TARDBP, FUS, ANXA11, ARPP21, and C9ORF72 genes, alongside healthy controls. To demonstrate the utilisation of these lines for ALS disease modelling, a subset of FUS-ALS iPSCs were differentiated into functionally active motor neurons. Further characterisation revealed an increase in cytoplasmic FUS protein and reduced neurite outgrowth in FUS-ALS motor neurons compared to the control. This proof-of-principle study demonstrates that these novel patient-derived iPSC lines can recapitulate specific and early disease-related ALS phenotypes. This biobank provides a disease-relevant platform for discovery of ALS-associated cellular phenotypes to aid the development of novel treatment strategies.Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting the upper and lower motor neurons, causing patients to lose control over voluntary movement, and leading to gradual paralysis and death. There is no cure for ALS, and the development of viable therapeutics has proved challenging, demonstrated by a lack of positive results from clinical trials. One strategy to address this is to improve the tool kit available for pre-clinical research. Here, we describe the creation of an open-access ALS iPSC biobank generated from patients carrying mutations in the  Since their discovery in 2007, human induced pluripotent stem cells (iPSCs) have broadened our understanding of basic biology and transformed the field of developmental biology . iPSCs hiPSCs hold great promise for drug screening and personalised medicine  and can ALS is a neurodegenerative condition which affects the upper and lower motor neurons , with a SOD1-ALS, Tofersen, was recently approved by the FDA; a milestone in drug development for ALS [SOD1, C9ORF72, FUS, and ATXN2 hold potentially beneficial clinical outcomes [ALS is characteristically complex and heterogeneous which has likely contributed to the overriding failure of ALS-CTs. ALS subclassification and stratification may increase the success rate of CTs ,22, whic for ALS . Additiooutcomes , and patoutcomes ,27,28,29Patient-derived cell biobanks allow us to determine disease-related phenotypes and test the toxicity and effectiveness of novel compounds. Such a biobank was created in 2003 by The Motor Neurone Disease Association, named The UK MND Collections. This biobank contains more than 3000 blood samples including lymphoblastoid cell lines (LCLs) and peripheral blood lymphocytes (PBLs) from healthy volunteers and ALS patients, alongside clinical and epidemiological information . LCLs arFUS-ALS patient-derived iPSC-neurons to demonstrate the prevalence of disease-specific early phenotypes in patient-derived cells. Thirty-five iPSC lines were generated as part of The UK MND Collections, which can be openly accessed through The Motor Neurone Disease Association.Here, we describe the generation of this ALS-iPSC biobank alongside proof-of-concept data generated with 2. LCL media consisted of RPMI 1640 , supplemented with 1× GlutaMAX™ (Thermo Fisher), and 20% fetal bovine serum (Thermo Fisher). Erythroblasts were enriched in mixed PBL cultures following a previously published protocol [LCLs were cultured in upright non-adherent T25 tissue culture flasks (Nunc) at 37 °C and 5% COprotocol . Briefly6 cells with plasmids expressing OCT3/4, L-MYC, KLF4, SV40LT, LIN28, SOX2, and shRNA-p53  with the Amaxa™ Human B Cell Nucleofector™ Kit . After electroporation, cells were transferred onto a feeder layer of inactivated mouse embryonic fibroblasts in LCL media. Cells were maintained in LCL media for five days, then media was transitioned to reprograming media (RM) (DMEM/F12 with GlutaMAX™ (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), 1× N2 Supplement (Thermo Fisher), 1× B27™ Supplement (Thermo Fisher), 0.1 µM 𝛽-mercaptoethanol (Thermo Fisher), 100 ng/mL bFGF , 1000 U/mL hLIF , 0.5 µM PD-0325901 , 3 µM CHIR99021 , 10 µM HA-100 , and 0.5 µM A83-01 (BioVision)). RM was then replaced every 1–2 days. iPSC colonies were identified around two weeks later, and media was transitioned to Essential 8™ Flex medium (Thermo Fisher) until colonies were large enough for manual picking. Established colonies were cultured on Geltrex™ (Thermo Fisher) in Essential 8™ Flex. Two LCL-derived iPSC lines were derived using CytoTune™ iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher), following the manufacturer’s protocol, with an additional spinfection step of 2250 RPM for 90 min in a large benchtop centrifuge immediately after the virus was added to LCLs. LCLs were maintained in LCL media for 48 h, with a media change at 24 h. LCLs were transferred onto inactivated mouse embryonic fibroblasts, maintained in LCL media until day 5, and then transitioned to RM as described above. iPSCs were routinely screened to ensure the absence of contamination with mycoplasma before cryopreservation using the MycoAlert® Mycoplasma Detection Kit .iPSCs were reprogrammed from LCLs and PBLs as previously reported ,35,36. i® High-Fidelity 2× Master Mix  with 35 cycles of 95 °C for 30 s, 61 °C for 30 s, and 72 °C for 30 s. PCR products were separated by gel electrophoresis in 4% agarose gels with 1% ethidium bromide, then visualised and photographed inside a UV transilluminator. STR profiling was outsourced to Source BioScience ; 16 STR loci were analysed and matched between iPSCs and parent LCL or PBL lines.iPSCs were subject to quality control (QC) experiments, including pluripotency immunocytochemistry and embryoid body assay (see below). Patient-derived lines were sequenced at the mutation region by sending PCR products and primers to Source BioScience . Genomic integrity was assessed by G-band or digital karyotyping: G-band karyotyping was outsourced to TDL Genetics  or to the Genome Editing and Embryology Core , and digital karyotyping was completed with KaryoStat™ Karyotyping Service (Thermo Fisher). To confirm the loss of EBV genes, iPSC lines were serially passaged, and genomic DNA was screened for EBV genes with PCR. DNA was extracted using the DNeasy Blood and Tissue Kit , and PCR was performed using primers targeting EBV genes   and the iPSCs were dissociated with Versene and transferred to poly-HEMA (Sigma) coated plates in Essential 8™ Flex medium (Thermo Fisher) with 10 μM Rock inhibitor (BioVision). The next day, media was changed to embryoid body media ((KnockOut™ DMEM (Thermo Fisher), 10% knock-out serum replacement (Thermo Fisher), 5% fetal bovine serum (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), 12 ng/mL hLIF (Merck Millipore), and 55 µM 𝛽-mercaptoethanol (Thermo Fisher)) after which media was replaced every 2–3 days. After one week, embryoid bodies were transferred to glass coverslips coated with 0.1% gelatine for spontaneous differentiation. Media was changed every 2–3 days for three weeks until fixation.Motor neurons were generated using a pre-established protocol . First, Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, permeabilised in 0.1% TritonX-100 for 15 min, and blocked with 10% normal donkey serum (Sigma-Aldrich) for one hour. Primary antibodies were diluted in 5% donkey serum and incubated overnight at 4 °C , Nanog , SMA (Abcam), AFP , OLIG2 (Millipore), NKX6.2 (Millipore), TUJI (Sigma-Aldrich), Islet 1 (BD bioscience), Hb9 , and FUS ). The next day cells were washed three times with PBS and incubated with anti-donkey Dylight™ secondary antibodies (Thermo Fisher) diluted 1:400 in 5% donkey serum for one hour at room temperature. Nuclei were stained with 1.25 μg/mL Hoechst (Thermo Fisher) for 5 min, washed with PBS three times, and mounted with FluorSave™ Reagent (Merck Millipore).® High-Content Screening System . Motor neuron quantification was performed in the Columbus™ Image Data Storage and Analysis system . Approximately 8–10 fields from two separate wells of a 96-well plate were quantified in three biological replicates. Neurite outgrowth was quantified in individual neurons using the ImageJ plug-in NeuriteTracer.Images were acquired with a Leica CYR5000 light microscope , a Leica TCS-SP5 microscope , or an Opera Phenix3, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose, and 10 mM Na-HEPES (pH 7.25)) and 0.02% Pluronic-F27 (Thermo Fisher) for 15 min at 37 °C. Subsequently, neurons were rinsed in fresh external solution for another 15 min at 37 °C. Live image collection was performed with an Opera Phenix® High-Content Screening System with a 20× water objective. Data were collected for 2 min, with one image taken every 2.8 s, and data were processed in ImageJ. Spontaneous calcium fluctuations were calculated as relative Fluo4-AM fluorescence intensity normalised to the background (F-F0/F0) across 10 regions of interest (ROI).Motor neurons were cultured in 96-well plates and aged for 108 days. Cells were incubated with 2 mM Fluo4-AM in an external solution , three with TARDBP mutations , four with C9ORF72 GGGGCC intronic expansions, three with mutations in FUS , and four with mutations of unknown significance in ARPP21 . The remaining line is derived from a sporadic ALS patient with no known genetic mutation. An additional ten control lines were generated from healthy individuals . Five PBL-derived iPSC lines were generated from the same donor cohort, including two healthy control and three ALS patient-derived lines: Two with mutations in TARDBP  and one with a mutation of unknown significance in ARPP21 (1× P529L). Healthy donor and ALS patient demographic information for the newly generated lines is included in Twenty-four novel iPSC lines were generated from a selection of healthy control and ALS patient LCLs, and five iPSC lines were derived from PBLs . An addiNewly generated LCL- and PBL-derived iPSC lines were subject to standard QC testing. Example data are included in Cell line identity was confirmed using STR profiling to ensure that daughter iPSC lines correctly matched parent LCL or PBL genetic profiles (in line with patient data protection). In addition, ALS patient-derived lines with known genetic mutations were directly sequenced and their genotypes were verified F. To conMeasures of genome stability included either G-band karyotyping D or digiFUS-ALS patients. iPSCs carrying FUS R521H and R522G and two control lines were differentiated into motor neurons using small molecule mediated differentiation. iPSCs were first differentiated into OLIG2-positive motor neuron progenitors via an intermediate neuroepithelial stage ) alongside QC data for each cell line. In addition, uncharacterised clones from the same cell lines may be available to those interested in the comparison of clonal lines (FUS-ALS patients recapitulate key pathological features observed in ALS patient tissue and other FUS models. Hence, these newly generated lines will aid ALS disease modelling, contributing to pre-clinical research on developing novel therapeutics.In summary, we have generated an open-access iPSC biobank, including multiple lines generated from ALS patients and controls. These can be accessed through The Motor Neurone Disease Association (al lines . As an e


Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna.Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated.The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF.Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer. Abnormasurvival . Tissue survival . The blosurvival . The expsurvival . Furthersurvival . Tumor csurvival .Dissemination of tumors is effectuated by circulating tumor cells (CTCs) which detach from primary lesions, intravasate, and generate metastases after having survived in the bloodstream . CTCs exSCLC26A was established at the Medial University of Vienna from a pleural effusion of an SCLC patient before treatment. The following four SCLC cell lines have been supplemented by the Department of Radiation Biology, the Finsen Centre, Copenhagen, Denmark: the permanent cell line GLC16 has been derived from a patient after chemotherapy, DMS153 was established from a liver lesion of a pretreated patient, and NCI-H526 from bone metastasis, respectively, whereas NCI-H417 stems from primary SCLC before treatment. The SCLC CTC cell lines BHGc7, BHGc10, BHGc16, BHGc26, and UHGc5 were established from peripheral blood of SCLC patients with the extended disease after second-line therapy . With exProteins involved in angiogenesis and coagulation were analyzed using the Angiogenesis Proteome Profiler Array  according to the manufacturer’s instructions. Experiments were done in duplicate and the different tests were calibrated using the six reference spots included for each individual membrane. A pixel intensity value of 500 corresponds to background levels (pixel intensity of empty positions). Arrays were evaluated using ImageJ  and Origin 9.1 software .Cell culture supernatants were assayed using the human coagulation FIII/TF Quantikine enzyme-linked immunosorbent assay . This test has a sensitivity of 2.05 ng/L TF and a measurement range of 7.8–500 ng/L.4 cells in 100 μL medium were distributed to wells of flat-bottom 96-well microtiter plates , and ten 2-fold dilutions of the test compounds were added. Assays were at least performed in triplicate. The plates were incubated for four days under tissue culture conditions  and viable cells were detected using a modified 3--2,5-diphenyltetrazolium bromide (MTT) assay . Half-maximal drug inhibitory concentration (IC50) values were determined using Origin 9.1 software.Aliquots of 1 × 102 tissue culture flasks in standard medium with the respective concentrations of epirubicin (250 nmol/L), topotecan (200 nmol/L), or DFX (25 μmol/L) for three days. Supernatants were used for the angiogenesis arrays. Additionally, cells were collected, washed with standard medium, counted, and used in MTT viability assays as described.Cells were incubated in 75 cmRNA was extracted using TRIzol . Starting from 1 ug total RNA, polyadenylated fragments were isolated, reverse transcribed, and converted into indexed sequencing libraries using the KAPA stranded messenger RNA (mRNA)-Seq kit . The first 51 bases of these libraries were sequenced on an Illumina HiSeq 2500 platform. The raw single-end sequenced reads were aligned to the reference transcriptome and genome using the Bowtie TopHat pipeline . Mapped reads were assigned to ensemble gene IDs by HTSeq and further normalization was performed with the EDASeq package.t-tests and P < 0.05 was regarded as a significant difference using Origin 9.1 software.Statistical significance was tested by Cell lines tested for angiogenetic factors were DMS153, NCI-H417, NCI-H526, GLC16, and primary SCLC line SCLC26A . All celFurthermore, angiogenetic proteins were determined in western blot arrays for the 5 SCLC CTC cell lines BHGc7, BHGc10, BHGc16, BHGc26, and UHGc5 . SignifiIn vitro exposure of the cells to epirubicin resulted in increased expression of angiopoietin-2 and uPA, whereas VEGF showed a marked reduction. Similarly, the application of topotecan resulted in a slight increase in angiopoietin-2 and uPA as well as a still greater reduction of the release of VEGF compared to epirubicin.The expression of angiogenetic proteins of BHGc16 SCLC CTCs was assayed after treatment of the cells with two chemotherapeutics, namely epirubicin, and topotecan, which are clinically administered for second-line treatment of disseminated SCLC . ConcentA hypoxia-like state was induced in BHGC10 and BHGc7 by preincubation of the cells with DFX. The concentration of DFX was selected to provide 80% viability of the tumor cells, as assessed in MTT assays. Angiogenin was increasingly expressed in both CTC lines tested and angiopoietin-2 in BHGc10, respectively. uPA was markedly decreased in BHGc7, whereas the expression of VEGF showed a significant increase . TF is eConditioned media (CM) of all SCLC CTC cell lines preincubated for 3 days were checked in an ELISA assay with a sensitivity of 2.05 ng/L. The cell lines were tested either in form of single cells or as compact tumorospheres and yielded TF values ranging from 38.3 ng/L to 265.22 ng/L . The difRNA was isolated from SCLC lines and transcripts was evaluated by next-generation sequencing (NGS). The number of reads was normalized between the individual cell lines of the panel. The results show that HGF, uPA, FGF basic, and TSP-1 (with exception of BHGC7) are expressed in the SCLC CTC lines . TF/coagGDPDH ENSG00000111640 as standard.The In cancer patients, TF expression has been correlated with tumor grade, increased vascular density, and a worse prognosis . TF promIn circulation, only a tiny fraction of cells survive and eventually form metastases after becoming trapped in the microvasculature, possibly in aggregates with platelets . Small c® kit has been used for the measurement of TF antigen in plasma from healthy controls and patients with cancer, reporting median levels of TF antigen of 39.1 ng/L (range 16.8 ng/L–87.3 ng/L) for healthy individuals, 47.2 ng/L for patients with cancer without thrombosis, and 56.0 ng/L (range 37.6 ng/L–318.7 ng/L) for patients who developed thrombosis [Unfortunately, it is not clear which kind of TF is recognized by the commercial ELISA assays—either alternatively spliced TF, microvesicle-associated TF, or degraded TF fragments . The Quarombosis . This asrombosis . VEGF isrombosis .An SCLC CTC cell line, namely BHGC16, was precultured with epirubicin and topotecan, which are typically administered for the second-line treatment of metastatic SCLC . Both drIn conclusion, the thromboembolic disease is observed in 6.8% and 11.5% of SCLC patients and is associated with reduced survival. Although TF plays an essential role in coagulation and other functions, SCLC CTCs seem to lack expression of functional TF and the expression of forms of TF in these cells is not inducible by hypoxia-like conditions. Therefore, venous thromboembolism events (VTEs) observed in a minority of SCLC patients are most likely initiated via microparticles released by primary tumors and platelets but not via a contribution of TF by CTCs . Treatme


Bruton’s tyrosine kinase inhibitors (BTKi) and CAR T-cell therapy have demonstrated tremendous clinical benefits in mantle cell lymphoma (MCL) patients, but intrinsic or acquired resistance inevitably develops. In this study, we assessed the efficacy of the highly potent and selective MCL-1 inhibitor AZD5991 in various therapy-resistant MCL cell models. AZD5991 markedly induced apoptosis in these cells. In addition to liberating BAK from the antiapoptotic MCL-1/BAK complex for the subsequent apoptosis cascade, AZD5991 downregulated inhibitor of apoptosis proteins (IAPs) through a BAK-dependent mechanism to amplify the apoptotic signal. The combination of AZD5991 with venetoclax enhanced apoptosis and reduced mitochondrial oxygen consumption capacity in MCL cell lines irrespective of their BTKi or venetoclax sensitivity. This combination also dramatically inhibited tumor growth and prolonged mouse survival in two aggressive MCL patient-derived xenograft models. Mechanistically, the augmented cell lethality was accompanied by the synergistic suppression of IAPs. Supporting this notion, the IAP antagonist BV6 induced dramatic apoptosis in resistant MCL cells and sensitized the resistant MCL cells to venetoclax. Our study uncovered another unique route for MCL-1 inhibitor to trigger apoptosis, implying that the pro-apoptotic combination of IAP antagonists and apoptosis inducers could be further exploited for MCL patients with multiple therapeutic resistance. Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma that is incurable because patients frequently relapse from their therapies . Bruton’The evasion of apoptosis that is associated with MCL pathogenesis and therapy resistance frequently involves dysregulated members of the B-cell lymphoma 2 (BCL-2) family . The proInhibitor of apoptosis proteins (IAPs) are a family of negative apoptosis regulators that interfere with the activated caspases to block apoptosis and contribute to pro-survival signaling pathways . Eight IHerein, we investigated the anti-MCL efficacy of the highly potent and selective MCL-1 inhibitor AZD5991 in various resistant MCL models. We found that AZD5991 induced dramatic MCL cell death, independent of resistance to ibrutinib and venetoclax. Moreover, the combination of AZD5991 with venetoclax was effective against therapy-resistant MCL models in vitro and in vivo. This enhanced cell lethality was accompanied by remarkable downregulation of IAPs, suggesting that development of AZD5991 plus venetoclax, or venetoclax plus IAP antagonist combinatorial treatments for therapy-resistant patients with MCL could improve treatment durability for this incurable disease.2. Antibodies to MCL-1, BCL-2, BCL-XL, GAPDH, poly (ADP-ribose) polymerase (PARP), BAK, BAX, NOXA, XIAP, c-IAP1, c-IAP2, c-MYC, and cleaved Caspase 3 (c-Caspase 3) were purchased from Cell Signaling Technology . Antibodies to β-actin, MCL-1 and BCL-2 were purchased from Santa Cruz Technology . Normal Rabbit Immunoglobulin (IgG) was purchased from PeproTech .AZD5991 was obtained from AstraZeneca . Venetoclax was purchased from Selleck Chemicals . S63845, MIK665 and BV6 were purchased from TargetMol Chemicals . The MCL cell lines Mino, Rec-1, JeKo-1, Z-138, and MAVER-1 were obtained from the American Type Culture Collection . The venetoclax-resistant cell line Mino-VEN-R (Mino-venetoclax-R) was generated by chronic venetoclax treatment of parental Mino cells. JeKo-IBN-R (JeKo-ibrutinib-R) was generated from JeKo-1 cells treated with escalating doses of ibrutinib. The JeKo BTK KD_1 and JeKo BTK KD_2 cell lines were generated through BTK knockdown using CRISPR/Cas9 editing by the MD Anderson Core Facility and has been previously verified and published . Cells tMCL1 gene and assessed the significance of differences between two groups using the default Wilcoxon Rank Sum test.To investigate MCL-1 expression at the single-cell level, we integrated two of our previously published single-cell RNA-seq datasets , 20. We 4 per well in 96-well plates and treated with drug for 72 h. Drug concentrations ranged from 0.02 to 1000 nM. Cell viability was assessed using CellTiter-Glo . Primary MCL samples were obtained after informed consent, following Institutional Review Board-approved protocols and in accordance with the Declaration of Helsinki, from patients of the Department of Lymphoma and Myeloma at MD Anderson Cancer Center. Purified peripheral blood mononuclear cells were isolated from apheresis samples and treated with drug for 24 h. Dose–response curves for viability were plotted and analyzed for IC50 using GraphPad Prism. Data from the cell viability assays were normalized to samples with DMSO treatment.MCL cell lines were plated at 1 × 10MCL cells were seeded in 24-well plates overnight prior to treatment. The seeded cells were incubated with single agents or combinations for 24 h at 37 °C. After centrifugation for 5 min at 1500 rpm, cells were washed with PBS, then resuspended in annexin V binding buffer  and stained with annexin V-FITC (BD Biosciences) and propidium iodide (Invitrogen) for 15 min. Flow cytometric analyses were conducted with NovoCyte Flow Cytometer .MCL cell lines were treated for 16 h with either single agent or their combination prior to harvest. Cells were then lysed on ice in lysis buffer  containing a complete protease inhibitor cocktail (Roche) for 30 min, then centrifuged at 4 °C for 10 min at 14,000 g; 4 × loading buffer was added to the lysates after protein concentrations were measured. All original western blots are available in the MCL cell lines were electroporated following the protocol of kit V  with the Amaxa nucleofector system according to the manufacturer’s instructions. MISSION® siRNA universal negative control #1 was used as control Table . Cells woC with a slurry of Protein A/G (1:1) magnetic beads . The pre-cleared lysates were incubated with anti-MCL-1 antibody  and protein A/G magnetic beads overnight at 4 °C with rotation. Beads were washed four times with cold lysis buffer, and the proteins were then eluted with 2× loading buffer. Input samples or IP samples were heated at 95 °C for 10 min after adding the loading buffer. Input samples for western blotting from MCL cell lines were probed with antibodies against MCL-1, BCL-2, BCL-XL, BAX, BAK, NOXA, c-Caspase 3, PARP, and β-actin. IP samples were probed with antibodies against MCL-1, BAK, BAX, and NOXA.MCL cell lines were treated with AZD5991 or DMSO for 0.25, 2, 4, or 24 h. Cells were then harvested and lysed for 30 min in the aforementioned lysis buffer with the protease inhibitor cocktail. Protein concentration of the lysates was measured. 25 μg of protein lysate was loaded as input. To obtain IP samples, lysates were diluted to 1 mg/ml in 2 ml, then pre-cleared for 30 min using rotation at 4 JeKo BTK KD_2 cells were used to detect the interaction between BAX with MCL-1 or BCL-2. Cells were processed as mentioned above. IP samples of BAX  or IgG  were obtained as mentioned above after pre-clearing. 25 μg of protein lysate was loaded as input. Input and IP samples were probed with antibodies against MCL-1, BCL-2 and BAX.The OCR in intact cells was measured using an XF Cell Mito Stress Test kit . Cells were treated with AZD5991, venetoclax, or their combination at indicated concentrations for 2 h and then seeded in XF cell culture microplates. The OCR was measured by Seahorse XF96 flux analyzers. The basal level was attained by subtracting non-mitochondrial respiration and the maximal OCR was recorded after adding FCCP to induce maximal respiration. Detailed procedure was described previously .n = 4 or 5 for each group) were treated with AZD5991 at 60 mg/kg  weekly, or S63845 at 30 mg/kg (iv) twice per week, or BV6 at 20 mg/kg (ip) (three times per week) or venetoclax orally at 5 mg/kg or 10 mg/kg once daily. Tumor volumes, survival duration, and body weight were monitored. Tumor diameters were measured biweekly starting on Day 0 of tumor cell inoculation until the maximal tumor diameter reached 15 mm, and tumor volumes were calculated as V = (L × W × W)/2. Mouse blood was collected on day 35 post-tumor cell inoculation. Human beta-2-microglobulin (β2M) level was detected by ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.Mino-VEN-R mouse model was established by subcutaneously inoculating Mino-VEN-R cells into NSG mice. Patient-derived xenograft (PDX) mouse models were generated by inoculating freshly isolated PDX cells (from successfully established PDX) subcutaneously into NSG mice. When the tumors became palpable, the mice  was used to calculate statistical significance. Where appropriate, the data are presented as mean ± SD of triplicate samples. The ICMCL1 expression and therapeutic resistance in MCL. The ibrutinib-resistant samples had higher MCL1 expression compared to the ibrutinib-sensitive samples and the dual ibrutinib-/CAR T-resistant samples had the highest MCL1 expression, implying a positive correlation between MCL1 and therapeutic resistance , and Mino exhibited a particularly high sensitivity to AZD5991   Fig. . StrikinnM) Fig. .Fig. 1AZTo investigate the mechanisms in overcoming resistance in MCL, we performed in vitro studies of JeKo-1 ibrutinib-sensitive cells, JeKo BTK KD_2 cells with intrinsic resistance to BTK inhibitors, and JeKo-IBN-R cells with acquired resistance to ibrutinib, Mino venetoclax-sensitive cells, and Mino-VEN-R cells with acquired resistance to venetoclax. AZD5991 effectively triggered cell apoptosis in these resistant cell lines in a dose-dependent manner, albeit with lower potency compared to the sensitive cell lines Fig. . ImmunobTo validate the influence of MCL-1 on the downregulation of IAPs, we examined the effects of two additional MCL-1 inhibitors, namely S63845 and MIK665. Consistent with AZD5991, S63845 and MIK665 resulted in a downregulation of IAPs in Mino and JeKo-1 cells Fig. . In addiTo interrogate mitochondrial dynamics during apoptosis in response to AZD5991, co-IP assays were performed to determine the interactions of MCL-1 with NOXA and BAK in JeKo-1 and its isogenic cell lines JeKo-IBN-R and JeKo BTK KD_2 cells. Treatment with AZD5991 for 15 min dissociated BAK and NOXA from MCL-1 in a dose-dependent manner Fig. , and theTo investigate the dependence of IAPs downregulation on BAK, electroporation of siRNA was employed to knockdown BAK expression in JeKo-1 cells before AZD5991 treatment. As shown in Fig. L remained largely unaffected. Strikingly, the anti-apoptotic MCL-1 was synergistically diminished in the combination treated cells, accompanied by significant downregulation of the negative regulator of apoptosis proteins, XIAP, c-IAP1, and c-IAP2, indicative of augmented apoptosis processing. Moreover, compared to DMSO control and both single treatments, the combination eradicated the expression of oncogenic c-MYC, which contributes to intrinsic ibrutinib resistance in MCL [Given that MCL-1 overexpression confers resistance to venetoclax and that there is favorable synergy in combinatorial targeting of MCL-1 and BCL-2 in multiple myeloma and acute myeloid leukemia –26, we he in MCL .Fig. 3Thn = 14, Fig. The augmented potency prompted us to further investigate the combination in a panel of MCL patient primary samples, including treatment-naïve, ibrutinib- or venetoclax-sensitive, and the BTKi- and/or venetoclax-resistant samples Fig. . As expeDysregulated mitochondrial oxidative phosphorylation (OXPHOS) contributes to both venetoclax resistance and ibrutinib resistance in lymphoma , 23. Givn = 5, p = 0.0002 for AZD5991, p = 0.0191 for venetoclax), showing the superior in vivo anti-tumor activity of the combination. Moreover, mice tolerated the combinatorial treatment without body weight loss (data not shown). To determine the safety of simultaneously targeting MCL-1 and BCL-2, we assessed the MCL-1 inhibitor S63845, whose affinity for mouse MCL-1 is higher than that of AZD5991, in combination with venetoclax in the Mino-VEN-R mouse model. As anticipated, we observed synergistic effectiveness against MCL. No significant weight loss or hair loss were observed in the mice with this combination  mouse model was established by inoculation of Mino-VEN-R cells into immunodeficient NSG mice Fig. . Treatmeion Fig. .Fig. 6CoThe AZD5991 and venetoclax combination was further tested in a PDX mouse model established from a patient with dual resistance to venetoclax and the BTKi zanubrutinib (VEN-R/ZAN-R). AZD5991 and venetoclax, either alone or together, reduced tumor burden and prolonged mouse survival significantly compared to vehicle Fig. . StrikinWe further investigated the combination in an aggressive PDX mouse model from an MCL patient with dual ibrutinib- and anti-CD19 CAR T-cell therapy resistance (IBN-R/CAR T-R) Fig. . CompareIn addition to the canonical pro-apoptotic role for AZD5991, the amplified apoptotic signal via IAP downregulation prompted us to examine IAP antagonists in MCL. The protein expression of the three prominent IAPs, XIAP, c-IAP1, and c-IAP2, was measured by western blotting in a panel of MCL cell lines. XIAP was widely expressed with altered expression patterns across the cell lines, whereas considerable variation was observed for c-IAP1 and c-IAP2 expression levels Fig. .Fig. 7ThExtensive studies are being carried out to develop IAP antagonists that mimic endogenous IAP antagonist SMAC (second mitochondria-derived activator of caspase), which blocks caspase activation by disrupting interaction to XIAP, c-IAP1, and c-IAP2 . The SMAAs an antiapoptotic protein, MCL-1 has become an attractive target and is implicated with venetoclax resistance in hematologic malignancies , motivatMetabolic reprogramming toward OXPHOS was implicated in both ibrutinib resistance in MCL  and veneMCL has a limited number of established cell lines to cover the full spectrum of phenotypes found in the clinic. Accordingly, we included several cell lines derived from those with differing therapy resistances, along with many patient-derived primary samples with multi-resistance phenotypes. Inclusion of these additional MCL cell lines and patient samples will certainly facilitate the translation from preclinical study to clinical outcome.Evading apoptosis is a cancer hallmark. To date, several IAP antagonists have entered clinical trials, and are being investigated in combinatorial protocols . The BAKSupplementary informationOriginal Data FileReproducibility checklist.


TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies.MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs.GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination.The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.The online version contains supplementary material available at 10.1186/s13046-023-02667-4. Renal cell carcinoma (RCC), one of the ten most common cancers, occurs in both sporadic and inherited forms. MiT family translocation RCC, is an aggressive form of the disease –3 which PRCC–TFE3 fusion protein [SFPQ–TFE3 fusion, inv(X) leading to NONO–TFE3 fusion, t leading to ASPSCR1–TFE3 fusion, and t leading to CLCT–TFE3 fusion [TFE3-fusion RCC, the most common form of MiT translocation RCC, is characterized by Xp11.2 chromosomal translocations involving TFE3 that result in a fusion of the C-terminal portion of TFE3 with the N-terminal portion of specific partner genes. Consequently, activity of the TFE3 C-terminus is dysregulated resulting in constitutive nuclear localization and increased transcriptional activity of the protein. The most common Xp11.2 translocation is t, resulting in a  protein ; other r17)p11;q2 leading MiT translocation RCC is characterized by heterogeneous architectural and cytologic features that overlap with clear cell and papillary RCC as well as oncocytic tumors , and havTFE3 translocation RCC [TFE3-fusion RCC is a highly aggressive form of renal cancer and affected patients often present with metastasis at initial diagnosis. There is currently no clinical standard of effective systemic treatment and no effective targeted drug therapies have been identified to date. Activity of tyrosine kinase inhibitors (TKI) against TFE3-RCC has recently been reported in in vitro organoid-based experiments , and a ction RCC . A few rtion RCC , 12. Destion RCC , 14.GPNMB is overexpressed in a number of cancer types [A known biomarker and potential therapeutic target for TFE3-fusion RCC is glycoprotein nonmetastatic melanoma B (GPNMB), a highly glycosylated transmembrane protein that regulates a variety of physiological processes, notably osteoclast differentiation and melanosome maturation . GPNMB ier types , 17 and er types , 19, buter types , 21.Unbiased high-throughput drug screens are a potential first line approach to identifying key signaling pathways for the development of therapies. Herein we have conducted quantitative high-throughput screens of multiple TFE3-fusion RCC cell lines derived from TFE3-fusion renal tumors and identified classes of drugs with cytotoxicity against TFE3-fusion RCC. We further validated the cytotoxicity and increased apoptosis of selected agents from each class and their effects on cell cycle and downstream pathway members in 2D and 3D in vitro assays and TFE3-fusion derived xenograft models. Notably, we identified the TFE3 transcriptional target GPNMB as a cell surface biomarker that is upregulated in TFE3-fusion RCC, and evaluated a clinically relevant antibody-drug conjugate (ADC) that targets this protein in in vitro and in vivo assays. Finally, combinations of these drugs with the ADC were assessed for potential synergy as therapeutic agents in the TFE3-fusion cell lines and xenograft models.Patients were seen at the Urologic Oncology Branch (UOB) of the National Cancer Institute (NCI), National Institutes of Health (NIH) for clinical assessment. This study was approved by the Institutional Review Board of the National Cancer Institute and patients provided written informed consent on either Urologic Oncology Branch protocol NCI-89-C-0086 or NCI-97-C-0147.2 and were cultured in high glucose (4.5g/l) Dulbecco’s modified Eagle’s medium  supplemented with L-glutamine (4 mM), sodium pyruvate (110mg/l), and 10% fetal bovine serum .The TFE3-fusion RCC cell lines UOK109, UOK120, UOK124, UOK145, and UOK146 were developed within the Urologic Oncology Branch (NCI) from surgically resected specimens , 23. TheDNA was extracted from tumor tissue as above using the Maxwell Tissue Kit . PCR reactions to amplify the sequences across the gene fusions were performed using KAPA2G Fast DNA polymerase  according to the manufacturer’s specifications. Bidirectional Sanger DNA sequencing of the PCR products was performed using the Big Dye Terminator v.1.1 Cycle Sequencing Kit  according to the manufacturer’s specifications and run on an ABI 3130xl or 3730 Genetic Analyzer (Applied Biosystems). Sanger Sequencing was conducted at the CCR Genomics Core at the National Cancer Institute, NIH, Bethesda, MD 20,892. Forward and reverse sequences were evaluated using Sequencher 5.0.1 . Fusion partner-specific primers to characterize the gene fusions were as follows: TFE3_Rv: GCAGGAGTTGCTGACAGTGA; NONO_Fw: ATCAAGGAGGCTCGTGAG; PRCC_Fw1: AGGAAAGAGCCCGTGAAGAT : PRCC_Fw2: ATGCCGCTGGTGCTTATTAT (UOK124); SFPQ_Fw: CTTTTGCGCCAAGATCTGA.In collaboration with the National Center for Advancing Translational Sciences, NIH, quantitative high-throughput screening (qHTS) of 1912 pharmacologically defined small molecule compounds was conducted on three TFE3-fusion cell lines bearing three different fusion partners  as outlined in 2D cell viability following 48–72 h drug treatment was evaluated using CellTiter-Glo reagent (Promega) according to manufacturer’s instructions. Assays were performed in triplicate and experiments were repeated three times in all five cell lines. Spheroids were successfully developed in three of the cell lines, UOK109, UOK120, and UOK124, using a previously described methodology , accordiCommercially available reagents and resources included Mithramycin A (Tocris) and NVP-BGT226 . EC-8042 (EntreChem SL), antibody-drug conjugate CDX-011 and non-conjugated antibody CR011 (Celldex Therapeutics) were obtained from the manufacturers through collaborative agreements. All other compounds were generously provided by the NCI Development Therapeutics Program, NIH.Cell cycle and cell apoptosis analyses were performed by flow cytometry as previously described  using anSP1 reporter assay was performed with Cignal Luciferase Reporter Assay Kit (Qiagen) following manufacturer’s instructions and outlined in Supplementary Methods.Protein lysates were prepared from cell lines or frozen tissues using RIPA buffer  supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). Western Blot analyses were performed with standard techniques using the following antibodies: β-Actin, total Src, phosphoSrc (T416), phospho-ERK1/2 (T202/204), total and phospho-Akt (S473), total and phospho-S6 (S240/244), phospho-mTOR (S2448), phospho-4EBP1(T37/46), LC3B , goat anti-human Osteoactivin/GPNMB antibody, p62 (both R&D Systems). Protein bands where visualized with IRDye secondary antibodies diluted in Odyssey blocking buffer containing 0.2% Tween and 0.1% SDS and signals were visualized with an Odyssey imager and analyzed with Image Studio software .Hematoxylin and eosin staining was performed by standard methods. Histology was reviewed by a pathologist experienced in evaluating kidney cancer. Immunohistochemistry for TFE3 and GPNMB was performed as previously described . PrimaryGPNMB (Hs01095669_m1) and BIRC5 (Hs04194392_s1), using ACTB (Hs01060665_g1) as reference gene.RNA was extracted from frozen tumor tissue and from cell lines with TRIzol™ Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions and converted to cDNA with SuperScript IV VILO cDNA synthesis kit (Invitrogen). Gene expression was measured by Real-Time PCR using TaqMan® Gene Expression Assays on a ViiA7 Real-Time PCR system (Thermo Fisher Scientific) per manufacturer’s instructions. All assays were run in triplicate and gene expression was calculated as comparative CT (ΔΔCT) values. Gene expression was evaluated for Tumor xenografts were generated by injecting UOK124 or UOK146 cells subcutaneously into flanks of athymic nude mice (Charles River). Mice were randomized into treatment groups (n = 10 mice/group) based on tumor volume and treated with the following agents, singly or in combination: (1) NVP-BGT226, (2) Mithramycin A, (3) Dasatinib, (4) Carfilzomib, (5) CDX-011, (6) respective vehicle. Details are outlined in Supplementary Methods. Body weights and tumor measurements were taken to determine response. Mouse survival was calculated as Log-rank test in Graphpad Prism and xenograft growth rates were compared based on rate-based T/C metric according to Hather et al.  and as ot (parametric) test or Mann-Whitney (non-parametric) test, with a p value < 0.05 considered significant. All experiments were performed three times, with exception of the animal studies which were performed once using 2 cell lines.Values are expressed as mean ± standard deviation. Where appropriate, data were analyzed using a two-tailed PRCC-TFE3 fusions , one line is characterized by a NONO-TFE3 fusion (UOK109) and one by an SFPQ-TFE3 fusion (UOK145)  , 23. The45) Fig. . In addi45) Fig. .The variable nature of the fusion partners and chromosomal alterations within the TFE3-fusion RCC tumors led us to perform a broad spectrum drug screen in 3 TFE3-fusion RCC cell lines  with 3 different fusion partners to identify agents that would be effective across all fusions. In collaboration with the National Center for Advancing Translational Science (NCATS), a high-throughput drug screen was performed utilizing a library of 1912 pharmacologically defined small molecules with clinical relevance in cancer . Hit comNVP-BGT226, Carfilzomib, Dasatinib, and Mithramycin A, representative agents from each drug class, were selected for further validation in mouse xenograft studies using the TFE3-fusion RCC cell lines UOK124 and UOK146 that consistently grew well as xenografts. Treatment with NVP-BGT226 and Mithramycin A caused significant tumor growth inhibition compared to vehicle controls in both xenograft models Fig. A and sigThe efficacy of PI3K/mTOR inhibitors against TFE3-fusion RCC cell lines confirms the importance of the mTOR pathway as a driver of tumor cell growth Fig. . To demoDue to dose-limiting toxicities of NVP-BGT226 in clinical trials , we testGiven the significant effect of Dasatinib in the initial in vitro and in vivo studies Fig. , we evalBIRC5 (survivin), was highly upregulated in TFE3-fusion RCC  represented a plausible pharmacological candidate. Notably, Mithramycin A scored high in the quantitative high-throughput screen. Additionally, TCGA gene expression data demonstrated that a known inhibition target of Mithramycin A, Mithramycin A shows relatively high toxicity in patients at therapeutic doses, and analogs with a better toxicity profile have been developed. We therefore tested the Mithramycin A analog with lowest reported toxicity, EC-8042 (EntreChem SL) , for itsGPNMB, a transcriptional target of TFE3, was significantly higher in TFE3-fusion RCC compared to clear cell RCC, papillary RCC or normal kidney [GPNMB expression was elevated in TFE3-fusion RCC at the mRNA and protein level compared to very low expression in corresponding normal kidney tissues as confirmed by real-time PCR and western blot  and papshown in . While tIn vivo efficacy of the ADC was tested in UOK124-derived xenografts treated with four doses of CDX-011  or vehicle . TreatedRecent preclinical and clinical data suggest that drug combinations can benefit cancer patients. We combined the most promising drugs from our screening effort, Mithramycin A, NVP-BGT226 and the antibody-drug conjugate CDX-011 in vitro and in vivo to test if drug combinations could enhance therapeutic response against MiT-RCC. Cell culture experiments demonstrated that combining Mithramycin A with NVP-BGT226 led to a synergistic decrease in viability as compared to single agents. Similarly, Mithramycin A and NVP-BGT226 increased the drug efficacy of CDX-011 in vitro in most TFE3-fusion RCC cell lines tested Fig. A. The coMiT-RCC constitutes a subset of primarily sporadic RCC characterized by genomic translocations leading to tumor-driving fusion proteins involving members of the MiT family of transcription factors . AlthougThe development of treatments for advanced rare tumor types always presents a challenge due to the scarcity of patients and, in this case, the severity of the disease and its rapid progression. For MiT-RCC patients, surgery remains the most effective treatment for localized disease since an effective systematic treatment for advanced, metastatic disease is still lacking. We have developed a series of TFE3 translocation RCC derived cell lines that represent different TFE3 fusions , 22, 23,The initial high-throughput drug screen and subsequent 2D and 3D spheroid-based validations highlighted the PI3K/mTOR inhibitor NVP-BGT226, the RNA synthesis inhibitor Mithramycin A, and the SRC inhibitor Dasatinib as promising therapies for MiT-RCC. On-target effects were confirmed for all three agents in the in vitro cell line models. Evaluation of these drugs in mouse xenografts derived from two different TFE3-RCC cell lines demonstrated growth suppression in both models with NVP-BGT226 and Mithramycin A, but growth suppression in only one model with Dasatinib. The efficacy of the PI3K/mTOR inhibitor NVP-BGT226 correlates well with recent studies that proposed Akt/mTOR inhibition as a promising therapeutic target for TFE3-fusion RCC . In lineFLCN-deficient tumor cells as well [BIRC5 (survivin) [BIRC5 expression in response to Mithramycin A treatment. The treated TFE3-RCC cells demonstrated a G2/M phase block in the cell cycle and increased apoptosis as was previously observed in ovarian carcinoma cells in response to a Mithramycin A analog [This study confirms growth suppression by Mithramycin A in MiT-RCC cells. A selective growth-inhibitory effect of Mithramycin A has been observed in  as well ,which ar as well , suggest as well  or E2F [ as well , therebyurvivin) , was uprA analog . MithramA analog . AlthougGpnmb silencing in a mouse model of this disease inhibited cell migration, suggesting a role in metastasis [The unbiased high-throughput drug screen identified pharmacological targets for TFE3-RCC from an array of known anti-tumor agents but was not directed against specific dysregulated pathways in the tumor. Improved understanding of MiT-RCC tumor biology may elucidate specific and untested therapeutic targets or vulnerabilities. In this study the observed tumor specific expression of the cell surface marker GPNMB led to the preclinical evaluation of Glembatumumab vedotin (CDX-011), a fully human antibody-drug conjugate (ADC) that targets GPNMB and delivers a cytotoxic dolostatin-like tubulin inhibitor, Monomethyl auristatin E (MMAE) , 37. CDXtastasis .While NVP-BGT226, Mithramycin A and CDX-011 induced growth inhibition as single therapies in this study, dual agent combinations demonstrated improved responses compared to single agents in both TFE3-RCC xenograft models studied. Recent advances in treating RCC have placed a substantial emphasis on combination therapies, even as a first line therapy . CombinaA limitation of this study is the use of cell line models in evaluating potential therapies. These cell lines models are artificially cultured, may have acquired additional genetic alterations, and may not accurately represent the wide variety of tumors classified as MiT-RCC. For MiT-RCC, this is particularly notable as the tumors are driven by gene fusions involving several members of the MiTF gene family fused to a diverse number of other partner genes. To partially counteract some of these disadvantages, this study utilized multiple cell lines that represented three different TFE3 fusion partners, NONO, PRCC, and SFPQ. Several other fusion combinations have been identified and it is possible that these other gene fusions may not respond in the same way to the potential therapies described in this study. Even the subset of TFE3-RCC tumors show high clinical and histological variability, which may in part correlate with the TFE3 fusion partner. Successful drug design and treatment strategies will therefore require a personalized medicine approach involving the molecular identification of the MiT-RCC subtype and potentially additional biomarkers  for each patient. Further investigations using patient derived xenografts (PDXs), potentially in humanized mice, may provide a more comprehensive representation of MiT-RCC in preclinical studies.In this study multiple TFE3-RCC tumor-derived cell lines representing different TFE3 fusions were utilized to generate in vitro and in vivo preclinical data supporting the efficacy of PI3K/mTOR inhibitor NVP-BGT226, transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The results of this study should provide the basis for designing future clinical trials for patients with MiT-driven RCC.Below is the link to the electronic supplementary material.Supplementary Material 1: Figures S1–S9Supplementary Material 2: Table S1Supplementary Material 3: Table S2Supplementary Material 4: Supplemental Methods


The analysis of sperm shape abnormalities is commonly used to assess the toxicity of pollutants and drugs and their genotoxic effects. This methodology is widely used based on counting the number of abnormal spermatozoa; however, in the literature, there is a wide variety of sperm counts, and standardization for cost-effectiveness and robust results remains essential. In this study, we reviewed the literature on the number of sperm counts in such assessments involving rodents, published from 1969 to 2023. A new dataset involving the analysis of two wild rodent populations was produced to infer the number of counts that provides the highest robustness of assay results. A range of 100 to 6000 was recorded in the literature review, and for each animal in the populations herein studied, 300, 500, 1000, and 2000 cells were counted sequentially, and anomalies were recorded. We propose that studies addressing sperm shape abnormalities should standardize counts to an optimal value of 1000 cells per animal, ensuring statistical power and better cost-effectiveness.Rattus norvegicus) captured in two port cities, aiming to detect the impact of differential sperm cell counts in the obtained results. During necropsy, the fresh epididymis tail of adult male rats was excised, and sperm cells were fixated in slides. For each animal, a total of 300, 500, 1000, and 2000 cells were sequentially counted, and head abnormalities were registered. Counting 300 sperm cells failed to detect significant differences between groups and 500 counts resulted in marginally significant differences. Only when 1000 or 2000 sperm cells were counted, significant differences emerged between groups. We propose that studies addressing sperm shape abnormalities should standardize counts to an optimal value of 1000 cells per animal, warranting robust statistical results while providing the best compromise concerning labor time.Rodents have been the preferred models for the evaluation of the toxicity of pollutants and drugs and their genotoxic effects, including sperm shape abnormalities. The scientific literature is dominated by studies conducted with model animals in laboratory conditions, but a generally accepted and standardized protocol addressing the optimal number of sperm cells to count is still lacking. In this study, we reviewed the literature regarding the number of counted sperm cells in such assessments, published from 1969 to 2023. To infer the number of counts providing the best cost/benefit regarding the robustness of the assay results, a new dataset involving the analysis of two populations of wild rodents was produced. We evaluated the frequency of sperm shape abnormalities in a total of 50 wild brown rats ( The toxic effects of pollutants and drug agents in organisms can be evaluated by genotoxicity studies . DiffereStudies involving small non-volant mammals ,14,15,16Studies investigating sperm abnormalities as a tool to evaluate the toxicity of chemicals and drugs, such as anticancer and immunosuppressants, have commonly employed animal models, particularly rodents ,25,26. TStudies evaluating teratogenic effects on wildlife using abnormal sperm cell assessment are limited ,31. ThesIn this study, we reviewed the literature to evaluate the protocols of sperm abnormality assays, in particular the number of cells counted. We analyzed the evolution of such measurements since 1969, along with the robustness of the results provided.Rattus norvegicus) inhabiting two port cities, Lisbon and Ponta Delgada , Portugal. We hypothesized that the level of urbanization influences the level of environmental toxicity, and therefore, genotoxic effects on animal populations. Differences in the average Air Quality Index (AQI) in both study cities were used as proxies of environmental exposure to pollutants. Moreover, we aimed to compare different cell counts and evaluate the cost/benefit ratio and the robustness of the data, enabling safeguards, mainly, in studies carried out in wild environments. This study handles information for researchers to make informed decisions when designing genotoxic studies.To validate our results, we studied two populations of wild brown rats .Using multidisciplinary research engines Web of Science (WoS), Scopus, and Google Scholar, we compiled studies using sperm shape abnormality assay in rodent species, by manual (non-automated) approaches, observed with a microscope. Review papers were excluded, as well as publications on reproductive improvement and with unclear methodological information. The keywords used for the search were “sperm abnormality assay”, “sperm abnormalities”, “sperm morphology”, and “genotoxicity”. For each selected study, several parameters were recorded: (i) number of sperm cell counts per individual; (ii) publication year; (iii) study’s country of origin; (iv) model species; and (v) type of environment (lab versus wild). Publications not clearly stating the number of sperm cells counted or reporting methods to previous publications were removed from the analysis.Rattus norvegicus) in two port cities, Lisbon and Ponta Delgada, Portugal. These cities differ in their level of urbanization and therefore, levels of air pollution, as determined by the European Environmental Agency . Trapping involved baited Tomahawk traps, distributed along a 10 km radius from both cities’ ports. A total of 50 adult male brown rats were used in this study . After capture, the animals were transported to the laboratory and euthanized by intoxication with isoflurane, in accordance with international guidelines.Animals were obtained in the scope of a project (PTDC/SAU-PUB/29254/2017) involving the live-trapping of brown rats , for the 500 sperm counting presented some significance , and the 1000 and 2000 sperm counting were statistically significant . The results obtained in the samples collected in Lisbon and Ponta Delgada are shown in t = 7.72; p < 0.001) and the total number of abnormalities detected , while the city of origin did not have a significant effect . The maximum number of cell classes per animal was six, registered only when 2000 cells were counted. However, seven out of eight classes were found with the minimal number of cell counts  and hook with wrong angle (in Ponta Delgada). The number of classes of abnormal cells identified were highly influenced by the number of counted cells , while counting 500 cells unveiled marginally significant differences (p = 0.04). Only when 1000 cells were counted did a highly robust difference emerge between groups (p = 0.002), which was even higher for the 2000 cell counts (p = 0.0006). Although in laboratory-based studies, inter-individual variance is reduced, and environmental factors highly controlled, in studies involving wild-caught animals, individuals are subject to various biotic and abiotic constraints (that contribute to data variance). Among these restrictions, we highlight the putative influence of wild animals’ age, known to increase levels of sperm abnormalities [In the case study presented herein, we observed that the number of sperm cells counted per animal was crucial for the interpretation of the obtained results, either detecting or not detecting statistically significant differences between groups. When counting 300 sperm cells, no statistically significant differences were observed between the two rat groups , by redmalities . Abnormamalities ,72, becomalities , comprommalities .Our power analysis suggests that previous knowledge of the expected genotoxic influence may lead to an optimal choice of cell counting. However, in most scenarios, such effects are unknown and, thus, unpredictable, in particular when assessing genotoxic effects on natural environments, with unknown levels of contamination or exposure. Considering the total sperm abnormalities observed in our case study, between ~2% in Ponta Delgada and ~4% in Lisbon, we validate the choice of 1000 cell counts per animal to accommodate different levels of contamination scenarios as calculated by the power analysis . Robust This case study shows that, in a dataset obtained from wild-caught rodents inhabiting moderately polluted urbanized cities , 300 celAn optimal number of cell counts is not only relevant concerning the total number of sperm anomalies detected in study groups but also regarding the type of anomalies identified. To our knowledge, different types of anomalies have not been connected with sperm cell activity, as all are potentially hazardous and compromise sperm function. However, we noticed that rarer anomalies may become undetected if a smaller number of cell counts is considered. This was the case for three out of eight classes of observed sperm abnormalities among our dataset. When only 300 cells were counted, no amorphous sperm heads were detected among the Lisbon rat population, nor swollen acrosomes or swollen hooks among the Ponta Delgada population .Our literature compilation returned only seven publications using wild rodent populations as bioindicators of environmental pollution. The outcomes of these publications have a wide range of cell counts: 100 cells , 200 celWe suggest that studies addressing wild animals in natural conditions, by standardizing cell counts to 1000, would provide sufficiently robust and repeatable results, allowing existing statistical differences to emerge, considering frequencies of abnormalities occurrence below 2%. Standardizing cell counts to 1000 would also be the most cost-effective option as cell counts > 1000 severely stretches the analytical time with no improved return. We also consider that increasing the cell pool by counting elevated numbers of cells from single animals should be avoided to account for inter-individual variation. When compared to those in laboratory settings, genotoxicity studies in natural environments are rare. The study of reproductive parameters in wild species is key not only in toxicology assessments but as a proxy of overall health of wild populations . KnowledThis review highlights the need for an optimized approach regarding research on genotoxicity assessment involving  counts of sperm head abnormalities in rodents. By reviewing the scientific literature and using a dataset from wild-caught rodents as a case-study, we concluded that counting 1000 sperm cells per animal constitutes the optimal trade-off between statistical robustness and operational costs. If a common and optimized methodology is followed by most researchers, the obtained results of genotoxic assessments will be comparable, even inter-laboratories, which is currently not the case.


Olive oil TPEs of determined OOPs’ content, originating from three different harvests of autochthonous olive cultivars in Greece, were evaluated as an attempt to investigate the efficacy of OOPs to reduce cancer cell numbers as part of olive oil consumption. Most combinations of OOPs showed strong synergistic effect (CDIs < 0.9) in their efficacy, whereas TPEs strongly impaired cancer cell viability, better than most individual OOPs tested herein, including the most resistant cancer cell lines evaluated. Several individual olive oil phenols (OOPs) and their secoiridoid derivatives have been shown to exert anti-proliferative and pro-apoptotic activity in treatments of human cancer cell lines originating from several tissues. This study evaluated the synergistic anti-proliferative/cytotoxic effects of five olive secoiridoid derivatives  in all possible double combinations and of total phenolic extracts (TPEs) on eleven human cancer cell lines representing eight cell-culture-based cancer models. Individual OOPs were used to treat cells for 72 h in half of their EC Oleaceae, Valerianaceae, Gentianaceae and Pedialaceae. They are abundant in the European olive tree (Olea europaea L.) and they comprise the main bioactive polyphenols in olive oil and drupes  were used as control groups. Concerning the evaluation of the anti-proliferative/cytotoxic properties of TPEs, three extracts of different phenolic composition were used  DMSO].Cell viability was evaluated using the MTT colorimetric assay as previously described ,31. BrieThe combined effect of the OOPs in the combinations’ treatments was assessed by the coefficient of drug interaction (CDI). CDI = AB/(A × B), where A is the percentage (%) of viable cells after treatment with substance A used alone and Β is the percentage (%) of viable cells after treatment with the 2nd substance used alone. AΒ corresponds to the percentage (%) of viable cells after treatment with the combination of the two compounds. The absorbance ratio of each group was normalized to the value obtained for the control group. Thus, CDI < 1, =1 or >1 indicates drugs with synergistic, additive or antagonistic action, respectively. Drugs with a calculated CDI < 0.7 exert a strong synergistic effect ,33.50 determination and statistical analyses were performed using the GraphPad Prism v8  and Office Excel 365 . For the cell-viability assays, the data were normalized to the average absorbance of the control group, which was considered as 100% viability.All data were derived from two or three independent experiments performed in triplicates. ECp values were estimated using the GraphPad algorithm.Statistical analysis to determine whether OOPs’ anti-proliferative/cytotoxic effect is significant or not was performed by calculating the mean values from the independent experiments and the S.E.s  = 55 μM]. The best reduction of cell numbers with the lowest total [OOPs] was observed for the combination oleocanthal/oleuropein aglycone . Double combinations of ligstroside or oleuropein aglycones with oleomissional also exhibited synergistic effects  similar to what was observed for the combinations of oleacein with either one of the two aglycones A. OleaceFrom the colon and gastric carcinoma cell models, the AGS stomach cancer cell line appeared to be the most sensitive to double OOP combination treatments. More specifically, the AGS cells were very sensitive to combinations of οleomissional with oleacein or ligstroside aglycone, showing the strongest synergistic effect . However, at lower total [OOPs], the best effect on the AGS cells was observed for the combination of οleocanthal with either οleuropein aglycone or ligstroside aglycone . Although the HT-29 colon cancer cells appeared to be more resistant than the AGS to the majority of the OOP combinations tested in the study, the oleacein/ligstroside aglycone combination dramatically reduced the cell numbers  in this cell line, with a very strong synergistic effect (CDI = 0.3) also observed for the MCF-7 breast cancer cells.The HepG2 liver cancer cell line responded well  to the treatment by most of the OOP double combinations A. The olThe PANC-1 pancreatic cancer cell line was the most sensitive of all cancer cells studied herein to all the double combinations of OOPs used in the treatments  . This paThe selectivity indices of the combinations for the cell lines (cancer and non-tumorigenic transformed cells) originating from the same tissue summarized in 50 values were determined in our previous study [Τhe observations on the synergistic, additive or in a few cases antagonistic effects of certain OOP double combinations paved the way for proceeding in testing samples of total phenolic extracts isolated from olive oil produced from olives of different cultivars. Two of the TPEs used had higher contents in oleocanthal  and contained all the other OOPs for which the ECus study , while tus study  and tyro50 values of the two TPEs for each cell line tested are presented in For the evaluation of the anti-proliferative/cytotoxic efficacy of the three TPEs, all the breast cancer  and melanoma  cell lines available for this study were selected for testing, as well as the cell lines from each other cancer type studied herein which were less sensitive to the treatment by the individual OOPs contained in the TPEs . As shown in 50s of TPE I < EC50s of TPE II). With respect to the cells treated with all three TPEs, it seems that in all cases they respond less to the treatment with TPE III as compared to treatment with the other two TPEs. The difference in the bioactivity of TPEs I and II was significant (p ≤ 0.05) in the AGS and HepG2 cells, while between the TPEs Ι and ΙΙΙ a significant difference (p ≤ 0.05) was observed in the MDA-MB 231 and AGS cell lines.As expected from each different composition of TPEs in OOPs, the levels of sensitivity to the treatments differed in the different cell lines . For exa50 TPE I < ΕC50 TPE ΙΙ; 50 of TPE ΙΙΙ > ΕC50 of TPEs Ι and ΙΙ) despite its higher content in the other two of the most bioactive OOPs . The EC50 value of oleocanthal, which is the most abundant OOP in the two TPEs (44.6% in TPE I and 33.9% in TPE II), was lower than all the EC50 values of TPEs I and II when used alone as a single compound (2.8–13.6 μg/mL). This could be explained by the antagonistic effect observed amongst oleocanthal and some of the OOPs in the double OOP combination experiments described in It is possible that the higher content in oleocanthal and oleacein, as well as the presence of tyrosol in TPE I , might h50 values of the TPEs were compared with those of each individual phenol for the same cell line, it was observed that in about 53–64% of cases the TPEs were more effective—even by more than two-fold—or in some cases they had at least equal efficacy. The case in which the exact opposite was observed mainly concerned oleocanthal, and in some cases the aglycone of oleuropein. It is interesting to highlight that the EC50 value of oleocanthal as a single phenol [50 values of TPEs I and II in which it is the most abundant phenol (44.6% in TPE I and 33.9% in TPE II) for all cell lines. The sole exception was the EC50 value of TPE II for HepG2 cells. A similar pattern was observed in the Hela cell line for all phenols as compared to TPEs I and II and in the MDA-MB 231 cell line for TPE III. These results confirm the existence of the antagonistic action observed between some of phenols in the experiments described above with the double OOP combinations . In two cell lines (MCF-7 and HepG2) TPE II was more effective, while in four others  the EC50s of the two TPEs were very close  as observed for the double OOP combinations, while the skin cell lines appeared to have a similar response to the melanoma cell lines. As for the MCF 10A cells, it was observed that they were more sensitive to treatment with TPE I than with TPE II (ECectively B). In adCalculations of the selectivity indices (SIs) for the cell lines of breast and skin origin showed that TPE II was less toxic (SI = 1.7–2.1) for non-cancer cell lines of both skin and breast origin . The hig50s of the two TPEs were compared to the EC50 values of every single OOP component tested on the same cell line, it was observed that in 64% of the cases the TPEs were more effective, even by more than two-fold, or were at least of equal efficacy (50 (ratio 0.6). Moreover, in the HepG2 cells oleuropein aglycone had almost equal efficacy to TPE II (ratio 1.1) and a two-fold higher efficacy than TPE I (ratio 1.9)  .In summary, olive oil TPEs of different composition in OOPs have different efficacies in inhibiting proliferation/viability of different cancer as well non-cancer cell lines. However, in all cases except for the HepG2 hepatoma cells, treatment with oleocanthal as a single compound showed the strongest effect in reducing cell numbers/viability. Interestingly, the HeLa cells seemed to be the least sensitive cell line to the two TPEs tested on them , responding better in all the OOPs tested as individual compounds .Pre-clinical studies on the anticancer effect of polyphenols from different plants have demonstrated that combinational treatment of two or three polyphenols inhibited cancer growth more effectively than a single compound treatment. The synergistic action between multi-nutrients can modulate natural products’ bioavailability and affect in a pleiotropic way multiple metabolic pathways involved in carcinogenesis simultaneously .The study presented herein demonstrates strong synergistic effects in most of the possible double combinations of five olive oil secoiridoids in treatments of eleven cancer cell lines from eight tissue origins. The existence of synergy between the OOPs under study is very important as it allows the reduction of the active concentrations for each substance and possibly the reduction of its toxic effect on normal cells in protocols for in vivo study of their anticancer efficacy. In addition, evaluation of the anti-proliferative/cytotoxic activity of olive oil TPEs, as compared to individual phenols, is a field of great interest for anticancer therapy, given that the microenvironment of TPEs could act as a means of easier uptake of OOPs by the cells, modify their bioavailability and enhance the efficacy of individual phenols with complementary or synergistic interactions.50 value for each substance, inhibited cell viability in percentages ranging from 35% to 80%, depending on the cell line and the OOP used in the combination. In fact, the CDI coefficient was less than one—in the majority of cases—indicating synergistic interactions between the tested OOPs. The combination of oleomissional which stood out for its synergy was that with oleacein . Treatment with oleomissional and oleacein inhibited cell viability by up to 80% in some cases. It is interesting to note that this particular OOP combination caused a greater than 60% reduction in cell viability in seven out of the eleven cancer cell lines tested, with the AGS stomach cancer and H1299 lung cancer cell lines proven to be the most sensitive. In addition, good synergistic action was observed between oleacein and the two aglycones. In particular, incubation with the combination of oleacein and ligstroside aglycone appeared to have stronger efficacy in the breast, colon, stomach and pancreas cell lines (inhibition > 70% at concentrations ≤ 25 µM), surpassing the efficacy of the individual OOPs.Some very interesting conclusions emerged in this study regarding the synergistic action of OOPs in double combinations. Specifically, two of the least bioactive OOPs were highlighted for their synergistic action with the other four OOPs tested. Oleomissional, when combined with each of the other four OOPs at concentrations of ½ the ECThe most bioactive double OOP combinations were tested in non-cancer cell lines. The MCF 10A breast cell line proved to be very sensitive to all dual OOP combinations, while the non-cancerous skin cell line HaCaT was relatively less sensitive to the combination therapy. The highest selectivity index was obtained for the combination of the two aglycones (oleuropein and ligstroside) in the skin cell lines, while the melanoma cell lines were slightly more sensitive to the combinations of oleocanthal—oleacein and oleacein—oleomissional as compared to the non-cancerous HaCaT cells.In general, the AGS, PANC-1 and H1299 cell lines were found to be the most sensitive lines to most of the double combinations applied, while phenols with lower efficacy, when used in the treatments as single compounds , showed a strong synergistic effect with all the rest. Finally, in most cases there was synergy between the OOPs studied, with the exception of some of the combinations with oleacein and oleocanthal in breast cell lines, where either additive or antagonistic effects were observed.Evaluation of the bioactivity of the double OOP combinations was followed by the study of the anti-proliferative/cytotoxic bioactivity of olive oil TPEs with different contents of the five OOPs under study and of other OOPs. As expected, differences in the composition of each TPE in terms of bioactive phenols resulted in differences in their efficacy to reduce cell numbers in the cell lines incubated with them. In particular, TPE I, which contained more oleocanthal and oleacein than TPE II , was more active in most cell lines tested, suggesting that these two OOPs contribute to the higher efficacy of TPE I to reduce cancer cell viability. It is also possible that this difference is due to the presence of tyrosol in TPE I. Tyrosol is a phenolic compound of olive oil mostly known for its antioxidant and anti-inflammatory properties and for the ability to act as radical chelator. Chronic inflammation and alterations of normal cellular redox status are two main factors that promote neoplastic transformation, so tyrosol may act as an anticancer or chemopreventive agent in different human malignancies . An exceTPE III, which lacked the oleocanthal and oleacein present in the other two TPEs, appeared to have equal or slightly lower efficacy in all cell lines tested. In fact, the difference in the bioactivity of TPE III as compared to TPE I appeared to be significant only against the breast cell line MDA-MB 231 and the stomach AGS cells. This could be due to the presence of oleocanthal, which is the most bioactive molecule of all the tested OOPs in these cell lines .50 values calculated for the three olive oil TPEs in the present study for the specific cell line . An explanation for this quantitative difference between the results reported herein and those by Reboredo et al. 2018 [Overall, in evaluating the results reported in this study regarding the anti-proliferative/cytotoxic activity of the three TPEs for each cancer type tested and comparing them with results already reported by other researchers, a number of interesting observations emerged. Regarding the MCF-7 breast cancer cells, the study by Reboredo-Rodríguez et al. 2018 showed that an olive oil extract with higher levels of the two aglycones (ligstroside and oleuropein) than of oleocanthal and oleacein inhibited cell viability by 50% at a concentration of about 500 µg/mL in 48 or 72 h treatments . This coal. 2018  may be d50s in the A431 epidermal cancer cells after 72 h incubation  [50 values calculated in that study were four- to five-fold higher than the EC50s reported in this study for the melanoma cell lines A2058 and SK-MEL-28 for TPE I and TPE II . This may be due to the difference in the cell lines tested, which, although originating from the same tissue, are derived from different kind of cancers. Μoreover, as observed for the breast cancer cell lines, a significant difference in the efficacy of TPEs may be due to the different concentrations of the secoiridoid polyphenols in the three different extracts and especially the absence of oleocanthal in the TPE III as well as to the method by which the OOP concentrations in the TPEs were determined.In this study, the melanoma cell lines SK-MEL-28 and A2058 showed a similar response to all three TPEs tested. Previous studies  have als50 ~ 20 µg/mL) compared to the other cancer cell lines tested. Nevertheless, other ex vivo and in vivo studies with olive oil TPEs on colon cancer have shown inhibition of certain critical steps in the development of carcinogenesis and in the invasive and migratory capacity of cancer cells [Furthermore, the results of the present study showed that the HT-29 colon cancer cells responded to higher concentrations of all three olive oil TPEs , while the effect of each individual OOP on these cells was stronger compared to that of the TPEs. Finally, an evaluation of the selectivity of TPEs I and II on breast and skin cell lines showed that TPE II had a higher selectivity index (SI = 1.1–2.1) as compared to TPE I (SI = 0.3–1.5). However, some individual OOPs have been shown to have higher Sis [Comparison of the TPEs’ ECgher Sis .The results presented herein demonstrate synergistic interactions between several OOPs in double combinations and in the context of olive oil TPEs, enhancing their anti-proliferative/cytotoxic efficacy in treatments of several cancer cell lines. As the existing chemotherapeutic approaches present problems of side effects in normal tissues and resistance development in cancer cells, there is a constant need for more and alternative anticancer agents and new strategies in current cancer treatments. Combinations of anticancer drugs with plant polyphenols in chemotherapeutic regimens hold promising value. Certain OOPs can be added to the list of promising polyphenols for this approach. Combinations of OOPs or olive oil TPEs enriched with the most bioactive OOPs represent good candidates as well. The results of the present study form a base for further in vivo studies in cancer animal models and clinical trials investigating the chemopreventive and anticancer value of OOPs and TPEs.


Glioblastoma (GBM) is the most lethal adult primary brain tumor, and has no cure. This study investigated the membrane protein sortilin as a prognosis biomarker for glioblastoma (GBM). We found that sortilin is overexpressed in GBM tumors and can be detected in the blood of GBM patients. In addition, in cell cultures, targeting sortilin resulted in the inhibition of GBM cell invasion. These data highlight the value of sortilin as a potential clinical biomarker and therapeutic target for GBM and warrant further translational investigation.Glioblastoma (GBM) is a devastating brain cancer with no effective treatment, and there is an urgent need for developing innovative biomarkers as well as therapeutic targets for better management of the disease. The membrane protein sortilin has recently been shown to participate in tumor cell invasiveness in several cancers, but its involvement and clinical relevance in GBM is unclear. In the present study, we explored the expression of sortilin and its potential as a clinical biomarker and therapeutic target for GBM. Sortilin expression was investigated by immunohistochemistry and digital quantification in a series of 71 clinical cases of invasive GBM vs. 20 non-invasive gliomas. Sortilin was overexpressed in GBM and, importantly, higher expression levels were associated with worse patient survival, pointing to sortilin tissue expression as a potential prognostic biomarker for GBM. Sortilin was also detectable in the plasma of GBM patients by enzyme-linked immunosorbent assay (ELISA), but no differences were observed between sortilin levels in the blood of GBM vs. glioma patients. In vitro, sortilin was detected in 11 brain-cancer-patient-derived cell lines at the anticipated molecular weight of 100 kDa. Interestingly, targeting sortilin with the orally bioavailable small molecule inhibitor AF38469 resulted in decreased GBM invasiveness, but cancer cell proliferation was not affected, showing that sortilin is targetable in GBM. Together, these data suggest the clinical relevance for sortilin in GBM and support further investigation of GBM as a clinical biomarker and therapeutic target. Glioblastoma multiforme (GBM) is the most common and lethal malignant primary brain tumor in adults, accounting for 45% of brain cancer cases , with a Sortilin (SORT1), also known as neurotensin receptor-3 (NTR3), is a membrane receptor that belongs to the VPS10P (vacuolar protein sorting 10 protein) family of receptors . The bioIn this study, we have explored the clinical relevance of sortilin in GBM. Using GBM patient samples and patient-derived cells, we have shown that sortilin expression is elevated in GBM compared to lower-grade glioma, and that sortilin was also detectable at varying concentrations in the blood of GBM and lower-grade glioma patients. In addition, sortilin inhibition was able to inhibit the invasion of GBM cells. These data point to sortilin as a potential biomarker and therapeutic target for GBM.http://gepia.cancer-pku.cn (accessed on 1 December 2022)) was used to explore GBM data in TCGA [SORT1. One-way ANOVA was used for differential analysis of gene expression, using disease states  as variables for the box plots. Log-rank tests for both disease-free survival and overall survival analyses were used.Gene Expression Profiling Interactive Analysis 2 (GEPIA2) . The ELISA kit  was from Aviscera Bioscience . The assays were performed as recommended by the manufacturer and as previously described . The WilGlioblastoma cancer cell lines A172 (CRL-1620) and U87MG (HTB-14) were obtained from the American Type Culture Collection . Patient-derived GBM cell lines BAH1, MN1, WK1, RN1, RKI1, HW1, PB1, SB2b, and SJH1 were a generous gift from Dr Bryan Day . Human astrocytes (HA) were from ScienCell Research Laboratories . GBM cell lines and patient-derived GBM cell lines, including their MGMT methylation status, have been described previously ,22. The Conditions for protein extraction and Western blotting were as previously published  with an ®  according to the manufacturer’s instructions and as previously reported [Cell growth assays were carried out using Cell Titer-Bluereported . Cells w® 8.0 µm Pore Polycarbonate Membrane Insert  as previously described [Invasion assays were carried out using the 6.5 mm Transwellescribed . Cells wt-test with unpaired two-sided was used for single comparisons. One-way analysis of variance (ANOVA) or two-way analysis of variance with Dunnett’s or Tukey’s correction were used for multiple comparisons. When data was not normally distributed, the non-parametric Kruskal–Wallis test was performed. Pearson’s correlation test was used to determine correlations. A p value less than 0.05 was deemed statistically significant. All experiments were performed at least in triplicate. All materials used and results generated were included for statistical analyses, with no exclusion of data points. All data are included in this publication and are presented as mean ± standard deviation (SD).GraphPad Prism  was used. H-scores were analyzed as continuous variables, with summary statistics presented as group-level medians and interquartile ranges (IQR). Student’s p = 0.0023 and p = 0.0004 respectively): patients with low SORT1 expression survived longer (We first performed a data mining of sortilin gene (SORT1) expression using GEPIA2  and acced longer C. Howeved longer D.p = 0.0016). The receiver operating characteristic (ROC) curve [p = 0.0157)  and lower-grade (1–3) glioma (20 cases); the results are presented in C) curve  indicate 0.0157) G. While  0.0157)  and GBM  0.0157)  show no p = 0.2315)  B. Intere 0.2315) ; however 0.2315) .p = 0.0823). Additionally, when we separated nine patient-derived GBM cell lines by their subtype [p = 0.0091 and p = 0.0131, respectively)  .p = 0.0039) (p = 0.0227), 53% in A172 (p = 0.0140), 48% in BAH1, 38% in RKI1 (p = 0.0150), and 52% in PB1 (p = 0.0005)  A compare 0.0039) A or MGMT 0.0039) B did not 0.0039) A. In the 0.0005) B. These The heterogeneity of GBM is complex and remains to be fully elucidated, not only to identify new effective targets for treatment, but also to identify noninvasive biomarkers that could enable accurate prognosis and help formulate appropriate treatments. The current study expands on previous observations of the expression and biological effects of sortilin in GBM ,28 and fIn terms of gene expression, our data mining, using GEPIA2 to access the TCGA GBM and LGG datasets, contradicted other studies  and reveThe upregulation of sortilin protein has been reported in several cancers, including breast cancer , pancreaOur study is the first to quantify soluble sortilin in the plasma of GBM patients. Outside GBM, soluble sortilin has already been reported and is presumably formed after proteolytic cleavage . The ELIAside from tissue expression and plasma release of sortilin in GBM, an interesting finding of the present study was the inhibition of GBM cell invasion induced by sortilin targeting. Sortilin targeting by knockdown and inhibition with AF38469 has previously been shown to reduce cancer cell invasion in a few GBM cell lines. Here, we confirm that AF38469 not only reduces cell invasion in the traditional GBM cell lines (U87MG & A172), but, importantly, also reduces cell invasion in the more recently developed patient-derived cell lines . In terms of GBM treatment, the use of a drug like AF38469 is more clinically relevant than a molecular knockdown, and AF38469 is the only blood-brain-barrier-crossing drug currently available to target sortilin. It is noteworthy to mention that the main issue with GBM recurrence is the invasive capacity of GBM cells, which can invade the brain tissue surrounding the tumor and thus escape surgery or radiotherapy. GBM rarely metastasises, and the main issue is the local invasion of GBM cells in the neural tissue. Interestingly, our data show that the pharmacological inhibition of sortilin, using the orally available blood-brain-barrier-crossing drug AF38469, resulted in the inhibition of patient-derived GBM cell lines. A statistically significant inhibition of GBM cell invasion by AF38469 was observed for tested patient-derived GBM cells, indicating that sortilin is a potential therapeutic target for inhibiting GBM invasiveness. In contrast, sortilin targeting with AF38469 was not found to have any effect on GBM cell survival, and was not found to potentiate the activity of TMZ. However, in one patient-derived cell line (RKI1), potentiation of the cytotoxic effect of TMZ by AF38469 was observed. It is unclear at this stage why RKI1 shows an increased sensitivity to TMZ in presence to AF38469, and this leaves open the possibility that in some GBM, sortilin inhibition could be a way to potentiate the efficacy of TMZ.It is important to note that the mechanism of action for AF38469 is currently not clearly understood. The structural data published by Schrøder et al.  show thaIn conclusion, the present study points to sortilin as a new potential clinical biomarker for predicting GBM aggressiveness and patient survival. In addition, targeting sortilin seems to decrease GBM cell invasion, and could therefore be used in GBM therapy. Although further in vivo studies are needed, it should be noted that sortilin targeting in human cancers has recently entered a clinical trial phase. In breast , thyroid