Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB.Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET  protein inhibitors , and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses.Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy.Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.The online version contains supplementary material available at 10.1186/s12885-021-08782-9. MYC (MYCN) oncogene, which occurs in 20–30% of all NB tumors and nearly 50% of the high- risk cases, remains a key predictor of poor outcomes. MYCN-amplified NB tumors typically exhibit high malignancy, metastatic properties, and treatment resistance [Neuroblastoma (NB) is the most common extracranial pediatric solid tumor of neural crest origin and accounts for approximately 10% of childhood cancers and 15% of cancer-related deaths in children. Approximately 50% of NB patients are diagnosed with high-risk disease, and despite intensive multimodal therapy options , effective treatment for these patients remains elusive . Particusistance . TherefoOne of the most frequently deregulated oncogenic pathways in cancers, is the protein synthesis (translation) pathway that drives increased cell proliferation and cancer progression/resistance , 6. SimimTOR signaling is another key regulator of protein synthesis, which is frequently deregulated in cancers including NB –20. MTORWe hypothesize that combined inhibition of transcription (by BET-protein inhibition) and translation (by mTOR inhibition) will synergistically blockade global protein synthesis and proliferation in MYC-driven NB tumor cells. Using small molecule/pharmacologic approaches, we tested this hypothesis by targeting BET with JQ1 or OTX-015 and mTOR with temsirolimus, in NB cell lines.Non-MYCN-amplified NB cell lines  and MYCN-amplified NB cell lines  were purchased from American Type Culture Collection (USA). Non-MYCN-amplified NB cell line CHLA-255 was provided by Dr. Kishore Challagundla (UNMC). The identity of cell lines was confirmed by their respective cell bank using STR analyses. Cell lines were also verified for mycoplasma-free condition using the MycoSensor-PCR assay kit . Cell lines were cultured in Eagle’s Minimal Essential Medium (EMEM) or Roswell Park Memorial Institute (RPMI)-1640 media containing 10% fetal bovine serum and 1% penicillin-streptomycin . Experiments were performed under 8–10 passages for each cell line. Small molecule inhibitors  and cisplatin (a chemotherapeutic drug) were purchased from Sellekchem LLC (USA).Effects of inhibitors on NB Cell growth/viability were assessed using the MTT assay as previously described , 23.Effects of inhibitors on NB spheres were performed using the neurosphere/sphere assay as previously described .Analysis of cell cycle distribution in NB cells was performed using a propidium iodide (PI staining) flow cytometry kit  according to manufacturer’s instructions. Apoptosis in NB cells was assessed using an Annexin-V flow-cytometry assay kit  following the manufacturer’s instructions. The flow cytometry analysis of Annexin-V/PI stained cells was determined using the FACSCalibur cell sorter system .4 cells/well) for 24 h. After treatment, culture media was replaced with fresh media containing O-propargyl-puromycin (OPP) and incubated for 30 min at 37 °C to be incorporated in translating polypeptide chains. Cells were then fluorescently stained with 5-FAM-Azide. The detection of fluorescent-labelled OPP was performed using the Protein Synthesis Assay Kit , according to the manufacturer’s instructions.NB cells were treated with inhibitors alone or in combination in 96-well plates  was analyzed using two-tailed Student’s t-test or analysis of variance (ANOVA) and p-values > 0.05 considered significant. GraphPad Prism-V6 software was used to determine IC50 values and dose-response curves of inhibitors in NB cell lines. The Chou-Talalay combination index (CI) method was used to analyze synergy/interaction between inhibitors by using CalcuSyn software . CI < 0.9 indicates synergism, 0.9–1.1 additivity and > 1.1 antagonism.Each experiment was repeated at last an additional three times and the mean ± standard error values calculated. Statistical significance  to target MYCN transcription (BET proteins) and mTOR signaling, respectively, , 25 in NWe next tested the efficacy of combined JQ1/TEM to explore potential for synergistic growth inhibition on NB cells. NB cell lines, including three MYCN-amplified and two non-MYCN-amplified NB cell lines, were treated with increasing concentrations of JQ1 and TEM alone or in combination for 72 h. As shown in Fig. To determine combined effects of JQ1 and TEM on cell cycle and apoptosis, two highly MYCN-amplified NB SK-N-BE2, SK-N-DZ) cell lines were treated with a suboptimal concentration of each inhibitor alone or in combination and subjected to cell cycle and apoptosis analyses using propidium-iodide and Annexin-V staining, respectively, followed by flow cytometry. The cell cycle analyses in both MYCN-amplified cell lines revealed that JQ1 and TEM alone slightly caused cell cycle arrest in G1 phase, while co-treatment with JQ1 and TEM drastically arrested the cells in G1 phase . Co-treatment with JQ1 and TEM further downregulated the expression of the above mentioned mTOR signaling components, as well as MYCN expression, compared with single agent treatments. These data suggest that concurrent inhibition of MYCN transcription and mTOR signaling cooperatively suppresses the protein synthesis pathway, justifying why this combined inhibition exerts the greatest antitumor effects in MYCN-amplified NB.To establish the molecular mechanism(s) associated with JQ1/TEM anti-NB activity, we examined the expression/activation of key components of MYCN and mTOR signaling pathways by western blotting in SK-N-BE2 and SK-N-DZ cell lines. Single agent treatments of MYCN-amplified cells with JQ1 or TEM significantly suppressed expression of MYCN and BET protein BRD4 and downregulated the levels of phosphorylated/activated signaling proteins  of the mTOR (translation) pathway Fig. a and b. Because of the key role of MYCN/mTOR signaling in controlling global protein synthesis, we further tested whether the inhibition of MYCN/mTOR represented a global blockage of protein synthesis. To this end, we performed protein synthesis assay using a robust chemical method based on a cell permeable analogue of puromycin, O-Propargyl-puromycin (OPP), in SK-N-BE2 and SK-N-DZ cell lines treated with JQ1 and TEM alone or in combination. Incorporation of OPP to nascent polypeptide chains can be labeled via copper catalyzed click-chemistry using 5-FAMAzide in order to quantify total protein synthesis. In this assay, we used cycloheximide as the positive control for protein synthesis inhibition. This fluorescence-based assay displayed high protein synthesis in control solvent (DMSO)-treated cells and strong inhibition of protein synthesis when blocked with cycloheximide Fig. c. AlthouWe next investigated the effect of JQ1 and TEM, alone or combined, in a neurosphere model of MYCN-amplified NB cells. Figure Given the limited success of current therapies, we next sought to determine whether JQ1 or TEM could enhance the anti-NB efficacy of chemotherapy by sensitizing NB cells. Cisplatin is one of the most common chemotherapeutic drugs in the treatment of NB patients. To evaluate the enhanced efficacy of inhibitors on cisplatin-mediated NB cytotoxicity, we treated NB  cell lines with cisplatin and either JQ1, TEM, or the combination cisplatin alone or in combination, in stepwise doses. At 72 h we determined cell growth using the MTT assay. Results shown in Fig. In previous experiments, the rationale for using JQ1 was its advantages over other BET inhibitors in preclinical cancer studies. Preclinical studies with JQ1 offered a great opportunity to better understand the biology of BET proteins and validate these proteins as the anti-cancer targets , 24. In Despite the availability of intensive multimodal therapy, the prognosis for patients with MYCN-amplified NB remains extremely poor . Since MBET protein inhibitors exert biological effects by competitively binding to the acetyl-lysine pockets and displacing BET proteins from chromatin, leading to the repression of oncogene transcription. BET protein inhibitors have been shown to target MYC/MYCN transcription in several cancers, including NB, demonstrating their potential as preclinical anticancer agents –17. In cIn addition, our data provide insights into the molecular mechanism(s) underlying the antitumor efficacy of JQ1/TEM we observed in NB. We show that co-treatment with JQ1 and TEM synergistically reduce the expression of key component proteins of the activated mTOR translation pathway, along with suppression of MYCN and BRD4 (a key member of BET family proteins) protein expression in NB cells. We further show that this co-inhibition of MYCN/mTOR by JQ1/TEM results in significant blockade of global protein synthesis in NB cells. These observations in NB are consistent with our previous study in medulloblastoma where we have shown similar combined activity of JQ1/TEM on MYC/mTOR targets, leading to inhibition of global protein synthesis. Overall, these findings suggest a synergistic and cooperative activity of JQ1/TEM on MYCN and mTOR pathways in aggressive NB.While our data show that although individual or concomitant inhibition of BET/mTOR has greater antitumor efficacies against MYCN-amplified NB cells, the combination also efficiently inhibits cell growth and induces apoptosis in non-MYCN-amplified NB cells, pointing to a broader relevance of our combined approach in NB. Because non-MYCN-amplified NB cell lines, including SK-N-AS, express MYC  which isIn NB and other cancers, MYCN and mTOR signaling have been shown to play key roles in cancer stem cells and contribute to relapse and drug-resistance. NB cells express neural stem cell markers such as CD133 and Nestin and have the ability to form neurospheres –33. Our Since overexpression or amplification of MYC/MYCN and activation of mTOR signaling are often associated with chemoresistance in many cancers including NB –38, we aIn clinical trials studies, TEM alone or in combination with other clinical agents, such as chemotherapeutic drugs temzilomide and irrinotican, has shown no significant benefit for relapsed NB patients , 43. ThiOur study demonstrates that targeting dysregulated protein synthesis pathway by pharmacologic dual-inhibition of MYCN transcription (by BET protein inhibition) and mTOR signaling has significant preclinical anti-NB efficacies in inducing cell growth inhibition, cell cycle arrest and apoptosis in vitro. Combination of JQ1 or OTX with TEM synergistically inhibited global protein synthesis by downregulating the key components and downstream targets of MYCN/mTOR signaling. Thus, this study is the first to demonstrate synergy in the combined inhibition of BET protein and mTOR signaling in NB. Our study also revealed that inhibition of MYCN/mTOR not only inhibits cell growth and survival in NB cells, but also chemosensitizes NB cells. Beyond NB, this therapeutic approach can be of broader relevance for therapy of MYC/MYCN-addicted cancers, as we have previously shown a synergistic antitumor efficacy of BET/mTOR inhibitors in MYC-driven medulloblastoma . While fAdditional file 1.


Wolbachia bacteria, carried by many arthropods, as well as some nematodes, interact in many different ways with their hosts. These include male killing, reproductive incompatibility, nutritional supplementation and suppression or enhancement of the transmission of diseases such as dengue and malaria. Consequently, Wolbachia have an important role to play in novel strategies to control human and livestock diseases and their vectors. Similarly, cell lines derived from insect hosts of Wolbachia constitute valuable research tools in this field. During the generation of novel cell lines from mosquito and sand fly vectors, we isolated two strains of Wolbachia and demonstrated their infectivity for cells from a range of other insects and ticks. These new insect cell lines and Wolbachia strains will aid in the fight against mosquitoes, sand flies and, potentially, ticks and the diseases that these arthropods transmit to humans and their domestic animals.Diverse strains of Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.Endosymbiotic intracellular bacteria of the genus  Wolbachia are endosymbionts of a wide range of invertebrate taxa, including many species of arthropod and parasitic nematodes. The nature of the relationships between Wolbachia and their hosts cover a wide spectrum, from reproductive manipulations such as cytoplasmic incompatibility (CI) and male killing to nutritional supplementation [Wolbachia transinfections in mosquitoes to reduce vector competence (population replacement) [Wolbachia are currently classified on the basis of gene sequence similarity into ~18 “supergroups” [Obligate intracellular bacteria of the genus entation . CI-induacement) . Wolbachrgroups” .Wolbachia can be cultured in vitro in arthropod cells, facilitating a variety of studies on bacterial–host cell interactions, antibiotic susceptibility and influence on the replication of coinfecting arboviruses or insect-specific viruses [Drosophila melanogaster cell line [Wolbachia were cultured directly from the host during primary cell culture initiation [Wolbachia-infected primary cultures [ viruses ,14,15,16 viruses ,21,22,23 viruses , and a nell line . While tWolbachia with contrasting characteristics in naturally-infected host insect cell cultures during cell line generation. A strain of wPip was isolated in both of two new cell lines derived from Culex pipiens complex mosquito embryos and larvae, respectively, and a strain of wPap was isolated in a new larva-derived Phlebotomus papatasi sand fly cell line. We tested the infectivity of both Wolbachia strains for a panel of heterologous insect and tick cell lines, including novel cell lines derived from the sand fly Lutzomyia longipalpis sensu lato.Here, we report the isolation of two strains of C. pipiens complex mosquitoes  originally supplied by the Walter Reed Army Institute of Research for distribution by BEI Resources, NIAID, NIH, Manassas, VA, USA) were provided by the Liverpool School of Tropical Medicine. Eggs from laboratory colonies of the Jacobina  and Sobral  strains of the sand fly L. longipalpis species complex, originating from Brazil [Eggs from a laboratory colony, established in 2011, of rrey, UK ) were orm Brazil ,28, wereC. pipiens eggs . The culture medium comprised L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth (TPB), 20% foetal bovine serum (FBS), 2mM L-glutamine (L-glut), 100 units/ mL penicillin, 100 µg/ mL streptomycin (pen/strep) and 50 µg/ mL amphotericin B . The sealed culture tube was incubated in ambient air in a dry incubator at 28 °C. The medium was changed weekly by the removal and replacement of 1/2–2/3 of the volume; amphotericin B was omitted after the first medium change, and the total volume was gradually increased to 2 mL.ens eggs a laid wiC. pipiens egg batch was processed in the same way, but not all the eggs were crushed, and larvae emerged within the first 24 h in vitro. On the day after initiation, 7 larvae were removed from the culture, chopped into several pieces in a drop of L-15 using a scalpel and returned to the tube with an additional 0.5 mL of medium. On the following day, the contents of the tube were centrifuged at 200× g for 5 min, the supernate was saved and the pellet resuspended in 0.5 mL of trypsin solution (500 µg/ mL in PBS). The larval pieces were chopped into smaller fragments and incubated with an additional 0.5 mL trypsin solution for 10 min at 37 °C; 0.5 mL of the saved supernate was added, and the tissue suspension was centrifuged at 400× g for 5 min. The tissue pellet was resuspended in the remaining 0.8 mL saved medium and returned to the tube, which was incubated at an angle of ~10°. Thereafter, the medium was changed weekly, gradually increasing the total volume to 2 mL.A second P. papatasi eggs , 20% FBS, L-glut and pen/strep (H-Lac) was added, the tissue suspension was centrifuged at 400× g for 5 min and the resultant pellet was resuspended in 1 ml H-Lac and placed in a new culture tube at 28 °C. Thereafter, this culture was maintained in a 1:1 mixture of L-15B and H-Lac media.asi eggs a laid ap2 culture flasks. Cells were cryopreserved in medium with 20% FBS and 10% DMSO and resuscitated as described previously for Culicoides nubeculosus cell lines [C. pipiens and P. papatasi cultures and young cell lines were periodically examined by preparation of Giemsa-stained cytocentrifuge smears as described previously [Mycoplasma in all young cell lines was confirmed using two commercial tests, the Mycoalert Mycoplasma Detection Kit  and the PCR Mycoplasma Test Kit , following the manufacturers’ instructions.The primary cultures were monitored by weekly inverted microscope examination. When clumps of proliferating cells appeared, the cultures were reseeded by vigorous pipetting to encourage cell growth. When cells were clearly growing, and the pH of the culture medium became acidic (orange or yellow colour) indicating a high level of active metabolism, an equal volume of fresh medium was added to the culture, the cells and tissue clumps were resuspended by vigorous pipetting and half of the resultant suspension was transferred to a new flat-sided culture tube. After several subcultures, cells were passaged into sealed, 25-cmll lines . Primaryeviously . AbsenceC. pipiens and P. papatasi cells using a DNeasy Blood and Tissue kit , following the manufacturer’s instructions for cultured cells. To confirm the species origin, a 696-bp fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene was amplified by PCR (forward primer: 5′-GGATTTGGAAATTGATTAGTTCCTT-3′ and reverse primer: 5′-AAAAATTTTAATTCCAGTTGGAACAGC-3′) from the mosquito cells as described previously [DNA was extracted from resuspended eviously , and an eviously .C. pipiens cells, conventional PCR targeting the CQ11 microsatellite locus was carried out, based on a method and the primers described previously [C. p. pipiens, C. p. molestus and hybrid mosquitoes as the positive controls. The PCR reactions consisted of 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.16-µg/µL bovine serum albumin, 1 U Platinum™ Taq DNA polymerase , 0.24-µM forward primer CQ11F, 0.32 µM reverse primer PipCQ11R, 0.14 µM reverse primer MolCQ11R and 3 µL DNA, made up to 25 µL with nuclease free water. The thermal cycling of the reaction consisted of 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min and a final elongation step of 72 °C for 10 min. The PCR products were visualised by agarose gel electrophoresis.To determine the ecotype of the eviously , on DNA Wolbachia, DNA extracted from cultured cells was subjected to conventional PCRs amplifying fragments of the pan-bacterial 16S rRNA gene and the Wolbachia wsp gene following the respective published protocols [To detect and identify rotocols ,35. The rotocols .Wolbachia sequences was carried out using sequences from the 16S rRNA and wsp genes. Initial multiple alignments for the 16S rRNA and wsp genes were constructed using MAFFT v7.480 [Phylogenetic analysis of the T v7.480  with theT v7.480  to selecT v7.480  was run T v7.480 . The resT v7.480 .Lutzomyia longipalpis cell line LLE/LULS45 was derived from embryos of the Jacobina strain following the same procedure for processing eggs as described above for P. papatasi, which yielded a culture of viable embryonic cells and tissues that gave rise to the cell line. The L. longipalpis cell line LLL/LULS52 was derived from eight larvae of the Sobral strain, 13 days after culture initiation, following the same procedure as described above for P. papatasi larvae derived from processed and cultured unhatched eggs. Establishment of the cell lines LLE/LULS45 and LLL/LULS52 was achieved with successful cryopreservation at 12 (passage 6) and 11 (passage 4) months, respectively. The origins of both cell lines were confirmed as the L. longipalpis species complex by sequencing cox1 PCR products amplified using the primers LCO1490 and HCO2198, as described previously [Lutzomyia cruzi  and 99.00% identical to a sequence from L. longipalpis , both from Mato Grosso State, Brazil [L. longipalpis species complex, Lutzomyia alencari, and 99.08% identical to a sequence from L. longipalpis , both from Espirito Santo, Brazil [Mycoplasma using two commercial kits, as described in The established tick cell lines BME/CTVM23 , ISE6 4 and IRE/eviously . A 601-bWolbachia-infected C. pipiens and P. papatasi cultures were centrifuged at 2000× g for 5 min to remove any intact cells; the absence of contaminating host cells was confirmed by examination of Giemsa-stained cytocentrifuge smears, and 0.3–0.5 mL aliquots were added to cultures of heterologous tick and insect cell lines (Wolbachia was confirmed by DNA extraction and subjecting the DNA to quantitative PCR (qPCR) targeting the Wolbachia 16S rRNA gene as described previously [Supernates removed from ll lines  in sealeeviously , except eviously .wPap on growth rate. Uninfected cells, and cells infected with wPap at passage 1, were seeded in 2 mL of medium in three replicate flat-sided tubes at a density of 1.41 × 106 cells/mL and incubated at 28 °C. The medium was changed on days 7 and 13 by removal and replacement of 1.5 mL. The cells in each tube were resuspended and counted using a haemocytometer on days 6, 10 and 14. Two-way ANOVA comparing the cell counts was performed on GraphPad Prism version 9.2.0 for Windows  as a main effects-only model.In a preliminary experiment, the tick cell line IRE/CTVM20, selected because it comprises cells easily resuspended by pipetting, was used to assess the effect of infection with Wolbachia-infected C. pipiens and P. papatasi cultures were treated weekly with tetracycline hydrochloride  added to the culture supernate following a medium change to give a final concentration of 5 µg/mL, over a period of at least 12 weeks. Absence of Wolbachia was determined by extracting DNA as described above from treated cells at least 1 week after the cessation of tetracycline treatment, and by subjecting the DNA to qPCR targeting the Wolbachia 16S rRNA gene as described above.C. pipiens eggs. Patches of twitching tissues resembling muscle fibres were visible after 8 days in culture, and small patches of growing round cells were seen after three weeks. These increased rapidly in number, and the first passage from the primary culture was carried out at 9 weeks. The early subcultures comprised attached round cells of varying size and granularity, sometimes forming clumps and accompanied by floating multicellular vesicles ; a further ecotype analysis revealed that the cell line contained a mixture of C. p. pipiens and C. p. molestus cells and possibly cells derived from hybrids of the two ecotypes  following the cessation of tetracycline treatment were negative for Wolbachia. Wolbachia-free CPE/LULS50 cells were cryopreserved at passage 12, and, at the time of writing, the cell line has reached passage 25. CPE/LULS50 comprises predominantly attached, round and spindle-shaped cells of varying sizes and granularities that form occasional three-dimensional clumps  to Wolbachia endosymbionts of the spittlebug Cosmoscarta heros, the weevil Curculio okumai and the ant Anoplolepis gracilipes . As with the Wolbachia detected in C. pipiens cells, examination of the sequence chromatogram indicated good sequencing quality and suggested absence of any other contaminating bacteria; screening of the PPL/LULS49 cells for Mycoplasma gave negative results. A Wolbachia-specific PCR targeting the wsp gene yielded a 316-bp sequence, confirming that the PPL/LULS49 cells harboured wPap  after the cessation of the tetracycline treatment and found to be free of Wolbachia. The original line of Wolbachia-infected cells, henceforward redesignated as PPL/LULS49/wPap, was maintained in parallel. At the time of writing, PPL/LULS49/wPap had reached passage 23, while the Wolbachia-free subline of PPL/LULS49 had reached passage 15; both sublines comprise sheets and clumps of attached cells composed of a mixture of small, rounded cells, some fibroblast-like and epithelial-like cells and floating, multicellular vesicles.When the PPL/LULS49 cells were nearly a year old, it became evident that bacteria were growing in the cultures, manifest as very small, faintly stained, rod-shaped and pleomorphic organisms both intra- and extracellular c. Pan-ba237882.1 ). In conwsp gene sequences from the novel wPip and wPap isolates were compared with published sequences to determine their relationship with other Wolbachia strains. A concatenated tree prepared using sequences from both genes placed our wPip strain in supergroup B, together with other Wolbachia strains isolated from C. pipiens complex mosquitoes, and close to strains from Aedes and Anopheles spp. mosquitoes and Drosophila spp. fruit flies , MZ577351(PPL/LULS49 18S rRNA), MZ577353 (LLE/LULS45 cox1), MZ577352 (LLL/LULS52 cox1), MZ577347 (wPip 16S rRNA), MZ577346 (wPip wsp), MZ577348 (wPap 16S rRNA) and MZ577354 (wPap wsp).The novel gene sequences generated in this study were deposited in GenBank under the following accession numbers: MZ577349  and nine (C6/36) weeks later  but prolonged survival of the culture as a whole. The infection was slow to develop in C6/36 cells, but the infected cells could be split 1:1 successfully and were taken through eight passages over a 9.5-month period before being cryopreserved.Cell-free CPE/LULS50 supernate containing and ISE6 . All recks later a,b. The wPip bacteria was inoculated into the tick cell lines BME/CTVM23 and IRE/CTVM20 and the insect cell lines CNE/LULS44, LLE/LULS45 and LLL/LULS52 (Wolbachia between two (tick cells) and three (insect cells) weeks later; development of CPE ensued rapidly in the BME/CTVM23 cells  than uninfected cells over a 14-day period  of the same colony than CPE/LULS50 (n = ~100), was found to comprise only C. p. molestus cells. There have been several previous reports of the misidentification of arthropod cell lines, in most cases those established prior to the general availability of molecular screening techniques. The RML-12 cell line, originally described as Aedes aegypti [Wolbachia strains [Ae. albopictus [Dermacentor variabilis [Rhipicephalus sanguineus ticks [Orconectes limosus [As part of the process of establishing the cell lines, we used order-specific PCR assays and amplicon sequencing to confirm the species origin of the cells, especially important for studies involving arthropod–symbiont relationships. For all the novel cell lines reported here, the species origin was confirmed, and the CPE/LULS50 cell line was found to contain cells derived from both S50 n = ~0, was fo strains , was latbopictus . Similarriabilis , was latus ticks . Most al limosus , was lat limosus .C. pipiens complex mosquitoes and P. papatasi sand flies, we isolated two strains of Wolbachia with contrasting characteristics. Although the wPip strain was initially tolerated by both C. pipiens primary cultures and early subcultures derived from them, it began to cause CPE after 8 months in vitro, and it was necessary to treat all surviving cultures  with tetracycline to save the cell lines. In contrast, wPap did not become manifest until the P. papatasi cells had grown for a year and was tolerated with little or no CPE to at least passage 22 (27 months post-initiation). Tetracycline treatment resulted in a Wolbachia-free subline, currently maintained alongside the original PPL/LULS49/wPap cell line. This difference in pathogenicity between the two Wolbachia strains was also seen in heterologous cell lines, with wPip causing rapid CPE in tick, midge and sand fly cell lines, while CPE caused by wPap took longer to develop in two of the tick cell lines and was negligible in the third tick cell line and both sand fly cell lines, allowing the infected cells to be subcultured.Although the main aim of setting up primary arthropod cell cultures has usually been to generate cell lines, the coincidental isolation of arthropod-borne bacteria has often been an unexpected but beneficial by-product. In addition to generating novel cell lines from wPip was previously isolated into an Ae. albopictus cell line from C. pipiens eggs of unspecified geographical origin [wPip in C. pipiens cell cultures. The C. pipiens complex is infected ubiquitously with wPip worldwide, and the symbiont is divided into five clades (wPipI–wPipV), generating the most complex pattern of CI recognised in nature to date [wPip strain isolated in the present study belongs. Interestingly, a recent report demonstrated that wPip is unusual in failing to impede dengue virus dissemination when transinfected into Ae. aegypti, providing opportunities to unravel the mechanisms of viral inhibition [wPap, which is a common symbiont of wild P. papatasi populations [wPip and wPap in multiple different arthropod cell lines will facilitate the genome sequencing and phenotypic characterisation of these Wolbachia strains. The ability to propagate multiple different strains of Wolbachia in the same cell line will also allow the comparison of their properties, as previously proposed [wPip killed its parental host C. pipiens cells, whereas wPap was tolerated by its parental host P. papatasi cells, we found differences in morphology, growth rate and habit and pathogenicity between wPip (supergroup B) and wPap (supergroup A) in the heterologous tick and insect cell lines. Intriguingly, in a preliminary experiment, infection with wPap appeared to confer an advantage on IRE/CTVM20 tick cells, enabling them to grow to a higher density than uninfected cells. Further characterisation and comparison with other Wolbachia strains from the same and different supergroups grown in the cell lines used in the present study and other cell lines derived from the same genera, such as the Culicoides sonorensis KC cell line [Ixodes ricinus IRE11 cell line [Wolbachia genomes and phenotypes and their effects on host cells. Notably, the wStri strain, originally derived from the planthopper Laodelphax striatellus and maintained routinely in the Ae. albopictus cell line AeAl-2, was also found to be pathogenic to tick (ISE6) cells [Wolbachia strain was recently introduced stably into a heterologous planthopper, the destructive rice pest Nilaparvata lugens, in which the wStri caused both CI and inhibition of rice ragged stunt virus [While a strain of l origin , the pre to date . Furtherhibition . The preulations ,75,76 anulations , but indulations ,79. The proposed . As wellell line  and the ell line , will he6) cells . This Wont virus .Wolbachia, especially if the cultures are monitored from an early stage and the presence of bacteria is expected. Equally, proliferation of unanticipated and undetected Wolbachia could result in loss of primary cultures or young cell lines, as would probably have happened with CPE/LULS50 and CPL/LULS56 if the tetracycline treatment had not been initiated. Persistent infections with Wolbachia in insect cell lines may be unstable, as reported for the Ae. albopictus line Aa23 [Wolbachia persists in PPL/LULS49/wPap cells at higher passage levels. As observed with insect cells, the presence, not always expected, of bacterial symbionts such as Rickettsia spp. and Spiroplasma spp. has resulted in either the loss of primary tick cell cultures but concomitant isolation of the microorganisms into heterologous tick cell lines [Confirming previous reports ,21,22, oine Aa23 ; it willll lines ,52, the ll lines  or a perll lines .Wolbachia [Wolbachia strains of differing insect origins [wPip grew in insect cell lines derived from the lepidopteran S. frugiperda, the mosquito Ae. albopictus, the biting midge C. nubeculosus and the sand fly L. longipalpis. Other strains of Wolbachia have previously been propagated in the Sf9 and C6/36 cell lines used in the present study [L. longipalpis and Culicoides sp. cell lines [wPap did not detectably infect the C. nubeculosus cell line CNE/LULS44, although it grew vigorously in all the other insect and tick cell lines tested. In all cases, infection with Wolbachia was achieved by simply adding a cell-free supernate to the recipient heterologous cultures, confirming previous results [Although ticks are not currently considered to be natural hosts of olbachia ,86,87, o origins . As wellnt study ,19 and ill lines ,88. Inte results , without results ,80,88.Wolbachia strains reported in the present study will facilitate research not only into interactions between the parent arthropods and the bacteria at the cellular level but, also, into human and veterinary pathogens carried by C. pipiens, P. papatasi and L. longipalpis and the influence of Wolbachia on their transmission [The new insect cell lines and cultured smission ,90.


Seven out of nine cell lines were susceptible to BLV infection as determined by at least one positive liquid-phase PCR result in the 3-month culture period. iSLK and MCF7 cell lines were able to produce a stable infection throughout the 3-month period, with both cytoplasmic and/or nuclear BLV-DNA visualized by IS-PCR. Our results support experimental evidence of BLV infection in humans by demonstrating the susceptibility of human cells to BLV infection, supporting the hypothesis of a natural transmission from cattle to humans.Evidence of the presence of bovine leukemia virus (BLV) in human beings and its association with breast cancer has been published in the literature, proposing it as a zoonotic infection. However, not enough evidence exists about transmission pathways nor biological mechanisms in human beings. This study was aimed at gathering experimental evidence about susceptibility of human cell lines to BLV infection. Malignant and non-malignant human cell lines were co-cultured with BLV-infected FLK cells using a cell-to-cell model of infection. Infected human cell lines were harvested and cultured for 3 to 6 months to determine stability of infection. BLV detection was performed through liquid-phase PCR and visualized through  Viral agents have been linked to approximately 20% of human cancer types, and some causative relationships have been established . The mosBovine leukemia virus is an exogenous retrovirus, grouped with human T-cell leukemia virus (HTLV) in the deltaretrovirus genus. These viruses cause leukemia/lymphoma both in cattle and humans, respectively . Cancer Previous research has reported the presence of BLV biomarkers in humans, such as gene segments, viral proteins, and antibodies against BLV, which provides clear evidence of the presence of the virus in this host . Studiesin vitro infection has been evaluated in cell lines of different origins/sources including other bovine tissues, and some other animal species  cells, constitutively infected with BLV, served as a repository of the virus. Minimal Essential Medium (MEM) was used for cell passage every 3–4 days after 80% confluency. Human cell lines used and growing conditions are described in To recreate a plausible scenario of infection of BLV, TransWells (Costar Corning Inc.) with 0.4-μm polyester pore membrane were inserted above a 12-well plate to co-culture BLV-infected cells with human cell lines. The TransWell pores do not allow whole cells to pass through the pores but do allow extensions of cells  to make contact below the insert, allowing a cell-to-cell transmission of the virus. Before infections, cell lines were verified to be negative to BLV .5 cells/ml until 70–80% confluency was reached in their respective media .cytochrome C oxidase housekeeping gene was used to verify that FLK cells did not trespass the membrane of the TransWells and human cell lines were not contaminated with FLK.After 48 h post-infection (hpi) with the TransWells, an aliquot of the cells in the bottom plate was recovered to perform DNA extraction with the DNeasy Kit from QIAgen following the manufacturer’s instructions. Total DNA recovered was stored at −20°C until further use. Human GAPDH housekeeping gene was used as a validation control of the DNA extraction. Sheep Viral genome was detected by PCR (see next section). Successful infected human cell lines were considered those in which BLV-DNA was detected in the initial DNA extraction post-infection and were maintained in cell culture with its respective conditions . A folloLTR, gag, pol, env, and tax were tested by liquid-phase nested PCR (nPCR) with GoTaq Promega to verify the presence of the complete genome of the virus at time 0 post-infection. Primers used for the viral detection and PCR conditions were used from previous studies  was performed after 16 weeks of infection to confirm the presence of the virus inside the cells using methodology adapted from For those cell lines that showed a stable infection of BLV during the total time of the follow-up, an 2, 0.4 mmol/L dNTPs, 1 μmol/L primers , 0.06% bovine serum albumin, 8 μmol/L digoxigenin-11-dUTP (dig) , and 0.053 U/μl of HotStart Amplitaq Gold DNA Polymerase . IS-PCR was directed to a segment of the tax region of the BLV genome , with an expected product of 373 bp. PCR mix was placed into the chambers of the slides and was sealed with the plastic cover of BioRad. Slides were placed into an IS-PCR machine  for amplification. Thermal profile was used as previously described . Unlike retroviruses such as HIV, translocation of the viral genome into the nucleus is weak and mainly relies on cell division processes   are protin situ PCR could be associated with higher amounts of viral genome. The localization of BLV in the nucleus in iSLK versus the cytoplasm for MCF-7 based on the results from IS-PCR could be interpreted as differences in the integration profiles of the BLV in the form of provirus. It could be associated with more copies integrated in iSLK in contrast with MCF-7; however, in this study, we did not look for the integration sites of the virus, as it was not the main objective of the article. Finally, stable and persistent infections could also be related with the cell cycles of the different cell lines, in which initial cells that were infected with BLV could die before its mitotic cycle or could be displaced by uninfected cells replacing the cell populations.The cell lines in which BLV genome was detected for a longer term support the hypothesis of a stable infection in MCF-7 (breast), iSLK (kidney), and DLD-I (colon) cell lines. Even if in our study we did not measure active viral production, nor functionality of the viral genes, the identification of these cell lines with the presence of viral DNA after a long-term infection suggests that a stable infection in the human body may occur as well. Although the present study does not suggest a mechanism as to why certain cell lines were unable to maintain a stable BLV infection, we hypothesize that it could be related with the process of integration in the host genome. Considering that BLV does not have a specific profile for integration within the host genome, and that it occurs randomly in different regions, with different patterns and in several chromosomes in cattle without a specific association with disease progression , this alC, which makes it unlikely that positive nested PCR results were due to FLK cell-line contamination. Furthermore, FLK control DNA and cell line were handled in different areas, separate from the human cell culture samples to avoid cross-contamination while processing and to ensure the positive results obtained from DNA within the human cell culture samples. Two different detection techniques were used as well to ensure reliability of our results. IS-PCR visualization showed presence of the viral genome inside the cells, which could be both in the cytoplasm of the cells or in the nucleus. Even if liquid-phase PCR is unable to provide any information as to the location of the source of the DNA, it showed the presence of viral genome after a total DNA extraction, indicating that early steps of the viral cycle were performed.One of the strengths of this study were the steps taken to ensure that the results obtained were free from cross-contamination. All DNA samples extracted from the human cell cultures were negative in PCR amplification of sheep cytochrome tax region, which codes an auxiliary protein with oncogenic potential, was used for IS-PCR. Those regions were chosen because they are the most highly conserved regions of BLV genome  promoter region of BLV was used for liquid-phase PCR, and V genome . It is tV genome . DetectiV genome . In furtA limitation of our current study is that the positive results from the liquid-phase PCR do not provide information as to the integration status of BLV with respect to the host genome. While these results show that viral retro-transcription occurred , we coulin vitro experimental evidence consistent with the hypothesis of BLV as an exogenous etiological agent of human breast cancer, obtained through a zoonotic infection (Of special interest, the mammary epithelial cell line MCF-7 was both susceptible to BLV infection and able to maintain a stable infection over the 3-month culture period. Both conditions seem to be important in the progression of the disease in its host . This renfection . NeverthBovine leukemia virus was able to infect human cell lines through a cell-to-cell model. BLV biomarkers were identified in the cell lines as evidence of a stable infection. iSLK and MCF-7 cell lines were those that showed positive for a longer term, even after freezing and thawing. Our results support the hypothesis of BLV being a zoonotic infection and are the basis for further studies for a better understanding of BLV mechanisms in human beings. Stable infected cell lines will be useful for future studies to be performed under controlled conditions at the laboratory level.The original contributions presented in the study are included in the article/NO-G and GB: conceptualization and visualization. NO-G, KT, SB, and HS: methodology. NO-G: software. GB: validation, resources, project administration, and funding acquisition. NO-G, SB, HS, and GB: formal analysis. NO-G, MG, and GB: investigation. NO-G and SB: writing—original draft preparation. NO-G, MG, and GB: writing—review and editing. MG and GB: supervision. All authors have read and agreed to the published version of the article.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malignant glioma cell line models are integral to pre-clinical testing of novel potential therapies. Accurate prediction of likely efficacy in the clinic requires that these models are reliable and consistent. We assessed this by examining the reporting of experimental conditions and sensitivity to temozolomide in glioma cells lines.50).We searched Medline and Embase (Jan 1994-Jan 2021) for studies evaluating the effect of temozolomide monotherapy on cell viability of at least one malignant glioma cell line. Key data items included type of cell lines, temozolomide exposure duration in hours (hr), and cell viability measure , 223.1 μM (IQR 92.0–590.1 μM) and 230.0 μM (IQR 34.1–650.0 μM), respectively. The median IC50 at 72 h for patient-derived cell lines was 220 μM (IQR 81.1–800.0 μM).We included 212 studies from 2789 non-duplicate records that reported 248 distinct cell lines. The commonest cell line was U87 (60.4%). Only 10.4% studies used a patient-derived cell line. The proportion of studies not reporting each experimental condition ranged from 8.0–27.4%, including base medium (8.0%), serum supplementation (9.9%) and number of replicates (27.4%). In studies reporting ICTemozolomide sensitivity reported in comparable studies was not consistent between or within malignant glioma cell lines. Drug discovery science performed on these models cannot reliably inform clinical translation. A consensus model of reporting can maximise reproducibility and consistency among in vitro studies.The online version contains supplementary material available at 10.1186/s12885-021-08972-5. Malignant glioma is the most common primary cancer of the central nervous system . TreatmeCancer drug discovery utilises pre-clinical models to predict the likely effect of a novel compound on cancer tissue. In vitro cell cultures are readily available from repositories  and in sTransparency in clinical research has received increasing attention in the past two decades. There has been an expansion of reporting guidelines for clinical studies facilitated by the Enhancing the Quality and Transparency Of health Research (EQUATOR) network . Most guThere is no suitable repository for systematic review of in vitro studies in which our protocol could be registered. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.We included studies published after 1993 that evaluated the effect of temozolomide monotherapy on cell viability of at least one malignant glioma cell line. The evaluation of temozolomide could be a control experiment within a study. There was no limit on the type of cell viability measure. We excluded studies that utilised a drug-resistant cell line, a modified preparation of temozolomide, or an animal model. Studies using cell line-based xenotransplant animal models or reporting outcome measures other than cell viability were excluded.We searched Ovid Medline and Embase between January 1994 and January 2021 using a combination of search terms relating to gliomas, cell lines, temozolomide, and cell death. The full search strategies are available in The online tool Covidence was our platform for conducting the primary screening and data extraction in this review. We applied the built-in deduplication function on the platform to records retrieved from our database search. One reviewer (MB) screened titles and abstracts of all records, of which 10% excluded records were reviewed by a second reviewer (MTCP). We used the same review approach for full-text eligibility assessment. One reviewer (MB) extracted data from all eligible articles and a second reviewer (MTCP) independently extract data from 40% of these studies. We resolved disagreements by discussion between the two reviewers or by seeking resolution from a third reviewer (PMB).We collected data on study characteristics, experimental setup and cell viability measures. Study characteristics included year of publication, country of primary affiliation, the primary aim of study, and types of cell lines used. ‘Primary aim of study’ had three categories: “therapeutic evaluation” referred to studies comparing the effects on cell lines of another therapeutic agent alone against temozolomide; “pathway modification” referred to studies that altered cellular pathways with a molecularly targeted compound and assessed the effect of temozolomide on the cell lines; “gene or protein measurement” referred to studies that measured levels of molecular markers in response to temozolomide. We categorised cell lines into human cell lines, murine cell lines, and cancer stem cell-like patient-derived cell lines. Patient-derived cell lines were developed from patients with malignant glioma at the investigators’ institute. Experimental setup data included temozolomide concentration, temozolomide exposure duration, base medium, addition of serum, cell density, cell passage number, use of hypoxic environment, and temperature and percentage of carbon dioxide in the ambient incubator environment.50), half-maximal effective concentration (EC50), dose reduces initial population to 10% (LD10), dose reduces survival number of cells to 37% (D37), approximate concentration of drug tolerated without lethality (DT), and median-effect dose (Dm). To assess the internal validity, we assessed whether a study reported the number of replicate plates and the number of replicates per plate.Cell viability was our outcome of interest. Data on cell viability included the assay, quantification technique used and cell viability measurement. Cell viability assays included 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), sulphondamine B (SRB), annexin V, propium iodide (PI), trypan blue, bromodeoxyuridine (BrdU) and luciferase-based techniques. Quantification techniques included flow cytometry, colorimetric or fluorometric plate reader, microscopy with manual counting, and microscopy with automated counting. Where reported, we collected drug sensitivity measures on half-maximal inhibitory concentration (ICThere was no suitable risk of bias tool for the type of studies included in this review. The completeness of reporting of the data items described above was instead used to indicate the transparency of the studies to permit summarisation of comparable studies.tidyverse’ (v1.3.1), ‘gtsummary’ (v1.4.1), and ‘car’ (v.3.0–10) packages.We used descriptive statistics to summarise the data items. Our reporting of temozolomide sensitivity measures was stratified for the two most common cell lines  and patient-derived cell lines. We divided studies into groups that had the same cell lines exposed to temozolomide for the same duration, in a non-hypoxic environment, and reported the same drug sensitivity measures. We tested the homogeneity of variances in temozolomide sensitivity across cell lines using Levene’s and Fligner-Killeen tests. There was no plan for meta-analysis because studies were anticipated to be heterogeneous and drug sensitivity measures without a measure of variance are not amenable to meta-analysis. These reasons also precluded the assessment of reporting bias. We performed all analyses in R version 4.1.0 using ‘s1–213 using malignant glioma cell lines that reported cell viability measures associated with temozolomide  were published in or after 2015. Countries accounting for over 10% of the included studies were China (35.4%) and the United States (17.9%). The proportion of studies published in or after 2015 from China and from the United States was 78.9 and 57.9%, respectively. Most studies (85.4%) evaluated therapeutic agents against temozolomide in their cell lines as the primary aim. There were 19 (9.0%) studies measuring gene or protein levels in their cell lines in response to temozolomide and 12 (5.6%) studies assessed the cell line response to temozolomide after modification of a cellular pathway. A human glioma cell line was at least one of the cell lines used in 193 (91.0%) studies. Twenty-three (10.8%) studies used at least one murine cell line and 22 (10.4%) studies used at least one patient-derived cell line , U251 (41.0%), and T98G (26.4%). Their use did not appear to change over time (Table Dulbecco’s Modified Eagle Medium (DMEM) was the commonest base medium, used in 142 (66.5%) studies (Table The range of temozolomide concentrations used in determining drug sensitivity was not determinable from 30 (14.2%) studies  studies Table . Thirty-50 of temozolomide for their cell lines , 223.1 μM (IQR 92.0–590.1 μM) and 230.0 μM (IQR 34.1–650.0 μM), respectively  and 176.50 μM (IQR 30.0–470.0 μM), respectively. The median IC50 of temozolomide at 72 h for T98G and patient-derived cell lines was 438.3 μM (IQR 232.4–649.5 μM) and 220 μM (IQR 81.1–800.0 μM), respectively.We examined studies together that used the same temozolomide exposure time and reported ICnes Fig. . Descrip50 values demonstrated in Fig. 50, we then included studies that used a normoxic culture environment (excluding hypoxic culture condition) and that used MTT as the cell viability assay, the most common composite conditions. The IC50 was 155.1 μM (IQR 37.8–640.5 μM) for U251 cell line at 48 h and 230.0 μM (IQR 58.0–650.0 μM) for U87 cell line at 72 h .The asymmetrical distribution of the ICThis systematic review identified 213 studies using cell culture models of malignant gliomas reporting drug sensitivity to temozolomide. Over 700 studies were excluded because they did not report cell viability measures. There were a wide variety of cell lines, and we did not observe an increase over time in patient-derived cell lines despite the implications of the cancer stem cell hypothesis for glioma biology. The reporting of experimental conditions and temozolomide specifications were variable, which made interpretation difficult. Temozolomide sensitivity was not consistent within each cell line. Furthermore, the range of concentrations used across the selected publications is not physiologically or clinically relevant to glioblastoma treatment  mutations, telomerase reverse transcriptase (TERT) mutations, 1p/19q co-deletion and alpha-thalassemia mental retardation syndrome (ATRX) mutations [We drew our results only from malignant glioma cell line models. There are other related experimental models such as drug resistant cell lines, genetically modified cell lines, tumour spheres, organoids and xenotransplant models . Our finutations . While iutations . StudiesTemozolomide sensitivity reported in comparable studies was not consistent between and within individual malignant glioma cell lines. This raises concerns about the reliability and translational value of drug discovery studies that use glioma cell lines. Appraisal and interpretation of the results can only be achievable if studies report key experimental conditions. While there will be variations of opinion on what the optimal design is, a consensus model of a reporting structure is the only rational way to maximise the yield from in vitro studies to find novel therapies for our patients.Additional file 1.


O-methyl-13α-oestrone series (9), exerted selective cell growth-inhibitory activity against UPCI-SCC-131 and T47D cell lines with low micromolar IC50 values. Moreover, it displayed good tumour selectivity property determined against non-cancerous mouse fibroblast cells.Microwave-assisted phospha-Michael addition reactions were carried out in the 13α-oestrone series. The exocyclic 16-methylene-17-ketones as α,β-unsaturated ketones were reacted with secondary phosphine oxides as nucleophilic partners. The addition reactions furnished the two tertiary phosphine oxide diastereomers in high yields. The main product was the 16α-isomer. The antiproliferative activities of the newly synthesised organophosphorus compounds against a panel of nine human cancer cell lines were investigated by means of MTT assays. The most potent compound, the diphenylphosphine oxide derivative in the 3- Consequently, development of additional 13α-oestrone derivatives with potential antiproliferative activities would be of particular interest.Literature reveals the existence of OPs of natural compounds, including those of steroids. Natural oestrone has a wide range of applications in the development of potent enzyme inhibitors and anticancer agents,,. The latter simple, but efficient strategy facilitates the convenient late-stage modification of biomolecules.The phospha-Michael addition is an important tool for the synthesis of OPs,Having developed an experience in microwave-assisted steroid synthesis2.Chemical syntheses, characterisation data of the reported compounds and selected 2 D NMR spectra, as well as experimental conditions of antiproliferative assays performed are described in the Supporting Information. Computational details are also explained in the Supporting Information.3.3.1.4 or 5 was carried out from 16-hydroxymethylidene derivatives 1 or 2, using formalin as a reagent and sodium carbonate as a base  served as starting materials in the phospha-Michael addition reactions.The efficient one-step synthesis of the known steroidal α,β-unsaturated ketones s a base 43,44. Th6) was reacted with 3-methoxy-16-methylene derivative 4 in acetonitrile as solvent. The mixture was irradiated in a microwave reactor at 100 °C for 60 min without addition of a base. Thin layer chromatography indicated full conversion of the starting material and the formation of two reaction products. The attack of the P-nucleophile at C-16a resulted in the formation of phosphorylated 16α- and 16β-diastereomers. The 16α:16β = 2.3:1 diastereomeric ratio was established from the 1H NMR spectrum of the crude product, containing solely the two diastereomers. After microwave irradiation, the reaction mixture was allowed to cool to room temperature. The majority of the 16α-isomer (9) was obtained in pure form as a white precipitate. The solid was filtered, and the solvent was removed from the filtrate by evaporation in vacuo. The remaining diastereomeric mixture from the filtrate was separated by flash chromatography and/or preparative RP-HPLC using a Phenomenex Biphenyl column. After evaporation of the solvent minor diastereomer 10 was isolated as white crystals. The simple microwave-assisted synthetic methodology elaborated for the reaction of compound 4 with diphenylphosphine oxide (6) was extended to the transformations depicted in O)H reagent and the 3-O-substituent. Transformations of the 3-methyl ether phosphine oxide 7 as nucleophilic partner required longer reaction times at the same temperatures (6 (8) required elevated reaction temperatures . The orientation of 16-H was deduced from the NOESY spectrum of compound 20  displayed substantial antiproliferative action against human cancer cell lines of gynaecological origin, with IC50 values in the range of 0.3‒0.9 μM (22 and 23 exerted activities similar to that of the 17-ketone24‒27)28)50 values in the low micromolar range (27 b) and 16β-phenyltriazolyl-17α-hydroxy (28) compounds reveal that the nature of the C-16 substituent strongly influences both the effectiveness and the selectivity. One of the most potent compounds was triazole 28, which induced cell cycle blockade at the G2–M transition and apoptosis via the intrinsic pathwayWe have recently described the development of a number of potential anticancer compounds based on the hormonally inactive 13α-oestrane core3‒0.9 μM 33. Howevar range . The oriin vitro antiproliferative capacity of twelve newly synthesised, 16-substituted, 13α-oestrane-based γ-ketophosphine oxides (9‒20) and their precursors (4 and 5) on a panel of human adherent cancer cell lines. The compounds were tested against breast , ovarian (A2780), cervical  and oropharyngeal (UPCI-SCC-131 and UPCI-SCC-154) carcinoma cell lines. Additionally, their tumour selectivity was also determined by using non-cancerous mouse embryo fibroblast (NIH/3T3) cells.Here we evaluated the 1 and 2) bearing an exocyclic 16-methylene group (4 and 5). Based on their calculated IC50 values determined on all tested cancer cell lines, these parent substances can be considered as highly effective antiproliferative agents (IC50 = 2.0‒7.0 µM)  . On most17‒20. Compounds 17 and 18 are 3-methoxy derivatives differing in the orientation of their C-16 substituents. Although these compounds exerted negligible growth inhibitory effect on most of the tested cell lines, they displayed the strongest effect against UPCI-SCC-131 cells. The other two substances are 3-benzyloxy derivatives, substituted at the 16α (19) or 16β (20) position. Their cell growth-inhibitory capacity was demonstrated to be more pronounced than that of their 3-methoxy pairs. Both benzyl ethers (19 and 20) inhibited cell division with the highest activity on T47D breast cancer cells. None of them was able to exhibit significant inhibitory effect on cell proliferation of MDA-MB-231 cells.According to the substituents on C-16, the tested twelve phosphine oxide derivatives can be divided into three main groups. The di analogues are para-tolyl analogues contains two 3-benzyloxy (15 and 16) and two 3-methoxy (13 and 14) derivatives, which form epimer pairs. The benzyloxy compounds exerted moderate antiproliferative effect on the tested cell lines except SiHa, MCF-7 and MDA-MB-231 cells, where their activities were evaluated to be insignificant. Both compounds demonstrated the strongest antiproliferative activity on UPCI-SCC-131 and T47D cell lines. The 3-methoxy derivatives (13 and 14) belong to the four most effective phosphine oxides. The IC50 values of 13 and 14 are between 10 and 25 µM on all tested cell lines. Similar to the previous test compounds (15 and 16), these analogues possess the highest cell growth-inhibitory activity against UPCI-SCC-131 and T47D cells. They exerted the weakest effect on the proliferation of MDA-MB-231 and A2780 cells. Moreover, their tumour selectivity was also determined on mouse embryo fibroblast cells. Their IC50 values on non-cancerous cell lines  are higher than their IC50 values on most of the tested cancer cell lines; therefore, these compounds possess better tumour selectivity compared to their parent compound 4.In a similar manner, the group of di-9 and 10) or benzyloxy (11 and 12) functional groups at C-3. Between the methoxy epimers, the 16α-isomer (9) demonstrated significantly higher antiproliferative activity against all tested cancer cell lines compared to that of its epimer pair (10). Compound 9 exerted the most significant cell proliferation inhibitory effect on UPCI-SCC-131 and T47D cell lines. These IC50 values are in the low micromolar range like that of cisplatin. Tumour selectivity of 9 can be considered as good since its IC50 value determined on non-cancerous NIH/3T3 cells is four times higher than its IC50 value measured on UPCI-SCC-131 cells. The benzyloxy compounds (11 and 12) of this group also displayed significantly different antiproliferative effects against the tested cancer cell lines. Unlike the methoxy epimer pair, the benzyloxy analogue with 16β-substituent (12) exerted more marked inhibition on cancer cell division than its epimer (11). On the other point of view, 11 demonstrated the highest inhibitory values on UPCI-SCC-131 and T47D cell lines, but this pattern of antiproliferative action cannot be observed in the case of 12. However, the IC50 value of 12 is 30.85 µM determined on non-cancerous NIH/3T3 cell line, its tumour selectivity is weaker than that of 9 due to its lower antiproliferative activity (IC50 = 13.62–29.83 µM) measured on all tested cancer cell lines.The third group of test compounds consists of four diphenylphosphine oxide derivatives bearing methoxy (9), which displayed markedly different antiproliferative effect on the HPV-negative oropharyngeal cancer cells in comparison to the HPV16-positive oropharyngeal cancer cells. On the other hand, this connection has not been supported by the results measured on HPV-positive and HPV-negative cervical cancer cells. Therefore, it can be concluded that the HPV-status of the tested cancer cell lines has no substantial impact on the antiproliferative activity of the phosphine oxide derivatives of 13α-oestrone. Moreover, UPCI-SCC-131 cells seem to be the most sensitive cell line when compared to most of the tested phosphine oxide derivatives.During the selection of the utilised cancer cell lines, the HPV-status of the cell lines was taken into consideration because we wanted to compare the antiproliferative effect of the test compounds on HPV-positive and on the corresponding HPV-negative cell lines. Among the tested substances, there is only a single compound  of the twelve newly synthesised 13α-oestrone analogues modified at the A- and D-ring have been identified as promising cell proliferation inhibiting agents. Their antiproliferative activity proved to be lower than that of their parent compounds. However, their tumour selectivity was better due to the modification of their chemical structure. Outstanding cell proliferation inhibitory activity of 9, a diphenylphosphine oxide analogue, has been revealed on UPCI-SCC-131 oropharyngeal and T47D breast cancer cell lines. In both cases, the IC50 values of 9 and cisplatin, our positive control, are comparable. Furthermore, observation with respect to the structure–activity relationship of the tested compounds can also be determined. The UPCI-SCC-131 cells derived from oropharyngeal carcinoma and T47D cells with breast cancer origin are the most sensitive cancer cell lines to our phosphine oxide derivatives. Bulky substituents (e.g. naphthyl) in either position at C-16 eliminates the antiproliferative activity of the test compound. In contrast, the orientation of functional groups at C-16 seems to have no significant impact on cell growth-inhibitory capacity if smaller substituents (e.g. para-tolyl or phenyl) are present on the phosphorus atom. Finally, based on the chemical structure of the most effective diphenylphosphine oxide analogue (9), it can be concluded that its substituent at C-16 in α position is preferred regarding its antiproliferative activity on UPCI-SCC-131 and T47D cells. Finally, since 9, like the other two promising compounds (13 and 14), belongs to the group of 3-methoxy derivatives, it suggests that 3-methoxy group can be an advantageous modification on certain 13α-oestrone derivatives.In summary, four phosphine oxide derivatives , as the diastereomer with the lowest total energy was not always the same stereoisomer.Considering the experimental results, computational investigations were performed for the epimer pair In general, simulations could not support any preference which was found in the synthetic work. Nevertheless, in Taking into account these results, we assume that most probably there is a reactionkinetic reason behind the diastereoselective preferences found in our study.4.9) exhibited impressive selectivity for HPV-negative oropharyngeal cancer cell line UPCI-SCC-131 with modest action on non-cancerous fibroblasts. Our results in connection with the antiproliferative capacity of the tested compounds might underlie the importance to design and synthesise more organophosphorus steroid analogues expecting that they show higher tumour specificity and better tumour selectivity.We carried out microwave-assisted phospha-Michael addition reactions in the 13α-oestrone series. Phosphorylation at C-16a resulted in two diastereomeric products (16α- and 16β) in high yields. A simple and efficient microwave-assisted methodology was elaborated for the synthesis of organophosphorus steroidal compounds representing an undervalued but promising family of potential anticancer agents in chemical space. One of the presented compounds (Click here for additional data file.


Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21Cip1/WAF1 and, inhibition of the NF-κB pathway.The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome  Among the different kinds of cancer, melanoma is the deadliest form of skin cancer for its aggressiveness and resistance to current therapies. The number of new cases of melanoma reported by the World Health Organization was approximately 324,000 with an associated 57,000 deaths worldwide in 2020 . It is ec release to the cytosol and caspase-9 activation , BD Pharmingen , anti-caspase-3  from Enzo ; anti-caspase-7 , anti-caspase-8  and anti-caspase-9  from Cell Signaling Technology ; anti-cytochrome v/v) heat-inactivated fetal bovine serum and 100 units/mL penicillin and 100 µg/mL streptomycin and maintained at 37 °C and 5% CO2 in a humidified atmosphere. Cell viability was assessed using 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded at a density of 5000 cells per well into a 96-well plate and incubated with increasing concentrations of HY-CHAL for 72 h. The supernatant was removed and 0.5 mg/mL MTT was added and incubated for 4 h at 37 °C. The reaction products were solubilized with sodium dodecyl sulfate (10% w/v) in 0.05 M HCl overnight under dark conditions. Absorbance was measured at 570 nm with a reference wavelength of 570 nm using an ELISA reader . Each experimental condition was analyzed in triplicate. The IC50 values were determined graphically for each experiment by a nonlinear regression using the curve-fitting routine implemented within the software Prism 5.0 .Human melanoma cell lines SK-MEL-1 (ACC-303) and MEL-HO ACC-62) were obtained from the German Collection of Microorganisms and Cell Cultures . SK-MEL-1 are mostly round cells growing singly or in clumps in suspension . They gr were obtTM cytometer  were performed as previously described . After 15 min on ice, cells were lysed by pushing them several times through a 22-gauge needle, and the lysate was centrifuged at 1000× g for 5 min at 4 °C to eliminate nuclei and unbroken cells. This pellet was used as a nuclear fraction. The supernatant fraction was centrifuged at 105,000× g for 45 min at 4 °C, and the resulting supernatant was used as the soluble cytosolic fraction.Immunoblot analysis of whole cell lysates and for cytosolic fraction was performed as previously described. For whole cell lysates, cell pellets were resuspended in lysis buffer , containing the protease inhibitors phenylmethylsulfonyl fluoride , leupeptin, aprotinin, and pepstatin A (5 μg/mL each) for 15 min at 4 °C. Lysates were homogenized by a sonifier (five cycles) and centrifuged at 11,000× g for 5 min at 4 °C and washed with PBS, and the cell pellets were kept on ice. The cells were resuspended in lysis buffer  and held on ice for 5 min. Lysates were centrifuged at 17,000× g for 10 min at 4 °C and the supernatants were collected. The total protein concentration in the supernatants was determined using the Bradford dye-binding assay. Equal quantities of protein from different treatments were used, and the assays were set up on the ice. The net increase of absorbance at 405 nm after incubation at 37 °C was indicative of enzyme activity. Specific colorimetric substrates for caspase-3/7, −8 and −9 activities were DEVD-pNA , IETD-pNA (N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline) and LEHD-pNA (N-acetyl-Leu-Glu-His-Asp-p-nitroaniline), respectively.Caspase activity was determined in cell lysates using specific colorimetric substrates. Briefly, cells were treated with increasing concentrations of HY-CHAL for 12 or 24 h, harvested by centrifugation at 1000× 2-DCF-DA, 10 μM) was used to determine ROS.The membrane potential and intracellular ROS production were determined by flow cytometry. The fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide  was used to measure mitochondrial membrane depolarization. The fluorochrome 2′,7′-dichlorodihydrofluorescein diacetate  of 0.05 was used. The GraphPad Prism 5 software was used.c and a mechanism dependent of caspases. Futures studies will be necessary to determine whether additional pathways of cell death are involved in SK-MEL-1 and MEL-HO melanoma cells. The sensitivity of melanoma cells to HY-CHAL suggests that it should be considered for further preclinical and in vivo testing. The exploration of the signal transduction pathway of cell death using SK-MEL-1 cells as a model revealed that this compound induced (i) cell death by a mechanism that involves the intrinsic and the extrinsic pathways, (ii) activation of the MAPK pathway and the blockade of MEK/ERK cascade amplified the cell death, and (iii) downregulation of β-catenin, c-myc, p21Cip1/WAF1, and Mcl-1 and (iv) inhibition of NF-κB. In addition, cell death was dependent on the generation of reactive oxygen species since HY-CHAL induced an increase in ROS levels, and pre-incubation with glutathione suppressed the increase of ROS and inhibited significantly the cell death. The studies described here may open new avenues of research to understand the safety and full potential of HY-CHAL in human health.We have evaluated the effects of a specific chalcone containing indole and pyridine heterocyclic rings on cell viability and apoptotic pathways in human melanoma cell lines. This hybrid chalcone, HY-CHAL, was a potent cytotoxic compound and an apoptotic inducer in SK-MEL-1 and MEL-HO melanoma cells. The fact that both melanoma cell lines were sensitive to HY-CHAL might be interesting given that melanoma is the most aggressive and lethal form of skin cancer and frequently resists chemotherapy. Here we explored the mechanism of cell death by investigating apoptotic pathways. In both cell lines, the antiproliferative effect of HY-CHAL was associated with the release of cytochrome


The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database. Xia et al. report the development and optimization of a high-throughput screening platform to systematically determine cancer cell sensitivity to pharmacological and genetic perturbations, BMS-PRISM, based on PRISM and high-throughput CRISPR/Cas9 loss-of-function screen technologies using cell line barcoding. However, these cancer cell lines, which are passaged for many years in culture dishes, may no longer fully recapitulate their original genetic and/or epigenetic characteristics owing to clonal selection3. It is not rare that scientific findings from one laboratory cannot be reproduced by other researchers using different strains of the same cell line that underwent genomic evolution. As such, a better approach to faithfully capture cancer vulnerability specific to a tissue lineage or genetic background is to assess the response to inactivation of a candidate disease-driving protein across a large number of cell lines covering common and distinctive genetic characteristics. This practice is usually impractical in small laboratories with limited resources, thus calling for the need for a reliable platform that allows the screening of hundreds of cell lines in a high-throughput manner.Human cancer cell lines serve as a powerful tool for defining the mechanism of cancer growth and metastasis, as well as for developing therapeutic interventions to attack cancer vulnerabilities. More than 1000 cancer cell lines from various tumor types have been established in the past decades and are widely used by the cancer research community5. The dependence on individual genes was determined using a predefined algorithm in cancer cell lines under standard nutrient-rich culture conditions for 10–12 cell doublings, resulting in the identification of potential therapeutic targets for various cancer indications. Although CRISPR/Cas9-based gene knockout has proven to be a powerful tool for inactivating gene function with greatly improved precision over traditional RNAi-mediated knockdown7, follow-up validation studies are needed to confirm whether knockout of an indicated target gene leads to loss of cell fitness via an on-target mechanism in a panel of cell lines. This effort would still take a large amount of time and resources if performed in dozens of cell lines individually.Broad Institute’s Project Achilles through the DepMap portal offers the assessment of gene essentiality for the majority of protein-coding genes in >700 human cancer cell lines based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9- as well as RNA interference (RNAi)-mediated gene perturbation8. The PRISM technology enabled the profiling of compound activity on cell viability in dozens to hundreds of cell lines in a single run to define genotype-specific cancer vulnerabilities. Recently, PRISM was successfully applied to evaluate the growth-inhibitory activity of 4518 drugs across 578 human cancer cell lines, revealing the potential application of non-oncology drugs for the treatment of cancer9. However, the PRISM platform is not readily amenable for high-throughput interrogation of cancer vulnerability using genetic tools, such as CRISPR/Cas9-mediated gene editing.In 2016, researchers from the Broad Institute reported the development of PRISM—a multiplexed cell line screening platform combining a DNA-barcoding technique and a Luminex microsphere detection systemIn order to enable the rapid validation of the therapeutic value of a given target of interest across a large panel of human cancer cell lines using both CRISPR and pharmacological perturbations, we developed BMS (Bristol Myers Squibb)-PRISM platform combining CRISPR/Cas9-mediated gene editing capability and DNA-barcoding multiplexing technique. We carried out a focused run with epidermal growth factor receptor (EGFR) and a full library run with KRAS as proof-of-principle studies. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with public database.Streptococcus pyogenes-derived Cas9 (spCas9) and a blasticidin-resistant gene (blast), with a unique 26-base pair (bp) DNA barcode between them. We integrated these constructs into cell lines of interest via lentiviral transduction and then selected with blasticidin to establish pooled stable lines. The Cas9 editing efficiency was then validated using a lentiviral Cas9 activity reporter  protocol for quantification of the percentage of each unique barcode in a mixture, which should faithfully represent the relative cell number of the corresponding cell line in a mixed pool with or without genetic and/or pharmacological manipulation. In brief, the barcode regions integrated into the genomic DNA were PCR amplified with a pair of universal primers. A second PCR reaction was performed on the first PCR product to incorporate dual-indexed Illumina primers into the final barcode library. The abundance of unique barcodes in each library was quantified via NGS , with each group containing 4 barcoded plasmids. Next, equal volumes of these diluents were mixed to generate the test plasmid pool, in which the relative representation of each of the 20 unique barcodes was quantified using the NGS protocol. As shown in Supplementary Fig. 10. To establish the EGFR dependence of these 20 NSCLC cell lines, we first evaluated the sensitivity of each cell line to Erlotinib treatment (CellTiter-Glo (CTG) cell viability assay) or EGFR knockout (flow cytometry-based CRISPR competition assay). HCC827 (E746-A750del), HCC4006 , and PC9 (E746-A750del) cells harboring typical EGFR exon 19del mutations were most sensitive to Erlotinib or EGFR knockout, while others showed little to no response to EGFR perturbation  cell lines with wild-type or mutant EGFR in the BMS-PRISM collection  cell culture). Nevertheless, most of the 17 KRAS G12C mutant cell lines showed their consistent response to AMG-510 and KRAS CRISPR PRISM screen  in the PRISM screen pool, the autocrine effects observed in cell lines when cultured as a homogenous population may not be detected in the PRISM screen, which could lead to differential addiction to genes affecting cell growth via an autocrine signal. Lastly, cell seeding density is another plausible factor affecting the readout of gene dependency in the PRISM screen. Notably, cell density is relatively higher in the PRISM screen than in standard CTG or CRISPR competition assays, so cell–cell and cell–matrix interactions may vary slightly from traditional cell culture conditions. Maintaining the cells in a monolayer in the PRISM screen helps to overcome this limitation, since cells may acquire some features of 3-D culture conditions once they outgrow the monolayer. For example, since we were able to maintain cells in a monolayer in the PRISM setting, we did not observe increased dependency for KRAS of any cell lines, which has previously been shown under 3-D culture conditions. Nevertheless, further investigation is needed to assess the difference in cell culture condition on gene dependency, especially in cell lines showing differential addiction to a given gene of interest. Moreover, since tumor cells in a human body typically grow in a nutrient-limited microenvironment where complex autocrine and paracrine cell–cell and/or cell–matrix interactions control the tumor growth and survival, the true dependence on any candidate gene needs to be validated in a panel of cell line or patient-derived xenograft models in vivo.One of the major differences between the PRISM screen and individual assays that may introduce inconsistency is the cell culture condition. First, nearly 400 cell lines were mixed together and cultured in a single pool for 5–10 days in the PRISM screen settings, and therefore paracrine signal(s) may affect the dependence on a gene of interest in certain cell populations. Although it is conceivable that the paracrine effects of genes involved in regulation of cell proliferation signals, such as EGFR and KRAS, should become more dominant in the PRISM screen, we observed minimal discrepancy between the PRISM screens and their individual assays when EGFR and KRAS were tested in a focused screen format and full-library screen format, respectively. We reasoned that the percentage of cell lines that could potentially affect the cell fitness of others via a paracrine effect controlled by any gene of interest in the PRISM screen is probably <5%, thus this paracrine effect, if existed, should not affect the overall scoring of gene dependency in the PRISM screen. This hypothesis is strongly supported by our data shown in this study as well as in the PRISM screen result published by the Broad InstituteIt is worth mentioning that normalization of raw reads is critical for the PRISM platform data interpretation, as hundreds of cell lines were cultured together as a mix and their proliferation rate varies. In BMS-PRISM, we did not quantify the barcode copy number of each cell line so the initial abundance of each barcode is not equal. For normalization purpose, we collect control samples (dimethyl sulfoxide (DMSO) treated or sgNT (non-targeting control single-guide RNA (sgRNA)) infected) at every time point as references. The abundance of each barcode in the treatment groups are normalized by that in the control group. For CRISPR screen, there is another critical factor that needs to be considered—different cell line may have different sensitivity to Cas9-induced DNA damage response (double-strand breaks). Therefore, we always include two control sgRNAs: sgNT and sgNC. sgNT does not match any genomic sequence so it does not introduce double-strand breaks, while sgNC matches the non-coding sequence (intron) of HBG1 gene. HBG1 is only expressed in the fetal liver, spleen, and bone marrow, so targeting its intron sequence in cancer cell lines is expected to have minimal on-target effect. Therefore, sgNC serves as a control for cell line’s sensitivity to DNA damage response. Interestingly, we did not observe apparent change of cell abundance upon the infection of sgNT and sgNC in most cell lines  or RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1× sodium pyruvate (Invitrogen), 1× non-essential amino acids (Invitrogen), 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). PRISM cell mix was cultured in complete RPMI-1640 medium as described above. Lentiviral vectors expressing Cas9 and unique 26-bp DNA barcode or Cas9 activity reporter were custom-synthesized at Genscript. EGFR inhibitor Erlotinib and KRAS G12C inhibitor Sotorasib (AMG-510) were purchased from Selleckchem.Human cancer cell lines cultured in the complete growth medium were seeded into 96-well plates containing DMSO or test compounds. The seeding density for each cell line  was optimized to allow the cell growth in the linear range during culture period (5–7 days). After the culture period, cell proliferation was assessed using CTG Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Individual compound test AUC was calculated with 10-point CTG assay data .Gene essentiality was evaluated with CRISPR-based competition assay. Briefly, human cancer cells were infected with red fluorescent protein (RFP)-sgNT or RFP-targeting sgRNA (two sgRNAs per gene) and then mixed 1:1 with cells infected with green fluorescent protein (GFP)-sgNT on Day3 . The ratios of RFP/GFP cells were monitored on Day3, Day7, Day10, and Day14. RFP-sgTargeting/GFP-NT ratio normalized by RFP-sgNT/GFP-sgNT  was used to evaluate the gene essentiality in this human cancer cell line.The sgRNA sequences used in this study were: sgNT ; sgNC ; sgEGFR ; and sgKRAS .Detailed screen protocol is described in 6 cells from each sample consisting of mixed PRISM cell lines using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions, with an RNase A treatment added upon cell lysis to eliminate cellular RNA. Total genomic DNA was visualized and quantified on the TapeStation 4200 using the Genomic DNA tape (Agilent). To amplify the 26 bp DNA barcode portion of the Cas9 construct transduced into each cell line, PCR was performed on 1 µg of genomic DNA per sample using custom primers that flanked the barcode region  A total of 368 cell lines were used to demonstrate the good correlation between PRISM methodology and individual assays. (2) For each treatment condition (and time-course point), three biological replicates were included. All data are shown in dotplots to demonstrate data distribution and represent individual data points. Data are expressed as mean ± SEM.Statistical analysis was performed using the GraphPad Prism 8 software. Two-group analysis was performed using unpaired two-tailed Student’s Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary