Lung cancer is one of the leading forms of cancer in developed countries. Interleukin-8 (IL-8), a pro-inflammatory cytokine, exerts relevant effects in cancer growth and progression, including angiogenesis and metastasis in lung cancer. Mesoporous silica particles, functionalized with newly extracted fish oil (Omeg@Silica), are more effective than the fish oil alone in anti-proliferative and pro-apoptotic effects in non-small cell lung cancer (NSCLC) cell lines. The mechanisms that explain this efficacy are not yet understood. The aim of the present study is therefore to decipher the anti-cancer effects of a formulation of Omeg@Silica in aqueous ethanol (FOS) in adenocarcinoma (A549) and muco-epidermoid (NCI-H292) lung cancer cells, evaluating cell migration, as well as IL-8, NF-κB, and miRNA-21 expression. Results show that in both cell lines, FOS was more efficient than oil alone, in decreasing cell migration and IL-8 gene expression. FOS reduced IL-8 protein release in both cell lines, but this effect was only stronger than the oil alone in A549. In A549, FOS was able to reduce miRNA-21 and transcription factor NF-κB nuclear expression. Taken together, these data support the potential use of the Omeg@Silica as an add-on therapy for NSCLC. Dedicated studies which prove clinical efficacy are needed. Lung cancer is one of the most common diagnosed and harmful forms of cancer, above all in developed countries . AmountiIn this context, we recently reported that mesoporous silica particles, functionalized with newly extracted fish oil (Omeg@Silica), significantly inhibit (in vitro) NSCLC cell growth . In closd-limonene is a biobased solvent endowed with several health-benefitting properties [The new Omeg@Silica material is made of a whole fish oil (AnchoisOil) sustainably sourced from anchovy fillet leftovers with citrus limonene , encapsuoperties .The molecular mechanisms that explain this remarkable efficacy of said Omeg@Silica submicron particles in aqueous ethanol, not yet understood, need to be urgently identified in light of the numerous promising characteristics of this new material. Its components  are indeed highly biocompatible and actually used in several commercial nutraceutical and pharmaceutical products.n-3) and eicosapentaenoic acid . In natural fish oil , said PUFAs are present in triglyceride form. By significantly inhibiting tumour growth, suppressing cancer cell viability, and inducing apoptosis in various cancer cells, these essential lipids exert a therapeutic role against certain types of cancer [The main polyunsaturated fatty acids (PUFAs) in fish oil are docosahexaenoic acid  is one of the key promoters of tumour progression given its ability to promote the angiogenic response of endothelial cells; recruit immunosuppressive cells (such as neutrophils); and induce the proliferation, survival, and migration of cancer cells . ClinicaIL-8 production is regulated by different signalling pathways. Nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) are the main triggers associated with lung inflammation . NF-κB, Thus, in this study, we explored the anti-cancer properties of Omeg@Silica microparticles using NSCLC cells lines which originate from different geographical districts of the lung, in particular adenocarcinoma (A549) and mucoepidermoid (NCI-H292) lung cancer cell lines, by evaluating cell migration, as well as the expression/release of IL-8, NF-κB, and miRNA-21.d-limonene as an extraction solvent [3 .Fish oil was obtained from anchovy fillet leftovers gently given by an anchovy fillet company , according to a circular economy process introduced in 2019, using biobased  solvent . An oil,v/v% ethanol–PBS Dulbecco’s phosphate-buffered saline . In particular, the Omeg@Silica mother suspension was prepared by adding 10.1 mg of material to 1 mL of ethanol and 9 mL of PBS. This formulation of Omeg@Silica microparticles in aqueous ethanol is named FOS. The AnchoisOil and the MCM-41 mother suspensions had a concentration of the single components equivalent to that of the Omeg@Silica mother suspension. Then, 5 mg of AnchoisOil was suspended in 10 mL of 10 v/v% ethanol–PBS and 5.1 mg of MCM-41 was suspended in 10 mL of 10 v/v% ethanol–PBS.The Omeg@Silica particles used in this study were MCM-41 silica particles loaded with 50 wt% anchovy fish oil. Their synthesis via the microencapsulation of AnchoisOil, following the dropwise addition of oil to the mesoporous silica, was carried out, as previously described . The aqul-glutamine, and 250 μg/mL of gentamicin, at 37 °C in a humidified atmosphere containing 5% CO2. In the sub-confluent cell monolayer, the serum concentration in the medium was reduced to 1%, and then was treated with oil. Following this, FOS (10 μg/mL) was performed.A549 and NCI-H292 cells were purchased from Interlab Cell Line Collection . The cells were kept in RPMI with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 1% MEM non-essential amino acids, 2 mM of In order to evaluate possible effects due exclusively to microparticles, we treated cells with silica alone. The concentration of FOS (10 μg/mL) used in the present work was identified in a previous work on A549 where cell viability, necrosis, and cell apoptosis were assessed [A549 and H292 cells were seeded in a 6-well plate and were cultured to confluence. Three circular wounds were prepared in each well using a 200-µL pipette tip as previously described . After wThe total RNA was extracted by A549 and H292 cell lines, treated or not with oil, FOS, and silica, using the RNAspin mini kit , according to the manufacturer’s protocol. The RNA concentration was assessed using the SpectroStar Nano reader . For this study, we only used the RNA with a ratio of A260/280 from 1.9 to 2.From the total RNA, the cDNA was synthesized using the iScript cDNA synthesis kit (Biorad), according to the manufacturer’s protocol. IL-8 gene expression was evaluated with specific FAM-labelled probe and primers, as part of the TaqMan gene expression assay for IL-8  with qRT-PCR using a Step One Plus real-time PCR system . The gene expression was normalized to GAPDH with a Taq-Man gene expression assay for GAPDH  as a housekeeping gene. For the normalization of RT-qPCR data, the 2-ΔCT method was used.For the reverse transcription of miRNA-21-5p, 1 µg of total RNA was incubated with specific primers using the TaqMan™ microRNA assay  and the TaqMan miRNA RT kit , according to the manufacturer’s protocol. miRNA-21-5p expression was evaluated using specific TaqMan microRNA assays  with qRT-PCR and the Step One Plus real-time PCR system . MiRNA-21-5p expression was normalized with RNU6 using the TaqMan™ microRNA assay , as endogenous control. For the normalization of RT-qPCR data, the 2-ΔCT method was used.The results were expressed as a percentage of area reduction at time point 24 h (T24h) and 48 h (T48h), compared to time point 0 h (T0h).g, and stored at −80 °C until the assay was used. These experiments were performed in triplicate.The release of IL-8 was evaluated by the ELISA method with the enzyme-linked immunosorbent assays , according to the manufacturer’s instructions. Cell supernatants of both line cells were harvested after 24 h of stimulation, centrifuged for 15 min at 15,000× β-actin mAb . These experiments were performed in triplicate.Nuclear translocation of NF-κB was assessed by Western blot analysis. Three hours after treatment with oil, silica, and FOS, A549 and H292 pellets were collected, and cytoplasmic and nuclear proteins were extracted using a commercial kit , as previously described . Proteinp < 0.05 was regarded as statistically significant.The determination of statistical significance for differentially expressed signals and for the scratch test assay was performed using an analysis of variance (ANOVA) approach corrected with Fisher’s test. A value of Increased cancer cell motility is a feature of tumour aggressiveness . To evalA549 and NCI-H292 were cultured with fish oil, silica, and FOS (10 μg /mL). After 24 h, the total RNA was extracted and supernatants were collected. To assess IL-8 gene expression, real-time PCR was used. The IL-8 concentration in cell supernatants was evaluated with an ELISA kit. As shown in Since NF-κB activation can control IL-8 expression , it was In A549, IL-8 gene expression and release increased using miR-21 up-regulated expression . We treaLung cancer is a very aggressive cancer. About 80% of lung cancers are non-small cell lung cancer (NSCLC) which mainly consists of two subtypes: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). They arise from different cells and differ in prognosis, signalling pathways, and gene expression. Moreover, LUAD provides about 40% of all diagnosed lung cancers . NSCLC pSubmicron mesoporous silica particles, functionalized with whole fish oil extracted with limonene only (Omeg@Silica) and further dispersed in aqueous ethanol (FOS), have recently been demonstrated to exert in vitro anti-tumour effects on NSCLC cell lines, increasing mitochondrial ROS production and cell apoptosis, and reducing cell growth ability.Herein, we further investigated the anti-cancer effects of FOS in two LUAD cancer cell lines which originate from different districts: adenocarcinoma (A549) and mucoepidermoid (NCI-H292) cell lines. This involves evaluating IL-8, NF-κB, and miRNA-21 expression, as well as molecules associated with cell migration (a process with a relevant role in cancer progression).In lung cancer, it was demonstrated that ω-3 PUFAs and their metabolites regulate pathways controlling the progression of lung cancer. Trombetta et al. showed that in A549, ω-6 and ω-3 PUFAs have anti-proliferative effects, affecting the DNA-binding activity of AP-1 and inducing PPAR . In addiFurther, 17-oxo-DHA has antitumor effects in NSCLC cell lines and, in combination with gemcitabine, leads to stronger anti-cancer effects compared to gemcitabine alone .The connection between chronic inflammation and tumour growth is well established . It reprUpon dietary consumption, long-chain polyunsaturated fatty acids (PUFAs), especially ω-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), commonly found in fish oils, have helpful effects against inflammation. Clinical studies on the dietary intake of PUFAs and in vitro studies showed that DHA can significantly decrease the expression of inflammatory cytokines and the expression of genes involved in inflammatory pathways such as NF-κB . A pivotTaking these findings into account, we tested whether FOS exerted anti-cancer effects, decreasing cell migration via the scratch assay. For the first time, we found that, both in A549 and H292, FOS decreases cell migration, more than in fish oil alone, and the FOS effects were similar to the effect exerted by anti-IL-8. These findings prompted us to test the effect of FOS, oil, and silica treatments on IL-8 expression and release in both cell lines. As expected, FOS was able to reduce IL-8 gene expression and protein release both in A549 and NCI-H292. Only in A549, FOS was more efficient in reducing both IL-8 gene expression and protein release than fish oil alone. In A549 cells, ω-3 fatty acids significantly decreased IL-8 . Here, f3) [3 molecules entrapped in the silica pores are protected from chemical oxidative degradation due to the air’s oxygen and natural light.It is well known that the hydrophilic–lipophilic balance (HLB) of cancer cell membranes is altered, with an increase in cell permeability and enhanced hydrophilic character. Hence, for example, the uptake of hydrophobic curcumin microencapsulated by A549 cells is highest for submicron pluronic-stabilized gelatin particles with the lowest hydrophobicity . We make3)  of Ancho3) , is sign3) , we assuThe overexpression of IL-8 is present in different types of cancer, including multiple myeloma, hepatocarcinoma, chronic lymphocytic leukemia ,35,36,37NF-κB is the main regulator of pro-inflammatory gene expression, particularly IL-8 expression . In the Hence, we assessed the nuclear expression of NF-κB in both cell lines. Only in A549 cell line, FOS was more efficient than fish oil alone to induce a significantly impairment in NF-κB expression. In the H292 cell line, under the same conditions, NF-κB expression was unaltered. These findings suggest that in H292, the effect of FOS on IL-8 gene expression and release can be due to the modulation of different transcription factors and kinase cascades, such as C/EBP-1, CREB, or AP-1, which bind to the IL-8 promoter .E. coli induces a differential IL-8 gene regulation pathway [Remarkably, it has already been demonstrated that in different uroepithelial cells, uropathogenic  pathway , with ce pathway . Similar pathway . Further pathway .In this regard, in the same subgroup of lung cancer (NSCLC), Sorrenti and co-workers have shown that tin mesoporphyrin (SnMP) has different anti-cancer effects in A549 and NCI-H292, likely due to gene level regulation by miRNA [These differences can be explained by the fact that these cancer cells have a different origin: H292 originates from the minor salivary glands lining the tracheobronchial tree, while A549 is derived from bronchioalveolar stem cells or from alveolar type 2 (AT2) cells. A significant relationship between microRNAs and IL-8 exists: at the post-transcriptional level, IL-8 is controlled by the miRNA network . In addiMiRNA-21, an onco-miR, represents a fundamental link between cancer and inflammation, as its increased expression has been found in different cancers. In lung cancer, specifically in NSCLC, several studies report on the upregulated levels of miRNA-21 in diagnosed patients. The affected expression of miRNA-21 disturbs different pathways, including cell proliferation and angiogenesis, as well as chemo- and radio-resistance .The use of miRNA-21 antisense sequences as therapeutic modulators shows encouraging results in the management of NSCLC . Thus, wIn conclusion, after studying in vitro the effect of whole fish oil encapsulated in mesoporous silica formulated in aqueous ethanol on adenocarcinoma (A549) and mucoepidermoid (NCI-H292) lung cancer cell lines, we discovered that a formulation comprised of submicron mesoporous silica particles functionalized with whole fish oil extracted with limonene only (Omeg@Silica) dispersed in aqueous ethanol (FOS) reduces IL-8 release, affecting cell migration and cancer progression in lung cancer cells. In adenocarcinoma cells, FOS inhibits cell migration through the inhibition of NF-κB and miRNA-21 in a cell-type-specific manner. Offering the first mechanistic insight into deciphering FOS anti-cancer property, these findings delineate a novel pharmacological and therapeutic strategy for lung cancer patients based on whole fish oil derived from the most abundant fish species (the anchovy) across the world’s seas.


BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9+ EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying  BRCA1/2-mutation (g/sBRCA 1/2) and more than three prior lines of chemotherapy [+ for the catalytically active site of PARP molecules leading to the persistence of unrepaired DNA single-strand breaks by trapping PARP molecules and consequently resulting in DNA double-strand breaks by the collapse of stalled replication forks [BRCA1/2 mutations [The approval of the poly (ADP-ribose) polymerase inhibitor (PARPi) olaparib in 2014 as a treatment option in patients with germline or somatic on forks . Double-on forks . Thus, ton forks , 7. Thisutations , 9.BRCA1/2 [RAD51C, and RAD51D [BRCA mutations and HR deficiency tests represent important predictive markers for PARPi response, the extended benefit of PARPi in EOC patients was also reported in BRCA wild-type and HR-proficient tumors in several clinical trials [Several approaches have been suggested to detect HR-deficiency (HRD), including scores to measure the effects on genome stability, namely loss-of-heterozygosity, large-scale transitions, and telomeric allelic imbalance, collectively referred to as “genomics scars” –12. StilBRCA1/2 –15, RAD5d RAD51D  have beel trials –19, highHR deficiency is also associated with sensitivity to other DNA-damaging agents , 21, andCas9 mutagenesis assay with further validation in other PARPi and carboplatin chemotherapy.For this purpose, our study comprehensively assessed the current literature reporting on genes functionally linked to PARPi response. Identified and curated gene candidates were systematically examined on their role in cancer cell fitness and olaparib response upon genetic deletion using a functional CRISPR-https://www.ncbi.nlm.nih.gov/) was assessed to identify publications on ovarian cancer and PARP inhibitors during a 16-year period, from 2005 to July 2021. Thus, the literature search utilized the following terminologies: “ovarian cancer (cells)” and “PARP inhibitor”, “PARP inhibition”, “PARP”, “Olaparib”, “Niraparib”, “Talazoparib”, “Veliparib”, “Rucaparib”, “PARP inhibitors molecular mechanisms of action”, “gene mutation sensitivity”, “PARP inhibitors target in BRCA proficient cells”, “markers of response to PARP inhibition”, “PARP inhibitors in non-HR deficient tumor”, “off target PARP inhibitors toxicity”, “PARP inhibitors gene mutation”; AND: “gene silencing”, “knockout”, “knock out”, “deletion”; OR: “on target”, “off target”, “targeted therapies”, “targeted therapy”, “CRISPR”; “cancer genomic”, “cancer genomics”. Since our search aimed to identify genes that may be related to PARPi response, we decided to broaden our inclusion criteria to gynecologic carcinomas. Selected publications include human-derived cell lines or human clinical cancer samples of gynecological carcinomas including breast, cervical, endometrial cancer, and ovarian cancer.The PubMed database , Selleckchem . Compounds were dissolved in DMSO , or water carboplatin and stored as aliquots at −20.EOC cell lines OVCAR3, OVCAR5, OVCAR8, ES-2, TYK-nu, and IGROV1 were purchased from ATCC, JCRB cell bank, and Oncotest GmbH . All cancer cell lines were cultured in RPMI-1640 growth medium supplemented with 10% FBS , 100 U/mL penicillin, and 0.1 mg/mL streptomycin . Cell lines were STR-profiled  and regularly tested for the absence of 50 data relative to cell lines were obtained from the depmap portal https://depmap.org/portal/download/custom/ (accessed in January 2022). These data were filtered for the following compounds: olaparib, niraparib, talazoparib, and cisplatin to generate Fig. 53BP1, AAVS1, PDS5B, ARID1A, ATM, ATR, AURKB, BARD1, BCL2L1, BCL2L2, BRCA1, BRCA2, BRD4, BRD9, CBLC, CCND1, CCNE1, CDCA8, CDK12, CDK4, CDK5, CDK9, CHEK1, CHEK2, DCLRE1C, DDB1, DNPH1, SEM1, DYNLL1, E2F7, EHMT1, EHMT2, FBXO5, ERCC3, ERCC8, EZH2, FEN1, FOXM1, GPBP1, HMGA2, HMGB2, HSP90AA1, INPP4B, XRCC5, LIG1, MAPK12, MET, MIR-107/107, MIR-493/5 P, MIR-506/506, MIR-509-3/3 P, MIR-622/622, MIR-96/96, MLH1, MLH3, MRE11, MUS81, MYC, NBN, NF1, GABPA, NRP1, PALB2, PARG, PARP1, PARP2, PARP3, PIK3CA, PCNA, PLK3, PNKP, POLQ, PTEN, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, ATRX, RBBP8, REV1, RNASEH2A, RNASEH2B, RNF168, RPA1, RPA3, RPRD1B, SEM1, SHLD1, SHLD2, SRC, SSRP1, TSSK3, STK36, TIGAR, TOPBP1, TP53, TRIP12, TWIST1, USP13, USP15, XAB2, XPC, XRCC1, XRCC2, XRCC3, XRCC5, XRCC6; data was then combined using cell line id.IChttps://benchling.com). SgRNAs with high-quality scores were selected for editing of the target gene  for subsequent expression of the sgRNA together with either EGFP or mOrange fluorescent proteins. Ligations were transformed into Stbl3 E.coli following ampicillin selection, ZR Plasmid Miniprep—Classic plasmid purification , and Sanger DNA sequencing  to confirm insertion of respective sgRNA using the human U6 primer (5′- GAG GGC CTA TTT CCC ATG ATT-3′).Single guide RNAs (sgRNA) targeting protein-coding genomic DNA sequences of target genes were designed using Benchling  or LRG2.1_mOrange (Addgene #124772) encoding the sgRNA of interest or LentV-Cas9-puro (Addgene #108100), 2 μg of pMD2.G (Addgene #12259), and 2 μg of pCMVR8.74 (Addgene #22036) were co-transfected using 24 μL of jetPEI reagent in 1 mL of 150 mM NaCl solution . The growth medium was changed 24 h after transfection. Supernatant containing lentivirus particles was collected 48 h later and filtered with a 0.45 μm polyvinylidene fluoride filter , aliquoted in 1.5 ml cryotubes, and stored at −80 °C until further use. Transduced cells were selected with 1–3 µg/mL puromycin for 1 week. Selected Cas9 expressing cells were transduced with lentivirus containing sgRNAs targeting selected genes  and all following passages using the CytoFLEX Flow Cytometer . In each passage, cells were washed with FACS-wash (FW-1% FBS in PBS) in 96-well V-shape plates and stained with DAPI (0.1 µg/ml)  for 5 min. All investigated cell lines were gated individually to exclude debris, doublets, and dead (DAPI+) cells.Stable 10) [+ or mOrange+ cells at each passage, relative to passage 0 (3 days after transduction) and was used as a readout of effects on cell fitness or drug sensitivity conferred by the CRISPR-Cas9 mediated gene mutations. Data analysis was performed using FlowJo v10 BD  and R/Bioconductor scripts. Of note, due to the high number of guides applied in this study and the number of cell lines investigated, each assay was performed at least in two independent experiments with the negative control AAVS1  and at least one positive control (RPA3 or PCNA). The time for cell passage up to 24 days was defined as the minimal time to observe an increase in the dropout higher than 1.7, for essential genes across the different cell lines  and carboplatin using a concentration causing approximately 10% growth inhibition (IC10)  for addig for 15 min at 4 °C. Clarified lysates were boiled in 1x sample buffer  at 95 °C for 5 min and resolved by SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane  and blocked with 5% (w/v) bovine serum albumin in TBS-T  for 1 h at room temperature. The membrane was incubated with one of the listed primary antibodies . Original membranes can be found in Supplementary Fig. 2.EOC cell lines were lysed in 1x radioimmunoprecipitation assay buffer  containing proteinase inhibitor cocktails . Lysates were clarified by centrifugation at 18,000 × BRCA1-edited cells, DNA damage was induced with 10 Gy y-radiation by a Caesium-137 source using the Gammacell 40 exactor  and incubated for an additional 24 h followed by fixation with 4% paraformaldehyde. After fixation, cells were permeabilized with Triton X-100 0.25% in PBS for 5 min followed by incubation with blocking solution (5% FBS with 1% BSA in PBS 1% Triton-X 100) for 1 h. After blocking, cells were incubated with one of the listed primary antibodies  for up to 48 h, fixed with 4% paraformaldehyde, and rinsed three times in PBS for 5 min each. For the evaluation of DNA repair capacity in Cas9 expressing non-transduced (control) or transduced cells with sgRNAs targeting AAVS1 (mock) sg1_AAVS1_5′-ACT GTT GAC GGC GGC GAT GT-3′, sg2_AAVS1_5′-GCT GAT ACC GTC GGC GTT GG-3′; TP53 sg1_TP53_5′-AGA TGG CCA TGG CGC GGA CG-3; or RPA3 sg1_RPA3 5′-CCC AGG TCG CGC ATC AAC GC-3′ was extracted using the DNeasy Blood & Tissue Kit  5 and 8 days after transduction. Locus targeted by TP53, RPA3 guide RNAs were amplified using the primers listed in Supplementary Table Genomic DNA of Cas9 expressing cells were transduced with lentivirus supernatant encoding sgRNAs targeting AAVS1, ATM, BRCA1, and CDK12. The percentage of cells harboring sgRNAs targeting the genes of interest was evaluated 3 days after lentivirus transduction and can be found in Supplementary Fig. + cells. Complete growth medium with and without drugs was replaced every 3 days. Then the medium was removed, and the colonies were fixed at room temperature for 1 h and stained with 0.05% crystal violet (Sigma-Aldrich) in 4% formalin (Formafix AG), the plates were then rinsed 3–4 times with water, dried and images of the plates were taken using the Fusion FX7 Edge Imaging System (Witek AG).Drug sensitivity was determined by the MTT and the colony formation assays. OVCAR3 and OVCAR8 To evaluate proliferation, cells were seeded at 1500 cells/well density into 96 well plates and incubated for up to 4 days. At each time point, MTT dye was added at a final concentration of  0.5 mg/ml and incubated for 3 h and optical density was measured as mentioned above.www.R-project.org). All negative and positive controls were performed on multiple replicates at least 3 times in each cell line. All experiments were performed at least in duplicates and statistical evaluation was performed using Prism 9 software (https://www.graphpad.com/scientific-software/prism/) or R/Bioconductor. Where applicable, evaluation was done using two-way ANOVA with correction for multiple comparison tests or the Wilcoxon test. p-values of <0.05 were considered statistically significant and presented as a value or as **p < 0.01, ***p < 0.001, ****p < 0.0001.Statistical analysis and figures were obtained through the use of the software R Studio version 3.6.1   in EOC cells may affect cell fitness in a gene-dependent manner and consequently outcompete with non-gRNA transduced cells during continuous cell passaging for up to 24 days  targeting the gene of interest  analysis, which decomposes raw sequencing traces into linear combinations of indel mutations [RPA3 target locus co-occurring with a decrease in the percentage of fluorescent/ RPA3_gRNA positive cells  after irradiation as compared with the controls  targeting BRCA1 and BRCA2, the most frequently single gene alterations used to prescribe PARPi in EOC patients [BRCA1 mutations confer olaparib sensitivity in a cell line-dependent manner. Of note, loss of BRCA1 and BRCA2 already impacted cell fitness in the absence of olaparib in most of the cell lines investigated, including OVCAR8, OVCAR3, and IGROV1 with reported BRCA1 promoter hypermethylation, chromosomal alteration in BRCA2 and mutations in BRCA1/2, respectively  , and frequently altered genes in EOC (n = 4). Mutagenesis of candidates resulted in a heterogenous outcome regarding the effect on cell fitness and olaparib response  indicating cell line-dependency. However, the enhanced olaparib sensitivity also correlated with an impact on cell fitness for those genes  failed to alter PARPi response upon genetic deletion. The loss of PARP1 and PARP3 only altered olaparib sensitivity in OVCAR3 cells without any alteration observed for the loss of PARP2. We used additional cell proliferation and survival assays to confirm the data generated by the CRISPR-Cas9 competition assay regarding the essential role of BRCA1 and enhanced sensitivity to olaparib and niraparib upon loss of BRCA1 and ATM  for further validation in combination with niraparib, talazoparib, and carboplatin. The different PARPi were selected based on their clinical application and PARP trapping potency [PARP1 failed to induce resistance in all the compounds tested. In the same line, talazoparib, the most potent PARP1 trapping inhibitor [Defects in homologous recombination have been associated not only with PARPi sensitivity but also with DNA-damaging agents (e.g. platinum compounds) , 21 and  potency . We obsenhibitor  failed t50 of olaparib was strongly positively correlated with response to niraparib, talazoparib, and cisplatin. Taken together, our functional screening together with publicly available data suggests a general mechanism of response for drugs that depend on DNA repair capacity for their efficacy, demonstrating the difficulty to identify specific predictive biomarkers for PARPi response.To evaluate whether the observed effects were specific for ovarian cancer models or a more generalizable and cancer type-independent effect, we accessed publicly available data regarding drug responses on a broad spectrum of cancer cell lines. As shown in Fig. CDK12, a previously reported gene conferring PARPi sensitivity in ovarian [Cas9 competition assay  [BRCA-wt/HRD positive population. However, only PRIMA and VELIA show an increased benefit in patients without BRCA mutation or HRD. In the recurrent setting accompanying patients with relapse after one or more lines of chemotherapy, Study 19 was the first to demonstrate that olaparib maintenance significantly improves progression-free survival in BRCAwt platinum-sensitive high-grade EOC [, and ARIEL 3 [BRCA-mutated, BRCAwt/HRD positive, BRCAwt/HRD negative) with the greatest benefit for the BRCA-mutated tumors. Based on these results, PARPi were also approved as maintenance therapy for platinum-sensitive recurrent EOC, and importantly, irrespective of biomarker status. Thus, clinically defined platinum sensitivity appears as the most reliable predictive factor for enhanced PARPi response. However, in frontline therapy, when platinum sensitivity is unknown, the clinical decision is still driven by BRCA and HRD status. MyChoice® CDx and Foundation Medicine’s FoundationFocus® CDx the two clinically approved HRD tests, only reflect a snapshot of the HR status failing to evaluate the dynamic changes that may occur during tumor evolution and treatment [BRCA HR-related genes, current ESMO guidelines also recommend further research, since there is still not sufficient evidence to predict PARPi response based on panels of individual genes [Several clinical trials have shown that PARPi therapy can be beneficial to a wider group of patients beyond cy (HRD) . In the cy (HRD) , PAOLA-1cy (HRD) , PRIMA/Ecy (HRD)  and VELIcy (HRD) ) demonstrade EOC . Afterwarade EOC , SOLO2 [rade EOC , and ARI ARIEL 3  trials dreatment , 14. Conreatment . In regaal genes .RNASEH2A [PTEN [PTEN loss only for OVCAR3 and IGROV1 with already reported alterations in BRCA1/2 genes. On the other hand, the altered olaparib response upon genetic loss of RNASEH2A was not associated with the BRCA status of the cell lines in our screen, suggesting a cell line-dependent effect rather than the BRCA status.To shed light on the predictive power of single gene aberrations conferring PARPi sensitivity, we performed a literature review and identified 93 genes reported to be involved in altered PARPi responses in gynecological tumors. However, we found inconsistent results regarding the outcome of gene manipulation and altered PARPi responses for the RNASEH2A , 53 and 2A [PTEN –57 in ouCas9 mutagenesis assay, we identified ATM, MUS81, NBN, BRCA2, and RAD51B as genes significantly associated with olaparib sensitivity in a cell-independent manner suggesting that mutations in those genes leading to loss of function enhance PARPi response. ATM is a master regulator of DNA damage response [ATM results in increased PARPi sensitivity. Despite additional validation studies being necessary, our data suggest that besides BRCA1/2 the genetic screen of ATM, MUS81, NBN, and RAD51B has the potential to increase the number of EOC patients that could benefit from PARPi therapy. We also identified that Cas9-mediated loss of PALB2, RAD51, RAD51C, RNASEH2A, XRCC1, and XRCC3 conferred olaparib sensitivity in at least 3 out of 6 cell lines. These results suggest that to some extent the effect of those genes on olaparib sensitivity can be dependent on several factors intrinsic to the cell lines such as genetic and epigenetic backgrounds [From the 33 genes assessed by our CRISPR-response , and altresponse –61. Howekgrounds , 63, andkgrounds . Therefo53BP1, ARID1A, ATR, BARD1, BRD4, CCND1, CCNE1, CDK4, CDK12, FOXM1, MRE11, NF1, PARP1, PARP2, PARP3, PIK3CA, PTEN, RAD51D, RAD52, SHLD2, XRCC2 on olaparib sensitivity. Although we cannot rule out that the inconsistencies between our results and the literature may be linked to the selection of different cell lines with differential genetic backgrounds, BRCA, and HR status, a possible justification for these inconsistencies may also rely on the novelty of our study by addressing the effect of gene modification in cell fitness together with altered PARPi response. This bidirectional assessment of gene dependencies has been less addressed by previous studies, which we see as critical to clarify the specific role of the identified genes in altering drug responses and exclude a possible general effect of cell survival and proliferation upon gene editing. Moreover, in the CRISPR-Cas9 competition assay cells are analyzed shortly after gRNA transfection allowing them no time to adapt to the loss of the target gene, which can happen by gene downregulation assays using shRNA or classical gene knockout experiments using CRISPR-Cas9 which may also face the problem on clonal effects. In addition, we cannot rule out the possibility that the genetic loss of a single gene in an in vivo scenario alters the cell response to PARPi, either by interference with the tumor microenvironment or triggering immune response as pointed out by recent publications [We found apparent inconsistent results regarding the role of ications –68.CDK12 as a conservative vulnerability in EOC cell lines without altering the olaparib response. These results suggest CDK12 as a potential new target for targeted therapy in EOC being in line with the literature on other cancer types [CDK12 has been previously reported to be associated with altered PARPi response in ovarian cancer [CDK12 specific.Through a series of functional assays, we identified er types , 70. On n cancer , and sevn cancer , 72, 73.BRCA mutated HER2 negative metastatic breast cancer [The application of PARPi was demonstrated to be successful in several tumor types , 75, butt cancer . Moreovet cancer . In addit cancer , pharmact cancer , 80. Thet cancer , 49, 81.t cancer  rather tCas9 competition assay, testing gene-dependencies for PARPi response, and cell fitness excluding possible general effects on cell survival and proliferation upon gene-editing of the candidate genes. The non-consideration of the potential effects on cell fitness induced by gene editing can lead to a misinterpretation of the results and reduce the efficacy of promising “in vitro” validated candidates when transferred at a clinical level [To the best of our knowledge, this is the first study functionally testing the role of more than 30 genes regarding cancer cell fitness together with PARPi response. This study used a CRISPR-al level .Original Data FileReporting SummarySupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary figure 7Supplementary figure 8Supplementary figure 9Supplementary figure legendsSupplementary table 1Supplementary table 2Supplementary table 3


The compounds investigated in this study  were found to have antiproliferative activity against BxPC-3 and PC-3 cancer cell lines in micromolar concentrations (IC50 0.11–0.33 µM). Here, we studied the genotoxic potential of the tested compounds with alkaline and neutral comet assays, accompanied by immunocytochemical detection of phosphorylated γH2AX. We found that pyrazolotetrazolotriazine sulfonamides induce significant levels of DNA damage in BxPC-3 and PC-3 cells without causing genotoxic effects in normal human lung fibroblasts (WI-38) when used in their respective IC50 concentrations (except for MM134) and showed a dose-dependent increase in DNA damage following 24 h incubation of tested cancer cells with these agents. Furthermore, the influence of MM compounds on DNA damage response (DDR) factors was assessed using molecular docking and molecular dynamics simulation.Pyrazolo[4,3- DNA-damaging agents are frequently used in oncology to treat both hematological and solid malignancies. Platinum compounds , cyclophosphamide, chlorambucil, and temozolomide are some of the most frequently utilized agents in cancer treatment. The above-mentioned drugs trigger apoptosis in cancer cells by altering the chemical structure of DNA. Unfortunately, the efficacy of these drugs can be greatly diminished by a variety of factors that contribute to drug resistance development. When drug efflux and/or metabolism rise, the intracellular concentration of an anticancer agent can be reduced, which impairs its capacity to inflict sufficient DNA damage and cell death ,2.Many of the cytotoxic drugs that are routinely used to treat cancer induce significant levels of DNA damage, which causes cell cycle checkpoint activation and results in cell cycle arrest and/or cell death . HoweverA growing research area of heterocyclic compounds is still in the early stages of development, but it holds great promise for the discovery of new drugs. The development of new synthesis methods and the use of modern organic chemistry techniques have resulted in the rapid expansion of this area ,7. One oe]triazine class of compounds encompasses many unique chemical structures with broad biological activity, including inhibitory activity against carbonic anhydrase (CAs) tetrazolotriazine sulfonamides   is shown in Previously we have shown that the pyrazolotetrazolotriazine derivatives  examined with alkaline and neutral comet assays, accompanied by immunocytochemical detection of phosphorylated histone protein (γH2AX).In this paper, we present the genotoxic activity of pyrazolotetrazolotetrazolotriazine derivatives in IC50 concentrations descendent in the order of: MM134 , MM137 , MM139  and MM136 . Once again, an increase in DNA damage levels was observed with an increase in the compound concentration used (dose–response relationship), which is characteristic of genotoxic compounds. However, in most cases, the increase was not statistically significant . Of particular importance, the tested compounds induced lower levels of DNA damage in the normal cell line compared with cancer cell lines.In contrast, in the WI-38 cell line , MM136, The difference between the genotoxic activity of the tested compounds and positive control was more profound in the BxPC-3 cell line compared with the PC-3 cell line, where derivatives induced levels of DNA damage more closely similar to that induced by bleomycin.MM134, -6, -7, and -9 exhibited genotoxic activity in BxPC-3 and PC-3 cancer cell lines. The tested compounds exhibited varied DNA-damaging capacities across the cell lines used in the study. DNA damage observed after 24 h incubation with the compounds in IC50 concentrations was highest in the BxPC-3 cell line when cells were exposed to MM136 and MM139 derivatives, while exposure to MM139 and MM137 resulted in the most profound induction of DNA damage in the PC-3 cell line. Furthermore, the BxPC-3 cell line exhibited higher sensitivity to both compounds and bleomycin used in the experiment as a positive control. In contrast, incubation of normal human fibroblasts (WI-38 cell line) with the tested compounds resulted in the induction of lower levels of DNA damage compared with the cancer cell lines. Moreover, a dose-dependent increase in DNA damage was observed after a 24 h incubation of cancer cells with increasing concentrations of the tested compounds (dose–response relationship), which is considered a hallmark of genotoxic compounds.In summary, MM compounds resulted in a substantial heterogeneity of DNA damage, with values ranging from 0 to 100% tail DNA in the samples, which is consistent with the results obtained by other authors. This matter of producing 100% of tail DNA % can be attributed to some sort of bias using the software or the quality of the obtained images tetrazolotriazine sulfonamide—induced apoptosis of DLD-1 and HT-29 cells with observed down-regulation of mTOR kinase, soluble intercellular adhesion molecule-1 (sICAM-1), and cathepsin, together with up-regulation of beclin-1 B triazines investigated in this study was recently published by our group triazine derivatives  used in the subsequent assays were based on previously published MTT assay results and are shown in The concentrations of the pyrazolotetrazolotriazine sulfonamides, which were specific for the cell line and dependent on the compound used in the experiment. Genotoxicity testing confirmed the high antineoplastic properties of MM134, -6, -7, and 9. Moreover, these effects were restricted mainly to cancer cells, indicating selectivity in targeting malignant cells. The DNA damaging potential of the compounds may be associated with CHK1 kinase inhibition, as suggested by in silico investigations. However, this needs confirmation in further studies.The present findings showed diverse cellular responses to the tested pyrazolo[4,3-


Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain. For each cell line, the method used to generate the primary culture, a morphological description of the cells and species confirmation by sequencing of the partial 16S rRNA gene are presented. Further molecular analysis of the two new Ixodes ricinus cell lines and three existing cell lines of the same species revealed genetic variation between cell lines derived from ticks collected in the same or nearby locations. Collectively, these new cell lines will support research into a wide range of viral, bacterial and protozoal tick-borne diseases prevalent in Europe.Tick cell lines are important tools for research on ticks and the pathogens they transmit. Here, we report the establishment of ten new cell lines from European ticks of the genera  Anaplasma, Babesia, Borrelia, Ehrlichia and Rickettsia [Ixodes ricinus, which is distributed widely throughout the continent [Ixodes persulcatus in Eastern Europe, Dermacentor spp. in central and southern (and increasingly northern) Europe, and Hyalomma and Rhipicephalus spp. in southern Europe.Ticks are vectors of multiple viral, bacterial and protozoan pathogens of medical and veterinary importance, including tick-borne encephalitis virus (TBEV), Crimean-Congo hemorrhagic fever virus (CCHFV) and multiple species of the genera ckettsia . Within ontinent ,3. Amongontinent , other iRhipicephalus appendiculatus and Rhipicephalus microplus, and the North American species Ixodes scapularis. Prior to the start of the present study, cell lines were only available from two European tick species: the I. ricinus lines IRE11 [Rhipicephalus sanguineus line RSE/PILS35 [Tick cell lines form an increasingly valuable part of the toolkit for research on the biology and control of ticks and tick-borne pathogens ,7,8,9,10es IRE11  and the E/PILS35 .Argas reflexus and the ixodid (hard) ticks Dermacentor reticulatus, Hyalomma lusitanicum, Hyalomma scupense, I. ricinus and Rhipicephalus bursa. Species origin and absence of contaminating bacteria were confirmed by molecular analysis. This study follows and expands the preliminary report of the A. reflexus cell line and primary cultures derived from the two Hyalomma spp. and R. bursa [Part of the remit of the Tick Cell Biobank  is to geR. bursa .A. reflexus), submitted by a member of the public to a screening programme in Germany (D. reticulatus), or collected from mammalian hosts in the field, as detailed in Engorged female ticks were reared in a laboratory colony at the Friedrich-Loeffler-Institut (FLI) derived from individuals originally collected in Berlin (A. reflexus) or 28 °C . The ticks were examined daily for commencement of oviposition and subsequent embryonic development.The engorged female ticks were transferred to the Tick Cell Biobank where they were surface-sterilised in a horizontal laminar flow cabinet prior to the start of oviposition. Briefly, the ticks were immersed in 0.1% benzalkonium chloride for 5 min, 70% ethanol for 1 min and two changes of sterile deionised water, dried on sterile filter paper and transferred individually to sterile 35 mm plastic Petri dishes and incubated at 100% humidity in a sealed plastic box at room temperature . Using the flattened end of a glass rod, the eggshells were then gently crushed in HBSS, or complete culture medium if the egg batch comprised <100 eggs, to release the embryonic tissues. The resultant suspension was either transferred directly to a flat-sided culture tube  or, for large egg batches, centrifuged at 200× Four different complete culture media were used singly or in combination; all were supplemented with 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin, and all were prepared freshly each week. All components were sourced from Invitrogen  or Sigma , except where indicated. Complete L-15 comprised L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth (TPB) and 20% foetal bovine serum (FBS); H-Lac comprised HBSS supplemented with 0.5% lactalbumin hydrolysate and 20% FBS; L-15/MEM comprised equal volumes of L-15 (Leibovitz) medium and Minimal Essential Medium with Hanks salts supplemented with 10% TPB and 20% FBS; L-15B comprised L-15B medium [All cultures were examined weekly by inverted microscope prior to changing medium. Medium changes involved removal and replacement of between half and three-quarters of the medium volume. Subcultures, only initiated when significant cell multiplication was observed, were performed by adding an equal volume (2.2–2.5 mL) of fresh medium to the primary culture, resuspending the cells and tissue clumps by pipetting, and transferring half of the resultant suspension to a new flat-sided tube. The other half of the culture was retained in the parent tube. In some cases, two primary cultures were combined in one of the parent tubes after 6–44 months, to increase the total amount of cells and tissues in an attempt to stimulate cell growth.Subculture series were initiated from primary cultures exhibiting significant cell growth . In some cases, cell growth was stimulated by resuspending some of the cells and tissue clumps and allowing them to reattach. Subcultures were performed by adding an equal volume of fresh complete culture medium to the culture tube, resuspending the cells and tissues by pipetting, and transferring half of the resultant suspension to a new daughter tube.g for 5 min, the supernate was discarded, the cell pellet was resuspended in 1.5 mL of ice-cold complete medium, an equal volume of ice-cold complete medium with 20% dimethyl sulphoxide was added and the cell suspension was immediately divided in 1 mL aliquots into three ice-cold cryovials. The cryovials were frozen rapidly in dry ice and transferred within 30 min to the vapour phase of a liquid nitrogen refrigerator. Cryopreserved cells were resuscitated by rapidly thawing the cryovial in a 37 °C water bath; the contents were immediately added to 9 mL complete medium at room temperature and centrifuged at 200× g for 5 min. The cell pellet was resuspended in 2.2 mL of complete medium, transferred to a culture tube and incubated at 28 °C. When cryopreserving cells grown in culture medium with an FBS concentration below 20%, the FBS concentration in the freezing and thawing media was increased to 20%.When sufficient numbers of daughter tubes had been generated, the contents of 3–4 tubes were pooled and centrifuged at 200× All cell lines generated in the present study are available under Material Transfer Agreement from the Tick Cell Biobank.Ehrlichia ruminantium DNA was used as a positive control. PCR amplicons were visualised by agarose gel electrophoresis, positive PCR products were purified using a Qiagen PCR Purification Kit  or a PureLink™ Quick Gel Extraction and PCR Purification Combo Kit  following the manufacturer’s instructions, and Sanger-sequenced in both directions . Randomly-selected cultures of each cell line were screened for Mycoplasma spp. using two commercial tests, the Mycoalert Mycoplasma Detection Kit  and the PCR Mycoplasma Test Kit , following the manufacturers’ instructions.DNA was extracted from resuspended tick cells using a DNeasy Blood and Tissue kit , following the manufacturer’s instructions for cultured cells. To confirm the species origin, a 456 bp fragment of the tick 16S rRNA gene was amplified as described previously  was usedI. ricinus was conducted using PCRs targeting the tick coxI [trospA [I. ricinus cell lines IRE/CTVM19, IRE/CTVM20 and IRE11 [Further analysis of the species origin of the cell lines derived from eggs laid by ticks identified morphologically as ick coxI  and tros [trospA  genes fond IRE11  was alsocoxI and trospA gene sequences were generated using PhyML 3.0 [http://tree.bio.ed.ac.uk/software/figtree/) for visualisation and the final figures were produced using Inkscape 1.1.2 (https://inkscape.org).Pair-end sequences obtained from PCR amplicons were assembled to produce corrected consensus sequences using 4Peaks  and the MUSCLE alignment in AliView . The seqhyML 3.0  with 500hyML 3.0 . The modcoxI sequences and ON375792–ON375796 for trospA sequences.Sequences generated during this study that were not identical to already-published sequences were deposited in GenBank under accession numbers ON366971–ON366983 for tick 16S rRNA sequences, ON367517–ON367521 for Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain .D. reticulatus egg batch was processed in January 2019, ten days after the start of oviposition. The primary culture was initiated in L-15/H-Lac/L-15B medium and initially comprised clumps of embryonic tissues and individual semi-adherent haemocytes, which disappeared within a few weeks. Some of the tissues formed large assemblies of floating vacuoles, the contents of which exhibited a range of pH evidenced by colours varying from yellow (acidic) to pink   c. Haemoclkaline) d, reachelkaline) . The 16SMK671590 ), Spain MK671590 ) and GerMK671590 ).H. lusitanicum egg batches laid by engorged female ticks collected in February (n = 2) and May (n = 1) 2014 and processed 19–22 days after the start of oviposition. The culture media used initially were L-15, H-Lac, L-15/MEM, L-15/H-Lac, L-15/L-15B and L-15/H-Lac/L-15B. All nine primary cultures survived for at least 25 months, with subculture series initiated from most of them after 8–14 months. Two primary cultures in L-15/H-Lac and L-15/MEM died after 25 and 36 months, respectively, and three were lost to yeast contamination after 41 months. Three of the remaining four primary cultures yielded cell lines: HLE/LULS42 originally in L-15 and now in L-15/L-15B, and HLE/LULS43 and HLE/LULS48 originally in H-Lac and now in L-15/H-Lac/L-15B  to the H. lusitanicum sequence from Portugal (KU130444) and was 100% identical (96% query cover) to a H. lusitanicum sequence from Malta (MK946449 [Three primary cultures were initiated from each of three ac/L-15B . Establiemocytes a and laremocytes b. The 16KU130444 ) and 99. (Z97881 ). The 16H. scupense egg batches laid by engorged females collected in February 2016. The eggs were processed 24–35 days after the start of oviposition. Of the twelve resultant primary cultures, two were contaminated with mould and were discarded. The remaining ten cultures were maintained for at least 17 months, at which point two more were lost to yeast contamination. Cells began to grow in several of the primary cultures and subculture series were initiated, leading to establishment of two H. scupense cell lines, HSE/LULS51 and HSE/LULS59 in L-15 and L-15/H-Lac, respectively. HSE/LULS51, now at passage 11, comprises small, rounded cells and some areas of cells arranged as sheets or ropes, to which round cells may attach  and Russia  and 99.75% identical to a sequence from a Hyalomma detritum tick from China (KC203346 [H. detritum was recently reclassified as H. scupense [H. scupense ticks from Pakistan (MN726557 [Two or three primary cultures were initiated from each of five y attach c. In cony attach d. The 16KU130468 ). The HSKC203346 ), all wiscupense . The 16Skey MW5462 and 99.I. ricinus. The first, IRE/LUAP46, was derived from eggs laid by a tick collected in September 2015 and processed approximately three months later. The primary culture was initiated in L-15 and comprised tissue clumps and a few uncrushed eggs, some of which hatched into larvae that survived for several weeks in the culture medium as shown by the observation of limb movements and midgut peristalsis visible through the larval integument. Although the tissue clumps survived, no appreciable cell growth occurred for the first two years; during the last few months of this period, medium changes were conducted every 2–3 weeks as there was little evidence of metabolism. From 26 months onwards, metabolism resumed and from 29 months floating multicellular vesicles began to appear, accompanied by increased metabolism indicated by a drop in the pH of the medium (from red to orange-yellow colour). Weekly medium changes were resumed at 31 months and the first successful subculture was carried out at 34 months , Spain MK671589 ) and PorI. ricinus, IRE/LULS55, was derived from the progeny of three ticks collected in February 2018 and subsequently held at 15 °C instead of 28 °C for five months. Two primary cultures were initiated in L-15/H-Lac and L-15/L-15B from these eggs; as neither culture contained sufficient tissues to support cell line development, they were combined at six months in L-15/H-Lac/L-15B medium. Proliferation of very small round and spindle-shaped cells commenced at 18 months and the first subculture was carried out at 20 months  and an MW727263 ). HoweveKM211789  and GU07KM211789 ) and SpaKM211789 ). The IRI. ricinus prompted further examination of additional gene sequences amplified from these two lines and from DNA extracted from three other I. ricinus cell lines: IRE/CTVM19, IRE/CTVM20 and IRE11. The 16S rRNA sequences from IRE/CTVM19 and IRE/CTVM20, both derived from the same pool of four egg batches laid by UK ticks [I. ricinus sequences from Spain , SwedenMH645519 ) and ItaMH645519 ) among oGU074588 ) among oMF370649 ) and FraIxodes sp. ticks into two “haplogroups” based on the presence of AG or CT bases at position 184/185 in the 16s rRNA gene sequences from ticks reported as I. ricinus and I. inopinatus [I. ricinus cell lines and selected published sequences from ticks reported as I. ricinus, I. inopinatus or Ixodes sp. (I. inopinatus) in a single clade (87% support), while sequences from IRE/CTVM19, IRE/CTVM20, IRE/LUAP46 and IRE11 clustered in a separate clade (87% support) with ticks in the 184-CT haplogroup (predominantly ticks assigned to I. ricinus). The presence of the AG or CT bases for each haplogroup can be observed clearly in the alignment of the 16s rRNA sequences used in the analysis  to between 3 and 15 published sequences from ticks described as I. ricinus from Italy, Croatia, Slovakia and Finland, among others. The top eight matches for the cell lines IRE/CTVM19 and IRE11 were identical, but all differed from the top matches for the cell line IRE/CTVM20. The top ten matches for the two Spanish tick cell lines, IRE/LUAP46 and IRE/LULS55, were almost identical to each other, but nearly all different from those for the UK and German tick cell lines. BLASTN analysis limiting to only querying published I. inopinatus records (taxid:1538109) showed that coxI sequences generated from the I. ricinus cell lines were only 98.91% to 99.27%  identical to sequences derived from ticks assigned in GenBank as I. inopinatus from Tunisia, Morocco and Algeria  and not with the three I. inopinatus sequences in the phylogenetic analysis   b. This iountries ,41. Nuclo clades .R. bursa egg batch, 20 days after the start of oviposition in 2018. The culture in L-15/L-15B died after 10 months, but the primary culture in L-15/MEM survived and eventually began to proliferate. The first subculture was carried out at 31 months (n = 1) and 2019 (n = 2) using eggs laid by Spanish R. bursa ticks are also expected to yield cell lines. RBE/LULS58 comprises a mixture of small, fibroblast-like, relatively non-granular cells growing in spreading clumps, and larger clumps of rounded, sometimes vacuolated, cells  and Tur (Z97878 ) among oA. reflexus cell line ARE/LULS41 derived in less than two years from a single primary culture prepared with just nine embryos, to the H. lusitanicum lines obtained from three out of nine primary cultures that took 3.5–6.25 years to become established. These four cell lines, and the other new lines derived from D. reticulatus, H. scupense, I. ricinus and R. bursa, greatly extend the availability of European tick cell lines and thus the scope of in vitro research that can be conducted in natural pathogen–vector combinations.The establishment of continuous tick cell lines can be a long and challenging process, with a historically low success rate ,12. The Anaplasma, Bartonella, Borrelia, Coxiella, Ehrlichia, Francisella, Midichloria, Neoehrlichia, Rickettsia and Spiroplasma [D. reticulatus, Dermacentor marginatus and I. ricinus [Anaplasma phagocytophilum, Bartonella spp., Borrelia spp., Coxiella burnetii, Ehrlichia canis, Francisella tularensis, Neoehrlichia mikurensis and Rickettsia spp., as well as on symbiotic bacteria, such as Coxiella-like and Francisella-like endosymbionts and Spiroplasma. Further study, including the attempted amplification of additional bacterial genes or whole genome sequencing of the new cell lines, is necessary to determine whether or not any of them harbour bacterial DNA integrated in the tick cell genomes, as reported for Rickettsia africae sequences in two Amblyomma variegatum cell lines and suspected for Francisella sequences in some North American Dermacentor spp. cell lines [European ticks have been reported to harbour a wide range of pathogenic and symbiotic bacteria, including species of the genera roplasma ,46,47,48 ricinus ,50. In tll lines ,52.I. ricinus and I. persulcatus in Northern, Central and Eastern Europe, and CCHFV transmitted by Hyalomma spp. ticks in the Mediterranean and Balkan regions. Several studies on TBEV have used (directly or indirectly) the existing I. ricinus cell lines derived from UK and German ticks [I. ricinus lines derived from Spanish ticks will add a new dimension to research on TBEV–vector interactions. D. reticulatus is also suspected to be a natural vector of TBEV [I. ricinus, as higher TBEV titres were previously reported in cell lines from I. ricinus compared to those from non-vector tick species [Hyalomma anatolicum [Hyalomma spp. will broaden the scope of such studies. Whole genome and RNAseq screening of the new cell lines may also reveal hitherto undescribed novel tick-borne viruses [I. ricinus cell line IRE/CTVM19 [I. scapularis cell line ISE6 [European ticks are also involved in transmission of multiple zoonotic arboviruses of the families Flaviviridae and Reoviridae and the order Bunyavirales, mostly of importance for causing disease in humans , with than ticks ,58,59,60 of TBEV ,61, but  species . To dateatolicum ,63,64; t viruses , such asE/CTVM19  and the ine ISE6 ,68.D. reticulatus, I. ricinus and R. bursa are vectors of protozoan parasites of the genus Babesia causing disease in, depending on the species, humans and/or domestic animals. Despite several attempts using tropical bovine parasites [Babesia sp. in tick cells has yet been developed [Babesia spp. naturally undergo cycles of replication within infected ticks, the potential for development of such a system exists if suitable culture conditions and host cells can be identified. The new tick cell lines reported here could be tested for susceptibility to infection with a range of Babesia spp. prevalent in Europe, such as Babesia canis, Babesia divergens and Babesia ovis.arasites ,70, no ieveloped . As BabeI. ricinus from Germany [R. sanguineus from France [A. reflexus and D. reticulatus cell lines from Germany and the new H. lusitanicum, H. scupense, I. ricinus and R. bursa lines from Spain. The present study depended on the provision of ticks by co-authors and colleagues with access to laboratory colonies and field collection sites, and was therefore biased towards these countries. It is hoped that cell lines can be established in future from ticks originating from additional European regions to provide a more comprehensive coverage of the continent, thereby reflecting potential regional variations in genotype.The present study has increased the number of geographic locations within Europe from which ticks, giving rise to cell lines, have originated. Previously, cell lines were available from  Germany  and the  Germany , and from France . Both noI. ricinus cell lines derived from Spanish ticks collected at sites in La Rioja approximately 42 km apart. The 16S rRNA genotype of IRE/LUAP46, derived from progeny of a tick feeding in late summer (September), was most similar to archetypal I. ricinus sequences from many parts of Europe, and also to the sequences obtained from the cell lines IRE11, IRE/CTVM19 and IRE/CTVM20 derived from northern European ticks. In contrast, the 16S rRNA genotype of IRE/LULS55, derived from progeny of ticks feeding in early spring (February), was most similar to a different subset of I. ricinus sequences, as well as some described as I. inopinatus. The two species have been reported to be allopatric in Spain [I. inopinatus did not yet reach as far north as La Rioja. An extensive collection of ticks from this province was included in their study, based on 16S rRNA sequences, but they did not find any I. inopinatus amongst them [coxI and trospA, used in previous studies to differentiate between I. ricinus and I. inopinatus or to compare ticks from north Africa and Portugal with ticks from the rest of Europe [I. ricinus cell lines either. Several studies that demonstrate the genetic differentiation of European and North African I. ricinus populations [I. inopinatus in Southern Europe and North Africa, but call into question the numerous descriptions of this tick species in Central and Northern Europe. As can be observed in the present study and previously [I. inopinatus specimens, and the morphological identification can be challenging. Thus, an extensive study with accurate morphological description of tick samples and deeper genetic characterisation should be performed to investigate the occurrence of I. inopinatus across Europe.This issue is illustrated by the two new in Spain ; the autgst them . To invef Europe . Howeverulations  support eviously , the chaI. ricinus genotypes should help to resolve the issues surrounding the genetic structure of I. ricinus populations in Europe and the Mediterranean region [I. ricinus 16S rRNA genotype examined in the present study, the two cell lines IRE/CTVM19 and IRE/CTVM20, derived from the same pool of four egg batches originating from the same location in Southern England, were not identical. These two cell lines were previously found to locate separately in a phylogenetic analysis based on a smaller 16S rRNA gene fragment [coxI and trospA sequences from these two sister cell lines each matched most closely with a different set of published sequences.While it is outside the scope of the present study to determine whether or not the parent ticks that gave rise to the IRE/LULS55 cell line should be considered a separate species, the availability of cell lines with both archetypal and divergent n region ,36,72. Efragment , althougArgas species, and joins a small group of argasid tick cell lines [Carios capensis and Ornithodoros moubata cell lines reported previously [D. reticulatus, H. lusitanicum, H. scupense and R. bursa are also the first continuous cell lines to be established and reported from these species. Collectively, all the ixodid cell lines will have wide applications in research on the biology and control of tick-borne diseases of medical and veterinary importance in the European context, including tick-borne encephalitis, Crimean-Congo haemorrhagic fever, tick-borne fever, babesiosis and rickettsioses.In conclusion, the ten new tick cell lines reported here will expand the possibilities for studies on isolation, characterisation and control of European ticks and tick-borne microorganisms. ARE/LULS41 is the first continuous cell line derived from any ll lines ,74 that ll lines ,74,75. Aeviously ,74 in th


We propose a novel type of immunotherapy for bladder cancer using gold nanoparticles bound to a peptide of a bacterial toxin with anti-tumor capacities as listeriolysin O called Listeria nanovaccines. Here, we present the pre-clinical experiments on a mice model of bladder cancer and blood cells of patients with bladder cancer using these Listeria nanovaccines that activate the immune system, block the tumor immunosuppression environment, and reduce the tumor size. The impact of Listeria nanovaccines on the field of immunotherapies for solid tumors can be extended to other solid tumors containing lymphocyte infiltration. Therefore, we propose Listeria nanovaccines as immunotherapy for tumors such as melanoma, urothelial bladder carcinoma, non-small cell lung carcinoma, and glioblastoma.91–99 nanovaccines) as immunotherapy for bladder tumors. GNP-LLO91–99 nanovaccines showed adjuvant abilities as they induce maturation and activation of monocyte-derived dendritic cells (MoDCs) to functional antigen-presenting cells in healthy donors and patients with melanoma or bladder cancer (BC), promoting a Th1 cytokine pattern. GNP-LLO91–99 nanovaccines were also efficient dendritic cell inducers of immunogenic tumor death using different bladder and melanoma tumor cell lines. The establishment of a pre-clinical mice model of subcutaneous BC confirmed that a single dose of GNP-LLO91–99 nanovaccines reduced tumor burden 4.7-fold and stimulated systemic Th1-type immune responses. Proof of concept assays validated GNP-LLO91–99 nanovaccines as immunotherapy by comparison to anti-CTLA-4 or anti-PD-1 antibodies. In fact, GNP-LLO91–99 nanovaccines increased percentages of CD4+ and CD8+ T cells, B cells, and functional antigen-presenting DCs in tumor-infiltrated lymphocytes, while they reduced the levels of myeloid-derived suppressor cells (MDSC) and suppressor T cells (Treg). We conclude that GNP-LLO91–99 nanovaccines can work as monotherapies or combinatory immunotherapies with anti-CTLA-4 or anti-PD-1 antibodies for solid tumors with high T cell infiltration, such as bladder cancer or melanoma.This study presents proof of concept assays to validate gold nanoparticles loaded with the bacterial peptide 91–99 of the listeriolysin O toxin (GNP-LLO In the past 10 years, nanotechnology has been widely studied for cancer treatment, as nanoparticles can play a significant role in drug delivery systems ,3. In thAdjuvants are classical immunotherapies that activate the tumoral immune responses by acting on antigen-presenting cells (APCs) ; examples are inorganic nanoparticles, cancer vaccines, oligonucleotides, bacterial compounds, and antagonists of Toll-like receptors ,9. HowevListeria monocytogenes is a human bacterial pathogen also used for the treatment of several tumors such as prostate cancer, cervix carcinoma, or pancreatic cancer, using attenuated mutants that lack the C-terminal domain of the main virulence factor, listeriolysin O (LLO) [91–99 nanovaccines), to validate them as valid immunotherapies for bladder tumors and inducers of immunogenic apoptosis.Bladder cancer (BC) is the ninth most common malignancy diagnosed worldwide. As a tumor, BC is highly immunogenic and T cell-infiltrated. It has been postulated that a dysregulation of the immune system within the bladder promotes BC growth. However, to date, this immunosuppression within BC has not been characterized in detail . BC aris O (LLO) ,22,23. M O (LLO) . LLO see O (LLO) . The cla O (LLO) , LLO act O (LLO) ,6,7. HerB16.F10 murine metastatic melanoma, A375 human melanoma, MB-49 murine and human T-24 bladder tumor cell lines, TC-1 murine and A-549 NSCLC lung tumor cell lines, O627 murine glioblastoma cell lines, Hepa1-6 murine hepatocarcinoma cell lines, CHO hamster ovary cell lines and SV-40-induced IC-21 murine macrophage cell lines were obtained from ATCC . The RG-1 human glioblastoma cell line was a gift of J. L. Fernandez-Luna . C57BL/6 male or female mice  8–12 months of age were used in the study.Nine patients with advanced solid tumors were included in this study: three patients with stage IV melanoma before enrollment in immunotherapy (MEL-1 and MEL-3), a stage IIIB cutaneous melanoma after surgery resection (MEL-2), a lung and bladder carcinoma treated with cisplatin-etoposide (BC-1), two urothelial bladder carcinomas without treatment , a hepatocellular carcinoma treated with ablation by microwaves (HEP-1), a prostate adenocarcinoma treated with taxocel (PROST-1), and a multiform glioblastoma treated with temozolomide and radiotherapy (GLIO-1). MEL-1, MEL-3, BC-1, HEP-1, PROST-1, and GLIO-1 were diagnosed at the Medical Oncology Department; BC-2 and BC-3 were diagnosed at the Urology Department; and MEL-2, the stage IIIB cutaneous melanoma, was diagnosed at the Dermatology Department . The patients participated in the study voluntarily, signed an informed consent agreement at the physician consultation, and received an information document on the research study. Patients were able to revoke the informed consent at any time. Healthy donors were obtained from the blood bank at our hospital. Blood samples were obtained in heparin tubes at the Dermatology, Urology, or Medical Oncology Departments on the day of patient consultation and processed at the IDIVAL laboratory within the following 2 h.91–99 peptide with a C-terminal cysteamide (LLO91–99C(O)NHCH2CH2SH, 14 mg, purity 95%) was purchased from GenScript  and 5-(mercapto)pentyl-β-D-glucopyranoside (GlcC5SH) was prepared as reported previously by our group [91–99 peptide were prepared by the reduction of Au(III) salt with sodium borohydride following previously described procedures [91–99: 8.9 µg (LLO91–99)/0.182 mg of GNPs. In brief, LLO91–99C(O)NHCH2CH2SH  and GlcC5SH  were dissolved in deuterated water (750 µL). 1H NMR analysis of the mixture showed a Glc:LLO91–99 ratio of ~9:1 . The above-prepared solution of the ligands  was added with an aqueous solution of HAuCl4 , followed by an aqueous NaBH4 solution  in four portions under rapid shaking. The dark dispersion was shaken for 2 h and then filtered with 3 KDa MWCO tubes by centrifugal filtering. The black colloid was recovered with water and lyophilized (0.482 mg). The residue was re-dispersed in the minimum volume of water, loaded in SnakeSkin pleated dialysis tubing , and dialyzed against 3 L of water under gentle stirring, recharging with fresh distilled water every ~8 h over the course of 72 h. After lyophilization, 0.456 mg of GNP-LLO91–99 were obtained. The ratio of glucose/peptide in the GNP was determined by quantitative NMR (qNMR) in a Bruker AVANCE 500 MHz spectrometer: 0.456 mg of GNP-LLO91–99 was dispersed in D2O 99.9% (200 µL). Then, 80 µL of this solution was added to 40 µL of a 0.05% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP-d4) solution in D2O and 60 µL of D2O. 1H NMR analysis of the mixture allowed the calculation of the amount of peptide in the GNP-LLO91–99: 8.9 µg (LLO91–99)/0.182 mg (GNP). TEM (JEOL JEM-2100F working at 200 kV). A single drop (5 μL) of the aqueous dispersion (ca. 0.1 mg mL−1 in MilliQ water) of the GNPs was placed onto a copper grid coated with an ultrathin carbon film (Electron Microscopy Sciences). The grid was air-dried for 12 h at RT. Average gold diameter was 1.5 ± 0.5 nm, obtained by counting 600 particles in the TEM image as previously reported [2O, 0.1 mg/mL): The average gold core size was confirmed by UV-Vis spectra, which did not show an absorption maximum at around 520 nm, typical of gold nanoparticles with a larger core diameter [LLOur group . GNPs caocedures . Peptidereported . UV/Vis diameter .TM columns  to select monocytes (Mo) as CD14+ positive cells. Mo (1 × 106 of cells/mL) were cultured in 6-well plates  over 7 days using GM-CSF (50 ng/mL) and IL-4 (20 ng/mL) in RPMI-20% FCS medium. Differentiated MoDCs were 98% CD45+CD11c+HLA-DR+/−CD86−CD14− positive cells. Bone-marrow-derived dendritic cells  were obtained from C57BL/6 control or bladder-transplanted mice femurs and differentiated with GM-CSF (20 ng/mL) for 7 days. Differentiated DCs presented a phenotype of 98% CD11c+MHC-I+MHC-II+CD11b−/+CD40−CD86− cells.Leukocytes from whole blood cells were collected from the interphase of a Ficoll gradient and incubated with microbeads conjugated to a mouse IgG2a monoclonal anti-CD14 antibody and passed through MACS91–99 nanovaccines. Direct apoptosis was evaluated in the different tumor cell lines after incubation for 16 h with GNP-LLO91–99 (50 µg/mL). Immunogenic apoptosis implies the incubation of tumor cell lines with ½ supernatants of ex vivo differentiated DCs (for murine tumors) or MoDCs from healthy donors (for human tumors), pre-treated for 16 h with 50 µg/mL of GNP-LLO91–99 nanovaccines. Direct and immunogenic apoptosis was examined by FACS after double staining with the DNA marker, 7-AAD (7-AAD-PE), and the apoptotic marker, annexin V (annexin V-APC). The staining of cells with 7-ADD corresponded to necrotic cell death, whereas the staining of cells with annexin-V alone corresponded to early apoptotic programmed cell death (mean ± SD). Experiments involving human samples were performed three times and five times for mice assays. Results are expressed as the % of apoptotic cells ± SD of triplicate samples (p ≤ 0.5).Cell toxicity was first evaluated using Trypan blue staining and incubation for 16 h with GNP-LLO6 cells) in a volume of 100 µL (n = 10/group). At 14 days, tumor transplanted mice received or not (NT) a single intravenous (i.v.) inoculation of GNP-LLO91–99 nanovaccines (50 µg/mouse). On day 7, mice were sacrificed, and tumor sizes were measured with a caliper. Values shown for tumor volume (TV) were calculated using the following formula: (length × (width)2)/2, as reported [91–99 nanovaccines were performed as above, alone or in combination with the following antibodies: anti-CTLA-4 or anti-PD-1 (50 µg/mouse). Cell populations of TILs and spleens were analyzed by FACS. All experiments were performed at least five times.Murine MB-49 bladder cell lines  or B16.FCell surface markers of human MoDCs were analyzed by FACS using the following antibodies for human MoDC: anti-HLA-DR-FITC, anti-CD45-PerCP, anti-CD11c-APC, anti-CD14-PE, and anti-CD86-V450 (clone 2331). Murine spleens and TILs were analyzed by FACS using the following antibodies: biotin anti-IAb (clone AF6-120-1), anti-CD11c-PE (clone HL3), anti-CD40-APC , anti-CD86-V450 (clone GL-1), anti-CD4-FITC (clone RPA-T4), and anti-CD8-PE . All the samples were treated with propidium iodide to gate dead cells. Flow cytometry was performed with a FACSCalibur . Cytokines in mice sera, DC, or MoDC supernatants were quantified using multiparametric Luminex kits. In brief, IFN-gamma, IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IL-17A, KC/CXCL1, MIP-2, and TNF-α levels in mice serum samples were quantified using the Luminex 200 platform with a magnetic system  following the manufacturer’s instructions. Cytokine concentrations are expressed as the averages of three replicates in pg/mL ± SD. Human cytokines in MoDC supernatants were quantified using the multiparametric Luminex kit  following the manufacturer’s instructions.t-test was applied to mice with auto transplants; each group included 5 mice for all assays reported (p ≤ 0.05 was considered significant). Experiments in mice were performed at least five times each, and experiments with MoDCs from healthy donors or oncologic patients and tumor cell lines were performed three times each. ANOVA analysis was applied to cytokine measurements and flow cytometry analysis per the manufacturer´s recommendations (p ≤ 0.05 was considered significant). For statistical purposes, each flow cytometry sample was performed in triplicate. The GraphPad software was used for the generation of all the graphs presented.For statistical analysis, Student’s 91–99 nanovaccines might function as immunotherapies for bladder tumors and planned the following assays: adjuvant abilities using MoDCs from human donors, the in vitro immunogenic death of murine and human tumor cell lines, the in vivo anti-neoplastic action after the establishment of a mouse bladder cancer model, and immunotherapeutic actions in comparison to anti-CTLA-4 or anti-PD-1 immunotherapies.We initiated this study with the hypothesis that GNP-LLO91–99 nanovaccines to be used in all the experiments and performed the quality and toxicity controls in vivo and in vitro. GNP-LLO91–99 nanovaccines are formed by a gold core covalently linked to LLO91–99 peptide and β-D glucose (GNP-LLO91–99 nanovaccines schematic representation in 91–99 nanovaccines or LLO91–99 peptide (5–500 µM) were non-toxic for human MoDCs after incubation for 16 h, as determined by evaluating the cell viability with Trypan blue staining (91–99 nanovaccines or LLO91–99 peptide (5–500 µM)  or bladder carcinoma  from our institution  , patients with melanoma , lung and bladder tumors (BC-1), or urothelial bladder tumors (BC-2) (GNP-LLO91–99 bars in 91–99 peptides (data not shown) had no effect on the surface expression of MHC-I, MHC-II, or CD86 molecules, similar to MoDCs treated with saline  . GNP-LLO91–99 nanovaccines increased the production of Th1 cytokines in MoDCs of healthy donors, especially TFN-α and IL-12p70 , except for those reported with a low DC functionality such as hepatocellular carcinoma [MoDCs from patients with melanoma or lung and bladder carcinoma showed under non-stimulated conditions a clear Th2 cytokine pattern  that reflects the systemic cytokine pattern of the patient’s sera . Incubatarcinoma   or BC (murine MB-49 or human T-24 cell lines). We examined two types of apoptosis by FACS: direct apoptosis and immunogenic DC-mediated apoptosis. GNP-LLO91–99 nanovaccines induced low percentages of direct apoptosis onto BC or melanoma tumor cell lines, barely 5% in murine B16.F10 melanoma (black bars in 91–99 nanovaccines. GNP-LLO91–99 nanovaccines induced high percentages (35–55%) of immunogenic apoptosis in melanoma and BC , or glioblastoma (murine O627 or human RG-1 cell lines), while not in tumors with low T cell infiltration, such as in hepatocellular carcinoma (murine Hepa 1-6 cell lines) [91–99 nanovaccine induction of DC-mediated immunogenic apoptosis appears to explain their anti-neoplastic abilities.Once we evaluated the possibility that GNP-LLOl lines) ,13, ovar91–99 nanovaccines for BC, we established subcutaneous (s.c.) transplants of murine bladder MB-49 and melanoma B16.F10 tumor cell lines . Mice were intravenously (i.v.) inoculated in their tails with a single dose of GNP-LLO91–99 nanovaccines (50 µg/mouse), and 7 days post-treatment, mice were sacrificed, and sera and tumors were collected to examine the effects of nanovaccines  and blocked tumor growth at all times tested (TV columns).First, we performed experiments to examine the effects of a single dose of GNP-LLOvaccines b. GNP-LLvaccines a. GNP-LL91–99 nanovaccines in BC mouse models, analyzing the cytokines in mouse sera. We confirmed a Th2 cytokine versus a Th1 pattern in mice transplanted with bladder MB-49 cell lines  and lower levels of activated DCs (CD11c+MHC-II+) and macrophages (F4/80+)  of mice s.c. transplanted with melanoma or BC, and treated or not with GNP-LLO91–99 nanovaccine action reduced immunosuppressed cells  in both tumor models and increased activated DCs and macrophages  or in combination with anti-CTLA-4 (50 µg/mouse) or anti-PD-1 (50 µg/mouse) immunotherapies. GNP-LLO91–99 nanovaccines, alone or in combination with anti-CTLA-4 or anti-PD-1 antibodies, increased the percentages of DCs with an activated MHC-II+CD40+CD86+ phenotype of functional APC and the numbers of T and B cells , while these treatments reduced the percentages of suppressor innate cells such as MDSC (Ly6G+CD11b+ granulocytes) and Treg (CD25+ cells)  the antigen presentation capacities of MoDCs of patients with BC  and (ii) a Th1 cytokine pattern by releasing high levels of IL-12p70 and TNF-α with anti-neoplastic abilities.GNP-LLO91–99 nanovaccines presented anti-neoplastic abilities for BC, as they induced immunogenic apoptosis directed by dendritic cells and the cytokines released by them.In this study, GNP-LLO91–99 nanovaccines showed immunotherapeutic abilities for BC, as they blocked the immunosuppression status of BC, increasing the numbers of cytotoxic T cells and DCs within the tumors and decreasing the number of immunosuppressive cells . In fact, GNP-LLO91–99 nanovaccines adequately combine well and potentiate the action of ICI—both anti-CTLA-4 and anti-PD-1—supporting them as a novel nano-immunotherapy for BC.Moreover, GNP-LLOhttps://patentscope.wipo.int/search/es/detail.jsf?docId=WO2017144762 (accessed on 9 May 2022) and US20190045527 https://patentscope.wipo.int/search/es/detail.jsf?docId=US237388381&_cid=P21-KC8ZLF-10597-1 (accessed on 9 May 2022), entitled to Instituto de Investigación Marqués de Valdecilla (IDIVAL).F.R., S.Y.D. and C.A.D. participated as authors in the patent number WO2017144762


Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines  and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status. Anthracyclines belong to a class of natural cytotoxic antibiotics and represent highly potent antineoplastic agents widely used to treat a variety of tumor types . Since tDespite its efficacy, anthracycline therapy is associated with cumulative dose-dependent cardiotoxic side effects, such as ventricular contractility and cardiomyopathy, leading to heart failure . The othIn addition to the cardiotoxicity of anthracyclines, chemoresistance is a major reason for the failure of anthracycline therapy . In geneAnother promising strategy for overcoming Pgp-mediated drug resistance is based on combining DOX with thiosemicarbazones—di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)—to directly leverage lysosomal Pgp transport activity ,28. PrevIn this study, we focused for the first time on the combination of anthracyclines with thiosemicarbazones in pediatric solid tumors. Due to the improvement of therapeutic strategies, from aggressive surgical approaches to multimodal approaches with neoadjuvant chemotherapeutic treatments, almost 83% of children with a diagnosis of cancer become long-term survivors ,37. HoweABCB1 (the gene encoding Pgp) expression only in the SH-SY5Y neuroblastoma cells  and anthracyclines  using MTT assays with a subsequent analysis performed using Calcusyn software . The hyp50 dose of DpC and Dp44mT (for 24 h) was determined according to the micromolar concentration  varied according to the nanomolar concentrations , Daoy, and RD cells . In the In the next step, 200 nM Valspodar (VAL), a selective inhibitor of Pgp, was added to the consecutive design treatment to verify whether the mechanism of synergy was based on lysosomal sequestration of the compounds via Pgp B. We expAs an additional analysis of anthracycline sequestration into lysosomes, fluorescence microscopy was performed. DOX is an autofluorescent compound, which allowed us to observe its effect on cells 2 h after its addition to the culture medium. For this purpose, the DOX concentration was increased to 10 µM. The lysosomes were visualized by indirect staining using an anti-LAMP-2 antibody. KB-V1 cells served as positive controls for the expected lysosomal sequestration of DOX , whereasIn general, this analysis showed that DOX did not colocalize with LAMP-2 but was primarily localized to the nucleus in all five tested cell types –G. ThereBecause the initial hypothesis explaining the mechanism of synergy was rejected, we changed the design to the concomitant treatment C, which As synergy between both thiosemicarbazones (DpC and Dp44mT) and all three anthracyclines  was observed in all evaluated cell lines , Saos-2, Daoy, and RD cells) after both combined treatments (consecutive and concomitant designs), the possible mechanism of synergistic interaction between these compounds seemed to be universal. Therefore, we predominantly focused on the common mechanisms of action of both types of compounds used.2/M phase. To confirm this effect in our tested cell lines, propidium iodide staining with subsequent flow cytometry analysis was performed to identify the changes in the cell cycle after 3 days of treatment with IC50 doses of DOX, DAU, and MIT. Cell cycle arrest in the G2/M phase was detected in four of the tested cell lines: SK-N-BE(2), Saos-2, Daoy, and RD cells  in DNA molecules, which are associated with cell cycle arrest in the GRD cells . For thedetected . Arrest 2 phase and enter mitosis, which can lead to mitotic catastrophe [50 doses after 3 days. Immunoblotting revealed that both thiosemicarbazones effectively decreased CHEK1 levels in the SH-SY5Y, Saos-2, and Daoy cell lines, and a slight decrease in CHEK1 levels was also observed in the SK-N-BE(2) and RD cells  was investigated as a possible mechanism contributing to combination treatment synergy. The generation of DSBs after the selected treatment was verified by indirect immunofluorescence staining of the phosphorylated γ-H2AX protein, which is considered a biomarker of DSBs . Staininll lines –E. In gell lines –E. Enhanll lines . NeverthThe obtained results proved the synergism of the combined thiosemicarbazone and anthracycline treatments in all chosen cell lines derived from pediatric solid tumors but also demonstrated that overcoming drug resistance mediated by Pgp is not the main mechanism of the observed synergism, as initially hypothesized. Accordingly, another synergistic mechanism based on enhanced DNA damage and the inhibition of checkpoint kinases was suggested.Considering previous studies that demonstrated a positive correlation between Pgp expression and the synergistic action of thiosemicarbazones in combination with DOX ,30,33,41To evaluate interactions between compound combinations, we first used a consecutive design of treatment, which was reported as effective in the literature . Contrar50 doses from the micromolar to nanomolar level, but were also able to observe synergistic action of the compound treatments in all five cell lines. Therefore, it can be concluded that concomitant application is the most convenient administration regimen for the combined treatment of anthracyclines and thiosemicarbazones in the tested cell lines.To explore the least toxic regimen of treatment, we decided to change the combined treatment design from a consecutive to concomitant application of the drugs. With this change, we were not only able to reduce the thiosemicarbazone ICSince the suitable application of the selected compounds was determined, the next objective was to describe the possible mechanism of the observed synergy. The mechanism seemed to be universal because all the combinations of DpC or Dp44mT with DOX, DAU, or MIT acted synergistically in four different cell types . Therefore, we focused on the common features of thiosemicarbazone and anthracycline mechanisms of action.The cytotoxic activity of anthracyclines is exerted through several mechanisms. In addition to their ability to intercalate between base pairs of DNA and induce ROS generation, anthracyclines can inhibit TOP2 activity by covalently binding to the DNA–TOP2 complex . The potIn addition, ROS generated by anthracyclines cause DSBs in DNA and activate ATM-dependent pathways . ATM kinThere is evidence of cross-talk and functional redundancy between ATR-CHEK1 and ATM-CHEK2 after anthracycline treatment. It was proven that DNA damage caused by DOX elicits both check kinase pathways .The stabilization of the DNA–TOP2 complex, manifesting as DNA breaks, was observed after treatment with Dp44mT in a breast cancer cell type . AccordiOne promising strategy for enhancing the effect of anthracyclines is based on their combined application with drugs targeting cell cycle checkpoints (CHEK1 and CHEK2). In general, cell cycle arrest is a major mechanism of cellular resistance to drugs that cause DNA damage ,53. TherRegarding the obtained results, we can assume that the observed synergy between the selected thiosemicarbazones and anthracyclines is based on the following mechanism. Both types of compounds, thiosemicarbazones and anthracyclines, cause DNA damage by inducing replication fork stalling and ROS production. Therefore, their combined treatment leads to an increase in DSB induction. Moreover, thiosemicarbazones decrease CHEK1 expression and, thus, help cells with damaged bypass arrest, increasing the rate of cells undergoing mitotic catastrophe. Together, our results suggest a new mechanism of synergism between thiosemicarbazones and anthracyclines that is effective in tumor cells regardless of the Pgp expression.v/v), which has been shown to have no effect on cell proliferation relative to the control medium [The selected thiosemicarbazones Dp44mT (Cat. No. SML0186) and DpC (Cat. No. SML0483), the DOX (Cat. No. D2975000), DAU (Cat. No. D0125000), and MIT (Cat. No. M2305000), and Pgp inhibitor VAL (Cat. No. SML0572) were obtained from Sigma-Aldrich . All chemicals were prepared as stock solutions in DMSO  and then diluted in cell culture medium to achieve a DMSO concentration < 0.5% .RD rhabdomyosarcoma cell line (Cat. No. 85111502), SH-SY5Y (Cat. No. 94030304), and the SK-N-BE(2) (Cat. No. 95011815) neuroblastoma cell lines were purchased from the European Collection of Authenticated Cell Cultures . The Daoy medulloblastoma cell line (Cat. No. HTB-186) and Saos-2 osteosarcoma cell line (Cat. No. HTB-85) were purchased from the American Type Culture Collection . The KB-3-1 and KB-V1 cervical carcinoma cell lines were gifts from Prof. Nóra Kucsma . The selected cell lines were cultured in various culture media, as shown in 50 values for the anthracyclines and thiosemicarbazones in the SH-SY5Y, SK-N-BE(2), Saos-2, Daoy, and RD cell lines. The cells were seeded in 96-well plates at a density of 5 × 103 cells/well , Saos-2, and RD cells) or 2 × 103 cells/well (Daoy cells) in 100 µL of complete DMEM, and then allowed to adhere overnight. Different seeding densities for the cell lines were chosen to ensure that the cells remained in the log phase of growth during all 3 days of treatment. The IC50 values for the individual compounds were calculated from the growth inhibition curves obtained after treatment with increasing compound concentrations based on the initial estimated IC50 values . The IC50 values for both anthracyclines and thiosemicarbazones were obtained after 3 days of treatment. For thiosemicarbazones only, the IC50 values were also obtained after 1 day of treatment.MTT proliferation assays were used to determine the IC50 values .To determine the combination index (CI) value, three different experimental designs of compound application were chosen . The “coFor direct observation of DOX administration in cells, the cells were seeded onto coverslips in Petri dishes (35 mm in diameter) and allowed to grow to 80% confluence at 37 °C. All the cell lines were then treated with 10 µM DOX and observed after 2 h of incubation at 37 °C.50 doses of DOX, DpC, Dp44mT, or with their combinations (DOX + DpC or DOX + Dp44mT) and incubated at 37 °C for 2 days.An indirect immunofluorescence assay was employed to reveal DSBs in DNA by staining for phosphorylated histone γH2AX. The cells were seeded onto coverslips and allowed to grow to 80% confluence as described above. The cell lines were then treated with the respective IC5/dish. The cells were then treated with the respective IC50 doses of DOX, DAU, and MIT and incubated at 37 °C for 3 days.To analyze the cell cycle using propidium iodide staining, all the tested cell lines were seeded in Petri dishes (90 mm in diameter) at a density of 1 × 1050 doses of DOX, DpC, Dp44mT, or with their combinations DOX + DpC or DOX + Dp44mT and incubated at 37 °C for 3 days. Immunoblotting was performed to determine the levels of CHEK1 and CHEK2. The cells were seeded in the same manner as in the cell cycle analysis described above. The cells were then treated with the respective ICA colorimetric MTT assay was performed to evaluate the cell proliferation rate as previously described . BrieflyThe CI values for the compound combinations were calculated to quantitatively compare the dose–effect relationship of each compound individually and in combination to determine whether a selected combination acts synergistically. CI values were obtained from growth inhibition curves using the constant ratio of the compounds in combination (1:1) as previously described . The CI The relative expression of selected genes was determined using RT-qPCR. Total RNA was isolated using a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and reverse transcribed into cDNA as described previously . qPCR waProtein extracts were obtained using LB1 lysis buffer . Total proteins (25 μg/well) were loaded onto 8%  or 10%  polyacrylamide gels and electrophoresed. The separated proteins were then blotted onto PVDF membranes . The membranes were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20 at RT for 1 h. Subsequently, the blocked membranes were incubated overnight with primary monoclonal antibodies. The following day, the membranes were washed in TBS-Tween and incubated with secondary antibodies at RT for 1 h. The primary and secondary antibodies are listed in Indirect fluorescence analysis was performed as described previously . The celThe treated cells were harvested from Petri dishes using Accutase  and fixed in cold 70% ethanol (Sigma-Aldrich) at 4 °C for 30 min. The fixed cells were washed with PBS, and the pellets were carefully resuspended in 100 µL of Vindelov’s staining solution (1 M Tris pH 8 (Sigma-Aldrich); 10 mM NaCl (Sigma-Aldrich); 5 µM ribonuclease A (Sigma-Aldrich); and 75 µM PI) . After 3t-test followed by the Games–Howell post hoc test. * p < 0.05 indicates significant differences compared with the respective control group.All experiments were performed in biological triplicates (unless otherwise specified). Numerical data are presented as the means ± standard deviation (SD). The data obtained by qPCR or immunoblotting were analyzed using SPSS Statistics software  by unpaired Welch’s