Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. During the last few years, comparing genomic data of most cancer cell lines has corroborated this statement and those that were observed studying the tumoral tissue equivalents included in the The Cancer Genome Atlas (TCGA) database. We are at the disposal of comprehensive open access cell line datasets describing their molecular and cellular alterations at an unprecedented level of accuracy. This aspect, in association with the possibility of setting up accurate culture conditions that mimic the in vivo microenvironment  coculture), has strengthened the importance of cancer cell lines for continuing to sustain medical research fields. However, it is important to consider that the appropriate use of cell lines needs to follow established guidelines for guaranteed data reproducibility and quality, and to prevent the occurrence of detrimental events . Cancer cell lines are valuable in vitro model systems that are widely used in cancer research and drug discovery . Their uThe establishment of a new cell line is a very complex process that is still not well understood. The success rate for the establishment is low and unpredictable for any specimen of origin . This stRecent findings that were obtained by the characterization of hundreds of cell lines with omics technologies  reinforced the concept of cell line usefulness in medical research. Indeed, for the majority of the existing cancer cell lines, these data have been published and made available through online datasets, which made it possible to explore detailed molecular and cellular alterations, such as mutations , copy nuThe history of cancer research and the establishment of continuous cell lines are closely related . DiffereOverall, each new cell line has been necessary, over time, to understand step by step a new feature of cancer disease and to test the efficacy of anticancer drugs . The impHuman cancer cell lines continue to play a critical role in modern cancer research. Indeed, they are widely used as preclinical model systems for gaining mechanistic and therapeutic insight. Notably, with the advent of -omics technologies , recent http://www.broadinstitute.org/ccle. Following this comprehensive approach, an important preliminary result that was obtained by Barretina et al. revealed the possible association between Schlafen family member 11 (SLFN11) gene expression and sensitivity to topoisomerase inhibitors. In the paper by Garnett et al., by performing a similar integration between genomic and pharmacological data, it was possible to disclose the association between the EWS-FLI1 gene translocation, which is frequently found in Ewing’s sarcoma, and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors, which are a class of drugs currently used in clinical trials for other cancer types. Both resources that were provided by Barretina et al. [http://www.cancerRxgene.org.In 2012 (and for the first time), two independent research groups that were led by Barretina et al.  and Garna et al.  and Garna et al.  are extra et al. . In the a et al. . The samDespite the enthusiasm generated by the aforementioned important works, it is important to consider that cell lines have important limitations, especially due to the differences in terms of gene expression, as compared to in vivo tumor tissues. Specifically, cell lines, when cultured in vitro, do not have interactions with other cell types; additionally, their growth is not under the influence of cytokines and other cell signaling molecules, and the native tissue architecture is lost. Moreover, the effects of in vivo drug distribution and metabolism are not easily matched in vitro . All of Klijn and colleagues  improvedhttps://tcpaportal.org/mclp/#/) for the research community to investigate tumor behaviors in a quantitative and efficient way and to compare the differences in protein expression across cancer cell lines and to in vivo tumor tissues.In parallel to advancements in the knowledge of genetics and transcriptomics of cancer cell lines, The Cancer Genome Atlas (TCGA) database  revealedhttps://depmap.org/portal/achilles/) to identify critical gene functions in 501 cancer cell lines. The authors identified genes whose expression is required for the proliferation or survival of subsets of these cell lines and developed an approach to identify the features that predict these gene dependencies. This cancer dependency map provides an innovative approach for defining and predicting genes that are essential for cell viability, which thereby facilitates the identification of cancer targets.The generation of these extensive datasets highlighted the need for functional assays to identify novel targetable genes. In this sense, it is important to consider the work by Tsherniak A et al.  that is https://portals.broadinstitute.org/ccle), where unbiased association analysis can be performed by linking the cancer metabolome to genetic alterations, epigenetic features, and gene dependencies [More recently, in 2019, data from two research papers added value to knowledge regarding the biology of the cancer cell lines that were included in the CCLE. The first study was published by Li et al. and focused on the metabolic diversity of 928 cancer cell lines derived from 20 cancer types . The autndencies . Overallndencies . Moreovehttps://cellmodelpassports.sanger.ac.uk/) interface developed at the Sanger Institute (UK)) due to the complexity of the newly derived datasets [Finally, wet laboratory researchers should have a friendly interface to explore the data on each cell line;  a list of 360 known cross-contaminated cell lines to address the important problem of cross-contamination. Most contaminants arise within the same species, with HeLa (as reported by Nelson-Rees approximately 40 years ago) still the most frequently encountered  among human cell lines, while interspecies contaminants account for a small, but substantial, minority of cases . In addition to the list of cross-contaminated cell lines, two important practical suggestions are provided: (i) Check the literature by searching the PubMed database while using the cell line name and “cross-contamination”; and (ii) Check the cultured cells; a cell line should be tested on arrival in a new laboratory, and all the cultures should be periodically tested while in use, before cryopreservation, and when thawed from liquid nitrogen [https://iclac.org/) created a website that is dedicated to the authentication of cell lines [https://iclac.org/databases/). The improper use of misidentified human cells can be avoided when carrying out research projects by performing regular checks on the cell lines used, given that these resources are now available to the scientific community.Cape-Davis et al. published  and madenitrogen . In thisnitrogen . An incrll lines . A list Mycoplasma contamination is the second critical issue to be discussed. This problem was first proposed in 1956, when it was discovered as a cell culture contaminant . MycoplaIn conclusion, the main reasons for both cross-contamination and mycoplasma infection are the same, namely, faulty cell culture techniques, inappropriate handling of cell lines, and a lack of knowledge and information regarding the consequences and effects of contaminants. Undoubtedly, according to current guidelines, it is good practice to obtain a cell line of interest from a qualified source, such as international cell line banks ,27,72. TAs discussed in the section dedicated to the history of cancer cell lines, over time, a number of different cell lines have been published in the scientific literature, especially in the 1970s, 1980s, and 1990s, with the aim of providing innovative in vitro model systems for experimental purposes. Recent findings highlight that the derived cell lines maintain most of the genetic alterations of the original in vivo tumor. Conversely, the transcriptomic and proteomic signatures of cell lines in most cases are completely different from their in vivo counterpart ,50. Subshttps://iclac.org/) that were taken to help the scientific community make false or misidentified cell lines more visible and to promote awareness and authentication testing as effective ways to combat the problem, authentication and mycoplasma infection testing are not universally practiced in preclinical research.In research laboratories worldwide, uncountable in vitro assays are performed daily while using cancer cell lines ,2,3,25. The importance of using authenticated and mycoplasma-free cell cultures is related to the fact that cancer cell lines, under the right conditions of use, still maintain most of the cellular and molecular alterations of the original cancer ,15,16,17Prospectively, we believe that cell lines will continue to support the scientific community by helping to obtain important results that can be translated from the laboratory bench to the patient bedside and by improving public health care. As discussed in this article, the growing use of massive sequencing technologies provides a wealth of information for each disease at an unprecedented level of accuracy . In this


Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in The online version of this article (10.1007/s11240-018-1398-5) contains supplementary material, which is available to authorized users. Catharanthus roseus (Madagascar periwinkle) is a medicinal plant which produces bioactive terpenoid indole alkaloids (TIA) such as the antihypertensive drugs ajmalicine and serpentine, as well as the antineoplastic agents, vinblastine and vincristine. However, most TIA especially the dimeric TIA are produced at low levels in the plant, difficult to extract or isolate, and unfeasible for total chemical synthesis due to their complex structures, which explain the high market prices of TIA  and vacuole  pathway  which is associated with the nucleus , thylakoid membrane of chloroplasts (N-methyltransferase [NMT]) and vacuole (peroxidase [PRX1])  is mainly operated to yield tryptamine , TDC, STR, G10H or G8O, and apoplastic peroxidase in C. roseus cells , 1-deoxy-C. roseus. The enzyme that catalyzes the conversion of GPP into geraniol, i.e. geraniol synthase (CrGES) has been isolated and characterized from C. roseus  and non-targeted metabolites were analyzed to determine changes in metabolic profiles of the transformed C. roseus cell suspension cultures.In the present study, we overexpressed plastidial Catharanthus roseus cell suspension cultures (cell-line MP183L) were subcultured weekly by transferring 10 ml culture into 50 ml of Linsmaier and Skoog (LS) medium (Linsmaier and Skoog 2/s) at 70% relative humidity.geraniol synthase gene of Catharanthus roseus (CrGES) consists of a 1770 bp DNA sequence that encodes a protein of 589 amino acids in length. Geraniol synthase is localized in plastids as suggested by the leader peptide encoded in its cDNA  were constructed. Primers were designed for different vectors employed for different purposes, i.e., pRT101 for constitutive expression and pTH2-ΔEcoRI containing sGFP for protein localization studies. The pTH2-ΔEcoRI plasmid used in this study is a derivative of pTH2 . Fragments were amplified by PCR  with following procedures: 98 °C, 1 min; 35 cycles, 98 °C, 15 s; 57 °C, 20 s; 72 °C, 1 min; 72 °C, 5 min. The PCR products were cloned into a pJET1.2/blunt cloning vector  and sequenced for confirmation. Subsequently, the verified CrGES and ΔplCrGES fragments were ligated into pTH2-ΔEcoRI plasmid excised with SalI  were harvested at day 7 after subculturing. Harvested cells were washed three times with deionized water and frozen in liquid nitrogen. Aliquots of the samples were stored at − 80 °C for RNA extraction and the remaining biomass was lyophilized for 72 h prior to metabolite analysis.Frozen cells were ground to a fine powder in liquid nitrogen. Total RNA was extracted with two volumes of hot phenol buffer  and one volume of chloroform. RNA was precipitated overnight (4 °C) with LiCl at a final concentration of 2 M, washed twice with 70% ethanol, and suspended in water.32P-labeled DNA probes. After overnight hybridization, blots were washed twice at 42 °C for 30 min with 0.1 × SSPE  and 0.5% SDS. Finally, the blots were washed briefly with 0.1 × SSPE at room temperature. Blots were exposed to X-ray films .Northern blot analysis was performed as described by Memelink et al.  with somReverse transcription was carried out using the Revert Aid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) following to the manufacturer’s instruction. The cDNA synthesized from each sample was used as template in PCR. Negative controls were performed by excluding reverse transcriptase enzyme in the reaction.C. roseus cell suspensions constitutively overexpressing geraniol synthase in the cytosol (ΔplCrGES). The cells were harvested after 24 h, freeze-dried for 72 h and stored prior to TIA analysis.The cell suspension cultures were subcultured weekly. Four days after subculturing, mevalonic acid  was fed to the C. roseus cell suspensions constitutively overexpressing geraniol synthase in the plastids (CrGES) and cytosol (ΔplCrGES), and the control culture on the 5th day after subculturing. The elicited cells were harvested after 24, 48, and 72 h. Samples were freeze-dried for 72 h and stored prior to TIA analysis.For elicitation, jasmonic acid (Sigma) at a final concentration of 100 µM was added to the Freeze-dried cells (100 mg) were extracted with 5 ml methanol, vortexed, sonicated for 20 min, and centrifuged for 30 min (3500 rpm). The dried supernatant was suspended in 1 ml phosphoric acid (1 M). Samples were subjected for terpenoid indole alkaloid and precursor analysis using high performance liquid chromatography-diode array detector (HPLC-DAD)  as described by Saiman et al. .Freeze-dried cells (100 mg) were extracted with 5 ml dichloromethane, vortexed, ultrasonicated for 10 min, and centrifuged for 10 min . The eluent was concentrated under a flow of nitrogen and 1 µl of concentrated extract was injected into gas chromatography-mass spectroscopy (GC–MS) (Agilent Technologies Inc.) equipped with a DB-5 capillary column  . The initial oven temperature was 45 °C for 1 min, and was increased to 300 °C at a rate of 10 °C/min and held for 5 min at 300 °C. Geraniol standard compound (Sigma) was used for identification.3OD and KH2PO4 buffer. The latter was prepared in D2O, adjusted pH to 6.0, and added 0.01% (w/w) trimethylsilyl propanoic acid (TMSP) as internal standard. The mixture was vortexed for 10 s, sonicated for 10 min, and centrifuged for 15 min . Samples were analyzed using 500 MHz NMR . NMR spectra were manually phased, baseline corrected, and calibrated to TMSP resonance at 0.0 ppm using XWIN NMR version 3.5 (Bruker). AMIX software (Bruker) was used for bucketing (width δ 0.04) and data reduction of the 1H-NMR spectra (δ 0.40–10.00) using total intensity scaling. Multivariate data analysis was performed with the SIMCA software version 12.0 . Analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) was performed on IBM SPSS Statistics 20  to determine statistical differences (P < 0.05) between means of groups.Freeze-dried cells (25 mg) were extracted with a ratio 1:1 of CDC. roseus’s geraniol synthase . CrGES or ΔplCrGES was constructed into the aforementioned plasmid vectors and transferred into C. roseus cells by particle bombardment. For the constitutive expression construct using pRT101 vector, an average of 10 putatively transformed calli were obtained per plate of 50 mm diameter size.A full fragment of CrGES and ΔplCrGES, the expression of the construct pTH2-ΔEcoRI::CrGES-GFP and pTH2-ΔEcoRI::ΔplCrGES-GFP transformed into C. roseus cells was analyzed. The results show that the truncated CrGES without plastidial leader peptide fused with GFP (ΔplCrGES-GFP) was displaying fluorescence in the cytosol . Northern blot analysis showed expression of GES in some of the putatively transformed cell lines. The reverse transcriptase PCR also showed GES expression and expected band sizes of the CrGES or ΔplCrGES fragments in the positive transformed cell lines. Therefore, these results confirm the overexpression of the CrGES (plastid) and ΔplCrGES (cytosol) in some of the transformed C. roseus cell suspensions cultures.Expression of CrGES and ΔplCrGES) and control lines (transformed with empty vector) of C. roseus cells were analyzed for the levels of TIA, iridoid and indole precursors using an HPLC-DAD. Neither TIA nor iridoids were detected in the transformed or control C. roseus cell cultures. Only tryptophan and tryptamine were detected in all transformed cell lines, similar to the non-TIA producing C. roseus cell-line (MP183L). This result indicates that geraniol synthase overexpression in the plastid or cytosol of C. roseus cells does not directly enhance the flux towards iridoids and TIA.The overexpression lines (CrGES) was overexpressed in the C. roseus cell cultures to overcome a limitation of geraniol transport from plastids to the cytosol. It is further hypothesized that by feeding mevalonic acid in the ΔplCrGES overexpression line, the pool of C5 precursors in mevalonate pathway could be increased, and the presence of geraniol synthase gene in cytosol may open a metabolic link between GPP available in the cytosol or GPP derived from the mevalonate pathway to the iridoids and subsequently increase the production of TIA. However, feeding mevalonic acid to the ΔplCrGES overexpression line did not result in production of iridoids or TIA. Despite the additional feeding, the level of free cytosolic GPP in these cells is presumably too low to allow conversion by the cytosolic GES to support detectable changes in metabolite composition towards the iridoids and TIA. Cytosolic GPP might be increased by co-transformation with a cytosolic localized GPPS, thus creating and improving a metabolic bridge between the mevalonate pathway and the iridoid/TIA pathway.The geraniol synthase gene without its plastidial leader peptide (ΔplC. roseus MP183L cell lines (non-producing TIA line) did produce neither TIA nor iridoid precursors in both the CrGES and ΔplCrGES lines. Our previous study showed that the non-TIA producing CATL cell line (a derivative of MPL183L line) has lower gene transcript levels of DXR (sevenfold), GPPS (threefold) and G8O  compared to the TIA-accumulating CRPP cell line  in C. roseus hairy roots and plants, respectively, increased the levels of several TIA which indicates that the G8O (G10H) availability is critical for TIA production. It is thus important to consider both the metabolic and overall pathway-related gene expression profiles of the GES overexpressing C. roseus MP183L cell line when evaluating TIA productivity and to compare such data to a cell line or other system accumulating iridoids and TIA, e.g. the earlier identified TIA producing CRPP line  lack of carbon-five precursors because of limiting activity of upstream biosynthesis enzymes, (2) competing pathways acting on GPP, e.g., leading to FPP and sterols or to GGPP and carotenoids, or (3) conversion of geraniol to glycosylated or other derivatives. Dong et al.  predominantly accumulated geraniol glycoside. Further work is required to analyze glycosylated geraniol in the transformed C. roseus cell lines.As overexpressing geraniol synthase in the plastids and cytosol of g et al.  reportedVoGES to the cytosolic mevalonate compartment resulted in 30% lower geraniol glycoside than the plastidial targeted VoGES  in the cytosolic mevalonate pathway increased production of limonene sixfold compared to the single overexpression of LS. However, the targeted subcellular compartment for GPPS expression in the mevalonate pathway needs to be evaluated since the IPP isomerase (IDI) and FPPS, which were generally regarded as cytosolic enzymes, were reported to be localized in peroxisomes  accumulated geraniol produced from the available GPP pool  is a supervised multivariate data analysis that uses information in another matrix. In addition to the X-matrix of NMR data, three groups  were assigned for the Y-matrix in PLS-DA. Figure 1H-NMR spectra can be observed among the samples (Supplement 5).Principal component analysis (PCA) which is an unsupervised clustering method shows 45 and 33% variation of PC1 and PC2, respectively  was performed by comparing the mean value of the relative levels of metabolites between the groups of observation. The result suggests that valine, leucine, isoleucine, tryptophan, phenylalanine, and tyrosine are the metabolites that significantly differ (P < 0.05) between the groups  in plastidial-CrGES overexpressing lines compared to the control (empty vector) cell lines. These results indicate that overexpressing CrGES in the plastids increased the levels of some primary metabolites including aromatic amino acids phenylalanine and tyrosine which derive from the shikimate pathway. Tyrosine and phenylalanine are essential components for plant growth and development. The latter is precursor for lignin, the component of plant cell wall. In addition, phenylalanine-derived compounds play diverse roles in plant adaptation such as plant defense, UV protection, and reproduction. The reason of the increase of metabolism in shikimate pathway after overexpressing plastidial geraniol synthase is not clear but could be related to the localization of shikimate pathway in the plastids. A study by Sung et al.  compared to the control and the CrGES overexpressing cell lines. This indicates that the different subcellular localization of geraniol synthase results in different metabolic effects, thus a suitable targeted compartment is crucial to determine the production of the compounds of interest. Alteration of unrelated biosynthetic pathways may occur upon genetic modification of the targeted pathway. For example, Nagel et al.  or the cytosol (ΔplCrGES). The study confirmed the expression of CrGES and ΔplCrGES in both subcellular compartments. Its product, geraniol was not detected in the transformed cells or present below the detection limit, which either indicates low production and accumulation of geraniol, or a rapid metabolization. Further analyses are needed to reveal if the GES product is formed and subsequently derivatized to glycosylated geraniol or other products. No accumulation of TIA or iridoid pathway precursors was detected in the C. roseus cell suspension cultures overexpressing geraniol synthase. Addition of mevalonic acid as precursor did not directly stimulate TIA production in the ΔplCrGES transformed cell lines; presumably the level of free cytosolic GPP in these cells is too low to support detectable changes in metabolite composition. Co-transformation with a cytosolic localized GPPS may increase the GPP availability in cytosol, thus improving a metabolic link between the mevalonate pathway and the iridoid/TIA pathway. Jasmonic acid elicitation stimulated the TIA production in most transformed cell-lines, but did not cause clear differences in TIA production between overexpression and control cultures. NMR-based metabolomics combined with multivariate data analysis revealed alteration of some primary metabolites in both plastidial and cytosolic CrGES overexpressing C. roseus cells. In contrast to a higher level of several metabolites of which some are associated to shikimate pathway in the constitutive CrGES overexpressing C. roseus cells, a lower level of these metabolites was detected in the ΔplCrGES overexpressing cultures compared to the control, thus suggesting different metabolic effects related to the subcellular compartmentation of geraniol synthase.In this study, we describe the development of Below is the link to the electronic supplementary material.Supplementary material 1 (PDF 275 KB)Supplementary material 2 (PDF 191 KB)Supplementary material 3 (PDF 189 KB)Supplementary material 4 (PDF 331 KB)Supplementary material 5 (PDF 301 KB)


MGMT) promoter methylation, resulting in increased MGMT expression. We established the NGT16 cell line with HIST1H3B K27M and ACVR1 G328E gene mutations from a DMG patient and used this cell line and other DMG cell lines with H3F3A gene mutation  to analyze MGMT promoter methylation, MGMT protein expression, and response to TMZ. Three out of 4 DMG cell lines  had unmethylated MGMT promoter, increased MGMT expression, and showed resistance to TMZ treatment. SF7761 cells with H3F3A gene mutation showed MGMT promoter methylation, lacked MGMT expression, and sensitivity to TMZ treatment. NGT16 line showed response to ALK2 inhibitor K02288 treatment in vitro. We confirmed in vitro that MGMT expression contributes to TMZ resistance in DMG cell lines. There is an urgent need to develop new strategies to treat TMZ-resistant DMGs.Diffuse midline gliomas (DMGs) show resistance to many chemotherapeutic agents including temozolomide (TMZ). Histone gene mutations in DMGs trigger epigenetic changes including DNA hypomethylation, one of which is a frequent lack of O6-methyl-guanine-DNA methyltransferase ( Diffuse midline gliomas (DMGs), comprised of diffuse intrinsic pontine gliomas (DIPGs) and thalamic gliomas, have very poor prognoses with a median survival of 8–11 months for DIPGs  and abouH3F3A gene encoding histone H3.3 protein or in HIST1H3B gene encoding histone H3.1 protein  in K27M in H protein –12. Epig protein , 14.MGMT promoter methylation inhibits the transcription of MGMT, leading to the silencing of MGMT  inhibits the killing of tumor cells by alkylating agents such as TMZ . MGMT tr of MGMT , 15, 16.e to TMZ . New stuhylation , 19. The therapy .HIST1H3B, the rarer form of histone H3K27M, from a diffuse intrinsic pontine glioma (DIPG) patient. Using this cell line and other DMG cell lines, we have demonstrated for the first time in vitro that MGMT expression contributes to resistance to TMZ in H3K27M mutant DMG cell lines.We set out to investigate this hypothesis in the preclinical setting using DMG cell lines. We established a cell line that has H3K27M mutation of Human DIPG specimens were obtained during surgery in accordance with institutional review board approvals (Niigata University #2583) after obtaining written consent from the family.The surgical specimens were fixed with 20% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-μm-thick sections stained with hematoxylin and eosin, and the paraffin-embedded sections were processed for immunohistochemistry using methods previously described . The hisThe NGT16 cell line was derived from surgical specimen taken from a DIPG patient  during t2. NGT16, SF8628, U87MG, T98G cells were grown as adherent monolayer cultures in 10% FBS DMEM. SF7761, JHH-DIPG1 cells were grown as sphere cultures in EF20 medium composed with Neurobasal Medium (Thermo), 20 ng/ml EGF , 20 ng/ml FGF (Peprotech), 2% B27 supplement (Thermo), 0.25% N2 supplement (Thermo), 3 mM L-Glutamine (Thermo), 2 μg/ml Heparin (Sigma), and 1% Antibiotic-Antimycotic (Thermo)  , 31.H3F3A gene or HIST1H3B gene including the mutational region of H3K27M. Primer sequences for the H3F3A were 5′- GAT TTT GGG TAG ACG TAA TCT TCA−3′ (forward) and 5′- TTT CCT GTT ATC CAT CTT TTT GTT−3′ (reverse) and for the HIST1H3B were 5′- GGG CAG GAG CCT CTC TTA AT−3′ (forward) and 5′- ACC AAG TAG GCC TCA CAA GC−3′ (reverse) as described previously , and DNA from surgically obtained formalin-fixed paraffin embedded (FFPE) tissue by using QIAamp DNA FFPE Tissue Kit  , 32. PCReviously . Polymereviously , 32. PurMGMT gene including CpG-rich promoter region. Primer sequences of MGMT were for the unmethylated reaction 5′- TTT GTG TTT TGA TGT TTG TAG GTT TTT GT−3′ (forward) and 5′- AAC TCC ACA CTC TTC CAA AAA CAA AAC A−3′ (reverse) and for the methylated reaction 5′- TTT CGA CGT TCG TAG GTT TTC GC−3′ (forward) and 5′- GCA CTC TTC CGA AAA CGA AAC G−3′ (reverse) as described previously (DNAs were subjected to bisulfite modification by using MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Takara Bio Inc.), and PCR was performed to amplify fragment of eviously .2MoO4, 1 mM Sodium orthovanadate). Fifteen microgram of protein from each Lysate sample were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck). After probing with primary antibodies, the membranes were incubated with horseradish peroxide-conjugated secondary antibody , and visualized by ECL prime Western Blotting Detection Reagent  , 4% SDS, 20% Glycerol) containing protease inhibitors  and phosphatase inhibitors  . Mouse mOne thousand five hundred cells per 100 μl medium were incubated on a 96 plate for 1 day and applied 10 μl of TMZ (Sigma) dissolved in dimethyl sulfoxide (DMSO) at concentrations of 0, 62.5, 125, 250, and 500 μM and incubated for 72 h, and ALK2 inhibitor K02288  dissolved in DMSO at concentrations of 0, 12.5, 25, and 50 μM of. As a background control (0% control), TritonX-100  was added to the control wells. Ten microliter of WST-8  was added to each well, and the cells were incubated for 4 h. Absorbance at 450 nm was measured in each well using ELx808  plate reader .MethylFlash global DNA methylation (5-mC) ELISA easy kit  was used to measure DNA methylation of cell lines. Briefly, 100 ng of DNA extracted from each cell line was placed in assay wells, washed then detection complex solution was added. After incubation, wells were washed and color developer solution was added to each well and absorbance was measured at 450 nm with ELx808 plate reader. Positive and negative controls were used to demonstrate a standard curve.post hoc Tukey's multiple comparison test. Error bars represent standard error of means (SEM)  . All staHIST1H3B (left), a rare mutation variant in histone, and G328E mutation in ACVR1 (right), a frequently encountered mutation in patients with DIPGs with HIST1H3B mutation  mutations are frequently found in HIST1H3B-mutant, but not H3.3 H3K27M-mutant DMGs (ACVR1 encodes for the type I bone morphogenic protein (BMP) receptor ALK2, and mutation of this receptor causes constitutive activation of BMP signaling pathway  and unmethylated (U) bands. The GBM control cell line U87MG showed only the M band whereas T98G cells showed both M and U bands . IC50 was 403.5 μM and 206.6 μM for U87MG and SF7761, respectively. Presence of histone mutations, MGMT promoter methylation, MGMT protein expression and sensitivity to TMZ for the 6 cell lines are shown in Finally, we evaluated the sensitivity of DMG cell lines to TMZ in association with MGMT promoter was methylated and was sensitive to TMZ, showed highest percentage of 5-mC of all cell lines (5-mC/(5-mC+C) = 2.63%).To determine the global DNA methylation changes of each cell line, levels of 5-methylcytosine (5-mC) were assessed in NGT16, SF7761, SF8628, JHH-DIPG1, T98G, and U87MG cell lines. DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-mC. Thus, global DNA methylation can be analyzed by determining 5-mC levels. Although not all H3K27M mutant cell lines showed decreased 5-mC expression compared to wildtype cell lines , NGT16 cH3F3A encoding histone H3.3 or in HIST1H3B encoding histone H3.1 ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.HA, MOk, RH, and MN designed the study. HA, MOk, JW, and YK performed experiments. YK and AK performed pathological evaluation. YT, JW, MN, JY, and MOi provided patient care and obtained written consent. YF approved the study design. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The great advances in the studies on metal complexes for the treatment of different cancer forms, starting from the pioneering works on platinum derivatives, have fostered an increasingly growing interest in their properties and biomedical applications. Among the various metal-containing drugs investigated thus far, ruthenium(III) complexes have emerged for their selective cytotoxic activity in vitro and promising anticancer properties in vivo, also leading to a few candidates in advanced clinical trials. Aiming at addressing the solubility, stability and cellular uptake issues of low molecular weight Ru(III)-based compounds, some research groups have proposed the development of suitable drug delivery systems  able to enhance their activity compared to the naked drugs. This review highlights the unique role of Ru(III) complexes in the current panorama of anticancer agents, with particular emphasis on Ru-containing nanoformulations based on the incorporation of the Ru(III) complexes into suitable nanocarriers in order to enhance their bioavailability and pharmacokinetic properties. Preclinical evaluation of these nanoaggregates is discussed with a special focus on the investigation of their mechanism of action at a molecular level, highlighting their pharmacological potential in tumour disease models and value for biomedical applications. This exchange reaction was also accompanied by reduction of the paramagnetic Ru(III) to the diamagnetic Ru(II) ion, as revealed by electron spin resonance (ESR) spectra, and explained by the well-known autoxidation ability of polysorbates .The evaluation of the in vitro anticancer properties performed on colon carcinoma SW480 and hepatoma Hep3B cell lines indicated a modest increase of the antiproliferative activity for the stable NPs (stored at 4 °C), along with a markedly higher cytotoxic activity for the NPs left at r.t. c,d. IndeFor KP1019, the same research group also proposed a strategy alternative to the physical encapsulation, based on the chemical conjugation of the ruthenium complex with a selected polymer . In part50 values between 1.6- and 22.7-fold lower compared to the free Ru compound  -2-methylpropanoic acid, and the subsequent chain extension with poly(ethylene glycol)methyl ether acrylate (PPEGMEA) to obtain a biocompatible amphiphilic block copolymer capable to self-assemble into polymeric micelles and incorporate NAMI-A (indicated as P(NAMI-A)-PPEGMEA, The polymerization reaction was followed by SEC analysis monitoring the retention time changes due to the increased polymer size over time and the resulting micelles were then characterized by DLS and TEM experiments a 115]..115].When tested on ovarian (A2780 and Ovcar-3) and highly aggressive pancreatic AsPC-1 cancer cell lines, a 1.5-times increase in cytotoxicity was found in all the tested cell lines for the polymeric NAMI-A-containing micelles compared to the naked ruthenium complex b 115]..115].Notably, polymeric micelles significantly improved also the NAMI-A antimetastatic potential, with increased inhibitory effects on both the migration and invasion of human breast cancer cells . In part1H NMR spectroscopy, showed a faster hydrolysis in aqueous solution with respect to its parent compound, with formation of the respective monoaqua complex, probably favoured by the hydrogen-bonding properties of the nitrogen atoms in the triazole moiety facilitating the access of water molecules to the metal Cl) evidenced a ca. two-fold higher activity when loaded into Pluronic® F-127 micelles compared to its naked form. This occurred also for the mononuclear PDT complex, showing a three-fold increased anticancer activity 5As in the case of low molecular weight Ru(III) compounds, also the nucleolipid- or aminoacyl lipid-based complexes incorporating AziRu, here described in paragraph 1.3, when dissolved in aqueous solutions, showed tendency to hydrolyse over time, even if sensibly reduced compared to naked AziRu ,105,110.Liposomes, discovered in the 1960s, consist in phospholipid-based nanovesicles having a morphology very similar to cellular membranes, with an aqueous core surrounded by lipid bilayers ,120,121.Thus, POPC and DOTAP lipids were chosen as Ru(III) complexes stabilizing agents on the basis of their proved biocompatibility ,131,132.The use of phospholipids—forming ordered bilayers in which the Ru complexes can be easily lodged—allowed obtaining Ru(III)-containing formulations stable for months in physiological media, in which the incorporated metal amount can be finely modulated.On the other hand, the selected lipids allowed exploiting different interactions for the Ru-complex embedding , thus providing a basis to evaluate a structure-activity rationale.I ..100].The fluorescence detection suggested an intracellular release of the ruthenium derivative from the liposomes: at longer incubation times , a significant decrease in the dansyl-dependent fluorescence signal intensity occurred b, supporAll these findings were thus corroborated in MCF-7 cells by fluorescence experiments using confocal microscopy coupled to Red E-cadherine-associated fluorophore, a 120-kDa transmembrane glycoprotein able to highlight adherens junctions and cell-cell contacts in epithelial clusters .Overall, these data highlighted the crucial role played by the cellular uptake process in determining the anticancer efficacy of Ru(III)-based drugs, showing both POPC and DOTAP-based liposomes as very efficient nanocarriers for the stabilization of Ru complexes in aqueous media and their effective transport in cells. Particularly relevant was their clear detection in the nucleus, differently from other known Ru(III)complexes, able to accumulate in the cytoplasm but not in the nuclear region . NotablyRuthenium intracellular amount was determined by ICP-MS analysis performed on subcellular fractions  of MCF-7 cells, after 24 h in vitro treatment with HoThyRu/DOTAP nanoaggregates . AziRu wFurthermore, analysis of the isolated subcellular fractions indicated for the HoThyRu/DOTAP formulations a broad ruthenium distribution among the intracellular compartments, above all in the nucleus. Almost 50% of the ruthenium administered as liposomal nanosystem was found bound to nuclear DNA vs. <10% of the metal from the naked AziRu detected in the nuclear fraction bound to DNA .To further support the relationship between cell viability and ruthenium-induced cytotoxicity, subconfluent cultures of the selected cancer cell lines throughout the in vitro experiments were also examined by phase-contrast light microscopy to monitor the dynamic morphological changes occurring during cell death. Thus, confluent MCF-7, WiDr and C6 cancer cells were evaluated after incubation with ToThyRu/POPC liposomes and MCF-7, WiDr and HeLa cells were examined after in vitro treatments with ToThyCholRu/DOTAP formulations, as representative example of POPC and DOTAP-based nanoaggregates, respectively.In all cases, after 48 h in vitro treatments of the selected cancer cell lines, morphological modifications of the cell monolayers were clearly evident ,79, withI hosted in DOTAP liposomes, not only malignant cells such as MCF-7, HeLa and C6 but also normal cell lines as 3T3-L1 (mouse embryo fibroblast) and HaCaT (human keratinocytes) were analysed by phase-contrast light microscopy in the absence and presence of the drug. Notably, while the cell distribution and cytomorphology were substantially unaffected on the healthy cells after 48 h of in vitro treatment, under the same conditions the cell monolayers integrity was strongly perturbed, with distinctive hallmarks of apoptotic cell death, such as membrane blebs, cell shrinkage, formation of balloon-like structures and cell contraction ; w; w79]; w50 doses of Ru(III)-containing formulations strongly indicated fragmentation, accompanied by typical internucleosomal DNA laddering. The DNA cleavage extent was very close to that induced by cisplatin, used as positive control . S. S100]. Starting from the promising in vitro bioactivity of Ru(III)-containing POPC- and DOTAP-based liposomes, able to selectively and significantly reduce cancer cell survival, further improvements of these systems were recently proposed. In particular, two alternative approaches were reported based on varying the platform used to deliver the nucleolipid-based Ru(III)-complexes.In a first approach, niosome vesicles were used to replace liposomes.In detail, vesicles prepared from non-ionic surfactants ,214, curOwing to their vesicle structure, niosomes are able to incorporate both hydrophilic and hydrophobic drugs, just like liposomes do ,220,221.5′GGTGGTGGTGGTTGTGGTGGTGGTGG3′, entered in Phase II anticancer clinical trials, selectively targets nucleolin, a multifunctional protein involved in cell survival, growth and proliferation, overexpressed on the outer membrane of cancer cells -based complexes, also of an active targeting agent, i.e., the oligodeoxyribonucleotide AS1411 . This G-s et al. ).Recently, AS1411 has been largely employed as a selective nucleolin-targeting agent, successfully combined with chemotherapeutic agents -based chemotherapeutic drug to improve its selective delivery via specific recognition of cancer cells .In a second approach, superparamagnetic iron oxide NPs (SPIONs) with a core/double shell architecture were exploited as platform for Ru incorporation, proving to be effective contrast agents for in vivo Magnetic Resonance Imaging (MRI) investigations and thus potential tools in theranostic nanomedicine, intended to provide simultaneous diagnosis and treatment of a disease, with early detection, easy monitoring, targeted therapy and minimal toxicity ,247.With the aim of exploring niosome vesicles as versatile scaffolds for effective loading of multiple anticancer drugs to be used in combination therapies, Riccardi et al.  devised The morphology, average size, zeta potential, electrophoretic mobility and stability over time of the functionalized niosomes were analysed using different biophysical techniques.In particular, both unfunctionalized and functionalized niosomes gave monodisperse vesicle-like aggregates, with typical size of ca. 100 nm, stable upon storage for one month, also able to fully incorporate both the nucleolipid Ru(III) complex HoThyRu and the oligonucleotide AS1411. The functionalization was driven by electrostatic interactions, in the case of the negatively-charged oligonucleotide, and by a combination of hydrophobic and electrostatic interactions for HoThyRu encapsulation .Then, the antiproliferative activity of the niosome formulations was evaluated using the MTT assay on a selected panel of human cells including a human cervix adenocarcinoma (HeLa) and two colorectal cancer cell lines , as well as a non tumoural embriogenic human cell line (HEK293T). HeLa cells were selected as a relatively drug-sensitive cell line with high nucleolin content, already proved to be responsive to niosome formulations  as well Niosome formulations containing both AS1411 and HoThyRu showed increased bioactivity compared to those carrying only HoThyRu, proving the ability of the nucleolin-targeting aptamer to markedly enhance the bioactivity of the Ru(III)-containing niosomes , and an inner amphiphilic layer (composed by oleic acid and oleylamine), covering the surface of the NPs . . 108]. IIn analogy to previous investigations , subconf2) reduction or transverse proton relaxation rate (R2) increase [Once assessed for their biocompatibility, 18LPC/SPIONs were also injected in the tail vein of a healthy rat, chosen as a small animal model to evaluate their in vivo pharmacokinetics and applicability as negative contrast agents for MRI analyses d. This eincrease ,256.2 relaxation time decrease was recorded in the liver of the animal, consistently with the nanoparticle capture by the reticuloendothelial hepatic tissue (Kupffer cells) [2 reached a maximum value 60 min after injection [Ten minutes after injection, a notable Tr cells) , withoutnjection .Thus, 18LPC/SPIONs demonstrated to favour long circulation times with a measurable reduction in the MR signals in the rat liver, indicating a very promising potential for use as MRI negative contrast agents .More recently, as additional ingredients for the SPIONs surface decoration, suitable lipophilic AS1411 conjugates were also studied  in orderCancer is one of the leading causes of death in the world, representing a key topic in scientific research. Cancer cells are able to accumulate multiple mutations allowing their uncontrollable growing, invasiveness, and eventually spreading to other parts of the body causing tumour metastasis formation. In addition, malignant cells typically exhibit aberrations in the physiological signalling pathways regulating various cellular processes, such as cell growth and division, as well as cell-to-cell communication. Therefore, the genetic and phenotypic heterogeneities of tumours form, making cancer a very difficult disease to treat.Stimulated by the success of cisplatin, intense efforts have been devoted to the development of alternative transition metal-based anti-tumour compounds. Pioneering studies in this field led to promising lead Ru(III)-complexes, such as NAMI-A, KP-1019 and NKP-1339, which entered clinical trials, even though none of these have been approved yet for human use.The difficulty of progressing lead compounds from animal studies to human clinical trials and/or effective clinical use highlights the complications involved in overcoming the pharmacokinetic and pharmacodynamic barriers in living animals. Metal compounds with proved selective anti-tumour in vitro activity are presumably able to reach the target tumour xenograft without causing adverse effects to the health of the animal. Nevertheless, this is not always verified since a compound active in vitro against cancer cell lines can fail under in vivo testing for several reasons. For example, the lipophilicity of complexes is determinant for effective cellular uptake. Secondly, the compound may be too unstable in human plasma or may be cleared too quickly by the liver. Alternatively, a compound may be too toxic to a particular organ, even though it may not show cytotoxicity in vitro against normal cell lines.In this context, the encapsulation of Ru(III) complexes into nanostructures is an increasing research area, particularly useful to address the uptake and stability issues of these anticancer agents; therefore, some ground-breaking advances are expected in the near future. Nanoformulations can be used to enhance the efficacy of Ru complexes in cancer treatment by targeted delivery of drugs into cancer cells and reduce their adverse effects and systemic toxicity by optimization of their pharmacokinetic properties. Notably, examples have been here discussed showing the ability of Ru(III)-functionalized liposomes to activate multiple cell death pathways, which represents a very interesting perspective in order to develop new intervention strategies in TNBC with limited chemotherapeutic options.Although nanostructured materials functionalized with Ru complexes  have shown great potential in the treatment of cancer, there are still many challenges in translating basic research into clinical applications.To reach these challenging goals, understanding the pharmacokinetic and pharmacodynamic parameters of a drug is essential for the design of dosage administration regimens, as well as to help reducing toxicity effects and enhancing target drug delivery. A more in-depth understanding of the mechanism of action of the hit/lead compounds could also potentially help to improve the potency and selectivity of the candidate drugs through rational design of optimized drugs.Finally, significant efforts must be invested in developing extensive in vivo screenings in models that mimic human pathophysiology in order to achieve higher predictability for successful Ru(III)-drug development.Overall, research on the encapsulation of Ru(III) complexes will undoubtedly continue to develop and, owing to the high structural variety of nanosystems and Ru derivatives, new delivery platforms will certainly be realized in the near future for anticancer therapies.Based on the continuous efforts that worldwide researchers have invested in this field, our feeling is that it is only a matter of time before new metal complexes with superior efficacy and low toxicity will be approved as anticancer therapeutics, finally replacing longstanding metal-based anticancer drugs as cisplatin and its derivatives.In this context, the next years will witness progress in elucidating real targets and mechanisms of action that will provide solid scientific basis for extensive preclinical development of selected Ru(III)-containing formulations.


Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions , we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures  the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture . Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton ( The oxygen consumed at the maximum incubation time , the data were not included in the analysis. An analysis of covariance (ANCOVA) was used to compare the CRO2-INTR regression lines obtained from each plankton culture.The CRpothesis  is that O2-INTR relationship of the cultures was compared with the published relationship derived from natural plankton populations Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The work described in this manuscript addresses the important issue of the validity of the INT method to estimate respiration. The experiments were well planned, well conducted and well interpreted.However, a number of points need attention.Lines 32-33. A quantitative comparison is required of the times needed to get an estimate of respiration for the two methods.Lines 37-40. Does this toxic effect include alteration to the function of the plasma membrane?Line 44. ‘a marine bacterium’ .Line 51. A salt is, by definition, composed of a cation and an anion. Replace by ‘INT is a cell permeable cation [15]…..’.Line 54. Prokaryotic plankton organisms, e.g. cyanobacteria, do have a cell wall, defined as structure outside the plasma membrane that can resist turgor Lines 58-59. Clarify ‘intracellular diffusion’. To me, intracellular means ‘within the cell’. The implication here seems to be the diffusion into, not within, the cell.Line 71. See my comment on Line 54. Are the cell coverings of the two coccolithophores and the two dinoflagellates turgor resistant? See Sikes and Wilbur 1982 Limnology and Oceanography 27: 18-26.Line 80. ‘were’, not ‘was’; ‘media’ is plural.Line 394. These references do not adequately distinguishing adsorptionon extracellular structures and entry into the cell. For the uptake of essential trace metal uptake into eukaryotic marine microalgal cells, there is a large body of information from the laboratory of F M M Morel in Princeton NJ. For the organic compound NADH, the permeability of the cell wall of chlorophytan freshwater microalgae depends on the presence or absence of a sporopollenin layer in the cell wall (Syrett and Thomas 1973 New Phytologist 72: 1307-1310).Lines 396-398. Is there any mechanistic basis for the allocation of electrons between the reduction of INT and the reduction of oxygen?Reviewer #2: In this paper the authors report on the use of the tetrazolium salt INT to determine respiration rates in microalgal cultures. The use of INT is well established as a mechanism to estimate electron transport activity but the present paper has the novelty of careful attention to the effects of time of incubation and potential toxicity on apparent rates of respiratory activity. As such it does represent a useful contribution to the literature. However there are a number of issues that require attention before the manuscript is suitable for publication.line 29: I would hardly call use of INT 'recent' as its use as a measure of respiratory electron transport goes back a long way  33: 119 for microalgae and Owens, T.G. & King, F.D. Mar. Biol. (1975) 30: 27. for zooplankton).line 58: I am not convinced about the arguments that different cell wall structures represent a major barrier to the entry of INT. For metals at least the cell wall components act less as a permeability barrier than a binding site for ions . The plasmalemma would be more of a barrier to transport of a changed molecule such as INT into the cell.line 79: Here and elsewhere 'media' is the plural of medium, so only use this (with 'were' ) if you used more than one type of mediumline 85: At what stage of growth were these cultures at the time of the experiments?This is important if experiments were done over 2 days as physiological characteristics can change rapidly in batch cultures. There is a brief reference to exponential stage cultures in the Discussion but this needs to be better defined early on.lines 89/90: which type of PAM? Walz make lots of different forms of the instrument. More details such as saturating pulse intensity are neededline 102-105: this looks like pseudoreplication rather than the use of independent biological replicates. Please clarify.lines 122-123: Consider showing the equation for the 3-parameter ‘exponential increase to a limit’ functionlines 208-9: This is fair enough as an estimate of steady state dark respiration, but how does this relate to the fact that respiration rates immediately after a light exposure can be up to x10 those at steady state in the dark? See e.g. Beardall et al a994 Journal of Plankton Research Vol.16 no.10 pp. 1401 -1410. This is also relevant to the Discussion.line 366: Stating this is 'for the first time' is not strictly accurate given the mention to reference 12 cited below (line 374)line 386: choice of incubation time is further complicated by the enhanced post illumination respiration shown by many phytoplankton species (see comment above)Reviewer #3: The manuscript "INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure" focuses on an alternate method for measuring plankton respiration with the help of INT. Overall the manuscript is well written and has applicable objective, however the authors would need to address the following concerns:1. Tetrazolium salts can also be reduced by reactive oxygen species in the cell, therefore if the plankton is under oxidative stress, use of this technique can lead to over-estimation of the respiratory rates. The authors need to either compare the reduction of INT in the presence and absence of oxidative stress or at least discuss the possibility of interference by ROS and advise caution with the interpretation.2. Fig. 3 shows extreme variation in results even within the same species, for example the oxygen consumption patterns are very different between the two figures of T. pseudonana. This suggest extreme variability in this method, not just between species but within the same species. Can the authors provide an explanation for this?Also, it looks like E. huxleyi and Scrippsella sp. were excluded from this measurement, is there a particular reason?3. Measurement of respiration with the help of Clark-type oxygen electrode usually takes about 10-15 mins? How does the authors just the use of INT as a better way to measure respiration? Moreover, the authors themselves have used optodes to compare the INT measurements. What advantages does INT measurements have over clark type electrodes and optodes? Consider discussing this in terms of cost, time, and sample volume?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  31 Oct 2019Journal Requirements:1. When submitting your revision, we need you to address these additional requirements. The manuscript meets PLOS ONE’s requirements2. In order to enhance reproducibility, please clarify the origin of the strains used, and provide any further details available on the origin and composition of the seawater used.More information on the origin of the strains and seawater are provided (lines 75-81 and lines 84-86).https://collections.plos.org/s/extreme-environments. If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.3. Our internal editors have looked over your manuscript and determined that it may be within the scope of our Life in Extreme Environments Call for Papers. The Collection will encompass a diverse range of research articles to better understand life and biogeochemistry in extreme environments. Additional information can be found on our announcement page: Thank you for the offer to include the manuscript in the “Life in Extreme Environments” Call. We consider that this study does not address any of the topics proposed in the call. Our study deals with eukaryotic plankton grown in healthy, nutrient rich, non hostile conditions. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.All data have been sent to BODC on the 28th October 2019. I have been notified that they will need two weeks to process the data and provide a DOI for it. I will forward it to the journal as soon as I have it. 5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository  and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.All data have been sent to BODC repository and a DOI number will be inserted upon reception___________Response to ReviewersWe are happy to read that the reviewers share our opinion that the manuscript contributes to the existing literature. We would like to thank the three reviewers for their comments and suggestions for additional references, which has helped to improve the manuscript. All the points have been dealt with in the text and new references have been included. These changes are indicated in our responses below specifying the lines in the latest submitted manuscript (after track changes). We have also addressed additional journal requirements. Reviewer #1: The work described in this manuscript addresses the important issue of the validity of the INT method to estimate respiration. The experiments were well planned, well conducted and well interpreted. However, a number of points need attention.Lines 32-33. A quantitative comparison is required of the times needed to get an estimate of respiration for the two methods.We have indicated (lines 32-33) that the incubation time for the INT method is from minutes to hours, while for the Winkler titration method is usually 24 h. Lines 37-40. Does this toxic effect include alteration to the function of the plasma membrane? As far as we are aware, reports of the toxicity of INT and other tetrazolium salts, are based on the inhibition of bacterial growth , inhibition of oxygen consumption , and inhibition of other metabolic activities . There is no mention in the literature of any alteration to the function of the plasma membrane. Line 44. ‘a marine bacterium’ . Corrected (line 43). Line 51. A salt is, by definition, composed of a cation and an anion. Replace by ‘INT is a cell permeable cation [15]…..’. Corrected (line 50).Line 54. Prokaryotic plankton organisms, e.g. cyanobacteria, do have a cell wall, defined as structure outside the plasma membrane that can resist turgor . Corrected. We have changed this sentence to focus only on the diversity of eukaryotic plankton cell wall composition (line 53). Lines 58-59. Clarify ‘intracellular diffusion’. To me, intracellular means ‘within the cell’. The implication here seems to be the diffusion into, not within, the cell. We have modified the sentence to indicate that the diffusion is into the cells (line 57). Line 71. See my comment on Line 54. Are the cell coverings of the two coccolithophores and the two dinoflagellates turgor resistant? See Sikes and Wilbur 1982 Limnology and Oceanography 27: 18-26. Unfortunately, we cannot comment on the turgor resistance of the species. We did not check the turgor pressure or the osmotic properties of any of the cultures, so we cannot respond to the reviewer with any certainty. From our results with the calcified and noncalcified organisms, we can indicate that the coccosphere of the calcified organisms seems to not affect diffusion into the cell, as the relationship between oxygen consumption and INT reduction was similar for the two species. If the coccosphere were negatively affecting diffusion into the cell, then there should be a different slope between the CRO2 and INTR, assuming that once inside the cell, the INT has a similar behaviour. The study of the similar or different CRO2 – INTR relationship is one of the main objectives of this study as it will indicate whether a single moles of INT to moles of oxygen conversion equation is adequate for all natural plankton populations which encompass a mixture of different eukaryotic organisms. Line 80. ‘were’, not ‘was’; ‘media’ is plural. Corrected in several part of the manuscript .Line 394. These references do not adequately distinguishing adsorptionon extracellular structures and entry into the cell. For the uptake of essential trace metal uptake into eukaryotic marine microalgal cells, there is a large body of information from the laboratory of F M M Morel in Princeton NJ. For the organic compound NADH, the permeability of the cell wall of chlorophytan freshwater microalgae depends on the presence or absence of a sporopollenin layer in the cell wall (Syrett and Thomas 1973 New Phytologist 72: 1307-1310). Thank you for the reference suggested. We now refer to these studies as the reviewer suggests (lines 57 and 406).Lines 396-398. Is there any mechanistic basis for the allocation of electrons between the reduction of INT and the reduction of oxygen? Four electrons are needed to reduce one mol of oxygen while only 2 electrons are needed for a mol of INT, but we are not aware of any mechanistic basis for the allocation of electrons between the INT and the oxygen. We are currently studying the enzymes involved in the INT reduction method with several marine plankton microorganisms in order to better understand the reduction of the INT by the ETS and other intracellular compounds. We may be able to answer this question in the future.Reviewer #2: In this paper the authors report on the use of the tetrazolium salt INT to determine respiration rates in microalgal cultures. The use of INT is well established as a mechanism to estimate electron transport activity but the present paper has the novelty of careful attention to the effects of time of incubation and potential toxicity on apparent rates of respiratory activity. As such it does represent a useful contribution to the literature. However there are a number of issues that require attention before the manuscript is suitable for publication.line 29: I would hardly call use of INT 'recent' as its use as a measure of respiratory electron transport goes back a long way  33: 119 for microalgae and Owens, T.G. & King, F.D. Mar. Biol. (1975) 30: 27. for zooplankton).This sentence has been changed and now it says: “Several studies have proposed…” (line 28). line 58: I am not convinced about the arguments that different cell wall structures represent a major barrier to the entry of INT. For metals at least the cell wall components act less as a permeability barrier than a binding site for ions . The plasmalemma would be more of a barrier to transport of a changed molecule such as INT into the cell. There is evidence that the cell wall can be a physical barrier to different molecules (ref 17). In addition, there are studies that reported a lack, or low, INT reduction by specific organisms and suggested differences in the cell wall as one of the possible causes . We considered that if true, this could create a bias when applying the method to a natural mixed plankton population. We agree with the reviewer that the plasmalemma could also be another barrier. Nevertheless, the significant similarities between the CRO2 – INTR regression lines for each culture suggest that the INT penetrates and it is reduced at a similar rate in the organisms tested.line 79: Here and elsewhere 'media' is the plural of medium, so only use this (with 'were' ) if you used more than one type of medium. The text has been corrected accordingly (line 81). line 85: At what stage of growth were these cultures at the time of the experiments? This is important if experiments were done over 2 days as physiological characteristics can change rapidly in batch cultures. There is a brief reference to exponential stage cultures in the Discussion but this needs to be better defined early on. As indicated in line 100, the experiments were done in exponential growth phase during both days (day 1: maximum incubation time experiments and day 2: oxygen consumption-INT reduction). lines 89/90: which type of PAM? Walz make lots of different forms of the instrument. More details such as saturating pulse intensity are needed We have added more information about the Phyto-PAM equipment. Now it reads: “The Fv/Fm ratio of the cultures was measured with a Walz PHYTO-PAM Phytoplankton Analyzer fluorometer  after cultures were kept for 10 minutes in the dark. The pulse amplitude modulated fluorescence measurements were completed at high repetition rates at four excitation wavelengths . The intensity of the measuring light was 64 μmol quanta m-2 s-1, and the saturating pulse was supplied at the maximum level specified by the Phyto-PAM (intensity level 10) at a pulse of 500 ms.” (Lines 93-99) line 102-105: this looks like pseudoreplication rather than the use of independent biological replicates. Please clarify.The maximum incubation time experiment was done with one cubitainer so there were no biological replicates. Experimental triplicate bottles were taken for each incubation time. We acknowledge that biological replicates would be ideal but the complexity of our experiments precludes this. Each maximum incubation time experiment, as designed here or in other studies , implies around 14 hours of work. Our main concern was to perform the maximum incubation time experiment as close to the oxygen - INT reduction experiment as possible. To achieve this, we could not perform biological replicates.lines 122-123: Consider showing the equation for the 3-parameter ‘exponential increase to a limit’ functionThe equation has been added (line 132).lines 208-9: This is fair enough as an estimate of steady state dark respiration, but how does this relate to the fact that respiration rates immediately after a light exposure can be up to x10 those at steady state in the dark? See e.g. Beardall et al a994 Journal of Plankton Research Vol.16 no.10 pp. 1401 -1410. This is also relevant to the Discussion. The reviewer is correct when he/she says that there is evidence of an enhancement of respiration following a period of light illumination and that this could bias the respiration rates measured in natural populations over 24 hours, or the respiration measurements from cultures after the light incubation period. Our measurements of respiration are dark respiration: the subsampling was done at the end of the dark cycle and the cells were maintained in dark during all the different processes . From our results, we have no information regarding the possible stimulation of respiration after the exposure of cells to light. Based on this, we have not included any speculation in the discussion section to as not to distract from the main conclusions: INT did not negatively affect the oxygen consumption when incubated at short incubation times, and there was a similar oxygen consumption – INT reduction relationship for eukaryotic plankton with different cell walls. line 366: Stating this is 'for the first time' is not strictly accurate given the mention to reference 12 cited below (line 374). Our work is the first to show with different marine eukaryotes that there was no difference between the oxygen consumption of a culture with and without INT during the time when the production of formazan remained linear. Villegas-Mendoza et al. 2015 showed no toxicity (change in the oxygen consumption rate) with only one eukaryotic organism in one experiment, but there was no concurrent estimation of the formazan production. line 386: choice of incubation time is further complicated by the enhanced post illumination respiration shown by many phytoplankton species (see comment above). We agree with the reviewer. Our results indicate that the choice of the incubation time for the INT reduction methodology depends not on the cell biomass, but on the activity of the sample, and that it is important to calculate the maximum INT incubation time to prevent biased the measurements. Reviewer #3: The manuscript "INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure" focuses on an alternate method for measuring plankton respiration with the help of INT. Overall the manuscript is well written and has applicable objective, however the authors would need to address the following concerns:1. Tetrazolium salts can also be reduced by reactive oxygen species in the cell, therefore if the plankton is under oxidative stress, use of this technique can lead to over-estimation of the respiratory rates. The authors need to either compare the reduction of INT in the presence and absence of oxidative stress or at least discuss the possibility of interference by ROS and advise caution with the interpretation.We are afraid we cannot quantify the proportion of INT reduced by ROS as we did not estimate the reduction of INT in the presence and absence of oxidative stress. We have commented on the possibility of biases due to the presence of ROS in the text and have suggest caution in interpreting the results (lines 444-457). 2. Fig. 3 shows extreme variation in results even within the same species, for example the oxygen consumption patterns are very different between the two figures of T. pseudonana. This suggest extreme variability in this method, not just between species but within the same species. Can the authors provide an explanation for this?Also, it looks like E. huxleyi and Scrippsella sp. were excluded from this measurement, is there a particular reason?We agree with the reviewer that there are different patterns for the oxygen consumption between species and within the same species, for example T. pseudonana. The two examples in Figure 3 are from different experiments with different cell abundances and different respiration rates. Different trends in oxygen consumption estimated with oxygen sensors have been previously reported  which indicate that dark respiration does not always follow a linear decrease. However, there was a linear decrease during the incubation time employed for the estimation of the oxygen consumption by Winkler, and therefore, our oxygen consumption results should not be biased by a non-linear trend. This is indicated in lines 241-246. We are afraid we cannot provide any oxygen consumption data measured with the optodes during E. huxleyi experiments. There was a reaction between the metallic needle of the optode and the medium, which precluded accurate measurements. The optode sensors were returned to the manufacturer for servicing and re-coating with a non-reactive film. The optodes were not available for the INT maximum incubation time experiments with Scrippsiella sp. 3. Measurement of respiration with the help of Clark-type oxygen electrode usually takes about 10-15 mins? How does the authors just the use of INT as a better way to measure respiration? Moreover, the authors themselves have used optodes to compare the INT measurements. What advantages does INT measurements have over clark type electrodes and optodes? Consider discussing this in terms of cost, time, and sample volume?The reviewer is correct when he/she comments that Clarke type electrodes and oxygen optodes are sensors that can provide oxygen concentration measurements in short periods of times (0.5-2 hours). However, despite their fast response, they still have limitations regarding the response time needed when applied to low plankton respiration rates (~ 0.5 μmol L-1 d-1 as occurs in open ocean oligotrophic environments). In these situations, the time needed to record the change is longer, and always longer than the time needed for the incubation in the INT reduction technique. Advantages of the INT technique (as mentioned in lines 31-35) over optodes and electrodes include sensitivity and the ability to determine the respiration of size classes of the plankton community without pre-incubation filtration.We do not agree that a detailed comparison of the different methods used to estimate plankton respiration  in terms of cost, time and sample volume is appropriate in this manuscript. We would prefer to focus on the two conclusions: INT did not negatively affect the oxygen consumption when incubated at short incubation times, and there was a similar oxygen consumption – INT reduction relationship for eukaryotic plankton with different cell walls. 18 Nov 2019INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structurePONE-D-19-27198R1Dear Dr. Garcia-Martin,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Antonietta Quigg, PhDAcademic EditorPLOS ONEAdditional Editor Comments :In submitting the final manuscript, please consider inserting, as appropriate, additional parts of your responses to the reviewers concerns. for example it will be helpful for readers to know why you had no biological controls, the issues with the optodes and why some data is missing, etc... in each case... a carefully placed sentence in the methods and results could further enhance the paper. 2 Dec 2019PONE-D-19-27198R1 INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure Dear Dr. Garcia-Martin:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Antonietta Quigg Academic EditorPLOS ONE


Four TOP1 poisons are clinically used for tumor treatment now. In spite of their effectiveness in solid tumors, these camptothecin (CPT) poisons suffer from many shortcomings. Therefore, many investigations have focused on the discoveries of non-CPT poisons and catalytic inhibitors. Herein, we systematically study the antitumor activity of CYB-L10, a novel indolizinoquinolinedione TOP1 catalytic inhibitor discovered in our laboratory. The results indicated that CYB-L10 mainly acts on TOP1 in cancer cells and is not a substrate of the P-glycoprotein. In addition, CYB-L10 can induce apoptosis of HCT116 cells, shows high cytotoxicity against 60 human clinical cancer cell lines (NCI60) with the mean-graph midpoint for growth inhibition of all cancer cell lines of 0.050 µM concentration and obvious antitumor efficiency  TOP1 poisons are able to trap TOP1cc to prevent further religation and thus leading to irreversible DNA strand breaks,,,,in vitro against 60 clinical cancer cell lines and in vivo in HCT116 xenograft mice model.In spite of their effectiveness in solid tumors, CPT poisons suffer from many shortcomings, including bone marrow dose-limiting toxicity, severe gastrointestinal toxicity26)The human wild-type cancer cell lines HCT116 and DU-145, the resistant cell lines HCT116-siTop1 and RC0.1 were a kind gift from Dr. Y. Pommier . The human wild-type cancer cell lines MCF-7, and HepG2, and the resistant cell lines MCF-7/ADR and HepG2/ADR were a kind gift from Dr. X. Z. Bu . CYB-L10 (molecular weight: 405.41) was synthesized according to our reported method .For the drug treatment experiments, the cancer cells were treated with CYB-L10 (predissolved in DMSO) at a five-dose assay concentration of 0.01, 0.1, 1, 10, and 100 µM. After incubation for 72 h at 37 °C, the MTT solution  in PBS (PBS without MTT as the blank) was fed to each well of the culture plate (containing 100 µL medium). After 4 h incubation, the formazan crystal formed in the well was dissolved with 100 µL of DMSO for optical density reading at 570 nm. The GI,,50) for each cell line at which the concentrations of compound that resulted in 50% cell growth inhibition. The overall antiproliferative potential is quantified as a mean-graph midpoint (MGM).The NCI60  tumor cell drug discovery panel was developed as a tool to assess the anticancer activity of compounds against a range of cell lines derived from nine cancer cell types, including hematological malignancies, lung, central nervous system, melanoma, colorectal, renal, breast, ovarian, and prostate5 cells/mL) were grown in culture medium on 6-well plates and incubated in the presence or absence of CYB-L10  for 24 h. And then, the cells were harvested and washed with cold PBS buffer, resuspended in 1 × binding buffer, and then stained with 5 µL Annexin V-FITC and 5 µL propidium iodide  for 15 min in dark. The stained cells were analyzed by using flow cytometry  within 1 h. The experiments were repeated independently for three times.HCT116 cells (3.0 × 10n = 2) were treated with CYB-L10 predissolved in 10% DMSO and 10% Kolliphor® HS15 (a nonionic solubilizer) by intravenous injection  and intragastric administration , respectively. Blood samples (200 µL) were collected into heparinized tubes via the jugular vein at the following times: 0.083, 0.25, 0.5, 1, 2, 5, 7, and 24 h after dosing. Plasma samples (100 µL) were obtained after centrifugation for 10 min at 3000 rpm and stored at −20 °C until used for analysis. The plasma was detected through LC-MS/MS.Male Sprague-Dawley rats  was implanted. When the implanted tumors had reached a volume of about 80 mm3, the mice were randomly divided into three groups (n = 5) and administered by i.p. injection at a frequency of once every 3 days. The testing groups were treated with CYB-L10 at 20 mg/kg and 6.7 mg/kg dose, respectively. The control group was treated with an equivalent volume of saline. Tumor volumes (V) were monitored by caliper measurement of the length and width, and calculated using the formula: V = (larger diameter) × 2/2, and growth curves were plotted using average tumor volume within each experimental group at the set time points. At the end of treatment, the animals were sacrificed, and the tumors were removed and weighed. The tumor weight inhibition (TWI) was calculated according to the formula: TWI =  × 100%. Statistical comparisons were conducted using a one-way analysis of variance, followed by Tukey’s test.Athymic nude mice bearing the nu/nu gene were obtained from Laboratory Animal Center of Sun Yat-sen University and maintained in pathogen-free conditions to establish the model of xenografts of HCT116. All animals were used under the Policy on the Care and Use of Laboratory Animal of Sun Yat-sen University. Male nude mice 4–5 weeks old weighing 12–15 g were used. HCT116 tumor preinduced in the mice by subcutaneously injecting of HCT116 cells . It was selected and submitted to National Cancer Institute  for a developmental therapeutics assay against the 60 clinical tumor cell lines representing nine tissue types (NCI60)50) was calculated relative to the control without compound. As shown in 50 values below the tested minimum concentration (10 nM) for 15 tumor cell lines.CYB-L10  was pote,The cytotoxicity of CYB-L10 in drug-resistant cell lines was evaluated by using the MTT assay and summarized in ,The prostate cancer cell RC0.1 has an R364H mutation in the TOP1 relating to the wild-type parental cell DU-145P-glycoprotein (Pgp) mediated drug efflux is generally responsible for classical multiple drug resistanceannexin V-FITC/PI was carried out for HCT116 cells. As shown in To estimate the effect of CYB-L10 on apoptosis, flow cytometry analysis using double staining with in vivo pharmacokinetic (PK) study of CYB-L10 was conducted in SD rats. The SD rats were divided into two groups (n = 2) and treated by i.v. injection at 1 mg/kg dose and i.g. at 5 mg/kg dose, respectively. The plasma samples were collected up to 24 h postdose and measured for the concentration of CYB-L10. The PK parameters were calculated and summarized in 0→∞ was 241 ± 40 h·ng/mL, and 1/2T was 18.4 ± 5.12 h. After oral administration, the maxT, maxC and AUC0→∞ were 0.46 ± 0.10 h, 24.7 ± 6.59 ng/mL and 76.0 ± 16.5 h·ng/mL, respectively. The bioavailability (F) was 6.3%.The in vivo. Mice were randomly divided into three groups and administered i.p. injection at a frequency of once every 3 days. Two groups were treated with CYB-L10 at 20 mg/kg and 6.7 mg/kg dose, respectively. The negative control group was treated with an equivalent volume of saline. As shown in p < 0.05) and 27.6% (6.7 mg/kg), respectively  with MGM value of 0.050 µM and induce apoptosis of HCT116 cells in the late apoptotic stage, implying CYB-L10 is a good anticancer candidate. The drug-resistant cell assays indicated that CYB-L10 may mainly act to TOP1 in tumor cells and is not a substrate of the Pgp, a drug efflux protein involving in multiple drug resistance. CYB-L10 was also evaluated on PK and antitumor efficiency


In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG •PODXL-knock out cell lines were established using HSC-2.•The growth of HSC-2/PODXL-KO cells were lower than that of parental HSC-2.•Tumor volumes of HSC-2/PODXL-KO cells were lower than that of HSC-2.•in vitro and in vivo.PODXL has an important role in tumor growth  BINDS-01 and BINDS-02 can be obtained from Kato's lab in Tohoku University Graduate School of Medicine (http://www.med-tohoku-antibody.com/topics/001_paper_cell.htm).HSC-2 cells were obtained from Japanese Collection of Research Bioresources Cell Bank . HSC-2/PODXL-KO cells (BINDS-02) were produced using CRISPR/Cas9 plasmids (Target ID: HS0000056763), targeting human PODXL . Three clones were selected for this study: HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3. Parental HSC-2 and HSC-2/PODXL-KO cells were cultured in Dulbecco's Modified Eagle's Medium , which was supplemented with 10% heat-inactivated fetal bovine serum , 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B , at 37 °C in a humidified atmosphere containing 5% CO2.2HSC-2/PODXL-KO cell lines were harvested following a brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid . After washing with 0.1% bovine serum albumin in phosphate-buffered saline , cells were treated with primary mAbs for 30 min at 4 °C, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG . Fluorescence data were obtained using SA3800 Cell Analyzer .2.3In vitro cell proliferation was measured using Cell Cloning Kit-8 , which can assess cell viability. Cells were plated  in quintuple wells in 96-well plates and were incubated for 48 h. After adding 10 μL of CCK-8 to each well, the plates were incubated for 4 h at 37 °C. Subsequently, the absorbance was recorded at 450 nm using iMark microplate reader . The mean absorbance of the 5-well set was obtained at 48 h after cell seeding. All data were expressed as the mean ± SEM. Statistical significance was analyzed using Tukey–Kramer's test. P-values < 0.05 were considered statistically significant.2.4in vivo proliferation assay. Cells (0.1 mL of 5 × 107 /mL in DMEM) were mixed with 0.1 mL of BD Matrigel Matrix Growth Factor Reduced . A 200 μL suspension (containing 5 × 106 cells) was subcutaneously injected into the left flanks of nude mice. In this experiment, the following four cell lines were used: HSC-2, HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3. Each group included eight mice. The tumor diameter was measured at day 7, 14, and 21 after injecting cancer cell lines using calipers, and the tumor volume was calculated using the following formula: volume = W2 × L/2, where W is the short diameter, and L is the long diameter. All mice were euthanized 21 days after cell implantation, and the tumor weight was measured. All data were expressed as mean ± SEM. Statistical analysis was performed using Tukey–Kramer's test. P-values < 0.05 were considered statistically significant.Five-week-old, female BALB/c nude mice were purchased from CLEA Japan . Seven-week-old mice were used for the 2.5After euthanizing mice at day 21, histopathological specimen sections  were deparaffinized in xylene, rehydrated, and stained with hematoxylin and eosin.3For this study, CRISPR/Cas9 plasmids targeting human PODXL were used to generate three PODXL-KO OSCC cell lines: HSC-2/PODXL-KO #1, #2, and #3. Before initiating the study, PODXL expression was confirmed in these cell lines. As shown in in vitro OSCC cell growth. At every plated cell density , the growth of HSC-2/PODXL-KO #1 and #2 cells was significantly lower than that of HSC-2 cells , JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number 17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.).This research was supported in part by