Pendrin (SLC26A4) is an anion exchanger expressed in the apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However its molecular structure has remained unknown, limiting our understanding of the structural basis of transport. Here, we determine the cryo-electron microscopy structures of mouse pendrin with symmetric and asymmetric homodimer conformations. The asymmetric homodimer consists of one inward-facing protomer and the other outward-facing protomer, representing coincident uptake and secretion- a unique state of pendrin as an electroneutral exchanger. The multiple conformations presented here provide an inverted alternate-access mechanism for anion exchange. The structural and functional data presented here disclose the properties of an anion exchange cleft and help understand the importance of disease-associated variants, which will shed light on the pendrin exchange mechanism. Pendrin SLC26A4 plays an important role for anion balance. Here, authors resolve cryo-EM structures of pendrin performing co-incident uptake and secretion of anions, providing a structural basis of this anion exchange mechanism. SLC26A4 disease-associated mutations varying by ethnicity3. Encoded by the gene SLC26A4, pendrin belongs to the solute carrier 26 (SLC26) family4. Originally, pendrin was identified as a sodium-independent transporter of Cl− and I− in heterologous cell expression systems (Xenopus laevis oocytes or insect Sf9 cells)5. Later, it was widely considered as a sodium-independent electroneutral Cl−/HCO3− and Cl−/I− exchanger expressed in the apical membrane of the inner ear, thyroid, and kidney epithelial cells9  and among these 3–5% is attributed to ls9 Fig. . In huma6. Briefly, pendrin is expressed in the epithelial cells of cochlear spiral prominence, endolymphatic sac, saccule, utricle, and ampulla12. The transport and exchange function of pendrin is taken to be critical in regulating the composition and pH stability of the inner ear’s endolymph. Pendrin knockout in mice leads to the development of an enlarged vestibular aqueduct (EVA), causing hearing impairment13, and affects the acid-base balance of the inner ear which causes sensorineural hearing loss15. In the kidney, pendrin localizes to type B and non-A, non-B intercalated cells (ICs) within the connecting tubule (CNT) and cortical collecting duct (CCD), mediating renal Cl− absorption and HCO3− secretion9. Within these apical plasma membranes, the abundance and activity of pendrin are significantly stimulated by angiotensin II and aldosterone, therefore it would be beneficial to correct the alkalosis and maintain NaCl homeostasis to regulate blood pressure in the kidney18. In the thyroid, pendrin is involved in I− transport, however, its precise function is still under debate19.Numerous genetics studies on Pendred syndrome patients and various mouse models have provided insight about pendrin. In the inner ear, pendrin was observed in the regions where endolymphatic fluid reabsorption occursDespite the importance of pendrin, the exchange mechanism has remained unknown. Here, we investigate the structural and functional properties of mouse pendrin using single particle cryo-electron microscopy (Cryo-EM) and anion exchange assays. We observe that the structures of pendrin vary in the presence of different anions, including three distinct states: a symmetric inward-open dimer, a symmetric outward-open dimer, and a characteristic asymmetric dimer composed of one inward-open protomer and one outward-open protomer. Moreover, clear anion densities are observed in both inward-open and outward-open protomers. This unique asymmetric homodimer architecture aids understanding of the pendrin exchange process as an inverted alternate-access mechanism, with one protomer transporting in- and another transporting out- to provide the structural basis to describe electroneutral anion exchange.25  in the presence of 150 mM NaCl. The purified protein was concentrated to 1.7 mg/mL and immediately used for cryo-EM grid freezing and subsequent data collection. Cryo-EM data processing showed that pendrin forms a dimer as other family members27, with 14 TMDs divided into two inverted repeats representing the core (helices TM 1-4 and 8-11) and gate regions (helices TM 5-7 and 12-14)  forms a domain-swapped homodimer Fig. . Each pr4) Figs. .Fig. 2Th− density is observed and defines the anion-binding site 29, among which S408F mutation almost abrogated transport activity31. In addition, R409 inside of the pocket has four disease-associated mutations 29, among which R409H showed a reduction of exchange and transport function33. Though histidine does not abolish the positive charge, it alters the multiplex interaction and charge environment mediated by R409. Hydrophobic A406, P140, F141 and P142 residues also have a few missense variants 29. P140H was reported to cause a loss of Cl−/I− exchange activity34 and P142R caused a loss of Cl−/HCO3− exchange activity35. P142L induced Cl−/I− exchange reduction36. In addition, N457 located at the gate region also has four mutations 29, and N457K reduced both Cl−/I− and Cl−/HCO3− exchange37. Taken together, the local pocket architecture is crucial for anion binding and therefore important for pendrin’s anion transport and exchange function.Around the anion-binding site, several disease-associated missense variants have been identified in patients and recorded in Deafness Variation Database [3– bound Pendrin (Pendrin-HCO3), pendrin in Cl− buffer was exchanged to a buffer containing HCO3− and frozen for cryo-EM data collection. The cryo-EM structure was determined to 3.5 Å resolution with C2 symmetry. Similar to pendrin-Cl, pendrin-HCO3 also forms a symmetric homodimer with inward-open TMD clefts. Anion HCO3− was clearly observed as well in the binding pocket, which is about 2 Å away from Cl− binding position  at both inward-open and outward-open states. Therefore, we investigated pendrin in a buffer containing two different anions, such as Cl−/HCO3−, Cl−/I−, or HCO3−/I− anion pairs.In the presence of a single anion, Cl3, resulting in a buffer containing 50 mM NaCl and 150 mM NaHCO3. The sample was then concentrated and used for cryo-EM data collection. The data was processed without symmetry. Surprisingly, the cryo-EM structural determination of pendrin in the presence of Cl−/HCO3− (pendrin-Cl/HCO3) mixture revealed three distinct states  form asymmetric homodimers composed of one inward-open protomer and the other outward-open protomer. In this asymmetric dimer, a clear anion density was observed in the inward-open protomer, which is assigned to the identical Cl− occupying position as that of pendrin-Cl symmetric structure  (pendrin-Cl/I) was prepared and used for structure determination. Symmetric inward-open and asymmetric homodimers were observed with approximate percentage of 75 and 25%, respectively. Finally, the structures of pendrin in HCO3−/I− (1:3) (pendrin-HCO3/I) was determined, which also revealed two distinct states: 75% symmetric inward-open and 25% asymmetric states  is co-precipitated with pendrin in immunoprecipitation, and it can guide internalization with pendrinate Fig. . The movate Fig. . Interes28 and SLC26A922, however, the atomic cryo-EM structures of pendrin reveal its intrinsic features of asymmetric homodimer as an exchanger, given the symmetric homodimers resolved for all other SLC26 family members28. The structures of mouse SLC26A9 have two states: inward-open (PDB 6RTC) and intermediate (PDB 6RTF) states21. The dolphin prestin structures include several states, including an inward-like prestin-SO42− (PDB 7S9C), intermediate-like prestin-Cl− (PDB 7S8X) and several other states in between these two states23. When superimposing the inward-state protomers, the TMD regions of pendrin, prestin, and SLC26A9 are relatively similar , and most remain to be examined experimentally  was synthesized and subcloned into the pEZT BacMam vector, with an N-terminal 3X Flag tag and a 3C protease cleavage site. Primers: F: 5’-tttttttttttctgttccaggggcccatggcagcgcggggcggcag-3’; R: 5’-tgcagtcgcggccgctcagtgatggtgatggtgatgggaagcaagtctacgcatggcctcatcc-3’. For fluorescence assay, full-length mouse pendrin was shuttled into a pcDNA 3.1 vector and N-terminal tags were replaced by a mCherry or EGFP fluorescent protein sequence. Primers: F: 5’-gacgagctgtacaagatggcagcgcggggcggcag-3’; R: 5’-tctagactcgagcggccgctcagtgatggtgat-3’. Single point mutations were PCR amplified and subcloned based on wild-type plasmids. Primers: P142A-F: 5’-ttggccctttcgccgtggtcagt-3’; P142A-R: 5’-ccattaaactgaccacggcgaaaggg-3’; Y105A-F: 5’-atggctgctgccctgctggca-3’; Y105A-R: 5’-agggcagcagccatcccttgca-3’; Y105F-F: 5’-ggatggcttttgccctgctggc-3’; Y105F-R: 5’-cagggcaaaagccatcccttgca-3’.DNA encoding full-length mouse pendrin , HEK293-E , HEK293T .Sf9 cells were cultured in ESF921 medium at 27 °C and used for BacMam virus amplification. The P3 virus used for infection was precipitated by PEG 6000 and re-suspended with 1/10 volume of OPM medium. HEK293E cells used for protein expression and purification were cultured in OPM medium at 37 °C and 5% CO6 /ml were infected with resuspended BacMam virus at a volume ratio of 100:1. A final concentration of 10 mM sodium butyrate was added 12 h after infection, and cells were cultured at 37 °C for another 48 h. For fluorescence assays, adherent HEK293T cells were cultured in 6-well plates with a cell density of ~70% and transfected with plasmids-polyethylenimine  mix.HEK293E cells cultured at 37 °C with a density of ~2.3 × 10g for an hour, the supernatant was filtered through a 0.45 μm filter and incubated with anti-flag affinity resin. The resin was washed with 20 mM Tris HCl pH 8.0, 150 mM NaCl, 5 Mm Mg-ATP, 0.02% (w/v) glycol-diosgenin (GDN) to remove heat shock proteins, and eluted with 0.3 mg/mL Flag peptide.All subsequent purification procedures were carried out at 4 °C. Two liters of HEK293E cells were harvested and resuspended in buffer containing 20 mM Tris pH 8.0, 150 mM NaCl and protease inhibitors , and 1.5% (w/v) digitonin power was added into cell suspension and incubated for three hours. After centrifugation at 39,000 x 3 and pendrin-Cl/I, an SEC buffer containing 20 mM Tris pH 8.0, 100 mM NaCl, 0.02% GDN was used. And an equal volume of 300 mM NaHCO3  or NaI was added into peak fractions before concentrating. For pendrin pendrin-HCO3 and pendrin-HCO3/I, an SEC buffer containing 20 mM HEPES pH 8.0, 60 mM Na2SO4, 0.02% GDN was used to replace Cl−, and a final concentration of 10 mM NaHCO3 was added into peak fractions during the concentration and the final pH of sample pendrin-HCO3 was 8.3. Sample pendrin-HCO3/I was added with a final concentration of 33 mM NaI based on sample pendrin-HCO3.For pendrin-Cl, the concentrate was loaded onto a size-exclusion chromatography (SEC) column (Superose 6 Increase 10/300 GL) equilibrated with a buffer containing 20 mM Tris HCl pH 8.0, 150 mM NaCl, 0.02% GDN , then the grid was blotted for 2.0 s with blotting force −2 and flash-frozen in liquid ethane with Vitrobot Mark IV (Thermo Fisher Scientific®).3, and pendrin-HCO3/I) were collected on Titan Krios (Thermo Fisher Scientific) operated at 300 kV, equipped with K2 Summit direct electron detection device (Gatan) and BioQuantum energy filter (Gatan) set to a slit width of 20 eV. Automated data acquisition was carried out with SerialEM software47 through the beam-image shift method48. Others  were collected on the same microscope with the camera upgraded to K3 Summit (Gatan®) using a slit width of 20 eV.Some pendrin cryo-EM datasets  were estimated by using Gctf51. Bad images were excluded upon ice condition, defocus range, and estimated resolution. The remaining good images were imported into cryoSPARC52 for further patched CTF-estimating, blob-picking, and 2D classification. Several good 2D classes were selected as the template for template-picking. From the 2D classification classes, good particles from blob-picking and template-picking were merged and deduplicated. Two rounds of 3D classification were done in RELION, using an initial model generated by cryoSPARC as a reference. The particles from the high-quality classes were selected and re-extracted without binning. Auto-refinement was performed with C1 symmetry, with further CTF-refinement and particle polishing, yielding consensus maps at the range of 3.4–3.8 Å. Then the well-aligned particles were imported back into cryoSPARC for 3D variability analysis (3DVA) regarding the conformational change. Surprisingly, these samples containing two anions all had different conformations including inward-open state and outward state. Preliminary data processing was finished so far, and variant strategies were employed to classify different conformations. To further investigate possible anion densities in the binding pocket of the outward-open state, we did local refinement of outward-open protomer from pendrin-Cl/HCO3 io data.The routine processing of all datasets was carried out with the same procedure. Movie stacks were binned 2 × 2, dose weighted, and motion-corrected using MotionCor253. The above procedures of data processing are summarized in Supplementary Fig. 54 and then “vop zflip” to get the correct handedness in UCSF Chimera for subsequent model building and structural analysis.The reported resolutions are all based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion. All the visualization and evaluation of 3D density maps were performed with UCSF Chimera55 based on the high-resolution 3.3 Å pendrin-Cl cryo-EM map. Most residues were clearly resolved in our cryo-EM map, and total 665 (18-737Δ596-650) amino acid residues were constructed for each monomer. N-terminal 1-17, IVS 596-650, and C-terminal 738-780 were not modeled because the corresponding density was absent in the map. Structure refinement was performed with PHENIX56 with secondary structure and geometry restraints to prevent structure overfitting. Model building of the outward state with C2 symmetry was refined based on the 3.6 Å pendrin-Cl/HCO3 cryo-EM map with the same workflow above. Other C1 symmetrical models were refined from C2 models based on their own maps. Statistics associated with data collection, 3D reconstruction, and model refinement can be found in Supplementary Table Model building of inward-open state with C2 symmetry was refined from the predicted model in Alpha Fold with Coot3, 10 mM glucose, and 5 mM HEPES pH 7.5, and BCECF fluorescence was recorded at the excitation wavelengths of 488 nm and the emission wavelengths of 530 ± 10 nm by confocal microscope (Leica TCS SP8) at 37 °C with a 5% CO2 gassing. Then cells were treated alternately by Cl–free buffer (NaCl is replaced by sodium gluconate) and Cl–containing buffer and photographed under the same conditions. Cl−/HCO3− exchange activities were estimated from fluorescence intensity change.Fluorescence assays were taken 48 h after transfection. Measurements of intracellular pH in HEK 293 T cells transiently transfected with mCherry-pendrin plasmids were performed using a pH-sensitive fluorescent probe BCECF −5-(and-6)-carboxyfluorescein). After dye loading , the cells were perfused with a buffer containing 110 mM NaCl, 25 mM NaHCO−/I− measurements, HEK 293 T was transiently co-transfected with EYFP and mCherry-pendrin plasmids. After titration, we selected the Cl−/I− concentration tuning the fluorescence intensity within the appropriate range of fluorescence microscopy. Cells were treated alternately by Cl–containing buffer  and I–containing buffer . The EYFP fluorescence was recorded at the excitation wavelengths of 488 nm and the emission wavelengths of 530 ± 10 nm by the same confocal microscope at 37 °C with a 5% CO2 gassing. Cl−/I− exchange activities were estimated from fluorescence intensity change57.For in vivo ClThe fluorescence intensity was measured by ImageJ and calculated by GraphPad Prism 8.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Reporting SummaryPeer Review File


Pancreatic neuroendocrine neoplasms (pNENs) are a rare and understudied cancer. Some of this low knowledge base is due to a historical lack of study models. Study models have tremendous implications for validating data for a range of cancer research topics, including treatment development. Therefore, correctly choosing a model is imperative and needs to consider a range of factors pertinent to the research question. In this review, we summarized the current field of models in pNENs. We considered factors, including complexity, accuracy, and cost, in models ranging from cell line cultures, 3D cultures, and whole organismal models, including mice and zebrafish. Improving the number and quality of models available will lead to new breakthroughs in treating pNENs and may lead to findings beneficial for other cancers.Pancreatic neuroendocrine neoplasms (pNENs) are a heterogeneous group of tumors derived from multiple neuroendocrine origin cell subtypes. Incidence rates for pNENs have steadily risen over the last decade, and outcomes continue to vary widely due to inability to properly screen. These tumors encompass a wide range of functional and non-functional subtypes, with their rarity and slow growth making therapeutic development difficult as most clinically used therapeutics are derived from retrospective analyses. Improved molecular understanding of these cancers has increased our knowledge of the tumor biology for pNENs. Despite these advances in our understanding of pNENs, there remains a dearth of models for further investigation. In this review, we will cover the current field of pNEN models, which include established cell lines, animal models such as mice and zebrafish, and three-dimensional (3D) cell models, and compare their uses in modeling various disease aspects. While no study model is a complete representation of pNEN biology, each has advantages which allow for new scientific understanding of these rare tumors. Future efforts and advancements in technology will continue to create new options in modeling these cancers. Pancreatic neuroendocrine neoplasms (pNENs) are a rare class of tumors which derive from neuroendocrine cells located in the islets of the pancreas. They are the third most common neuroendocrine tumor subtype in the gastro–entero–pancreatic (GEP) system, accounting for 1.2 cases per 100,000 people and showing a steady rise over the last few decades globally [Classification of pNENs falls into several phenotypic and genomic categories . GradingpNENs comprise a heterogeneous background of subtypes . There aMEN1 is the most common altered gene and is found mutated in 40–50% of pNEN patients and is associated with both a germline condition causing numerous endocrine tumors as well as with sporadic biallelic inactivation [MEN1 is involved in a wide range of pathways, including regulation of DNA transcription and replication, maintenance of genome integrity, and control of cell cycle, and it is believed mutations in MEN1 can prevent the nuclear localization of menin, the protein encoded by the MEN1 gene, losing these important functions [DAXX (22–25%) and ATRX (10–18%) [DAXX has a wide range of interactions, including cell cycle and apoptosis control, while ATRX is involved in chromatin remodeling during the cell cycle [PTEN loss is found in 7% of all cases [TSC members 1 and 2 are found to be mutated in a small percentage of cases (2% each); however, they form an autosomal dominant phenotype (TSC syndrome) as the loss of one copy is sufficient for tumorigenesis. TSC1 (hamartin protein) and TSC2 (tuberin protein) negatively regulate activation of mTORC1 through inhibition of the small GTPase Rheb, a positive regulator of mTORC1. Finally, DEPDC5, a Rag inactivating protein, is observed to have biallelic inactivation in 2% of cases. Mutations to DNA damage repair proteins have a threefold effect on tumorigenesis; they promote further genomic instability, prevent checkpoint activation, and increase treatment resistance. MUTYH, a protein involved in base excision repair, was found to have both germline mutations and biallelic inactivation (5%) in patient specimens. Mutations to checkpoint kinase 2 (CHK2), regulator of the G1-S phase transition, have also been observed in a subset of patients . Finally, mutations to chromatin-modifying proteins, including histone methyltransferases SETD2  and MLL3 (5%), are observed in patients. Alterations to INK4A/ARF locus, which encodes p16INK4a and p14ARF, can also limit the expression of tumor suppressor Rb1 and p53 by failing to block cell cycle activating proteins. Evidence has shown that although these genes are not often mutated in WD pNETs, hypermethylation may cause inactivation of these genes, in particular for gastrinomas [Rb1 and p53, demonstrating loss of tumor suppression in addition to the oncogenic gains [The genomic characterization of pNENs from initiation to cancer has only been covered in the recent literature ,12. Desptivation . MEN1 isunctions . There a10–18%) . DAXX hall cycle . Controlll cases . It prevtrinomas ,18. In f% . DAXX trinomas ,20. In ctrinomas . Similarns [DAXX –25% and Treatments of pNENs remain limited. The current gold standard and only curative treatment is surgical removal . In mostTo better understand pNENs from initiation, progression, and treatment, proper study models are required. Choosing the correct model is imperative to create an accurate representation of this cancer . Each moIn scientific research, there is often a need to create large scale, high-throughput experiments to address a variety of hypotheses. Cell lines have allowed for fast, reproducible results for tissue types throughout the entire body. Isolation of the tumor cell populations often requires specialized culture techniques including substrate and cell culture medium selection. They are a useful model for cancer as the process to create stable, “immortalized” cultures can often mimic the development of tumors. There are often mutations in tumor suppressor genes, including p53, and they may have alterations to hTERT, conferring the long-term ability to reproduce without cell forced senescence or apoptosis, also known as the Hayflick limit ,34. Thesp53 and Rb1, as seen in pNECs [Cell lines derived from animals are advantageous as animal models have less stringent requirements when compared to human studies for cellular acquisition. They can be generated from parent organisms through indirect genetic manipulation, such as radiation bombardment or by targeted viral infection to induce oncogenesis. Many of the animal pNEN cell lines are derived from early-stage insulinomas and were developed to create cell models for diabetes research, making stable long-term insulin-producing and responding lines . Oncogenin pNECs . It is aThe most common host species for animal cell lines are mice. Mouse models for pNENs have been extensively developed, and work to establish cell lines for study has persisted for some time. Early work included attempts to create cell lines isolated from beta cells in insulinoma-like pancreas islets for the study of diabetes using the SV40 large T antigen. These cell lines, including βTC, MIN6, BTC-5, NIT-1, and TCP61, have been utilized in a wide range of research for prediabetes and for the study on early stage insulinomas ,40,42,43There are currently four cell line models of pNENs from animal species other than mice. Two rat cell line models, RIN and INS1, were derived from the same rat line, the inbred NEDH  rat ,46,61. TEfforts to establish pNEN cell lines from human tumors has been an important experimental objective. It involves taking cells from primary or metastatic tissues to create long-term study models to mimic the disease process while providing a more biologically relevant model when compared to animal cell lines. This approach is not without its pitfalls; while the mimicry of the disease state is important, slow growth characteristics of pNENs can mean poor establishment success along with long cell-doubling times, which makes therapeutic testing difficult ,55. ChanThere has been considerable subtype diversity in human-generated cell lines. The earliest published human cell line, QGP-1, was derived from an aggressive somatostatinoma which also produces carcinoembryonic antigen (CEA) . This ceIn addition to the long-term cultured cells described above, there have been recent efforts to culture primary cells taken by biopsy for experimental study. The goal for doing so is to more accurately model pNENs by controlling the potential of genetic drift in long-term culture. A comparison of treatment combinations involving everolimus and somatostatin analogues performed using tumor cultures of primary pNET cells cultured with bovine ECM has been described by Mohamed et al. ,75. ManyIn modeling cancer, animal models are important to study the whole-body effects of cancer. As entire organisms, these models maintain the tumor and non-tumor components, including stromal, immune, and non-cellular elements, organized into tissues and organs, allowing for important crosstalk between functional systems . AnimalsXenograft models of cancer use animals to propagate tumor cells over the course of multiple passages. They are one of the oldest methods for the culture of tumor cells, with studies reported for over 50 years . They caThe first published attempt to form pNEN PDX models was reported in non-obese diabetic (NOD)-SCID subcutaneously flank implanted mice during the attempt to create stable lines from several NEN tumors . The autThe most developed model system of pNENs is the genetically engineered mouse model (GEMM). Mouse models have been a valuable tool in scientific research for decades; indeed, there may be upwards of thousands of mouse strains developed for research . Early wp53 or Rb1, which are common pNEC genes. One of the earliest methods of induced oncogenesis in mouse models is using oncogenic viruses. The introduction of virus into a target sequence is followed by injection into embryonic cell lines. These cells are then introduced to embryos and can be bred to form stable populations. Commonly, the simian virus 40 large T-antigen (SV40 Tag) sequence is altered to remove the ability for viral reproduction before being incorporated downstream of a tissue-specific promoter of interest. This gene of interest should be “targeted” to the organ-specific function of the cells in question, as limiting the number of cells expressing the SV40 Tag is important in eliminating off-target effects. The promoter of the tissue-specific gene will drive transcription in specific cells which allows for the expression of the SV40 large T-antigen, causing oncogenesis. Mutations which arise from this process often included −/− adaptive-immunity-depleted mice crossed with RT2, anti-angiogenic treatment would lead to smaller tumors more capable of metastases [Of the major developed hybrid target sequences of SV40 Tag, the Rat Insulin Promoter (RIP) model has the most extensive publication record. First described in 1985, the RIP-Tag mouse lines were developed by incorporating the SV40 Tag downstream of the rat insulin promoter (RIP) to create a pancreatic islet beta-cell only transcription sequence . The restastases . In conttastases ,97. Of ntastases . RT5 is tastases ,101. RIPThere have also been other SV40 target hybrid sequences utilized for the study of pNEN development . Based op53, Rb1, and MEN1. They are also useful for modeling genes which cannot experience germline knockouts, such as MEN1 [MEN1 mice [Rb1 has also been studied in conjunction with other inactivated mutations, and although Rb1 heterogenous KOs can form pNENs, work has shown it to not augment tumorigenesis in MEN1 KOs [P53 KO work has not shown the ability to form pNENs alone, although they do create more aggressive pNECs in combination with Rb1 heterozygous KOs [Cul9, a ubiquitin ligase of p53, demonstrated a case of pNEN formation in a cohort of mice at 21 months [In modeling cancer development, the use of global heterogenous knockouts (KOs), also known as conventional KO models, have found a niche in many organisms . The KOs as MEN1 . If the EN1 mice ,118  using an inactivating neomycin-resistant gene insertion result in normal births but cause alpha and delta cell hyperplasia at three months followed by tumor development at six months [SPC2-null mice, mice are born normally, but within 2–3 months have alpha cell hyperplasia, with tumors present at 10–12 months [In contrast to heterozygous KOs, homozygous KOs are achieved through the complete targeted deletion of a functional gene. Targeted deletions are created through the breeding of heterozygous KOs to create the genotype. The genes must not be essential for embryogenesis or normal animal function, as these offspring would be non-viable. A common target in pNENs is hormone production related to the function of the pancreas . As witnx months ,124. Oth2 months ,126. Fin2 months . HomozygMEN1 heterogeneity that did not affect the increased CDK5 expression [MEN1 expression to show how the two would create shorter survival in mice, in particular for homozygous KO vs. heterozygous KO [Models of induced activation are utilized as a selective way to analyze the effect of potential oncogenes on tumor development . Methodspression . Most reygous KO . The micCDK (R24C) demonstrated that cells became immortal, and a double mutation resulted in a wide range of tumors, including several pNEN subtypes in mice [Gcg) locus, creating an easily visualized model of Gcg-expressing cells in mice [Homozygous knock-in models are advantageous in the study of pNENs, where mutations have caused altered function . The kno in mice . Another in mice . The douMEN1. Importantly, a system of controlled expression would allow for both a negative control and experimental model in a single animal, ensuring the change in expression is responsible for any alterations in the model organisms. The Cre-LoxP system was developed from the bacteriophage P1 and uses the Cre recombinase enzyme to create a recombination of a target sequence present between two LoxP sequences inserted into the genome, deleting the sequence [Prdx1, or insulin promoters. The target sequence can be either important in the repression or activation of genes. Importantly, this system can allow for the controlled activation by several methods, including tamoxifen induced (CreERT) or doxycycline (Dox). In generating Cre-LoxP mice, two parental strains need to be generated: one with the Cre-driver strain in which Cre recombinase will target the cell or tissue of interest and another with the loxP flanked (floxed) DNA. The two are then bred to create the conditionally transformed mice. Since the inception of the technology, it has been used in pancreatic cell origin, diabetes, and pNEN research [While models of heterozygous or homozygous KOs have created numerous discoveries, the limitations of modeling germline mutations essential for organism development can prevent the analysis of certain genes, including homozygous inactivation of sequence . A “guidresearch .MEN1  have many of the same advantages, including a well-developed genetic modification toolbox [Rb1, rbl1, and p53 in CRISPR-Cas9 KOs demonstrated that p53 did not enhance tumor growth in the Rb1/rbl1 KOs for pNEC SC tumors [MEN1, ATRX, and in SSTR2 expression [Outside of genetic mouse models, a variety of other species have been utilized in pNEN research . Rats arebrafish . Over th toolbox . A studyC tumors . Finallypression . Indeed,pression . OverallThree-dimensional tumor models are the intermediate step in terms of complexity and utility when compared to both traditional 2D cell line and animal models. They represent a middle ground in terms of accuracy, function, cost, and throughput. Three-dimensional models of tumors can be broadly summarized into three categories: spheroids, extracellular-matrix (ECM)-supported, or bioengineered hybrid models . Herein,Spheroids consist of adhered cells forming a collective, spherical unit. They are typically formed through low-gravity environments such as the hanging-drop technique or with special plates to mimic low gravity . SpheroiMEN1 in the expression of apoptosis markers was performed in BON-1 and QGP-1 spheroids, demonstrating a negative link between MEN1 expression and TP53 and CDKN1A, which could be remedied with staurosporine [The overarching theme of spheroid usage in pNEN research is in drug screening applications. The first 3D modeling of human pNENs was performed in 2012 by Wong et al., who described spheroid formation using BON-1 and QGP-1 cells in agarose-coated plates for usage in a single-drug therapeutic screen, Trichostatin A (TSA), and demonstrated a smooth spheroid formation for BON-1 but not for QGP-1 . The samosporine  Finally,ECM-supported constructs, including organoids and tumoroids (PTOs), can form larger and more complex cultures using ECM, which provide an increase in nutritional support while better mimicking cell–cell interactions . They fuTP53, RB1, DAXX1, and KRAS, with large chromosomal alterations to the pNET PTO group. Finally, the group analyzed growth factor dependency based on PTO mutational profiles and used CRISPR/Cas9 technology to solidify these findings. The second published study modeled pNENs as tumoroids using cryopreserved specimens including insulinomas and NF pNENs, demonstrating the potential for a multicenter approach [There are currently four published works describing PTO work with two preprints in press. Kawasaki et al. described the first study using PTOs to represent pNENs in a larger study of neuroendocrine tumors . They weapproach . The groapproach . pNEN suapproach . The autTP53 mutation which was derived from the parental tumor but were also able to determine the pNEC PTOs showed large intra-tumor diversity compared to other neuroendocrine tumor PTOs in this study. Finally, drug screening applications were demonstrated using paclitaxel, everolimus, and navitoclax, demonstrating the suitability of these PTOs for this application. The second preprint demonstrated the continued work of April-Monn et al. on tumoroids [DAXX, ATRX, and Rb1, with all containing >75% Ki67 expression. The study also confirmed the maintenance of gene expression pathways, including Myc, p53, and EGF/VEGF, between tumoroids and PTOs. Interestingly, QGP-1 and NT-3 spheroids were also created in this study and compared to the primary tissues, demonstrating differences in gene expression and showing the tumoroids were more accurate. Tumoroids were then screened with cisplatin or temozolomide, illustrating patient specific responses which could match the clinical courses of the patients who were treated similarly. Further analysis of treated tumoroids would suggest IFNB1 and KDM5A as targets for combinational therapy, which the authors confirmed with inhibitors for each target as well as with cisplatin in tumoroids and in a QGP1 xenograft in zebrafish. Current advances in 3D ECM supported constructs have demonstrated notable promise in modeling cancers throughout the body, and new research continues to advance our understanding of pNENs.Two preprints further demonstrating robust value to PTO research are currently in press. Dayton et al. has described the establishment of a variety of neuroendocrine PTOs, which include one large-cell pNEC . They noumoroids . The groBioengineered hybrid 3D models contain both biological and artificial components to functionally mimic the tumor microenvironment. These components may include microfluidics to represent circulation or channels to create tumor “invasive” potential . They maOne group has described using bioreactor technology to keep patient tissue slices of neuroendocrine tumors, BON-1 cells, and BON-1 PDX tumors alive for over a month. Herring et al. created a joint hydrogel and polydimethylsiloxane (PDMS) bioreactor attached to inflow and outflow channels, which allowed for flow of cell culture media to mimic vascularization. In their first study, they confirmed viability for each tissue group and demonstrated the ability to track therapeutic response in pretreated tissues . The respNENs have experienced profound interest from the clinical community due to the rise of cases and lack of mechanistic understanding. Combined, the outcomes for these patients remain poor, with subtype heterogeneity confounding treatment prognosis and preventing clinical trial accrual. To improve these issues, preclinical models must create an accurate and reproducible system to analyze the biology behind these rare tumors. Researchers must balance model complexity with high-throughput capability. Advancements in technology have allowed for focused, controlled study systems which have improved our understanding of individual subtypes; nevertheless, there is ground to be made to improve the outlook of pNENs. MEN1. As whole organisms, the interactions between the pNEN and the entire body, including nearby and distal organ systems along with non-cellular ECM components, can be observed. These relationships also allow for more accurate therapeutic testing, as the effects of the therapy, from dosing to on-target tumor and off-target healthy tissues can be measured. Patient-derived xenograft establishment also provides another method of long-term propagation of primary tumor cells, allowing for continued study. Current interest in non-mouse models, including zebrafish and frogs, demonstrates opportunities for models of advanced genetic manipulation and have shown this promise in other tumor types. However, being non-human models, there can never be complete translational comparison as animals often contain different versions of the genes present in humans. Many methods of genetically manipulated animal models suffer from either slow, unpredictable tumor development, off-target effects, or low success rates. Additionally, high institutional upkeep and lower throughput prevent large scale implementation of many models as a universal standard. Considerations for study model choice must include both advantages and pitfalls of each option. Cell line models of pNENs carry the advantages of high throughput and low cost. Experience with lines has carried through decades of pNEN research and they have been involved in the development of many clinical agents. However, there are significant shortcomings in the modeling of pNETs, as most derived cell lines undergo significant oncogenic transformation resulting in a highly proliferate, mutation-heavy profile seen in pNECs. Cells grown in 2D culture are not representative of cells grown in the body as they respond to different spatial stimuli. Additionally, there has been recent debate about the evolution of established lines, with some lines falling out of favor as more advanced analyses finds tumor characteristics related to non-pNEN diseases or the discovery of shifted mutational profiles in established cell lines ,67. AnimThree dimensional cellular models are the newest arrival to the pNEN research benchtop. These models allow for spatially accurate systems of cellular interactions, which has been shown to improve tumor signaling along with cell phenotype and genomics . RecentlFurther advances in technology have shown promise in continuing to better model pNENs. Most importantly, recent advances in our understanding of genetics, proteomics, and epigenetics have allowed for a significant increase in model fidelity and number. Several recent cohorts of genetic analyses in pNEN patient populations have increased our knowledge level, identifying new genotypes and potential targets ,21,201. pNENs are a complex family of tumors due to their slow growth and rarity. Despite this, pNEN model development has progressed substantially, with increasingly complex models being developed. With new technological advances, future models will continue this trend.


The estrogen receptor acts as a transcription factor and triggers cell proliferation and differentiation. Hence, investigating ER-DNA interaction genomic regions can help identify genes directly regulated by ER and understand the mechanism of ER action in cancer progression.The largest group of patients with breast cancer are estrogen receptor-positive . Also, the meta-analysis results were compared with the results of T47D cell lines for more precision. Enrichment analyses were also employed to find the functional importance of common meta-differentially bound sites and associated genes among both cell lines.In the present study, we employed a workflow to do a POU5F1B, ZNF662, ZNF442, KIN, ZNF410, and SGSM2 transcription factors were recognized in the meta-analysis but not in individual studies. Enrichment of the meta-differentially bound sites resulted in the candidacy of pathways not previously reported in breast cancer. PCGF2, HNF1B, and ZBED6 transcription factors were also predicted through the enrichment analysis of associated genes. In addition, comparing the meta-analysis results of both ChIP-seq and RNA-seq data showed that many transcription factors affected by ER were up-regulated.Remarkably, meta-analysis of ChIP-seq data of estrogen-treated MCF7 cell line leads to the identification of new binding sites of ER that have not been previously reported. Also, enrichment of the meta-differentially bound sites and their associated genes revealed new terms and pathways involved in the development of breast cancer which should be examined in future in vitro and in vivo studies.The The online version contains supplementary material available at 10.1186/s12920-023-01655-z. The majs (ESR1) , 3. Whens (ESR1) . Hence, meta-analysis supplies more exact results [meta-analysis of ChIP-seq data. A meta-analysis study was conducted by Kolmykov and colleagues on ChIP-seq datasets through the rank aggregation approach, and the significant TFBSs in BC were identified [meta-analysis, including: (1) it is necessary to consider the same cell line, dose, and treatment period (known batch effects), (2) selecting the appropriate treatment period, i.e., after which treatment period, treated and untreated samples show the most differentiation, (3) removing the effects of differences laboratory conditions (unknown batch effects) seems to be vitally important to obtain more precise meta-analysis results, (4) since each study may have been analyzed with different platforms and genomic references, it is necessary to re-analyze the samples with the same tools and references, and (5) it is noteworthy that even if the same platform is applied, a different number of peaks and regions are obtained for each sample. Therefore after combining the datasets, they must be prepared to obtain the same number, regions, and scores of TFBSs for all samples. Next, the unknown batch effects must be removed before meta-analysis. Despite the necessities mentioned above, no meta-analysis study has already been performed on integrated TFBSs to obtain significant binding sites in BC.Chromatin immunoprecipitation sequencing (ChIP-seq), as a next-generation sequencing technique, is applied to find the transcription factor binding sites (TFBSs) . In the  results . Few stuentified . Some cometa-analysis results were compared with the results of T47D cell lines, and the intersection of both cell lines was obtained. By enriching the shared meta-DBSs and their associated genes, TFs and pathways that had not been previously reported in BC were identified. Moreover, the results of the ChIP-seq data meta-analysis were confirmed by comparing the results of the RNA-seq data meta-analysis.In the current study, to obtain meta-differentially bound sites in estrogen receptor-positive breast cancer cell lines, we employed an innovative workflow for the meta-analysis of ChIP-seq data. Given the ER's essential role in BC, MCF7 and T47D cell lines stimulated with E2 were collected to find genomic regions directly interacting with ER. MCF7 and T47D cell lines, representing luminal A subtype, are extensively utilized as experimental models in breast cancer research, particularly for the study of hormone-dependent BC. These two ER + BC cell lines serve as valuable tools in understanding the molecular mechanisms and exploring potential therapeutic interventions for BC, particularly luminal A subtype. In the MCF7 cell line, concerning the same cell line, dose, and treatment period (removing known batch effects), public ChIP-seq datasets were selected and re-analyzed. Next, samples were integrated, and the same number, regions, and scores of TFBSs were obtained in doses of 10 nM and 100 nM separately. Finally, unknown batch effects were removed, and several ChIP-seq datasets were meta-analyzed based on TFBSs scores. Hence, we take advantage of the term meta-differentially bound sites (meta-DBSs) for the first time. For more precision, the meta-analysis was performed, and meta-DBSs were identified in the MCF7 cell line treated with 10 nM and 100 nM E2 separately. Then, Peak Annotation was applied for DBSs and meta-DBSs, and the results of both cell lines were compared. Peak set functional enrichment analysis (PSFEA) and ChIP enrichment analysis (ChEA) were implemented for the common meta-DBSs and their associated genes, respectively. Also, the meta-analysis results of both ChIP-seq and RNA-seq data were compared. The general steps of the present study are summarized in Fig. After collecting data from SRA-NCBI  and ENA-+ cell lines; MCF7 and T47D cell lines, (2) availability of both treated with vehicles (as controls) and treated with E2 samples, (3) datasets without any knocked out genes, and (4) datasets with the same and appropriate dose and period of treatment. Consequently, GSE94023, GSE99626, GSE67295, GSE115607, GSE80367, GSE23893, GSE54855, and GSE59530, including MCF7 and T47D cell lines stimulated with 10 nM and 100 nM E2 for 40 or 45 min were selected. The details of the datasets are described in Table A comprehensive search was conducted on SRA-NCBI  and ENA-https://galaxyproject.org) [The selected ChIP-seq datasets were re-analyzed under the Galaxy platform (ect.org)  to homogect.org)  and Trimect.org) , respectect.org) .q−value) > 2; fold-enrichment > 2). Then, the ChIPseeker (version 1.26.2) [meta-analysis. During peak calling with MACS2, the input samples were also considered to reduce noise. The Blacklist regions were also considered to improve the signal-to-noise ratio. The maximum tags were set to keep one tag at the same location (–keep-dup = 1). In the individual dataset analysis, replicates were combined using the rmspc  package [Within each individual study, DBSs were identified in MCF7 and T47D cell lines treated with E2 compared to treated with vehicle (as control) through MACS2  (version 1.26.2)  package  package .A. ER-ChIP treated with E2 vs. input = corrected ER-ChIP treated with E2.B. Vehicle vs. input (for vehicle) = corrected vehicle.C. Corrected ER-ChIP treated with E2 vs. corrected Vehicle = detection of enriched regions in ER-ChIP treatment E2 (using MACS2).Typically, analysis of treatment, vehicle, and input samples on ChIP-seq data is performed in the following steps:meta-analysis workflow was utilized to compare cells of treated with vehicle versus treated with E2 at the TFBSs score levels  > 2 to select meta-DBSs. Finally, the ChIPseeker package was utilized for peak annotation. All packages were implemented in R software. The parameters in the ARSyNseq and the metaSeq packages were set as follows:To find meta-DBSs, a els Fig. . First, els Fig. . In ARSymydata2corr1= ARSyNseq  result<- meta.oneside.noiseq , studies = flag2)http://go.cistrome.org/) [P-value ≤ 0.05 was applied to select significant GO terms and KEGG pathways. Also, a comprehensive ChIP Enrichment Analysis (ChEA) was performed on meta-DBSs-associated common genes using ChEA3 (the updated version of 2019) (https://amp.pharm.mssm.edu/ChEA3) [P-value ≤ 0.05 was considered significant.Gene ontology (GO) and pathway enrichment analyses were performed with Cistrome-GO (the updated version of 2019) (me.org/)  to determe.org/)  enrichedu/ChEA3) , which cmeta-analysis was performed on the ChIP-seq data based on TFBSs scores to identify meta-DBSs. Here, known batch effects were first removed. Since the treatment period with E2 can affect the results, several treatment periods were investigated to select the appropriately treated cell lines. In the GSE94023 study, there were various periods of treatment with E2, including 0 (mock treated as control) and 5, 10, 20, 40, 80, 160, 320, 640, and 1280 min (cases). An appropriate sampling time reveals the maximum distinction between treated and untreated samples. Hence, a heat map correlation matrix was drawn for all samples. According to the matrix, the samples after 40 min showed the most significant difference compared to the control  > 2) were identified   was performed. By enriching meta-DBSs and considering adjusted Also, enriched CCs terms were linked to the cortical actin cytoskeleton (GO:0030864), cortical cytoskeleton (GO:0030863), and cytoskeleton (GO:0005856). Significant GO terms were found for MFs with regulatory region nucleic acid binding GO:0001067), glucose binding (GO:0005536), lipid binding (GO:0008289), RNA polymerase II regulatory region DNA binding (GO:0001012), activating transcription factor binding (GO:0033613), DNA-binding transcription factor binding (GO:0140297), and transcription coregulator activity (GO:0003712) , Rap1 signaling pathway (hsa04015), Tight junction (hsa04530), Neurotrophin signaling pathway hsa04722), Fluid shear stress and atherosclerosis (hsa05418), MAPK signaling pathway (hsa04010), Thyroid cancer (hsa05216), Bladder cancer (hsa05219), and Pathways in cancer (hsa05200)  identified through the meta-analysis but not found in individual studies. For the strict selection of meta-DBSs, MCF7 cell lines meta-analysis results were compared with the results of T47D cell lines and initial individual studies, and common genes and the associated meta-DBSs were determined. With the enrichment of meta-DBSs correlated with common genes, several pathways with presumably clinical importance in BC were identified. Meta-DBSs-associated common genes were also enriched, and TFs were identified that have been predicted as potential key regulators in the progression of BC. In addition, we compared the meta-analysis results of both ChIP-seq and RNA-seq data.Breast cancer is the most incident cancer among women worldwide. The largest group of patients with BC is ERntiation , 3. Henceriments . To the meta-analysis of RNA-seq data based on those methods [meta-analysis, we faced two problems: (1) none of the mentioned methods removed unknown batch effects, and (2) the number of peaks and their associated genomic start and end intervals were different among samples, and batch effects could not be removed. Therefore, sample preparation was required. To this end, we obtained the same number, regions, and scores of TFBSs using the DiffBind package.Some of the differentially expressed genes (DEGs) identification methods in the RNA-seq context, such as DEseq2  and edge methods , 55, 56. methods , in whicPOU5F1B, ZNF662, ZNF442, KIN, ZNF410, and SGSM2 TFs were found in the meta-analysis of MCF7 cell lines treated with 10 nM E2 but not in individual studies. POU5F1B, a processed pseudogene highly homologous to OCT4, was recently shown to be transcribed in ER+ BC [POU5F1B is specifically expressed in mammalian totipotent embryonic stem and germ cells and has a crucial role in regulating and maintaining pluripotency and self-renewal [ZNF662, ZNF442, and ZNF410 were involved in the regulation of transcription by RNA polymerase II [GPER1) is significantly associated with adverse survival of BC patients [ZNF662 was identified as one of the unique factors related to GPER-DNA binding [ZNF442 plays a role in the strategy adopted by ER+ BC and triple-negative breast cancer (TNBC) cell lines for maintaining zinc homeostasis [ZNF410 uniquely activates CHD4, one of the catalytic components of the Nucleosome Remodeling and Deacetylase (NuRD) complex [+ BC [KIN) promotes apoptosis of TNBC [SGSM2) is involved in ER+ BC metastasis by enhancing migrator cell adhesion via interaction with E-cadherin [Herein, n ER+ BC . POU5F1B-renewal . The Zin-renewal . ZNF662,erase II . It has patients . ZNF662  binding . ZNF442 eostasis . ZNF410 ex [+ BC , 64. Rec of TNBC . Small GPCGF2, HNF1B, and ZBED6 were predicted as regulatory factors involved in BC progression. Based on our findings, no peaks associated with those TFs were found in individual studies and meta-analysis results. Maybe it is because all TFs do not always regulate their own expression. Polycomb group proteins (PcG) play a critical role in cancer development, proliferation, senescence, and carcinogenesis [PCGF2 serves as a tumor suppressor in BC, gastric cancer, and colon cancer, probably for the negative regulation of Akt activation [HNF1B, plays a critical role in BC's insurgence or progression [ZBED6) has been shown to repress insulin-like growth factor 2 (IGF2) transcription [ZBED6 has only been reported in TNBC but is not well understood in ER+ BC so far [With enrichment analysis of meta-DBSs-associated common genes by ChEA3, TFs including ogenesis . PCGF2 stivation . Recent gression . Zinc Ficription . The rolC so far .meta-analysis, and the top 50 TFs obtained from ChEA3  [+ BC. Tissue development (GO:0009888) [+ BC previously. Regulation of anatomical structure morphogenesis (GO:0022603) [+ BC. Glucose binding (GO:0005536) [Interestingly, the significant GO terms and KEGG pathways obtained from PSFEA in the present study were enriched by many predicted TFs by ChEA3 Figs.  and 7. T0070268) , anterio0070268) , embryon0070268) , respons0070268) , and neg0070268)  have bee0009888)  was enri0009888) , and cor0009888)  in pancr0009888) , 83 in o0022603)  in color0022603)  in hepat0022603)  in chron0022603)  in Alzhe0022603) , RNA pol0022603) , activat0022603) , DNA-bin0022603) , transcr0022603)  have bee0005536)  was enri+ BC. MAPK signaling pathway (hsa04010) [The nine enriched KEGG pathways include Pathways in cancer (hsa05200) , Estrogesa04010)  was enrisa04010) , and Blasa04010) , 102 havsa04010)  is relatPOLRMT and TFAM TFs. Especially TFAM increased expression has been reported in a meta-analysis of MCF7 cell lines treated with E2 [According to the cases mentioned, GO terms and pathways previously approved concerning BC were enriched Fig. . Moreove with E2 . This mameta-analysis results showed that many TFs predicted by ChEA3 were up-regulated in RNA-seq meta-analysis results. There can be several reasons why some TFs are down-regulated. FOXA1 is a determinant of drug resistance in breast cancer cells, and it has different functions in response to treatment in ER+ and TNBC cell lines [FOXA1 [The comparison of ChIP-seq and RNA-seq data ll lines   The Heat map Correlation matrix and (B) PCA plot for the GSE94023 study. MCF7 cell line was treated with E2 for different times, including 0 (treated with vehicle as control), 5, 10, 20, 40, 80, 160, 320, 640, and 1280 minutes. According to the matrix, after 40 minutes, E2-treated samples showed a more significant difference compared to the control sample. Figure S2. The Heat map Correlation matrix for GSE94023, GSE99626, GSE67295, and GSE115607 studies. The default binding affinity matrix was obtained from the DiffBind package. The datasets with MCF7 cell lines that were treated with 10nM E2 for 40 or 45 minutes were shown (A) before and (B) after normalization and unknown batch effect correction. Batch effect removal was performed using the ARSyNseq package in R. Figure S3. The PCA plot for GSE94023, GSE99626, GSE67295, and GSE115607 studies. The default binding affinity matrix was obtained from the DiffBind package. The datasets with MCF7 cell lines that were treated with 10nM E2 for 40 or 45 minutes were shown (A) before and (B) after normalization and unknown batch effect correction. Figure S4. The Heat map Correlation matrix for GSE23893, GSE54855, and GSE59530 studies. The default binding affinity matrix was obtained from the DiffBind package. The datasets with MCF7 cell lines that were treated with 100nM E2 for 40 or 45 minutes were shown (A) before and (B) after normalization and unknown batch effect correction. Batch effect removal was performed using the ARSyNseq package in R. Figure S5. The PCA plot for GSE23893, GSE54855, and GSE59530 studies. The default binding affinity matrix was obtained from the DiffBind package. The datasets with MCF7 cell lines that were treated with 100nM E2 for 40 or 45 minutes were shown (A) before and (B) after normalization and unknown batch effect correction.Additional file 2: Table S1. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 10nM E2 for 45 minutes in GSE94023 study. Table S2. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 10nM E2 for 45 minutes in GSE99626 study. Table S3. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 10nM E2 for 45 minutes in GSE67295 study. Table S4. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 10nM E2 for 45 minutes in GSE115607 study. Table S5. Differentially bound sites (DBSs) obtained from T47D cell line treated with 10nM E2 for 45 minutes in GSE80367 study. Table S6. Differentially bound sites (DBSs) obtained from T47D cell line treated with 100nM E2 for 45 minutes in GSE23893 study. Table S7. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 100nM E2 for 45 minutes in GSE23893 study. Table S8. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 100nM E2 for 45 minutes in GSE54855 study. Table S9. Differentially bound sites (DBSs) obtained from MCF7 cell line treated with 100nM E2 for 45 minutes in GSE59530 study. Table S10. Default binding affinity matrix of 6 samples by the 63,612 sites that overlap in at least two of the samples using DiffBind in  MCF7 cell line treated with 10nM E2 for 45 minutes. TableS11. Default binding affinity matrix of 6 samples by the 23,517 sites that overlap in at least two of the samples using DiffBind in  MCF7 cell line treated with 100nM E2 for 45 minutes. Table S12. Meta-differentially bound sites (meta-DBSs) obtained from a meta-analysis on  MCF7 cell line treated with 10nM E2 for 45 minutes. Table S13. Meta-differentially bound sites (meta-DBSs) obtained from a meta-analysis on  MCF7 cell line treated with 100nM E2 for 45 minutes. Table S14. literature_ChIP-seq. Table S15. Enrichr. Table S16. ARCHS4—Coexpression. Table S17. ENCODE--ChIP-seq. Table S18. ReMap--ChIP-seq. Table S19. GTEx—Coexpression. Table S20. Integrated_topRank. Table S21. Integrated_meanRank. Table S22. Gene Ontology (GO) for 7,308 meta-DBSs related to 617 common genes among MCF7 & T47D cell lines using Cistrome-GO. Table S23. KEGG pathways analysis for 7,308 meta-DBSs related to 617 common genes among MCF7 & T47D cell lines using Cistrome-GO. Table S24. Differentially expressed genes (DEGs) identified from GRO-seq data in the MCF7 cell line treated with 100nM E2 for 40 minutes in the GSE27463 study.


Glioblastomas (GBM), also known as glioblastoma multiforme, are the most aggressive type of brain cancers. Currently, there is no real treatment for GBM and thus there is a compelling need for new therapeutic strategies for such type of cancers. Recently, we demonstrated that specific combinations of epigenetic modifiers significantly affect the metabolism and proliferation rate of two most aggressive GBM cell lines D54 and U-87. Importantly, these combinations exhibited minimal effect on normal stem cells growth. In this study we demonstrated that the combinations of modulators of histone and DNA covalent modifying enzymes that synergistically suppress D54 and U87 cell lines growth, also impair the viability of a patient freshly-derived GBM stem cell line. These data suggest that epigenetic modifiers alone or in specific combinations exhibit cytotoxic effect on established and low passage patient derived GB cell lines and thus could be a promising therapeutic approach for such type of brain cancers. Glioblastomas (GBM) are an aggressive type of cancers that can occur in the brain and/or spinal cord, with a median survival of 12.6 months GBM , howeverOver decades, basic and preclinical cancer research has been based on the use of established, commercially available cell lines, originally derived from patients’ samples but adapted to grow indefinitely in specific culture conditions . The mosThe effort to find preclinical models able to better predict the clinical outcome led to the generation of patient-derived cancer models . CurrentIn our previous study we showed that specific combinations of histone methyltransferases inhibitors (BIX01294 and DZNep), histone deacetylases inhibitor (Trichostatin A) and DNA methyltransferase inhibitor in relatively small concentrations suppress the growth of D54 cells, but exhibited minimal effect on bone marrow derived mesenchymal stem cells (BM-MSCs) proliferation . Based oThus, all our recent studies showed that revealed combinations of epigenetic modifiers exhibited similar cytotoxic effect on two different GBM cell lines. These data led us to test whether these unique combinations will affect also a patient derived glioblastoma stem cell line. To this end, we tested the most effective combinations of chromatin modifying agents on patient derived cell line such as GBM965 developed by Dr.Quinones-Hinojosa group –19.™ Supplement without Vitamin A , 20ng/ml bFGF  and 1% Pen/Strep .Expansion of Patient-derived Glioma. GBM965 glioma cell line was derived, characterized, and cultured as previously described –19. For 2 culture flasks in α-MEM supplemented with 10% FBS, 1% Pen/Strep, and 1% GlutaMAX and incubated at 37°C in 5% CO2. The medium was changed every 4 days until the cells reached confluence.Expansion of MSCs. Fat tissue was obtained from the Medical College of Wisconsin’s Tissue Bank (patient remaining anonymous). For purification of MSCs from pre-adipocytes and other cell types, fat tissue was digested in a 1% Collagenase  solution for 1 h then centrifuged at 50 g for 1 min. The pellet containing AD-MSCs was washed three times in 1x PBS. These cells were then plated in 25-cmTreatments with chromatin modifying agents.2. After 4 h of attachment, cells were exposed to combinations of different concentrations of three epigenetic modifiers. The following concentrations were used for each modulator in low and medium doses: BIX01294–100nM and 1uM ; Trichostatin A (TSA) – 10nM, 50nM . The following concentrations were used for RG108  in low, medium, and high doses: 50uM, 100uM, and 500uM.Cells were seeded in 96 and 24-well plastic plates at a density of 15000/cmCell viability assay with MTT. Cells grown in 96-well-plates for 120 h were tested for viability with the MTT assay as described previously (35). Briefly, MTT tetrazolium salt (5 mg/mL)  was added to each well, and incubated for 4 h at 37°C. The formazan crystals resulting from mitochondrial enzymatic activity on the MTT substrate were solubilized with 12mM SDS . Absorbance was measured at 570 nm using a microplate reader . Cell survival was expressed as absorbance relative to that of untreated controls.Cell Count. For cell count, cells grown in 24 well plates for 120 h were collected and counted using trypan blue stain. Control and each treatment was carried out in triplicate.All data was expressed as mean ± SEM for the number (n-3) of independent experiments performed. Differences among the means for all experiments described were analyzed using one-way analysis of variance. Newman-Keul’s post hoc analysis was employed when differences were observed by analysis of variance testing (p < 0.05).In this study we aimed to investigate, whether the specific combinations of epigenetic modulators, that had demonstrated synergistic activity on U87 and D54 cells would exhibit a similar effect on patient-derived GB cell line (GBM965). To this end, different combinations of BIX01294, TSA and RG108 at  small (S), medium (M) and high (H) concentrations that exhibited the highest cytotoxic effect on U87 and D54 cell lines but showed minimal effect on normal stem cells were tested on GBM965 cells growth. We also tested small, medium and high concentrations of RG-108 individually, on GBM965 cells growth. For cell viability assays (metabolism and proliferation) the MTT test and trypan blue cell count approaches were used.Results showed that while individually the RG108 at the high concentration (500 uM) had significant effect on GBM965 cell viability it also displayed a significant effect on normal stem cells (such as AD-MSCs) proliferation and metabolic activity ,b. MediuSimilar to the previous studies on U87 and D54 cells , 12, theOne of the strategies to develop new therapeutics for GBs is the use of established GB cell lines such as U251, U87, U373MG, D54,T98G and others. However, recent several studies showed that the cell lines only partially reflect the gene expression profiles of tumors found in GB patients. In addition, with long term passaging these cell lines undergo spontaneous mutation which further change their gene expression and properties. Thus, freshly developed, or low passaged glioblastoma cell lines could be more clinically relevant cellular models for basic research and drug screening.Recently, we demonstrated that specific combinations of modulators of chromatin covalent modifying enzymes significantly affect the viability of two most aggressive glioblastoma cell lines such as D54 and U-87.With this study we aimed to test the effect of these specific combinations of epigenetic modifiers in patient derived glioblastoma cell line. Results demonstrated that combination of two histone covalent modifiers that significantly suppress D54 and U-87 cell lines viability exhibit also cytotoxic effect on patient derived glioblastoma cell line GBM965. These studies revealed that the combination of TSA(M) + BIX(M) displayed the highest cytotoxic effect on GBM965 while exhibiting the lowest effect on AD-MSCs metabolism and proliferation. These studies also showed that DNA methyl transferase inhibitor which individually at high, medium and low concentrations exhibit only minor effect on D54 and U87 cells viability, significantly suppress GBM965 cells growth. Importantly, low and medium concentrations of RG108 only slightly affect AD-MSCs growth. Adding RG108 to TSA + BIX affect AD-MSCs growth without significantly augmenting the cytotoxic effect of TSA + BIX on GBM965 cells.These data suggest that epigenetic modifiers alone or in specific combinations can exhibit high cytotoxicity against established and low passage patient derived glioblastomas while displaying minor effect on normal-stem cells growth and thus could be a promising therapeutic approach for such type of brain cancers.


Introduction: In an anterior cervical discectomy and fusion (ACDF), various types of graft materials including autograft, allograft, and synthetic graft have been used to achieve adequate spinal fusion. Allograft spacer is mainly used in cervical fusion, especially in the anterior approach. The synthetic bone graft material BGS-7 can bind with surrounding bone tissue by forming a hydroxyapatite layer bone bridge, leading to faster graft osseointegration. This study was conducted to compare long-term clinical outcome of BGS-7 spacer and allograft spacer for anterior cervical discectomy and fusion surgery.Materials and Methods: From September 2014 to December 2016, Consecutive anterior cervical discectomy and fusion surgeries using a BGS-7 spacer (N = 18) and Allograft spacer (N = 26) were compared for postoperative clinical outcomes. Radiologic assessments were performed, and Instrumental failure, including breakage, cage migration, subsidence were observed and Fusion status were analyzed. Finite element analysis was performed for simulating mechanical stress between the vertebral body and implant. Clinical outcomes were evaluated using neck VAS, NDI, and JOA on the patient’s final follow-up visits.Results: Among the 44 patients who underwent an anterior cervical discectomy and fusion surgery using the BGS-7 spacer and Allograft spacer, there were 30 men and 14 women. The average age at the operation was 47.69 ± 10.49 in allograft spacer and 51.67 ± 11.03 in BGS-7 spacer. The mean follow-up period was 89.18 ± 5.44 months. Twenty three (88.46%) patients in allograft spacer and 20(100%) patients in BGS-7 spacer were demonstrated radiologic evidence of interbody fusion in last OPD, which accounts for fusion grade 4 or 5. Peak stresses were 343.85 MPa in allograft spacer, and 132.55 MPa in BGS-7 spacer. Long-term clinical outcomes including neck VAS, NDI, and JOA didn’t show statistical differences between the two groups. There were no adverse events related to the BGS-7 spacer.10.3389/fbioe.2023.110046.Conclusion: The BGS-7 spacer demonstrated reliability as a spacer in anterior cervical discectomy and fusionF surgery without instrumental failure. Early stabilization with a bony bridge formation was observed at the intermediate follow-up period, and the long-term clinical outcome was favorable at more than 60 months after surgery without any adverse events. Thus, the BGS-7 spacer is a safe and effective alternative to the allograft spacer in anterior cervical discectomy and fusion surgery. In an anterior cervical discectomy and fusion (ACDF) surgery, Allograft cervical spacer is the second most used material after autogenous bone because of its many advantages, such as decreased operation time, low donor site morbidity, and stable bone union rate. . HoweverTitanium has shown high strength and biocompatibility, but it requires filler material and does not fuse with adjacent bone tissue. In addition, the subsidence rate is high, and the metal artifact on an MRI is profound. A polyether ether ketone (PEEK) cage is the most widely used spacer due to its reasonable strength, biocompatibility, and low subsidence rate; however, it also requires filler material and does not fuse with the bone. Hydroxyapatite (HA) has several advantages, including acceptable biocompatibility, osteoconductivity, a low subsidence rate, and fusion ability with bone. . But theIn vivo model, the dense cylindrical shaped specimen demonstrated better bone bonding to adjacent bones compared with hydroxyapatite  and allograft spacer (CERVICAL SPACER C+), including finite element analysis for biomechanical properties of each implant.), and the patients provided written informed consent.From September 2014 to December 2016, Consecutive ACDF surgeries (N = 44) using a BGS-7 spacer  or allograft spacer  were performed in a single medical center by a single expert surgeon. We compared fusion rate and clinical outcomes between the two groups of patients at more than 60 months after surgery. The disease entity was a degenerative cervical disease from the C3/4 to C6/7 levels, including cervical disc herniation and foraminal stenosis. The use of patient data for research purposes was approved by the institutional review boards (IRB No. 3-2021-0437The inclusion criteria were 1) adult men and women over the age of 19, 2) patients with bioactive ceramic spacer or allobone for cervical anterior intervertebral disc removal in single or multiple segments fusion from September 2014 to December 2016, 3) a patient who voluntarily signed a written consent form. The exclusion criteria included 1) patients who voluntarily did not agree to participate in the study, 2) Patients with other factors influencing clinical trial product effectiveness assessment.The surgical techniques followed a standard ACDF surgery procedure, which includes a plate augmented interbody fusion, using a BGS-7 spacer or allograft. Linear anterior incisions were made on the opposite side of the main neural compression. For example, if the patient had a left-side protrusion at C5/6, the incision was made on the right side. The Smith-Robinson anterior cervical approach was employed to expose the anterior surface of the cervical disc. The disc material was removed with a No 15. blade and forceps. Meticulous curettage was performed to remove the cartilage portion of the upper and lower endplate, leaving the bony structure intact. The uncinate process was removed with a high-speed drill to obtain a wide decompression of the foraminal space, if needed. The posterior longitudinal ligament was resected only when it was needed for decompression, such as for the removal of a ruptured disc material that exited through the ligament. After confirmation of a complete neural decompression, a proper sized BGS-7 spacer or allograft was impacted into the empty disc space. Plate augmentation was applied for all patient’s entire surgical levels.Radiologic assessment was performed using a cervical spine X-ray series at the preoperative, immediate postoperative, and long term follow-up time points. A breakage of the cage was defined as the presence of a crack within the inserted cage or a destructive change to the cage’s shape on the postoperative cervical spine X-ray. Subsidence was defined as a decrease in interbody height of more than 3 mm on the follow-up plain film. The difference of subsidence was based on the length between the center of the upper and lower endplate in a lateral cervical spine X-ray. Fusion status was evaluated by the Brantigan and Steffee fusion grade using an X-ray at the final follow-up visit  Brantig, and alsThe mechanical performance of the BGS-7 spacer was confirmed using finite element analysis. The comparative model is the allograft spacer. Implant models and a three-dimensional cervical spine vertebral body model were constructed. The region of interest between implants and bone-implant interface was set up using different element sizes that could be distinguished from other parts. The Young’s modulus and Poisson’s ratio were applied to each moiety using previously published data in the literature.  KumaresTwo types of surgical models were constructed by placing each implant between the vertebral bodies using Abaqus software.  Sliding contact was applied as the boundary condition between the vertebral body and the implant. The inferior endplate of the most caudal vertebra (C6) was fixed in all degrees of freedom, while 1,200N loads were applied to the superior endplate of the most cephalic vertebral level (C6). . The peaStress and contact pressure of the vertebral body were used to determine the risk of subsidence, and the risk of implant yield was confirmed using the stress of the implant.t-test or paired t-test. For the comparison of categorical variables, chi-square tests and Fisher’s exact tests were performed between two independent groups. All p-values less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 26.0  by a statistical professional.Descriptive data are represented as mean ± standard error of the means, and statistically compared using two-sample Among the patients who underwent an ACDF surgery using the BGS-7 spacer and Allograft spacer, there were 30 men and 14 women. . The aveBGS-7 spacer showed no adverse findings at 60 months after surgery when compared with allograft spacer . AverageThe result of fusion according to Brantigan and Steffee grading is compared allograft group and BGS-7 group at The sagittal and coronal reconstruction of CT 1 year postoperatively revealed a detailed image of a bony bridge formation, which surrounded the spacer . An examp = 0.706). And the VAS at the immediate postoperative status and long-term postoperative status for BGS-7 spacer and allograft spacer were 1.8 ± 2.0 and 2.1 ± 2.1 (p = 0.662), and 2.6 ± 2.5 and 3.4 ± 2.9 (p = 0.365), respectively.The result of clinical outcomes for the BGS-7 group and the allograft group are shown in p = 0.064). And the NDI at the immediate postoperative status and long-term postoperative status for BGS-7 spacer and allograft spacer were 5.6 ± 4.4 and 7.3 ± 5.9 (p = 0.309), and 6.9 ± 7.1 and 9.3 ± 7.1 (p = 0.293), respectively.Neck disability index (NDI) showed 12.3 ± 6.8 and 16.7 ± 7.50 at pre-operative status for BGS-7 spacer and allograft spacer, respectively (p = 0.853). And the NDI at the immediate postoperative status and long-term postoperative status for BGS-7 spacer and allograft spacer were 15.8 ± 1.4 and 16.5 ± 0.6 (p = 0.037*), and 15.4 ± 2.1 and 16.2 ± 1.6 (p = 0.157), respectively.Japanese orthopedic association (JOA) score showed 15.4 ± 1.5 and 15.5 ± 2.2 at pre-operative status for BGS-7 spacer and allograft spacer, respectively  and contact pressure of the vertebral body/implant were analyzed by finite analysis . The peaBioactive ceramics include calcium phosphate compounds  and bioaIn the present study, the BGS-7 spacer improved its durability without permitting any breakage of in the spacer. Neither breakage nor migration was found in this study. And also the follow-up period at least 60 months can conclusively prove the durability of the BGS-7 spacer, it is impressive to show no instrumental failure rate considering that cage breakage is relatively common problem in allograft spacer. Because all patient enrolled in this study had a plate augmentation, basic stability of the spacer can be further evaluated in the future study.In the BGS-7 group, the average subsidence was 1.33 mm and the subsidence rate was 0%, while the average subsidence in the allograft spacer group was 2.27 mm and the subsidence rate was 26.6%, which was statistically significantly higher than BGS-7 spacer group. The subsidence rate in ACDF surgery with plate augmentation is reported as 9.6–55.6%. . In a meThe long-term results of interbody fusion appeared to be acceptable. Eighteen of 18 patients who were followed for more than 60 months had enough fusion results with grades 4,5; and there were no patients with fusion grade 2, 3, rather few patients in allograft group showed grade 2, 3 fusion status. According to previous studies, the fusion rate seems to differ depending on the features of the space. . The usePseudarthrosis is known to be associated with unfavorable outcome, including arm pain and axial pain. . TherefoAt first, a limited number of enrolled patients are weak point of this study. More accumulated data are required although this study revealed the long-term clinical result of ACDF surgery with BGS-7 interbody fusion spacer. And stand-alone cage insertion without a plate augmentation might be better surgical technique to reveal the direct effect of spacer and maximize the difference from previously commercialized cervical spacer. Because this study was the first long-term clinical trial using BGS-7 glass ceramic spacer, a plate augmentation had to be applied for the safety of enrolled patients.BGS-7 spacer demonstrated reliability as a spacer used in ACDF surgery without instrumental failure. Although the high mechanical strength of the spacer, no subsidence was observed at over 60 months of the follow-up period, while significant subsidence was noted in 22.2% of the patients in the allograft group. Moreover, the early stabilization with a bony bridge formation was observed at the intermediate follow-up period, and the clinical outcomes were favorable consequently in the BGS-7 group more than 60 months after surgery without any adverse events. Therefore, the results suggested that theBGS-7 spacer is a safe and effective alternative to allograft for ACDF surgery.