Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine. Microvascular endothelial cells (ECs) form a continuous monolayer lining the inner face of vascular walls throughout the body and play crucial roles in the regulation of vascular functions  ,2. The pVascular endothelial cells also constitute target cells for microorganisms, in particular for vector-borne pathogens. Pathogens that target ECs can compromise endothelial monolayer integrity and barrier permeability, which is a way for them to cross the vascular barrier, either to reach the blood and disseminate throughout the body or to penetrate inside an organ. Even if infected, ECs are not always the main target cells of pathogens but might represent the site of initial replication after vector-borne inoculation, and before infection of blood cells. Moreover, they could constitute a reservoir for the pathogen during persistent infection by playing the role of niche cells, protecting the pathogen from the immune system . Flaviviridae such as dengue, West Nile or Zika viruses [Reoviridae such as African horse sickness virus or Bluetongue (BT) virus (BTV) [Culicoides midges and is notably characterized in domestic ruminants by vascular injury with hemorrhage and ulceration of the oral cavity and upper gastrointestinal tract, tissue infarction, and widespread edema [Ehrlichia ruminantium, a tick-borne bacterium responsible for heartwater in cattle, can also infect ECs [Picornaviridae member, the agent of a highly contagious vesicular disease of cloven-hoofed animals (Artiodactyla order) with a tremendous economic impact at the global level [Endothelial cells are often targeted by viruses, including members of  viruses , or membus (BTV) , which cad edema . Endothead edema ,10. In tad edema . Additiofect ECs . On the al level , prefereal level ,14. Howeal level . Thus, mPathogens can express a selectivity towards endothelial cells from a specific organ. In fact, a strong heterogeneity exists between endothelial cells depending on their belonging to the macrovasculature or to the microvasculature but also on their location in the organism. Microvascular ECs isolated from blood vessels of various tissues vary structurally, phenotypically, and functionally according to the organ they are coming from and to their exposure to the microenvironment ,17. TheiDespite a constant increase in the number of publications in the fields of veterinary medicine, including cell–pathogen interactions, inflammation, and cancer, valuable biological models are still lacking compared to research tools developed concerning humans and rodents. The use of primary cells requires the use of animals but also displays several disadvantages. First, they stop dividing after a finite and limited number of passages. Second, as batches do not come from the same animal, they differ from one to another and do not provide repeatable and reproducible data. In contrast, immortalized cell lines, established in a controlled and identical manner, can divide infinitely in long-term in vitro culture allowing a large cell production for scientific studies and represent a good alternative to overcome these problems ,18,19. E. ruminantium [® CRL-2048™) from cornea [® CRL-8659™) from the adrenal gland, can be purchased. In addition, only a few immortalized microvascular ECs are described in the literature: bovine brain ECs [Only few bovine immortalized cell lines are available up to now, whatever the cell type considered, and cells of human origin, such as human umbilical vein ECs (HUVECs) are even sometimes used as a model of cattle endothelial cells. The most used bovine endothelial cells are originated from the macrovasculature, such as bovine aortic endothelial cells or bovine umbilical cord ECs that allow the growth of the bacterium inantium ,21,22. Ninantium . Only twm cornea  and EJG rain ECs ,26, bovirain ECs , and bovrain ECs . Bartonella henselae, which can lead to bacillary angiomatosis and/or peliosis in immunocompromised human patients, showing a differential pro-angiogenic effect on human but not feline microvascular ECs. This was consistent with the absence of bacillary angiomatosis in cats and highlighted the fact that the outcome of endothelial cell infection by B. henselae is governed by species specificity [To palliate the lack of bovine microvascular endothelial cell lines for veterinary studies, allow the growth of pathogens in adapted cell lines, close to the in vivo conditions, and avoid the regular use of cattle for primary cell needs, there is an urgent need to establish immortalized bovine endothelial cell lines from the microvasculature of various organs representing potential targets for infectious agents. Such organospecific cell lines were previously developed in mice , in humacificity .In the present paper, our aim is the establishment of bovine endothelial cell lines from various organs as new models to study cattle viral infections. We present the isolation, immortalization, and characterization of six microvascular and one macrovascular bovine endothelial cell lines, that were immortalized in the same way as previous human, murine, and feline ECs, their successful infection by two already mentioned viruses, BTV and FMDV, and their ability to produce viral proteins upon DNA transfection. Six bovine endothelial cell lines from the microvasculature of various organs  and one cell line from the macrovasculature  were established in culture . All theAll cell lines displayed endothelial cell characteristic morphologies. Nevertheless, slight morphological differences were observed between the seven cell lines during their growth . Under mBovine endothelial cell lines were assessed for their ability to form capillary-like structures in vitro in a Matrigel™ assay and compared with the human endothelial cell line (HSkMEC) and the human endothelial progenitor cell line-cord blood (HEPC-CB1) displaying an angiogenic potential. gusb, hprt1, and ppia) after a normalization step. The seven bovine cell lines were analyzed for the RNA expression of several markers, including endothelial cell markers, progenitor cell markers, and non-endothelial cell markers, using bovine primers . All marConcerning mature endothelial cell line markers, CD31, CD143, CD146, VEGFR-2, and von Willebrand factor (vWF) mRNAs were weakly expressed in all bovine cell lines, compared to the mature bovine aortic primary endothelial cells (BAEC), with some differences of expression between the cell lines. In particular, BBrMEC and BMLNMEC displayed the highest expression of VEGFR-2 mRNA and CD146 mRNA, respectively, among all bovine cell lines. In addition, vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 1 (VEGFR-1) mRNAs were more expressed on bovine cell lines than on BAEC. Endothelial hyaluronan receptor 1 (LYVE-1) mRNA, a marker of lymphatic endothelial cells, was strongly expressed in the seven bovine cell lines, with a very high relative expression in BOvMEC and BLuMEC, whereas it was very little expressed in BAEC. For the three markers of progenitor cells, the relative expression of CD117 mRNA was high in BBrMEC compared to other cell lines and to BAEC. CD133 mRNA was more expressed in bovine cell lines than in BAEC, particularly in BMLNMEC. Furthermore, chemokine receptor type 4 (CXCR4) mRNA, which can be found both in progenitor and mature endothelial cells, was slightly expressed in the seven cell lines, compared to its high level in BAEC. Finally, CD38 mRNA, a marker of hematopoietic cells, was only slightly expressed, except in BIntMEC.To complete the phenotypic study, bovine cell lines were analyzed by flow cytometry for the expression of specific markers at the protein level . As mostFrom those results, it was not possible to establish a close correlation between RT-qPCR and flow cytometry data when comparing the expression of markers and their corresponding mRNAs in bovine endothelial cell lines, suggesting post-transcriptional regulations of protein expression.The secretion of the major endothelial and proangiogenic growth factor, VEGF, was investigated in the culture supernatants of the seven endothelial cell lines as well as of BAEC using three different specific bovine, one murine, and one human VEGFA ELISA detection kits. No VEGFA secretion was detected, whether the cells were grown in normoxia or in hypoxia, a condition well-known to increase VEGFA production.Apart from BOvMEC which were not tested, the six other cell lines were infected with BTV at multiplicity of infection (MOI) 0.1. Cells were collected/lysed at 18 h post-infection (hpi) and the expression of the VP5 viral protein was measured by Western blot. As shown in −3 and 10−5) and monitored for cytopathic effect (CPE) apparition during 48 h. Highly sensitive ZZ-R127 cells were inoculated as positive control. The results are summarized in In another set of experiments, five endothelial cell lines  were inoculated with FMDV strain of O serotype (MOI 10Madin-Darby bovine kidney cell line (MDBK) was used in our laboratory as a model of viral infection since these cells have been notably demonstrated to be permissive to BTV and FMDV infections ,30. HoweBartonella henselae, were then developed and allowed to reproduce at the cell level some of the major differential effects induced by the bacteria in humans versus cats [The isolation and maintenance of ECs from specific vascular beds are important to study physiological and pathological processes such as inflammation, wound healing, leukocyte homing and transmigration, tumor development and invasion or metastasis , as wellsus cats .In the field of veterinary medicine, despite the need of bovine microvascular endothelial cell lines for research purposes, only few microvascular endothelial cell lines from cattle have been established from brain ,26, adreHere we present the successful isolation, immortalization, and characterization of six endothelial cell lines from the microcirculation of various organs or tissues with potential contact with pathogens  and one cell line from the macrovasculature , obtained from the same bovine fetus. The rapid cell proliferation of the seven selected cell lines in endothelial specific medium and their endothelial cell characteristic morphology were in favor of the successful isolation of the correct cell type. The cell lines’ characterization confirmed that they have kept their blood vessel phenotype, with the expression of, at least, several typical EC phenotypic markers such as CD31, CD143, CD146, CXCR4, VEGFA, VEGFR-1, VEGFR-2, and vWF, either at the mRNA and/or at the protein level. The lack of labeling for CD31 and CD105 on both mature endothelial cells, BAEC, and on all the cell lines, may come from the low affinity of the antibody (targeting the human protein) for the bovine form, precluding from concluding about the presence or the absence of the protein. The lack of available bovine cell lines, of anti-bovine specific antibodies, and of valuable bovine ELISA kits emphasizes the paucity of biological tools when researchers work on species other than humans, rats or mice and confirm the importance to develop tools for other animal species, at least for domestic animals, in order to allow advances in veterinary medicine applications. However, CD31, an important marker of ECs, was not detected even when using anti-bovine antibodies, but the corresponding mRNA was present in cell lines, at a level much lower than in BAEC. These results highlight the utility to detect markers at two different levels, especially when antibodies against the species of interest are lacking. LYVE-1, a marker of lymphatic vessels, was detected in all ECs, with a very high expression in BOvMEC and BLuMEC, and to a lesser extent in BBrMEC and BIntMEC, suggesting that these cell lines may be of lymphatic origin or not yet totally differentiated endothelial cells inside the embryo ,35. The As compared to BAEC, the seven cell lines expressed to a lower extent the markers that are expressed at a high level in the mature bovine endothelial cells . Conversely, they expressed to a higher extent the markers that are expressed at a low level in BAEC , mainly the markers of non-mature endothelial cells. This suggests that the bovine cell lines are not totally differentiated and that they still keep part of their progenitor status. This could be linked to the origin of the cells since the cultures were derived from a calf at six months gestation. At this stage, different endothelial cell populations co-exist, including stem endothelial cells, early or late endothelial progenitor cells, circulating endothelial cells, and mature endothelial cells, displaying a large panel of markers (CNRS patent WO2011086319A1) ,36,37. All the established bovine endothelial cell lines can be cultivated long-term in the laboratory, contrary to primary cells that stop proliferating after a finite number of divisions, and without losing their phenotypic characteristics as primary cells can do. Consequently, they allow the acquisition of repeatable and reproducible experiments. Furthermore, they display their own phenotype, according to the organ they are coming from, allowing to identify their specificity and, thus, providing a relevant in vitro model for organospecific endothelium studies.In addition to their phenotype, endothelial cell lines can also be characterized by their functional activities. Apart from BOvMEC, whose aptitude to angiogenesis was limited, the six other cell lines demonstrated their ability to form pseudovessels on Matrigel™, confirming their endothelial origin. The fact that cell lines retained their angiogenesis capacity in vitro is capital information, as these cell lines could be used for in vitro studies in cancerology, as a model of hemangiosarcoma, or for anti-angiogenesis applications. The interest of these cell lines is thus not restricted to in vitro infections and host-pathogen interactions, but can be extended to many applications concerning physiological or pathological processes involving endothelial cells.In order to evaluate the susceptibility of the cell lines to pathogens, their capacity to be infected by two viruses, BTV and FMDV, involved respectively in two ruminant diseases, Bluetongue and foot-and-mouth disease, were compared.Such cells could constitute good substrates for determining if the significantly higher susceptibility of sheep to BTV as compared to cattle and if the differences in clinical manifestations when cows can become sick (BTV-8 serotype) could be the consequences of variations in endothelial cell susceptibility to BTV infection . MoreoveFMDV preferentially targets epithelial cells, in particular in the respiratory tract. In vitro work is rarely based on immortalized bovine cell lines from the primary organs targeted in vivo due to the lack of available cell lines. Bovine cell lines used for FMDV in vitro investigations are indeed epithelial cells derived from kidney (i) MDBK cells used by Kopliku and colleagues  or (ii) This work highlights the potentially high susceptibility of BMLNMEC, along with BLuMEC and BUcEC, to pathogen infection or at least to viral infection. This should be further confirmed by testing other pathogens, in particular those targeting endothelial cells at certain infectious stages. In addition, as only some cell lines are permissive to viral infection, it would be interesting to investigate if there is a link between the expression of specific markers of endothelial cell lines and their susceptibility to viral infection.Ehrlichia ruminantium [Furthermore, these cells would be very useful for studying other pathogens that target specifically bovine endothelial cells, like the intracellular bacterium inantium . In addition to the direct pathogen infection, BBrMEC could bring an interesting solution to study in vitro the blood-brain barrier (BBB) integrity and functionality. Indeed, they could be co-cultivated with other cell types in order to reconstitute an in vitro BBB, which could permit to study the consequences of pathogen infection on the BBB permeability, as was already studied in human cells , and morIn conclusion, these seven established bovine endothelial cell lines, mainly originating from the microvasculature of various organs, will enrich the available biological tools in cattle research. They constitute a valid and suitable model for in vitro studies as they retain their phenotype after successive passages and can be grown in large quantities. Their organospecificity allows to work with cell lines very close to in situ conditions. They can thus constitute an excellent alternative to animal experimental testing, either as monolayers models or as more complex structures like organoids. Their scope of application will cover not only host-pathogen interaction studies, as we demonstrated here with viral infections, but also pharmacological studies in order to test new chemicals before their in vivo use and cancerology research  as they 2) at 37 °C and were tested weekly as negative for mycoplasma.Bovine aortic primary endothelial cells (BAEC), isolated from an Aubrac-cattle aorta, were used as a bovine endothelial cell positive control. This cow was humanely euthanized due to very bad health at the School of Veterinary Medicine of Toulouse, France. BAEC were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% non-essential amino acids solution, 100 U/mL penicillin and 100 µg/mL streptomycin , 16 U/mL heparin and 1 µg/mL hydrocortisone . The human skin microvascular endothelial cell line (HSkMEC)  and the Bos taurus) calf fetus of six months of gestation was humanely removed during a therapeutic cesarean section at the School of Veterinary Medicine in Maisons-Alfort, France. No ethical approval was required in this case, as our experiment joined a cesarean that was performed due to life-threatening condition of the cow. Tissue biopsies were placed in sterile Roswell Park Memorial Institute (RPMI) medium supplemented with 15% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.8 µg/mL fungizone (Gibco-Thermo Fisher Scientific). Isolation and culture of microvascular and macrovascular endothelial cells from several organs  were performed and cell immortalization was undertaken by transfection using the pSV3-neo plasmid containing the SV40 early region of the large T-antigen gene, as already described [2) at 37 °C. Transfected cells were selected by addition of 40 to 800 µg/mL G418 in the culture medium. Once established, cell line growth was checked at least twice a week using an inverted Leica DMi1 microscope equipped with digital camera Leica MC120 HD. All cell lines were checked weekly for potential mycoplasma contamination and were tested as negative.A Montbéliarde female (escribed  and CNRSv/v) in OptiMEM basal medium at 4 °C. Then, 40 µL were distributed in a 96-well plate and allowed to polymerize during 1 h at 37 °C. Cells (1.5 × 104 in 100 µL OptiMEM for each cell line) were seeded on Matrigel™ and cultures were grown at 37 °C in the incubation chamber of the video station equipped with a video microscope  and with an Axiocam camera using the Zeiss Axiovision acquisition software. The direct real-time visualization of the formation of pseudovessels was monitored every 30 min for 24 h. As the kinetics of angiogenesis was not the same for all cell lines, the best time was selected for each of them. The experiments were performed twice, each in triplicate.Matrigel™ matrix  was diluted  were used for normalization. The list of genes analyzed, and the corresponding primer sequences are described in After washing with phosphate-buffered saline (PBS), confluent cell monolayers were scraped, lysed, and stored at −80 °C. RNA extraction was performed using NucleoSpin RNA kit  following the manufacturer’s instructions and elution was done in RNase-free water. RNA amounts were determined using the NanoDrop ND-1000 spectrophotometer. Reverse transcription from 1 µg of RNA was performed with random hexamers/Oligo(dT) using Maxima First Strand cDNA synthesis kit for RT-qPCR (Fermentas–Fisher Scientific) according to the manufacturer’s instructions. Specific primers  were designed using KiCqStart™ Primers (Sigma-Aldrich). Three reference genes  supplemented with 0.5% of bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% azide (Sigma-Aldrich). Cells were detached using 0.5 mg/mL type I collagenase (Gibco) and washed. Part of the cells was permeabilized with BD Cytofix/Cytoperm . Cells (5 × 105) were then incubated with either fluorochrome labeled-antibodies for 60 min or with primary antibodies for 60 min, followed by an additional incubation with the fluorochrome-labeled secondary antibodies for 30 min, as described in Cells were grown until 90% confluence and washed using complete PBS (PBS with 1 mM of CaCl2) or in a hypoxia chamber  in 6-well plates in complete medium. The complete medium was replaced by medium without FBS 48 h before the collection of supernatants and cultures were grown up to 90% confluence. Supernatants of three independent cultures for each cell line were collected and immediately frozen at −80 °C. VEGFA production was measured using either the human VEGFA kit , the murine VEGFA kit  or three different bovine VEGFA specific ELISA kits , according to the manufacturer’s instructions. Each ELISA kit was tested twice with three independent cultures from each cell line. Cells were grown either in normoxia ) supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics) and phosphatase inhibitors . To quantify viral replication, VP5 protein was detected by Western blot analysis using the corresponding monoclonal antibody . Secondary anti-mouse HRP-conjugated antibody was from GE-Healthcare. Results are presented as one representative experiment out of three.Wild-type field strain of serotype 8 (BTV8) is from the National Reference Laboratory collection  and was isolated in the Ardennes (France) in 2006 . Then, 5® (Invitrogen), 45% DMEM/F-12 (1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12) (Invitrogen), supplemented with 10% FBS. Cells were grown at 37 °C in a 2.5% CO2 atmosphere and routinely passaged once a week in 75 cm2 tissue culture dishes. Bovine endothelial cell lines were grown as already described. FMDV reference strain O Manisa TUR/8/69 /mL) used in this study was provided by the World Vesicular Disease Reference Laboratory .ZZ-R127  , fetal g4 cells per well then incubated at 37 °C under 2.5% (ZZ-R127) or 5%  CO2 for 24 h. The day after, monolayers were washed twice with serum-free culture medium before inoculation. Each sample was inoculated in duplicate . After 1 h of adsorption at 37 °C under 5% CO2, inoculums were discarded and replaced by growth medium. Cells were then incubated at 37 °C under 5% CO2 and monitored for CPE for 48 h using an inverted LEICA Statif DM IL LED microscope. For each cell line, CPE was estimated qualitatively, by comparison with ZZ-R127 cell line which is very susceptible to FMDV infection [Cells were seeded into 24-well plates at a density of 5 × 10nfection . If no C4 cells were dispensed in each well of a 24-well plate . One day later, cells were transfected with 0.5 µg/well of pEGFP-C1 expression vector encoding BTV8-NS3  according to the manufacturer’s instructions. At 24 h post-transfection, the cells were washed three times with PBS, incubated with a 4% paraformaldehyde (PFA) solution  for 30 min at room temperature and then treated with PBS-glycine (0.1 M) for 5 min at room temperature to quench the cells. The cells were washed three times with PBS and incubated for 30 min at room temperature in a PBS solution containing Hoechst 33258 dye. Preparations were visualized using an Axio Observer Z1 fluorescence inverted microscope (Zeiss). Next, 5 × 10CNRS/EnvA Patent FR 19 15307: Lignées immortalisées de cellules endothéliales bovines et canines issues de différents organes. Anne-Claire Lagrée, Fabienne Fasani, Clotilde Rouxel, Claudine Kieda, Henri-Jean Boulouis, Nadia Haddad and Catherine Grillon.


In this work, we studied the metabolic reprogramming of same-patient-derived cell lines with increasing metastatic potential to develop new therapeutic approaches against metastatic colorectal cancer. Using a novel systems biology approach to integrate multiple layers of omics data, we predicted and validated that cystine uptake and folate metabolism, two key pathways related to redox metabolism, are potential targets against metastatic colorectal cancer. Our findings indicate that metastatic cell lines are selectively dependent on redox homeostasis, paving the way for new targeted therapies.With most cancer-related deaths resulting from metastasis, the development of new therapeutic approaches against metastatic colorectal cancer (mCRC) is essential to increasing patient survival. The metabolic adaptations that support mCRC remain undefined and their elucidation is crucial to identify potential therapeutic targets. Here, we employed a strategy for the rational identification of targetable metabolic vulnerabilities. This strategy involved first a thorough metabolic characterisation of same-patient-derived cell lines from primary colon adenocarcinoma (SW480), its lymph node metastasis (SW620) and a liver metastatic derivative (SW620-LiM2), and second, using a novel multi-omics integration workflow, identification of metabolic vulnerabilities specific to the metastatic cell lines. We discovered that the metastatic cell lines are selectively vulnerable to the inhibition of cystine import and folate metabolism, two key pathways in redox homeostasis. Specifically, we identified the system xCT and MTHFD1 genes as potential therapeutic targets, both individually and combined, for combating mCRC. Colorectal cancer is the second leading cause of cancer mortality with over 800,000 deaths per year . SurgeryRecently, metabolic reprogramming has emerged as an enabling feature of metastasis and cancer stemness . BrieflyG12V-mutant cell lines derived from the primary tumour and a lymph node metastasis, respectively, of a patient with a Duke’s type B colorectal cancer -glucose incubation, indicated a higher glucose contribution into the TCA in the metastatic cell lines  had a stronger Warburg effect than SW480, as they consumed more glucose and produced more lactate a,b. Subsll lines c,j. Consll lines  indicati13C]-glutamine into the TCA cycle through both oxidative and reductive pathways in the metastatic cell lines  compared to SW480 . The titTo complete the characterisation of metabolic traits associated with metastatic capacity, we measured intracellular metabolite concentrations as well as metabolite exchange fluxes between cells and cell culture media using targeted metabolomics and HPLC/MS/MS . IntraceAdditionally, acyl-carnitines and free carnitines were detected at significantly higher concentrations in SW480 compared to metastatic cell lines , potentiA significant concern when working with in vitro models is that their metabolic phenotype can be significantly different from those encountered in vivo . To dete13C]-glucose or [U-13C]-glutamine was higher in vivo in comparison with in vitro  algorithm  and idenThe simultaneous inhibition of the two main cystine transport systems, the cystine/glutamate transporter system xCT (coded by the genes SLC7A11 and SLC3A2)  and the 50 values for the metastatic cells than for SW480. Moreover, erastin exhibited IC50 values up to three orders of magnitude lower than those of sulfasalazine  . MethotrFinally, as folate metabolism plays a key role in nucleotide synthesis, we evaluated whether the enhanced activity of folate metabolism in the metastatic cell lines could be attributed to their increased proliferation rate. We determined that, similarly to the Warburg effect, the enhanced fluxes through folate metabolism, particularly through the cytosolic branch, could not be solely attributed to the increased proliferative capacity of the metastatic cell lines .Having explored the inhibition of two major vulnerabilities of the metastatic cell lines predicted by the computational model , we evaluated whether the inhibition of these pathways could be synergetic. Indeed, both pathways have been associated with antioxidant defence as cystine contributes to glutathione synthesis, and MTHFD1 contributes to ROS detoxification by regenerating NADPH .The results showed that the combination of erastin and LY345899 synergistically affected the proliferation of the metastatic cell lines from concentrations of 0.5 μM of erastin and 20 μM of LY345899 . In lineThe cumulative evidence generated to date suggests that both the Warburg effect and glutaminolysis are associated with metastatic potential in several cancer types ,9,40,41.Interestingly, the metabolic reprogramming we observed correlates with high levels of both MYC and E-cadherin in the metastatic cell lines compared to the primary tumour cell line. These results concur with the extensive existing literature on the metabolic changes driven by MYC in cancer cells ,44 and w13C resolved metabolomics, transcriptomics and gene dependencies). This allowed the construction of highly accurate cell line specific GSMMs, which facilitated the identification of potential metabolic vulnerabilities associated with metastatic progression. Our cell line specific GSMMs predicted that the metastatic cell lines were dependent on cystine uptake from the extracellular media. Indeed, we determined that inhibition of the cystine transporter system xCT with sulfasalazine or erastin was highly specific for the metastatic cell lines, which could make it a potentially effective therapeutic strategy against metastasis in colorectal cancer. Indeed, the system xCT is overexpressed in several cancer types including colorectal tumours with mutant KRAS -glucose, glutamine 50% enriched in [U-13C]-glutamine, or unlabelled substrates. Cells and media were obtained for metabolite extractions at 6 and 24 h after the labelled substrates were added. The metabolites analysed and the procedures are specified in Quantities of 2.5 × 1013C]-glucose (1 bolus of 20 mg/35 g) or [U-13C]-glutamine . Fifteen minutes after the last bolus, the mice were terminally anaesthetised, and blood was taken by cardiac puncture. Tumour tissue was snap frozen in liquid nitrogen. Then, tumour samples were ground in liquid nitrogen to form a powder, which was lyophilised. Polar intracellular metabolites were analysed as specified in After cell line injections into immunocompromised NOD/SCID mice (see Xenograft experiments section) and once tumours reached 8–10 mm in diameter mice were given a bolus of either [U-p > 0.05). For comparisons between two conditions , we used Student’s t test with p < 0.05, and we used asterisk to indicate significant differences. Further statistical analyses are explained in All experiments were performed at least in triplicates and repeated two or more times. Statistical analyses of experimental measures were performed using the Agricolae package for R. More specifically, for comparisons that are between the cell lines (SW480–SW620–LiM2) we used a one-way ANOVA for the factor “cell line”, and Scheffe’s test for multiple comparisons. Groups that are indicated with the same letter are not significantly different  were determined by combining gene expression data with the GPR rules of Recon 2. A reaction was considered to be inactive if when a gene was inactivated , the mapped gene expression value decreased at least 16-fold. Then, the effect of reaction KOs was simulated using the reference flux distribution computed for each cell line as input for running MOMA  in the fUsing a novel systems biology strategy to integrate metabolic and transcriptomic data, we unveiled the metabolic reprogramming related to cystine transport and folate metabolism as druggable metabolic vulnerabilities of mCRC in the SW480-SW620-LiM2 same-patient-derived model. We demonstrated that the combined inhibition of xCT and MTHFD1 is synergetic and is specific for metastatic colon cancer cell lines.


Cell lines are widely used for research, but even commonly used cell lines may not be well characterized, making validation of published data and generalizing the results to the clinic problematic. Here, we have established a head and neck squamous cell carcinomas (HNSCC) cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between e.g., hypoxia and radiosensitivity in HNSCC.KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in  Head and neck squamous cell carcinomas (HNSCC) are a malignant disease occurring in the mucosal regions of the oral cavity, hypopharynx, oropharynx, larynx, and other sites in the upper respiratory tract. It is a heterogeneous group of malignancies that, depending on disease type and location, has a relatively poor prognosis ,2. Five-We collected 22 HNSCC cell lines frequently used in in vitro and in vivo studies . For SCCThe short tandem repeat (STR) profiles of the UT-SCC-19A, UT-SCC-24A, UT-SCC-45, UM-SCC-6, UM-SCC-47, 93-VU-147T, UPCI: SCC090 and UPCI: SCC154 cell lines were identical to previously reported profiles  10,22,2,24,25,26In addition to genetic characterization, information about cellular morphology is important to establish the authenticity of a cell line. For example, prolonged passaging and exposure to different culture media could potentially lead to changes in morphology, polarity, epithelial-mesenchymal transition, and therefore cellular behavior ,30. In oFurthermore, from a radiobiological perspective, the colony-forming properties of these cell lines is of particular interest, as the colony-forming assay remains currently the gold standard for radiosensitivity experiments . UT-SCC-We characterized the presence of mutations in well-known oncogenes and tumor suppressor genes using single molecule Molecular Inversion Probe (smMIP) sequencing of 23 oncogenes and tumor suppressor genes. This method was previously established to test for clinical mutations in patient-derived samples and covers 41 hotspot regions required for cancer diagnoses and/or predictive diagnostics  32]..32].In our HNSCC cell line panel we detected a number of mutations, mostly in the TP53 gene. Strikingly, all of the TP53 mutations that were detected were localized in the DNA and/or rendered a significant part of the DNA binding domain (DBD) dysfunctional through frameshifts, premature stop codons, or inappropriate intron retention . In fourThe mutations in the TP53 gene, or lack thereof, were found to be identical to the original reports for UT-SCC-5 , UT-SCC-HPV status and p16 status have been shown to be important factors that determine how well tumors and patients respond to treatment. Characterization and documentation of these factors is therefore important. While HPV status and p16 status are commonly used interchangeably in literature, they have different effects on treatment outcome. We previously reported the p16 and HPV status and summarize them here . Previou2). As hypoxia , and especially severe hypoxia (<0.1% O2) is one of the best described variables to negatively affect radiosensitivity, we also included cellular sensitivity to low oxygen tensions. In addition, we summarized the hypoxic fractions that have been reported to occur in in vivo xenografts of individual cell lines, as measured using the hypoxia-marker pimonidazole . FaDu cells were kindly provided by Michael Baumann . 93-VU-147T was kindly provided by Dr. Dorsman, Amsterdam University Medical Center, Amsterdam, the Netherlands). UPCI:SCC090 and UPCI:SCC154 were purchased at DSMZ. All cell lines were cultured in DMEM medium  supplemented with 4.5 g/L glucose, GlutaMAX, 10% FBS, 100 u/mL penicillin/streptomycin, non-essential amino acids (Gibco), HEPES (Gibco), and sodium pyruvate (Gibco). The SCCNij lines were derived from patient biopsies obtained between 1996 and 2006 at the Radboud university medical center  and passaged as xenograft models in BALB/c /nu mice ,44. CellAll cells (except SCCNij) were grown to approximately 70% confluency. Phase-contrast images were taken using a Zeiss AXIO microscope and Zeiss AxioCam ICc5 camera  at 100× magnification. Software used was Zen 2012 (Blue edition).g for 3 min. Lysates bound to the column were then repeatedly washed with 100% ethanol and subsequently eluted into a nucleic acid free tube. gDNA was quantified by spectrophotometric analysis. For SCCNij tumors, ex vivo frozen chunks were thawed and homogenized using sonification. Genomic DNA was then isolated in a similar manner as described above.HNSCC cell lines (aside from SCCNij) were cultured to approximately 70% confluency and then harvested by cell scraping. Genomic DNA extraction was performed by using a genomic DNA extraction kit . Cells were lysed using a digestion buffer, followed by an RNAse A treatment (10 kU per sample). Proteinase K treatment was performed for approximately 1 h at 55 °C. Samples were washed with 100% ethanol and transferred to a binding column and centrifuged at >5000× Mutation analysis of a panel of cancer-related oncogenes and tumor suppressor genes was performed by single molecule Molecular Inversion Probe (smMIP) sequencing. Sequencing was performed on 100 ng genomic DNA at the department of Pathology, Radboud University Medical Center , according to a previously described protocol . TargeteShort tandem repeat (STR) profiling was performed by Eurofins genomics  on gDNA extracted from the HNSCC cell lines. STR analysis was performed for 16 markers, including CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA, and amelogenin. Where possible, STR profiles were compared to previously published consensus profiles, as well as the Cellosaurus database of existing STR profiles. STR analyses were not performed for the SSCNij lines, as these are grown in mice, which would result in mixed STR profiles.Radiosensitivity data were acquired by colony-forming assays. Data on all cell lines were available from previously published work, except for UT-SCC-9 and FaDu ,42. In bTaken together, we have established an informative HNSCC cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between hypoxia and radiosensitivity in HNSCC.


O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC–ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their The online version of this article (10.1007/s00018-020-03504-z) contains supplementary material, which is available to authorized users. With over 18 million new cases worldwide in 2018, colorectal cancer (CRC) is the third most common cancer in the world  using siVarious genetic studies have confirmed that cancer cell lines recapitulate the molecular features of the tumours , 6 and tNext to genetic, metabolic and proteomic signatures, protein glycosylation is a major factor in colon differentiation and CRC development . It has N-glycosylation of a set of CRC cell lines has been characterized, revealing association of antennary fucosylation with differentiation and caudal type homeobox 1 (CDX1) expression  allowed sequential release of both N- and O-glycans from cell lysates in a higher throughput manner using 96-well plates. Cell lines were harvested after they were grown up to 80% confluence. Cell lysates from 500,000 cells were used for protein immobilization on PVDF membrane. Proteins were denatured by guanidine HCl and DTT followed by an overnight N-glycan release by PNGase F digestion. After removal of the released N-glycans, O-glycans were released by reductive β-elimination, purified by cation exchange chromatography (CEX) and graphitized carbon solid phase extraction (SPE) packed in a 96-well filter plates and finally analysed by PGC nano-LC-ESI-MS/MS. Supplementary material 2(.pdf): Figure S2 Technical and biological variation. PC1 and PC2 score plot (a) together with PC2 and PC3 score plot (b) based on monosaccharide average compositions of all technical (marked with 1 and 2) and biological replicates  of cell lines and fetuin O-glycan standard (control) display close clustering of scores showing robustness of the method. The top three principal components explain 86.85% of the variation within the data. Cell lines cultured at VUMC are marked with “VU”. Supplementary material 2(.pdf): Figure S3 Structural diversity of CRC cell line O-glycans. Relative abundances of individual O-linked glycans released from 26 CRC cell lines. Glycans are numbered according to Supplementary Table 2: S1. The most abundant O-glycans in different cell lines are displayed in the legend. Sulphate modification is indicated with “S”. Reduced reducing end is indicated by a circle on the reducing end of the glycan. Different classifications of the cell lines are displayed with colour codes based on gene expression . Supplementary material 2(.pdf): Figure S4 Abundance of structural glycan features per cell type. Geometrical tile of the rescaled relative abundance (%) of the calculated structural glycan features (x-axis) and cell line type (y-axis). Different classification of the cell lines is displayed with colour code based on gene expression (colon-like in red and undifferentiated in light blue) and consensus molecular subtypes . Non assigned cell lines were marked in grey for the gene expression as well as CMS status. All variables have been rescaled fitting within a 0 up to 1 scale. Supplementary material 2(.pdf): Figure S5 Glycan structure determination of major fucosylated glycan structures from LS180 cell line. (a) In order to reveal structural information about all three isomers at m/z 1186.40 in the sample α2,3 Neuraminidase digestion was performed. (b) Digestion resulted in a loss of two peaks (RT= 30 and 32 min) carrying α2,3-linked sialic acid(s), and one remaining isomer Gal(Fuc)GlcNAcβ1-3Galβ1-3(NeuAcα2-6)GalNAcol (RT= 23 min). (a) and (b) The loss of two isomers resulted a significant increase in the abundance of the neutral isomer Galβ1-3 [Gal(Fuc)GlcNAcβ1-6]GalNAcol upon digestion. (c) The terminal Lewis epitope was confirmed by a characteristic Z/Z ion at m/z 551.23 and Z/Z-CH2O at m/z 521.22. (d) The structure of the remaining isomer at m/z 1186.40 (RT= 23 min) was further confirmed by the fragmentation spectra. Supplementary material 2(.pdf): Figure S6 Glycan linkage determination of Lewis epitopes expressed by LS180 cell line. (a) Additional enzymatic cleavage of the α2,3 neuraminidase digested sample was performed with combined α1,3/4 fucosidase and β1,4 galactosidase digestion in order to distinguish between  Lewis x and a epitopes. (a) A significant increase in intensity of the remaining structures Galβ1-3(GlcNAcβ1-6)GalNAcol at m/z 587.30 and GlcNAcβ1-3Galβ1-3(NeuAcα2-6)GalNAcol at m/z 878.32 compared to control (b) suggest the presence of terminal sialyl-Lewis x epitope Galβ1-4(Fucα1-3)GlcNAc-R on the isomer eluting at RT=34 min. (d) The fragmentation of GlcNAcβ1-3Galβ1-3(NeuAcα2-6)GalNAcol revealed the diagnostic ion at m/z 495.21 for the α2,6-linked sialic acid to the reducing end GalNAc structures. Supplementary material 2(.pdf): Figure S7 Glycan linkage determination of major fucosylated glycan structures from CaCo-2 cell line. (a) The structures of two isomers present at m/z 1186.40 in the sample could be determined from the fragmentation spectra of the sialylated species, by the presence of the diagnostic fragment ion at 0,2A3α-H2O at m/z 409.08 (b), confirming the blood group antigen type 2 in the isomer eluting at 52.4 min. (c) The 0,4A0α fragment at m/z 424.04 of the reducing end GalNAc revealed the composition of the 6 arm of the core 2 isomer at RT 63.8 min, together with the Y1α ion at m/z 530.25 indicating the blood group antigen type 3 on the 3 arm. Supplementary material 2(.pdf): Figure S8 Supplementary figure S8 Differentially expressed glycosyltransferase and transcription factor genes in colon like versus undifferentiated cell lines. Volcano plot displays a selection of genes involved in O-glycan biosynthesis (depicted by circles) and transcription factors (depicted by triangles) which show the highest fold change in expression (loge > 0.5) when comparing colon-like and undifferentiated cell lines with high statistical significance . Genes upregulated in colon-like cell lines are marked in red, while genes upregulated in undifferentiated cell lines are marked in blue [8]. The genes that do not pass the thresholds are marked in grey. Supplementary material 2(.pdf): Figure S9 Correlation between the expression of glycosyltransferase encoding genes and transcription factors. A Spearman’s nonparametric correlation was performed with significance α=0.05. Crosses indicate non statistically significant correlations [8] (PDF 2746 kb)Supplementary Table 1 (.xlsx): S1- Calculated monosaccharide average compositions (MSAC) per technical (1 or 2) or biological replicate . The MSAC represents the normalized number of sugar residues and modifications per glycan molecule . The intensities of corresponding structures were multiplied by the monosaccharide compositions and summed over the entire sample. N: N-acetylhexosamine, H: hexose, F: fucose, S: N-acetylneuraminic acid, Su: sulphate residue. Supplementary Table 1: S2-S31 (.xlsx) - O-linked glycans present in fetuin standard (S2), and different cell lines (S3-S31) with their retention times (min), peak areas, relative abundances with standard deviation and relative standard deviation (CV), and calculations of monosaccharide average compositions for each technical (1-3) replicate (XLSX 372 kb)Supplementary Table 2 (.xlsx): S1 - Overview of individual O-linked glycans present in colorectal cancer cell lines, with their retention time (min), relative abundances per cell line, standard deviation and relative standard deviation (CV). The glycan relative abundances for each cell line were averaged for two technical and 3 biological replicates . Identification of glycans was performed based on PGC retention time, known biosynthetic pathways and manual inspection of fragmentation spectra following known MS/MS fragmentation pathways of O-glycan alditols in negative-ion mode. Glycan sequences and linkages were confirmed by the analysis of neutral glycans upon α2-3 neuraminidase digestion. Annotated glycan structures were used for calculations of structural glycan features calculated by summing relative intensities of each glycan multiplied by the number of epitopes per glycan. This was performed for the structures which could be unambiguously annotated. Blue square: N-acetylglucosamine, yellow circle: galactose, red triangle: fucose, pink diamond: N-acetylneuraminic acid, N: N-acetylhexosamine, H: hexose, F: fucose, S: N-acetylneuraminic acid, Su: sulphate residue. Supplementary Table 2: S2 (.xlsx) - Characteristics of cell lines used in this study together with their calculated structural glycan features. Information about the cell lines was obtained from ATCC and referenced literature. Structural glycan features were calculated and averaged per cell type (XLSX 3311 kb)Below is the link to the electronic supplementary material.


Despite the important role of radiotherapy in cancer treatment, a subset of patients responds poorly to treatment majorly due to radioresistance. Particularly the role of radiotherapy has not been established in gastric cancer (GC). Herein, we aimed to identify a radiosensitivity gene signature and to discover relevant targets to enhance radiosensitivity in GC cells.q-value < 0.025) were identified between radiosensitive and radioresistant cell lines, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for validation. Gene set and pathway analyses were performed using Ingenuity Pathway Analysis (IPA).An oligonucleotide microarray  was performed in 12 GC cell lines prior to radiation. A clonogenic assay was performed to evaluate the survival fraction at 2 Gy (SF2) as a surrogate marker for radiosensitivity. Genes differentially expressed .Radiosensitive (SF2 < 0.4) and radioresistant cell lines (SF2 ≥ 0.6) exhibited a marked difference in gene expression. We identified 68 genes that are differentially expressed between radiosensitive and radioresistant cell lines. The identified genes showed interactions via We identified a GC-specific radiosensitivity gene signature and suggest that the Akt signaling pathway could serve as a therapeutic target for GC radiosensitization. Gastric cancer (GC) is the third leading cause of cancer-related death worldwide , 2. In oA combination of radiotherapy and chemotherapy has been widely investigated in preoperative as well as postoperative settings as a strategy to improve GC treatment –5. HowevGene expression profile analysis to predict response to radiation therapy has been performed in various types of cancers –14. It hIn this study, we measured the radiosensitivity, analyzed the mRNA expression profile of GC cells before radiotherapy, and identified differentially-expressed genes and relevant signaling pathways involved in the development of radiosensitivity by comparing radiosensitive and radioresistant GC cell lines. Especially, the Akt pathway was evaluated as a crucial pathway in determining radioresistance of GC cells, and an Akt inhibitor markedly increased radiosensitivity in radioresistant GC cell lines.Twelve GC cell lines  were used. The SNU-series of cell lines were obtained from the Korean cell line bank, and the YCC-series represented cell lines that were established using samples from Korean GC patients at the Songdang Institute for Cancer Research . Cells were incubated in RPMI supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified atmosphere, following the institutional protocol .Radiosensitivity was defined as the survival fraction at 2 Gy of radiation (SF2). To evaluate radiation sensitivity in GC cells, cells were seeded in triplicate and were incubated overnight at 37°C to enable them to adhere to the wells. Cells were then irradiated with X-rays (2 Gy), and cell viability was evaluated 7–10 days post-irradiation. After fixation, colonies containing over 50 cells were calculated. Experiments were replicated three times independently, and the average number of colonies was used. SF2 was determined by the following formula:seedingefficiency is defined as the number of colonies formed divided by expected colony number. SF2 ranged from 0 to 1, wherein a lower SF2 represents higher radiosensitivity.where GSE39747).Microarray data were obtained from GC cells in the unirradiated condition. The total RNA was extracted from each cell line using the TRIzol reagent  according to the manufacturer’s instructions. The Yonsei reference RNA was prepared as previously described . The qua1 for gene identification and gene set analysis. For normalization, the linear models for the microarray data (LIMMA) package were applied using R (version 3.6.1). Genes for which less than 20% of expression data exhibited at least a 1.5-fold change in either direction from the median value of the gene were excluded in the filtering process. Genes differentially expressed between radiosensitive and radioresistant cells were identified by a two-sample t-test using a random-variance model . The P value was adjusted for multiple hypothesis testing using the q-value as suggested by Story . Differentially expressed genes (DEGs) were overlaid onto a global molecular network developed based on the information contained in the Ingenuity Knowledge Base. Networks of network-eligible molecules were then algorithmically generated based on their connectivity. In the genetic network, molecules were represented as nodes, and the biological relationship between two nodes was represented as an edge. All edges were supported by at least one reference from the literature, a textbook, or canonical information stored in the Ingenuity Pathways Knowledge Base.The genetic network was generated through the use of Ingenuity Pathways Analysis  performed on the 12 GC cell lines. In brief, 4 μg of total RNA from each sample was reversely transcribed using the SuperScript II Reverse Transcriptase . Two hundred nanograms of synthesized cDNA were PCR-amplified using QuantiTect SYBR Green PCR . Each reaction was run in a Stratagene MX3005P . Expression values for each gene were determined using a standard curve constructed from Human Genomic DNA . The house-keeping gene hypoxanthine phosphoribosyltransferase  was selected for the normalization and construction of the standard curve. Non-template control wells without cDNA were included as negative controls. The primer sets designed for PCR amplification are shown in TP53, KRAS, and PIK3CA, whole exome sequencing (WES) data of the 12 GC cell lines were obtained from the genome database of the SICR and the Yonsei University College of Medicine . Briefly, single nucleotide variants (SNVs) were evaluated using WES data as previously described  or the vehicle control for 1 h, followed by 2 Gy or mock irradiation. Immediate growth medium exchange was not performed after radiation. Plates were incubated to allow for colony formation (7 d). Colonies containing over 50 cells were then calculated and experiments were repeated three times.A highly selective AKT inhibitor, MK-2206 was purchased from Selleckchem . To assess the radiosensitizing effect of AKT pathway inhibition in combination with radiation, clonogenic assays with or without the AKT pathway inhibitor MK-2206 were performed using radioresistant YCC-16 cells, which harbor the PIK3CA mutation, and YCC-2 cells, which harbor the t-test was applied when comparing two groups which are non-paired. When comparing continuous variables among more than two groups, analysis of variance (ANOVA) was applied and Tukey’s post hoc analysis was performed to identify which groups exhibit significant difference among the multiple groups. The statistical tests used are specified in the figure legends. A Pearson’s correlation analysis was used to evaluate the correlations between parameters. P values < 0.05 indicated statistical significance.Statistical analyses were performed using the GraphPad Prism 7 and R software (v.3.5.1). For continuous variables, student’s The study design is summarized in t-tests between radiosensitive (SF2 < 0.4) and radioresistant cell lines (SF2 ≥ 0.6) identified 613 genes showing expression levels with differences greater than 2-fold and q-value of less than 0.025  for analysis by qRT-PCR. Similar to the microarray results, these genes exhibited a greater than 6-fold difference between radiosensitive and radioresistant GC cells  to assess which pathways are involved in the development of radiosensitivity in GC cells. and TP53 . Functioand TP53 . The Aktand TP53 .Considering the potential role of the Akt pathway in determining the radiosensitivity based on gene network analyses, we further investigated whether the Akt pathway could serve as a druggable target to decrease radiosensitivity in GC cells. The radioresistant cell lines, YCC-2 and YCC-16, were subjected to combinatorial treatment with 2 Gy of radiation and allosteric Akt inhibitor, MK-2206. Although a decrease in SF2 was observed after monotherapy with either MK-2206 or radiotherapy, a combinatorial treatment of MK-2206 and radiotherapy resulted in a significant decrease in SF2 compared to that observed on using either treatment alone .Herein, we describe the gene signature of radiosensitive and radioresistant GC cells, and demonstrate the role of the Akt pathway in determining the radiosensitivity of GC cells. We used 12 GC cell lines and found that radioresistant GC cell lines exhibit contrasting gene expression profiles compared with radiosensitive GC cell lines. Further gene network analyses revealed Akt as a central pathway regulating radiosensitivity. Blocking the Akt signaling pathway through a clinically available Akt inhibitor, MK-2206, significantly enhanced radiosensitivity in radioresistant GC cell lines, which implies that Akt pathway could serve as a potential therapeutic target.This is the first study to describe the SF2 of multiple GC cell lines and correlate gene expression profiles with radiosensitivity. From our gene network analyses, we discovered major signaling pathways, including molecules such as Akt, VEGF, HIF-1α, TGF-β, and p53. These gene networks are known to regulate cellular growth, proliferation, movement, and cell cycle. We further focused on the role of Akt as the Akt-centered network exhibited the highest IPA score. Akt is a serine/threonine protein kinase and a major signaling molecule of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway, which activates downstream molecules involved in cell survival, cell cycle, and proliferation . Amplifiin vitro data from our study, where MK-2206 treated as a single agent induced similar tumor cell killing effects as radiotherapy. The discrepancy may come from the lower efficacy of in vivo drug delivery to tumor compared to in vitro treatment, which may result in a suboptimal drug dose in tumor to kill cancer cells. This implies that although Akt itself may be a therapeutic target in GC, development of combination treatments is needed considering the poor therapeutic efficacy as monotherapy. The combination of radiotherapy and Akt inhibitors has mostly been tested in preclinical models, with Akt inhibitors having a radiosensitizing effect . We identified other candidates responsible for the development of radiosensitization among the 68 genes. Expression of DIRAS3 and CDKN2B was markedly increased in radiosensitive cell lines. DIRAS3 expression has been shown to be associated with chemosensitization to paclitaxel via cell cycle arrest at the G2/M stage in breast cancer cells  has been used as a cancer stem cell marker and is associated with chemoresistance in ovarian cancer .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Gene Expression Omnibus (GEO) database (accession number: KK, HK, SK, and SR contributed conception and design of the study. HK, SK, and DK collected the data. KK, HK, and SK performed the statistical analysis. KK, HK, and SR wrote the manuscript. YK and HC discussed the hypothesis and contributed to data interpretation. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although not a replacement for the whole organism, tick cell lines enable studies at the cellular and molecular level and provide a more accessible, more ethical and less expensive in vitro alternative to in vivo tick feeding experiments. Here we review the role played by tick cell lines in studies on acaricide resistance, mode-of-action of acaricides, identification of potential novel control targets through better understanding of tick metabolism, and anti-tick vaccine development, that may lead to new approaches to control ticks and tick-borne diseases.Ticks and the diseases they transmit are of huge veterinary, medical and economic importance worldwide. Control of ticks attacking livestock and companion animals is achieved primarily by application of chemical or plant-based acaricides. However, ticks can rapidly develop resistance to any new product brought onto the market, necessitating an ongoing search for novel active compounds and alternative approaches to tick control. Many aspects of tick and tick-borne pathogen research have been facilitated by the application of continuous cell lines derived from some of the most economically important tick species. These include cell lines derived from acaricide-susceptible and resistant ticks, cell sub-lines with  Rhipicephalus (Boophilus) microplus, is also the most notorious for rapid development of resistance to every new acaricidal product brought onto the market  are of huge veterinary, medical and economic importance due to their negative impact on the health of humans and domestic animals . Therefoe market . Acariciustralia , and areustralia .in vitro studies to examine and assess the impact of chemical molecules and vaccines against ticks and TBPs, as well as to investigate the mechanisms of resistance and tick metabolism which can lead to the development of novel approaches to control ticks and TBPs  of the R. microplus embryo-derived cell line BME26  than the R. microplus-derived line BME26 that did not survive after exposure to 12.5 μg/ml ivermectin , which are essential for cell cycle progression, and the results suggested that CDKs may present an alternative strategy for designing a novel acaricide that targets both oogenesis and embryogenesis processes.The BME26 cell line also played an essential and effective role in the first report on characterization of a cell cycle protein in arachnids, and the reversal of its functions with an inhibitor . The BMEI. scapularis embryo-derived tick cell line ISE6  are iron-storage proteins that act to store excess iron available in the cellular iron pool by the binding and oxidation of the ferrous ion (Fe2+) and the formation of ferric ion (Fe3+) in the FER core cavity  and lectin pathway inhibitor (AAY66632), and their I. ricinus homologs – constitute candidate protective antigens for the control of tick infestations as well as A. phagocytophilum infection. They showed that anti ISCW005600 and AAY66632 IgG significantly increased the percentage of apoptotic tick cells and decreased pathogen infection, which could help in the development of effective anti-tick and anti-pathogen vaccines.Vaccination with recombinant tick antigens represents a promising tick control strategy. Vaccines containing recombinant tick antigens demonstrated advantages including reduction in acaricide application, cost-effectiveness and reducing the prevalence of tick-borne diseases through reducing exposure of cattle to infected ticks. However, the efficacies of these vaccines vary considerably between tick species and geographic location . Tick ceris IDE8  and ISE6E/CTVM20 , Contrerin vitro-generated acaricide-resistant cell lines, cell lines derived from acaricide-resistant and susceptible ticks, genetically modified cell lines and cell lines supporting growth of intracellular TBPs all have a role to play. Experimental parameters can be easily controlled, effects at the cellular and molecular level can be determined, and cell lines reduce, while not ultimately replacing, the need for expensive in vivo studies involving maintenance of tick colonies and feeding on laboratory animals. As the numbers of cell lines derived from economically important tick species increase, so do the opportunities for exploiting them in a wide range of studies. For example, the potential for developing high-throughput preliminary screens of novel, potentially acaricidal compounds using cell lines from multiple tick species remains unexplored; such screens could greatly increase the number of active compounds identified for further testing at relatively low cost, and could be applied both to compound libraries held by pharmaceutical companies and to natural plant products. It is anticipated that gene editing of tick cell lines by technologies such as CRISPR will add another facet to the control portfolio. The Tick Cell Biobank (Ticks and TBPs have huge negative impacts on global livestock and public health and national economies. Scientific research has focused on creating and developing a variety of strategies to prevent and control ticks and TBPs. Tick cell lines represent essential tools in this area of research:  Biobank , which hAA-R drafted the manuscript, LB-S edited the draft and both authors produced and agreed the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer UM declared a past co-authorship with one of the authors LB-S to the handling Editor.


Multiple myeloma (MM) is the second most common hematologic malignancy. While major advances have been made in the disease, it is still incurable. Although antifolate-based drugs are not commonly used to treat myeloma, new generation analogs with distinct patterns of preclinical and clinical activity may offer an opportunity to identify new classes of potentially active drugs. Pralatrexate (PDX), which was approved for the treatment of relapsed or refractory peripheral T-cell lymphoma in 2009, may be one such drug. Pralatrexate exhibits a potency and pattern of activity distinct from its predecessors like methotrexate (MTX). We sought to understand the activity and mechanisms of resistance of multiple myeloma to these drugs, which could also offer potential strategies for selective use of the drug. We demonstrate that PDX and MTX both induce a significant decrease in cell viability in the low nanomolar range, with PDX exhibiting a more potent effect. We identified a series of myeloma cell lines exhibiting markedly different patterns of sensitivity to the drugs, with some lines frankly resistant, and others exquisitely sensitive. These differences were largely attributed to the basal RFC (Reduced Folate Carrier) mRNA expression levels. RFC mRNA expression correlated directly with rates of drug uptake, with the most sensitive lines exhibiting the most significant intracellular accumulation of pralatrexate. This mechanism explains the widely varying patterns of sensitivity and resistance to pralatrexate in multiple myeloma cell lines. These findings could have implications for this class of drugs and their role in the treatment of multiple myeloma. Multiple myeloma (MM) is a malignancy of immunoglobulin-secreting plasma cells, and is considered the second most common hematologic malignancy. Despite the introduction of many effective drugs over the past decades, the disease is widely considered incurable . IncomplPralatrexate  is a folate analogue rationally designed to have greater affinity (more than 10-fold greater affinity compared to methotrexate) for RFC, and has proven more potent than methotrexate (MTX) , 7. The A number of pharmacologic determinants that correlate with methotrexate resistance have been established, including DHFR, FPGS, gamma-glutamyl hydrolase (GGH) and RFC –30. The Drug screens in our laboratory indicate that some myeloma cell lines exhibit marked sensitivity to PDX and MTX, while others maintain a more resistant phenotype. Based on these findings, we sought to better understand the mechanisms of intrinsic resistance and sensitivity, in anticipation of identifying strategies to optimize the drug in myeloma and other malignant diseases.50: 1.7–9.7 nM vs MTX IC50: 22.7–40.9 nM; 50 concentrations or no sensitivity at all at the highest concentrations  cells. This effect was time and concentration dependent  and PDX did not cause a significant shift in the concentration dependent response, and did not change the ICcytokine . Similarcytokine . IL-6 di to IL-6 . It shouAs myeloma is highly dependent on cell - cell interactions and paracrine signaling provided by the bone marrow microenvironment, co-culture experiments were performed to determine the import of these variables on drug sensitivity. Co-culture with the transformed HS-5 BMSC line is thought to recapitulate aspects of the microenvironment influence and may mediate contact-mediated drug resistance in some settings. PDX-sensitive (MM.1s) and resistant (U266) myeloma cell line were plated onto a layer of HS-5 cells and co-cultured for up to 48 hours in the presence of increasing doses of PDX. Those myeloma cell lines plated without stroma served as a control. Myeloma cells were selected by staining for the expression of the lymphocytic surface markers, including CD38 (MM.1s) and CD138 (U266). Tumor cell viability was determined by the identification of the double positive CD138+/annexinV+ or CD38+/annexinV+ populations from the myeloma cell marker total subset . The kinTo identify discrete biomarkers of sensitivity and resistance to pralatrexate, we surveyed a panel of pharmacologic determinants established as possibly contributing to the phenotype. A subset of PDX-sensitive  and resistant  myeloma cells were treated with increasing concentrations of PDX and expression of DHFR protein were quantitated by western blot over a 48-hour period , 5B. BasRFC, GGH, FPGS and DHFR) were examined in eight myeloma cell lines, including four resistant lines  and four sensitive lines . While expression of each gene differed between the different cell lines (p < 0.0001). These data suggest RFC expression in myeloma tumor cells is the key biomarker of sensitivity to pralatrexate.To explore the significance of other pharmacologic and genetic determinants of resistance, we quantitated the expression levels of gene transcripts associated with antifolate sensitivity. The relative mRNA expression levels of four folate pathway genes  on MTX uptake  MTX was added to a final concentration of 1 μM and uptake was performed at a pH 7.4. Aliquots from the cell suspension were collected over time and the reaction was stopped by injection of 10 volumes of ice-cold HBS buffer. The cells were washed and digested with 500 ml 0.2 N NaOH at 65° C for 45 min. Lysates were assessed for tritium on a liquid scintillation β-counter and protein concentration was determined by BCA assay . Intracellular MTX was expressed as picomoles per milligram of protein.p values are obtained by implementing a paired Student’s t-test, all values yielding a p < 0.05 are considered significant values. Unless otherwise noted: *p < 0.05, **p < 0.01, ***p < 0.001.GraphPad Prism 5.0/6.0 was used to tabulate, chart and calculate all data and statistics. Unless noted all


Studies have suggested that type 2 diabetes (T2D) is associated with a higher incidence of breast cancer and related mortality rates. T2D postmenopausal women have an ~20% increased chance of developing breast cancer, and women with T2D and breast cancer have a 50% increase in mortality compared to breast cancer patients without diabetes. This correlation has been attributed to the general activation of insulin receptor signaling, glucose metabolism, phosphatidylinositol (PI) kinases, and growth pathways. Furthermore, the presence of breast cancer specific PI kinase and/or phosphatase mutations enhance metastatic breast cancer phenotypes. We hypothesized that each of the breast cancer subtypes may have characteristic PI phosphorylation profiles that are changed in T2D conditions. Therefore, we sought to characterize the PI phosphorylation when equilibrated in normal glycemic versus hyperglycemic serum conditions. Our results suggest that hyperglycemia leads to: 1) A reduction in PI3P and PIP3, with increased PI4P that is later converted to PIP2 at the cell surface in hormone receptor positive breast cancer; 2) a reduction in PI3P and PI4P with increased PIP3 surface expression in human epidermal growth factor receptor 2-positive (HER2+) breast cancer; and 3) an increase in di- and tri-phosphorylated PIs due to turnover of PI3P in triple negative breast cancer. This study begins to describe some of the crucial changes in PIs that play a role in T2D related breast cancer incidence and metastasis. In the United States, over 276,480 new female cases of breast cancer are expected in 2020 alone . Nearly . This suggests each cancer subtype should have specificity in their PI phosphorylation profile P2; PIP2; PIP2; PIP3) thus playing a role in their respective incidence rates and aggressiveness. Furthermore, several reports have shown a potential linkage between kinase and phosphatase mutations and enhanced/metastatic breast cancer phenotypes through changes in activity for cell adhesion proteins, delocalization of polarity lipids and proteins, and activation of invasion/migration signaling [A survey of literature suggests that phosphatidylinositol (PI) signaling is a key area of focus when analyzing the links between hyperglycemia, T2D, and the onset of breast cancer. Therefore, we suggest a possible role for PI phosphorylation as a marker for specific breast cancer phenotypes linked to T2D related onset. Cancer microenvironment growth factors increase glucose metabolism, generally thought of as being important for increased growth, migration, and proliferation rates ,18,19. Fignaling ,25,26,27Site-specific phosphorylation of PIs has been shown to regulate and stabilize the formation of local membrane heterogeneities thought to play a role in normal membrane protein signaling and cellular adhesion complex formation ,29,30,31There has been consistent effort to understand the role of mutations and changing expression levels of PI kinases and phosphatases, extending our knowledge of protein signaling. However, these enzymes have specific roles in maintaining the cellular lipidome prompting others to characterize the PIs, generally by their fatty acyl chain content. Dória et al. (2012) has shown that cancerous cell lines have higher levels of certain PIs in comparison to all other lipids . Kim et To study the effect of hyperglycemia on hormone positive breast cancer, we utilized the estrogen receptor (ER+)/progesterone receptor (PR+) MCF7 breast cancer cell line. This metastatic cell line has several subtype specific characteristic changes in PI related enzymatic activity, namely in 3′-kinase Class I PI3K and 4′-phosphatase INPP4B. This cell line expresses a mutation in the Class I PI3K catalytic subunit which leads to an increase in activity linked to unregulated growth signaling ,65,66,67p < 0.05) in the PI3P, di-phosphorylated, and PIP3 relative concentrations as a function of glucose serum concentration. There was also a statistically significant two-fold increase in the percentage of PI4P between 5.5 mM and 50 mM glucose serum concentrations (p < 0.05). In summary, these results suggest that hyperglycemia causes an overall depression of 4′-phosphatase activity in hormone responsive breast cancers.The whole cell lipidome percentage of each PI phosphorylation category in the MCF7 cell line across increasing glucose serum concentrations were obtained from the spot blots and summarized pictorially in After characterizing the whole cell PI phosphorylation lipidome, we further endeavored to characterize the hormone responsive breast cancer cell line surface. Representative images demonstrating the comparative response between normal and hyperglycemic conditions of glucose serum concentrations in MCF7s across specific PIs are shown in The cell surface expression of selected PI phosphorylation in the MCF7 cell line across increased glucose serum concentrations was obtained as fluorescent intensities from fixed cell surfaces . OverallOverall, these findings suggest that hyperglycemia led to a greater production of the whole cell PI4P while maintaining the di-phosphorylated and tri-phosphorylated PI content. Furthermore, the results suggested that hyperglycemia caused a decrease in PI3P and PIP3 and a greater production of cell surface PIP2. PI4P is generally in the Golgi apparatus and a reasonable assumption would be that it was phosphorylated by PI3K upon transportation to the cell surface thereby generating increased amounts of cell surface PIP2. However, this study also suggested that hyperglycemia leads to decreased relative amounts of PI3P, most likely due to an increase in PI4K activity.To study the effect of hyperglycemia on HER2+ breast cancer, we utilized the metastatic SKBR3 cell line. The literature has identified HER2+ subtype specific aberrancies in PI phosphatases and kinases; namely PI3K Class I and PI4K ,70. FurtWe analyzed the whole cell percentage of each PI phosphorylation in the SKBR3 cell line across increase glucose serum concentrations , while tCharacterization of the HER2+ whole cell PI phosphorylation lipidome was again followed with the characterization of the surface expression lipidome. Representative images demonstrating the comparative response between normal and hyperglycemic conditions of glucose serum concentrations in SKBR3s across specific PIs are shown in The cell surface expression of selected PI phosphorylation in the SKBR3 cell line across increased glucose serum concentrations are summarized in These findings were consistent with our hypotheses and suggested that hyperglycemia led to a greater overall production of PI3P, but a decrease in surface expression of PI3P. These findings could be due to the depressed activity of INPPB4 in this cell line. Furthermore, hyperglycemia led to a decrease in the PI4P which could be phosphorylated by hyperglycemic induced PI5K phosphorylation, followed by overactive PI3K phosphorylation to generate increasing amounts of PIP3 on the cell surface.To study the effect of hyperglycemia on TNBC, we utilized the metastatic MDA-MB-468 cell line. The literature has identified TNBC specific PI phosphatases and kinases aberrancies; namely PI3K, PTEN, and INPP4B. Through a metformin study, it was demonstrated that increasing serum glucose concentrations would increase the activity of glucose transporters and create lipid rafts in the cell surface of MDA-MB-468 cells . SignifiWe analyzed the whole cell percentage of each PI phosphorylation in the MDA-MB-468 cell line across increasing glucose serum concentrations , while tWe again followed characterization of the whole cell PI phosphorylation lipidome with the characterization of the MDA-MB-468 surface expression. Representative images demonstrating the comparative response between normal and hyperglycemic conditions of glucose serum concentrations in MDA-MB-468s across specific PIs are shown in The cell surface expression of selected PI phosphorylation across increased glucose serum concentrations are summarized in Overall, these findings suggest that TNBC in normal glucose conditions can largely be characterized by its PI3P content and that hyperglycemia leads to increased di- and tri-phosphorylated PIs due to a turnover of PI3P and decreased turnover of PIP3.p < 0.05). However, there were no differences in the PIP2 content for the three cell lines when in hyperglycemic conditions. PIP3 content at normal glycemic conditions showed that MCF7s had a statistically significant lower content than that of MDA-MB-468s . In this case, MCF7 PIP3 content at hyperglycemic conditions was also statistically different between it and SKBR3s and MDA-MB-468s.After characterizing the PI lipidome of these three types of cancerous cell lines, we further endeavored to find characteristic differences between them. We performed a 2-way ANOVA comparison of the MCF7, SKBR3, and MDA-MB-468 whole cell lipid components to find differences in their profiles. As a result, we have found that PIP2 and PIP3 show statistically significant differences in their whole cell expression profiles . While Pp < 0.005) in their profiles (p < 0.005). An additional comparison between MCF7, SKBR3, and MDA-MB-468 cells surface composition suggests that each cancer type is also statically different (p < 0.05). These results suggested that at normal glycemic conditions, hormone responsive breast cancers had a characteristically low PI3P/high PIP3 content while TNBC had a characteristically high PI3P/low PIP3 content. Hyperglycemic conditions also led to significant differences in their cell type specific profiles. Thus, we suggested that hyperglycemia led to hormone responsive breast cancer low PI3P/high PIP2/high PIP3 content and HER2+ breast cancer high PI3P/low PIP3 content.We then looked for characteristic differences between the cell surface expression of across all cell lines. The 2-way ANOVA comparison of the MCF10A, MCF7, SKBR3, and MDA-MB-468 cell surface components that PI3P, PIP2 and PIP3 all show statistically significant differences P2 best demonstrated potential as markers for changes related to hyperglycemic serum conditions. PI4P has been shown to impair Golgi-endosomal trafficking and create a pro-tumorigenic environment via interactions with receptors implicated in tumorigenesis and downstream regulation of the migratory and proliferation pathways . Though Alternatively, the trends from the HER2+ cell line demonstrated the exact opposite in regard to PI4P. Hyperglycemia led to a decrease in the PI4P, which we postulated to be due to hyperglycemic induced PI5K phosphorylation and overactivity of PI3K phosphorylation, thereby generating increasing amounts of PIP3 on the cell surface. This PI4P effect suggests that HER2+, unlike the hormone responsive breast cancer, cells undergo an increased Golgi-endosomal activity that should decrease migratory and proliferation pathways. However, PIP3 is generally localized to the basolateral membrane of polarized cells and an overabundance of this lipid causes the formation of apical membrane protrusions of over-accumulated basolateral proteins, a clear sign of a loss of polarity . These tFinally, hyperglycemia in TNBC in led to a decisive increase in PIP3 due to phosphorylation of PI3P and the associated di-phosphorylated PIs. As discussed within the context of HER2+ breast cancer, hyperglycemic related increases in PIP3 leads to overall dysregulation of the cellular polarity causing extensions suggestive of transition to a more mesenchymal cell type. This would be further exasperated with the loss of the PI3P, which is known to lead to a loss of apical membrane proteins localized within the plasma membrane. While T2D has been linked to the onset and aggressiveness of the other breast cancers in this study, T2D premenopausal breast cancer patients are more often diagnosed with hormone receptor negative, HER2-negative, TNBC . TNBC alThough the different breast cancer types may have similar proteomic level changes, i.e., the activating mutation or overexpression of PI3K across subtypes, a lipidomics approach demonstrates that each subtype can have distinct PI phosphorylation profiles that are drastically different upon the introduction of hyperglycemic conditioning. Even though it has been previously proposed that the mechanisms associated with hyperinsulinemia and inflammation act as an exclusive driver of breast cancer in T2D, our study suggests that through lipidomic remodeling hyperglycemia independently encourages tumorigenic processes. Rising glucose levels without a high insulin state were associated with the tumor’s acquisition of varying degrees of invasive and proliferative character in hormone receptor positive and TNBC cell lines. This finding elucidates the multi-modal effects of the diabetic state on breast cancer tumorigenesis. More significantly, however, our study considers an emerging role for lipid-based biomarkers to distinguish between subtypes of breast cancer; as a case in point, each cell line demonstrated a distinct lipidomics profile. Our results suggested that marker(s) for hyperglycemic related breast cancer should be: 1) reduction in PI3P and PIP3 with increased PI4P and PIP2 in HR+; 2) reduction in PI3P and PI4P with increased PIP3 in HER2+; and 3) increased PIP3 with decreased PI3P in TNBC. Human breast tissue cell lines  were gifts from Dr. Clark Wells . Newborn Calf Serum (NCS) was purchased from Peak Serum . Antibodies against PI3P (Z-P003), PI4P (Z-P004), PIP2 (Z-P034b), PIP2 (Z-P035), PIP2 (Z-P045), and PIP3 (Z-P345) were obtained from Echelon Biosciences . All other materials were purchased from Fisher Scientific .2 (v/v) as previously described [v/v NCS and a 0.5 kg/L glucose stock solution for final solutions at 5.5 mM , 10 mM (transition), and 25 and 50 mM (hyperglycemia) [Breast cancer cell lines were normally cultured in DMEM with 10% NCS at 37 °C in 5% COescribed ,119,120.lycemia) .2O, dried completely, and sealed with mounting media to slides. Imaging was done using Zeiss Axiovision (v. 48) on a Zeiss Axio Observer Z1 confocal microscope  using a 60× oil objective [n > 20 cells in each replicate.Cells were rinsed twice with phosphate buffered saline solution (PBS). The cells were then fixed on the cover slips with 4% PFA in PBS, washed 3 times with PBS, permeabilized by 0.5% saponin, and again washed with tris buffered saline (TBS). The cells were blocked with 10% goat serum in TBS at 4 °C overnight followed by incubated for 1 hr at 37 °C with antibodies previously conjugated to Alexa Fluor 350, Alexa Fluor 488, and Alexa Fluor 594  and washed 3 times with TBS . The celbjective ,118,119 4, 2 mM β-glycerol phosphate, and protease inhibitor mixture . Lysates were blotted onto the Ambersham Hybond ECL membrane and dried. The membrane was then blocked at room temperature for 30 min in phosphate buffered saline buffer containing 0.1% Triton-X and 1% (w/v) nonfat dry milk. The membrane was then incubated for an additional hour with the specific PI primary antibody, washed in phosphate buffered saline with 0.1% Triton-X, and incubated with the secondary antibody fluorescently labeled with Alexa Fluor 790 (1/1000 dilution) . The membrane was rinsed in phosphate buffered saline with 0.1% Triton-X, and the bound protein was then analyzed using a LiCOR Biosciences imaging station and Odyssey v1.2 was used to quantify the fluorescence intensity previously reported [Cells were rinsed twice with phosphate buffered saline solution and then lysed in radioimmune precipitation assay buffer (50 mM Tris pH 8.0, 2 mM EDTA, 10% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate containing 1 mM NaVOreported ,120.The whole cell percentage of each PI phosphorylation in the MCF7, SKBR3, and MDA-MB-468 cell lines across increase glucose serum concentrations were analyzed with the two-tailed Student t-test, which assumed two sample equal variance using an alpha value of 0.05. The cell surface percentage expression of each PI phosphorylation in the MCF10A, MCF7, SKBR3, MDA-MB cell lines across increased glucose serum concentrations was compared to normal glycemic conditions using the One-Way ANOVA analysis. The Q-test was used as a post-hoc test to determine significance between each of the groups, where the alpha value was 0.05. Differences in the profiles of the whole cell lipid components of MCF7, SKBR3, and the MDA-MB-468 cell lines were determined using a Two-Way ANOVA was performed where the alpha value was 0.05. All statistical analysis was done using the Microsoft Excel Data Analysis Toolpak.