It is therefore necessary to design robust scalable methods to enable efficient expansion of bona fide CSCs in vitro. Here, we evaluated the applicability of computer-controlled bioreactors combined with 3D aggregate culture and microcarrier technology, widely used in stem cell bioprocessing, for the expansion and enrichment of CSCs isolated from different types of solid tumors—colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC) from two patients. Results show that these culture strategies improved cell expansion and CSC enrichment. Both patient-derived CSC lines were able to grow on microcarriers, the best results being achieved for PPlus 102-L, Pro-F 102-L, Fact 102-L, and CGEN 102-L beads . As for 3D aggregate culture strategy, the cell proliferation profile was donor dependent. NSCLC cells were the only cells able to form aggregates and proliferate, and the flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller was the most efficient configuration for cell growth (21-fold increase in cell concentration achieved in 8 days). Serum-free medium promotes CSC enrichment in both 3D aggregate and microcarrier cultures. The protocols developed herein for CSC expansion have the potential to be transferred to clinical and industrial settings, providing key insights to guide bioprocess design towards the production of enriched CSC cultures in higher quantity and improved quality.Cancer stem cells (CSCs) have recently raised great interest as a promising biological system for designing effective cancer therapies. The scarcity of CSCs  Cancer stem cells (CSCs) represent a promising target for effective anticancer therapies , 2 as thIt has been reported that CSCs exist within almost every solid tumor , 6 at a In this work, computer-controlled stirred tank bioreactors combined with 3D cell aggregate cultures as well as microcarrier technology were applied for the first time to expand and enrich CSCs from two different patient-derived cell lines—non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). The findings reported herein provide novel knowledge to guide cell bioprocess design towards the production of CSC in higher quantity and improved quality, which are key requisites for their application in drug discovery and in the development of new cancer therapeutics.CSC lines were established in Merck Biopharma, ImmunoOncology, following a proprietary protocol. Tumor cells were derived from lung and colorectal cancer patients and purchased from Indivumed . Classification of the tumors was large-cell carcinoma, NOS, and colorectal carcinoma. CSC lines (CRC and NSCLC) were routinely propagated in collagen I-coated T-flasks as described in supplemental online data.6 cell/mL and cultured during 8 days in two different bioreactor configurations—round-bottom bioreactor vessel equipped with a pitched 4-bladde impeller (BR-R/P4b) and flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller (BR-F/T) . Two culture medium formulations (serum-containing medium (SCM) and serum-free medium (SFM)) and four cell aggregate dissociation protocols were tested .CSC lines were inoculated as single cells in computer-controlled stirred tank bioreactors at a concentration of 0.25 × 104 cell/cm2) in ultra-low-attachment plates and cultured for 6 days at 37°C in a humidified atmosphere of 5% CO2 using SCM or SFM. Eight commercially available microcarriers were tested: Cytodex1™, Cytodex3™, PPlus 102-L, Pro-F 102-L, Fact 102-L, CGEN 102-L, Cytopore2™, and CultiSpher®-S as described in the supplemental online data.CSC lines were inoculated as single cells with empty microcarriers  (data not shown). For their expansion, different bioreactor configurations and aggregate dissociation protocols were evaluated. In addition, microcarrier-based cultures were investigated; eight different microcarriers  and non-small-cell lung cancer (NSCLC) from two patients, when routinely cultured in static adherent culture systems, show percentage of ALDHem cells , 11, supμm) [+ cells (>70%) , contrasting to SCM cultures where no increase was attained  (NSCLC cells were able to grow as aggregates in computer-controlled stirred tank bioreactors independently of the medium used (SCM or SFM) Figures . Importaμm) . Notewors (>70%) . Culture/T; SCM) .+ subpopulations (74-83%)   were obtained in less culture time (6 days) when compared to aggregate culture in bioreactors, the exception being Cytopore2™ beads . In addinoculum) .+ subpopulations in relation to the inoculum were observed for cultures using SFM (6 cells/mL) tested  was observed for PPlus 102-L, Fact 102-L, CGEN 102, and Pro-F 102-L beads . The culsing SFM . The twosing SFM , Table 1This work describes, for the first time, the successful application of computer-controlled stirred tank bioreactors combined with 3D aggregate cultures as well as microcarrier technology to expand and enrich human CSCs. Despite the fact that there is no universal culture strategy capable of embracing different types/patient-derived CSCs, the protocols developed herein for CSC expansion can be easily screened prior to their transfer to clinical and industrial settings. This study also provides key insights to guide bioprocess design towards scalable production of patient-derived CSCs with improved quality. This will potentiate their application in drug discovery and for the development of new cancer therapeutics.


The observation that some aspects of amoeba-fungal interactions resemble animal phagocytic cell-fungal interactions, together with the finding that amoeba passage can enhance the virulence of some pathogenic fungi, has stimulated interest in the amoeba as a model system for the study of fungal virulence. Amoeba provide a relatively easy and cheap model system where multiple variables can be controlled for the study of fungi-protozoal (amoeba) interactions. Consequently, there have been significant efforts to study fungal–amoeba interactions in the laboratory, which have already provided new insights into the origin of fungal virulence as well as suggested new avenues for experimentation. In this essay we review the available literature, which highlights the varied nature of amoeba-fungal interactions and suggests some unsolved questions that are potential areas for future investigation. Overall, results from multiple independent groups support the ‘amoeboid predator–fungal animal virulence hypothesis’, which posits that fungal cell predation by amoeba can select for traits that also function during animal infection to promote their survival and thus contribute to virulence. Cryptococcus neoformans in macrophages and the free-living amoeba Acanthamoeba castellani revealed remarkable similarities leading to the proposal that its capacity for virulence emerged accidentally from selection by amoeboid predators in soils [Aspergillus spp., Histoplasma capsulatum, Blastomyces dermatitides, Sporothrix schenkii, Cryptococcus gattii and entomogenous fungi [Fungi are major pathogens of plants, insects and ectothermic vertebrates but there are relatively few that are able to cause disease in mammals. The remarkable resistance of mammals to fungal diseases is attributed to a combination of endothermy, which creates a thermal restriction zone for survival of most fungal species and adaptive immunity ,2,3. Howin soils . Subsequus fungi ,7,8,9. Aus fungi ,11.In the past two decades, the concept that amoeba serve as selection mechanism leading to the emergence of virulence in different microbes has gained credence ,13,14 anCryptococcus spp. [A. castellanii was shown to feed on C. neoformans [Torula famata and Candida parapsilosis [C. parapsilosis was much faster than growth on the filamentous T. famata since the protozoa had difficulty ingesting hyphae [C. neoformans with amoeba and established that the protozoa preyed and devoured encapsulated cells with the emergence of hyphal cells as resistant forms, which were hypovirulent in mice [C. neoformans in the environment [A. castellanii was shown to rapidly kill cultures of S. cerevisiae, reducing more than half of the fungal cells after 90 min of co-incubation [The interaction of amoeba with animal pathogenic fungi has been studied for decades. In 1931, Castellani reported an amoeba growing in cultures of a cus spp. , providioformans  and subspsilosis . Interesg hyphae . Beginni in mice ,25. Furtironment , establicubation .Alternaria, Curvularia, Helminthosporium were internalized by Thecamoeba spp. and later egested in a viable condition, in a closer resemblance of nonlytic exocytosis [The focus of this review is on animal pathogenic fungi with particular emphasis on those that cause disease in mammals, but we note that other investigators have also explored the interaction of soil amoeba with other groups of soil fungi, including yeast and filamentous fungi acting in nature as saprophytes, entomopathogens and even as phytopathogens ,30,31,32ocytosis . DespiteC. neoformans is the most extensively studied species with regards to interactions with protozoa , a process discovered in macrophages [A. castellanii [D. discoideum [C. neoformans cells produce 3-hydroxy fatty acids, which inhibit amoeba phagocytosis and could have a similar role during infection for macrophages [In addition to the correspondence between animal and amoeba survival for selected fungal cells expressing certain virulence factors, there are also remarkable similarities in several cellular processes. response . The capresponse . The progal cell . Hence, gal cell , incapabrophages ,49, was tellanii  and D. dscoideum , providiscoideum . Anotherscoideum , which hscoideum  and to iscoideum . C. neofrophages .C. neoformans virulence comes from passage experiments of an attenuated cryptococcal strain with D. discoideum, which showed a rapid increase in virulence [D. discoideum mutant defective in myosin VII synthesis was susceptible to a highly attenuated acapsular C. neoformans strain [C. neoformans are Paramecium spp, which readily ingested and killed cryptococcal cells [More direct evidence for the ability of amoeba to affect irulence . This wos strain . Anotheral cells .C. neoformans, there is circumstantial evidence that amoeba and Aspergillus spp. interact in the environment. Analysis of the environmental microflora in moisture-damaged buildings revealed the co-habitation of molds and amoeba [Aspergillus spp. benefitted from the presence of the protozoa while amoeba were indifferent to the presence of molds [C. neoformans, the interaction of Aspergillus fumigatus conidia with A. castellanii was found to have remarkable similarities to their interactions with avian macrophages [A. fumigatus with another amoeba, Vermamoeba (Hartmannella) vermiformis, promoted fungal filamentation and growth of and the major reduction of amoeba viability, while in the same conditions the interaction of this fungus with A. castellanii had no influence in fungal growth [A. fumigatus with the social amoeba D. discoideum revealed concordance between virulence factors for animals and those needed for fungal survival and killing of amoeba [D. discoideum thus linking the production of mycotoxins to predator control and fungal environmental survival [Entamoeba histolytica [Aspergillus spp. have been shown to have toxic effects on Naegleria gruberi and to mediate anti-phagocytic effects on human neutrophils and macrophages [Aspergillus spp. DHN melanin, which inhibits phagolysosomal acidification also in macrophages interferes with phagocytosis by amoeba, suggesting that this virulence determinant has different functions for fungal cell survival with confronted by animal and protozoal phagocytic cells [Like d amoeba . Laboratof molds . In pararophages . In addil growth . The autf amoeba . Specifisurvival . Anothertolytica , and on tolytica . Substanrophages ,66. Aspeic cells .Candida spp. interactions. Although commensal Candida spp. limited to mammalian hosts would not be expected to be under predation by ameba in soils, there are some body sites that are co-inhabited by fungal and protozoal cells where they could potentially interact. For example, amoeba are present in the human oral cavity [Candida spp. are also commonly found in a commensal state. Steenbergen et al. reported that A. castellanii preyed on C. albicans and efficiently reduced colony-forming units consistent with fungal killing [V. vermiformis was shown to be able to internalize yeast cells of C. albicans, C. glabrata and C. parapsilosis and promote their proliferation in tap water with or without saliva traces [Candida spp. and S. cerevisiae with D. discoideum and other non-axenic social amoeba was studied, including mutant strains of both the fungal and protozoal spp. [Relatively little work has been done to explore amoeba-l cavity  where Ca killing . In anota traces . Recentloal spp. . This stoal spp. .B. dermatitides, H. capsulatum and S. schenkii (A. castellanii and reported that each of these fungal species was able to grow in the presence of the amoeba [Fusarium spp. with amoeba is of interest given that both are involved in corneal infections. Amoeba have been proposed to be major biotic control factors for Fusarium oxysporum in soils [Fusarium with two different types of amoeba, A. castellanii and V. vermiformis, showed that although fungal cells were ingested the interaction resulted in enhanced fungal growth [Fusarium spp. with A. castellanii can enhance the virulence of both organisms [Fusarium spp. and A. castellanii has been reported with the suggestion that this condition should be suspected in situations of refractory infection [One study has investigated the interaction of schenkii  with A. e amoeba . The intin soils . Given tin soils ,73. Incul growth . Moreovenfection .Candida parapsilosis [C. neoformans lawns resulted in the emergence of pseudohyphal forms, which were resistant to amoeba but less virulent in mice [B. dermatitides, H. capsulatum and S. schenkii yeast cells with A. castellani at 37 °C led to the rapid emergence of filamentous forms for each fungal species even at a temperature where the yeast form is preferred [A. fumigatus conidia with the free living amoeba V. vermiformis was reported to promote fungal filamentation and growth [C. albicans forms hyphae inside or outside D. discoideum, but no hyphae formation without amoeba [C. neoformans hyphae, pseudohyphae and yeast forms showed that the elongated forms were resistant to phagocytosis by both macrophages and amoeba [C. neoformans cells with hyphal and pseudohyphal morphology were much less virulent in mice and moths, indicating that resistance to one host may be associated with vulnerability in another [C. neoformans during amoeba predation of cryptococcal lawns in agar was considered an ‘escape hatch’ for the survival of some cells [An emerging theme in amoeba-fungal studies is that hyphal forms are more resistant to protozoal predation than yeast forms. This was noted as early as 1964 when Nero et al. reported that a mold was more resistant to amoeba than psilosis . Similar in mice . Analysi in mice , suggestd growth . C. albit amoeba . Comparid amoeba . However another . The difme cells .A. castellanii grown in axenic media. Amoeba that have been selected in laboratory conditions to grow in axenic conditions may exhibit very different physiology than wild amoeba. In general, amoeba recovered from the environment do not grow in culture conditions and thus are difficult to use for fungal–amoeba interaction studies. Given the tremendous variety of amoeba in the environment, the reliance on A. castellanii in laboratory studies raises the concern that insights gained with this protozoal species may not be representative of most fungal–amoeba interactions in nature. Similarly, the relatively few studies of fungal interactions with D. discoideum have also been done with laboratory strains. Hence, the limitations of current systems and the paucity of amoeba strains that have been examined suggest caution in extrapolating laboratory results from environmental conditions. One potential resource to overcome this problem could be the studies of fungal pathogens with mycophagous amoeba. Both A. castellanii and D. discoideum have been shown to be more adapted to have bacterial cells as food sources. Although we can find a few studies about the interaction of amoeba species that in nature feed primarily on fungal cells, there is a general lack of knowledge of the interaction of mycophagous amoeba with animal fungal pathogens [Most of the studies of fungal–amoeba interactions have been conducted with athogens ,80. One C. neoformans and other pathogenic fungi with environmental habitats were reported to be more resistant to A. castellanii than C. albicans or laboratory adapted S. cerevisiae strains [C. neoformans with A. castellanii in phosphate buffered saline result in fungal growth while confrontations in amoeba media result in fungal predation with reduction of cryptococcal colony-forming units [C. neoformans, recent findings suggest that the enhanced protozoal activity could have resulted from divalent cations amoeba media. In this regard, the presence of Ca2+ and Mg2+ have strong effects in potentiating A. castellanii antifungal activity [Which entity prevails in fungal–amoeba confrontations is highly dependent on the experimental parameters. As evident from the review of the available literature, fungi prevailed in some studies while amoeba were ascendant in others. Clearly, there are major differences in the ability of individual fungal species to resist amoeba predation, a finding that dates to early studies . In this strains ,6. Thereng units . Althougactivity , suggestAlthough from our anthropomorphic vantage point we tend to focus on animal and human pathogenic fungi, it is important to note that this is a minute part of fungal interactions with other hosts ranging from those with Protista, Plantae and Animalia. Indirect evidence for the longstanding nature of amoeba-fungal interactions comes from the finding that higher fungi synthesize lectins that are toxic to amoeba, a finding consistent with the development of inducible fungal anti-protozoal defense mechanism . There iCryptococcus sp. and mechanisms for iron acquisition and biofilm formation in Candida spp. Once adapted to the intracellular environment of amoebas, these pathogens could also be protected from the action of biocides and environmentally harsh conditions, making their survival and dissemination more effective. Therefore, the expression of these fungal virulence traits should be also considered in the extent to which amoeba predation could influence susceptibility to antifungal drugs. In this regard, passage of fungi from sites inhabited by birds in amoeba reduced their susceptibility to amphotericin B [C. neoformans triggers transcriptional changes to lipid genes that affect the susceptibility to antifungal agents [Due to the enormous genetic variety of the community of microorganisms in the soil, each possesses a unique combination of characteristics and distinct attributes to avoid the action of phagocytic predators, such as chemical defenses, including the expression of capsule in ericin B . Althougl agents . In a siMetarhizium anisopliae and Beauveria bassiana has also been proposed to involve interactions with soil amoeba selected for traits that allow survival in insect hemophytes, which are host phagocytic defensive cells [The idea that animal pathogenic fungi acquired many of their characteristics for virulence from interactions with soil protozoal is almost two decades old . The orive cells . DespiteC. neoformans as the polysaccharide capsule, melanin synthesis and phospholipase production have protective roles in the environment that are equally adapted to protection against macrophages during animal infection [Aspergillus spp., melanin and mycotoxins tripacidin, gliotoxin and fumagillin serve similar functions during its interaction with amoeba and macrophages [The amoeboid predator-animal virulence hypothesis posits that constant amoeboid predation over eons selected for fungal traits that also facilitate survival in certain animal hosts and thus confer on those fungal species the capacity for virulence. According to this view such virulence factors in nfection  (Figure rophages . AlthougCryptococcus spp., each of which have capsules, found that the capsule of C. neoformans was the most effective in protecting fungal cells against macrophages [A central feature of the amoeboid predator-animal virulence hypothesis is that it posits that the capacity for virulence can emerge independent of the final host. The phenomenon was termed ‘accidental virulence’ in prior essays . The notrophages . Alterna


Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito,  G-protein-coupled receptors (GPCRs) are cell surface, membrane-binding proteins that are responsible for signal transmission through extracellular signal binding to activate and regulate intracellular factors. Both the constitutive and spontaneous activities of GPCRs are critically involved in cell signaling responses , providiCulex quinquefasciatus [In the last decade, many studies have confirmed that GPCRs play a crucial role in regulating insect physiological processes such as development, behavior, metabolism, and reproduction. These conserved intracellular pathways are present in several insect species. Because of the importance of functional GPCRs  and theiasciatus ,12,13. Iasciatus .Sf9 cells via baculovirus-mediated insect expression in order to investigate the specific function of each effector in insecticide resistance and the P450-expressed regulation of insect cells, as well as their complex connection via second messenger (cAMP) and PKA activity. The findings of this study are expected to not only lead to exciting new insights into intracellular cascades in insecticide resistance, but also to provide useful information that will support the development of novel strategies and/or insecticides for pest control and resistance management in the future.This study focuses on the expression of the mosquito GPCR, Gαs, AC, and PKA in insect Culex mosquitoes [Previous studies have shown that cell signaling effectors of GPCR–Gαs–AC–PKA–P450 link up to form a functional transduction pathway in squitoes ,12,13. TSf9 cells, the PKA activity dramatically increased from 40 U/mL/mg protein at MOI = 0.2 to 60 U/mL/mg protein at MOI = 0.5, and 90 U/mL/mg protein at MOI = 1.0. In the Gαs006458 expression Sf9 cells, the PKA activity significantly increased from 17 to 22 U/mL/mg protein as the MOI ratio rose from 0.2 to 1.0, but there was no significance of PKA activity between MOI ratio of 0.2 and 0.5. In the AC007240 expression Sf9 cells, PKA activity significantly increased from 14 to 22 U/mL/mg protein as the MOI rose from 0.2 to 1.0, however, no significance of PKA activity between MOI ratio of 0.2 and 0.5 was detected. In the PKA018257 expression Sf9 cells, the PKA activity significantly increased from 70 to 95 U/mL/mg protein as the MOI rose from 0.2 to 1, as well as the PKA activity showed similar levels as ~70 U/mL/mg at MOI ratio of 0.2 and 0.5  in CAT expression cells . Followi and 0.5 . In addiCulex mosquitoes [CYP9A30, -A31, -A32, and CYP333B4) could be induced by insecticides in Sf9 cells indicating they may be involved in insecticide resistance [Sf9 cell lines, we detected the relative expression changes of CYP9A30, -A31, -A32, and CYP333B4 genes in GPCR020021, Gαs006458, AC007240, and PKA018257 expression cell lines. The results showed one out of four P450 genes (CYP9A32) was significantly increased expression in all the cell lines in comparison with the control cell line (CAT)  A. The rell lines A. Howevell lines A. Since ll lines B. Howevell lines B. Even all lines B.Sf9 cell lines. There was no difference in the chemical effect between Bupivacaine HCl/acetonitrile and DMSO on Sf9 cell viability among all cell lines, thus, it is not necessary to use DMSO and acetonitrile to treat the cell lines as a negative control. In the control cell line (CAT expression), there was a decrease in the cell survival ratio from 85% to 60% under permethrin treatment (50 to 400 µM) after inhibitor treatment in comparison with cells treated with the Bupivacaine HCl + acetonitrile (control) , followed by measuring the cAMP concentration in each treatment. The results showed that the cAMP concentration significantly decreased in the Bupivacaine HCl-treated cell lines (~1.5- to 2-fold) in comparison with the cell lines that were treated with the solvent  A. The sycontrol) B. In thecontrol) B. In thecontrol) B. The cAcontrol) B.Sf9 cells was examined in specific gene expression cell lines and the CAT expression cell line. In the CAT expression cell line, H89 2HCl-treated cells showed significantly increased susceptibility to permethrin  compared to cells treated with the H89 2HCl + acetonitrile  was utilized to treat the GPCR020021, Gαs006458, AC007240, and PKA018257 expression cell lines, and the CAT gene expression cell line. The H89 2HCl-treated cell lines all showed significantly decreased PKA activity compared to cell lines that were treated with the solvent (DMSO) A. The syonitrile B. The GPonitrile B.G8 were treated with a serial of concentrations of Bupivacaine HCl ranging from 25 to 200 µM and simultaneously exposed to serial concentrations of permethrin ranging from 0.1 to 3 ppm. The HAmCqG8 larvae showed significantly decreased resistance to permethrin at 100 and 200 µM of Bupivacaine HCl, but lower concentrations of Bupivacaine HCl (25 and 50 µM) had no apparent effect, with treated mosquitoes exhibiting the same resistance level to permethrin to the controls exposed to permethrin alone or DMSO plus permethrin  using LR Clonase Enzyme mix . The LR reaction was analyzed by PCR with primers of Polyhedrin forward primer and V5 reverse primer according to the manufacturer’s instructions. Sf9 insect cells were purchased from Invitrogen  and the Sf9 cells were grown and maintained in SF 900 III SFM medium  supplemented with 10% FBS . The cell lines were cultivated either in monolayers or in suspension at 27 °C, shaken at 120 rpm. Transfections with recombinant virus following the required multiplicity of infection (MOI) were performed in suspension at a cell density of 1.0 × 106 cells/mL. Specifically, GPCR, Gαs, AC, and PKA recombinant with P2 MOI of 0.2, 0.5, and 1 were expressed in Sf9 cells for 72 h prior to cell lysis preparation. Chloramphenicol acetyltransferase (CAT) expression cells with the same serial MOI served as controls. Cell harvest was determined at the peak of production (72 h post-infection) for tests of cAMP production, PKA activity, and P450 gene expression.The cDNAs for GPCR020021 (Gene ID: CPIJ020021), Gαs006458 (Gene ID: CPIJ006458), AC007240 (Gene ID: CPIJ007240), and PKA018257 (Gene ID: CPIJ018257) were amplified by polymerase chain reaction (PCR) using forward primers containing 4 nucleotides (CACC) prior to the start codon (ATG) that stabilize the PCR product cloning into the Gateway entry vector pENTR/D-TOPO following the procedure described in the pENTR/D-TOPO Cloning Kit manual . Details of the primer pairs are listed in Sf9 cell lines was examined using the Detect X Direct Cyclic AMP Enzyme Immunoassay Kit (Arbor Assays) following the instructions in the kit manual. 1 × 108 cells/mL were washed by PBS and then directly treated with Sample Diluent for 10 min at room temperature, followed by centrifugation at ~1000 rpm at 4 °C for 15 min, after which the supernatant was assayed. Samples containing cAMP were bound by the cAMP antibody and cAMP-peroxidase conjugate. After 2 h of incubation at room temperature with shaking, the reaction was terminated and the intensity of the generated color at 450 nm was measured via a Cytation 3 imaging reader. Moreover, a cAMP production inhibition test was conducted by treating the recombinant gene expressed cells (MOI = 0.5) with 40 µM Bupivacaine HCl (in DMSO)  [To identify the critical function of cAMP and its connection with each effector in the GPCR regulation pathway, the cAMP production in gene expressed TX, USA)  for 2 h Sf9 cells. CAT expression cells served as the control. The Detect X PKA (Protein Kinase A) Activity Kit  was used to determine the PKA activity following the kit manufacturer’s instructions. Briefly, suspension Sf9 cells were prepared at a cell density of 1.0 × 108 cells/mL after 72 h expression with recombinant gene  then lysed in Activated Cell Lysis Buffer  for 30 min on ice with occasional vortexing. The resulting cell lysate mixture was then centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant utilized for PKA assay. Lysate containing PKA was used to phosphorylate the immobilized PKA substrate on a microtiter plate using the kit-provided ATP to produce a phosphor-PKA substrate that could be bound by a specific rabbit antibody and then detected by peroxidase-conjugated Goat-anti-Rabbit. The absorbance of the reaction was determined using a Cytation 3 imaging reader at 450 nm in order to determine the amount of PKA in the samples. To further confirm the involvement of PKA in the GPCR regulatory pathway, recombinant gene-expressed Sf9 cells were treated with a potent PKA inhibitor . The GPCR, Gαs, AC, PKA expression (MOI = 0.5) Sf9 cells were pretreated with 60 µM H89 2HCl (in DMSO) at 27 °C for 1 h avoiding light, then 10 µM forskolin (in DMSO)  [To investigate the relationships linking GPCR–Gαs–AC–PKA, a PKA activity test was performed based on the GPCR, Gαs, AC, and PKA recombinant expression in TX, USA)  and 30 µSf9 cell lines (MOI = 0.5) using the E.Z.N.A. HP Total RNA Kit (Omega). Prior to cDNA synthesis, genomic DNA was removed from the total RNA using a TURBO DNA-free kit . cDNA was synthesized by SuperScript® IV Reverse Transcriptase  following the instructions in the kit manual. qRT-PCR was performed using the All-in-OneTM qPCR Mix  and CFXTM 96 Real Time system. Each qRT-PCR reaction (25 µL) consisted of SYBR Green master mix, specific primer pairs for four Sf9 P450 genes [CT−∆∆ method [Total RNAs were isolated from GPCR020021, Gαs006458, AC007240, PKA018257, and the CAT expression 50 genes  -2,5 diphenyltetrazolium bromide MTT (Sigma) colorimetric assay [Sf9 cells after 48 h infection by a construction baculovirus, either GPCR020021, Gαs006458, AC007240, or PKA018257 (MOI = 0.5), were transferred to a 24-well plate  at a cell density of 1.0 × 106 cells/mL. CAT-infected Sf9 cells served as control. The cells were then treated with permethrin  at a series of concentrations ; cells treated with acetonitrile alone served as solvent controls (CK) in each cell line treatment. The cell survival rate was evaluated by MTT assay after 48 h permethrin treatments. Briefly, the culture medium was removed and 200 µL MTT (5 mg/mL) added to each well. The MTT-treated cells were then incubated avoiding light at 37 °C for 4 h. After incubation, 1 mL of DMSO was added to each well. The absorbance of the resulting solution was determined using a Cytation 3 imaging reader at 540 nm. To test the regulation pathway through cAMP and PKA activity, each effector expression cell line was treated with either 40 µM of the cAMP production inhibitor (Bupivacaine HCl) or 60 µM of the PKA activity inhibitor (H89 2HCl) and simultaneously treated by permethrin, as described above. Inhibitors with acetonitrile was served as control treatment. The cell survival ratio was calculated as  * 100%. Each treatment was repeated at least 3 times with independent infections of the construction baculovirus.The relative number of viable ic assay . TypicalCx. quinquefasciatus, HAmCqG8 and MAmCqG6, were utilized in this study. HAmCqG8 is the eighth generation of permethrin-selection HAmCqG0 offspring that was collected from Mobil County, AL; MAmCqG6 is the sixth generation of permethrin-selection MAmCqG0 offspring that was collected from Madison County, AL. These two counties are located more than 600 km apart. HAmCqG8 and MAmCqG6 have obtained ~2700- and ~590-fold higher levels of resistance than lab-susceptible mosquito strains, respectively. To maintain the resistant levels, HAmCqG8 and MAmCqG6 strains were treated with certain concentrations of permethrin every 6 months [Cx. quinquefasciatus in 6 oz. Sweetheart ice cream cups  containing regular tap water, inhibitors, and 1% permethrin solutions in acetone at four or five concentrations that yielded >0% and <100% mortality. Serial concentrations of the inhibitors were applied simultaneously with permethrin. Control groups received either permethrin alone or DMSO plus permethrin solution. All tests were performed at 25 °C and replicated at least four times on different days. Mortality was assessed after 24 h. Bioassay data were pooled, and a probit analysis was conducted using Abbott’s correction for control mortality.Two insecticide resistant mosquito strains of 6 months . The most-test for all 2-sample comparisons and a one-way analysis of variance (ANOVA) for multiple sample comparisons using Statistical Package for the Social Science (SPSS) software with both least significant difference (LSD) and Tukey tests to analyze the significance of the means; a value of p ≤ 0.05 was considered statistically significant. All significances were present by alphabetic letters .The statistical significances for the cell viability, PKA activity, cAMP production, and gene expression were calculated using a Student’s


Cancer cell lines (CCLs) have been widely used to study of cancer. Recent studies have called into question the reliability of data collected on CCLs. Hence, we set out to determine CCLs that tend to be overly sensitive or resistant to a majority of drugs utilizing a nonlinear mixed-effects (NLME) modeling framework. Using drug response data collected in the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC), we determined the optimal functional form for each drug. Then, a NLME model was fit to the drug response data, with the estimated random effects used to determine sensitive or resistant CCLs. Out of the roughly 500 CCLs studies from the CCLE, we found 17 cell lines to be overly sensitive or resistant to the studied drugs. In the GDSC, we found 15 out of the 990 CCLs to be excessively sensitive or resistant. These results can inform researchers in the selection of CCLs to include in drug studies. Additionally, this study illustrates the need for assessing the dose-response functional form and the use of NLME models to achieve more stable estimates of drug response parameters. Moreover, using in vitro CCL models to study cancer pharmacogenomics2 can be helpful to understand the resistance and sensitivity to therapy currently in use in cancer treatment, explore genomic factors associated with drug response, and develop more anticancer drugs3. Recently, two independent large-scale studies, the Cancer Cell Line Encyclopedia (CCLE)4 and the Genomics of Drug Sensitivity in Cancer (GDSC)5, were completed in which drug response information was collected on a number of therapeutic agents in addition to extensive molecular information . The use of large-scale drug studies on CCLs depends on the reliability and reproducibility of drug response assessments.Over the past decades, the cancer cell lines (CCLs) have been widely used to study the biological processes in cancer, as well as 7. Multiple factors are likely to contribute to these observed inconsistencies in the drug-response data, including methodological and analytical challenges due to the differences in assay types, maximum tested drug concentration, range of tested drug concentrations, and drug sensitivity measurements employed by different studies8. In response, the authors of CCLE and GDSC reported a significantly better agreement for the differences between these two pharmacological data by incorporating both analytical and biological considerations9. However, discrepancies in the measured drug sensitivities still persist10. Furthermore, consistency can be achieved when biologically grounded analysis methods are incorporated using the standardization of assay methods and laboratory conditions12.Despite the wide use of CCLs for drug response studies, inconsistency in drug-response data and poor concordance between mutational profiles of CCLs compare to patient tumors have been reported13. Analysis of dose-response data typically focuses on the EC50 14, which is a description of shape of the dose-response curve at its midpoint, by which cell lines are often determined to be either sensitive or resistant. However, the dose-response curves can differ in other aspects, such as the slope of the curve or the area under the curve. Analyzing each cell line individually across a drug combination is highly problematic since the variation exhibited across the cell lines is ignored and this information could be harnessed inform the response profile of any single cell line . Incorporating variation across all the cell lines in dose-response curve fitting can reduce noise and lead to more reliable inferences for any single CCL. Recently, the nonlinear mixed-effects (NLME) model has become an important approach to improve the accuracy of EC50 estimates (or similar parameters), through the borrowing of information across all CCLs16. Such models allow one to account for the repeated measures aspect in the data  through the inclusion of random effects in model, along with fixed effects  as included in traditional nonlinear regression models.Testing of drug sensitivity has been a routine procedure in clinical and laboratory researches. Dose-response data collected on CCLs often are sigmoidal in shape and thus nonlinear logistic models are often used to model the data for each cell line individuallyThe objectives of this study are first to determine difference in model functional form  for drug-response data using data from the CCLE and GDSC. Then, for each cancer type and drug combination, a NLME model was fit to the drug-response data collected on the CCLs that had the same nonlinear functional form. Using the CCL specific estimated random effects from the NLME model, a set of cell lines were determined to be consistently sensitive or resistant to a large number of drugs. These findings can aid cancer researchers in the selection of cell lines to include in their experiments by eliminating CCLs that are always sensitive or resistant to drugs as results may not be generalizable. Moreover, this study illustrated the need for assessing model functional form for drug-response data and the ability to model all cell lines simultaneously using a NLME model to provide more stable estimates of drug response parameters.http://broadinstitute.org/ccle was downloaded (February 2015) and processed. In total, the analysis consisted of in vitro drug response data for 8-point dose were collected on 24 compounds assessed on 504 cancer cell lines. Supplemental Fig. The drug sensitivity data available at 1. Drug sensitivity data included 213,605 drug-response series distributed over 990 cell lines and 265 drugs were downloaded from http://www.cancerrxgene.org (July 2016). The drug-response data were generated over 9 drug concentrations (2-fold dilution series) or 5 drug concentrations (4-fold dilution series). Supplemental Fig. The drug sensitivity data are generated by the Cancer Genome Project at the Wellcome Trust Sanger Institute (WTSI) and the Center for Molecular Therapeutics at Massachusetts General Hospital using a collection of >1000 cell lines6.As both the CCLE and GDSC used existing CCLs, the two studies had a number of cell lines and drugs represented in both studies. In particular, dose-response data across 354 cell lines for 15 drugs were in common between the CCLE and GDSC. However, it should be noted that the two studies used different experimental protocols such as differences in the pharmacological assay types and the range of drug concentrationsin vitro dose response data for each CCL individually for a given drug. The shape of the dose-response curves vary between CCLs for a given drug  or four-parameter (4P) logistic regression13. Although the functional form of the model remains the same for all cell lines , the parameter values vary from cell line to cell line.Typically, an individual dose-response model is fitted to the 17 for repeated measures dose-response data have become popular due to their flexible covariance structure which allows for the joint modeling of multiple in vitro measurements taken off a set of CCLs. An advantage of using the NLME model is that both within-cell line and between-cell line variation are accounted to improve the statistical estimation of parameters. Moreover, the problem of extreme estimates caused by considering a single drug-cell line is reduced by using the NLME model where the information across cell lines is borrowed, with individual cell line parameters shrunk towards the population-level parameters17.Recently, nonlinear mixed-effects (NLME) modelyij represent the jth dose-response at ni drug doses, ith cell line, xij denote the corresponding jth drug dose assayed for the ith cell line. The total number of measurements collected is equal to eij is a random error term reflecting uncertainty in the response, given the ith cell line. In Eq. , and βi4 is the slope of the dose-response curve for the ith cell line18. Collecting the errors for the ith cell line into (ni × 1) vector, eij in Eq.  were fit with models assessed with the Akaike’s information criterion (AIC)22 to account for the complexity of the model . The best fitting model was then determined for each CCL drug combination. Then, for each cancer site and drug combination, the model that fit most of the CCLs the best was selected as the best fitting model.Choice of the 4P logistic regression model is fairly typical in dose-response data analysis, while for some dose-response data, the 4P logistic regression model does not provide an adequate fit. In these cases, often a 3P logistic regression model is used by setting 0 in Eq. . The non0 in Eq. , can be 17. To model the drug response data for all CCLs for a given drug simultaneously, a NLME model is considered where the function form was either 3P or 4P logistic regression model. Although, the form of regression function f is common to all CCL dose-response data for a given drug, the parameter vector βi may vary across CCLs. Variation among different cell lines is accounted for through the cell line regression parameters where a model for the dependence of βi on random components is taken into account. This can be accomplished with a hierarchical model framework by adding the following second-level model to the nonlinear model outlined in Eqs (βi asβ0 is (r × 1) vector of fixed-effects parameters representing the parameter values for the population, bi is a (q × 1) vector of random effects, and Ai (p × r) and Bi (p × q) are the design matrices associated with the fixed-effects and the random effects, respectively. The within-cell variation in Eq.  was used to compare both the estimates of EC50 from the single cell line model and the standardized random effect estimates (SREs) from the NLME. Similar to Haibe-Kains, Benjamin, et al.6, the qualitative descriptions of correlation values are defined as; poor (rs < 0.5), fair (0.5 ≤ rs ≤ 0.6), moderate (0.6 ≤ rs ≤ 0.7), substantial (0.7 ≤ rs ≤ 0.8), and perfect (0.8 ≤ rs).As a different NLME model was fit for all cell lines of a given cancer type and drug, we standardize the estimated cell lines random effects for each drug to enable comparison across drugs. For CCLE and GDSC datasets, the Spearman’s rank correlation coefficient  and a linear model (LM) to the dose-response data for each cell line, with the best fitting model determined based on AIC ; Type II or “moderate” outlier cell line if SREs fall between ; and Type III or “extreme” outlier cell line if SREs fall outside of ±3. CCLs with SREs fall between  are neutral. These outlier types (I-III) are defined based on the boundaries of 95%, 99.4%, and 99.9% confidence intervals, respectively. CCLs with SREs < −1.65 or SREs > 1.65 across greater than or equal to 20% of drugs are sensitive or resistant, respectively, while the rest of CCLs were considered neutral. The second approach we employed to determine problematic CCLs or outliers was based on the distribution of the SREs. CCLs with SREs < 0 or SREs > 0 across greater than or equal to 80% of drugs are sensitive or resistant, respectively, while the rest of CCLs are neutral.To determine the cell lines that are consistently sensitive or resistant to a number of drugs assayed in the CCLE and GCSC, and therefore potential cell lines to remove from future drug studies, the estimated random effects were examined for each cell line and drug combination determined from the NLME model, as outlined in previous sectionNonlinear models with functional forms of 4P and 3P logistic and a linear model (LM) were fit to the 497 CCLs in the CCLE, with best functional form based on AIC for each CCL and drug combination presented in Supplemental Tables Similar to the comparison of model fit statistics for the CCLE, we completed the assessment of functional forms  for the 979 cell lines in the GDSC  which fit the majority of CCLs was used in fitting a NLME model defined in Eqs . As an example, Fig. 26.Similar to analysis completed on the CCLE, we completed the identification of outlier cell lines in the GDSC across 29 out of 54 cancer types with more than 10 cell lines. Similar to the CCLE analyses, all GDSC cell lines were not included in each model. The median number of NLME models that the cell lines in GDSC were included in was 74 drugs out of 242 drugs (range 3 to 153 drugs). Supplemental Figure rs = 0.75; PLX4720 with rs = 0.74), along with moderate correlations for drugs TAE684 (rs = 0.56) and AZD0530 (rs = 0.50)  and Paclitaxel (rs = 0.64) were observed with moderate correlations and drug Sorafenib (rs = 0.52) had fair correlation between estimates  across 354 CCLs and 15 drugs. For each drug, the Spearman’s rank correlation between SREs and nonlinear estimates of EC50 were computed , and two drugs with fair correlations  between studies. The majority of drugs (7 out of 11) yielded poor concordance (rs < 0.50).We then assessed the consistency between drug response data using SREs for EC50 across 196 CCLs and 11 drugs in both GDSC and CCLE, while the two studies used different experimental protocols12 Fig. . We obsein vitro drug response data generated on cell lines and second to determine using a nonlinear mixed-effects model cell lines that appear to be sensitive or resistant to a majority of the drug tested in either the CCLE or the GDSC studies. As the model for curve fitting has a direct impact on the measurement of drug potency , we compared the appropriateness of the 4P logistic, 3P logistic, and linear models for the 497 cell lines and 24 drugs in the CCLE and the 979 cell lines and 265 drugs in the GDSC. We observed that the best fitting dose-response relationships is often a 3 or 4 parameter logistic (3P logistic or 4P logistic) nonlinear model. However for some dose-response data, neither the 4P or 3P logistic models provided an adequate fit to the data. These results illustrate the need to assess the functional form for in vitro studies to ensure the most accurate and precise estimates of drug response, such as the EC50 or the area under the dose response curve. Further work is needed to assess other non-linear models besides the 3P and 4P logistic, such as the Cedergreen-Ritz-Streibig five-parameter model used in Moyer et al.27, for modeling drug-response data in the CCLE and GDSC.The goals of this study were to first determine if there was a common functional form for 28 .The use of NLME model allows the joint modeling of all the CCL drug response data collectively with the ability to model the cell line-to cell line variation along with the within-cell line variation. Moreover, the flexible covariance structure allows both within-cell line and cell line-to-cell line variations are accounted to improve the statistical analysis. Such NLME model uses the information across the cell lines to adjust the extreme estimates caused by considering a single drug-cell line . Therefore, a NLME model was used to detect sensitive or resistant cell lines with the SREs for the EC50 parameter using two approaches, one based on determination of outliers using the boundaries of confidence intervals and one based on the distribution of SREs for a CCL. We found 17 cell lines based on the CCLE data to be overly sensitive and resistant to many drugs (see Table in vitro drug screens are generalizable. Additionally, this study illustrated the need for assessing functional form for the drug-response data and the ability to model all cell lines simultaneously using a NLME model to provide more accurate estimates of drug response parameters.In conclusion, the results from this study can aid basic scientists in the selection of cell lines to include in experiments to ensure that results from the Supplemental InformationSupplemental Tables 1 - 10


Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets.Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study.  Most patients present with locally advanced or metastatic disease, or develop recurrent disease following curative surgery2. While many systemic treatment options are available, the prognosis of advanced gastroesophageal cancer remains poor, with median survival of less than 1 year3. Greater than 90% of gastric and gastroesophageal junction cancers are adenocarcinomas, with gastrointestinal stroma tumours (GIST), lymphomas, and neuroendocrine tumours found in a small minority of cases4.Gastroesophageal cancers are among the most common and lethal cancers worldwide3. There is an increasing recognition of the limitations of using primary tumour features to guide systemic cancer treatment, due to tumour heterogeneity and the frequent disparity observed between primary and metastatic sites5. Metastatic biopsies are rarely undertaken however, due to both inaccessibility of metastatic sites and procedure morbidity. Circulating tumour cells (CTCs) are the likely intermediates of metastasis dissemination of cancer, and as such, can be expected to include the subpopulations which are responsible for disease progression6. While CTC enumeration has an established prognostic role, the true promise of CTCs is to provide a ‘real time’ view of the cancer using only peripheral blood samples, avoiding the need of repeat invasive biopsies7.Most patients with gastroesophageal cancer will require systemic treatment at some point in their treatment8.Moreover, while most cancer deaths are due to the haematological spread of metastases, research into the mechanism of metastasis initiation, formation, and propagation has been hampered by limited access to cancer cells within the various stages of the metastatic cascade. CTCs additionally provide a unique window into the biology of cancer as it spreads through the blood stream. The rarity of CTCs compared to normal blood cells has provided significant technical challenges in developing isolation methods which are sufficiently sensitive and specific10, and long-term primary CTC cultures provide an ideal laboratory tool for the investigation of the biology of metastasis formation11. However, establishment of long-term primary CTC cell cultures has proved to be challenging. To date, despite intensive efforts, only several long-term CTCs cultures have been reported worldwide, including in colorectal12, breast13, and prostate14 cancer, all established with modest success rates, with 1–16% of blood samples producing stable cultures. To improve culture rates, initial expansion of the CTC population using xenotransplantation into immunodeficient mice prior to in vitro culture has been trialled15.CTC culture provides an expanded cell population for expression analysis, functional assays, and drug sensitivity testingIn this current work we describe establishment of two novel and distinct CTC cell lines derived from patients with metastatic gastroesophageal cancer.A total of 41 blood samples were processed for CTC enumeration, with 23 samples processed for culture using the optimised protocol (15 ml blood sample with negative selection using the CTC Enrichment Cocktail). CTCs were detected in 38/40 samples (93%) by EpCAM  based capture using the IsoFlux system (one specimen clotted and was not processed for CTC enumeration), with ≥10 CTCs found in 22 (54%) of the samples. Numbers of CTCs detected ranged from 0–150, with the mean number of CTCs 27.3  who had a high CTC count of 109. This patient presented with diffuse bone and peritoneal metastasis. Endoscopy demonstrated a large ulcerated gastric mass confirmed on biopsy to be a gastric adenocarcinoma. A matched culture was established simultaneously from the ascitic fluid sample from the same patient (UWG02ASC). Patient 41 also progressed rapidly prior to receiving any anti-neoplastic treatments and passed away.in vitro growth characteristics. UWG01CTC is adherent and requires trypsinisation for passaging, although a loose adherent spheroid phenotype is inducible in a hypoxic environment and serum free media , reaching tumour size endpoints (>10 × 10 mm) within 4 weeks for UWG01CTC Fig.  and 3 weDetailed DNA analyses were limited by poor DNA quality from both source patients’ formalin fixed paraffin embedded (FFPE) samples. However, by STR analysis, 24/28 (85.8%) alleles of UWG02ASC and UWG02CTC were identical to those of the primary tumour from patient 41, confirming the source of the cell lines  stain Fig.  with a hThe primary tumour of patient 41 and UWG02CTC both demonstrated high grade appearances by H + E stain Fig.  with a h20 by IHC  did not change the phenotype of any cell line as detected by IHC (data not shown). Neither CTC cell line expressed mesenchymal markers (vimentin or N-cadherin) or urokinase plasminogen activator receptor (uPAR), a key receptor for the initiation of proteolytic cascadesIHC Fig. . UWG02CTIHC Fig. . No cellIHC Fig. .in vitro culture. These include neuroendocrine markers in UWG01CTC such as chromogranin A, CD56, and synaptophysin  growth was observed on NanoString gene expression profiles using unsupervised hierarchical clustering, though monolayer (2D) cultures grouped together and away from spheroids (3D) and xenografts, which grouped together  relative to UWG01CTC (−log2 9.4 fold). Interestingly, UWG02CTC when compared to UWG01CTC, had a higher expression of genes known to be involved in Helicobater pylori mediated carcinogenesis such as AKT24, ETS225 and MYC26  and PLA2GA, an important regular of metastases in gastric cancer and expressed with constitutively active Wnt29. UWG02CTC also showed higher expression than UWG01CTC of targetable pathways including EGFR, FGFR2, HER-2 (ERBB2), and MET, as well as key genes in the JAK/STAT pathway, genes which overexpression are frequently reported in gastric adenocarcinomas but not gastrointestinal neuroendocrine cancers31.The IHC and STR findings, showing two highly distinct CTC lines, were further explored by NanoString analyses. As expected the sample cohorts derived from patient 20 versus patient 41 grouped together by unsupervised hierarchical clustering  genes in UWG02CTC compared to UWG02ASC and overall these two Patient 41 lines demonstrated very similar gene expression profiles.in vitro sensitivity of both CTC and UWG02ASC cell lines to commonly used DNA-damaging chemotherapeutics with varying mechanisms of action was evaluated. UWG01CTC and UWG02CTC varied in their sensitivities to these drugs as demonstrated by the dose response curves shown in Fig. 50 value for etoposide at 0.036 µM is ~3-fold lower than the minimum value reported for other gastroesophageal cancer cell lines (0.136 µM)32, suggesting that patient 41 may have benefited from treatment with this drug even though etoposide is more commonly used for neuroendocrine tumours. The IC50 values for the other compounds otherwise fall within the ranges reported for other cancer cell lines32. Expectedly, UWG02CTC and UWG02ASC cell lines were very similar in their drug sensitivities  significantly enhanced the radiosensitivity of cells resulting in decreased mean surviving fractions compared with cells exposed to radiation alone, especially after 1 Gy  gastroesophageal cancer cell lines derived from CTCs. These two cell lines are genotypically and phenotypically distinct, reflective of differing tumour biology in the donor patients. Similar to other reported CTC lines, UWG02CTC displayed an epithelial phenotype, with strong expression of EpCAM and pan-cytokeratin. In contrast, UWG01CTC, isolated from a patient with neuroendocrine tumour, showed variable and weak cytokeratin (Cam5.2) immunostaining, with strong expression of neuroendocrine markers . UWG01CTC had no detectable EpCAM, mesenchymal , or cancer stem cell marker . This phenotype makes UWG01CTC a unique EpCAM negative CTC cell line.in vitro and in vivo growth with a high grade histological appearance supportive of a stem cell (CSC) phenotype. It was interesting to detect differences in CD44, ALDH1 protein and STAT3 gene expression between the UWG02CTC and UWG02ASC, which probably reflects the different pathways of tumour cell dissemination. Of note, higher expression of CSC markers and key stem pathways was also found on UWG02CTC versus UWG01CTC, despite ALDH1, CD44, and CD133 reported as CSC markers in both adenocarcinomas and high grade neuroendocrine tumours34.Both CTC derived cell lines recapitulated the phenotype of the source patient’s tumour, highlighting that CTCs are a representative tumour source. The RNA expression profiling confirmed these data, with each cell line’s expression clustered with the source patient’s tumour and the corresponding mouse xenograft. Both CTC derived cell lines demonstrated rapid The preserved overall RNA expression profile and IHC staining pattern seen in tumours excised from the mice, and the cell cultures  derived from these tumours, highlight that these procedures, at least in short term cultures, do not grossly alter the original tumour characteristics, making CTC derived cell lines an excellent model to study tumour biology. Together with the confirmed tumourigenicity of the cell lines by the rapid development of tumours (within 4 weeks of inoculation) in all xenografted mice, and presence of key mutated and amplified genes that are characteristic of gastric adenocarcinomas or high grade neuroendocrine tumours, confirms the establishment of two novel CTC derived immortalised cell lines.35. All cell lines were very sensitive to radiotherapy, with a synergistic effect of carboplatin seen in UWG01CTC. This is consistent with the clinical experience of the radiosensitivity displayed by high grade neuroendocrine cancers, driven by low expression of key DNA damage response enzymes, such as ATM and ATR38. We also explored potential synergistic effects of drug combinations as this may allow clinical dose reduction, which in turn reduces toxicity while retaining the therapeutic efficacy.We also undertook cytotoxic and radiotherapy assays of the CTC cell lines based on standard cytotoxic agents used for these cancers. We found drug sensitivities were generally similar to those previously reported for gastroesophageal cell linesEGFR, FGFR2, ERBB2, and JAK/STAT pathway genes, suggesting potential sensitivity to treatments directed at these validated targets, while UWG01CTC had high expression levels of the DLL3 gene, which is proposed as a potentially druggable target39. While these data show that CTC derived cell lines can be used to define personalised drug sensitivities for gastric cancer patients, it is important to note the rapid disease progression and clinical decline of the patients that yielded CTC cultures. The test results in our study would have been too late to guide management, and faster protocols are needed to inform personalised therapy in a clinically meaningful way. Nevertheless, more comprehensive drug sensitivity testing will be performed on the established CTC lines, and may help to define better drug screening libraries for faster future protocols.Multiple other potential therapeutic targets were identified by RNA expression profiling. UWG02CTC had high expression of 12. While we agree with this requirement, this issue is complicated by the inherent biases when selecting the isolation method and epithelial marker based CTC definition used for enumeration. We employed the standard EpCAM based isolation and cytokeratin based CTC identification in the matched blood sample used for CTC enumeration. Due to the biological differences discussed above, our successful cultures derived from samples with high (109) and low (3) CTC counts, subsequently developing EpCAM positive and negative cell lines, respectively. This finding highlights a key issue in the CTC field. While patient 20 has a low EpCAM+ CTC count, which is within the range of background cells found to express CTC markers  detected from healthy donor blood samples with the IsoFlux system (data not shown), the successful establishment of a CTC cell line argues that this patient had a high number of EpCAM− CTCs with the ability to establish metastatic deposits. This hypothesis is also supported by the clinical picture. CTC numbers are known to increase with disease burden40, and at the time of blood draw patient 20 had high volume and rapidly progressive disease. It is reasonable to suggest this patient had a high number of biologically relevant CTCs which were missed using standard CTC isolation techniques. The negative selection used for CTC culture (CD45 depletion), rather than the positive selection used for CTC enumeration (EpCAM capture) was critical to detect these cells. The optimal isolation method of CTCs continues to evolve in the face of these issues, and while more emphasis has been given to epithelial to mesenchymal transition (EMT) phenotype CTCs in recent years41, the data presented here highlight the persistent challenges identifying the optimal markers to isolate CTCs.Other studies have suggested a high CTC count (>300) is necessary for successful culture12. It is unclear whether this finding is indicative that a high proliferation rate is a requirement for successful cultures, or is a result of selection pressures for aggressive clones in the culturing process.Both CTC cell lines displayed a highly aggressive phenotype, with rapid growth, high grade histological appearance, and high Ki67 expression. In both cases, this phenotype mirrored the rapid clinical progression seen in the corresponding patient. Similar results have been seen in other CTC cell lines28, while UWG02ASC was negative for CD44. This is consistent with other results showing that CTC cultures exhibit a stem cell phenotype, and further supports the notion that CTC cultures develop from the CTC population which are able to establish metastases42.The matched culture derived from ascitic fluid (UWG02ASC) simultaneously established from patient 41 as UWG02CTC demonstrated an identical phenotype, with the exception of cancer stem cell marker expression. UWG02CTC was strongly positive for CD44, a key CSC marker in gastric cancerIn conclusion, we report the first two long-term CTC cell lines developed from patients with metastatic gastroesophageal cancer, providing a high fidelity platform for high-throughput gene expression, drug response profiling and functional genomics studies. The two CTC cell lines displayed distinct profiles which faithfully recapitulate the source patient’s tumour. Our results support the developing role of CTC culture as an essential laboratory resource for the understanding of the biology of metastases and importantly undertake personalised screening for therapeutic strategies.Peripheral venous blood samples were collected from patients with metastatic gastroesophageal cancer prior to treatment. Patients had histologically confirmed gastric or gastroesophageal cancer treated at the Illawarra Cancer Centre, Wollongong Hospital, NSW Australia. Informed consent was obtained from each patient prior to enrolment, and the study was approved by South Western Sydney Local Health District Human Research Ethics Committee (Project Number 15/072). All experiments were performed in accordance with relevant guidelines and regulations.43. Enumerated CTCs were defined by the standard EpCAM/Cytokeratin/DAPI positive and CD45 negative phenotype40.Initially 7.5 ml of blood was collected in EDTA tubes. This was increased to 15 ml after seventeen patients were enrolled for higher CTC capture to improve culture success rates. Blood samples were transported immediately at room temperature for CTC isolation. A separate matched 7.5 ml EDTA blood sample was collected at the same blood draw for EpCAM based capture for CTC enumeration using the IsoFlux system (Fluxion Biosciences), and processed as per manufacturer instructions44, in both cell lines successful cultures were established in both media formulations, and we did not find any phenotypic differences between media conditions (data not shown). All media was supplemented with antibiotics  prior to a density gradient separation with Leucosep tubes (Stemcell Technologies) to isolate a peripheral blood mononuclear cell (PBMC) layer. We initially found excessive lymphocyte contamination prohibiting CTC growth with the RosetteSep Human CD45 Depletion Cocktail alone. This improved with changing to the CTC Enrichment Cocktail. The PBMC layer was washed twice, then immediately plated into 24-well ultra-low attachment plates (Corning) with serum free Advanced DMEM/F12  supplemented with epidermal growth factor , fibroblast growth factor  and N2 supplement (Life Technologies), or ADMEM/F12 with 10% foetal calf serum (FCS) in hypoxic conditions. Separate CTC cultures were maintained in 10% FCS or serum free media. While serum free media is reported to support a cancer stem cell phenotypePatient 41 had marked peritoneal disease and gross ascites from gastric adenocarcinoma. At the same time as the blood draw for CTC isolation, 200 ml of ascitic fluid were collected from a peritoneal catheter. This sample was transferred immediately to the laboratory, centrifuged to collect a cell pellet, washed twice in ADMEM/F12 with 1% antibiotic/antimycotic (Sigma Aldrich), and cultured as per conditions listed above. Cultures derived from the patient’s CTC and ascites samples were maintained independently under identical conditions. Once established as cell lines, for all subsequent routine cell culture and experimental assays, cells were maintained in ADMEM/F12 with 10% FCS supplemented with EGF.6 cells from early passages of each cell line were injected subcutaneously into the flank of NOD scid gamma (NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ) mice. Mice were monitored for tumour growth and sacrificed when the tumour grew greater than 10 × 10 mm, or the animal demonstrated signs of stress (such as >15% weight loss) or evidence of impedance of tumour on movement. Tumours were collected from the sacrificed mice for subsequent culture, RNA and DNA extraction, and histological analysis. For culture from xenografts, tumour tissues were cut into approximately 1 mm pieces and then incubated with tumour dissociation enzymes  in ADMEM/F12, after which the tumour homogenate was centrifuged, the pellet resuspended, and plated in serum-free media and hypoxic conditions as above. All procedures were carried out in accordance to the Australian Code for the Care and Use of Animal for Scientific Purposes 8th edition 2013, and approved by the University of Wollongong’s Animal Ethics Committee (study AE15/17).For confirmation of tumourigenicity, 1–2 × 10Expression of key proteins on the cell lines, mouse xenografts, and representative sections from the matching patient’s tumour specimen were compared using immunohistochemistry (IHC). For cell lines, cells were collected, centrifuged with supernatant removed, then clotted with plasma and commercially prepared thrombin (Fibri-Prest Automate from Stago) to prepare a cell block. All samples were fixed in 10% formalin and paraffin embedded, with 4 µm sections cut for staining. Antigen retrieval and development was performed on the fully automated Bond system according to manufacturer’s instructions  or AllPrep DNA/RNA/Protein Mini Kit , respectively, according to the manufacturer’s instructions. All samples were quantified using the NanoDrop . DNA/RNA samples had A260/280 ratios between 1.8 and 2.1.45. As recommended, genes with expression levels at or below the level of the negative controls were removed from analysis. Differential expression of key transcriptomic pathways were compared between cell lines, with fold change and P values calculated using nSolver default settings. With the remaining list of genes, a filter cutoff of fold change ≥ ±2 and P value < 0.05 were used to identify the significant gene expression changes46.Total RNA extracted from fresh-frozen (25 ng) and FFPE (150 ng) specimens were run on the NanoString nCounter Sprint system using the 770 gene PanCancer Pathways panel with additional cancer stem cell and proteolytic genes as per the manufacturer’s instructions (NanoString Technologies). RNA extracted from patient 20 FFPE specimen had low RNA integrity and did not meet the threshold for further analysis. Results were analysed using the NanoString nSolver 4.0 and Advanced Analysis Module, which normalizes gene expression to a set of positive and negative control genes built into the platformSTR profiles for the cell lines and their matching patient FFPE tumour tissue were verified by the PowerPlex 18D System, using the following 18 markers (seventeen STR loci and Amelogenin): D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWA, D8S1179, TPOX, FGA, D19S433 and D2S1338. As per standard practice, cell lines were considered to match if profiles are more than 80% identical to source patient sample.DNA of the cell lines and xenografts were sequenced using the Oncomine Comprehensive v3 Assay on an Ion S5 Sequencer (Thermo Fisher Scientific). The assay detects specific hotspots as well as coding sequences in particular genes. STR and Oncomine assays were performed by KCCG Sequencing Laboratory & Cancer Diagnostics, Garvan Institute of Medical Research, Sydney Australia.50 . Data is presented as a mean ± standard error of the mean (SEM) from ≥2 independent experiments.Approximately 10,000 cells were seeded per well in triplicate into a 96-well plate 48–72 h prior to drug treatment. Cells were incubated with serial dilutions of each drug for 72 h with drug vehicle  and the dilution kept constant across all drug concentrations and controls . The viability of cells were assayed using CellTitre 96 Aqueous One Solution Cell Proliferation Assay  using a SpectroMax 250 UV plate reader using SoftMax Pro software . Cell viability of treated cells was normalized against vehicle controls. This data was analysed using a logarithmic sigmoidal dose–response curve using the variable slope parameter to determine IC50 values determined as monotherapies and ranged from 0.125×, 0.5×, 1×, 2× and 4× the IC50 of each drug, as optimal for the Chou-Talalay median-effect equation47. To determine synergism/antagonism, the combination index (CI) and dose response index (DRI) was calculated using CalcuSyn , with Monte-Carlo simulation to assign confidence. By the Chou-Talalay method, a CI = 1 represents an additive effect, a CI value > 1 represents antagonism and a CI value < 1 represents synergism.For combination drug assays cells were seeded as above and then treated with the drug combinations specified. The drug concentrations were kept at a constant ratio based on their approximate IC48. UWG02CTC and UWG02ASC were not suitable for this assay as they formed loose and small colonies. Briefly, cells were first acclimated for at least a week in normoxic conditions, then plated into 12.5 cm2 tissue culture flasks in regular cell culture media (as above) in order to reach ~60% confluency 3 days later. Cells were then pre-sensitized with 0, 1, 2 and 5 µM carboplatin for 48 h prior to exposure to 1 or 2 Gy X-Ray radiation delivered in a single fraction at room temperature. Cells were then seeded into triplicate tissue-culture petri dishes  in 10 mL of ADMEM/F12 with 10% FCS, pencillin/streptomycin, EGF and L-glutamine, at different cell densities per dish (ranging from 1000 to 20000 cells per dish). After approximately 15 doubling times, petri dishes were washed with PBS and adherent cell colonies fixed and stained with a 1:3 crystal violet:ethanol solution. Colonies (≥50 cells/colony) were manually counted and presented as Mean Plating Efficiency  and Surviving Fraction  as previously described49. Unirradiated control samples (with and without drugs) were handled under the same conditions as the irradiated samples.The sensitivity of the UWG01CTC cells to 10MV x-ray irradiation with drug pre-treatment was investigated using clonogenic survival as the radiobiological endpoint as previously described50. Cells were irradiated in T12.5 cm3 flasks using a Varian 2100iX linear accelerator. Prior to irradiation flasks were completely filled with medium. Flasks were located at midpoint in a 23 cm thick Solid Water phantom . Flasks were surrounded with Super-Flex bolus  to ensure electronic equilibrium. Doses of 1 or 2 Gy were delivered using parallel opposed beam geometry, in a single fraction at room temperature. Briefly, cells (2.0 × 104) were stained with PI master mix  for 30 min at 37 °C. DNA content was then measured using a Becton Dickinson BD LSR II FACSort flow cytometer  and the proportion of cells in G0/G1, S and G2/M phases of cell cycle were calculated on the basis of DNA distribution histograms using FlowJo software as previous described 51.Measurements of cell cycle distribution after irradiation were performed essentially as previously describedt test, Fisher’s exact test, one-way analysis of variance test, and Dunnett’s test were performed. P values < 0.05 were considered statistically significant.Apart from the NanoString analyses, all the experimental data were expressed as means ± SEM and statistically analyzed using GraphPad Prism . For comparisons, two-tailed Student’s Supplementary Information.


R), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1’(R), and 6’(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1( Acute myeloid leukemia (AML) is one of the main causes of death related to leukemia, with 70% of patients either not responding to treatment or eventually relapsing to secondary chemoresistant AML. Standard therapy has been almost unchanged for the last 40 years, and new drugs or drug combinations targeting the resistant fraction of patients are actively researched .www.fda.gov), and is under evaluation for AML treatment [B-cell lymphoma (Bcl)-2 and myeloid cell leukemia (Mcl)-1 are two major anti-apoptotic proteins, which are frequently deregulated in cancer. The Bcl-2 family of proteins is the target of recently developed synthetic BH3 mimetics, namely ABT-263 (Navitoclax) and ABT-199 (Venetoclax) or natural compounds [reatment . Howeverreatment . Recentlreatment .Aplysina aerophoba to act as a cytostatic drug against a panel of cancer cell lines and sensitizes lymphoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [Pseudovibrio denitrificans Ab134 isolated from the sponge Arenosclera brasiliensis [Marine organisms and sponges in particular constitute a precious source of biologically active compounds, which provide potential agents against cancer ,13,14,15 (TRAIL) . Fistula (TRAIL) ,25,26,27iliensis , which e1H and 13C NMR spectra of fistularin stereoisomers [R), 6(S) for C1 and C6, the absolute configuration at the two secondary carbinols C11 and C17 is rather complicated. This high molecular weight molecule presents four secondary alcohols that hampered the co-determination of their absolute configuration by chemical modification and NMR studies. Suitable crystals for X-ray studies have never been obtained for one of the stereoisomers of fistularin-3. Moreover, the carbon configuration also seems to vary depending on sponge species, and even mixtures can occur [Aplysina oroides, which was recognized as a 11R isomer of fistularin-3 extracted from Aplysina cauliformis, attributing to this latter the 11S configuration [Due to the similarities of oisomers  confusioan occur . The namguration . FinallyR), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3 -11(R), 17(S)-fistularin-3, and abbreviated here as RS-F3), isolated from the sponge Suberea clavata. We characterized the complete absolute configuration of RS-F3, revealing that this compound is in fact the (+)-11(R), 17(S)-fistularin-3 isomer. We confirmed the potential of this isomer to interfere with DNA methyltransferase (DNMT) 1 and assessed its anticancer potential on a panel of AML cell lines and show that it induces differential proliferation arrest and cytotoxicity. We also found that RS-F3 induces an early decrease of Mcl-1 protein levels in sensitive AML cell lines, which was independent of Noxa, but could be rescued by proteasome inhibition. Moreover, combination treatments with ABT-199 resulted in strong induction of apoptosis in AML cell lines resistant to Bcl-2 inhibition, which was not observed in healthy human progenitor/stem CD34+ cells or platelets.Here, we report the activity of the stereoisomer (+)-1(Suberea clavata (200 g) was extracted at room temperature with methanol/dichloromethane (MeOH/DCM) (1/1) to give 57.8 g of dried extract. The crude extract was submitted to n-butanol/H2O partition to obtain a desalted butanol extract (30 g). A portion (5 g) of the later extract was submitted to filtration on Phenomenex Sepra C18  silica gel and eluted with a gradient H2O/MeOH (100/0 to 0/100) and MeOH/DCM (100/0 to 0/100) to give 6 fractions (f1 to f6). The fraction f3 (1.3 g) was submitted to reversed phase (Sunfire C18) HPLC chromatography with H2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH: 85/15 to 0/100 in 40 min and yield 13 subfractions (F1 to F13). The fraction F6  was pure (+)-11(R), 17(S)-fistularin-3 (1H and 13C data of the compound (+)-11(R), 17(S)-fistularin-3   E.To investigate involvement of molecular markers well-known to contribute to AML cell survival in the effect of RS-F3, we assessed the expression of the anti-apoptotic protein Mcl-1, associated with resistance to chemotherapy in AML ,30. KineWe previously showed that isofistularin-3 induced endoplasmic reticulum (ER) stress in RAJI Burkitt lymphoma cells  and ER s50 above 1 µM [Since combination treatments targeting Mcl-1 and Bcl-2 are considered a promising anticancer strategy against AML ,7,34, weove 1 µM ,35. We pInterestingly, in the presence of the caspase inhibitor, U-937 cells shifted to a caspase-independent apoptotic-like cell death, whereas apoptosis was fully prevented, and a small fraction of necrotic cells appeared in the case of OCIAML-3 cells . Under t+ cells from healthy donors, a reduction of cell viability of about 30% was observed, which is less than a half of the cell death occurring in the Bcl-2-positive AML cell lines tested  6(S), 1’(R), and 6’(S) configuration (R)-C17(S) exhibited lower global energy score than other stereoisomers (R) 6(S), 1’(R), 6’(S) configuration, RS-F3 showed the lowest global energy score (−44.34) when DNMT1  [Four C11-C17 diastereoisomers for fistularin-3 can exist in theory, but not all the diastereoisomers have been discovered in Nature so far. To gain structural insights of these fistularin-3 stereoisomers on DNMT1 activity, we implemented simulated docking experiments on DNMT1 with four C11-C17 diastereoisomers with established 1  was usedS-adenosyl methionine (methyl donor) concentration did not affect the inhibition of isofistularin-3 against DNMT1 activity, the methyl donor binding site would not be an isofistularin-3 binding site [To further support the predicted binding modes of RS-F3 on DNMT1, we used a different docking program, AutoDock 4.2 , which iing site . Taken aIn the case of most favorable docking pose, RS-F3 penetrated the negatively charged cleft that is surrounded by CXXC and RFTS domains and consolidated the interaction between CXXC and RFTS domains. Furthermore, RS-F3 stretched to the DNA binding loop on CXXC domain, SKQ, that could influence to the interaction between CXXC domain and DNA . Given tWe then assessed the ability of RS-F3 to inhibit DNMT1 activity in vitro. We used epigallocatechin-3-gallate (EGCG), a well-known in vitro DNMT1 inhibitor, as a positive control. Results showed that 50 µM RS-F3 reduced the activity of purified DNMT1 by about 25% C.Finding new effective treatments against AML is one major challenge of current anticancer research. Several marine compounds have already shown promising activity against AML cells ,44 and cR, 17S-fistularin-3, briefly RS-F3. RS-F3 was tested for its anticancer effect on a panel of AML cell lines. Three AML cell lines decreased proliferation and viability at 5–15 µM. These results are similar to those obtained previously with the compound isofistularin-3 from Aplysina aerophoba, a stereoisomer of fistularin-3 with a non-specified configuration at C17 [We report here an interesting cytostatic and cytotoxic effect against AML cell lines of a brominated sponge-derived compound of the fistularin family, named (+)-11n at C17 . In thisWe thus investigated the anti-proliferative molecular mechanism of RS-F3 by assessing levels of Mcl-1. We showed that sub-toxic doses of RS-F3 reduced Mcl-1 levels in AML cells lines at early time points. However, in HEL cells, which continued to proliferate and survive to RS-F3 treatment, we detected no Mcl-1 decrease. The JAK2 V617 mutation status of this cell line could be implicated in this differential behavior; however, this hypothesis remains to be tested. Overall, these data suggest an important role for Mcl-1 in the anti-proliferative effect of RS-F3. Mcl-1 downregulation could result from transcriptional inhibition, increased degradation or from the combination of these two phenomena. When U-937 cells were treated with two different proteasome inhibitors together with RS-F3, Mcl-1 levels were rescued. Our data suggest a contribution of proteasomal degradation to RS-F3-induced Mcl-1 decrease.+ cells), to sub-micromolar concentrations of ABT-199. These data attest for a promising sensitizing activity of this marine compound towards Bcl-2 inhibitors, which could be exploited in the treatment of AML with high Mcl-1 expression and resistance to BH3 mimetics.Importantly, in this study we found that combination treatments of RS-F3 with the Bcl-2 inhibitor ABT-199 sensitized U-937 and OCIAML-3 cell lines, but not Bcl-2 negative TF-1 cells or healthy platelets , 6(S), 1’(R), 6’(S) fistularin-3 stereoisomers and to bind to the interface between CXXC and RFTS domains. In addition, RS-F3 exhibited an inhibitory effect on the in vitro DNMT1 activity. Altogether, these data support the increasing knowledge about the potential of marine bromotyrosines to modulate epigenetic enzymes and lead cancer cells to proliferation arrest or to death.Bromotyrosine derivatives comprise several biologically active molecules ,24,27,59Suberea clavata , which was collected by hand using SCUBA diving in Solomon Islands, Russel Group, off Lologhan Island on the 30/06/2004 between 6 m and 12 m deep. A voucher sample is deposited at the Queensland Museum under the accessing number G322641 and was identified by Dr. J.N.A. Hooper as Suberea clavata . The sponge was deep frozen on board until work up.Compound RS-F3 was extracted from the freeze-dried sponge 2O partition to obtain a desalted butanol extract. A portion of the extract was submitted to filtration on Phenomenex Sepra C18 silica gel and eluted with a gradient H2O/MeOH (100/0 to 0/100) and MeOH/DCM (100/0 to 0/100) to give 6 fractions. The fraction f3 was submitted to reversed phase (Sunfire C18) HPLC chromatography with H2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH: 85/15 to 0/100 in 40 min and yield 13 subfractions (F1 to F13). The fraction F6 was pure (+)-11(R), 17(S)-fistularin-3.The lyophilized sponge was extracted at room temperature with methanol/dichloromethane (MeOH/DCM) (1/1). The crude extract was submitted to n-butanol/HR), 17(S)-fistularin-3 was established using the Mosher method and intensive purification by RP-HPLC, LCMS and NMR analysis .The absolute configuration of (+)-11; bortezomib, Venetoclax (ABT-199), and ABT-263 were purchased from Selleckchem . All drugs were dissolved in DMSO.+ progenitor/stem cells were isolated from cord blood of healthy donors and cultured as previously described [2.The AML cell lines U-937, HL-60, HEL, THP-1, and TF-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) were cultured in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal calf serum  and 1% antibiotic–antimycotic (Lonza); TF-1 cells were also supplemented with 5 ng/mL granulocyte–macrophage colony-stimulating factor; PeproTech, London, UK). The AML cell line OCIAML-3 was cultured in MEM-ALPHA (Lonza) supplemented with 15% FCS and 1% antibiotic–antimycotic. PBMCs and platelets  from human healthy donors were isolated and cultured as previously described . Human pescribed . All celCell number and viability were determined using a semi-automated image-based Cedex cell counter  hinged on the Trypan Blue (Lonza) exclusion assay. Colony formation assays were performed as previously described , with a Lymphocyte subpopulation from PBMCs was identified by flow cytometry  based on cell size and granularity. Data were recorded statistically (10.000 events/sample) using the CellQuest 4.0.2 software  and analyzed using Flow-Jo 8.8.7 software .Platelet viability was assessed by measuring ATP levels with the Cell Titer-Glo Luminescent Assay , according to the manufacturer’s instructions. Data were recorded using an Orion Microplate Luminometer . In parallel, viability was evaluated by measuring phosphatidylserine exposure by FACS analysis of cells stained with FITC Annexin V . The platelet-toxic compound ABT-263 and a challenge with 50% DMSO for 1 min were used as a positive control for platelet mortality.The modality of cell death was assessed performing nuclear morphology analyses by fluorescence microscopy , after staining with Hoechst 33342 and propidium iodide.Enzymatic activity of caspases-3/7 was determined using the Caspase-Glo 3/7 Assay . The assay was performed according to the manufacturer’s instructions and luminescence was measured using an Orion Microplate Luminometer .®  supplemented by 1× protease inhibitor cocktail  according to manufacturer’s instructions. Western blots were carried out, as previously described [Whole cell extracts were prepared using M-PERescribed , using tCHOP gene expression levels were evaluated by Q-RT-PCR, as previously described . XBP1 spDNMT1 activity was measured using the in vitro DNMT activity/inhibition assay  according to manufacturer’s instructions. The methylation reaction was performed by incubating 25 ng of purified DNMT1 with compounds for 2 h at 37 °C in the presence of 0.01% Triton X-100. The methylated DNA was then recognized by the His-tagged methyl-CpG binding domain protein 2b. The addition of a poly-histidine antibody conjugated to horseradish peroxidase then provided a colorimetric readout quantified with a spectrophotometer  at the wavelength of 450 nm.p-values below 0.05. All histograms represent the mean ± SD of at least 3 independent experiments.Statistical analyses were carried out using the GraphPad Prism 7.0 software . One-way or two-ways ANOVA, followed by the Holm-Sidak multiple comparison tests were used for statistical comparisons. Statistical significances were evaluated at R), 17(S)-fistularin-3 (RS-F3). We showed RS-F3 to induce an interesting anticancer effect against AML cell lines. Our data suggests an important role for Mcl-1 in mediating the anti-proliferative activity of the drug. Importantly, we show that this fistularin-3 stereoisomer synergizes with the Bcl-2 inhibitor ABT-199 in killing Bcl-2-positive AML cell lines. Moreover, our docking and in vitro studies support the increasing knowledge about the potential of marine bromotyrosines to modulate DNMT1 activity.We determined the absolute configuration of the fistularin-3 stereoisomer (+)-11(


Carnosic acid was used as starting material to synthesize royleanone derivatives featured C11–C14 para quinone. The importance of C-20 group of royleanone derivatives was verified by the cytotoxicity assay of royleanonic acid, miltionone I and deoxyneocrptotanshinone. Following our synthetic route, 15 amide derivatives were synthesized and 8 compounds exhibited moderate cytotoxic activities against three human cancer lines in vitro.The online version of this article (10.1007/s13659-018-0173-y) contains supplementary material, which is available to authorized users. Royleanones, possessing a characteristic 11,14-para benzoquinone 12-hydroxy abietane skeleton, were first isolated by Handa’s group in 1945 from the roots of Inula royleana as yellow pigment [ pigment –5. These pigment .para benzoquinone moiety in royleanones could not act as Michael acceptor, the cytotoxic activity of royleanones would be weak. In fact, royleanones has IC50 of 32.5 µM to MIAPaCa-2 cell line and deoxyneocryptotanshinone was inactive (IC50 > 100 µM) to the same cell line [para benzoquinone derivatives might lead to more potent analogues than columbaridione.Our research interests are largely related to the antitumor molecules, especially tricyclic terpenoids derivatives –12. Tansell line . Royleanell line . The difpara benzoquinone. As a result, 15 new amide derivatives were synthesized , intramolecular esterification occurred to afford lactone 2 with DCC. The 12-hydroxy group was then protected as MOMO ether. After hydrolysis, the resulting free phenol 10 was oxidized into para benzoquinone 11. Liberation of MOMO ether by HCl afforded the desired royleanonic acid.To verify the hypothesis of the importance of C-20, we synthesized royleanonic acid as indicated in Scheme p-TsOH in MeOH, Deoxyneocrptanshinone was obtained in 70% yield  (Table 7 had a significant cytotoxicity toward Hep G2 and MCF 7 human cancer lines in vitro (IC50 1.02–2.03 μM).All the synthesized compounds were evaluated for cytotoxicity against three human cancer cell lines . Eight royleanone derivatives showed potent cytotoxicity against three human cancer cell lines IC50 < 0 μM . These results may attribute to mechanism other than Michael addition of DNA, RNA etc. [7 gave the best result. All derivatives are active towards HpeG2 cell line. These findings may serve as new clues for discovering antitumor molecules with new structure scaffold.Our experiments demonstrated that, for the first time, modification of orthoquinone into RNA etc. . The exiSupplementary materials such as experimental procedures, MS and NMR spectra of the synthesized compounds are available in the online version of this article free of charge. Supplementary material 1 (DOC 5423 kb)Below is the link to the electronic supplementary material.