Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 –substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties. Protein disaggregases engage and dissolve proteins that aggregate and form amyloid. Protein aggregation is implicated in a range of diseases, and so it is important to understand the structure and mechanism of disaggregases. Hsp104, a member of the Hsp100 family, is a hexameric AAA+ protein disaggregase from yeast that is highly conserved in bacteria and eukaryotes, though it is absent in metazoa. Hsp104 assembles into a ring structure with a central channel through which substrate is threaded. ATP hydProtein misfolding underpins numerous disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and diabetes, 11. BecC. elegans model of PD[To enhance Hsp104 activity, efforts have been made to tune Hsp104 to boost its activity and modulate its substrate-specificity–23. Thesdel of PD. A majordel of PD, 19, 20.H641A showed a loss of ~50% activity relative to ClpBWT[In order to apply rational protein design principles to further engineer Hsp104, it is important to comprehensively understand its structure and mechanism. Recently, a series of near-atomic level resolution cryo-electron microscopy (cryo-EM) structures of Hsp104 were reported. These sto ClpBWT. Howeverto ClpBWT. It is tHere, to investigate the functional role of this putative substrate binding region, we employed a series of yeast-based assays. Using a combination of thermotolerance, temperature sensitivity, and disease-substrate toxicity assays, we find that removal of the tyrosine 650 side chain from this pore loop does not ablate Hsp104 function as occurs when the canonical pore loop residues are mutated. However, the Y650A mutation does subtly modulate the function of a potentiated Hsp104 variant. Our results suggest that modulation of this site may prove useful for tuning Hsp104 activity.Fig 1)[Fig 1)[Substrate binding by Hsp104 is mediated primarily by Y257 and Y662, located in nucleotide binding domain 1 (NBD1) and NBD2, respectively. These 12 residues project into the central channel of Hsp104 to bind substrate and mediate its translocation via a ratchet-like mechanism , 28. An Y650A would display impaired thermotolerance. We constructed Hsp104Y650A, Hsp104Y662A, and Hsp104Y257A-Y662A in W303Δhsp104 yeast with Hsp104 expression under the control of the heat-shock element (HSE), which is present in the promoter region of Hsp104. This promoter allows low basal expression of Hsp104 that is significantly enhanced following stress. Following a brief pre-treatment at 37°C to induce the heat shock response and enhance Hsp104 expression, we exposed the cells to a severe heat shock at 50°C for 0 to 30 minutes. Following outgrowth, we noted that the strain expressing Hsp104Y650A displayed thermotolerance nearly identical to that of Hsp104WT. Strains expressing Hsp104Y662A and Hsp104Y257A-Y662A did not confer thermotolerance, and displayed growth similar to the vector strain, which does not express Hsp104 . We confirmed expression of each variant via immunoblotting, and observed that the expression pattern was consistent with Hsp104 expression being driven by the HSE promoter . Our findings demonstrate that residue Y650 plays little role in conferring thermotolerance, suggesting that if the Y650 residues contact substrate, this interaction is not essential for maintaining substrate remodeling activity.Upon exposure to environmental stress, Hsp104 expression is upregulated to broadly counter protein aggregation and promote cell survival, thereby conferring thermotolerance, 7. FollY257F-A503V-Y662F, in which the Y257F and Y662F pore loop mutations are introduced into the potentiated Hsp104A503V background, still rescues the toxicity of TDP-43, FUS, and α-syn[A503V. Hsp104Y257F-A503V-Y662F confers thermotolerance at levels similar to Hsp104WT, while Hsp104A503V is impaired in thermotolerance. Additionally, Hsp104Y257F-A503V-Y662F confers diminished temperature sensitivity relative to Hsp104A503V. Enhanced thermotolerance and diminished temperature sensitivity in yeast likely reflects decreased off-target effects and suggests that modulation of the Hsp104 pore loops is a viable way to tune disaggregase properties[Potentiated Hsp104 variants confer a therapeutic gain of function and robustly rescue the toxicity of TDP-43 and FUS (implicated in ALS) as well as α-synuclein (implicated in PD), 29. It and α-syn. Howeverroperties.A503V background: Hsp104A503V-Y650A, Hsp104A503V-Y662A, and Hsp104Y257A-A503V-Y662A. We chose to introduce alanine rather than phenylalanine mutations, anticipating that this strategy would more robustly demonstrate if these residues played a role in substrate binding. While a Y650F mutation would likely maintain a rescue regardless of the role of Y650 in substrate binding, ablating Y650 via mutation to alanine should impair the capacity of Hsp104A503V to confer a rescue if Y650 binds substrate. We employed the 416GAL vector to drive galactose-inducible expression of the genes, and we co-expressed these variants with TDP-43, FUS, and α-synuclein. Potentiated Hsp104A503V robustly rescued toxicity of all three substrates, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A did not rescue the toxicity of any of these substrates. Hsp104A503V-Y650A still strongly rescued toxicity, indicating that the variant retained substrate binding, and supporting our results in the thermotolerance assays. However, we did note a modest decrease in rescue of TDP-43 toxicity when compared to Hsp104A503V. Here the rescue decreases from a score of 3 for Hsp104A503V to a score of 2 for Hsp104A503V-Y650A . However, Hsp104A503V-Y650A shows only a subtle decrease in rescue of FUS and α-synuclein toxicity as compared to Hsp104A503V, and the average score for this variant is equal to that of Hsp104A503V against FUS and α-synuclein . We confirmed consistent expression of each of the Hsp104 variants and disease substrates using immunoblotting . Our findings suggest that while the Y650 residue is not crucial for mediating substrate remodeling, modulation at this position could be a useful way to tune the properties of potentiated Hsp104 variants.We therefore designed experiments to both corroborate our findings that Y650 does not drive substrate remodeling, and also test if modulation of Y650 might be a way to tune Hsp104 substrate-specificity. We constructed a series of variants in the potentiated Hsp104A503V background subtly diminishes the toxicity rescue conferred by Hsp104A503V, we decided to perform a final functional test to assess the temperature sensitivity of these variants. While Hsp104A503V confers thermotolerance nearly similar to Hsp104WT  [A503V also confers a temperature sensitive phenotype at 37°C, displaying impaired growth when Hsp104A503V is expressed during a prolonged heat stress treatment. This temperature sensitive phenotype is diminished for Hsp104Y257F-A503V-Y662F [A503V, as do the verified substrate binding residues Y257 and Y662. However, we found that Hsp104A503V-Y650A displayed a similar temperature sensitive phenotype to Hsp104A503V, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A displayed diminished temperature sensitivity . These results support the idea that Y650 does not play a crucial role in substrate remodeling in contrast to the essential roles played by residues Y257 and Y662.Because the introduction of Y650A into the potentiated Hsp104 , 30, Hsp3V-Y662F , 18. We Y662A and Hsp104V663G are defective in thermotolerance[A503V background to generate Hsp104A503V-E648A and Hsp104A503V-K649A. We tested these variants for suppression of TDP-43, FUS, and α-Syn toxicity and found that, like with Hsp104A503V-Y650A, ablating the side chains of E648 and K649 only subtly modulates the potentiation of Hsp104A503V . Our findings suggest that while modulation of this pore loop region might be a useful way to tune the properties of Hsp104A503V, this pore loop region does not play a major role in Hsp104 –substrate remodeling.It has been demonstrated that residues neighboring key substrate-binding residues Y257 and Y662 play an important role in Hsp104 substrate binding. For instolerance. To asseY650A is fully functional in thermotolerance, indicating that Hsp104 can still remodel substrate without the Y650 side chain. To investigate the role of this site further, we tested Hsp104A503V-Y650A in toxicity suppression assays. We find that the Y650A mutation does not abolish activity, supporting our findings in the thermotolerance assays, but that it does subtly decrease the rescue of TDP-43, FUS, and α-syn toxicity by potentiated Hsp104A503V. Our findings are in contrast to recent work with the bacterial homolog of Hsp104, ClpB. ClpBH631A, in which this third pore loop is ablated, shows impaired activity in a luciferase reactivation assay[Amyloid and aggregate dissolution by Hsp104 is driven by substrate binding and translocation through its central pore. Binding is mediated via residues Y257 and Y662 leading to translocation via a ratchet-like mechanism[ion assay. These dhsp104 [All yeast were WT W303aΔ, ade2-1). Yeast w, ade2-1). pRS416G600nm = 1.5, serially diluted, and spotted in duplicate onto synthetic dropout media containing glucose or galactose. Plates were analyzed after growth for 2–3 days at 30ºC. Each experiment was repeated with three independent transformations.Yeast were transformed according to standard protocols using polyethylene glycol and lithium acetate. Hsp104 hsp104 yeast were transformed with the indicated pHSE-Hsp104 plasmid. Yeast were grown to saturation overnight at 30°C in glucose dropout media. Cultures were normalized to A600nm = 0.3 and grown in glucose dropout media for an additional 4h. Cultures were then normalized to A600nm = 0.6 and incubated at 37°C for 30 min. Cultures were then heat shocked at 50ºC for 0-30min and then cooled for 2min on ice. Cultures were serially diluted and spotted on glucose dropout media, and the plates were incubated at 30ºC for 2–3 days. Experiments were repeated with three independent transformations.W303Δhsp104 yeast were transformed with the indicated pRS416GAL-Hsp104 plasmid. Yeast were diluted and grown in synthetic raffinose medium overnight. Cultures were grown in synthetic raffinose medium to A600nm = 2.0 and spotted onto SD-Ura or SGal-Ura media and incubated at 30ºC or 37°C. Plates were analyzed after 48-72h of growth. Experiments were repeated with three independent transformations.W303Δ600nm = 0.6, 3ml cells were harvested, treated in 0.1M NaOH for 5 min at room temperature, and cell pellets were then resuspended into 1x SDS sample buffer and boiled for 4min. Lysates were cleared by centrifugation at 14,000 rpm for 2min and then separated by SDS-PAGE , and transferred to a PVDF membrane. Membranes were blocked in Odyssey Blocking Buffer (LI-COR). Primary antibody incubations were performed at 4ºC overnight. Antibodies used: anti-GFP monoclonal (Roche Applied Science), anti-TDP-43 polyclonal (Proteintech), anti-FUS polyclonal (Bethyl Laboratories), anti-Hsp104 polyclonal (Enzo Life Sciences), and anti-PGK monoclonal (Invitrogen). Membranes were imaged using a LI-COR Odyssey FC Imaging system.Yeast were grown and induced in galactose containing medium for 5h from overnight cultures supplemented with raffinose. For plasmids under the control of the HSE promoter, cells were grown as for the thermotolerance assay and harvested after the incubation at 37ºC. Cultures were normalized to AS1 Supporting Information(DOCX)Click here for additional data file.


Here, we have investigated the differential expression of nAChR subunits in human breast cancer cell lines and breast epithelial cell lines at mRNA and protein levels and the effects of the αO-conotoxin GeXIVA, antagonist of α9α10 nAChR, on human breast cancer cells. Reverse transcription polymerase chain reaction (PCR) demonstrated that all nAChR subunits, except α6, were expressed in the 20 tested cell lines. Real time quantitative PCR (QRT-PCR) suggested that the mRNA of α5, α7, α9 and β4 nAChR subunits were overexpressed in all the breast cancer cell lines compared with the normal epithelial cell line HS578BST. α9 nAChR was highly expressed in almost all the breast cancer cell lines in comparison to normal cells. The different expression is prominent ( Cancer is one of the three principal diseases which lead to human mortality and was responsible for 9.6 million deaths worldwide in 2018 . Nicotinic acetylcholine receptors (nAChRs), the ligand-activated neurotransmitter receptors in mammals, are divided into muscle and neural nAChRs. The nAChRs are either homomeric or heteromeric pentamers and are comprised of α and non- subunits ,5,6,7,8.Conus generalis, which potently blocks α9α10 nAChR [As competitive antagonists of nAChRs, α-conotoxins (α-Ctxs), have been particularly useful in developing ligands that selectively target one nAChR subtype over another. Some of them have therapeutic potential ,23,24,2510 nAChR . In vivo10 nAChR ,28. Rece10 nAChR . αO-conoThe present study aimed to detect the differential expression of nAChRs in human breast cancer and human normal breast epithelial cells and the nAChR subunits that may be potential targets for breast cancer, to further evaluates the effects of αO-conotoxin GeXIVA on cellular proliferation.Previous studies demonstrated that nAChRs as ligand-gated ion channels were regulated by binding with acetylcholine (ACh) or nicotinic agonists, such as nicotine and nicotine-containing products . In addiThe LBD cDNAs for α3, α4, α5, α7, α9, α10, β2, β3 and β4 neuronal-type nAChR subunits were amplified with expected size respectively in all the 17 breast cancer cell lines and the three human normal mammary gland epithelial cell lines . It meanAs described above, all the tested cell lines were found to express neuronal nAChR subunits of α3, α4, α5, α7, α9, α10, β2, β3 and β4. Moreover, previous studies showed that nAChRs play a crucial role in the course of human cancer development ,12,32. IFor α5 nAChR subunit C, the hiCompared with normal cell line of HS578BST, α7 nAChR subunit had the highest expression level of mRNA in HCC1806 cancer cell line with ~600-fold higher than in the normal cell line D. The seFor α9 nAChR subunit E, the hiThe most significant upregulation for β4 nAChRs mRNA level was observed in the breast cancer cell line AU565, which had ~1500-fold more than the normal cell line HS578BST. There were three breast cancer cell lines, SK-BR-3, HCC1806 and MDA-MB-231 with 120~300-fold of β4 subunit mRNA higher than that of the HS578BST. Six breast cancer cell lines, MDA-MB-361, Bcap37, HCC1395, ZR-75-30, MCF-7 and HCC1937 showed 25~60-fold of β4 mRNA higher than that of the HS578BST. There was 2~10-fold increase in the amount of β4 nAChRs mRNA in MDA-MB-453, BT549, HS578T, BT483, BT20 and MDA-MB-157 cell lines compared to normal cell line HS578BST I.Compared with human normal mammary gland epithelial cell line HS578BST, there were 14 breast cancer cell lines expressing 2~20-fold higher levels of α3 nAChR subunit, including MDA-MB-453, BT549, HS578T, Bcap-37, HCC1395, BT483, SK-BR-3, BT20, ZR-75-30, HCC1806, AU565, HCC1937, MDA-MB-231 and MDA-MB-157. MDA-MB-361 and MCF-7 did not show significant changes in α3 nAChR expression from cancer cells compared to normal epithelial cells A. For α4 nAChR subunit B, the hiThe mRNA expression level of α10 nAChR subunit in five breast cancer cell lines was similar with the normal cell line respectively, which included MDA-MB-453, BT549, SK-BR-3, ZR-75-30 and HCC1937 F. The otCompared to normal cell line HS578BST, there were nine breast cancer cell lines including MDA-MB-453, Bcap37, HCC1395, BT483, BT20, ZR-75-30, HCC1806, MDA-MB-231 and MDA-MB-157 with expression of β2 nAChR subunit mRNA 4~25-fold higher than HS578BST G. There For β3 nAChR subunit mRNA expression H, there It is shown from quantitative PCR results that only four nAChR subunits that is, α5, α7, α9 and β4 nAChRs are highly expressed in all tested human breast cancer cell lines compared to normal mammary gland epithelial fibrocystic cells .p < 0.001) except MDA-MB-453 and HCC1395 cell lines ) and analyzed by flow cytometry (FCM). The specificity of these antibodies was confirmed by Western blotting in some cell lines, such as MCF-7, MAD-MB-157, MDA-MB-231, BT20, SK-BR-3, BT483, HCC1395, MDA-MB-157 α9 KO, HEK-293T, Hela, A549 . Take MDll lines .p < 0.01). There were three cancer cell lines, BT483, MDA-MB-231 and MDA-MB-157 cell lines that were statistically significant p < 0.05. In our study, the mRNA and protein expression differences for the α9 nAChR subunit in different human cancer cell lines have shown in the above results. Thus, further research is warranted to determine the expression differences of α9-containing nAChR protein in these cell lines by Western blot . Using aPrevious experiments have established that the over-expression of α9-nAChR at mRNA and protein level in almost all breast cancer compared to normal epithelial cells HS578BST  and αO-c50 values ranging from 35 to 130 μM. However, TxID had no effect on breast cancer cell line BT20. Co-treatment of BT20 cells with GeXIVA and TxID resulted in no significant difference of these cells to GeXIVA. Compared with normal cells treated with the same drug at the same concentration, the inhibition of cancer cells at any concentration of GeXIVA used was significantly higher than that of normal cells. The effects of different treatments on cells proliferation in breast cancer cells were determined by CCK-8 assay. In breast cancer cells, GeXIVA significantly inhibited the proliferation of cancers . AnalysiRecent gene expression profiling studies have categorized breast cancer into four groups including luminal subtypes A and B, HER2+/ER-, Basal-like ,33  belonged to HER2 over-expression subtype. Three cell lines  belonged to Luminal A subtype. Two cell lines (BT-474 and MDA-MB-361) fell within subtype of Luminal B. Twelve cell lines  fell within the Basal-like subtype. We designed the ten neuronal nAChR subunits gene specific primers and demonstrated for the first time that normal human mammary glands epithelial cells and mammary cancer cells express the α3, α4, α5, α6, α7, α9, α10, β2, β3, β4 neuronal nAChR subunits and examined the expression level of each subunit. All the existing neuronal nAChR subunits mRNA were analyzed by RT-PCR, which presented in almost all the cell lines except for α6 nAChR subunit . But, th2+ permeability [2+ influx into cancer cells and trigger membrane depolarization, which activates voltage-gated Ca2+ channels and subsequently activates the MAPK pathway, which may result in inhibition of apoptosis [p < 0.001) except MDA-MB-453 and HCC1395 cell lines  according to the manufacturer’s protocol. The concentration and purity of RNA were determined by NanoDrop 2000 spectrophotometry . Equal amounts of RNA (1 μg) from each sample were reverse-transcribed into first-strand cDNA with High Capacity cDNA Reverse Transcription Kit , then the first-strand cDNA subsequently amplified by RT-PCR using specific primers. Based on the reported mRNA sequence of ligand binding domain of human (homo sapiens) nAChR subunits, the specific primers for each subunit were designed , which w2O with total volume of 20 µL. The cycling conditions were 95 °C for 10 min  followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 40 s and extension at 60 °C for 60 s. To correct minor variations in mRNA extraction and reverse transcription, the gene expression values were normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative quantification of the mRNA level was computed by the comparative Ct (2−∆∆Ct) method [Quantitative real-time PCR (qRT-PCR) was performed using Analytikjena qTOWER3G Real-Time PCR system to amplify various nAChR subunits in each cell line, including α3, α4, α5, α7, α9, α10, β2, β3 and β4 nAChR subunits. Each qPCR reaction mix contained 5 µL of SYBR Green I Master , 2 µM of each primer, 1 µL cDNA and DNase and RNase-free HThe cultured cells were washed with cold phosphate buffer saline (PBS) for three times and harvested using a cell lysis buffer containing protease inhibitors PMSF . Equal amounts of protein from control and treated cell lysates were separated using a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducing conditions and transferred onto polyvinylidene fluoride (PVDF) membranes  that were subsequently probed with primary antibodies . In all Western blot experiments, membranes were additionally probed with an antibody for GAPDH  to ensure equal loading of protein among samples. Horseradish peroxidase-conjugated secondary antibodies  were used with enhanced chemoluminescence reagent  to visualize the protein bands. Images of the films are captured using the Alpha FluorChem E .A guava easyCyteTM flow cytometer with InCyte software  was used to validate the presence of the nAChRs. The cultured cells were harvested and washed with PBS for twice, fixed the cells with 4% PFA for 20 min, washed the cells twice by centrifugation at 1000 rpm for 5 min each time. Suspend the cells in 0.1% Triton X-100 in 1 × PBS buffer for 10 min and washed the cells twice with PBS by centrifugation at 1300 rpm for 5 min each time. The cells were blocked with 3% BSA in 1 × PBS for 30 min and then the cells were washed twice with PBS by centrifugation at 1300 rpm. Add primary antibody at an appropriate dilution and incubate for 2 h at room temperature, washed and incubated with secondary antibody (FITC-conjugated Goat anti-rabbit Ig (G + L)) for 1 h at room temperature. Finally, the cells were washed twice and re-suspended in 300 μL 1 × PBS and the expression of nAChRs was analyzed by flow cytometry. The changes of mean fluorescence intensities of each antibody were calculated and it represent the protein expression level of nAChRs in each cell line. The primary antibodies used were as follows—CHRNA3 Rabbit Polychonal antibody , anti-Nicotinic Acetylcholine Receptor α4 antibody , anti-CHRNA5 antibody , CHRNA6 Rabbit Polychonal antibody , anti-CHRNA7 antibody , CHRNA9 Rabbit Polychonal antibody , anti-CHRNA10 antibody , anti-nicotinic acetylcholine receptor β2 antibody , anti-CHRNB3 antibody  and CHRNB4 antibody . H-tetrazolium, monosodium salt (WST-8) assay kit . Exponentially growth cells were seeded onto 96-well plates (1 × 10 4 cells per well) and allowed to grow overnight. The cells were treated with five different concentration of αO-conotoxin GeXIVA . After 24 h, WST-8 was added into each well for 4 h before the measurement according to the manufacturer’s instruction. The absorbance 450 nm was measured by a microplate reader .The effect of αO-conotoxin GeXIVA on all tested breast cancer cell lines and normal cells HS578BST proliferation were examined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2t-test was used and a p-value < 0.05 was considered significant. Analysis was performed with the SPSS software package (Version 17.0).Three to five independent repeats were conducted for all experiments. Error bars represent these repeats. Statistical comparisons between groups were performed using ANOVA (Prism GraphPad Software and SPSS 17.0). A Student’s It is well known that the nAChRs are involved in processes of oncogenesis and inflammation. All the compounds mentioned above provided molecular evidence for the possible chemotherapeutic ability of them for smoking-mediated tumorigenesis through nAChRs pathway. Specifically, α-conotoxins as a common tool in the studies of nAChRs, which blocked various subtypes of nAChRs selectively and potently. In this article, expression of neuronal nAChR subunits in each cell line was delineated through different methods, which suggested α-conotoxins targeting various nAChR subtypes may display potent and additional therapeutic effects on related cancers. This study, the high expression of α9-nAChR in cancer cells was correlated with the pathogenesis. Our study has confirmed αO-conotoxin GeXIVA significantly inhibit growth of cancer cells compared with normal cells HS578BST in vitro. Due to its high potency, GeXIVA may possess great potential as anticancer agent for α9α10 nAChRs overexpressed breast cancer cell lines.


In recent years, lots of studies have reported that m6A modification and m6A RNA methylation regulators were involved in cancer progression. However, the m6A level and its regulators in esophageal cancer (ESCA) remain poorly understood. In this study, we analyzed the expression of m6A regulators using The Cancer Genome Atlas data and found 14 of 19 m6A regulators are significantly increased in ESCA samples. Then we performed a univariate Cox regression analysis and LASSO (least absolute shrinkage and selection operator) Cox regression model to investigate the prognostic role of m6A regulators in ESCA, and the results indicated that a two-gene prognostic signature including ALKBH5 and HNRNPA2B1 could predict overall survival of ESCA patients. Moreover, HNRNPA2B1 is higher expressed in high-risk scores subtype of ESCA, indicating that HNRNPA2B1 may be involved in ESCA development. Subsequently, we confirmed that the level of m6A and HNRNPA2B1 was significantly increased in ESCA. We also found that HNRNPA2B1 expression positively correlated with tumor diameter and lymphatic metastasis of ESCA. Moreover, functional study showed that knockdown of HNRNPA2B1 inhibited the proliferation, migration, and invasion of ESCA. Mechanistically, we found that knockdown of HNRNPA2B1 inhibited the expression of de novo fatty acid synthetic enzymes, ACLY and ACC1, and subsequently suppressed cellular lipid accumulation. In conclusion, our study provides critical clues to understand the role of m6A and its regulators in ESCA. Moreover, HNRNPA2B1 functions as an oncogenic factor in promoting ESCA progression via up-regulation of fatty acid synthesis enzymes ACLY and ACC1, and it may be a promising prognostic biomarker and therapeutic target for human ESCA.N6-methyladenosine (m Esophageal cancer (ESCA) is one of the major malignant cancers that threatened human health worldwide , 2. Esop6A) modification is the most abundant modification involved in the progression of different cancers  or removed by m6A demethylases (erasers). In addition, the specific RNA-binding proteins (readers) could recognize and bind to m6A motif, regulating RNA metabolism, including RNA stability, degradation, splicing, transport, localization, translation, and others  dataset, as well as their association with the clinicopathological characteristics. After a comprehensive analysis, we found that HNRNPA2B1 may play a key role in ESCA development. Subsequently, we found that the levels of m6A and its regulator HNRNPA2B1 were significantly increased in ESCA, and HNRNPA2B1 acts an oncogenic role in the progression of ESCA cells, indicating that it may be a promising prognostic biomarker and therapeutic target for human ESCA.In the present study, we systematically analyzed the expression of 19 m6A-related genes in gastric cancer . All 19-gene expression data are downloaded via the R package “TCGA-Assembler.” The expression of 19 m6A-related genes in 160 ESCA tissues and 11 normal esophageal tissues was analyzed via limma package. Next, the violin map was used to visualize the expression of 19 genes in 160 ESCA tissues and 11 normal tissues. We then used the STRING database (http://string-db.org) to analyze the protein–protein interaction (PPI) among 19 m6A regulators. The correlation analysis of 19 m6A regulators was further analyzed by Pearson correlation analysis. To evaluate the association between the expression of m6A regulators and prognosis of ESCA patients, the ESCA cohort was clustered into different groups through consensus clustering analysis with “ConsensusClusterPlus” in R  Cox regression algorithm. The formula of the risk score for ESCA patients' prognosis prediction was as follows: risk score = the sum of each multivariate cox regression coefficient ratio of mRNA multiple each expression of mRNA. Based on the median risk score, we divided the patients into high- and low-risk subgroups. Each patient's survival status, death time, and gene expression profile in two subgroups were presented via “heatmap” and “survival” R packages , were obtained after signed informed consent. The ESCC tissue microarray  was obtained from servicebio . Immunohistochemistry (IHC) was performed according to standard procedures as described previously , 23. InsThe two specific HNRNPA2B1 siRNAs were designed and synthesized by RiboBio : the sequence of si-HNRNPA2B1#1: GGAGAGTAGTTGAGCCAAA and the sequence of si-HNRNPA2B1#2: GCTACGGAGGTGGTTATGA. The HNRNPA2B1 siRNAs and corresponding control siRNA were transfected into the ESCC cells by DharmaFECT4 .https://en.bio-protocol.org/e2095). The experiments procedures have been described previously . Reverse transcription (RT) was performed with HiScript Q RT SuperMix for qPCR . RT–polymerase chain reaction (PCR) was performed with an SYBR Green PCR Kit  on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems), with triplicate reactions. The primers used are listed in For CCK8 assay, after the cells were transfected for 48 h, the cells were plated at a density of 2,000 cells per well in 96-well-plates. After 72 h, cell viability was determined using CCK-8 assay according to the manufacturer's instructions .For clonogenic assay, after the cells were transfected for 48 h, the cells were seeded at a density of 500 cells per well in 12-well-plates and incubated at 37°C for 10–14 days. Then, cells were fixed with methanol for 30 min and stained with crystal violet  for 30 min.For EdU assay, the cells were plated at a density of 10,000 cells per well in 96-well-plates, and the cells were transfected for 48 h. The cell immunofluorescence was determined by the EdU kit according to the manufacturer's instructions . Images of the cells were acquired with a Leica DMi8 system.For wound-healing assay, the transfected cells were grown to confluence in a 12-well-plate. Next, the cells were cultured in RPMI-1640 with 1% FBS for 12 h. The confluent monolayer was then disrupted with a cell scraper and filmed at the indicated hours via Leica DMi8 system. The rate of wound closure was calculated as the ratio of the average distance between the two wound edges and the total cell duration of migration.For Transwell assay, the transfected cells in 100 μL of serum-free medium were seeded in the upper chambers coated with or without 50 μL of Matrigel (BD Biosciences), and 600 μL of culture medium containing 10% FBS was placed in the lower chambers. After 12 h of incubation at 37°C, cells that migrated to the bottom of the membrane were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet  for 30 min, and imaged.The transfected cells were fixed in 4% paraformaldehyde solution on the 12-well-plates, stained with 0.05 μg/mL Nile red  for 10 min, washed with phosphate-buffered saline twice, and then stained with DAPI . Images of the cells were acquired with a Leica DMi8 system.6A-related genes in ESCA tissues and normal tissues from TCGA dataset was analyzed via one-way analysis of variance. Kolmogorov–Smirnov test was used to analyze the relationship between m6A-related genes and clinicopathological characteristics. The median risk score was used as a cutoff value to divide into a high- and a low-risk group. OS was analyzed between different groups through the Kaplan–Meier method. The relationship between the risk score and clinicopathological variables was analyzed through χ2-test. Functional experiments were performed at least three times. The representative data shown are means ± SD; P < 0.05 was considered statistically significant.The expression of 19 m6A RNA methylation regulators  in ESCA tissues and normal tissues from TCGA dataset  (P = 0.064)  . Moreove= 0.064) .6A regulators in the prognosis of ESCA patients, univariate Cox regression was used to analyze the expression of m6A-related genes associated with OS in ESCA TCGA dataset. The results demonstrated that high expression of ALKBH5 was significantly correlated with good OS , but high expression of HNRNPA2B1 was associated with poor OS  (http://gepia.cancer-pku.cn/). The results showed that the expression of HNRNPA2B1 was significantly positively correlated with the expressions of ALKBH5 (P = 0.05501)  than individual ALKBH5 or HNRNPA2B1 by univariate Cox regression, suggesting that the risk signature of them is more reliable for OS prognosis (P = 0.016), whereas whose with a high level of HNRNPA2B1 suffer a poor OS  . Further2–1.022) . Then, wf ALKBH5 . In orde0.05501) . Howeverrognosis . FurtherTo better understand the clinical relevance of two-gene signature in ESCA, we first selected the patients with clinical characteristic variables and divided these patients into low- and high-risk groups, which were assessed by two-gene expression and clinical characteristic variables . Intereshttp://ualcan.path.uab.edu/index.html). Compared with the normal subgroup, the HNRNPA2B1 expression was significantly up-regulated (P < 0.05) in cancer patients with different clinical characteristics . The results showed that the expression of HNRNPA2B1 was significantly positively correlated with the expressions of MKI67 and PCNA, which were the classic biomarkers of proliferative cancer cells  and nine readers  were significantly increased in ESCA tissues, whereas no significant difference was found for the two erasers (FTO and ALKBH5), and most of RNA m6A regulators in our study were overlapped with the previous study (6A regulators, which could be systematically and directly helpful to analyze the interaction between these regulators. Herein, we found there are direct or indirect interactions among the 19 m6A regulators, and the writers METTL3, METTL14, WTAP, and KIAA1429 and the erasers FTO and ALKBH5 may localized in the center of regulatory network. It was also demonstrated that the relationship between most of the m6A RNA methylation regulators is positively correlated, and the KIAA1429 and YTHDF3 genes are most relevant. Subsequently, we confirmed that the RNA m6A levels were significantly higher in 18 cancerous tissues than corresponding normal tissues in ESCC patients via a dot blot assay. These results suggest that RNA m6A modification may be involved in the ESCA development.The mg cancer , 34. Thethylases , 35. Mea cancers . Previouer types . Howeverus study . Meanwhi6A regulators and OS in ESCA via the consistent clustering analysis, and the results showed that cluster 1 had a shorter OS than cluster 2. In addition, the univariate Cox regression analysis and LASSO Cox regression data indicated that a two-gene prognostic signature including ALKBH5 and HNRNPA2B1 could predict OS of ESCA patients. Moreover, high expression of HNRNPA2B1 and low expression of ALKBH5 were indicated as the risk factor for the survival of ESCA, and the combination of these two factors showed more predictive potential than the alone, although the ROC curve did not show robust prediction within 4 and 5 years, which because of that there are too few patients in the fourth and fifth years, which may lead to the instability of the ROC curve. It is reported that HNRNPA2B1 could selectively bind to m6A-containing transcripts via the “m6A-switch,” a mechanism in which m6A weakens Watson–Crick base pairing to destabilize the RNA hairpin structure and thereby exposes the single stranded hnRNP binding motif (We then analyzed the relationship between RNA mng motif . HNRNPA2ng motif –40. HNRNng motif . It alsong motif . In thisde novo synthesis and oxidative metabolism of fatty acids, which provide the necessary raw materials for tumor cell proliferation (de novo fatty acid synthesis, fatty acid β-oxidation, and fatty acid uptake, revealing that de novo fatty acid synthetic enzymes ACLY and ACC1 were markedly positively regulated by HNRNPA2B1. However, the expression of FASN, fatty acid uptake, and fatty acid oxidation genes is inconsistent in the two ESCC cell lines with HNRNPA2B1 deficiency, which may be due to the heterogeneity of the two different ESCC cells. In addition, knockdown of HNRNPA2B1 suppressed cellular lipid accumulation. Collectively, the results reveal that HNRNPA2B1 could accelerate fatty acid synthesis via up-regulation of de novo fatty acid synthetic enzymes ACLY and ACC1.Further, we analyzed the KEGG enrichment of genes, which were correlated with HNRNPA2B1 expression in ESCA patients using TCGA data. The data indicated that HNRNPA2B1 may be involved in fatty acid metabolism of ESCA. We also found that the HNRNPA2B1 level was significantly higher in extreme obese subgroup than other subgroups. The abnormal lipid metabolism of tumor cells is mainly manifested in the activation of feration , 42. Theferation , 44. Sub6A and its regulator HNRNPA2B1 were significantly increased in cancerous tissues of ESCA, and overexpression of HNRNPA2B1 promotes ESCA progression via up-regulation of de novo fatty acid synthetic enzymes ACLY and ACC1. Therefore, HNRNPA2B1 may be a promising prognostic biomarker and therapeutic target for human ESCA.In summary, our findings reveal that the levels of mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Ethical Committee of Nanjing Drum Tower Hospital. Written informed consent to participate in this study was provided by the patients.SW, QW, and XZ provided the direction of this manuscript. HG, BW, and KX collected and analyzed the data. LN and YF analyzed and evaluated the IHC of TMA. KX and ZW performed the experiments. QW wrote the manuscript. SW revised this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to current insufficient therapies, evaluating novel therapeutic agents for late-stage PCa is of considerable interest for both species. PDA indolylmaleimides showed anticancer effects in several neoplastic cell lines. Herein, a comparative characterization of PDA-66 and PDA-377 mediated effects was performed in human and canine PCa cell lines, which is also the first detailed characterization of these agents on cells derived from solid tumors in general. While PDA-377 showed only weak growth inhibition on human PCa cell lines, PDA-66 inhibited proliferation and induced apoptosis in human and canine cell lines with concentrations in the low micromolar range. Morphological characterization and whole transcriptome sequencing revealed that PDA-66 induces mitotic death through its microtubule-depolymerizing ability. PDA-66 appears to be a worthwhile anti-mitotic agent for further evaluation. The similarities in cellular and molecular response observed in the cell lines of both origins form a solid basis for the use of canine PCa  Prostate cancer (PCa) is the most common malignancy diagnosed among males in almost all western countries . PCa emeCanine tumors are valuable naturally occurring models helping to reveal mechanisms in cancer development, behavior and treatment , 7. ProsVinca alkaloids, are used in various cancers  was cultivated in DMEM/Ham's F-12 medium . The human LNCaP cell line (RRID:CVCL_0395) was grown in RPMI 1640 medium (Biochrom GmbH). The canine cell line TihoDProAdcarc1258 (RRID:CVCL_W737) was established by our group and cultivated in medium 199 . All media were supplemented with 10% heat-inactivated fetal bovine serum  and 2% penicillin/streptomycin (Biochrom GmbH). All cells were cultivated at 37°C in a humidified atmosphere of 5% CO2.Two human and one canine prostate carcinoma cell line were used. The human PC-3 cell line   were applied (0.1% DMSO as control). The cells were observed for 72 h in 150 μl incubation medium under a live cell imaging microscope  at 37°C with 5% CO2. An image of each well was captured every 15 min during the incubation period and these single images were combined to create time-lapse movies.PC-3, LNCaP and CT1258 cells were seeded in 96-well plates with a cell density of 1 × 10Morphological changes mediated by PDA were analyzed by May-Grünwald-Giemsa staining.2 cell culture dishes and covered with 10 ml of culture medium. Per dish and per cell line, 1 x 106 cells were seeded. On the following day, the microscope slides were transferred to new dishes with incubation medium or 0.15% DMSO for the control cells. The slides were exposed to the PDA agents in two different settings. For the first setting, the slides were incubated with 15 μM PDA-66 or PDA-377 for 24 h, an application equal to the cells used in the transcriptomic analyses. For the second setting, the slides were incubated with 5 μM PDA-66 or PDA-377 for 72 h, a concentration that showed moderate effects in all cell lines in live cell imaging. Microscope slides were washed with PBS and air dried at room temperature. The slides were stained in May-Grünwald's eosine-methylene blue solution  (undiluted) for 5 min and rinsed with tap water. Afterwards, they were stained for 15 min in Giemsa's azur eosin methylene blue solution (Merck KGaA) (1:10 dilution) and rinsed thoroughly with tap water again. The slides were left to air dry before analysis.Microscope slides were placed in 60 cmProliferative index of cells in response to PDA-66 and PDA-377 exposure was evaluated using the assay Cell Proliferation ELISA, BrdU (colorimetric) . The assay measures the incorporation of the thymidine analog 5-bromo-2-deoxyuridine (BrdU) into newly synthesized DNA of replicating cells using an anti-BrdU monoclonal antibody.4 cells per well and incubated in 150 μl culture medium for 24 h to allow the cells to attach to the surface. Afterwards, the cells were exposed to different concentrations of the PDA derivatives. The human cell lines were treated with 0.25–10 μM of the derivatives and 0.1% (v/v) DMSO as control. CT1258 cells were exposed to 0.25–25 μM of both derivatives (0.25% DMSO as control) as no significant effect was displayed after 24 h with 10 μM PDA-66. The proliferation assay was carried out in accordance with the manufacturer's protocol for adherent cells with the exception that BrdU was added simultaneously with the PDAs. The reaction products were quantified by measuring the absorbance at 370 nm minus the absorbance at 492 nm over a 30 min period using the Multi-Mode Reader Synergy 2 . Each single experiment was performed 8-fold.All three cell lines were seeded in 96-well plates with a density of 1 × 10After this point, no further tests were performed with PDA-377 due to its limited effect on the human cell lines.The concentrations used for cell count analysis and apoptosis are based on the 72 h values of the BrdU assay. Concentrations were chosen, that lead to 50% inhibition (2.5 μM PDA-66 in PC-3 and 5 μM PDA-66 in LNCaP). These values were chosen to ensure that there would be some margin in both directions to calculate possible significances, even if the other tests would show higher or lower efficiencies. Based on this criterion, 5 μM PDA-66 should have been used for the CT1258 cell line. However, this concentration showed no effect after 24 h. As it was crucial to be able to detect effects early after exposure for the transcriptomic analysis, 15 μM was used for this cell line, the first concentration to show a significant effect in the BrdU assay after 24 h of exposure.4 cells per well in 1 ml culture medium and incubated overnight. Based on the proliferation assay, PC-3 cells were treated with 2.5 μM PDA-66 and 0.025% DMSO, LNCaP with 5 μM PDA-66 (0.05% DMSO), and CT1258 cells with 15 μM PDA-66 (0.15% DMSO). After 24 h, 48 h and 72 h, the cells were detached by TrypLE™ Express  and counted via automatic cell counter . The experiment was carried out in biological replicates three times.Cells were seeded in 24-well plates with a cell density of 5 × 10RRID:SCR_014489).Apoptosis rates were determined by staining cells with the fluorescence dyes Annexin V FITC and Propidium Iodide (PI)  in accordance with the manufacturer's protocol and determined by flow cytometry using a FACSCaliburTM . In addition to the cell specific concentrations used in the cell count analysis, two lower concentrations were used to demonstrate that the induction of apoptosis already starts at lower concentrations. The data were evaluated using BD CellQuest software , LNCaP to 1.0, 2.5, and 5.0 μM PDA-66 (0.05% DMSO), and CT1258 to 7.5, 10 and 15 μM PDA-66 (0.15% DMSO). After the drug exposure period, the medium was saved to collect the non-adherent cell fraction to count all treated cells regardless of their condition. Remaining adherent cells were detached by TrypLE™ Express  and combined with the medium cell fraction. In addition to the double stained samples, for each cell line and each incubation time point, single stained controls were measured. Exposure of the cells to PDA-66 and DMSO as reference was conducted in biological replicates three times.All three cell lines were seeded in 12-well plates with a cell density of 2 × 10As PDA-66 is a strong inducer of apoptosis, the transcriptomic analysis was performed on early time points (12 and 24 h). This early time points, before all the apoptosis signaling cascades are fully active and most transcripts would be cell death related, were chosen for the best chance to detect the direct effects of PDA exposure. As 15 μM was the first concentration to show significant effects in CT1258 after 24 h, this concentration was used for all cell lines to keep the transcriptomic data comparable (0.15% DMSO for the control groups). After 12 and 24 h, the cells were harvested and total RNA was extracted using the AllPrep DNA/RNA Mini Kit  in accordance with the manufacturer's protocol. For each treatment condition (DMSO control and PDA-66 exposed cells), three independent biological replicates were prepared and sequenced.RRID:SCR_010910). For each of the 24,581 annotated protein-coding canine genes (EMBL gene ID nomenclature), the aligned reads were counted using the R package GAGE (RRID:SCR_017067). For the human PC-3 and LNCaP, mapping and read counting were conducted using the RNA Express (version: 1.0.0) workflow within the Illumina basespace environment . In brief, after mapping to the human HG19 reference genome using the STAR (2.3.1s) aligner  read counts for 23,710 annotated RefSeq genes were generated.RNA integrity numbers (RIN) were determined using a Bioanalyzer 2100 . Sequencing libraries were prepared using 1 μg total RNA with RIN > 7. PolyA RNA was enriched and ligated to sequencing adapters using the NEBNext Ultra RNA preparation kit  in accordance with the manufacturer's protocols. Single-read sequencing (75 bp) was conducted on an Illumina NextSeq500 . Sequences were aligned to the canine genome  using the Burrows-Wheeler Aligner (BWA)   using SAS enterprise guide 7.1 . Differences were considered statistically significant for p < 0.05. IC50 values were calculated using GraphPad Prism 7.02 .Within each experiment, results were described using the mean values of the replicates. The graphs show the mean ± standard deviation. Significant differences between treatment and control were calculated by Dunnett's multiple comparison test for the confidence levels 95, 99, and 99.9%  or student's RRID:SCR_012802). The average total read counts mapped within annotated genes were 12.2M (STD: 0.7M), 15.8M (STD: 1.1M), 13.5M (STD: 0.9M) for CT1258, PC-3 and LNCaP samples, respectively. Each PDA-66 treatment group (12 or 24 h) was compared to the respective control cells treated with DMSO. After multidimensional scaling and plotting of the data, one PC-3 DMSO treated control sample was identified as outlier and was excluded from further analyses so that the PC-3-24 h-DMSO group consisted of only two independent samples. Genes with a false discovery rate adjusted p-value (FDR) of < 0.001 were considered to have significantly different expressions compared to the control samples. These genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for functional enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool (RRID:SCR_001881).Differential gene expression analyses for canine and human samples were conducted using the BioconductorR package edgeR 3.14.0   indicated the induction of cell death.Consistent with live cell imaging observations and the proliferation assay, a significant change in the total number of cells was observed 48 h after PDA-66 application in all tested cell lines compared to their tested control cells . While tPDA-66 significantly induced apoptosis in all three PCa cell lines compared to the DMSO-exposed controls at different concentrations . ApplicaAll cell lines displayed a higher number of differentially expressed genes  after 12 h compared to the 24 h treatment groups , correlaIn the present study, the effects of PDA-66 and PDA-377, two indolylmaleimide derivatives, were comparatively evaluated on three prostate carcinoma cell lines with a diverse genetic background. The human PC-3 and LNCaP cells are among the most used and best described PCa cell lines , 35, botBoth PDA derivatives caused two distinct morphological changes in all tested cell lines. Live cell imaging and May-Grünwald-Giemsa staining revealed clear apoptotic features through observed pyknosis and karyorrhexis in May-Grünwald-Giemsa staining as well as the formation of apoptotic bodies in live cell imaging. These features match the proposed mechanism of induced mitotic arrest and subsequent MD. Secondly, some enlarged multinucleated cells formed. Cells can overcome the mitotic arrest before MD is triggered; a process known as slippage. After DNA replication in the S phase and escaping the mitotic arrest, these cells fail cytokinesis and/or the equal distribution of chromosomes, leading to enlarged multi-nucleated cells similar to those we observed , 43, 44.Proliferation was measured via BrdU assay to quantify growth inhibition seen in live cell imaging. PDA-66 caused significant and dose-dependent anti-proliferative effects in all tested PCa cell lines. The herein demonstrated doses to inhibit proliferation significantly by at least 50% were between 5 and 10 μM PDA-66 within 72 h. The analyzed PCa cell lines were slightly less sensitive compared to other neoplastic cell lines like human neuroblastoma, human acute lymphoblastic leukemia or canine lymphoma –17. ConsPDA-377 also induced anti-proliferative effects in all tested cell lines. However, for both human cell lines, the inhibition of proliferation was limited and most likely dose-independent, as a comparable decrease in proliferation was measured for almost all tested concentrations after 48 h. The proliferative index did not drop below 50%. In the canine cell line, the proliferation inhibition was clearly dose-dependent. After a 72 h incubation period, the proliferative index dropped below 10% with an IC50 of 3.14 μM. PDA-377 may be suitable for the treatment of prostate cancer in dogs. Nevertheless, no further testing with this substance was conducted, as the limited effect on the human cell lines contradicts the cross-species approach with dogs as a model to generate transferable findings to the benefit of both species.Vinca alkaloids or colchicine-binding site drugs, lead to failures in mitotic spindle formation and therefore chromosome segregation, which triggers the spindle assembly checkpoint . SAC activation causes mitotic arrest in the metaphase of the M phase of the cell cycle. Within the enriched KEGG cell cycle pathway, several important genes involved in SAC are upregulated after PDA-66 exposure, e.g., BUB1B, CDC20, CDK1, and MAD2. These DEGs are commonly involved in MD , a gene that plays an important role in interphase checkpoint activation (Interestingly, there was also an upregulation of genes involved in G1/S phase cell cycle progression and DNA replication, especially within the two human cell lines. Given the M phase cell cycle arrest induced by SAC, an enrichment of S phase transcripts is unexpected. Moreover, G1/S phase transcripts were downregulated in canine lymphoma cell lines after PDA-66 exposure . Howevertivation .The observed morphological changes and the transcriptome analysis match other anti-mitotic drugs causing MD. While most cells die as a result of this intrinsic cell death, some cells remain in a polyploid state after mitotic slippage. These cells are usually senescent and therefore incapable of further proliferation. However, they bear the risk of reinforcing the tumor, as their potential proliferation can lead to aneuploid cells, a hallmark of cancer , 50, 52.PCa cell lines of human and canine origin showed comparable cellular and molecular reactions, including significant inhibition of proliferation and induced apoptosis after PDA-66 incubation. Furthermore, efficacy of PDA-66 was not influenced by the androgen status of the cell line. As PDA-66 mediates its effects in a hormone-independent manner through microtubule destabilization, the compound is suitable to be used in androgen-dependent as well as -independent PCa subtypes. Based on the findings of this study, PDA-66 appears to be a worthwhile agent for further evaluation and potential treatment of PCa in dogs and, if successfully introduced, also in humans.in vitro characterization of PDA-66 effects on PCa cells encourage, as a next step, to initiate an early clinical trial in dogs to evaluate the compound in naturally occurring PCa in the presence of an immune system. First, however, a pharmacokinetic study in dogs needs to be performed. An early study in mice revealed that i.p. application of 100 mg/kg PDA-66 once per day was well-tolerated (The cellular and molecular olerated . The valolerated –57. Treahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162832.The original contributions presented in the study are publicly available. This data can be found at: JS carried out partial BrdU assay, May-Grünwald-Giemsa staining, cell treatment and RNA extraction for whole transcriptome analysis, performed data analysis, interpretation and visualization, and drafted the manuscript. IN provided the resources for all cellular analyses, supervised the study, and revised the manuscript. JB performed transcriptomic sequencing and revised the manuscript. DJ carried out partial BrdU assay, live cell imaging, cell count, and flow cytometry analyses. CR helped with primary study design. AP-D synthesized the PDA compounds. AR participated in initial compound generation. LH evaluated the May-Grünwald-Giemsa staining. MB supervised PDA compound design and synthesis. BB and ES provided the resources for the transcriptomic analysis and supervised the transcriptomic work packages. CJ and HM performed the primary study design. In addition, HM supervised the study and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors HM.


Granulosa cell tumor treatment is challenging as there are few effective options besides surgery. In this study, we obtained tumor tissue from patients at surgery and cultured tumor cells in the laboratory. After sufficient expansion, we tested the effects of current treatments, such as chemotherapy and anti-hormonal treatment, and novel anti-cancer treatment options on cell survival. Results were generated within three weeks after tissue collection. We found that all drugs were ineffective when used as single treatments; however, some combinations were very effective. The PI3K protein inhibitor alpelisib was effective in combination with chemotherapy and achieved 50% cell death at assumed tolerable patient plasma concentrations. In conclusion, this study shows an approach to rapidly establish patient-derived cell lines for drug screens. The effectiveness of combined treatment with alpelisib and chemotherapy in granulosa cell tumors should be further investigated and may be a promising novel treatment option in patients with a granulosa cell tumor.Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 < Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients. FOXL2 c.402C > G (C134W) mutation testing can be performed. This specific mutation is a hallmark of AGCTs and is present in 90–97% of patients . Su. Su® memeatments , includieatments . Next, t2 values were used to indicate how well the model fit the data  was plotted against the drug concentration range of each drug (in log10[μM]) using GraphPad Prism 8.3.1. Values that exceeded 100% survival  were normalized to 100% survival. Similarly, values below 0% cell survival  were normalized to 0% survival. Values exceeding this deviation were excluded. Dose–response curves were obtained using the model “log(inhibitor) vs. variable slope ” to fit the dose–response curves and Rthe data . We inteIn addition to the 11 monotherapies, 12 drug combinations were tested  simultanSimilar to monotherapies, the safety for in vivo use of drug combinations as assessed by comparison to Cmax or Css values of the individual drugs . The dosThis study shows that rapid, systematic, patient-specific AGCT drug screens are feasible and can be used to test individual responses to existing monotherapies and novel combinations. The findings from a set of 13 cell lines demonstrated synergistic growth inhibition of the PI3K inhibitor alpelisib combined with the current first-line chemotherapeutic agents carboplatin and paclitaxel at potentially tolerable concentrations in vivo. Therefore, alpelisib, carboplatin, and paclitaxel may be a promising novel combination for treatment of recurrent AGCTs.


Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer. Therefore, new therapeutic modalities are urgently required, particularly for tumors with high genetic heterogeneity and poor response to current treatments. Cancer is caused by the accumulation of dozens of mutations which lead to uncontrolled cell proliferation, but the precise biochemical changes and genetic background is unique to each individual cancer [Despite decades of research and an unprecedented advancement of therapeutic options , cancer l cancer . Even wil cancer . Cancer l cancer .MicroRNAs (miRNAs) are naturally occurring short (~22 nucleotide) double stranded RNA molecules with a unique nucleotide sequence (seed sequence) that binds, at least partially, to complementary mRNA sequences. This leads to mRNA degradation or suppressed translation which then silences gene expression , 5. The CCND1, KRAS, RASSF1, STMN1, and MCL1 [The therapeutic potential of miR-193a was identified in a high-throughput functional lentiviral-based screen of miRNAs in tumor cells and the 3p arm (miR-193a-3p) was subsequently demonstrated as the relevant active entity in screening assays . The tumand MCL1 , 24–26. in vivo. To overcome the challenge of delivering 1B3 to tumors upon systemic administration in experimental tumor models, it has been formulated in a novel lipid nanoparticle (INT-1B3). We recently evaluated the pharmacodynamic and pharmacokinetic profile of INT-1B3 and showed that it is efficiently delivered to tumor tissues and has a favorable safety profile (manuscript in preparation). In addition, RNA-sequencing and subsequent pathway analysis in a panel of cell lines confirmed the multi-targeted nature of 1B3 by revealing hundreds of target genes with key roles in tumor cell survival, proliferation, and migration [in vitro by silencing different 1B3 target genes. We also confirm the anti-tumor activity of INT-1B3 in experimental human tumor-bearing mouse models, indicating its potential as promising novel modality for therapeutic intervention in oncology.1B3 is a synthetic chemically modified miR-193a-3p mimic with 100% sequence homology to the mature miR-193a-3p guide (antisense) strand and a fully complementary passenger (sense) strand. The molecule incorporates chemical modifications on its passenger strand to improve strand-selection and prevent inflammatory pathway activation igration . In the TP53 deletion or loss of function  and KRAS activating mutations . The level of cell-associated oligonucleotide (1B3) measured after transfection with 10-nM 1B3 was 100- to 1,000-fold higher compared to mock-transfected cells , demonstrating significant transfection efficacy. As indicated in the Materials & Methods section, the 2-tailed RT-qPCR protocol detects both transfected 1B3 and endogenous miR-193a-3p. Notably, the increase after 1B3 transfection significantly exceeds the differences in basal expression among the tested cell lines. Further, 1B3 overexpression downregulated expression of the 1B3 target cyclin D1 (CCND1) by ~50% at the mRNA level  and completely abolished expression at the protein level , strongly demonstrating not only drug uptake, but efficient target engagement. These observed effects were 1B3-specific, as transfection of ‘negative’ miRNA control (3A1) did not lead to any significant changes in CCND1 mRNA or CCND1 protein expression. 3A1 is based on a commercially available random sequence miRNA that has been validated to not produce identifiable biological effects and is routinely used as a ‘negative’ miRNA control.To characterize the effect of 1B3 in cell-based assays, 1B3 was transiently transfected in a panel of human tumor cell lines from six different cancer types including colon (HCT116), liver , non-small cell lung carcinoma , skin (A2058), pancreatic (PANC-1), and breast (BT-549). The selected cancer cell lines had a variety of genetic backgrounds including Overexpression of 1B3 reduced cell number in every cell line by 25–93% compared to the mock control 96 h after transfection . The ‘neKRAS wild type, TP53 mutant, lung carcinoma cells (H460 and A549) had a very high caspase activation of 9- and 4.5-fold, respectively. Conversely, 1B3 induced only a mild caspase 3/7 activation (~1.3-fold) in the hepatocellular carcinoma (HCC) cancer cell lines SNU-449, HUH-7, and Hep3B. As with the effect on nuclei count, a milder effect was observed after treatment with 1-nM compared to 10-nM 1B3 . To validate the effect of 1B3 on apoptosis, 1B3 was overexpressed in the two cell lines with the highest caspase 3/7 activation  and the two cell lines with the lowest caspase 3/7 activation , and the level of cleaved poly (ADP-ribose) polymerase (PARP) was measured by Western blot. As expected, A549 and H460 had a strong increase in cleaved PARP compared to mock and 3A1-transfected cells, while no cleaved PARP was detected in SNU-449 or HUH-7 cells . Additionally, the presence of apoptotic cells after 1B3 overexpression in A549 and H460 cells was confirmed by flow cytometric analysis of annexin V/PI-stained cells (data not shown). These data show that 1B3 causes apoptosis in some, but not all, of the cancer cell lines tested, indicating that it is one of the anti-proliferative mechanisms of 1B3.As several biological processes can contribute to reduced cell number, including cell death, the effect of 1B3 on caspase 3/7 mediated apoptosis was investigated. Overexpression of 1B3 induced caspase 3/7 activity by more than 2-fold compared to the mock control in eight of the 11 cell lines tested . The effPremature cell cycle arrest can also reduce cell number over time, therefore the effect of 1B3 on the cell cycle was investigated using image based DNA-content analysis . As showBecause 1B3 overexpression reduced cell number but did not always lead to a strong caspase 3/7 induction (apoptosis), the effect of 1B3 on cell senescence (type of growth arrest that primarily occurs in aged cells) was investigated in a subset of cell lines. The cell lines selected represent a range of apoptotic response to 1B3 including SNU-449, Hep3B, A2058, and H460. To ensure that the effect of 1B3 on cell count did not bias the results, a total of 500 cells was counted for each condition. Overexpression of 1B3 increased the number of cells expressing the senescence marker, SA-β-gal, compared to mock and 3A1-transfected cells in all cell lines tested . A repreBoth G2/M cell cycle arrest and cell senescence are associated with an accumulation of DNA replication errors , therefoFinally, the effect of 1B3 on cell migration was investigated in a range of cancer cell types including A549, A2058, and SNU-449. The strongest effect on transwell cell migration was observed in A2058 and SNU-449 cells , while a 43% decrease was observed in A549 cells when compared to mock . AlthougTaken together, these data shows that transfection of 1B3 induces a range of anti-cancer phenotypes in a panel of cancer cell lines, supporting its potential as a novel cancer therapeutic by targeting multiple mechanisms .CCND1, CDK6), proliferation (KRAS), apoptosis , and cytoskeletal organization (STMN1). As the effects of 1B3 varied depending on cellular context, the effects were investigated in several human cancer cell lines representing three different cancer types.To further investigate the mode of action through which 1B3 exerts its effects on tumor cells, selected individual 1B3 target genes were silenced using siRNAs, and their effect on key phenotypes measured. MiRNAs suppress the expression of numerous different target mRNAs. We therefore tested siRNAs against several known and validated 1B3 mRNA targets which have been documented as critical for its tumor suppressive functions. These include regulators of cell cycle .Furthermore, the siRNAs tested had a diverse effect on cell cycle arrest in five different cell lines (data summarized in Supplementary Table 1). Again, no siRNA mimicked the exact effect of 1B3 in all cell lines. For example, siCCND1, siCDK6, siMCL1, and siYWHAZ phenocopied the effect of 1B3 by inducing a G0/G1 arrest, but only in one cell line (H1299). Nonetheless, and consistent with the effect of 1B3, siSTMN1 induced a G2/M arrest in three cell lines . These data suggest that although some of the 1B3 targets tested play a role in cell cycle arrest, this effect is cell line-dependent and does not always replicate the effect of 1B3.KRAS and MCL1 to induction of cellular senescence was measured. Neither siRNA mimicked the exact effect of 1B3 in all cell lines tested  (versus the 11-fold increase by 1B3). siMCL1 on the other hand did not induce senescence in any of the cell lines (data not shown). This shows that in some contexts, silencing of KRAS may be responsible for 1B3 induced senescence, although this effect was not observed in all cell lines.Next, the contribution of 1B3 targets nd H460) . AlthougKRAS and STMN1 in reduction of migration was assessed  and transfected them with combinations of siRNAs, then measured the effect on cell count  was observed between individual animal plasma AFP levels and tumor weights . Notably, INT-1B3 treatment resulted in a significant reduction in median AFP levels over time, highlighting its anti-tumor activity in the Hep3B model (p = 0.0005), reaching the same growth inhibition as the kinase inhibitor sorafenib  in the tumors .To evaluate INT-1B3 in the experimental human HCC Hep3B tumor model, Hep3B tumor fragments were implanted into the left liver lobe (orthotopic tumor model) of SCID/Beige mice. After tumor establishment, animals were treated intravenously with 10 mg/kg/administration INT-1B3 or vehicle control (PBS) for three consecutive days in the first week and maintained on a twice weekly treatment schedule for an additional three weeks . As it i3B model . INT-1B3orafenib  used as 3 treatment with 3 mg/kg/administration INT-1B3 or vehicle control (PBS) was administered intravenously daily for five days during the first week then twice weekly for the next two weeks (p = 0.0062) . This clearly indicates that 1B3 is effectively delivered and functional in experimental tumor models and leads to significant anti-tumor activity.To extend these findings, efficacy of INT-1B3 on tumor growth was further investigated in the human melanoma A2058 subcutaneous tumor model. Once tumors reached a median volume of ~150 mmwo weeks . Tumor vwo weeks . The eff 0.0062) . Four anin vitro and in vivo. 1B3 consistently reduced cancer cell proliferation, through a range of mechanisms, in a variety of cancer cell lines. This confirms our recent transcriptome-wide analysis of differential gene expression demonstrating 1B3 as a strong phosphatase and tensin homolog (PTEN) pathway activator which governs various cell processes including tumor cell survival, proliferation, and migration [Cancer utilizes an array of mechanisms to escape cell death and proliferate without control . MiRNAs igration . Our resin vitro are in agreement with data from a miR-193a-3p mimic screen in a panel of 122 human cancer cell lines from 13 different cancer types [The anti-proliferative effects of 1B3 er types . In thater types . In lineer types , 33 and CCND1, CCND2, CCNE2, CDK2, CDK4, and CDK6 [TP53 expression deficient repair could lead to further DNA damage accumulation and G2/M arrest. Nevertheless, the cell cycle is controlled by several factors and further investigation is required to dissect the specific role of 1B3 on this phenotype in different genetic contexts.MiR-193a-3p has been described as a cell-cycle regulating miRNA due to its silencing of several important cell cycle regulators, including and CDK6 , which wand CDK6 . As expeand CDK6  demonstrand CDK6  observedKRAS mutant cell lines HCT116, H460, A549, and PANC-1 cells were among those with the strongest response to 1B3, but proliferation was also reduced in wild-type KRAS expressing cells such as A2058, H1299, and SNU449. Expression of oncogenic KRAS is linked to aggressive disease and therapeutic resistance, yet despite years of effort no effective inhibitor has reached the clinic [KRAS-driven tumors, but also for tumors with wild-type expression of this oncogene. This broad application of 1B3 in different oncogene-driven tumors is supported by the 50% tumor growth inhibition of BRAF-mutated A2058-derived tumors after INT-1B3 treatment in vivo.An important observation is that some effects of 1B3 differ between human cancer cell lines. As illustrated by the different cell cycle arrest patterns and caspase 3/7 activation, the genetic background of the cancer cell may influence the exact phenotype induced by 1B3 overexpression. Despite different upstream events, 1B3 overexpression reduced proliferation in every cell line, regardless of genetic background. For example, the e clinic , 36. ConCCND1, cKIT, HMGB1, GRB7, PLAU, RASSF1, PAK4, S6K2, STMN1, and PTP1B [The exact mechanism through which miR-193a-3p induces an anti-cancer effect has been widely studied and although it is almost universally described as a tumor-suppressor miRNA, the genes which it targets are not always consistent. While some studies connect the anti-proliferative effect of miR-193a-3p mimics to a single gene target, our data suggests this effect is the result of unique, simultaneous, regulation of multiple mRNAs. In support of this, our recent transcriptome wide analysis of six human cancer cell lines uncovered activation of the PTEN tumor suppressor pathway as one of several key mechanisms behind the 1B3 mode of action . 1B3 exend PTP1B , 38–43. nd PTP1B . This cond PTP1B , 46.in vitro data suggest that in human cancer cells: 1) 1B3 overexpression leads to an anti-cancer effect, although the precise downstream mechanism varies between cell lines; 2) the anti-cancer effect of 1B3 is due to simultaneous targeting of multiple target genes, rather than one individual gene; and 3) the target genes responsible for the anti-cancer effect of 1B3 vary between cell lines. Thus, the broad anti-cancer effect of 1B3 lends it to a potentially useful and effective agent for therapeutic intervention in oncology. Cancer is a complex, multigenic, and heterogeneous disease, and a single patient will often carry cells with unique genetic origins. Therefore, a miRNA therapeutic which regulates multiple pathways, or multiple elements within a pathway, has the potential to destroy several different tumor subclones and might be less subject to drug treatment-acquired resistance [Overall, our sistance , 12. In sistance , 48.in vivo delivery remains a challenge. Indeed, most in vivo investigations of miR-193a-3p implant stably transduced tumor cell lines, rather than treating established experimental tumors. Our study extends the findings of several groups who have shown decreased tumor growth and metastasis of miR-193a-3p transduced cells in vivo [in vivo (manuscript in preparation). In the current study, we extend these findings by demonstrating that INT-1B3 significantly delays tumor growth in both HCC and melanoma mouse models. Interestingly, Seviour et al. [Despite growing evidence of the therapeutic value of miRNAs, effective  in vivo , 43, 49.r et al.  used a lin vitro. Some of these effects  can be broadly applied to all cancer cell lines, while others  are unique to specific cell lines and genetic backgrounds. Using both individual and combined siRNA(s) we showed that no individual 1B3 target reproduced the miRNA effect which suggests that multiple, simultaneous, gene repression by 1B3 is more effective at reducing tumor cell growth than single gene targeting. These data add to the growing body of evidence that miR-193a-3p has an anti-cancer effect through several mechanisms, and that this overall anti-cancer effect is conserved regardless of tumor origin or genetic background. Finally, in two different in vivo models we showed a significant reduction of tumor growth. Together, this evidence supports the further clinical development of INT-1B3 as an anti-cancer therapeutic molecule.In summary, we have shown that the miR-193a-3p mimic, 1B3, consistently suppresses several pro-tumorigenic phenotypes The miR-193a-3p mimic, 1B3, and miRNA control 3A1  were manufactured by BioSpring GmbH . The nucleotide sequences are as follows (with limited 2′-O-methyl nucleotide modifications on the passenger strand ):(3′)-TTUUGACCGGAUGUUUCAGGGU-(5′)1B3 passenger (sense) strand: (5′)-AACUGGCCUACAAAGUCCCAGU-(3′)1B3 guide (anti-sense) strand: (3′)-TTAAUGCAGCAGCGCAGCAAU-(5′)3A1 passenger (sense) strand: (5′)-UUACGUCGUCGCGUCGUUATT-(3′)3A1 guide (anti-sense) strand: in vivo. INT-1B3 represents 1B3 formulated in lipid nanoparticles [Small double stranded oligonucleotides, such as siRNAs or miRNAs targeting RNA, are easily degraded by nucleases and have low membrane permeability due to their high molecular weight and negative charges. Hence, an efficient drug delivery system is needed to improve their therapeutic efficacy articles  was manuIn addition, several siRNAs were used in the presented studies: siPOOL (negative siRNA control) and SMARTpool siRNAs  mRNA, respectively) were obtained from Dharmacon. Each SMARTpool contains four unique siRNA sequences targeting the same mRNA target.The human cell lines Hep3b (HCC), SNU-449 (HCC), H1299 (NSCLC), H1975 (NSCLC), H460 (NSCLC), A2058 (melanoma), BT-549 (TNBC), HCT116 (colon), PANC-1 (pancreatic), and A549 (NSCLC) cells were obtained from ATCC. Human HuH-7 (HCC) cells were obtained from the Japanese Cancer Resources Bank (JCRB). All cell lines were processed and cultured according to the vendor’s instructions. Cells were seeded at 80% confluency into 6-well culture plates 4 h before transient transfection. For single agent transfection the indicated concentrations of either 1B3, 3A1, siPOOL, or SMARTPOOL siRNAs were mixed with Lipofectamine RNAiMax (ThermoFisher) and added dropwise to the cell monolayer. For combination transfection, siRNAs were combined at the concentrations listed in For cell count and high content cell cycle analysis, transfected cells were seeded into triplicate wells of black-walled, clear bottom 96-well plates (Greiner) at the appropriate cell density to reach confluence 72 h later. Forty-eight, 72 and 96 h after transfection, media was removed and replaced with 100 μL staining mix containing 0.25% PFA, 0.075% saponin and 1 μg/mL Hoechst 33342 in PBS. Cell imaging was performed on the Thermo CellInsite Automated Imager using the inbuilt high content screening software to identify cell nuclei. Fifteen images/well using the 386_23 filter were captured at 10× magnification. High content cell cycle analysis was performed using nuclei intensity information as described previously . Taxol aFor Caspase-Glo 3/7 assay, transfected cells were seeded into triplicate wells of white-walled, clear-bottom 96-well plates (Greiner) at the appropriate cell density to reach confluence 72 h later. The Caspase-Glo 3/7 assay (Promega) was performed 48 and 72 h after transfection, according to the manufacturers’ instructions.For RNA extraction, 100–200 thousand transfected cells (dependent on the cell line) were seeded into 12-well tissue culture plates (Greiner). Twenty-four hours after transfection, media was aspirated from the 80% confluent cell monolayers, and frozen at –80°C. RNA was extracted using TRIzol (Thermo Fisher) according to the manufacturers’ instructions. RNA concentration was measured on NanoDrop One (Thermo Fisher).in vitro, the reverse transcription reaction of 20 ng of total RNAs was performed using 2-tailed RT primers designed by TATAA Biocenter and GrandScript cDNA synthesis Kit (TATAA Biocenter), according to the manufacturer’s instructions. Quantification of the miRNA expression levels were subsequently assessed by quantitative PCR (qPCR) using SYBR Green (Bio-Rad). Results were interpolated with a calibration curve to determine the amount of 1B3 per cell. It is of note that the guide (antisense) strand sequence of 1B3 is identical to the mature human miR-193a-3p. Therefore, primers cannot distinguish between endogenous miR-193a-3p expression and the level of exogenous (transfected) 1B3. All figures are labelled ‘1B3 level’ but it should be assumed that this also includes endogenous miR-193a-3p expression, which is determined in the mock-transfected conditions or PBS control treated animals.For the detection of miRNAs in vivo, the reverse transcription reaction of 100 ng of total RNAs was performed using in-house designed stem-loop RT primers (IDT). Quantification of the miRNA levels were subsequently assessed by quantitative PCR (qPCR) using SYBR Green (Bio-Rad). Cycle threshold (Ct) values were interpolated with a calibration curve to determine the amount of 1B3 per ng tumor tissue. The Stem loop primer sequences used in this study are provided in Supplementary Table 2. As indicated in the previous chapter, the primers used cannot distinguish between endogenous miR-193a-3p expression and the level of exogenous (transfected) 1B3. All figures are labelled ‘1B3 level’ but it should be assumed that this also includes endogenous miR-193a-3p expression, which is determined in the mock-transfected conditions or PBS control-treated animals.For the detection of miRNAs −ΔCt method. ΔCt was calculated by subtracting the geometric mean of the Ct of two reference genes from the Ct of the target genes. Values were normalized to mock to determine target downregulation.For the detection of target mRNAs, 100 ng of total RNA was first transcribed into single stranded cDNA. One μL synthesized cDNA was used in a 20 μL PCR amplification reaction using SYBR Green master mix (BioRAD) on CFX96 Real-Time qPCR machine according to the manufacturers’ instructions. Triplicate reactions were performed for each condition. The qPCR primer sequences used in this study are listed in Supplementary Table 3. Expression values were calculated using 2For protein extraction, cells were seeded into 6-well tissue culture plates at the appropriate cell density to reach confluence 72 h later. Cell pellets were collected 24, 48 and 72 h after transfection by trypsinization. One hundred to 200 μL RIPA buffer , supplemented with protease and phosphatase inhibitor cocktails, was added to harvested cells. Lysates were centrifugated at 15,000× g for 1 h at 4°C, and clarified by removing the cell debris pellet. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). Samples were separated at 120 V by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) on Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad). Proteins were transferred at 200 mA for 2 h to polyvinylidene difluoride (PVDF) membranes in transfer buffer . The membranes were blocked using 5% milk or 5% BSA in Tris-buffered saline with Tween  to prevent non-specific antibody binding. Blots were probed with primary and horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using enhanced chemiluminescence (ECL) reagents. Membranes were stripped by incubation in stripping buffer  for 30 min at 50°C and re-probed as appropriate. Band intensities were quantified by densitometry using ImageJ software and values normalized to the tubulin loading control and shown relative to the mock transfected cells.For the SA-β-gal assay, transfected cells were seeded in 6-well cell culture plates (Greiner) at the appropriate cell density to reach confluence 72 h later. One well of untreated cells was seeded in low-serum (0.1% FBS) media as a positive control for senescence. The SA-β-gal assay was performed 96 h after transfection using the SA-β-gal assay kit  according to the manufacturer’s instructions. Cells were imaged at 10x magnification using a Leica DMIL LED microscope. A total of 500 cells were counted from each well using the image analysis software Fiji .2 incubator. Forty-eight hours after transfection, unmigrated cells were removed from the chamber with a cotton swab and the chambers fixed with 70% ethanol then stained in a 1% Crystal Violet (Sigma) solution. Using a Leica DMIL LED microscope, 10 images per field at 20x magnification were obtained. The number of migrated cells was counted using the imaging software Fiji [For the migration assay, 16 h after transfection in 6-well plates, media was aspirated and replaced in low serum (0.2% FBS) media. After a further 6 h, cells were dissociated as described above and seeded into duplicate wells of 24-well cell culture migration inserts (Corning) at the appropriate cell density to reach confluence 24 h later. Complete media (10% FBS) was added to the bottom well. The cells were allowed to migrate for 24 h at 37°C in a humidified 5% COare Fiji . To cont3 piece of a subcutaneously grown Hep3B tumor into the left liver lobe (orthotopic tumor model). Tumor development was monitored by measurement of serum alpha-fetoprotein (AFP) which serves as tumor marker and indicator of liver tumor development [6 A2058 cells in 0.1-mL PBS. Once the mean subcutaneous tumor size reached ~150 mm3 , animals were randomized in the various experimental groups, and animals were treated with either vehicle (PBS) or 3 mg/kg/administration of INT-1B3 by repeated bolus injection (i.v) daily for 5 days during the first week, then maintained on a twice weekly (‘Monday/Thursday’) schedule for the next 2 weeks. Tumor volume was monitored by caliper-aided measurements twice a week throughout the study.Studies with the human HCC Hep3B and human melanoma A2058 experimental tumor models were performed at Crown Bioscience Inc  and Oncotest/Charles River , respectively. Study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, and were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care. For the experimental Hep3B tumor model, Hep3B tumor cells were first injected into the flank of Balb/c immune-compromized (Nude) mice to form subcutaneous tumors. Then, 7- to 8-week old female SCID/Beige mice were implanted with a single 2-mmelopment . When in


Mastoid surgery is an aerosol-generating procedure that involves the use of a high-speed drill, which produces a mixture of water, bone, blood and tissue that may contain the viable coronavirus disease 2019 pathogen. This potentially puts the surgeon and other operating theatre personnel at risk of acquiring the severe acute respiratory syndrome coronavirus-2 from contact with droplets or aerosols. The use of an additional drape designed to limit the spread of droplets and aerosols has been described; such drapes include the ‘Southampton Tent’ and ‘OtoTent’.To evaluate the use of a novel drape ‘tent’ that has advantages over established ‘tent’ designs in terms of having: (1) a CE marking; (2) no requirement for modification during assembly; and (3) no obstruction to the surgical visual field.During mastoid surgery, the dispersion of macroscopic droplets and other particulate matter was confined within the novel drape ‘tent’. Use of this drape ‘tent’ had no adverse effects upon the surgeon's manual dexterity or efficiency, the view of the surgical field, or the sterility. Hence, our findings support its use during mastoid surgery in the coronavirus disease 2019 era. An aerosol can travel over longer distances, and refers to a suspension of fine solid or liquid droplet in a gas, with a diameter of 10 μm or less.13 given its connection with the viral-laden nasopharynx14 via the Eustachian tube. However, despite other coronaviruses having been found in the middle ear,15 there have been no reported investigations of SARS-CoV-2 in this anatomical location to date. Regardless, mastoid surgery is considered to be high risk for the transmission of Covid-19 from an infected patient to healthcare workers via the drill-induced dispersion of a mixture of water, bone, blood and tissue.6It is highly feasible that the middle ear of a Covid-19 positive patient contains SARS-CoV-2,6 the ‘Ototent’16 and other methods.18 Recent pre-clinical studies have suggested that the use of such a drape ‘tent’ could reduce the dispersion of particulate matter during temporal bone drilling.19 However, all of these established and previously evaluated ‘tent’ designs require modifications during the assembly process, or are associated with a significant amount of ‘spare’ drape that can interfere with surgical efficiency.19Infection prevention control measures that aim to reduce aerosolisation and contain the spread of droplets and other matter during mastoid surgery have been described. These include the use of an additional or multiple drape(s) to create a ‘tent’ over the surgical field, such as the ‘Southampton Tent’,We evaluated the use of a novel drape ‘tent’ that has advantages over other designs in terms of having: (1) a CE marking; (2) no requirement for modification during assembly; and (3) no significant ‘spare’ drape gathering around the surgical field.The evaluation of the novel drape ‘tent’ occurred during a mastoid surgical procedure performed on a two-year-old child.The microscope  was initially covered with a standard sterile microscope drape (Glass Lens Micro-Kover (product reference: 09-GL902); Advance Medical Designs, Marietta, Georgia, USA). The drape ‘tent’ used in this case consisted of a CE marked sterile polyethylene drape with a custom-made hole that fit securely over the lens cap of the standard microscope drape ; Vital Care Industries, Tinley Park, Illinois, USA ). The dr18 In order to access the surgical field, the operating surgeon was required to pass their hands underneath the drape ‘tent’.No modifications or alterations to the drape were required during assembly, and the visual field was unobscured by the drape (a hinderance of previous designs).The time taken to assemble the drape ‘tent’ was 3 minutes and 30 seconds, and the time taken to disassemble the drape ‘tent’ was 2 minutes; hence, each process had a negligible effect on the total length of the surgical procedure.The surgeon reported that the drape ‘tent’ did not affect their mobility, dexterity or tactility during the surgical procedure. No macroscopic droplet spread or particulate matter outside of the drape ‘tent’ was visualised by the surgeon at any point during the surgery.19The findings in this report align with evidence from pre-clinical studies, in which the use of a protective barrier in the form of a drape ‘tent’ substantially reduced the dispersion of particulate matter during temporal bone drilling.18 The ‘OtoTent’ utilises a 3M™ Steri-Drape™ 1060 that requires an incision to be made in the drape before it can be secured to the microscope lens mount.16 The Great Ormond Street Hospital drape ‘tent’ involves the customising and attaching of four overlapping panels of a 3M Steri-Drape 1060 to the side of a draped microscope.18 In contrast, the use of our novel drape ‘tent’ does not require any modifications for assembly, and no significant amount of ‘spare’ drape gathers around the surgical field. Furthermore, it is already CE marked for clinical use in its current form. We advocate the use of such a drape ‘tent’ during mastoid surgery in the Covid-19 era.We report substantial advantages for this novel drape ‘tent’ over other designs. Specifically, the ‘Southampton Tent’, which involves the use of a second microscope drape to create a ‘tent’ over the patient, is reported to have a significant amount of ‘spare’ tent; this subsequently impacts on surgical efficiency in terms of instrument use.