ABCB1. We also identify functions for YB-1 in cell invasion, lipid metabolism and activation of the MYC oncoprotein. Importantly, through the generation of cell lines resistant to standard-of-care medulloblastoma therapies, we identify a drug-tolerant gene expression signature which may represent global, targetable mediators of medulloblastoma drug resistance. Together, our findings reveal important mechanisms and genes underlying therapy resistance in medulloblastoma and provide routes to their intervention.Medulloblastoma is the most common malignant childhood brain tumour. Under standard therapy, relapse occurs in 30% of patients and is almost universally fatal, accounting for 10% of all childhood cancer deaths. A barrier to effective medulloblastoma treatment is cellular resistance to standard-of-care therapies. Here, we investigate mechanisms surrounding medulloblastoma therapy resistance, both related to and independent from oncoprotein YB-1. Accordingly, we reveal roles for YB-1 in therapy sensitivity and the regulation of the multi-drug resistance gene ABCB1, as determined by chromatin immunoprecipitation. Whole transcriptome sequencing of YB-1 knockdown cells identified a role for YB-1 in the regulation of tumourigenic processes, including lipid metabolism, cell death and survival and MYC and mTOR pathways. Stable cisplatin- and vincristine-tolerant Group 3 and SHH cell lines were generated to identify additional mechanisms driving resistance to standard-of-care medulloblastoma therapy. Next-generation sequencing revealed a vastly different transcriptomic landscape following chronic drug exposure, including a drug-tolerant seven-gene expression signature, common to all sequenced drug-tolerant cell lines, representing therapeutically targetable genes implicated in the acquisition of drug tolerance. Our findings provide significant insight into mechanisms and genes underlying therapy resistance in medulloblastoma.Therapy resistance represents an unmet challenge in the treatment of medulloblastoma. Accordingly, the identification of targets that mark drug-resistant cell populations, or drive the proliferation of resistant cells, may improve treatment strategies. To address this, we undertook a targeted approach focused on the multi-functional transcription factor YB-1. Genetic knockdown of YB-1 in Group 3 medulloblastoma cell lines diminished cell invasion in 3D in vitro assays and increased sensitivity to standard-of-care chemotherapeutic vincristine and anti-cancer agents panobinostat and JQ1. For vincristine, this occurred in part by YB-1-mediated transcriptional regulation of multi-drug resistance gene  WNT), SHH , Group 3 (MBGroup3) and Group 4 (MBGroup4)—which can now be further categorised into second-generation sub-groups [Group3 is particularly poor due to the frequent occurrence of metastasis in this group, MBSHH and MBGroup4 have intermediate outcomes, whereas the outcome for MBWNT is generally good. Accordingly, improving our understanding of resistance mechanisms surrounding standard-of-care medulloblastoma chemotherapy represents an important area of medulloblastoma research [Medulloblastoma represents the most frequent malignant paediatric brain tumour. It comprises four principal molecular groups with different patterns of metastasis and overall prognosis—WNT  following therapeutic exposure, linking YB-1 expression to multidrug resistance mechanisms [Y-box binding protein 1 (YB-1), is a multi-functional protein encoded by the ively in ). Over-eively in , breast ively in , osteosaively in , head anively in , prostatively in , glioma ively in  and non-ively in . Frequenively in ). In tumively in ,13,14. Aively in ,16, whilively in ,18,19. Yively in ,21 and tively in , furtherchanisms .Group3 cell lines and subsequent transcriptomic analyses, the current study aimed to investigate the role of YB-1 in medulloblastoma tumourigenesis, with a focus on drug resistance. Bioinformatic analysis of publicly available medulloblastoma patient datasets was utilised to validate pre-clinical findings, whilst in vitro 3D invasion, cytotoxicity and chromatin immunoprecipitation assays provided key functional validation. Alongside this, we designed a global approach to identify alternative mechanisms driving resistance to standard-of-care therapies. Vincristine- and cisplatin-tolerant MBGroup3 and MBSHH cell lines were derived and mechanisms driving therapy resistance were identified by next-generation sequencing. Our findings, and the tools that we have created, identify key mediators of medulloblastoma drug resistance and routes to their intervention.The aforementioned studies highlight clear YB-1 involvement in cellular mechanisms surrounding stress response and drug resistance in multiple tumour types. However, how YB-1 is associated with such mechanisms in medulloblastoma remains to be explored. Through the creation of YB-1 knockdown MB2 and 37 °C. Mycoplasma testing was performed monthly using a PlasmoTestTM Mycoplasma Detection kit ; rep-pt1) as per the manufacturer’s instructions.DAOY and D283 cell lines were obtained from ATCC , ONS76 from Annette Künkele , UW-228-3 and D458 from John R. Silber , HD-MB03 from Till Milde  and CHLA-01 and CHLA-01R from Geoff Pilkington . DAOY, D283, D425, and D458 cells were cultured in DMEM with 10% foetal bovine serum , SH30541.03). UW-228-3 cells in DMEM/F-12 with 15% FBS and 1% sodium pyruvate. ONS76 and HD-MB03 cells were cultured in RPMI 1640 with 10% FBS. CHLA-01 and CHLA-01R were cultured in DMEM/F-12 supplemented with 2% B-27 , 17504044), 20 ng/mL recombinant human epidermal growth factor  and 20 ng/mL recombinant human basic fibroblast growth factor . All cell lines were grown under antibiotic-free culture conditions at 5% CO2 for 72 h. Following treatment, surviving cells were assayed with PrestoBlue , A13262) at a final dilution of 1:10 for 60 min at 37 °C and 5% CO2 and fluorescence was measured at 560/590 nm using a FLUOstar Omega ) microplate reader. The drug response was calculated as a percentage of the vehicle-treated control and dose–response curves and IC50 values were generated by non-linear regression analysis using GraphPad Prism 8 .For drug response analysis, cells were grown in standard culture conditions as previously described and harvested when they reached approximately 70% confluence. Cells were seeded at a density of 10,000 cells/well (D283), 5000 cells/well (HD-MB03 and D425) or 1000 cells/well (DAOY) in black-walled, clear-bottom 96-well plates and left to settle for 24 h. Cells were treated with cisplatin , S1166), vincristine , JQ1 , panobinostat  or an equivalent concentration of drug vehicle (dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO))—and incubated at 37 °C and 5% CO2 and 37 °C and the treatment dose escalated upon cell proliferation. Cells were passaged in T-25 flasks in duplicate and dosing was commenced at 1/100 IC50 in each cell line. Matched DMF-treated (DMSO-treated for DAOY) vehicle control flasks were passaged bi-weekly alongside drug-treated flasks to account for morphological and genetic changes occurring from long-term culture and vehicle exposure. Drug resistance was measured by drug response assays, with cells considered resistant when the IC50 value of the treated cells had exceeded the treatment dose and the cell lines exhibited a significant increase in fold resistance .To generate drug-tolerant cell lines a continuous model of selection was utilised by which D283, D458 and HD-MB03 cell lines were cultured continually in the presence of cisplatin, and the DAOY cell line in the presence of vincristine, in antibiotic-free media at 5% CO® RNA Plus kit ; 740984). RNA was converted to cDNA using superscript II reverse transcriptase ; 1080-044). Gene expression of the resultant cDNA template was assessed by quantitative reverse transcription PCR ) using iQ SYBR SuperMix . Primer sequences were utilised as follows: YB-1 forward (5′ AAG AAG GTC ATC GCA ACG AAG 3′) and YB-1 reverse (5′ CTC CTA CAC TGC GAA GGT ACT 3′); ABCB1 forward (5′ CCC ATC ATT GCA ATA GCA GG 3′) and ABCB1 reverse (5′ GTT CAA ACT TCT GCT CCT GA 3′); ABCC1 forward (5′ TTC TCG GAA ACC ATC CAC GA 3′) and ABCC1 reverse (5′ CCT GTG ATC CAC CAG AAG GT 3′); GAPDH forward (5′ ATG TTC GTC ATG GGT GTG AA 3′) and GAPDH reverse (5′ CTC TTC TGG GTG GCA GTG AT 3′). The housekeeping gene GAPDH was used as a control to normalize the data and the relative mRNA expression level was calculated using ΔCq/ΔΔCq methodology [RNA isolation of cell lines was performed using the NucleoSpinhodology .g for 15 min at 4 °C and the resultant supernatant was harvested. The supernatant was mixed with 4 × SDS loading buffer and boiled at 95 °C for 10 min and subjected to SDS-PAGE on 12% SDS-PAGE gels. Following electro-blotting, samples were probed with the following antibodies: anti-YB-1 at 1:1000 ; 4202S); anti-GAPDH at 1:1000 ; HRP-linked anti-Rabbit IgG at 1:2000 . To visualise proteins, membranes were incubated with Pierce™ ECL Western Blotting Substrate ; 32106). Chemiluminescence was then measured using a LAS Mini 3000.Cells were lysed in NP-40 lysis buffer containing 1 × cOmplete™ EDTA-free Protease Inhibitor Cocktail ) on ice for 30 min with regular vortexing. Lysates were centrifuged at 14,000× ® mouse collagen IV  3410-010-02). Following 24 h, 50 µL of 100 µg/mL Laminin I  was added to each coated insert, which was partially dried for 1 h. 1 × 105 viable cells were then plated in serum-free media within the coated inserts and the outer chamber was filled with complete medium (10% FBS). Plates were incubated for 48 h at 37 °C and 5% CO2. Media was then removed from the chambers and invaded cells dislodged from the bottom surface of the insert by the addition of 1× cell dissociation solution for 1 h at 37 °C and 5% CO2 ; 3455-05-03). A PrestoBlue cell viability assay was used as previously described to assess metabolic activity and quantify cell migration relative to a standard curve of known cell numbers.Prior to assay commencement, cells were starved in reduced-serum (2% FBS) media, and 8 µm 24-well plate transwell inserts ; 662641) were coated with 50 µL 10 µg/mL CultrexYBX1 ; VGH5526-EG4904) according to the manufacturer’s instructions.Cell lines with stable knockdown of YB-1 expression were generated through shRNA-mediated gene silencing using the GIPZ Lentiviral particle starter kit for ABCB1 promoter region was undertaken. Primer sequences were as follows: ABCB1 forward (5′ CAT GCT GAA GAA AGA CCA CTG C 3′) and ABCB1 reverse (5′ AGG CTT CCT GTG GCA AAG AG 3′).D283 and HD-MB03 cells were cross-linked in 1% paraformaldehyde for 10 min. Cell lysis, shearing, immunoprecipitation and DNA purification were undertaken using a Magna ChIP A/G immunoprecipitation kit ; 17-10085) according to the manufacturer’s instructions. Samples were sonicated using a water bath sonicator  to obtain 200–500 bp chromatin fragments. The following antibodies were utilised to pull down protein-DNA complexes, with Immunoglobin (IgG) 1 used as a negative control and histone H3K4Me3 used to confirm active promoter regions: YB-1 ; SC101198); IgG1 ; H3K4me3 ; 61379). To quantify target protein interaction with the gene of interest, qPCR using primers specific to an inverted CCAAT box within the 2 fold change ≥0.5 or ≤−0.5, p-value ≤ 0.05 and False Discovery Rate (FDR) p-value ≤ 0.1 (p < 0.05 with the Benjamini-Hochberg (B-H) method employed to control FDR. Where possible, Z-score activation predictions were calculated. Z-scores > 2 or <−2 were considered as significantly activated or inhibited, respectively.YB-1 knockdown and non-silencing control lines were pelleted in triplicate in consecutive passages and RNA was extracted using the RNeasy Mini kit  according to the manufacturer’s instructions. Whole transcriptome sequencing was performed by Qiagen Genomic Services . Library preparation was undertaken using the QIAseq Stranded Total RNA Library Kit  with QIAseq FastSelect rRNA/globin depletion according to the manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 instrument ; SY-415-1002) in a dual index 1 × 75 bp format at a sequencing depth of 30 M reads/sample. Sequencing data analysis was conducted using CLC Genomics Workbench  and CLC Genomics Server . Differentially expressed genes (DEGs) were defined as logue ≤ 0.1 . Ingenui2 fold change ≥0.5 or ≤−0.5 and B-H-adjusted p-value ≤ 0.05. Gene ontology enrichment analysis of differentially expressed genes was conducted with R package GOSeq (version 1.4.0) with default settings and a cut-off p-value of p ≤ 0.05.Drug-tolerant cell lines were pelleted in triplicate in consecutive passages and RNA was extracted as previously described. Libraries were prepared using the QuantSeq 3′mRNA-Seq library prep kit for Illumina in the forward read direction supplemented with unique molecular index (UMI) as per manufacturer’s instructions ; 015.96). The resultant library pool was sequenced on a NextSeq 500 High Output v2.5 75 cycle kit ; 20024906), to generate approximately 5 million 75 bp single-end reads per sample. Differential gene expression analysis was undertaken using the R statistical environment package DESeq2 (version 1.24.0) with default settings. DEGs were defined as LogResults are shown as mean ± SEM of the indicated number of independent experiments. The statistical significance of differences in group results was compared using one- or two-way analysis of variance (ANOVA) with multiple comparison testing as indicated. All statistical analyses and plots were carried out using GraphPad Prism 8  unless stated otherwise. The number of biological samples, corresponding statistical test and significance levels are indicated in each figure legend.http://r2.amc.nl (accessed on 6 April 2020)). A cut-off between high YB-1 gene expression and low YB-1 gene expression groups was selected, where p-values obtained from the log-rank test were minimized.Published medulloblastoma patient datasets as described by Cavalli et al. , NorthcoGroup3, MBGroup4 and MBSHH patients with high expression levels of YB-1 mRNA had significantly worse 10-year overall survival outcomes than those with low YB-1 expression levels -like barrier. Cells were seeded in serum-free media in the upper chamber of the assay and incubated for 48 h to facilitate migration (uncoated membrane), or invasion (coated membrane), towards a complete media-containing lower chamber C. AbundaComparatively, no alteration in migration was observed upon YB-1 knockdown in either cell line D, indicaGroup3 status of each line. Notable top 10 DEGs included those encoding extracellular matrix components COL8A1 and TNC and cell adhesion molecule CADM3, which have previously been associated with cellular invasion and migration in gastric cancer [We next wanted to build a picture of the global YB-1 transcriptome in Group 3 medulloblastoma. Accordingly, we performed whole transcriptome sequencing on each YB-1 knockdown line and the appropriate non-silencing control line. Analysis of the most significantly altered genes (up and down) confirmed YB-1 to be down-regulated in both cell lines  was employed to predict affected cellular biology from statistically significant differential gene expression patterns. Despite no commonality at a gene level, analysis of affected molecular and cellular functions identified cell death and survival processes to be dysregulated in both cell lines following YB-1 knockdown C,D. ExamIPA molecular and cellular functions analysis also revealed enrichment for metabolic processes, with alterations in carbohydrate metabolism specific to HD-MB03, vitamin and mineral metabolism specific to D283 and lipid metabolism affected across both cell lines, C,D. ExamMYC/MYCN amplification with treatment-refractory disease and relapse in medulloblastoma [MYC-amplified status of this line, which may act to mask such changes in MYC activation. Interestingly, upstream regulator analysis also revealed inhibition of rapamycin-insensitive companion of mTOR (RICTOR) and regulatory-associated protein of mTOR (RPTOR)—components of the mechanistic target of rapamycin (mTOR) signalling. Previous studies have demonstrated clear communication between YB-1 and the PI3K/AKT/mTOR pathway and thus this finding may indicate a similar association within paediatric brain tumours [Group3, revealing potential novel functions in cellular stress response, lipid metabolism, as well as MYC and mTOR pathway activity.To further analyse the YB-1 transcriptome in Group 3 medulloblastoma, we also performed upstream regulator analysis, a subset of IPA that allows the identification of upstream regulators that may be responsible for the gene expression changes in a particular dataset F. The beblastoma ,35. The  tumours ,37,38. TYB-1 appears to be implicated in cell death and survival processes and cellular stress mechanisms A–F. ThisGroup3 [ABCB1 exon 3 promoter region within both cell lines . Contrastingly, MBSHH cell line DAOY did develop vincristine tolerance (DT-DAOY-VIN), displaying a significant 2.8-fold increase in IC50 compared to the parental line  were generated utilising standard-of-care medulloblastoma chemotherapy agents: cisplatin and vincristine. Following continuous drug treatment and dose escalation following cell proliferation, three cisplatin-tolerant MBtal line D. It is is study . Due to ABCB1 compared to its vehicle-treated control sister line , despite cisplatin being a non-ABCC1 substrate; perhaps indicative of a function of ABCC1 in therapy resistance and/or cell survival independent of its role in cytotoxic drug efflux [Assessment of ABC transporter expression supported this theory, with the DT-DAOY-VIN line exhibiting significantly elevated expression of DAOY-VIN . InteresThe aforementioned drug-tolerant cell lines underwent 3′mRNA sequencing to elucidate transcriptomic alterations that occur following chronic drug exposure and the concurrent development of drug tolerance. Unsupervised hierarchical clustering analysis revealed significant alteration in the transcriptome following the acquisition of drug tolerance . InteresGroup3 lines , only one, LGALS1, had been researched previously in relation to medulloblastoma, where it is up-regulated in MBSHH patients and activated by the SHH signalling pathway [YB-1 independent mechanisms of drug resistance were revealed by 3′mRNA sequencing of three cisplatin-tolerant MB pathway . Thus, tABCB1 and provide evidence to support a function for YB-1 in intrinsic cellular resistance to various anti-cancer therapies, both related to and separate from its aforementioned role as a regulator of the ABCB1 gene. Finally, through the establishment and next-generation sequencing of drug-tolerant medulloblastoma cell lines, we identify targetable hits implicated in the acquisition of tolerance to standard-of-care agents, which may inform future pre-clinical investigations with a focus on hindering the development of therapy resistance.In conclusion, we demonstrate here an association between YB-1 expression and various aspects of medulloblastoma tumourigenesis, including cell invasion, MYC oncoprotein activity and lipid metabolism. Bioinformatic analysis of published datasets validated these pre-clinical findings, supporting a role for YB-1 in cell death and survival processes, cholesterol biosynthesis, response to therapeutics and MYC/mTOR signalling; as well as overall survival and metastasis in medulloblastoma patients. Importantly, we also identify YB-1 as a transcriptional regulator of drug resistance-related gene


Atypical teratoid rhabdoid tumor (ATRT) is a deadly type of human pediatric brain cancer without effective treatments. ATRT is mainly linked to the inactivation of a tumor suppressor gene, SMARCB1; however, additional biomarkers remain to be identified to develop novel therapeutic strategies. Therefore, different tumor antigens and extracellular matrix modulators were investigated in two human ATRT and one kidney malignant rhabdoid tumor cell lines and compared with the nonmalignant HEK293 cell line. Alpha-fetoprotein (AFP), mucin-16 (MUC16 or cancer antigen 125/CA125), osteopontin (OPN), and mesothelin (MSLN) are highly expressed in these human malignant rhabdoid cancer cell lines. Inhibiting MMPs using a small-molecule inhibitor decreased cell survival. This biomarker discovery process may lead to the identification of novel diagnostic and therapeutic strategies, such as the development of targeted and immunotherapies against cancer biomarkers, to treat cancer patients.Introduction: Atypical teratoid rhabdoid tumor (ATRT) is a lethal type of malignant rhabdoid tumor in the brain, seen mostly in children under two years old. ATRT is mainly linked to the biallelic inactivation of the SMARCB1 gene. To understand the deadly characteristics of ATRT and develop novel diagnostic and immunotherapy strategies for the treatment of ATRT, this study investigated tumor antigens, such as alpha-fetoprotein (AFP), mucin-16 (MUC16/CA125), and osteopontin (OPN), and extracellular matrix modulators, such as matrix metalloproteinases (MMPs), in different human malignant rhabdoid tumor cell lines. In addition, the roles of MMPs were also examined. Materials and methods: Five human cell lines were chosen for this study, including two ATRT cell lines, CHLA-02-ATRT and CHLA-05-ATRT; a kidney malignant rhabdoid tumor cell line, G401; and two control cell lines, human embryonic kidney HEK293 and HEK293T. Both ATRT cell lines were treated with a broad-spectrum MMP inhibitor, GM6001, to investigate the effect of MMPs on cell proliferation, viability, and expression of tumor antigens and biomarkers. Gene expression was examined using a reverse transcription polymerase chain reaction (RT-PCR), and protein expression was characterized by immunocytochemistry and flow cytometry. Results: All the rhabdoid tumor cell lines tested had high gene expression levels of MUC16, OPN, AFP, and MSLN. Low expression levels of neuron-specific enolase (ENO2) by the two ATRT cell lines demonstrated their lack of neuronal genotype. Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) and tissue inhibitor of metalloproteinases-2 (TIMP-2) were highly expressed in these malignant rhabdoid tumor cells, indicating their invasive phenotypes. GM6001 significantly decreased ATRT cell proliferation and the gene expression of MSLN, OPN, and several mesenchymal markers, suggesting that inhibition of MMPs may reduce the aggressiveness of rhabdoid cancer cells. Conclusion: The results obtained from this study may advance our knowledge of the molecular landscapes of human malignant rhabdoid tumors and their biomarkers for effective diagnosis and treatment. This work analyzed the expression of human malignant rhabdoid tumor antigens that may serve as biomarkers for the development of novel therapeutic strategies, such as cancer vaccines and targeted and immunotherapies targeting osteopontin and mesothelin, for the treatment of patients with ATRT and other malignant rhabdoid tumors. Malignant rhabdoid tumors are aggressive tumors that mostly appear in children under two years of age . AtypicaATRT has several histological features, including eosinophilic cytoplasm, large nucleoli, and filamentous cytoplasmic inclusions ; howeverCD44 is expressed on the surface of ATRT cells and tumor mesenchymal stromal cells from the rhabdoid tumor microenvironment . In addiCompared with medulloblastoma, ATRT has a stemness-like feature and expresses an embryonic stem cell gene profile of OCT4, NANOG, SOX2, and c-MYC . Hence, Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases/proteinases with the function of degrading extracellular matrix (ECM) proteins, including basement membrane and interstitial matrix . Their rThe expression levels of tissue inhibitors of metalloproteinases (TIMPs) and disintegrin and metalloproteinases (ADAMs) were also measured in the five cell lines. ADAMs are anchored on the cell membrane, functioning in processes of fertilization, restenosis, neurogenesis, and tumor invasion and metastasis by degrading ECM ,58, wherDiagnostic and therapeutic biomarkers with improved sensitivity and specificity are needed for ATRT due to its complexity ,61. ATRT® CRL-3020TM, ATCC) and CHLA-05-ATRT  cells were cultured based on the manufacturer’s protocol. Briefly, the cells were plated in suspension using Gibco Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12  supplemented with 2% B-27, 20 ng/mL of epidermal growth factor , and 20 ng/mL of fibroblast growth factor (FGF)-2 , in a non-treated 6-well plate at 37 °C, 5% CO2. The initial cell density was 1 × 106 cells/mL. The cell density was determined every 2 to 3 days using a hemocytometer (see 6 cells/mL), they were passaged at the ratio of 1:6 into a new vessel.Both CHLA-02-ATRT  and HEK293T  cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C at 5% CO2. The cells were passaged when they reached 80% confluence using trypsin . G401  was cultured based on the ATCC protocol. Briefly, the cells were cultured in McCoy’s 5A Medium  supplemented with 10% FBS. The cells were also passaged upon reaching 80% confluence.HEK293  was used to quench the reaction. A mixture of the cell solution with Trypan Blue 0.4%  at a 1:1 ratio was pipetted onto Countess chamber slides  using the Countess® II FL instrument . The total cell number and cell viability were reported.Both CHLA-02 and CHLA-05 were harvested from suspension and washed three times with PBS. Then, the samples were treated with Accumax  for 20 min at 37 °C at 5% COMatrigelTM-coated surfaces to let the cells spread outward from the spheroids. The images of these cells were also taken using an Olympus IX70 microscope.The images of CHLA-02 and CHLA-05 aggregates were taken using an Olympus IX70 microscope . In addition, some of the aggregates of CHLA-02 and CHLA-05 were replated on ® Total RNA Kit I  was used to isolate the total RNA from each cell line, and an RNA Clean & Concentrator-5 kit  was used to clean and concentrate the total mRNA samples. The reverse transcription required 2 μg of the total RNA, anchored oligo-dT primers , and Superscript III  and was performed according to the manufacturer’s protocol. The primers specific to target genes (ACTB) gene was used as an endogenous control for normalizing expression levels. RT-PCR was performed on an ABI7500 instrument  using SYBR1 Green PCR Master Mix (Applied Biosystems). The amplification reactions were 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 68 °C for 30 s. The fold variation in gene expression was quantified using the comparative 2−ΔΔCT method based on the comparison of expression of the target genes  among different conditions.An E.Z.N.A.et genes  were desMatrigelTM-coated 96-well plate and then fixed with 4% paraformaldehyde (PFA). For nuclear markers, the cells were permeabilized with 0.5% Triton X-100. The samples were then blocked in 2% FBS in phosphate-buffered saline (PBS) for 1 h and incubated with various mouse antibodies in a blocking buffer at 4 µg/mL . The data were analyzed against the isotype control using FlowJo software.Briefly, 1 × 106 cells/mL. GM6001  was dissolved in DMSO to a stock concentration of 8 mM and added to the culture at a final concentration of 10 µM [Matrigel. In addition, the cells were harvested for RT-PCR analysis for epithelial, mesenchymal, and important ATRT biomarkers .CHLA-02 and CHLA-05 cells were replated at a density of 1.6 × 10of 10 µM . After 4n = 3). To compare two conditions in t-test was used to determine the significance. A p-value < 0.05 was considered significant. On the other hand, for comparing more than two different conditions   A–D. The for ATRT , and theImmunocytochemistry experiments were performed to detect the protein expression of several markers . AFP wasMMPs), tissue inhibitors of metalloproteinases (TIMPs), and a disintegrin and metalloproteinases (ADAMs) were characterized for all five cell lines  and mesenchymal markers . The epithelial gene expression of CDH1 was higher in all the cell lines when compared with HEK293 cells, and it was highest in G401 cells   A,B. In aeatment) C. Despiteatment) D.CDH1) and most mesenchymal markers . The important ARTR biomarkers (MSLN and OPN) were also decreased by 16%. CHLA-05 showed an increase in the expression of VIM (~4 folds) and MUC16 (~0.14 folds).The effect of GM6001 on different biomarkers in both ATRT cell lines was also investigated using the RT-PCR method . The preThe two human ATRT cell lines, CHLA-02 and CHLA-05, showed distinct differences in several gene expression markers. In many cases, the investigated MMP, TIMP, and ADAM gene expression levels in CHLA-02 were higher than in CHLA-05 , suggestCD44 in CHLA-02 might suggest its invasive phenotype. AFP gene and protein expression may serve as a valuable biomarker for malignant rhabdoid tumors due to its much higher expression levels compared to nonmalignant cells.Among all the biomarkers investigated, OPN, AFP, MUC16, and MSLN may be the most important biomarkers for ATRT. OPN is a stem cell-promoting factor in glioblastoma , and itsMSLN mRNA, but only low levels of protein were detected by immunocytochemistry (ICC), indicating that MSLN protein expression might be low, the proteins might be shed into the cell culture media, or that the sensitivity of the antibody is not sufficient. Therefore, targeting the ERK and AKT pathways and disrupting MSLN–MUC16 interactions may become novel strategies for ATRT treatment.MUC16 acts as a signaling molecule and a lubrication barrier of the epithelial surface . The expNMP1 gene expression was lower in both ARTR cell lines compared with the nonmalignant HEK293 cells in this study. This could be due to the different isoforms of NMP1. OCT4 is expressed in other cancer types, such as glioma, hepatocellular carcinoma, rectal cancer, and pancreatic cancer [OCT4 mRNA in rhabdoid tumor cell lines, but the results were inconsistent with ATRT cell lines. Our results showed that gene expression levels were significantly higher in G401 than in HEK293 cells. At the protein level, all the rhabdoid tumor cell lines expressed OCT4, suggesting its important role in preserving the stem cell-like property. However, in other studies, the protein expression of OCT4 was not detected in ATRT or non-central nervous system malignant rhabdoid tumors [Our NPM1 and OCT4 expression results differed from those reported in the literature. Even though NMP1 has been suggested to be a potential target for ATRT treatment in the literature , NMP1 gec cancer ,77,78,79d tumors ,81,82,83MMP-26, an invasion biomarker in various cancers, including gliomas [MMP7 is a biomarker associated with the epithelial-to-mesenchymal transition (EMT) of tumor cells that are highly invasive [MMP7 was highly expressed in CHLA-02. The gene expression profile of CHLA-02 demonstrated this cell line’s aggressive and invasive phenotype.Investigating MMP and TIMP expression is important for understanding the invasiveness of ATRT cells. MMP1 is highly expressed in glioblastoma multiforme xenografts . Further gliomas , astrocy gliomas , colorec gliomas , and eso gliomas . High MM gliomas . MMP26 m gliomas ,90 and i gliomas ,92 to prinvasive ,94, as winvasive  and glioinvasive . MMP7 waTIMP1 or 2 [TIMP2 in both ATRT cell lines was significantly higher than in the nonmalignant HEK293 cell line. TIMP4, a biomarker for astrocytoma in glioma patients [LOX) and SNAI2 were highly expressed in both ATRT cell lines. LOX and SNAI2 can regulate the induction of TIMP-4, which can lead to tumor invasion, migration, and VEGF expression [In addition to their inhibitory abilities against MMPs, TIMPs may promote cell proliferation and inhibit apoptosis ; howeverMP1 or 2 . This stpatients , was higpatients . Lysyl Opression .ADAM9 was highly expressed in both ATRT cell lines, but ADAM10 was only expressed in CHLA-02. ADAM9 is an adhesive protein and can induce cell signaling, leading to increased proteolytic activities [tivities  and it itivities ,104. ADAtivities ,106,107,tivities ,108. ADAtivities ,110. It tivities ,112. Mortivities ,114.Matrigel-coated surfaces, further supporting this hybrid phenotype hypothesis. Recently, two chromatin-modifying complexes, polycomb repressive complex 2 (PRC2) and lysine methyltransferase 2D-complex of proteins associated with SET1 (KMT2D-COMPASS), have been suggested to govern epithelial–mesenchymal plasticity [Epithelial–mesenchymal transition (EMT) is a process in which cells change their phenotypes from polarized epithelial to mesenchymal ,116. Thiasticity . Thus, Aasticity . Therefoasticity .VIM expression in CHLA-05. The suppression of MYC protein expression in colorectal cells by knocking down MMP1 [GM6001 is a potent broad-spectrum MMP inhibitor. GM6001 treatment decreased the cell proliferation and gene expression of several important biomarkers in both CHLA-02 and CHLA-05, including MSLN, OPN, and many mesenchymal markers. The effect of GM6001 was more significant in CHLA-02, perhaps due to the higher MYC and MMP expression levels in CHLA-02 than in CHLA-05 . In addiown MMP1  may suggHuman embryonic kidney cell line HEK293 cells were used as nonmalignant controls for G401, a kidney-derived malignant rhabdoid tumor; however, HEK293 may not be the ideal control for ATRT cell lines. The origin of ATRT is still under investigation, but it might be in neural progenitor cells ; howeverThis comprehensive study investigated biomarkers of different types, epithelial, mesenchymal, MMPs, TIMPs, and ADAMs, as well as the effects of GM6001 on ATRT cells; however, these studies were limited to using two ATRT cell lines, one malignant kidney rhabdoid tumor cell line (G401), and two kidney control cell lines HEK293 and HEK293T. In the future, other ATRT cell lines and patient-derived xenograft samples need to be investigated to verify these ATRT antigens. Although CHLA-02 and CHLA-05 have different cellular pathways and metabolic regulations, they might not be able to represent all ATRT patient-derived cell lines and tissues. Thus, future large-scale studies are needed to verify our findings for therapeutic development to treat patients with ATRT and other malignant rhabdoid tumors.In summary, CHLA-02 and CHLA-05 may represent two different subtypes of ATRT based on their gene expression profiles. AFP, MSLN, MUC16, OPN, and MT1-MMP/MMP14 may serve as biomarkers for human malignant rhabdoid tumor detection and therapeutic targets. Due to their high MT1-MMP and other MMP expression levels, ATRT cells may be highly invasive. Treating ATRT with a broad-spectrum MMP inhibitor, GM6001, decreased cell proliferation with no significant changes in viability or several important markers in both ATRT cell lines. ATRT may utilize the MYC and ERK pathways as their central metabolic regulation, suggesting an important therapeutic intervention approach. OPN and MSLN may serve as targets for immunotherapies and cancer vaccine development to treat ATRT patients. This study may also advance our knowledge of the molecular features of malignant rhabdoid tumors and their aggressiveness.


Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof  are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS). During the last years, immortalized HSC lines have become an established experimental tool in biomedical research in place of primary cells. Compared to primary cells, immortalized HSC lines offer several advantages. They are cost effective, easy to handle, and provide an unlimited supply of biological material. Moreover, they can bypass ethical concerns that may arise by isolation of primary cells from animal or human tissue . In addiPresently, continuous HSC lines were established from mouse, rat, pig, and humans. Although widely applied, only several of these continuous lines have been genetically characterized, while numerous scientific journals and funding agencies now request cell authentication before considering publication or funding when working with cell lines. Some years ago, we described genetic characteristics of the human HSC line LX-2 . In addiNevertheless, based on the fact that results obtained from one specific cell type might require the reproduction in another cell line, we here established genetic characteristics for the cirrhotic fat-storing cell line (CFSC). This cell line and four different clonal sublines derived thereof, i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B, were originally established in the laboratory of Marcus Rojkind from a carbon chloride cirrhotic rat liver and proposed as a useful tool to study extracellular matrix production by HSCs ,8.In the present study, conventional chromosome analysis and multicolor spectral karyotyping were accomplished to gain insight into the genetic organization of CFSC-2G cells that has successfully uncovered numerous characteristic features of its chromosome organization. We further established a specific short tandem repeat (STR) profile for this cell line that includes 31 species-specific allelic variant sites. Bulk sequencing of mRNA isolated from CFSC-2G cells confirmed the expression of typical HSC markers associated with extracellular matrix synthesis and turnover. In addition, electron microcopy and Rhodamine–Phalloidin stain confirmed that CFSC-2G cells form a robust microfilament network, cytoplasmic fat droplets, and other cellular features typical for HSCs. In sum, our study provides genotypic and phenotypic characteristics of CFSC-2G cells that are useful for cell authentication.Papers using CFSC cells were identified by searching the PubMed database  using thCFSC cells were established and characterized over thirty years ago in the laboratory of Marcus Rojkind ,8. It is4)  solved in 25 mM sucrose buffer (Merck). Fixed cells were dehydrated and subsequently incubated in propylene oxide  in a mixture of Epon resin (Serva) and propylene oxide (1:1) and pure Epon. Finally, ultrathin sections (70–100 nm) were prepared and packed upon Cu/Rh grids . Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate . The samples were analyzed at an acceleration voltage of 60 kV using a Zeiss Leo 906  transmission electron microscope and depicted images were taken at magnifications of 2156× to 35,970×.Electron microscopic analysis of CFSC-2G cells was essentially conducted as described before . In briew/v) hypotonic potassium chloride solution and fixed with a mixture of methanol and acetic acid (3:1). Air-dried chromosome spreads were prepared, and slides were treated with 0.025% (w/v) trypsin solution followed by staining with Giemsa solution to visualize G-banding pattern. In addition, the heterochromatin in metaphase chromosomes was stained following an established procedure ..23].Rattus norvegicus reference transcriptome. The complete list of mRNAs detected with their abundances is given in −11  to 12,768.5 . In particular, we found strong expression of α-smooth muscle actin , collagen type I α1 , collagen type III α1 , Vimentin , two Fibronectin 1 transcripts , galectin 1 , secreted protein acidic and cysteine rich , tissue inhibitor of metalloproteinases 1 , Timp2 (TPM: 1030.84), Cd63 (TPM: 2732.07), transgelin 2 , cofilin 1 , caveolin 1 , and cysteine and glycine-rich protein 2 , respectively. All these markers indicate that CFSC-2G cells originate from primary HSCs. The expression of most of these markers was recently demonstrated in rat HSC line HSC-T6 cultured under the same culture conditions (Bst2), representing a component of the innate immune response to enveloped viruses. Similar to HSC-T6, the NGS data demonstrated that CFSC-2G cells express large quantities of ferritin heavy chain 1 , ferritin light chain 1 , and ferritin light chain 1-like protein , which are associated with the intracellular storage of iron. Moreover, in line with their activated phenotype, CFSC-2G cells express only low mRNA amounts of the peroxisome proliferation-activated receptor-γ (Pparg), representing one nuclear receptor whose activity is significantly decreased in activated HSCs [We next isolated mRNA from CFSC-2G cells cultured under basal conditions to 80% confluence and performed bulk mRNA sequencing (mRNA-Seq). The identity and abundance of each mRNA were determined using the nditions . FurtherEnumeration of Giemsa-stained chromosomes of CFSC-2G cells at different intervals consistently showed a shift from the standard diploid chromosome number that could be characterized as a hyperdiploid karyotype with varying number of chromosomes ranging from 53 to 65 . No cellConsistent with the GTG-banded chromosomes, large bi-armed rearranged chromosomes were noted in the CBG-banded metaphase plates cf. A. ThoughStrikingly, cytogenetic analysis in CFSC-2G cells further detected the sporadic occurrence of ring chromosomes in metaphases that appeared to differ in size and were partly di-centric C–E. In tDue to the specific color code assigned to individual chromosomes, the SKY analysis precisely allows authentic identification of both structural and numerical aberrations. p-) material in all these RNO1 derivatives. Both RNO2 and RNO7 are organized in multiple copies, but these two autosomes displayed unusual organization for a bi-armed chromosome that may arise through isochromosome formation, pointing to an overt duplication of the long arms for RNO2 and RNO7. Additionally, the SKY analysis disclosed translocations of different chromosomal segments in the derivative chromosomes of RNO2, RNO4, RNO6, RNO10, RNO14, RNO17, and RNO19. Both RNO9 and RNO18 are the only ones that are organized in two copies. On the other hand, three chromosomes  are present in two copies, but the SKY analysis revealed that additional parts of these chromosomes were also found in other derivative chromosomes. Likewise, RNO15 and RNO16 occurred in single copy, but multiple segments from these two chromosomes were found in different derivative chromosomes.However, the most striking observation was found for chromosome 1 (RNO1), which was found in five copies. All RNO1 copies are literally derivative ones in which the tiny, short arms are replaced by different chromosome segments, hinting to chromosome translocations and simultaneous loss of the short arm  DNA profiling using a set of 31 established polymorphic markers. The resulting PCR products were analyzed on a genetic analyzer resulting in a well analyzable electropherogram for each marker tested  that all4 cirrhotic liver [Continuous HSC lines resemble primary cultured HSCs in many ways and are valuable and useful tools to address issues of HSC biology and function. As a result, the number of studies which use HSC lines is steadily increasing. However, from the ~30 HSC lines available, only a few lines have found widespread use . In regaic liver ,8. SinceNevertheless, the chromosomal and genetic characteristics of this cell line were not established. It is well accepted that continuous cell lines are under permanent stress and selection due to the artificial environment in which they exist. Genetic mutation can occur during cell passaging and laboratory manipulation, which can lead to a heterogeneous population with a selective advantage. As a consequence, this could result in a final cell population that is completely different from the starting pool. Unfortunately, the factors provoking a genetic drift within a cell line can be different in various labs.A fundamental study has shown that established cell lines, generally thought to be clonal, are highly genetically heterogeneous, most likely resulting from clonal dynamics and continuous instability . It is sConsequently, it is necessary to identify resilient biochemical and genetic features of a continuous growing cell line to confirm the identity and genetic stability and to mark the ‘biochemical quality’.In our study, we describe several molecular features of CFSC-2G cells that are useful for cell authentication. The cell line has a fibroblastic morphology that is in agreement with the proposed fibrogenic phenotype . ElectroFurthermore, CFSC-2G cells have a prominent network of intracellular actin filaments  and are Acta2, Col1a1, Col3a1, Vim, Fn1, Lgals1, Sparc, Csrp2, Timp1, Timp2, Tagln2, retinol-binding proteins, and their nuclear receptors , matrix metalloproteinase-2 (Mmp2), Timp1, Timp2, and plasminogen activator inhibitor-1 (PAI-1/Serpine1) in CFSC-2G [Previous work demonstrated that the treatment with all-trans retinoic acid (ATRA) inhibits proliferation and diminishes mRNA expression of procollagen α1(I), procollagen α1(III), transforming growth factor-β1, connective tissue growth factor -induced cirrhotic male Wistar rats [4 is a non-genotoxic carcinogen known to be associated with intra-chromosomal recombination provoking liver cancer in rats and mice [4. The observation of multiple copies of chromosomes leading to a pseudo-triploid karyotype can be considered as a significant and specific feature of CFSC-2G cells, since similar multi-copy chromosomes are absent, for example, in the rat HSC-T6 cell line [It is noteworthy to mention that the recent analysis of the rat hepatic stellate cell line HSC-T6 that was established through SV40T transfection surprisingly showed relatively less chromosome changes without significant change in the modal chromosome number [tar rats ,8. CCl4 and mice . Therefoell line ,30. Inteell line ,32,33. Mell line .Essentially, the karyotype abnormality described in this study provides insight to the genomic instability of CFSC-2G. The numerical and sweeping chromosomal changes might contribute to the capacity of this cell line to proliferate indefinitely.Altogether, chromosome banding and multicolor SKY analysis revealed that CFSC-2G cells are characterized by extensive aneuploidy and striking chromosomal rearrangements that might be too complex to be used in daily routine for cell authentication. Therefore, we next performed STR DNA profile analysis involving the simultaneous amplification of 31 rat-specific STR markers. This analytical DNA technique is suitable to establish a characteristic fingerprint of these hypervariable microsatellite regions, allowing unambiguous cell authentication. The analysis revealed a unique STR profile in which 18 markers produced only one peak in the analysis and can be considered homozygous, while 13 markers gave rise to two peaks and can therefore be considered to be homozygous . The finIn sum, the presented biochemical characteristics, standard karyotyping analysis, SKY analysis, and the STR profile established in this study are certainly suitable means that can be used for cell authentication of CFSC-2G cells. In the future, this information will increase the biomedical value of CFSC-2G cells in liver research. In addition, our study helpful to fulfill the requirement of increasing the number of scientific journals and funding agencies that have started to require cell authentication prior to paper or grant submission when working with cell lines.CFSC-2G cells are an attractive cellular model to analyze aspects of HSC biology. Since the original establishment of these cells three decades ago in the Marion Bessin Liver Research Center , the cells have made their way into many laboratories worldwide. The identification of specific chromosomal alterations and the definition of a unique STR profile will now allow discriminating these cells from all other cell lines. The demonstration that CFSC-2G cells express typical markers of HSCs , have a robust microfilament network, and can uptake and store fats confirms the proposed activated HSC phenotype. Altogether, the identification of a highly characteristic genetic profile will further increase the biomedical value of CFSC-2G for liver research. Together with cell line HSC-T6 that was recently genetically characterized by our team, researchers have now two independent, genetically well-characterized cell lines in hand that can be mutually used to reproduce experimental findings in continuous growing HSC lines of rat origin.


The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.  The importance of circulating tumor cells (CTC) has recently been recognized. Although the cultivation of CTC is very promising as a preclinical model for human cancer, difficulties are associated with establishing CTC cell lines due to the low number of CTC in blood and the majority of these cells not being viable. Only a limited number of permanent cell lines of CTC are currently available and their management is more challenging than that of standard cell lines.CTC cell lines are important for examining the biology of CTC, but not for providing quick feedback to cancer patients. Furthermore, the translational relevance of cell lines is often questioned because prolonged cultures and multiple passages lead to phenotypes that are no longer representative of the original tumor in terms of cell epigenetics and gene expression profiles . TherefoWe previously established cancer cell lines from several tissue specimens, including esophageal and pancreatic cancer ,3. We idThe systematic detection of CTC was initially reported by other research groups. Racila et al.  used an The majority of conventional cancer cell lines generally proliferate on a two-dimensional (2D) environment, in contrast to the 3D environment of most CTC cell lines, such as spheroids or organoids. We established a small cell esophageal cancer cell line (TYUC-1) that actively grew with sphere formation in a standard culture flask from a tissue specimen . This exConcerning the approaches to long-term culture, we also examined the literature for short-term cultures. Our aim was to obtain a more detailed understanding of the characteristics of CTC and several methods that support the establishment of long-term cultures from short-term cultures. Based on previous findings and our own research, we herein discussed critical issues facing the development of CTC culture methodologies and proposed a rational strategy for the long-term culture of CTC.In this review, a long-term culture (CTC cell line) is defined as a culture period of longer than 1 year or continuous passages for more than 6 months. A short-term culture is defined as a culture period of longer than 1 week. We briefly discussed culture-related factors, such as methods for enriching CTC, the characteristics of CTC in the bloodstream, and stem cell biology. We excluded methods for ultra-short term (less than 1 week) cultures and expression assays because the focus of these methods is clinical evaluation, not long-term cultures. We mainly concentrated on the development of a reproducible protocol for establishing CTC cell lines. We also excluded details on the CTC capture system, clinical applications, genetic analyses, CTC-derived xenografts, and cultures of bone marrow disseminated tumor cells.Thirty-nine CTC cell lines were reported by 2021: 11 from colon cancer, 13 from breast cancer, 2 from prostate cancer, 2 from gastroesophageal cancer, 5 from small cell lung cancer, 2 from non-small cell lung cancer, and 4 from malignant melanoma. Establishment rates ranged between 1 and 17%, with most studies achieving a rate <10%  cells should survive in the blood because they negligibly express EpCAM. Therefore, EpCAM is not a standard marker for the establishment of cell lines. However, some cells express EpCAM and maintain epithelial characteristics. Additionally, many cells express stem cell markers, such as CD133, CD44, CD24, and ALDH1. These findings indicate that CTC have a mixed epithelial phenotype, an intermediate epithelial/mesenchymal phenotype, and stem cell-like properties.Stem cell concepts have been employed for long-term CTC cultures. CTC as non-adherent spheres may reflect the intrinsic properties of cancer stem cells that remain viable in the bloodstream after the loss of their attachment to the basement membrane . HoweverThe majority of CTC cell lines have been established using 6 to 18 mL of blood. A blood volume >10 mL is a suitable source for CTC cultures. Furthermore, since the establishment of cell lines was found to be slightly easier from patients with a high CTC count (>30/mL), a CTC count >300 is preferable for the success of cultures. However, a high CTC count only does not guarantee the establishment of a permanent cell line. Furthermore, a conventional CTC count is generally assessed using EpCAM(+) cells. Since EpCAM(−) CTC have been overlooked, CTC counts are not accurate. The most recent study on CTC used a 96 mL sample obtained from leukapheresis for a cell culture . Leukaph®). Although antibody-based approaches were employed in three studies, capture with antibodies against surface antigens is harmful, precluding its use for CTC cultures [An enrichment step for CTC is indispensable before cells are cultured. Viable CTC isolation may be divided into three types: antibody-based, physical property-based, and function-based approaches. The most successful enrichment method for long-term cultures is the negative selection method  has been used as the start medium. The culture medium is then changed to ordinal standard medium. The culture medium is important during the initial 2 weeks. The proliferation of most cells peaks after approximately 21 days. CTC with proliferative potential may then grow without special growth supplement, except for FCS .Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) are often listed in recipes for stem cell cultures. Recipes for serum-free CTC culture media mostly include proprietary B27 and N2 supplements, together with insulin, transferrin, and selenium. Y27632, a cell-permeable inhibitor of Rho-associated coiled-coil containing protein kinase, is also used. However, the optimal conditions for ex vivo CTC cultures have yet to be established.To appropriately culture CTC, it is important to understand the progression of CTC growth in each step. In this section, we focus on the growth progression of selected CTC cell lines.1.CTC-1, CTC2, and CTC3Zhang et al.  previous2.CTC-MCC-41In the case of CTC-MCC-41 cells, viable CTC were still observed after 4 days of culture in serum-reduced medium under hypoxic conditions. After 10 days, CTC started to proliferate and formed spheres. After a few weeks, the cell culture was switched to normoxic conditions. Single colonies from the CD45(−) population that appeared during the first week of the cell culture were transferred into a 24-well plate for further growth .3.BHGc7 and BHGc10BHGc cells initially grew as typical small spheroids that eventually showed the outgrowth of adherent tumor cells and shedding of apoptotic cell fragments . Prolife4.CTC-TJH-01During an ex vivo culture of CTC-TJH-01 cells, contaminating blood cells and a small number of CTC underwent rapid cell death. The majority of surviving CTC adhered to the plate, but remained dormant throughout the initial two weeks, suggesting the low in vitro clonogenic potential of CTC. CTC started to proliferate after two weeks, and the culture was switched back to normoxic conditions. CTC slowly proliferated, formed clusters, and then exhibited sustained proliferation ,20.5.CTC-ITB-01CTC-ITB-01 cells grew in parallel as adherent and non-adherent cell fractions with various cell sizes. Non-adherent cells grew from adherent cells. CTC showed an interchangeable adherent and non-adherent cell population similar to cancer stem cells .6.UWG01CTC and UWG02CTCIn the case of UWG01CTC and UWG02CTC cells, viable, relatively pure cultures were observed within 3 weeks and rapidly expanded. UWG01CTC is adherent and requires trypsinization for passaging; however, a loose adherent spheroid phenotype was induced in a hypoxic environment and serum-free media. In contrast, UWG02CTC grew in long mucinous, loosely aggregated, and weakly adherent strands that only required gentle mechanical dissociation for passaging .7.CTC-3CTC-3 cells began to proliferate and form clusters after 14 days of culture . HoweverSeveral conditions were used for the expansion of CTC. However, suitable conditions depended on the characteristics of each cancer cell. Furthermore, the optimal timing and culture conditions for CTC expansion have not yet been established. Representative cases are described below.1.CTC-1, CTC-2, and CTC-3Adherent plates were used during the first week with growth factor-enriched stem cell culture medium. The medium was changed to EpiCult-C medium between 8 and 21 days. Standard medium with 10% FCS was then used for maintenance .2.Mel-167, PEM-22, Mel-182, and PEM-78CTC were cultured in 24-well ultra-low attachment plates for between 4 and 8 weeks. A 3D fibrin Matrigel culture was then used, and this was followed by a switch to an anchorage-independent culture [3.CTC-MCC-41CTC were cultured in 24-well non-adherent plates for the 1st week. Cells were then transferred to new 24-well non-adherent plates in the 2nd week and maintained in a T25 flask .4.CTC-3A Matrigel-coated plate was used in the first 2 weeks, and cells were then transferred to a normal 6-well plate .To establish approaches to long-term culture, short-term culture was reviewed. Information that will contribute to the establishment of long-term cultures of CTC was obtained .We successfully performed a short-term culture of CTC using a magnetic cell sorting system (MACS) with anti-Human Epithelial Antigen (HEA) 125 in 1999 . We dete®). Few studies employed RBC cell lysis only. Under optimal conditions, the introduction of an RBC lysis step does not markedly affect the viability of the nucleated cell fraction [® (filtration using a porous polycarbonate membrane) selection [Similar to long-term cultures, most enrichment methods for cultures involve the negative selection method  [® detects cancer cells not only on the separating membrane, but also on the plastic bottom of the 6-well plate [The MetaCellof 8 μm) ,45,47,51ll plate . Membran2.On-chip cultureZhang et al.  describeKapeleris et al.  suggesteRegarding CD45, a significant population of “double positive” cells with hematopoietic and epithelial markers (CK+/CD45+) were detected in many patient samples . FurtherThus, most of the double positive cells may be the heterogeneous CTCs . From thKhoo et al.  maintainThe viability and morphology of expanded CTC were examined by LIVE/DEAD staining .Few studies have employed low attachment plates and hypoxic conditions. Therefore, short-term cultures are generally aimed at clinical use, not long-term cultures. Therefore, they are routinely conducted under adherent and normoxic conditions. Furthermore, most studies used simple standard culture medium with FCS.Tapered microwells are preferred over conventional cylindrical microwells for cluster formation. Cultures are sheltered within microwells with minimal disturbance during medium changes, which reduces shear stress and cell loss to allow for subsequent expansion . This meCarmona-Ule et al.  suggeste® was 65%. Despite the leukapheresis approach, CTC from only a few patients have been successfully cultured. A success rate of 100% (12/12 samples) was achieved by Ficoll-Paque selection [The success rate of cultures was previously reported to be more than 50%. The total success rate of MetaCellelection .The typical cell culture period is <14 days and few cells may be cultured for more than 3 months. Typical spheres were shown to increase in size over time from 7–21 days . Khoo etCarmona-Ule et al.  investigAlthough proliferation was not observed due to the challenging ex vivo growth environment, the maintenance of CTC viability was guaranteed for at least two months . EliasovKhoo et al.  showed tKhoo et al. ,63 demonThe morphological spheroid construct typically consists of three different presentable types: large-sized, cohesive round-shaped spheroids; small-sized cohesive irregular or round spheroids; and discohesive “grape-like” spheroids . Some sa® system, CTC grew through the membrane on the bottom of the well after 14 days in an in vitro culture exhibiting two different phenotypes, epithelial-like and stem cell-like. Stem cell-like cells formed visible cell clumps. Cells growing on the bottom showed a very plastic morphology [In the MetaCellrphology . CTC incrphology .Non-adherent blood cells provide a cushion layer that keeps CTC in suspension . CTC havAfter 1 week of a cell culture under hypoxic conditions, cells are generally switched to standard cell culture conditions. When 85% confluence is reached, growing cells are transferred to an ultra-low attachment 24-well plate . HoweverRegarding the establishment of CTC cell lines, the majority of cultured CTC lack the ability to proliferate. Therefore, 2 months is a sufficient time period to assess whether CTC exhibit the ability to continuously proliferate. Future studies that compare CTC lines with CTC that stop proliferating after a short-term culture will facilitate the development of optimal molecular conditions for the permanent survival and growth of CTC .Cell growth depends on the proliferative potential of each cell. Among 21 conventional esophageal cancer cell lines, the time after the initiation of the primary culture to the second culture varied from 2.5 weeks to 48 weeks, depending on cell growth patterns, such as the adhesion-dependent type, spheroid type, and mixed type. Adaptability to a new environment is also important [We previously reported the maintenance of several cancer cells in protein-free medium without other supplements for 2 years . These cBased on our experience and previous findings, we preliminarily performed a culture of CTC collected from patients with prostate cancer and renal cell carcinoma. We recently developed a CTC capture system . We firs®-pot (porous polycarbonate membrane selection) with Lymphoprep or negative selection using RosetteSep® CD45, CD36, or CD56. A short-term culture of CTC was successful for 16 out of 27 samples; however, expansion was very challenging to overcome. One reason is that we did not obtain a high number of viable CTC from patients because most had already received several courses of chemotherapy. The most important issue is that we have not yet established a specific protocol for individual carcinoma cells.We also used VIVANT-CELLRegarding double positive (CK+/CD45+) cells, we also detected these cells in the short-term culture of CTC. Some of these cells were large, polymorphic in shape and polynuclear . These mIn several trials, we developed three cell lines with the spontaneous immortalization of lymphocytes from different patients under conditions involving several growth supplements, such as Y-27632 and Z-VAD-fmk. We previously reported the incidental establishment of B-cell cell lines (ITSM), derived from EB virus spontaneous activation during an ordinary cell culture from pseudomyxoma peritonei . The lonCTC cell lines have unique intermediate characteristics between primary tumors and distant metastasis. CTC are not simple travelers in the bloodstream. Although most CTC are apoptotic and sensitive to stress, a few viable CTC survived.The CTC count of samples is the most important factor for the successful establishment of CTC cell lines. A CTC count of >100 cells/mL blood is favorable. Leukapheresis may be the most powerful method for enriching CTC; however, its success rate is low.There is currently no evidence to support the contribution of CD45+ cells to the success of long-term culture. However, a reconsideration of the parameters for CTC isolation is warranted .It is important to note that because optimal CTC culture conditions have yet to be defined, current research relies on stem cell culture methods that ensure maximal CTC expansion. Each protocol is designed based on a researcher’s own expertise and empirical results, and not by a rationale based on the biological features of CTC, which are presently not defined .Moreover, activated lymphocyte proliferation is transient, ending with programmed activation-induced cell death. Macrophages are the only cell type among peripheral blood mononuclear cells that are capable of surviving for weeks in culture. However, they are also a terminally differentiated cell type that is incapable of ex vivo proliferation .Other key technical limitations include the maintenance of the CTC phenotype and the composition of a stable population in culture. Previous studies suggested that normal human mesenchymal stem cells are prone to genomic changes and subsequent malignant transformation in long-term cultures .Therefore, the further optimization of CTC culture conditions and a more detailed understanding of the differences between non-adherent CTC cell lines and adherent cell lines as well as CTC cultures and patient samples is required .As described above, the recommended protocol for CTC cell lines is as follows . With caA careful review of previous studies suggested that CTC in long- and short-term cultures have different characteristics. Not only the culture method, but also the characteristics of CTC themselves affect the success of establishing CTC cell lines.In the circulation, CTC from any type of cancer encounter the same environment, which differs from their solid tissue location. Only cells with specific characteristics, such as stem cell-like cells, may survive in the bloodstream. CTC without these characteristics are protected by surrounding cells, such as leukocytes and platelets. However, it currently remains unclear whether only CTC with stem cell features are suitable long-term culture.Low attachment plates and hypoxic conditions may be the ideal conditions for the initial culture steps. A successful culture medium contains several growth factors and anti-apoptotic supplements. However, as described above, cancer cells have already acquired growth and survival autonomy. Therefore, CTC cultured in the presence of growth factors and small molecule inhibitors may not be ideal for modeling cancer and stromal interactions. The most important factors are the timing of CTC transfer to the next step and the careful management of medium changes.Short-term culture is the initial approach to long-term culture; however, prolonged protocols sometimes deviate from the main expansive route for establishing CTC cell lines. The success of short-term cultures is not always affected by a high number of CTC; however, long-term cultures generally require high numbers of CTC. Therefore, the culture method may only prolong cell survival; it does not affect permanent proliferative activity. The most important issue is obtaining CTC that are suitable for long-term culture; however, a method to identify these CTC has not yet been established.There is currently no gold standard method. The selection of an optimal method for each sample may be achieved through careful management and continued efforts. Extensive trials are needed to establish long-term cultures of CTC. Without these efforts, CTC that are suitable for expansion will not be identified.


Protein essentiality is usually accepted to be a conditional trait and strongly affected by cellular environments. However, existing computational methods often do not take such characteristics into account, preferring to incorporate all available data and train a general model for all cell lines. In addition, the lack of model interpretability limits further exploration and analysis of essential protein predictions.In this study, we proposed DeepCellEss, a sequence-based interpretable deep learning framework for cell line-specific essential protein predictions. DeepCellEss utilizes a convolutional neural network and bidirectional long short-term memory to learn short- and long-range latent information from protein sequences. Further, a multi-head self-attention mechanism is used to provide residue-level model interpretability. For model construction, we collected extremely large-scale benchmark datasets across 323 cell lines. Extensive computational experiments demonstrate that DeepCellEss yields effective prediction performance for different cell lines and outperforms existing sequence-based methods as well as network-based centrality measures. Finally, we conducted some case studies to illustrate the necessity of considering specific cell lines and the superiority of DeepCellEss. We believe that DeepCellEss can serve as a useful tool for predicting essential proteins across different cell lines.http://csuligroup.com:8000/DeepCellEss. The source code and data underlying this study can be obtained from https://github.com/CSUBioGroup/DeepCellEss.The DeepCellEss web server is available at Bioinformatics online. Essential genes are indispensable for the survival of a single-celled organism, a cell line or a multicellular organism  to capture long-range dependencies between residues. Finally, a fully connected layer with a sigmoid function performs the classification task.We conducted extensive experiments to evaluate the performance of DeepCellEss. In comparison, DeepCellEss shows greater effectiveness in predicting essential proteins than existing sequence-based methods. Compared to network-based centrality measures under cell line-specific networks, the results demonstrate that DeepCellEss effectively compensates for the limitations of network-based centrality measures. Furthermore, we performed some case studies which show the advantages of taking cell line-specificity into consideration. In addition, we carried out ablation studies to demonstrate the benefits of our proposed network architecture. Finally, we built a user-friendly webserver to expand our tool's accessibility.To construct a practical cell line-specific prediction model, we collected protein essentiality data in extremely large-scale cell lines. We downloaded the essentiality data generated by the Wellcome Sanger Institutes (Release 1) from the Project Score database (We collected sequence information from the Consensus CoDing Sequence (CCDS) database (Release 22)  Xeon(R) Gold 5220 CPU @ 2.20 GHz, 256GB memory and a Nvidia GeForce RTX 2080 Ti GPU. The hyper-parameter settings were determined by grid search techniques.We evaluated our models on the independent test sets of different cell lines. The model performance was assessed by the area under the receiver-operating characteristic curve (AUROC) and the area under precision-recall curve (AUPRC), which can measure the ranking ability for prediction models. It should be noted that AUPRC is more sensitive to the positive samples i.e. essential proteins and thus can provide more comprehensive evaluation.To evaluate the performance of DeepCellEss, we trained and tested DeepCellEss on a large collection of benchmark datasets across different cell lines. Specifically, DeepCellEss was trained independently on 323 cell line benchmark datasets using the same model optimization settings. After all training processes are completed, we carried out the tests on the corresponding independent test set of each cell line model. The detailed performance results of all cell line models are listed in In this section, we carried out comparison experiments to investigate the effectiveness of DeepCellEss for essential protein prediction. We compared the performance of DeepCellEss with five sequence-based baseline methods (described in Section 3.4) on the independent test set of HCT-116 cell line. The comparison results are shown in In addition, we can see that Pheg gets AUROC and AUPRC scores of 0.427 and 0.450, respectively, which are lower than the other methods. This can be explained by the fact that Pheg web server only provides a general human gene essentiality predictor, and it ignores specific differences in the essentiality of genes and the encoded products across cell lines, resulting in poor prediction performance on cell line-specific test datasets. Such results indicate the difficulty of identifying cell line-specific essential genes and proteins using a general model trained on common essential samples in cell lines. Thus, training cell line-specific models is necessary for discovering specific essential genes and proteins in different cell lines.Moreover, we observed that the performance of DeepCellEss is better than DeepCellEss-nc, which means that protein sequence features are more effective than nucleotide sequence features for DeepCellEss model. The results may be thanks to that: (i) protein sequences are composed of 21 types of amino acids while nucleotide sequences are made up of four different types of nucleotides, resulting in protein sequences has a more diverse sequence information; (ii) the encoded protein sequence is much shorter than the nucleotide sequence for a gene, which can reduce computational consumption and processing complexity; and (iii) protein sequence features are more informative for essentiality prediction.Over the past two decades, many studies have reported that the essentiality of proteins is highly related to the topological properties of PPI networks. Extensive network-based centrality measures were developed for discovering new essential proteins. These methods can efficiently mine latent information from network topology and rank essentiality for proteins in PPI networks. However, the network-based methods suffer from several major drawbacks: (i) these methods cannot be directly used for proteins that are not in the PPI networks; (ii) their prediction ability for essential proteins with low degrees is greatly limited. As a sequence-based method, DeepCellEss is able to compensate for the shortcomings of network-based centrality measures.In order to investigate whether DeepCellEss can achieve promising prediction performance without interaction information, we designed the following experiments: We first downloaded protein interaction data of HCT-116 cell line from the BioPlex 3.0 database  of essential and non-essential proteins. We ranked the result scores predicted by the six network-based methods and DeepCellEss from highest to lowest and compared the cumulative counts of essential proteins in the top 10%, top 20%, 30% and top 40% proteins. The results in We further explored the prediction performance of DeepCellEss on the proteins with low degree in PPI networks. According to the Centrality-Lethality Rule . DDX59 is a member of the DEAD box helicase family proteins, which involves in all aspects of RNA metabolism and plays an important role in many cellular activities. Last, we explored the performance of DeepCellEss on the proteins that have no protein interaction information in our HCT-116 benchmark dataset. Based on our statistics, there are 438 essential proteins that do not appear in the HCT-110 network, which means they are not able to be identified by network-based approaches. Then, we tested them using our re-trained HCT-116-specific model. The results show that 63.9% (280) of them are correctly predicted, indicating that our model has practical and effective prediction ability for essential protein prediction without PPI information.Overall, these experiment results and comparative analysis of DeepCellEss and network-based methods confirm that DeepCellEss can achieve promising performance for essential proteins with no PPI information or low degree in PPI networks. DeepCellEss effectively compensates for the limitations of network-based methods and offers a more practical approach to essential protein prediction.A major advantage of our proposed model is the capability to learn and predict protein essentialities across different cell lines. To demonstrate the effectiveness of our model for cell line-specific prediction, we used ‘G1/S-specific cyclin-D1’  as an example to compar DeepCellEss with other two available servers (Pheg and EP-GBDT). From pre-existing biological experiments, CCND1 performs obvious differences within various cellular environments in terms of essentiality. For instance, it is identified as essential in CL-11 cell line while non-essential in RPMI-B226 cell line. Moreover, DeepCellEss leverages the advantage of the attention mechanism to assign residue-level attention scores for query proteins, and provides the visual heatmap for interpretation. The JAB1/MPN/Mov34 metalloenzyme (JAMM) motif is highly conserved, typically consisting of a canonical sequence of ‘H-[NST]-H-x(7)-S-x(2)-D’. JAMM-containing proteins are metal-dependent proteases and responsible for providing the active site for isopeptidase activity . ResearcAdditionally, we analyzed the performance of DeepCellEss on intrinsically disordered proteins (IDPs), which are widely distributed in eukaryotes and closely associated with human diseases. From the cancer-related protein dataset of DisProt database, we found a conditional essential IDP with 100% disorder content, called ‘nuclear factor erythroid 2-related factor 2’ . Several studies have revealed that NFE2L2 is highly related to lung cancers  indicateTo measure the contributions of individual components to DeepCellEss structure, we conducted ablation studies by re-training and validating DeepCellEss without different components. Specifically, four main components, including skip connection, CNN module, multi-head self-attention module and bi-LSTM module, were separately removed, and we obtained four variants of DeepCellEss model. Then, we trained and validated these four models. The strategies for dataset splitting and model training remain unchanged as the raw DeepCellEss. http://csuligroup.com:8000/DeepCellEss. The DeepCellEss web server provides cell line-specific essential protein prediction and visualization for a large amount of cell lines. To facilitate the access to DeepCellEss, we developed a user-friendly web server, The identification of cancer cell line-specific essential proteins is particularly relevant for the discovery of novel precision cancer drug targets. However, existing computational methods have not taken into account the specificity of essential proteins in different cell lines, and lack practical and interpretable tools for human essential protein prediction. In this study, we proposed DeepCellEss, a cell line-specific interpretable deep learning prediction method based on the attention mechanism. The main contributions of DeepCellEss are summarized as follows:To the best of our knowledge, DeepCellEss is the first computational method that supports cell line-specific essential protein predictions, which makes it possible to predict protein essentialities in different cellular environments;DeepCellEss implements an interpretable deep-learning model through residue-level attention scores from multi-head self-attention mechanism. The attention scores enable to locate the most important sequence regions for different prediction results, and further make more comprehensive analysis and comparison for cell line-specific essential proteins;For real practical applications of our cell line-specific model, we constructed extremely large-scale datasets across 323 cell lines. Moreover, we provided a user-friendly web server of cell line-specific essential protein predictions. It is expected to help discover potential diagnostic biomarkers and therapeutic targets for precision cancer therapy.Although the extensive results show that DeepCellEss is an effective predictor for cell line-specific essential proteins and outperformers existing sequence-based methods, we would like to point out its limitations. The main limitation is that we do not consider the relations of different cell lines under the same tissue or cancer type. In our reported results, the models of different cell lines under the same cancer type show varying prediction performance. For example, in the cancer type of Colorectal Carcinoma, SNU-C1 model yields the best AUROC (0.825) and AUPRC (0.826), while MDST8 model gets the worst AUROC (0.728) and AUPRC (0.731). Therefore, future efforts could be devoted to improving the poor performance for some cell lines by introducing the relations between different cell lines. One potential solution is to use transfer learning techniques (btac779_Supplementary_DataClick here for additional data file.