 4) ‐ B. CDH2, C. DSE, D. CPA4, E. FLNC, F. TUBB6, G. BICC1. NT1 and NT2 denote technical replicates of siNT (nontargeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of the candidate genes except TUBB6.Fig. S5. Effect of knockdown of candidate genes on NCI‐H1666 cell migration. A. Knockdown verification by real‐time qPCR (n = 3). Expression in cells treated with the nontargeting (NT) siPOOL was normalized to 100%. All genes show efficient knockdown. Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. CDH2 shows efficient knockdown but is not statistically significant. B‐G. Scratch assay results for knockdown of candidate genes (n = 4) ‐ B. CDH2, C. DSE, D. CPA4, E. FLNC, F. TUBB6, G. BICC1. NT1 and NT2 denote technical replicates of siNT (non‐targeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of all six candidate genes.Fig. S6. Effect of knockdown of candidate genes on HCC‐827 cell migration. A. Knockdown verification by real‐time qPCR (n = 3). Expression in cells treated with the non‐targeting (NT) siPOOL was normalized to 100%. All genes show efficient knockdown. Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. CDH2 knockdown was not statistically significant. B‐G. Scratch assay results for knockdown of candidate genes (n = 5) ‐ B. CDH2, C. DSE, D. CPA4, E. FLNC, F. TUBB6, G. BICC1. NT1 and NT2 denote technical replicates of siNT (non‐targeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of the candidate genes ‐ DSE, FLNC and BICC1.Fig. S7. Effect of knockdown of candidate genes on NCI‐H838 cell migration. A. Knockdown verification by real‐time qPCR (n = 3). Expression in cells treated with the non‐targeting (NT) siPOOL was normalized to 100%. All genes show efficient knockdown. Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. B‐G. Scratch assay results for knockdown of candidate genes (n = 3) ‐ B. CDH2, C. DSE, D. CPA4, E. FLNC, F. TUBB6, G. BICC1. NT1 and NT2 denote technical replicates of siNT (non‐targeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of the candidate genes except CDH2 and CPA4.Fig. S8. Effect of knockdown of candidate genes on NCI‐H1792 cell proliferation and migration. A‐B. Cell proliferation assessed with trypan blue counting at 0 h, 48 h and 72 h after siPOOL‐mediated knockdown of the six candidate genes (n = 3). F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. Knockdown of all genes show no effect on proliferation for all time points, except for a minimal decrease for siBICC1 condition at 72 h. C‐H. IncuCyte® cell migration assay with siPOOL‐mediated knockdown of candidate genes in the presence of Mitomycin C (7.5–10 µm) for ‐ C. CDH2, D. DSE, E. CPA4, F. FLNC, G. TUBB6 and H. BICC1. NT1 and NT2 denote technical replicates of siNT (non‐targeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of all six candidate genes.Fig. S9. Effect of knockdown of candidate genes on NCI‐H2009 cell proliferation and migration. A‐B. Cell proliferation assessed with trypan blue counting at 0 h, 48 h and 72 h after siPOOL‐mediated knockdown of the six candidate genes (n = 3). F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. Knockdown of all genes show no effect on proliferation for all time points. C‐H. IncuCyte® cell migration assay with siPOOL‐mediated knockdown of candidate genes in the presence of Mitomycin C (7.5–10 µm) for ‐ C. CDH2, D. DSE, E. CPA4, F. FLNC, G. TUBB6 and H. BICC1. NT1 and NT2 denote technical replicates of siNT (non‐targeting siRNA). Mean and SEM are shown. F‐test followed by t‐test was done for each time‐point, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001. A significant reduction in migration capacity was observed upon knockdown of DSE, CPA4 or FLNC, while a non‐significant trend to reduction was noted for the other candidates.Fig. S10. Survival and staging analyses of FLNA and FLNB expression in patient samples. A. Kaplan‐Meier survival plot of TCGA NSCLC patient samples with FLNA high versus low expression. Log rank‐test P‐value is not significant. B. Kaplan‐Meier survival plot of TCGA NSCLC patient samples with FLNB high versus low expression. Log rank‐test P‐value is not significant. C. Expression of FLNA in different stages of NSCLC patient samples from TCGA. D. Expression of FLNB in different stages of NSCLC patient samples from TCGA. Statistical test: One‐way ANOVA followed by Dunnett’s test not significant. P‐value for test‐of‐trend is not significant.Fig. S11. circRNA profiling of NSCLC cell lines. A. Pipeline for the identification of genes with differential circRNA expression  from RNA‐seq data. The analysis identified 28 candidate circRNAs based on expression, regulation and significance at the gene level. B. Volcano plot for 4072 genes that have a minimum expression of circRNAs detected in ≥ 50% of cell lines. The red dots represent genes with significantly upregulated circRNAs  and the green dots represent genes with significantly downregulated circRNAs . Candidates selected for validation are indicated with larger circles. C. Pipeline for the identification of differentially expressed circRNAs  from RNA‐seq data. The analysis identified 33 individual differentially expressed circRNAs based on expression, regulation and significance. D. Volcano plot for 5050 circRNAs that have been detected in ≥ 50% of cell lines. The red dots represent significantly upregulated circRNAs  and the green dots represent significantly downregulated circRNAs . Candidates selected for validation are indicated with larger circles. E‐G. RT‐qPCR validation of selected circRNA candidates at the back‐splice level in eight fast versus eight slow cell lines. The RNA‐seq level expression is also provided for the same cell lines. Statistical test: t‐test with Welch’s correction, P‐values: *P < 0.05, **P < 0.01, ***P < 0.001.Click here for additional data file.Table S1. siPOOLs. Sense and antisense sequences of 30 siRNAs used against each gene ‐ CDH2, DSE, CPA4, FLNC, TUBB6, BICC1, FLNA and FLNB.Table S2. Primers. Forward and reverse primer sequences for all genes tested by real time‐qPCR.Table S3. Summary of ORIS™ assay for 54 NSCLC cell lines. Values for the assay were calculated for each cell line grown on each matrix surfaces. The table contains average values from all biological replicates. Orange, blue and green represents slow, medium and fast cell lines.Table S4. RNA‐seq data of 54 NSCLC cell lines. RNA‐seq (log2FPKM) expression data for 58096 genes for 54 NSCLC cell lines. Fold change values, significance and correlations with different coatings and proliferation for each gene are also provided.Table S5. Candidate genes from RNA‐seq data regulated between fast and slow cell lines. List of 84 candidate genes obtained from RNA‐seq data through the filtration criteria described . Log2FPKM values provided for 18 fast and 26 slow cell lines.Table S6. RT‐qPCR validation of RNA‐seq data. Total of 41 genes were tested in 8 fast and 8 slow cell lines by RT‐qPCR. F‐test followed by t‐test was done for fast versus slow cell lines. All genes are significantly differentially regulated in the RNA‐seq data.Table S7. Mass spectrometry data of 54 NSCLC cell lines. Protein expression data for 9304 genes for 54 NSCLC cell lines. Fold change values, significance and correlations with different coatings and proliferation for each gene are also provided.Table S8. Candidate genes from mass spectrometry data regulated between fast and slow cell lines. List of 90 candidate genes obtained from mass spectrometry data through the filtration criteria described . Expression values provided for 18 fast and 26 slow cell lines.Table S9. Summary of analyses with candidate genes – CDH2, DSE, CPA4, FLNC, TUBB6 and BICC1.Table S10. circRNA expression analysis at the gene level from RNA‐seq data of 54 NSCLC cell lines. Back splicing reads per gene normalized to the sequencing library size for 12251 genes in 54 NSCLC cell lines [ll lines , 38. FolTable S11. Differentially expressed circRNAs at the gene level regulated between fast and slow cell lines. List of 28 genes obtained from RNA‐seq data with differential circRNA expression selected through the filtration criteria described (Supplementary figure 11A). Expression values are provided for 18 fast and 26 slow cell lines which showed these fast or slow properties on the majority of coatings.Table S12. circRNA expression analysis at the back‐splice level from RNA‐seq data of 54 NSCLC cell lines. Back splicing reads per individual circRNA (back‐splice site) normalized to the sequencing library size for 148811 circRNAs in 54 NSCLC cell lines [ll lines , 38. FolTable S13. Differentially expressed circRNAs regulated between fast and slow cell lines detected at the back‐splice level. List of 33 candidate circRNAs obtained from RNA‐seq data and selected through the filtration criteria described . Expression values provided for 18 fast and 26 slow cell lines which showed these fast or slow properties on the majority of coatings.Click here for additional data file.


Aquaporin 3 (AQP3) is the membrane channel of water and involved in fluid homeostasis. The aim of this study was to reveal the expression and significance of AQP3 in cutaneous lesions. We analyzed AQP3 mRNA levels using RT-PCR in 311 cutaneous lesions and confirmed AQP3 expression in these lesions by immunohistochemistry. AQP3 mRNA was detected in normal epidermis, seborrheic keratosis, solar keratosis, Bowen's disease, squamous cell carcinoma, eccrine poroma, apocrine carcinoma, and sebaceoma; however, AQP3 mRNA was absent in basal cell carcinoma, nevocellular nevus, or malignant melanoma. By immunohistochemistry, diffuse AQP3 expression was seen in all keratotic lesions including seborrheic keratosis, verruca vulgaris, molluscum contagiosum, solar keratosis, Bowen's disease, and squamous cell carcinoma. Diffuse AQP3 expression was also present in all extramammary Paget's disease. No AQP3 staining was obtained in basal cell carcinoma. Positive AQP3 staining was seen in sweat gland tumors including hidradenoma, eccrine poroma, and apocrine carcinoma. Among sebaceous tumors, AQP3 expressed diffusely in all sebaceous hyperplasia and sebaceous adenoma, but not in sebaceous carcinomas. Only focal AQP3 staining was seen in nevocellular nevus and no AQP3 staining in melanoma. Our findings indicate the function of AQP3 maintained in most skin tumors. AQP3 may be used for differential diagnosis in skin tumors. Aquaporin 3 (AQP3) is a water-transporting aquaglyceroporin and plays a significant role in physiologic functions of reabsorption and secretion in the kidney , 2, epidSkin cancer is one of the most common human malignancies with increasing incidence worldwide –16. SkinIn a recent study , we founOur study was approved by the local research ethics committee of Peking University Cancer Hospital & Institute. We included 311 surgically resected skin lesions from the routine surgical pathology file of Peking University Cancer Hospital & Institute, including 74 benign nonneoplastic lesions , 40 benign skin tumors , and 197 premalignant lesion and malignant tumors . The cases are summarized in Total RNA was isolated from various formalin-fixed and paraffin-embedded skin lesion tissues with Recover ALL™ Total Nucleic Acid Isolation Kit . We designed the specific PCR primers targeted for AQP3 and PGK1  as shown in The immunohistochemical staining was performed as described previously . The rabWe first detected and analyzed the expression pattern of the AQP3 mRNA in the normal squamous epithelium and skin lesions by RT-PCR. As shown in We used the sections from normal kidney tissue as a positive control. The expression of AQP3 was specifically localized in the cytoplasmic membrane of the collecting duct cells of the kidney  and squan = 14), verruca vulgaris (n = 16), molluscum contagiosum (n = 5), and sebaceous hyperplasia (n = 13) showed diffuse staining for AQP3 , one of the aquaglyceroporins, is responsible for transporting water and glycerol and maintaining fluid homeostasis in normal tissues –20. In oNo expression of AQP3 in basal cell carcinoma and sebaceous carcinoma might help to elucidate their histogenesis and/or pathogenesis. Basal cell carcinoma usually arises from the lowermost layer of the epidermis and less commonly from the outer root sheath of the pilosebaceous unit. Basal cell carcinoma cells share many common features with follicular epithelium such as hair bulbs, follicular bulges, and follicular matrix cells . In our The absent expression of AQP3 in basal cell carcinoma and sebaceous carcinoma also has some diagnostic value. In clinical practice, sometimes, it is a big challenge to distinguish basal cell carcinoma from basaloid squamous cell carcinoma or metaplastic basal cell carcinoma from poorly differentiated squamous cell carcinoma. AQP3 immunohistochemical staining is helpful in this situation. Since both basal cell carcinoma and sebaceous carcinoma show negative AQP3 staining, AQP3 is not useful to distinguish basal cell carcinoma with sebaceous differentiation from sebaceous carcinoma. As for sebaceous tumors, which consist of sebaceoma, sebaceous adenoma, and sebaceous carcinoma, their differential diagnosis depends mainly on histological architectural, cytological features, and mitotic rate. Only immunoreactivity of p53 has been reported to act as a useful marker to distinguish between benign and malignant sebaceous carcinomas , 23. In It is not surprising that malignant melanoma shows no expression of AQP3 as normal melanocytes within the epidermis do not show AQP3 expression as demonstrated by our prior study .In summary, our results showed that AQP3 is expressed in most skin tumors except basal cell carcinoma, sebaceous carcinoma, and malignant melanoma, reflecting the biological characteristic of skin tumors. AQP3 has some diagnostic utility in differential diagnosis of skin tumors.


The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar. The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I  and three class II genes , were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2, were identified. Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope–major histocompatibility complex interactions in pigs.Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens. We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase ( Antigen presentation by major histocompatibility complex (MHC) class I and II molecules is an essential process in adaptive immune interactions that triggers T cell proliferation and antigen-specific cellular immune responses . MHC claMHC genes are extremely polymorphic and generating distinctive structural variations, especially in the antigen binding pockets that interact with antigenic peptides by anchoring amino acids . A singlThe activation of primary T cells requires the integration of three distinct signals, namely the three-signal theory of activation including antigen recognition, costimulation, and cytokine-mediated differentiation and expression . In thesDetermination of MHC alleles is important to predict adaptive immune responses of an individual. The genomic sequence-based high-resolution typing methods for SLA genes have been developed . Howeverin vivo compared to the cell-free system . The disaggregated cells were washed in PBS and cultured in six-well plates using Dulbecco’s Modified Eagle’s Medium (DMEM)  with 10% fetal bovine serum (FBS)  and 20 μg/ml gentamycin . Subsequently, the cells were cultured in DMEM supplemented with 10% FBS and a 1% penicillin-streptomycin antibiotic mixture (Gibco). All experimental procedures were approved and performed accordance with the guidelines and regulations set by the Institute of Animal Care and Use Committee (IACUC) and the Center for Research Ethincs of Konkuk University (KU20229).Tissue samples (ear notch or lung tissue) were obtained from five purebred landraces on local farms, two NIH miniature pigs, one PWG micropig , and one Seoul National University miniature pig  . All ani6 cells using an AllPrep DNA/RNA Mini Kit  according to the manufacturer’s protocol. Genomic DNA-based high-resolution typing of SLA-1, SLA-2, SLA-3, SLA-DQA, SLA-DQB1, and SLA-DRB1 was conducted for each gene as previously described  following the manufacturer’s protocol. Cell selection occurred after 48 h when transfected cells were placed under selective pressure by adding 2 μg/ml puromycin  for 4 weeks. The cells were transferred and cultured in DMEM supplemented with 10% FBS and a 1% penicillin-streptomycin antibiotic mixture (Gibco) at 37°C and 5% CO2. Cells were frozen overnight at −80°C in DMEM containing 20% FBS and 10% dimethyl sulfoxide, without antibiotics, and then stored in liquid nitrogen. Before use, the cells were rapidly thawed in a water bath at 37°C.Cells were transfected with ERT-Puro  using Li5 cells/well) were seeded in a six-well plate and cultured in the same conditions as described for cell selection. Cells were trypsinized at 24, 48, and 72 h post-seeding, counted using the trypan blue exclusion method .Cells  and subjected to cDNA synthesis by SuperiorScript III Reverse Transcriptase  according to the manufacturer’s protocol. PCR was performed in a 20 µL reaction mixture containing 2 µL cDNA, 0.5 µM primers, 0.5 U of Supertherm DNA polymerase (JMR Holdings) in 1.2x PCR buffer (1.5 nM MgCl2), and 0.1 mM dNTPs using the ABI 9700 Thermocycler (Applied Biosystems). The amplification primers for hTERT and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were hTERT-F (5′- GCCGAGACCAAGCACTTCCTCTACT-3′) and hTERT-R (5′-GCAACTTGCTCCAGACACTCTTCCG-3′), and GAPDH-SP-F (5′-GCTACACTGAGGACCAGGTTG-3′) and GAPDH-SP-R (5′- AGGAGATGCTCGGTGTGTTG-3′), respectively. The PCR profile consisted of an initial denaturation cycle of 5 min at 95°C, 35 cycles for 30 s at 95°C, 30 s at 63°C, 45 s at 72°C, and a final extension of 5 min at 72°C for hTERT. The cycling conditions for GAPDH was similar to that of hTERT except that the denaturation cycle was 3 min (at the same temperature), the number of annealing cycles was 25 at 58°C, and extension was performed for 30 s at 72°C. Subsequently, PCR products were electrophoresed on 1% agarose gel and band intensity was compared between samples.Total RNA from 2 × 10SLA-1, SLA-2, SLA-3, and SLA-DRB1 were SLA1-e1F1 and SLA-e4R4, SLA2-e1F and SLA-e4R4, SLA1/3f#92 and SLA3r#121, CpDRB5a and CpDRB3b, respectively  treatment, and RNA quality was analyzed on 2% formaldehyde agarose gel. Reverse transcription was performed in a 20 μL reaction mixture using 1 μg of total RNA with oligo-(dT)15 and SuperScript® III Reverse Transcriptase , for 50 min at 50°C with inactivation for 15 min at 72°C. In contrast to the materials used in SLA typing, the target cDNA was amplified using 2 μL of cDNA with Pyrobest polymerase . The primers used to amplify the entire coding region of ectively   in PBS for 30 min at room temperature. The cells were incubated with a mouse monoclonal antibody specific to pig SLA Class I  at 1:250 dilution in PBS with 2.5% BSA at room temperature for 2 h. After washing in PBS, cells were incubated with goat anti-mouse antibody conjugated with Alexa Fluor 568 (Invitrogen) at a dilution of 1:500 in PBS containing 3% BSA for 1 h at room temperature in the dark. After washing in cold PBS, cover slips were mounted with Vectashield . The cells were observed under a fluorescence microscope .Cells were seeded on glass cover slips in 6-well plates at a density of 1.2 × 10SLA-1, SLA-2, and SLA-3, and three SLA class II genes, SLA-DQA, SLA-DQB1, and SLA-DRB1, from 10 individuals of five different breeds , by the ISAG SLA nomenclature committee.To establish a panel of cell lines with diverse SLA haplotypes, we conducted high-resolution typing of three SLA classical class I genes, t breeds . A totalSLA-1, SLA-2, SLA-3, SLA-DQA, SLA-DQB1, and SLA-DRB1 was 90%, 80%, 60%, 50%, 70%, and 60%, respectively, with SLA-1 and DQA showing the highest and the lowest heterozygosity, respectively. The typing results for individuals corresponding to iKUF cells 02, 04, 06, and 09 showed the presence of three alleles in SLA-1, indicating the presence of the duplication haplotypes. The allelic constitution for each SLA gene was consistent to the reported allelic frequencies of the corresponding breeds   by subtracting the identified haplotype of iKUF04 from the genotyping results of iKUF03. For the rest of the cell lines, we were unable to separate the combined genotyping results of the six SLA genes into individual haplotypes due to the unavailability of family typing results.In our cell panel, iKUF04, which originated from an inbred NIH miniature pig, was homozygous across all six loci (: 02:01) . Based ohTERT expression construct. After culturing for 4 weeks in the selection medium containing puromycin, ∼10–50% of cells survived. A small number of cells were found to grow rapidly from enlarged and flattened senescent cells when they were relaxed in a puromycin-free culture. These cells were isolated and cultured until passage number 20 to make a homogeneous population. The passage numbers of the established fibroblast cell lines ranged from 30 to 45 . All four cell lines grew well on soft agar, with PK15 forming numerous colonies. In contrast, the  culture .SLA-1, SLA-2, and SLA-3, and a class II gene, SLA-DRB1, using semiquantitative RT-PCR , we analyzed the expression of three SLA class I genes, e RT-PCR . Seven omulation .SLA-1, -2, and -3 between iKUF08, iKUF09, and iKUF10 cells before and after immortalization (SLA-1 expression. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The expression of SLA classical class I gene was also analyzed at the protein level by immunostaining with Pan-SLA classical class I-specific antibodies (We compared the expression levels of lization . In bothtibodies . AlthoughTERT-transformed cells have extended cellular lifespans and are effectively used to prepare cells in sufficient number while maintaining their biological properties (hTERT-transformed porcine fibroblast cell lines, conducted high resolution analysis of the major classical class I and class II SLA genes, including SLA-1, SLA-2, SLA-3, SLA-DQB1, SLA-DRB1, and SLA-DQA, and characterized the expression patterns of three class I genes and DRB1 in each cell line. However, owing to the unavailability of MHC haplotype reference information, we were unable to reliably characterize the haplotypes of most of the six SLA genes in pigs. Together with other publicly available pig cell lines (Epstein–Barr virus-transformed B-lymphoblastoid cell lines and extensive curation have been important developments in the molecular characterization of MHC genes and MHC-antigen interactions related to HLA . Similaroperties . In thisll lines , immortaSLA-1 duplication in certain haplotypes, are not well studied in pigs (Except for nucleotide substitutions, structural variations in the SLA gene region, including  in pigs . To coll in pigs . In pigs in pigs . Limited in pigs .The use of different typing methods between laboratories can contribute to heterogeneity in typing results . The useUnderstanding the kinetics of epitope binding to MHC molecules is important for the development of effective vaccines. However, resources for the systematic analysis of interactions between antigenic epitopes and SLA molecules are limited in pigs. We previously reported that the affinity of PCV2 ORF2 as an epitope to MHC class II molecules varied depending on the MHC haplotype  and showhTERT transfected porcine alveolar macrophages (Our immortalized fibroblast cell lines showed lower growth rates than PK15 and did not form colonies under the anchorage-independent culture condition, suggesting greater similarity in phenotype to primary cells than available ATCC fibroblast cell lines. This is consistent with the results of a previous study on rophages . These cin vitro analysis. In vivo studies are required to describe the natural variation of SLA class I gene expression among individual pigs of different SLA haplotypes. Consistent to our observation, haplotype-specific expression patterns of SLA class I genes have been previously reported (Resilience after pathogenic infection is likely to be affected by an individual’s immune capacity. Considering the functionality of SLA molecules, their expression should affect the consequence of adaptive immune responses and general animal health. The expression of MHC class I molecules varies depending on cell types and tissue origins . Our resreported . Our fibreported .HLA-DR and proto-oncogene c-myc. The expression of MHC genes could be influenced by the rate of cell proliferation and may explain the increased expression of SLA class I genes in the immortalized fibroblast cell lines.In this study, the expression levels of SLA class I genes were slightly higher in immortalized cells than in primary cells . Modo etThe development of diverse cell lines with various MHC haplotypes could be useful for immunogenetic analyses in pigs but the availability of these cell lines has been limited. Fibroblasts are relatively easy to isolate, culture, and immortalize compared to other livestock cell types and, therefore, appropriate to develop stable cell lines with confirmed MHC genotypes. Studies on variations in the genetic structures of SLA and their expression using reference cell lines have great potential to enhance our knowledge regarding the adaptive capacity and regulatory elements of the porcine immune system. Although MHC genes of mammalian species are evolutionary conserved, the organization of the MHC region in pigs differs from others in that MHC class II and I genes are separated by a centromere . The imm


Cell biology, plant-based extracts, structural chemistry, and laboratory in vitro or in vivo experiments are the principal aspects or interfaces that can contribute to discovering new possibilities in cancer therapy and to developing improved chemotherapeutics. Forestry residues can be used for their wealthy resource in polyphenols and other phytoconstituents known for anticancer properties. This review is designed to bring together information on the in vitro or in vivo anticancer potential of woody vascular plants especially the bark extracts (BE) and biosynthesized metallic nanoparticles (BMN) using bark extracts. Type of extracts, main phytoconstituents found in extracts responsible for the anticancer activity, and targeted cancerous cell lines were followed. The literature data were collected via Clarivate Analytics, Science Direct, PubMed, and Google Academic (2011–2021). The search terms were: bark extracts, metallic nanoparticles, silver nanoparticles, gold nanoparticles, anticancer, cytotoxic activity, antiproliferative effect, and antimetastatic potential in vitro and in vivo. All of the search terms listed above were used in different combinations. The literature data highlight the efficaciousness of the BE and BMN as anticancer agents in in vitro experiments and showed the mechanism of action and their advantage of nontoxicity on normal cells. In vitro testing has shown promising results of the BE and BMN effect on different cancer cell lines. In vivo testing is lacking and more data is necessary for drug development on animal models. Today, cancer, or malignant disease is an alarming situation because of the uncontrolled and abnormal growth of cancer cells. The most undesirable complication of cancer is metastasis and unfortunately this causes death. High mortality in cancer patients is still persistent and the statistics are not very encouraging . Drug thCancer is one of the pathological conditions in which apoptosis plays an important role, whether it is abnormal apoptosis or a defective one. That leads to unsuppressed proliferation, which results in cancerous cells that will not naturally die. For that, scientists focused their attention on anticancer strategies for targeting the uncontrollable production of malignant cells and the disorder of apoptosis ,8. The cTaxus brevifolia L., and Vincristine, Vinblastine, and Vinorelbine  extracted from Catharanthus roseus G. Don should remind researchers of the endless source of plants as anticancer agents [There is a real need to discover new botanical products for developing new anticancer agents. The chemical composition of plants is still poorly studied and this should be the most important source because of their potency in the pharmacological field. Because plants have been traditionally used by humans in healing processes for centuries, and many of them have success that should be an imperative calling for assiduous research in this field. It is necessary to develop new bioactive compounds to meet the need for new drug therapies. For example, Paclitaxel (Taxol) obtained from r agents .The current tendency in biotechnology is to recycle and use environmentally friendly products. In this context, forest waste is now widely appreciated as a rich source of natural polyphenols, with therapeutic potential . PolypheBesides classical plant bark extracts (BE), biosynthesized metallic nanoparticles (BMN) are synthesized using ecofriendly and green-chemistry ways. These BMNs present a new interest for nanomedicine, as they are being intensively studied. It was found that BMN are potent antioxidants and antimicrobial and anticancer agents .Cancer chemoprevention implicates the use of pharmacological intervention to prevent, inhibit, or reverse carcinogenesis with synthetic or natural chemical products. Less than one percent of an estimated 250.000 plants has been screened pharmacologically. The use of phytochemicals present in medicinal plants as antioxidants possess no toxicity as compared to modern drugs and are efficient against different types of cancers with no side effects ,15,16. TThe literature data were collected via Clarivate Analytics, Science Direct, PubMed, and Google Academic (2011–2021). The search terms were: bark extracts, metallic nanoparticles, silver nanoparticles, gold nanoparticles, anticancer, cytotoxic activity, antiproliferative effect, and antimetastatic potential in vitro and in vivo. All of the search terms listed above were used in different combinations.https://app.biorender.com/, accessed on 31 August 2021) [BioRender was used for drawing the figures from manuscripts (st 2021) .The plant kingdom includes a multitude of secondary compounds such as polyphenols, alkaloids, flavonoids, lignans, terpenes, and taxanes. The list continues with minerals, vitamins, oils, and other biomolecules with great anticancer potential. The anticancer potential is due to potent antioxidant activity, inhibition of cancer cell-activating proteins, activating the repair DNA mechanism, or stimulating the formation of protective enzymes of biomolecules present in BE .Malus baccata and Malus toringoides. The three main polyphenols found in BE were protocatechuic acid, gallic acid, and catechin. They exerted potent anticancer activity against breast cancer, cervical cancer, and Jurkat T cell leukemia cancer cell lines, also having antioxidant and antimicrobial capability. The same authors found in another study that Quercus species showed an increased profile of polyphenol acids such as ellagic acid, gallic acid, protocatechuic acid, vanillic acid, and caffeic acid. These bio compounds showed necrosis and apoptotic effect in cancerous cells after treatment in a dose-dependent manner [One of the important sources of bioactive compounds is forestry waste, especially bark ,20. Phent manner .Tabernaemontana johnstonii BE exhibited important cytotoxicity against the P-388 mouse lymphocytic leukemia cell culture system [The alkaloids showed a beneficial effect on normal cells and cytotoxic activity against cancerous cells. This chemical family is one of the most important biologically active secondary metabolites found in the plant kingdom with multilateral biological activities ,31. Gabue system . Paclitae system .Terpenes are another class of bio compounds in the alkaloid family with anticancer properties. Monoterpenes can act synergistically with polyphenols or alone with great potential in anticancer activity . Over 20Saraca indica L. (Fabaceae) alcoholic BE inhibited the proliferation of breast cancer cell lines. Its activity was more prominent in MCF-7 than in MDA-MB-231 cells.Being the most pronounced cancer among women, breast cancer is one of the most studied cancer types. MCF-7 and MDA-MB-231 human breast cancer cell lines are often used in experiments. Fagus sylvatica L. (Fagaceae) and Picea abies L. (Pinaceae) bark. In this study, the cytotoxic effect was not observed in the A549 lung carcinoma cell line but a slight modification was observed by increasing the concentration of the samples. In another study methanolic BE of Costus pictus D.Don (Costaceae) remarkably decreased cell viability in A549 lung carcinoma cell line in a dose-dependent manner [Human lung carcinoma cell migration was inhibited in a dose-dependent manner by an ultrasound-assisted extract from t manner .Aquilaria crassna. The effect was similar to one of 5-fluorouracil, betulinic acid, and tamoxifen. Dahham et al. [Aquilaria crassna bark had an antiproliferative effect mostly against HCT 116 and HT29 colon cancer cell lines. Because of the effect against colon cancer cell lines, they tested the efficacy of β-caryophyllene on the HCT 116 cell line. The recorded result was that β-caryophyllene can cease colonization with its cytotoxic effect.PANC-1 was one of the pancreatic cancer cell lines inhibited by the β-caryophyllene compound. This was obtained by Dahham et al.  from essm et al.  showed tBEs are increasingly used in metallic nanoparticle biosynthesis as bioactive compounds that have the ability to reduce metallic ions. Because these biomolecules are ecofriendly and safe for human therapeutic use, the low cost makes it suitable for large-scale production over other bioactive products ,62,63. BMore than 65% of nanoparticles used in general biological applications are silver NPs as earlier studies have shown. That is because they have the smallest dimension and have easy access to permeate the cellular membrane and affect cellular physiology. When the diameter of a nanoparticle decreases the touching surface increases, and that has an explicit influence on their cell entrance and efficacy . Beyond Elaeodendron croceum displayed an IC50 value of 138.8 µg/mL against the MDA-MB-231 breast cancer cell line, comparable with the IC50 value of 80 µg/dL obtained after treatment with Paclitaxel [Silver nanoparticles have an important role in therapy for wound healing, not only in skin but also in breast cancer. In this review, the majority of cited studies are on silver NPs and the principally tested cells are breast cancer cell lines. For example, the BE AgNPs from clitaxel .Toxicodendron vernicifluum (Stokes) F.A. Barkley (Anacardiaceae) AgNPs [Another study showed that approximately 82.5% of A549 lung cancer cells were dead after treatment with e) AgNPs . Table 2It has been reported that plant-derived extracts show a selective decrease in the growth activity of cancer cells compared to normal cells . This sePinus massoniana has a role in both pathways. After the treatment of HeLa cells the expression of Bax, a proapoptotic protein vigorously increased. Cell apoptosis was induced by releasing of cytochrome C [Cell death appears by two alternate modes apoptosis or necrosis. These two ways are different because the first one is programmed managed cell death without a local inflammatory response. Necrosis is characterized as a passive unexpected form of cell death dependent on environmental disturbance with the unrestrained release of inflammatory response. Apoptotic cells go through diverse morphological transformations such as membrane blebbing, chromatin condensation, nuclear membrane disruption, nuclear fragmentation, and dissolution . The firchrome C ,83.Spondias pinnata mediated apoptosis induction was observed in A549 lung carcinoma and MCF-7 breast cancer cell lines. The western blot results showed an increased expression of Bax (proapoptotic) protein and a decreased one for Bcl-2 (antiapoptotic) protein in A549 cells. The same expression was observed after treatment in MCF-7 cells. The only difference was in Bcl-2 expression that was not changed [Tecomella undulata caused apoptosis on the K562 chronic myeloid leukemia cell line. That was evident after DNA fragmentation assay, increase in FAS (cell surface death receptor), FADD (FAS associated death domain protein) levels, and activation of caspase 8 and 3/7 .The molecular mechanism of BE from  changed . Alvala  changed  found thAlstoria scholaris and Alstoria venenata BE showed important membrane blebbing, nuclear condensation, and damage, which are manifestations of apoptosis rather than necrosis. The cytotoxic effect of BE on DLA  cells and their cytoprotective effect on normal ones may be due to their potent antioxidant activity [activity .Azadirachta indica on the MG-63 osteosarcoma cell line after 24 h of treatment. They observed the formation of granulations in the nucleus, which is a measure of chromatin condensation. That reaction is well documented in nanoparticle treated samples before. The cytotoxic activity of metallic nanoparticles is due to their small size and affinity for the acidic pH of the cancer cells [Aquilaria crassna essential oil BE had a potent capacity in apoptosis-inducing. The fluorescence signal decreased in treated cancer cells PANC-1 pancreatic cancer, HCT-116, and HT29 colorectal cancer. The untreated cells showed a substantial fluorescence intensity. These results present the unaffected state of normal cells [Nayak et al.  studied er cells . It is kal cells . In the al cells .Acanthopanax sessiliflorus on MDA-MB-231 and MCF-7 breast cancer cell lines was shown. The intracellular ROS (reactive oxygen species) levels were determined by inspecting the fluorescent intensity of a redox reagent. It was observed that the BE elevated the proportion of green fluorescence in both cell lines dose-dependently. That suggests that the studied BE stimulates the production of intracellular ROS after the selective interruption of the mitochondrial respiratory chain. This is evidence of apoptosis with the involvement of mitochondrial membrane depolarization. In general, ROS contributed to cell death inducing the cell membrane lipid oxidation and the expression of genes associated with DNA disruption, which promotes DNA damage in apoptosis or necrosis at levels above the cellular antioxidant defense systems [In another study , the eff systems ,86.Euphorbia umbellata, showed that chloroform fraction promotes cell cycle arrest at G0/G1 phase. Moreover, treatment with this potent extract showed a decreased percentage of cells in S-phase [Another way for cancer cell extinction is interrupting the cell cycle. After the analysis of cellular cycles, BE from  S-phase . Another S-phase  revealed S-phase ,87,88.Toxicodendron vernicifluum BE was studied. The results indicated that NIH3T3 mouse embryonic fibroblasts growth was not significantly reduced and cell death was not observed at different concentrations of the extract [Toxicity on different normal cells was realized to observe the behavior of the BE. For example, cytotoxicity of the  extract .Aquilaria crassna bark exhibited low toxicity against 3T3-L1 and RGC-5 normal cell lines. The results showed that even the higher concentration of β-caryophyllene did not affect the morphology of a normal cell line (3T3-L1) [Fagus sylvatica and Picea abies did not show cytotoxic activity. Instead, BE stimulated normal cells’ proliferation [The β-caryophyllene found in essential oil from (3T3-L1) . In the feration .Elaedendron croceum was assessed on male Wister rats. The authors revealed that there was no mortality or any toxic reaction after the acute oral test. There was no toxicity observed in the heart, liver, and kidney after the morphological investigation [The in vivo toxicity of AgNPs bark extract of tigation . Anothertigation . In additigation .Accordingly, with data shown in studies, the tests effectuated on normal cells with BE and BEM exhibited no harmful or toxic effects.Cancer has become a front line between resistance to chemotherapy, aggressive invasion, secondary toxic effects, and scientists who struggle to find a better chemical bioactive extract to maintain homeostasis, to avoid the unwanted secondary toxic effects, and to prevent metastatic cascade. BEs and BMNs are a future promise source of antioxidant compounds, which can fight against cancerous cells. All standardized extracts, shown in the listed studies above, had an impressive antioxidant capacity that was linked with anticancer ability. For maximal certainty, researchers tested in vitro the BEs and BMNs cytotoxic effect on cancer cells and normal cells. Unfortunately, in vivo studies for the anticancer potential of BEs are few. The results are encouraging and in vivo experiments should be supported. BMNs showed also antioxidant potency with great in vitro cytotoxic effect on cancer cells but no in vivo experiment was found. The toxicity on normal cells was shown as harmless. This is a promising result for the application of BEs and BMNs on animal subjects and bypasses the secondary undesirable effect of chemotherapy. There remains a need for in vivo studies to confirm the anticancer action of BEs and BMNs.