Cell lines serve as tools for understanding molecular signatures of predictive biomarkers. However, the use of highly passaged commercial cell lines has to be questioned, as these do not closely resemble the originating tumor. Here, we established patient-derived cell lines from MPM tumors and performed whole exome and mRNA sequencing to understand genomic and transcriptomic differences between MPM tumors, patient-derived cell lines, and commercial cell lines. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines than with commercial cell lines. These findings are of major relevance for the scientific community in regard to using cell line models to study predictive biomarkers and to interpret preclinical results which cannot be translated in clinical practice.FBXW7, CDKN2A, CDKN2B, and MTAP, all known to drive MPM tumorigenesis. Patient-derived cell lines correlate to a high degree with their originating tumor. Gene expressions involved in multiple pathways such as EMT, apoptosis, myogenesis, and angiogenesis are upregulated in tumor samples when compared to patient-derived cell lines; however, they are downregulated in commercial cell lines compared to patient-derived cell lines, indicating significant differences between the two model systems. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines rather than commercial cell lines. These results are of major relevance for the scientific community in regard to using cell lines as an appropriate model, resembling the pathway of interest to avoid misleading results for clinical applications.Cell lines are extensively used to study cancer biology. However, the use of highly passaged commercial cell lines has to be questioned, as they do not closely resemble the originating tumor. To understand the reliability of preclinical models for Malignant pleural mesothelioma (MPM) studies, we have performed whole transcriptome and whole exome analyses of fresh frozen MPM tumors and compared them to cell lines generated from these tumors, as well as commercial cell lines and a preclinical MPM mouse model. Patient-derived cell lines were generated from digested fresh tumors and whole exome sequencing was performed on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples, corresponding patient-derived cell lines, and normal tissue. RNA sequencing libraries were prepared from 10 fresh frozen tumor samples, the 10 corresponding patient-derived cell lines, and 7 commercial cell lines. Our results identified alterations in tumor suppressor genes such as  Malignant pleural mesothelioma (MPM) is an aggressive and rare tumor with a poor prognosis arising from the pleural lining. For inoperable MPM, the current standard of care is a platinum-based chemotherapy or an immunotherapy combination, the later showing a benefit in overall survival ,2,3. DesTo study tumor biology and to discover targets for treatment, it is crucial to use reliable preclinical models. For cancer research, these include cell lines, 3D organoids, or systems in living organisms, in particular rodents (summarized in ). SyngenPreclinical testing of drugs on cancer cell lines is a common practice to study cancer biology and molecular pharmacology. However, the relevance of highly passaged commercial cell lines has to be questioned, as they might not closely resemble the originating tumor . In lungExtensive studies investigating transcriptomic and proteomic changes have been performed in various cancer types, such as breast cancer, ovarian cancer, glioblastoma, and hepatocellular carcinoma, indicating significant differences between commercial cancer cell lines and patient-derived (low-passage-number) cell lines compared to the originating tumor material ,10,11,12The aim of this study was to compare transcriptomic and genomic profiles of patient-derived cell lines and their originating tumors with commercial cell lines in order to understand which model system should be used to answer which research question. Moreover, we analyzed the transcriptome of a syngeneic MPM mouse model and correlated this to the human disease. Our data will help to understand the mesothelioma biology and how to choose appropriate preclinical models for future studies.Tumor tissue and clinical data were collected from 10 patients undergoing surgical resection before the start of chemotherapy between 2018 and 2020 at the University Hospital Zurich. Patients included in this study had a median age of 66 years (range 64–90 years). Nine (90%) male and one (10%) female patient were included with seven (70%) epithelioid and three (30%) biphasic histological subtypes. The progression free survival (PFS) from the start of treatment was 10.6 months (range 5.62–18.63 months) and the average overall survival (OS) was 13.88 months (range 6.24–38.73 months) . All pat2 in DMEM  or RPMI 1640  supplemented with 10% heat-inactivated FBS , 2 mM L-glutamine, 50 units Penicillin, and 50 µg/mL Streptomycin . All cell lines were negatively tested for mycoplasma using the VenorGeM Mycoplasma PCR detection kit  according to the manufacturer’s instructions.The following commercial cell lines were used in this study: MSTO-211 (ATCC CRL-2081), NCI-H28 (ATCC CRL-5820), Mero82 (ECACC 09100105), ACC-MESO1 (RCB RCB2292), ONE58 (ECACC 10092313), ZL55 (ECACC 12050301), and the MeT-5A control cell line (ATCC CRL-9444). Cells were cultured in a humidified incubator at 37 °C and 5% COThe patient-derived cell lines used in this study, MPM659 (biphasic), MPM665 (biphasic), MPM671 (epithelioid), MPM673 (epithelioid), MPM680 (epithelioid), MPM686 (epithelioid), MPM690 (epithelioid), MPM693 (biphasic), MPM700 (epithelioid), and MPM716 (epithelioid), were established in our laboratory.g for 5 min and plated out in 10 cm collagen-coated dishes . Amphotericin B  was supplemented until passage two. After the third passage, Human FibrOut 1  was supplemented into the MPM medium to deplete fibroblast contaminations.Tumor tissue was cut into 3 × 3 mm pieces and either frozen in FBS containing 10% DMSO and stored in liquid nitrogen until further use or digested using the human Tumor Dissociation Kit  according to the manufacturer’s instructions. In short, tumor pieces were incubated with the enzyme mix in RPMI 1640 three times for 30 min at 37 °C with thorough shaking by hand in between. After the incubation, the remaining tissue was smashed through a 100 µm cell strainer and the cells were washed with RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 50 units Penicillin, 50 µg/mL Streptomycin, 25 mM HEPES (Sigma-Aldrich), 1× MEM Non-Essential Amino Acids Solution (Gibco) (MPM media), and 0.25 µg/mL Amphotericin B (Gibco). The cell suspension was centrifuged at 450× Patient-derived cell lines were seeded in 8-well µ-Slides  to check for potential fibroblast contaminations. After 72 h of incubation, the slides were washed with TBS, fixed for 10 min in Histofix , blocked for 2 h at room temperature (RT) with 10% goat serum , and incubated with a rabbit anti-Calretintin antibody  and a mouse anti-TE7-fibroblast antibody  over night at 4 °C. The next day the slides were incubated with a goat anti-rabbit AlexaFluor594 antibody  and a goat anti-mouse AlexaFluor488 antibody  for 2 h at RT. The slides were mounted with VECTASHIELD Vibrance Antifade Mounting Medium containing DAPI , scanned on a CLSM–Leica SP8 CLSM, and analyzed using Fiji (version 2.9.0) [2O2, a primary antibody incubation for 30 min at RT, and heat-induced epitope retrieval using HIER2 solution with a Bond Polymer Refine Detection kit . The slides were imaged using a Zeiss Axio Scan.Z1 and analyzed using QuPath (version 0.4.2), tumor regions were assigned using H&E stainings, and positive tumor cells were grouped based on DAB  staining intensity into low, medium, and high expressing cells.Four µm thick sections were cut from paraffin blocks and mounted on positively charged glass slides. The tissue was stained for Calretinin , Mesothelin , NF2 , and Podoplanin  using an automated IHC platform (Ventana Benchmark). Stainings were performed using a pretreatment of 30 min with HProtein extracts were obtained by lysing cells in TNN buffer supplemented with 1× Complete ULTRA protease inhibitor  and 1× PhosSTOP . A total of 25 µg protein was separated on 4–10% Criterion TGX Stain-Free Protein Gels  or Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and immediately transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (BioRad) or Trans-Blot Turbo Mini Transfer Packs (BioRad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at RT followed by incubation with anti-human CDKN2A antibody , anti-human Mesothelin antibody , anti-human Podoplanin antibody , and anti-human NF2 antibody  overnight at 4 °C. The detection antibody HRP anti-rabbit IgG  was incubated for 1 h at RT. Membranes were imaged using either Clarity Western ECL Substrate (Biorad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) on a Fusion FX . GAPDH and α-Tubulin were used as loading controls, anti-human GAPDH antibody , and anti-human α-Tubulin antibody .DNA was isolated from 2–3 0.6 mm punches from tumor and healthy tissue regions from formalin-fixed, paraffin-embedded (FFPE) blocks at the Institute of Pathology and Molecular Pathology at the University Hospital Zürich using the Maxwell 16 FFPE Tissue LEV DNA Purification Kit . DNA was further isolated from patient-derived cell lines using the PureLink Genomic DNA Mini Kit . Library preparation and sequencing was performed at the Genomics Facility Basel. Initial DNA quality controls were performed using the Agilent NGS FFPE QC Kit  and a Genomic DNA ScreenTape Analysis (Agilent). FFPE samples were treated with the NEBNext FFPE DNA Repair v2 Module (NEB). Sequencing libraries were prepared using the Human All Exon V6+UTRs kit (Agilent) according to the manufacturer’s protocol (SureSelect XT Automated Target Enrichment for Illumina Paired-End) including QC steps of DNA shearing, pre-capture libraries, post-capture libraries, and pooling using the HS NGS Fragment Kit (1–6000 bp) (Agilent). Sequencing was performed on a NovaSeq6000 Sequencing system with one lane of a S4 FlowCell v1.5 with a 200 Cycle reagent kit v1.5 .The nf-core/sarek pipeline (version 3.0.1)  written Eight-week-old female C57BL/6 mice were purchased from Janvier Labs and housed under pathogen-free conditions at the Laboratory Animal Service Center (LASC) of the University of Zürich. All animal experiments were performed in accordance with the Swiss federal and cantonal regulations on animal protection and were approved by The Cantonal Veterinary Office Zurich (ZH147/2018).2 in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 units Penicillin, and 50 µg/mL Streptomycin [6 RN5 cells were injected s.c. in the flank of C57BL/6 mice. Mice were euthanized after tumors reached a size of 800 mm3.Murine mesothelioma RN5 cells were cultured in a humidified incubator at 37 °C and 5% COptomycin . Cells w+ endothelial cells, CD45+ immune cells, and Ter119+ erythrocytes using the human/mouse Tumor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. In brief, single cell suspensions were incubated with 20 µL Non-Tumor Cell Depletion Cocktail A and 20 µL Non-Tumor Cell Depletion Cocktail B for 15 min at 4 °C. After incubation, the total reaction volume was adjusted to 1 mL using PBS supplemented with 0.5% bovine serum albumin (Roth) and the suspension was loaded on pre-activated LS Columns (Miltenyi Biotec) on a magnetic rack. The tumor cells in the flow through were collected and used for RNA isolation. The isolation purity was analyzed by flow cytometry using anti-human CD45-FITC antibody , anti-human CD31-PE antibody  (Ten human MPM tumors and six murine RN5 tumors were dissociated using the human Tumor Dissociation Kit (Miltenyi Biotec) for human samples and 600 U/mL DNAse 1 (Roche) and 100 mg/mL Collagenase 1 for murine samples. Tumor cell suspensions were depleted from CD317, 1:50) , anti-mo7, 1:50) . Dead ce6 cells and tumor samples after magnetic tumor cell isolation using the RNeasy Mini Kit . The quality of the isolated RNA was determined using a Fragment Analyzer (Agilent). Samples with a 260 nm/280 nm ratio between 1.8 and 2.1 and a 28S/18S ratio between 1.5 and 2 were further processed. The TruSeq Stranded mRNA protocol (Illumina) was used in the succeeding steps for murine samples. Briefly, total RNA samples (100–1000 ng) were poly-A-enriched and reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired, and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using the Fragment Analyzer (Agilent). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10 nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.Total RNA was isolated from cell line pellets containing 10The Novaseq 6000 (Illumina) was used for cluster generation and sequencing. Sequencings were paired-end at 2 × 150 bp or single-end 100 bp. The human libraries were prepared following the Smart-seq2 protocol . FollowiThe raw reads were first cleaned by removing adapter sequences, trimming low quality ends, and filtering reads with low quality  using Fastp (version 0.20) . The reaGene expression values were computed using the function featureCounts from the R package Rsubread (version 2.8.2) . The optDifferentially expressed (DE) genes were identified using the R package edgeR (version 3.36.0)  from BioTo identify contaminations of the bulk sequencing data, deconvolution was performed using the granulator package for R (version 1.2.0) . The pacGenes were ordered based on their difference expression and enrichment scores (ES) were calculated for every predefined gene set and normalized to all dataset permutations to obtain the normalized enrichment score (NES). Here, we used the Hallmark gene sets  to identNF2 gene [We characterized all human MPM tumors, which were used to establish patient-derived cell lines: MPM671, MPM673, MPM680, MPM68, MPM690, MPM700, and MPM716 were identified as epithelioid and MPM665, MPM659, and MPM693 as biphasic based on hematoxylin and eosin (H&E) staining a. The exNF2 gene  which caNF2, were found in all patient-derived cell lines.To characterize the established patient-derived cell lines, Western blots for the MPM markers Mesothelin and Podoplanin were performed c. MesothMUC19 contains missense mutations in six patient-derived cell lines, which were also found in three corresponding tumors. An amplification of MUC19 could be found in MPM700. MUC4 missense mutations were identified in four patient-derived cell lines and two corresponding tumors. NF2 mutations leading to short variants and impaired protein function were found in two patient-derived cell lines and their corresponding tumors and two additional tumors. FBXW7 mutations were identified in three tumors and one corresponding cell line. BAP1 and LRP1B mutations were found in the MPM693 tumor and corresponding patient-derived cell line , and immune system response in tumor tissue.GSEA between tumors and corresponding patient-derived cell lines with a false discovery rate (FDR) cutoff at 25% revealed a significant upregulation of gene sets including the reactive oxygen species pathway, oxidative phosphorylation, PI3K/AKT/mTOR signaling, and MYC targets in patient-derived cell lines, while tumors showed an upregulation of TNFα signaling via NF-κB, KRAS signaling, Notch signaling, TGFβ signaling, hypoxia, IL-6 JAK STAT3 signaling, IL-2 STAT5 signaling, inflammatory response, and Hedgehog signaling d,e.The comparison between patient-derived and commercial cell lines (FDR < 25%) revealed an upregulation of gene sets including EMT, UV response inhibition, IL-6 JAK STAT3 signaling, IL-2 STAT5 signaling, inflammatory response, and hypoxia in patient-derived cell lines . In contDue to the significant differences of EMT signaling in commercial and patient-derived cell lines and no significant differences between patient-derived cell lines and tumors, we decided to investigate genes involved in EMT regulation. We specifically investigated if specific genes involved in this pathway were regulated differentially. The reversible transition from an epithelial to a mesenchymal phenotype is accompanied by the downregulation of epithelial markers such as E-cadherin, Collagen type 6, and Cytokeratin 5/6 and an upregulation of mesenchymal markers such as α-SMA, N-cadherin, Cadherin 11, and Vimentin . The traCDH1 is significantly downregulated in all cell lines compared to tumors, but this difference is significantly higher in commercial cell lines compared to patient-derived ones. This is also true for other known markers of an epithelial phenotype as KRT5, KRT6A, LAMA3, and TJP1 [We compared our data with 84 previously published EMT signatures from the EMTome project . The EMTand TJP1 ,41  signaling in patient-derived cell lines only. The transcription factors ZEB1 and ZEB2 are significantly downregulated in all cell lines compared to tumor tissue.EMT signaling through the transcription factor GREM1 (Gremlin 1) is highly upregulated in patient-derived cell lines. It is known to induce an invasive phenotype in MPM through SNAI2 [TGM2 (Transglutaminase 2) is upregulated in patient-derived cell lines and downregulated in commercial cell lines. TGM2 plays a role in tumor formation and generates a stem-cell-like phenotype in mesothelioma [gh SNAI2 . TGM2  are expressed ubiquitously in almost all types of cancer except renal cancer [Histone deacetylases class 1 . This process leads to polyubiquitination of HIF1A by VHL and results in proteasomal degradation. Further negative regulation of HIF1A includes NAA10 (ARD) and RACK1.In the tumor microenvironment of most solid tumors, a state of low nutrients, high interstitial fluid pressure, and hypoxia is predominant. The lack of sufficient oxygen supply to dense tumor regions occurs due to a slower rate of angiogenesis and vascularization compared to tumor cell proliferation in cancer . HypoxiaNAA10, RACK1, and VHL was observed in the transcriptome of all cell lines compared to tumor tissue, with a higher similarity between patient-derived cell lines and tumors  and ERN1 (IRE1) [Furthermore, hypoxia can downregulate mTOR signaling to reduce oxygen consumption by decreasing protein synthesis . Due to 1 (IRE1)  are upreFor hypoxia-related genes, we could identify closer similarities of patient-derived cell lines to tumors than commercial cell lines with significance only for genes involved in negative regulation.Lastly, we were interested in the transcriptomic profile of the syngeneic mouse mesothelioma model RN5, by analyzing the RN5 cell line and the matching tumor to understand the reliability of this mouse model for preclinical testing . We analGene set enrichment analysis between the RN5 cell line and corresponding tumors with a FDR cutoff at 25% revealed a significant upregulation of the mitotic spindle Hallmark gene set in the cell line, while in the tumors the P53 pathway, UV response, TGFβ signaling, adipogenesis, EMT, notch signaling, hypoxia, glycolysis, angiogenesis, cholesterol homeostasis, and reactive oxygen species were upregulated c.Mtap and Tet2. Low overall expression of Bap1, Cdkn2b, Dnmt3a, Fbxw7, Nf2, Rb1, Tert, and Tet2 could indicate potential genomic alterations in these genes with Bap1 and Nf2 deficiencies previously described by Rehrauer et al. , CDKN2A (42.2%), CDKN2B (36.0%), NF2 (31.3%), MTAP (27.3%), TP53 (17.3%), SETD2 (10.2%), and PBRM1 (9.1%) [BAP1 (20%), CDKN2A (20%), CDKN2B (20%), MTAP (10%), and RB1 (10%) in patient-derived cell lines and their corresponding tumors. Alterations of NF2 (40%) and FBXW7 (30%) were also identified in tumors only, indicating a heterogeneity of tumor cells and clonal selection process in some patient-derived cell lines during the expansion process in vitro. A study by Ben-David et al. showed that clonal selection, rapid genetic diversification, and even gains of whole chromosome arms can occur in well-characterized commercial cell lines [On the molecular level, MPM tumors are characterized by genomic alterations in , CDKN2B .0%, NF2 ll lines . Moreove), SETD2 .2%, and ll lines . Due to DNMT3A (10%) and LRP1B (10%) could be identified. These genes are known to drive stem cell expansion, hypomethylation, and proliferation in hematologic malignancies and lung cancer [DNMT3A alterations correlate with shorter survival [Further mutations in g cancer ,61. In Msurvival . Here, wsurvival . There asurvival ,65. HoweBAP1 and CDKN2A are conserved in patient-derived cell lines, we compared SNV and CNV frequencies across MPM tumors, patient-derived cell lines, and commercial cell lines used in this study and further commercial cell lines with genomic data listed in the cell model passport  have shown promising results with the use of commercial cell lines ,78 and iBap1 and Nf2 deficiencies have previously been described by Rehrauer et al., reflecting two of the most commonly altered genes in human MPM [Preclinical mouse models are widely used to translate in vitro results into a more complex living organism. However, pathogenesis mechanisms and the involved pathways differ between the human and mouse disease including processes like-cell duplication time, lifespan, and cancer susceptibility . Here, wuman MPM . Gene seuman MPM . Recent uman MPM ,84. The uman MPM ,86. Howeuman MPM .Our results indicate a different regulation of multiple pathways of the murine RN5 model compared to human MPM tumors. Based on these data, this in vivo model can be of use; however, for pathway regulations or drug interactions specific considerations must be taken into account for each research question.Here, we established and characterized low-passage-number patient-derived cell lines and demonstrated that these are a suitable tool for preclinical studies of MPM, as they closely resemble the genome and transcriptome of originating tumors. However, depending on specific pathways of interest, an appropriate model has to be chosen in regard to murine preclinical MPM models. These findings are of major relevance for the scientific community to use suitable models and avoid results which cannot be translated into clinical practice.


Molecular subtype (MS) is one of the most used classifications of breast cancer (BC). Four MSs are widely accepted according to receptor expression of estrogen, progesterone, and HER2. The impact of adipose tissue on BC MS metabolic impairment is still unclear. The present work aims to elucidate the metabolic alterations in breast cancer cell lines representing different MSs subjected to adipocyte associated factors. Preadipocytes isolated from human subcutaneous adipose tissue were differentiated into mature adipocytes. MS representative cell lines were exposed to mature adipocyte secretome. Extracellular medium was collected for metabolomics and RNA was extracted to evaluate enzymatic expression by RT-PCR. Adipocyte secretome exposure resulted in a decrease in the Warburg effect rate and an increase in cholesterol release. HER2+ cell lines (BT-474 and SK-BR-3) exhibited a similar metabolic pattern, in contrast to luminal A (MCF-7) and triple negative (TN) (MDA-MB-231), both presenting identical metabolisms. Anaplerosis was found in luminal A and TN representative cells, whereas cataplerotic reactions were likely to occur in HER2+ cell lines. Our results indicate that adipocyte secretome affects the central metabolism distinctly in each BC MS representative cell line. Breast cancer (BC) is a complex and heterogeneous pathology with several classifications such as pathological and clinical tumor stage, histological type, differentiation grade, and molecular subtype (MS). Each of these classifications impacts prognosis and treatment strategies. One of the most influent classifications is the MS which includes the expression assessment of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). The stratification of receptor status distinguishes four main MSs of BC: luminal A, luminal B, HER2 positive (HER2+), and triple negative (TN) . This isAdipose tissue accumulation is associated with ER and PR positive BCs and with worse prognosis . Such acTo accomplish these proliferative needs, cells shift their metabolism towards carbohydrate anabolic pathways and to the well-established Warburg effect, for nucleotides and NADPH biosynthesis, amino acids requirements, and basic energetics . In the Adipose tissue was obtained in São João Hospital Centre, Porto, Portugal (CHUSJ) according to the protocol submitted to the Ethics committee of CHUSJ with the reference n. 226/2020. All subjects gave their informed consent. Procedures were executed in accordance with the international recommendations of Good Clinical Practice, the Declaration of Helsinki, and the applicable national legislation.2 atmosphere at 37 °C on Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12  supplemented with 17.5 mM of glucose , 1% penicillin/streptomycin , 18 µM of pantothenic acid , 100 µM of ascorbic acid , 16 µM of biotin , and 10% newborn calf serum . When preadipocytes reached confluency, they were differentiated into adipocytes. Cells were incubated for 3 days with DMEM/F12 medium supplemented with 17.5 mM of glucose, 1% penicillin/streptomycin, 18 µM of pantothenic acid, 100 µM of ascorbic acid, 16 µM of biotin, 3% NCS, 10 µg/mL of insulin , 0.1 μM of dexamethasone , 0.5 mM of 3-isobutyl-methyl-xanthine , and 1 μM of rosiglitazone . Cells were incubated with DMEM/F12 medium supplemented with 17.5 mM of glucose, 1% penicillin/streptomycin, 18 µM of pantothenic acid, 100 µM of ascorbic acid, 16 µM of biotin, 3% NCS, 10 µg/mL of insulin, and 0.1 μM of dexamethasone until the fully differentiated phenotype was reached, at day 9. Cell medium was changed every 2/3 days. The pool of adipocyte secretomes was collected from 7 assays performed from adipose tissue samples of different patients. The purpose of this approach was to control individual variability. Secretome was preserved in −80 °C until used as treatment. Photographs were taken for each time-point using Nikon TMS with attached camera Nikon D40× . Subcutaneous adipose tissue was mechanically degraded and digested with collagenase  at 37 °C for 1.5 h. The stromal vascular fraction was washed and filtered to obtain isolated preadipocytes, which were cultured at a 5% COAdiponectin was used as an obesity-related biomarker in the differentiation of adipocytes . AdiponeA representative breast cancer cell line of each molecular subtype was used according to the specifications described in TM, ATCC, Manassas, VA, USA), and MDA-MB-231  were cultured in DMEM , supplemented with 10% Fetal Bovine Serum  or 20% in BT-474 cell line, 1% penicillin/streptomycin , and 3.7 g/L sodium bicarbonate  and maintained at 37 °C in a humidified atmosphere containing 5% CO2. Serum-free DMEM was used as control and treatments were performed using the secretome of differentiated adipocytes for 24 h. The nutrient composition of both cell media is presented in MCF-7 , BT-474 (authenticated in i3S), SK-BR-3 (HTB-301H-NMR) for metabolite quantification. Samples were prepared by adding 45 µL of sodium fumarate  in deuterated water solution to 180 µL of culture media. The samples were loaded into 3 mm NMR tubes  and analyzed using the NOR5X3INSB optimizer inserts . Solvent-suppressed 1D 1H-NMR spectra were acquired on a Bruker Avance III HD 500 MHz spectrometer equipped with a 5-mm TXI probe , at 298 K. A zgpr pulse sequence was used and a total of 16 scans were acquired for each sample. After the acquisition, 1H-NMR spectra were processed, and chosen metabolite peaks were integrated using the NUTS-Pro NMR software . Sodium fumarate was used as an internal reference (6.50 ppm) for the quantification of the following metabolites : valine , isoleucine , alanine , acetate , glutamate , pyruvate , glutamine , lactate , and glucose . Results were normalized to β-actin total RNA , on the AU 5800 analyzer  according to the manufacturer instructions and São João Hospital Center patronized protocol. Total cholesterol concentration was determined by an enzymatic, colorimetric method through a commercial kit  according to the manufacturer protocol. Results were normalized to β-actin total RNA and presented as the difference between pre and post treatment.Triglyceride quantification was determined by enzymatic colorimetric assay , and maintained at −20 °C. Afterwards, RNA later was removed by centrifugation. Cells were then washed and resuspended in 500 µL PBS with 7 µL of Poly(A) . RNA extraction was performed using the Lab-Aid 824 s Nucleic Acid Extraction System . Total RNA was quantified using Multiskan SkyHigh Microplate Spectrophotometer . Total RNA concentration was standardized to 5 µg/mL.3 instrument  and a SYBR Green commercial kit . Primer sequences  are described in Real time PCR was performed in sealed 96-well microplates using qTOWERt-test between controls and treatments. Significance was set at p < 0.05.Results were expressed as mean ± SD. Data were analyzed with GraphPad Prism 6.0 . Adipocyte differentiation was evaluated by ANOVA test. Metabolomic, lipid, and PCR results were evaluated by Student’s The levels of glucose as well as glycolysis end products, pyruvate, and their dual metabolites, lactate, and acetate are presented in Under adipocyte secretome exposure, MCF-7 did not achieve significant alterations in glucose consumption nor in lactate and acetate production. Yet, our results show a decreasing trend for both lactate and acetate production when subjected to adipocyte secretome. Pyruvate variation was also not significantly affected by adiposity; however, our results point to a shift in pyruvate utilization from a consumption in the control condition, to a release under the influence of adipocyte secretome, although with a high standard deviation.p-value = 0.024).When compared to the other MS cell lines, luminal B representative cell line (BT-474) revealed lower levels of these four metabolites. Subjected to adipocyte secretome, these cells exhibited a significant increase in acetate consumption , resulting in a decrease in lactate production , implying that the Warburg effect in this aggressive cell line slowed down. A significant switch from acetate production in control conditions towards acetate consumption in adiposity conditions was also found . Altogether, these findings suggest that under exposure to secretome from adipocyte differentiation, the cell lines present distinct metabolic pathways.Finally, MDA-MB-231 cell line representing triple negative MS showed a significant decrease in both glucose consumption and lactate production under adiposity when compared to control. Under exposure to adipocyte secretome, these cells did not depend much on glucose uptake  and MDA-MB-231 cell lines , while in SK-BR-3 cells  and BT-474, alanine release was reduced.Alanine and pyruvate, for instance, can be directly interconverted. When exposed to adipocyte secretome, alanine metabolism was significantly altered in MCF-7, SK-BR-3, and MDA-MB-231 cell lines. Alanine shifted from production to consumption in MCF-7 . No significant difference was found in HER2+ representative cell line. This amino acid is directly converted into glutamate by glutaminase. Therefore, we next examined glutamate levels in our experiment.Glutamine consumption was significantly decreased in luminal A, luminal B, and TN representative cell lines when exposed to adipocyte secretome  and TN , accompanying the decrease in glutamine consumption levels observed. Interestingly, glutamate release increases significantly in luminal B  and HER2+  representative cell lines.Glutamate production in adipocyte secretome condition decreases significantly in luminal A .The role of branched amino acids, such as valine and isoleucine, is not clear in BC progression. We next investigated the level of these two amino acids in our study. No significant difference was found in any of the cell lines except for MDA-MB-231 TN cells, where these two amino acids were significantly less consumed after exposure to adipocyte secretome (Triglycerides (TG) and cholesterol levels in culture media were then addressed .No differences were found in TG levels under adipocyte secretome exposure when compared to control, although a slight increase was observed in MCF-7 and MDA-MB-231.p-value MCF-7 = 0.0179; p-value BT-474 = 0.0301; p-value SK-BR-3 = 0.0086; p-value MDA-MD-231 = 0.0005).In contrast, a significant increase in cholesterol release was observed in every MS representative cell line when subjected to adipocyte secretome  and a decreased expression of Hexokinase (HK), and of the enzymes that catalyze the late steps of gluconeogenesis: Fructose-1,6-bisphosphatase (FBP), and Glucose-6-phosphatase (G6P).In contrast to MCF-7 fingerprint, SK-BR-3 displayed significantly increased levels of glycolytic and gluconeogenic enzymes: HK, Pyruvate kinase (PK), Phosphoenolpyruvate carboxykinase (PCK), FBP, and G6P. We also observed a tendency towards increased Pyruvate Carboxylase (PC) expression, but statistical significance was not achieved. PFK expression was not altered in SK-BR-3 cells.Triple negative MDA-MB-231 representative cell line enzyme expression profile was similar to MCF-7, with a significant decrease in HK, FBP, and G6P and a decreasing trend in all other enzymes except PFK, which is increased under adipocyte secretome exposure.Given the results obtained in PFK expression, we further quantified the expression of TP53 Induced Glycolysis Regulatory Phosphatase (TIGAR), an enzyme that modulates glycolysis by targeting PFK. Results are illustrated in Carbohydrate metabolism and the Warburg effect in BC have been widely and extensively studied. However, the metabolic associated pathways are far from being fully understood. Tumor cell plasticity and the ability to use multiple metabolic pathways to increase survival advantage is acknowledged and recognized . RecentlOverall, we found three main similarities across the different molecular subtypes when submitted to adipocyte secretome. Firstly, there was a decrease in lactate production, which led us to postulate that the Warburg effect is attenuated by adipocyte released factors. One possible trigger for this scenario is the presence of high levels of fatty acids in the adipocyte secretome, which feed the TCA cycle, providing a robust energy source to tumor cells. A mechanism similar to the “glucose–fatty acid cycle” or “Randle cycle” seems to occur. This involves a competition between glucose and fatty acids, where glucose oxidation is able to inhibit the conversion of triacylglycerol to long chain fatty acids (LCFAs) and LCFAs are able to inhibit glucose oxidation. The absence of significant alterations of TG levels corroborates this hypothesis. Among others, PFK has an important role in the extent of glycolysis inhibition . This meAlso consistent across the molecular subtypes was the decrease in glutamine consumption. The association between glutaminolysis and tumor proliferative needs is well established, glutamine being the second most used energy substrate of tumor cells . GlutamiAnother common feature is the significant increase in the cholesterol levels. Redundantly, cholesterol was already described as a risk factor in BC . InternaWhen exposed to adipocyte secretome, no significant alterations were found in glucose, pyruvate, lactate, acetate, isoleucine, valine, and triglyceride levels in luminal A representative cell line. Contrarily to the other cell lines, acetate levels in extracellular medium did not differ from control in MCF-7. Nevertheless, cholesterol biosynthesis is significantly enhanced, implying that part of the acetate is driven to this steroid synthesis. In addition, the slight increase in pyruvate release is probably caused by the extensive consumption of alanine, which is transaminated to this keto acid. Part of this metabolite can either be used for acetyl-CoA synthesis in the mitochondria, thus keeping TCA cycle active, or result in fatty acid, membrane phospholipids, and cholesterol synthesis. Glutamine is significantly less consumed when subjected to adipocyte associated factors. This amino acid is rapidly converted into glutamate by glutaminase. The significant decrease of this latter amino acid in the medium suggests it is metabolized to α-ketoglutarate, entering the TCA cycle through an anaplerotic reaction as PFK was the only upregulated glycolytic enzyme. PFK catalyzes the phosphorylation of Fructose-6-phosphate to Fructose-1,6-bisphosphate in glycolysis, but it is also associated with several moonlight functions such as survival, apoptosis inhibition by BAD inactivation, alteration of cell shape and motility by binding to cytoskeleton, and transcription regulation ,24. We sIn control conditions, we observed that BT-474 cell line exhibits both a lower secretion of lactate, pyruvate, glutamate, and alanine, and a lower uptake of glucose, glutamine, isoleucine, and valine from culture media, when compared to the other cell lines. BT-474 is characterized by its high proliferation rates, which in turn leads to higher energetic/biomass requirements. We hypothesized that, given the high proliferative state, the majority of the metabolites are used by the cell, and not released to the extracellular medium. Similar results were described previously, namely with a decrease in glucose uptake compared to other cell lines . We founFurthermore, almost all glycolytic and gluconeogenic enzymes were upregulated, although without statistical significance. This is the case with PCK and G6P. Both PCK and G6P function are dependent on the origin tissue (gluconeogenic or non-gluconeogenic tissue). In non-gluconeogenic tissues, as the case of BC, G6P is involved in cell cycle regulation, survival, and migration. The major described function of PCK is cataplerosis by enhancing TCA cycle intermediates’ disposal that are then used in other pathways, namely pyruvate synthesis, glyceroneogenesis, pentose phosphate pathway, and most importantly glutaminolysis . GlutamaInterestingly, we found that the metabolic influence of mature adipocytes in SK-BR-3 is similar to the one found in BT-474. One could speculate that HER2 could be the connection. Up until now, no specific ligand has been described for HER2, although it is known that HER2 is the most favorable dimerization partner for the three other members of the EGFR/ErbB family—1, 3 and 4 . SeveralSK-BR-3 was the only cell line overexpressing all glycolytic and gluconeogenic enzymes when exposed to adipocyte secretome, which is in agreement with the high proliferative profile of this cell line. PFK expression is somewhat increased, attributed to the TIGAR upregulation, as previously discussed.Triple negative representative cell line, MDA-MB-231, exhibited significant alterations in most metabolites studied. Glucose uptake and lactate production were decreased when exposed to adipocyte secretome, revealing a diminished Warburg effect. MDA-MB-231 also revealed the most accentuated acetate consumption correlated with a heightened cholesterol production. Alanine switched from release in control to uptake in adiposity mimicking conditions. The conversion of this amino acid into pyruvate is likely to occur, which then suffers oxidative decarboxylation originating acetylCoA. Upon adipocyte secretome exposure, this cell line does not depend on glutaminolysis, as we observed by the lower consumption of glutamine. Glutamate, in turn, originates a-ketoglutarate entering the TCA cycle by an anaplerotic reaction. Valine and isoleucine are significantly decreased in the MDA-MB-231 cell line. Elevated levels of these branched-chain amino acids (BCAA) are associated with body mass index, although no consensus has yet been reached regarding the potential risk in breast cancer . MetabolConcordant with the results obtained from BT-474 and SK-BR-3, MDA-MB-231 has an enzymatic pattern identical to MCF-7. Only PFK is increased, although without statistical significance, corroborating the unchanged TIGAR expression. HK, FBP, and G6P are significantly downregulated in accordance with the proposed attenuated dependence on glycolytic and gluconeogenic pathways.Lastly, we would like to mention the study limitations. We are aware that cell culture medium from control and treatment are two different references with different nutrient composition. We overcome this by presenting the results as the difference between pre and post treatment. Additionally, we would like to highlight that: (1) we could not change the required medium for adipocyte differentiation since alterations would affect the protocol, and (2) we did not have access to adipose tissue from normal weight individuals. Moreover, we would like to mention that the use of only one representative line is not sufficient to extrapolate the results.Our findings suggest that adipocyte-related factors impact breast tumor metabolism beyond estrogen/progesterone receptors. HER2 receptor seems to play a major role in central metabolism since molecular HER2 receptor positivity subtypes share common features that differ from those with HER2 negative receptors. Metabolic imbalance is more evident in triple negative BC representative cell line MDA-MB-231.Overall, our results highlight the metabolic differences among molecular BC subtypes, relative to one representative cell line, but also the influence of the secretome of differentiated adipocytes on the central biochemical metabolism in each BC molecular subtype representative cell line. Further studies are needed regarding the importance of adipocyte-related factors in breast cancer development, which ultimately will provide new therapeutic guidelines.


Absidia glauca AM177 strain. The lactones were transformed into the hydroxy derivative, regardless of the type of halogen atom in the substrate structure. For all lactones, the antiproliferative activity was determined toward several cancer cell lines. The antiproliferative potential of halolactones was much broader than that observed for the hydroxyderivative. According to the presented results, the most potent was chlorolactone, which exhibited significant activity toward the T-cell lymphoma line (CL-1) cell line. The hydroxyderivative obtained through biotransformation was not previously described in the literature.The microbial transformations of lactones with a halogenoethylocyclohexane moiety were performed in a filamentous fungi culture. The selected, effective biocatalyst for this process was the  Lactones are cyclic, intramolecular esters that may differ in the size of the lactone ring. They occur naturally, mainly as plants’ secondary metabolites , but canFilamentous fungi are known for their ability to transform organic compounds with a wide variety of carbon skeletons ,21. The cis-5-(1-iodoethyl)-4-phenyldihydrofuran-2- one, as well as separate enantiomers, were transformed by three fungal strains: Absidia cylindrospora AM336, Absidia glauca AM254, and Fusarium culmorum AM10. Each biocatalyst carried out hydrolytic dehalogenation. The biotransformation processes were highly stereoselective, and enantiomerically pure hydroxylactones were obtained nonan-8-one3, ee = 16%), ESISMS m/z 184.1 ; 1H NMR  δ: 1.30 , 1.31 , 1.39 , 1.48 , 1.55  1.57–1.69 , 1.8 , 1.93 , 2.08 , 2.63 , 2.67 , 4.21 ; 13C NMR  δ: 13.9 (C-11), 19.1 (C-4), 20.1 (C-3), 24.3 (C-2), 25.1 (C-5), 32.4 (C-7), 37.6 (C-6), 69.2 (C-1), 82.2 (C-10), 171.2 (C-8). IR : 3421 (s), 2936 (m), 1718 (s), 1246 (m), 1147 (m).αThe study involved a series of cell lines CLBL-1 , CNK-894 (NIH/3T3) or 1 × 105 (canine cancer cell lines) cells per mL were inoculated in a 96-well-plate . Evaluated lactones were added in the increasing concentrations range of 6.25–50 μg/mL in culture medium (DMSO concentration was less than 1% in each dilution). After incubation for 72 h, 20 µL of MTT solution (5 mg/mL) was added to each well. After the contents dissolved, the optical density of the wells was measured with a microplate reader  at a reference wavelength of 570 nm. The results were determined from more than three independent experiments (four wells each) and presented as mean IC50 value ± SD.Cell proliferation was determined by the MTT assay  according to the literature described above . In summAbsidia glauca AM177 strain was selected as an efficient biocatalyst in biotransformation processes. The substrates were iodo-, bromo-, and chlorolactones with the cyclohexane system. In each process, the biocatalyst removed the halogen atom from the compounds, and the hydroxylactone was obtained as a hydrolytic dehalogenation product. The products were received with a slight enantiomeric excess not exceeding 16%. The antiproliferative activity of all compounds was evaluated toward the selected normal and cancer cell lines. All lactones did not exhibit cytotoxicity toward normal cells. The halolactones were more active than the hydroxylactone. The most potent was chlorolactone 3.From a panel of filamentous fungi, the


Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) infections are widespread in human populations. Here, I describe single-cell RNA sequencing of two lymphoblastoid cell lines harboring both episomal EBV and inherited chromosomally integrated HHV-6. Rare instances of HHV-6 expression appear enriched with EBV reactivation. Herpesviruses have ubiquitously spread among humans. Epstein-Barr virus (EBV), a gammaherpesvirus, infects ~90% of the population . The cirin vitro generates lymphoblastoid cell lines (LCLs) (Transformation of primary B cells by EBV s (LCLs)  that havs (LCLs) . These rThe EBV-immortalized lines HG00362 and HG01277  contain iciHHV-6B and iciHHV-6A, respectively . LCLs weNC_007605.1), and either HHV-6A U1102 (NC_001664.4) or HHV-6B Z29 (NC_000898.1). Viral genomes were appended as extra chromosomes.A pipeline optimized for simultaneously measuring viral and human transcripts , 13 was HHV-6 RNA is enriched with high levels of EBV gene expression . The majGSE230239, which links to NCBI Sequence Read Archive entries SRX20032490 and SRX20032491.These data have been deposited in the NCBI Gene Expression Omnibus , 18 unde


Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1  and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance. High-grade osteosarcoma (HGOS) is a very aggressive malignant bone tumor arising in children and young adults. Conventional first-line treatment for HGOS is based on neoadjuvant chemotherapy with methotrexate, doxorubicin, and cisplatin, with the possible addition of ifosfamide . As clinSeveral studies have reported that the most relevant mechanisms for MTX resistance in HGOS are the decreased expression of the solute carrier family 19 member 1/replication factor C subunit 1 , which negatively affects the transportation of the drug inside the cell, and the increased expression of DHFR that limits the inhibitory efficacy of MTX .Pharmacogenetic and pharmacogenomic studies in HGOS have identified gene polymorphisms that play a fundamental role in MTX metabolism and transport  and suggSeveral studies reported methylenetetrahydrofolate reductase (MTHFR) rs1801133 as a polymorphism associated with the development of treatment-related adverse toxicity . FurtherIn this study, we have focused on single-nucleotide polymorphisms (SNPs) affecting genes involved in MTX transport, folate metabolism, MTX-related toxicity, and TP53 , and expThe U-2OS and Saos-2, MG-63, and HOS human HGOS cell lines were obtained from the American Type Culture Collection . The IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/OS20, IOR/MOS, and IOR/SARG human HGOS cell lines were established and characterized, as previously described , at the in vitro step-by-step increasing MTX concentrations. The in vitro continuous drug exposure resulted in the establishment of variants which are resistant to 3 ng/mL MTX (U-2OS/MTX3), 30 ng/mL MTX (U-2OS/MTX30 and Saos-2/MTX30), 100 ng/mL MTX (U-2OS/MTX100 and Saos-2/MTX100), 300 ng/mL MTX (U-2OS/MTX300 and Saos-2/MTX300), and 1 μg/mL MTX (Saos-2/MTX1µg). These MTX concentrations corresponded to 0.01 (3 ng/mL), 0.07 (30 ng/mL), 0.22 (100 ng/ul), 0.66 (300 ng/ul), and 2.20 µM MTX (1 μg/mL)  .2 at 37°C. All the cell lines were found to be mycoplasma-free using a MycoAlert Mycoplasma Detection Kit .MTX-resistant variants have been cultured in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 10% fetal bovine serum , and with an appropriate MTX concentration, and then maintained in a humidified atmosphere with 5% CODNA fingerprint analyses were performed for all cell lines using 17 polymorphic short tandem repeat sequences, confirming their identity .Both DNA and RNA were isolated from frozen cell pellets. The extraction of nucleic acids was performed using the AllPrep DNA/RNA Mini Kit , according to the manufacturer’s instructions. After isolation, DNA and RNA concentrations were determined by spectrophotometry , and the quality of all RNA samples was evaluated using the 2100 Bioanalyzer system  using the RNA 6000 kit .Library preparation was performed for all 20 cell lines, according to the QIAseq Multimodal Panel handbook v06/2020 , as previously reported . The priFor library preparation, the input amount was 40 ng of DNA and 100 ng of RNA. The libraries were quantified through the QIAseq Library Quant Assay kit  on a real-time PCR system . The libraries were then diluted to 1.2 p.m., pooled together, and sequenced on a NextSeq 500 system  using a Mid-Output Reagent Kit v2.5 (300 cycles).FastQ files were downloaded from the BaseSpace cloud  and imported into the CLC genomics workbench (GWB) v22.0.1 . Through the QIAseq Multimodal Analysis workflow, DNA variants, gene expression, and transcript fusions were identified. For the identification of RNA variants, a custom workflow was developed by the QIAGEN bioinformatics support.The detection of DNA and RNA variants was performed by aligning the reads to the human genome hg38 as a reference sequence and filtering using coverage of 100x and a variant allele frequency (VAF) higher than 3%. 2fold-change between the drug-resistant variants and their respective parental cell lines was evaluated using the differential expression tools of CLC GWB. A Bonferroni correction was used, and a p-value <0.05 was considered significant.For the analysis of differential gene expression, the transcripts per million data were considered, and a logThe hierarchical cluster tool of CLC GWB was used to create the heatmap setting, with the parameters Euclidean distance and complete linkage.The principal component analysis (PCA) was conducted using PCA for the RNA-seq tool of CLC GWB, considering the 12 genes for all 20 cell lines to identify the clustering of the cell lines.To validate the sequencing data, a genotyping analysis was undertaken by TaqMan SNP genotyping and drug-metabolizing enzyme (DME) assays , which are listed in For each polymorphism, the data obtained by the mmNGS approach were compared with the data obtained by TaqMan genotyping. From the comparison between the genotyping data and the sequencing results, we found that for 13/20 (65%) cell lines, the match was 100%, whereas for 7/20 (35%) cell lines the match was 95%. Overall, we found a high concordance between the data obtained by genotyping and sequencing approaches, and therefore, the performance of our custom panel for DNA variant calling can be considered reliable.The comparison of DNA variant calling data between U-2OS and its MTX-resistant variants identified a change in the polymorphism status of TP53 rs1642785. In the parental cell line, the genotype of TP53 rs1642785 was a homozygous variant, which changed during the acquisition of MTX resistance .TP53 gene were revealed to be near to the rs1642785 locus , with a VAF higher than 3% , while all four MTX-resistant variants were homozygous wild types (TT). Furthermore, These findings suggest that this transition emerged during the first phases of MTX resistance acquisition.The polymorphism status revealed on DNA was identified to be equal at the RNA level in 96% of the polymorphisms, indicating that these variants were also maintained during transcription. ABCB1 rs2032582 was found to be a heterozygous variant with a consequent amino acid change of a Ser-Ala in both parental and resistant cell lines . This geAnother polymorphism that was found to be a heterozygous variant in all cell lines was MTHFR r1801131, which caused a Glu to Ala amino acid change . This vaTP53 rs1042522 was found to be a homozygous variant in all cell lines, suggesting a possible biological advantage for cells carrying this allele. This SNP has been reported as a somatic mutation in HGOS, where it has been described to be associated with poor survival, supporting a more aggressive behavior of tumors carrying the variant allele . By contIn Saos-2 cell lines, we observed that the genotype status occurring on DNA was maintained in 94.7% at the RNA level. Most of the polymorphisms were homozygous wild type with the exception of ABCB1 and MTHFD1, which showed a heterozygous and homozygous genotype, respectively. These variations were also identified at the RNA level .By contrast, the status of one SNP, namely, MTHFR rs1801133, changed between DNA and RNA . The MTHRNA-derived libraries were synthesized with primers that are specific for the aforementioned 12 genes and were analyzed with the RNA expression tool of CLC GWB. In U-2OS/MTX-resistant variants, DHFR was significantly upregulated compared to U-2OS in parallel with the increasing MTX resistance. In addition, ABCB1 was also upregulated but did not reach significance.p-value <0.05 after Bonferroni correction.On the contrary, in Saos-2 resistant variants, SLC19A1 was significantly downregulated in all MTX-resistant variants, with a Heatmap and hierarchical clustering analysis including all the studied cell lines and considering the expression level of all 12 genes were performed. The resulting clusterization showed that 20 cell lines can be divided into four main groups based on different gene expression levels .Two groups included the sensitive cell lines, whereas the other two included the MTX-resistant variants. The U-2OS parental cell line and its relative resistant variants were grouped and characterized by the elevated expression of DHFR, ABCB1, and SLCO1B1. These data were in agreement with the fold changes shown in The clustering of the two groups of MTX-resistant variants similar to their parental MTX-sensitive cell lines was also observed by PCA . SensitiFastQ files of all RNA-derived libraries were explored for the presence of fusion transcripts using the QIAseq Multimodal Analysis of CLC GWB. One fusion transcript involving DHFR and MSH3 genes was identified in the U-2OS/MTX300 variant, the variant with the highest MTX resistance level . A graphThe intrachromosomal fusion transcript comprised exons 1 to 4 of DHFR, which were juxtaposed to exon 9 of MSH3 . InteresIn the present study, the pharmacogenomic profiling of MTX-resistant and -sensitive human HGOS cell lines has been performed by mmNGS targeting 22 SNPs with reported evidence to be related to the MTX-based chemotherapy response or toxicity development by mainly pharmacogenetic studies in HGOS patients. This innovative approach has provided novel biological insights and confirmed previously reported findings.The validation of the custom mmNGS panel for variant calling by TaqMan genotyping assays was satisfactory, confirming the reliability of this methodology. Interestingly, the genotype distribution of the 22 SNPs was very heterogeneous, particularly being a homozygous wild type for all transporter genes except ABCB1. Different from the transporter genes, MTHFR rs1801133 and TP53 rs1042522 were most frequently found with variant alleles.Germline and somatic TP53 mutations have been studied in patients with HGOS by several authors . HGOS ofThe data obtained in the present study have confirmed the high instability of TP53 in HGOS cell drug-sensitive cell lines and MTX-resistant variants. However, the revealed TP53 changes could not directly be attributed to the development of MTX resistance.One study reported a significant correlation between TP53 mutations and increased levels of DHFR mRNA in patients with acute lymphoblastic leukemia (ALL) , suggestAmong the 22 SNPs investigated in this study, MTHFR rs1801133 was found to be a variant in all eight MTX-resistant cell lines. This SNP caused an amino acid change which was reported to increase the thermolability of MTHFR protein, with a consequent decreased enzyme activity, leading to elevated plasma homocysteine levels and altered distribution of folate . In addiThe reduced expression of SLC19A1 was reported as a mechanism underlying MTX resistance in MTX-resistant HGOS cell lines . These dn = 49) and the homozygous AA genotype in 22.8%. According to this definition in relation to the MTX transport, the detected change of rs1051266 to TT on DNA and RNA levels observed in all four Saos-2-derived MTX-resistant variants could, therefore, be associated with the altered MTX transport. However, it must be underlined that In addition, the sequence alterations of the entire coding region of the SLC19A1 gene were studied in 162 patients with different types of osteosarcoma . InteresIn other cancers, many fusion transcripts have been reported in association with drug resistance , and it So far, two studies have reported that in MTX-resistant cell lines, the promyelocytic leukemia HL-60 cell line  and the Fusion genes and RNAs are a hallmark of cancer and are the result from either chromosomal rearrangements or splicing mechanisms . The funShoshani and co-workers recently showed how selective the pressure of MTX in HeLa S3 cells stimulated several rounds of chromothripsis, leading to the amplification of DHFR . HGOS arThe overexpression of MSH3 was not investigated because no primers were included in the mmNGS panel. However, our unpublished expression array data suggested that MSH3 was 1.99-fold upregulated in MTX-resistant variants compared to the parental U-2OS cell line, although these findings need to be further confirmed.In conclusion, although HGOS has been studied by whole-genome sequencing techniques, the customized mmNGS approach targeting 22 SNPs of 12 genes with pharmacogenomic evidence reliably identified allele changes by variant calling on DNA- and RNA-derived libraries, differentially expressed genes, and a novel fusion RNA in relation to MTX resistance development.


The problem of academic dropout in the first year of studies represents an important issue for higher education, in that it accounts for an important indicator of quality but also for the negative consequences it produces on individual, institutional and social level. The main aim of the study is to validate and evaluate a robust measure of overall academic maladjustment.The participants were 809 first-year students from various Romanian universities.The results showed a reliable version of the instrument with a factorial structure that did not deviate significantly from the authors’ initial model. The exploratory and confirmatory factor analysis revealed a unified score including six dimensions, procrastination, dishonesty – unethical behavior, test anxiety, machiavellian attitude, neuroticism, and somatization. Our results confirmed that besides academic achievement, personal factors are important indicators of adjustment, showing that personal resources management, emotional and behavioral strategies are components of adjustment. Our study revealed a medium and positive correlation between overall maladjustment and academic dropout intention, procrastination seemed to be the most relevant predictor of dropout intention.Academic adjustment acts as a safeguard against dropping out, and it is crucial to acknowledge that most students enter college with the intention of completing their studies. University represents a vital transition period for youths’ evolution as it includes numerous challenges which pertain not only to academic requirements but also to the change of a new social environment where adaptability is an important factor for academic achievement . First yThe problem of academic dropout in the first year of studies represents an important issue for higher education, in that it accounts for an important indicator of quality but also for the negative consequences it produces on individual, institutional and social level. Students who abandon college have few possibilities to develop professional competences which could offer them the chance of a good integration on the labor market. Statistics in USA show that dropout rate for young people between the ages of 16−24 years is of 5.2% in 2021 , in UE tAt personal level, dropout can lead to low expectations, negative self-evaluation , high riIn the present paper we focus on the main predictor of academic dropout which is academic maladjustment. The topic was previously approached, as we will present below, however, there isn’t a recent integrative framework of academic adjustment. Baker and Siryk’ s model (1984) and Tinto’s theory (1993) are still the most cited and with large empirical evidence, but recent studies argue that academic adjustment is more contextual and should be analysed as such . TherefoStudies in the literature confirm that the first year of study represents an important determinant in the appearance of dropout due to the transition period in the academic adjustment process . The traPersistence and the effects generated by the university dropout phenomenon, as well as the necessity to improve graduation rates have received increased attention in reference to first year students’ academic adjustment. It is arguable that identifying the causes and predictors of academic maladjustment could determine the development of efficient programs which, through early warning measures will help diminish risk factors in order to prevent academic dropout or absenteeism.Adjustment refers to the behaviors which allow each individual to deal with the demands and expectations of a new environment, to act and develop new coping strategies . AcademiResearch showed the fact that the easier the students get through this stage and adjust their expectations to the demands of the new environment, the more likely they engage in study and in academic life. For some students, getting over this process is easy and fast while for others changes are overwhelming and difficult, manifesting academic maladjustment . StudentAcademic adjustment is a process which results in academic achievement . The speAcademic maladjustment approach is less represented in the specialty literature than academic adjustment, the problematics being quite controversial mainly due to its conceptualization in opposition to academic adjustment. Not always low levels of the factors which determine academic adjustment explain the variation academic maladjustment. Academic maladjustment refers to the set of behavioral, social and emotional difficulties which prevent students from adjusting to the new learning environment, from meeting successfully academic demands, from obtaining performance and academic engagement . StudentDefining the term academic maladjustment is important because the manner in which it is defined can determine the manner in which it is approached, measured and analyzed . TheorieAcademic maladjustment is a multifaceted concept, being dynamic and multidimensional, which is explained by the fact that the student enters university with certain personal characteristics  which are challenged by the interaction with the new environment  in order to set a fit between their demands, values and expectations and the demands and regulations of university life; consequently, academic maladjustment lead to academic failure .As previously mentioned, the literature predominantly emphasizes academic adjustment, with relatively fewer studies focusing on academic maladjustment. Its relevance in understanding and approaching the dropout phenomenon is undoubted, as it leads to a series of negative consequences both at academic and emotional levels. For example, in a previous study academic dishonesty was found to be consistently associated with low levels of academic adjustment , but theTheoretical models in the field of academic adjustment and achievement affirm that academic adjustment predictors fall into three categories: academic, social/environmental and personality factors. Academic factors include a series of variables such as aptitude and ability, study skills and test anxiety, academic motivation, self-efficacy and attribution, social/environmental factors refer to variables such as social support, life stress, campus environment, work involvement, family variables, and academic environment, while personality factors include personality characteristics, self- esteem, locus of control and trait anxiety .Many studies in the specialty literature analyzed background characteristics in predicting adjustment and academic achievement . The effStudies showed the essential role of academic adjustment in the prediction of academic performance and of dropout occurrence. Pupils who had good results in high school or at standardized tests adapt easier to the requirements of the new academic environment. Scores at Scholastic Assessment Test (SAT) and American College Testing (ACT) reflect many general cognitive skills such as the capacity to process new information and to acquire new skills. High grades in high school reflect solid cognitive skills which facilitate academic adjustment. Persistence and academic achievement had been most frequently predicted by cognitive variables, respectively, grade point average (GPA) and SAT scores. The grades from high-school and scores at ACT and SAT maintained a more influential predictive relation than study skills, motivation and personality characteristics .Academic adjustment represents a mediating variable upon academic performance . AcademiA variety of individual traits can facilitate or hinder the process of academic adjustment . The terThe relation between personality factors and academic adjustment is not well represented in the literature. The role of personality factors can be better represented through the concept of academic achievement. Among personality traits, conscientiousness has a consistently positive association with academic achievement  and perfNeuroticism relates with poor academic performance , being aProcrastination, conceptualized as the behavioral tendency to unnecessarily postpone decisions or work tasks , which rTest anxiety refers to a set of phenomenological, physiological, and behavioral responses that accompany concern about possible negative consequences or failure on an exam or similar evaluative situation. Students who score high values of test anxiety manifest worry, stress, a tension which leads to a failure in regulation and focus. Test anxiety is associated with negative results of students’ academic achievement  and acadDishonesty represents unethical academic behaviors such as cheating, plagiarism, or unauthorized help, being a widespread phenomenon in the academic environment . Ajzen . AcademiWhile existing literature has identified these factors as predictors of academic maladjustment, there is still unclear how these predictors interact with each other and how their interactions could influence academic adjustment or achievement. For example, how do personality traits, especially neuroticism, interact with procrastination and with dishonesty? Or are there any differences in these factors due to demographics characteristics? These are some of the research gaps in the literature that warrant further investigations with more stable instruments that cover all relevant dimensions of academic adjustment.The concern for the manner in which students tackle the challenges of the new academic environment with the aim to improve their experience during the first year of study is reflected in the modality of evaluating academic adjustment. The problematics of academic adjustment conceptualization generated an array of modalities and techniques of measuring the construct. Some researchers focused on a single element to measure the whole construct, whereas others focused upon certain facets of the construct. The most popular instrument which measures students’ adaptation to college multi-dimensionally is the Student Adaptation to College Questionnaire (SACQ) . The insThere are few studies in Romania which measure academic adjustment; an instrument worthy of mention is the Academic Adjustment Questionnaire – AAQ , its firBased on our experience in developing academic maladjustment questionnaires, we decided to create a broader and more detailed concept of this phenomenon, the new version of the instrument for university students encompassing an internal dimension  as well as an external dimension . These dimensions will be combined to form a comprehensive and unified score.Given the potential benefits of academic adjustment, the current study aimed to validate and evaluate a robust measure of overall academic maladjustment.age = 20.91, SD = 4.84), male (N = 165), and female (N = 634), non-binary (N = 7), not declared (N = 3), covering six fields of studies: Mathematics and Natural Sciences (N = 62), Sports Science and Physical Education (N = 23), Biological and Biomedical Sciences (N = 62), Engineering Sciences (N = 184), Social Sciences (N = 638), Humanities and Arts (N = 58), tuition-free students (N = 423), and tuition-fee paying students (N = 386).The participants were 809 first-year students from various Romanian universities , the items being measured on five-point Likert scale (ranging from strongly disagree to strongly agree), Cronbach’s Alpha being high, 0.82, high scores indicating a higher intention to dropout.Dropout intention was measured through a five-item scale developed for this research  and 0.85, for the Neuroticism scale .Two personality traits - neuroticism and conscientiousness - were measured using the IPIP scales . Each scA factual questionnaire was also used to collect data about the educational background: profile of the high school, baccalaureate mean grade (GPA), previous degrees or diplomas, the elapsed time from high school graduation, range of admission to the study program applied , and previous university enrolments.N = 401) and confirmatory (N = 408) samples, the two halves not differing on gender , type of enrolment tuition-free and tuition-paying students) , or previous academic achievement . Exploratory factor analysis was computed using IBM SPSS 23.0, Promax with Kaiser Normalization being computed, and Confirmatory factor analysis was computed with IBM AMOS 23.0. Predictive validity was tested with linear regression, the dropout intention being the criterion. The Kaiser–Meyer–Olkin (KMO = 0.923) and Bartlett’s test of sphericity  indicated that the data were suitable for factor analysis. For the CFA, parameters were computed through the maximum likelihood estimation method.The questionnaires were administered during November – December 2022. Construct validity was estimated through Exploratory and confirmatory factor analyses. The convenience sample was randomly split into exploratory  but upon closer inspection, proved to be valid data points and, therefore, were retained in the data set.Multivariate normality of the data was determined by reviewing the absolute ranges for skewness and kurtosis, the values being less than 2.0, which indicates that the data is relatively normally distributed. The Mahalanobis distance was also checked, individual cases were analyzed , TLI (Tucker–Lewis index), AIC (Akaike information criterion), RMSEA (Root Mean Square Error of Approximation) were used to evaluate model fit . Factor Based on the eigenvalues, Kaiser’s rule, and the scree plot, a solution with seven to nine factors was assumed. The nine-factor solution covered 64.837% of the total variance. Although all the nine factors have eigenvalues higher than 1, the eighth and ninth factors included only 3, respectively, 2 items, with divergent meanings. Therefore, we decided to keep a seven-factor solution covering 57.748% of the variance . A totalThe seven factors were labeled as follows: F1 Procrastination, F2 Dishonesty – unethical behavior, F3 Test anxiety, F4 Machiavellian attitude, F5 Neuroticism, F6 Somatization, F7 Disengagement. There are two affective dimensions (neuroticism and test anxiety), a somatic dimension (somatization), four behavioral dimensions . However, the seventh factor has a low number of items, one of them having also a low loading (0.412) and a mixt structure (being saturated both in Factor 1 and 7). Therefore, the factor structure with and without factor seven will be tested in the confirmatory phase.Based on the EFA results, we confirmed the structure of the questionnaire through CFA. The fully correlated factor structure with all the factors converging to a general one showed good fit for some indices [root mean square error of approximation (RMSEA)] but low for others  was computed for the second-order CFA model, and the results were reported in p < 0.001), showing that the dimensions could be facets of the same construct (The correlations between the factors were positive and significant (between 0.16 and 0.69). The correlations of the scales with the total score were significant  correlated weakly with the maladjustment. On the other hand, the two personality traits (neuroticism and conscientiousness) correlated significantly with maladjustment, the correlations being positive for neuroticism and positive for conscientiousness.p < 0.001. The academic maladjustment is a significant predictor . Given the relatively high correlations between some of the maladjustment dimensions, we did not compute a multiple regression analysis .To test the concurrent validity of the scale, we used the dropout intention as criterion. All the dimensions and the total score of the maladjustment scale correlated positively and significantly with the dropout intention, procrastination and the total score being the strongest predictors. A simple regression analysis, using the overall maladjustment score as predictor led to a 15.5% of explained variance for the dropout intention, the regression model being statistically significant, F = 147.68, t tests showing that men had higher levels for Dishonesty – unethical behaviors and Machavellic attitudes, while women had higher levels of Test anxiety, Somatization and Neuroticism. There were no significant differences for Procrastination and the overall score  which are influenced by their interaction with the new environment  to establish a fit between their own demands, values, and expectations, and those of university life. The results further support partial measurement invariance across genders. Moreover, academic adjustment was substantially different but related to academic dropout intention. Furthermore, academic maladjustment was negatively associated with conscientiousness and was positively related to neuroticism. The correlation with academic performance was weak.Academic maladjustment remains a rather controversial issue, and even if it is defined in contrast to academic adjustment, the definitions reveal a multifaceted concept, representing a dynamic and multidimensional process, referring to a set of behavioral, social, and emotional difficulties that prevent students from adapting to the new learning environment, coping successfully with academic demands, achieving performance, and academic engagement , in its A new aspect is represented by the introduction of academic dishonesty into the model of academic maladjustment, which has been less discussed in the literature. Even though the initial concerns of the authors regarding the integration of the academic dishonesty dimension into the structure of academic maladjustment date back a few years  the resuIn the specialized literature in Romania, there are few concerns regarding the measurement of academic (in)adaptation, the most well-known being materialized in the creation of the Academic Adjustment Questionnaire – AAQ . The autNot surprisingly, procrastination seemed to be one of the best-represented factors of maladjustment, with the highest eigenvalue and the highest explained variance, convergent with previous studies which defined procrastination as a common phenomenon that disrupts academic responsibilities , academiNegative emotions as a dimension of academic maladjustment proved also to be a relevant component, operationalized as test anxiety and neuroticism. Studies have demonstrated a connection between emotions and academic achievement. Generally, positive emotions, like finding joy in learning, are associated with higher levels of achievement. Conversely, negative emotions, such as experiencing test anxiety, are linked to lower academic performance . The acaThe crystallization resulting from the factorial analysis of a factor synthesizing somatic complaints is not coincidental, previous studies suggesting that somatic complaints are often indicators of school maladjustment , somaticThe low correlations between previous academic performances and maladjustment are not surprising, previous studies confirming these results . Our res2 = 0.152); procrastination seemed to be the most relevant predictor of dropout intention, the correlation with dropout intention being higher than for the other maladjustment dimensions. Academic adjustment acts as a safeguard against dropping out, and it is crucial to acknowledge that most students enter college with the intention of completing their studies  and moral aspects (machiavellian attitudes) is highly relevant as it offers a more comprehensive understanding of students’ difficulties and challenges in adapting to the academic demands of the university environment. Previous studies have often approached dishonest behavior as being associated with an individual tendency to overlook norms, but the use of the new instrument might shed more light on the complex relationship between academic demands and academic dishonesty among first-year students. This has practical implications as well, higher education institutions can use the instrument as a toolkit to assess the tendency to engage in procrastination and unethical behavior in direct response to challenges and demands of the academic environment. The deeper understanding and acknowledgment of these behaviors can serve in designing and implementing interventions to prevent or diminish procrastination and unethical behaviors among students and in promoting a culture of ethics within the university.Secondly, the exploration of the six distinct dimensions of academic maladjustment, including procrastination, dishonesty, test anxiety, machiavellian attitudes, neuroticism, and somatization, reveals the multifaceted nature of this construct. This multifaceted approach can help researchers and university staff to better address the various aspects that can facilitate students’ successful adaptation to university life. By understanding both the behavioral and emotional dimensions of academic adjustment, universities can target better their interventions. By recognizing the specific aspects that students experience or struggle with, tutors, educational counselors and even teachers can personalize the support they offer to first-year students. For example, students who experience intense negative emotions in relation to evaluation may benefit more from targeted educational and psychological counseling, while students who confront more with somatization may also need medical support and long-term educational or psychological counseling.Finally, the Academic Maladjustment Questionnaire has significant implications for educational practice as it can be used to identify students at-risk of dropout. As all the dimensions and the unified score correlated positively with dropout intentions, universities can early recognize students experiences academic maladjustment and address the issue in early interventions. By doing this, they can potentially reduce the high dropout rates in first-year students and can also enhance academic performance of those students by addressing their difficulties early on in the course of their studies. Procrastination was the strongest predictor of dropout intention, sustaining once more the need for targeted intervention on multiple facets of academic maladjustment.In conclusion, our first attempt to conceptualize and measure academic maladjustment has shown promising results. Our results support the importance of measuring academic maladjustment as an indicator of dropout intention and academic success. The effectiveness of the newly developed instrument will be confirmed through its implementation in prevention, counseling, and academic stress management activities. Moreover, its use in transcultural studies will provide a more accurate representation of culturally specific constructs being measured. Preventing academic maladjustment could therefore be an important aim of alternative programs and strategies that researchers and practitioners could implement to sustain student efforts’ toward a successful transition to university and professional life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by the Ethics Committee in Social-Human Scientific Research at the Transilvania University of Brasov. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.A-MC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review and editing. MMS: Investigation, Methodology, Writing – original draft, Writing – review and editing, Conceptualization. AIC: Supervision, Validation, Writing – original draft, Writing – review and editing. CT: Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing, Methodology. CIM: Data curation, Formal analysis, Methodology, Software, Writing – review and editing.