The immune system has been shown to play a critical role in controlling tumor development. Therefore, therapeutic strategies based on targeting tumors from this point of view are becoming increasingly important. One of the approaches offered by nanotechnology and small molecules in this context is the induction of immunogenic cell death (ICD). In this work, a characterization of the antitumor response of paramagnetic iron nanoparticles conjugated with a platinum compound is presented. A highlight of this work is the significant improvement in the antitumor properties of the nanoconjugate comparing it with the platinum compound. Moreover, the conjugation has been shown to have crucial properties for the induction of ICD, such as the activation of cellular endoplasmic reticulum stress. Our results emphasize the relevance of nanotechnology as a fundamental strategy for the development of new approaches with onco-immunotherapy activity.From chemistry design to clinical application, several approaches have been developed to overcome platinum drawbacks in antitumoral therapies. An in-depth understanding of intracellular signaling may hold the key to the relationship of both conventional drugs and nanoparticles. Within these strategies, first, nanotechnology has become an essential tool in oncotherapy, improving biopharmaceutical properties and providing new immunomodulatory profiles to conventional drugs mediated by activation of endoplasmic reticulum (ER) stress. Secondly, functional proteomics techniques based on microarrays have proven to be a successful method for high throughput screening of proteins and profiling of biomolecule mechanisms of action. Here, we conducted a systematic characterization of the antitumor profile of a platinum compound conjugated with iron oxide nanoparticles (IONPs). As a result of the nano-conjugation, cytotoxic and proteomics profiles revealed a significant improvement in the antitumor properties of the starting material, providing selectivity in certain tumor cell lines tested. Moreover, cell death patterns associated with immunogenic cell death (ICD) response have also been identified when ER signaling pathways have been triggered. The evaluation in several tumor cell lines and the analysis by functional proteomics techniques have shown novel perspectives on the design of new cisplatin-derived conjugates, the high value of IONPs as drug delivery systems and ICD as a rewarding approach for targeted oncotherapy and onco-immunotherapies. Nanotechnology has emerged as a fundamental tool to improve the biopharmaceutical profiles of currently available drugs and small molecules as potential therapeutic drugs. These functionalization strategies have played a key role not only for novel drugs, but also for those conventional therapies that can be reintroduced or repurposed for different clinical applications . A notabNanotechnology and platinum therapies have demonstrated to converge in their antitumor profiles in immunogenic cell death (ICD) . Apart fWithin the wide variety of available nanocomplexes, iron oxide nanoparticles (IONPs) present most of the essential characteristics that make them suitable for onco-immunotherapy. Classical applications of IONPs are related to diagnosis and therapy, such as magnetic resonance images (as contrast agents) or in hyperthermia ,15,16,17® , and 6-well clear flat bottom plates were purchased from Corning . Further details about the reagents of the chemistry section can be found in previous reports   was slowly added dropwise. It was stirred in the dark at 60 °C for 24 h. Next, extraction with methanol was carried out using C-18 cartridges. The solvent was evaporated and the residue was extracted with 4 fractions of methanol (10 mL/fraction). The solution was then slowly applied to a preparative chromatography plate  using isobutyl acetate-methanol (35/65% v/v) as eluent. The compound was extracted with methanol, obtaining 19 mg (17%) of [PtCl(GUDCA)en]. Physical appearance: slightly yellowish solid. Elemental analysis of PtC28H50N3O5Cl : C, 45.49/44.85; H, 6.82/6.73; N, 5.68/5.60; Pt, 26.38/25.67. FT-IR (cm−1) (1H-NMR (3OD): 0.72 , 0.96 , 1.03 , 2.50 , 4.03 . 13C-NMR (3OD): 12.5 (C18), 19.88 (C19), 22.0 (C21), 24.1 (C19), 27.8 (C15), 49.2 , 180.1 (C24), 189.9 (C2′). 195Pt-NMR (3OD): −2269. 2D-NMR  of glycoursodeoxycholic acid (HGUDCA) was added to water (200 mL) and sodium carbonate to facilitate dissolution of bile acid. The solution was then heated at 60 °C until completely dissolved. To this solution, another aqueous solution (200 mL) of , thus obtaining a suspension of IONP@BCP@[PtCl(GUDCA)in] NPs.The platinum complex with glycoursodeoxycholic acid [PtCl(GUDCA)en] was incubated with IONP@BCP NPs for 24 h, incubating a 0.097 mg/mL suspension in a 4.95 × 103 overnight. Dynamic light scattering (DLS) and zeta potential measurements of ferrofluids were performed using a Malvern Zetasizer NS . The measurements were performed after dilution in ultra-pure water and were repeated at least three times on each sample to ensure consistency. Cryo Scanning Transmission Electron Microscopy (cryoSTEM) observations were carried out on a FEI Tecnai F30 microscope .Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) was performed in a plasma 40 ICP Perkin-Elmer spectrometer. Samples for ICP-OES were prepared by freeze drying the SPIONs suspensions and digestion of the solid in concentrated HNOThe incubated mixture of the IONPs with the platinum complex, IONP@BCP@[PtCl(GUDCA)en] (1 mL), was placed in an Eppendorf whose top had been replaced by a cellophane membrane . The system was placed in contact with 5 mL of ultrapure water, keeping the device closed to prevent evaporation. It was observed that only the released platinum complex crosses the membrane. The absorbance at 240 nm was then recorded against time. The data fitted a first-order kinetics and a half-life of t1/2 = ln2/k = 266.6 h.® HTB-37™); HT-29 (ATCC® HTB-38™); H460 (ATCC® HTB-177); A549 (ATCC® CCL-185)] were cultured at 37 °C in a humidified CO2 incubator (5% CO2) in complete RPMI media (Jurkat) or DMEM media  (both supplemented with 10% (v/v) FBS and 1% (v/v) P-S). When the cells reached 80% confluence, they were subcultured. For the adherent cells , 0.25% trypsin-EDTA was used to detach the cells. When necessary, the cells were counted using a Neubauer counting chamber and dyed with Trypan Blue.All the human cell lines  with IONPs was confirmed by UV-visible and fluorescence spectra. The visible spectra showed the band at 562 nm corresponding to rhodamine and a charge transfer that hides the characteristic shoulder of the platinum complex. The fluorescence spectrum of the IONP@BCP@[PtCl(GUDCA)en] incubated with the platinum complex displayed a notable increase in fluorescence intensity in rhodamine emission with respect to the NPs without the platinum complex, assigned to FRET mechanism. The proximity of both fluorophores could be calculated by overlapping the emission band of [PtCl(GUDCA)en], which acts as a donor, and the absorption band of rhodamine, which would be the acceptor. This overlap allowed us to calculate the most probable distance (Föster radius) between both fluorophores, which in this case is Ro = 40.3 Å. Values of the Föster radius in the range 20–90 Å indicate an efficient energy transfer between fluorophores  (named in this section as Pt) and novel IONP@BCP@[Pt(GUDCA)en] (named in this section as NP-Pt) were tested in several tumor cell lines. Viability was assessed in five different tumor cells: lung cancer cell lines (H460 and A549), colon carcinoma cell lines (Caco-2 and HT-29), and a T lymphocyte cell line (Jurkat). The time of incubation of the compounds with cells was also taken into consideration (24 h and 72 h) as well as drug concentration  to define an overall perspective of the activity of the platinum compound on its own and after conjugation to the NP . At firs50 values between NP-Pt and Pt were studied. As displayed in 50 value in the NP-Pt treated condition. It was detected that H460 cells showed a significant increase in cytotoxicity for NP-Pt with respect to the Pt. There is also a considerable increase, although not significant, in cytotoxicity in A549 and Jurkat cells between NP-Pt and Pt. However, the most interesting ratio is reached by NP-Pt in the Caco-2 line, where the IC50 value of NP-Pt is decreased up to nine-fold with respect to the reported IC50 of the platinum compound. These results suggest that the properties of the Pt have been improved in certain tumor cell lines by conjugation with NPs, and provide promising results for further research on signaling networks mediated by NP-Pt and Pt and their comparison.Here, the focus was on the effect analysis of the cytotoxicity of the conjugate with respect to Pt. For this purpose, the differences between the ICEvaluation of the intracellular pathways has been carried out by incubation of biotin labeled cell lysates on antibody microarrays as depicted in On one side, apoptosis triggered by DNA in the transition of G1/S phase of the cell cycle, in the DNA replication phase. On the other side, proteins involved in ER stress, a key feature for the development of ICD, have been analyzed B. IntracSubsequently, a more exhaustive analysis of the variations in intracellular protein levels concerning the Pt compound and the NP was conducted. It should be noted that the cells with significant variations were those in which the NP-Pt conjugate promoted greater cytotoxicity. These viability results can be correlated with the variation of intracellular protein concentrations D. AnalyzIn Caco-2 cells, there was a smaller number of significant proteins. In this case, proteins behaved similarly in the NP-Pt and in the Pt compound (SOD2 and IL2) and others that presented an opposite trend (TRAP1 and SOD2). Finally, in Jurkat cells, differences were only observed in two proteins, ABL1 and SOD2. In this case, the SOD2 protein was common to the Caco-2 line and presented different levels in Pt and NP-Pt.This new bile-acid platinum complex and novel nanoconjugate demonstrated cytotoxic properties and immunomodulatory potential profile when testing in different tumor cell lines. The viability of cells was analyzed in different tumor cell lines, demonstrating changes in the behavior of the platinum complex and the nanoconjugate between them. This fact highlights the need to always explore several pharmacological targets to prevent toxicity and to establish a structure–activity relationship of the new compounds synthesized.High-throughput functional proteomics analysis provided a correlation between intracellular signaling, cytotoxicity, and ICD patterns mediated by ER stress activation A. In theHSF1 and SRC proteins play a crucial role in the activation of ER stress and ICD. On the one hand, HSF1 is a transcription factor for the synthesis of “heat shock proteins” such as HSP90 or HSP70, chaperone-like proteins synthesized by the mitochondria in situations of cellular stress, and which are considered DAMPs when released in the cell death process . Along tA similar insight was observed in the Caco-2 cells by activation of mitochondrial signaling. This organelle plays its role in the management of reactive oxygen species through the activation of proteins such as SOD2. This protein converts ROS into non-reactive substances and detoxifies the cell through biochemical proton/electron exchange reactions. In this case, the SOD2 protein is also described as participating in the activation of the UPR . TRAP1, Nanoconjugates from platinum derivates and IONPs antitumoral properties have proved to be very interesting and selective in different tumor cell lines. The improvement of the properties of the platinum complex after inclusion in the nanosystem enhanced the activity significantly. In addition, promising immunogenic properties of the nanoconjugate have been described by ER stress activation. Thus, we can conclude that cisplatin-derived agents together with the high value of IONPs as drug delivery systems and immunogenic cell death are a promising approach to continue searching for novel strategies in nanotechnology and in antitumoral- and immunotherapy. High-throughput functional proteomics-based techniques also provide a suitable approach to deciphering intracellular pathways and slight differences in biopharmaceutical approaches.


Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [+PSI] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [+PSI] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.The yeast Sup35 protein misfolds into the infectious [ Saccharomyces cerevisiae, which has made yeast a model organism to study prion transmission  yeast prion, which forms when the translation termination protein, Sup35, misfolds into amyloid fibers or prion seeds [+PSI] prion [+PSI] prion [We have been studying the [on seeds . Since t+] prion ,12,13. H+] prion . It is k+] prion ,20,21. U+] prion . The yea+] prion .+PSI] prion occurs as different variants. Structural studies have shown that the different [+PSI] variants differ in their conformations and biophysical properties [+PSI] variants, which have fewer seeds and more soluble Sup35 than the strong [+PSI] variants, are cured faster by Hsp104 overexpression than the strong [+PSI] variants [+PSI] variants, but not the strong [+PSI] variants  was discovered more than 25 years ago, there is still no consensus on the mechanism of this curing. An understanding of the mechanism is confounded by the finding that other proteins, such as Hsp90 and the cochaperone STI1, also affect the rate of [+PSI] curing by Hsp104 overexpression [+PSI] by Hsp104 overexpression. One proposed mechanism is that this curing is caused by asymmetric segregation of the prion seeds [+PSI] seeds  seeds . However+] seeds . They thAnother model proposed by the Mogk laboratory is that when Hsp104 is overexpressed, it causes curing by binding to the seeds in a nonproductive conformation that inhibits the severing of the seeds . However+PSI] prion, which suggested that this curing is caused by an increase in the trimming of the prion seeds by Hsp104  by Hsp104 overexpression. One prediction is that, if curing is caused by asymmetric segregation of the seeds, then the daughter cells should cure faster than the mother cells, whereas if curing is caused by dissolution of the seeds, mother and daughter cells should cure at the same rate. Another key difference between these models is that curing by asymmetric segregation has been reported to require Sir2 [There are two key differences between the asymmetric segregation model and the dissolution model for the curing of [ire Sir2  and more+PSI] by Hsp104 overexpression. First, using a weak [+PSI] variant, we showed that mother and daughter cells cure at the same rate, consistent with our previous observations [+PSI] variant. In fact, we were able to almost completely block cell division and still cure [+PSI] by Hsp104 overexpression in 10% ethanol nor did curing by Hsp104 overexpression require Sir2. Interestingly, ethanol, which both arrests cell division and induces higher levels of Hsp104 [+PSI] without having to overexpress Hsp104 from a plasmid. These data show that [+PSI] can be cured without cell division and therefore, strongly support the dissolution model of curing by Hsp104 overexpression.Here, we have reexamined the curing of  curing by Hsp104 overexpression from the dissolution model of curing. One feature is the greater retention of seeds in the mother than the daughter cell, which, in turn, leads to faster curing of the daughter than the mother cell [+PSI] strain instead of the stronger [+PSI] variant that we used in our previous study [+PSI] variant was cured by Hsp104 overexpression, we obtained ~50% curing per generation when the weak L1758 [+PSI] variant was used  prior to separating the fluorescent mother cells from the non-fluorescent daughter cells via flow cytometry. These two populations were then plated on ½ YPD plates and the extent of curing was measured using the red/white colony assay. In this assay, only completely red colonies as opposed to red/white sectored colonies indicate cured cells. As shown in +PSI] by Hsp104 overexpression. The L1758 [SIR2 affected the curing of the L1758 [+PSI] strain. Sir2 is a protein that is required for the formation of the polarisome, which, in turn, functions in the transport of misfolded proteins from daughter to mother cell [+PSI] by Hsp104 overexpression would not be affected by Sir2 expression. As shown in +PSI] variant. As with the L1758 [+PSI] strain, the curing of the 779-6A [+PSI] strain by Hsp104 overexpression was not affected by Sir2 expression  in 10% ethanol, ~50% of the L1758 [+PSI] was cured in synthetic medium with glucose as a carbon source  from the SUP35 chromosomal locus. Next, we imaged GFP-labeled Sup35 in [+PSI] strain was cured without cell division by expression of high levels of Hsp104 in 10% ethanol, we first imaged the L2888 [+PSI] strain transformed with the empty vector in galactose and 10% ethanol. As shown in +PSI] strain. These results are consistent with our previous studies that showed that the L1758 [+PSI] strain cures more readily than the L2888 [+PSI] strain and higher level of Hsp104 expression is needed to cure stronger [+PSI] variants [Since the L1758 [variants .+PSI] strain, we transformed the L2888 [+PSI] strain with a plasmid to overexpress Hsp104 from the GAL1 promoter. When Hsp104 is overexpressed from the GAL1 promoter for extended times, there is greater than 10-fold Hsp104 overexpression [To achieve curing of the L2888 [pression . Galacto+PSI] by Hsp104 overexpression. When the cells were retrieved from the chamber slide after overnight imaging, we found that they grew a little more than one generation and during that time, the plating assay showed that 7% of the cells were cured and 44% of the cells were red/white sectored, indicating that the yeasts were cured shortly after being plated. Importantly, none of the cells that we imaged showed formation of large aggregates or tethering of the foci to a subcellular structure, mechanisms that have been proposed to account for asymmetric segregation of the seeds by causing their retention in the mother cell. Therefore, the imaging data showing a loss of foci in the absence of cell division are consistent with the dissolution model rather than the asymmetric segregation model for the curing of [+PSI] by Hsp104 overexpression can occur in the absence of cell division, we applied other methods that have been reported to inhibit cell division. Addition of nocodazole was not effective in inhibiting cell division, but we were more successful when we added the mating pheromone, alpha-factor [+PSI] strain in the absence of Hsp104 overexpression. However, when Hsp104 was overexpressed from the GAL1 promoter for 4 h in L1758 [+PSI], there was ~65% curing of the L1758 [+PSI] strain by Hsp104 overexpression. Since alpha-factor confers at least partial inhibition of growth, it would be expected to affect the extent of curing according to the asymmetric segregation model. However, it did not affect the curing of L1758 [+PSI] yeast by Hsp104 overexpression indicating that this curing is due to the dissolution of the prion seeds. To further confirm that the curing of [a-factor . Based o+PSI] in the absence of Hsp104 overexpression  of the mother–daughter pairs showed no significant difference in prion seed number . In fact+PSI] strain than predicted by the asymmetric segregation model. In ethanol, we effectively completely arrested cell division and still were able to cure the L1758 [+PSI] strain. As a control, since the curing of the [URE3] yeast prion by overexpression of Btn2 has been reported to be due to asymmetric segregation of the [URE3] seeds  seeds ,47, we e+PSI] strain in ethanol. Ethanol stress has been shown to both arrest cell growth and increase Hsp104 expression from the HSP104 promoter [+PSI] yeast, indicating ethanol stress increases the amount of soluble Sup35 [+PSI] variant, it did not cure stronger [+PSI] variants; e.g., L2888 [+PSI]. Furthermore, our results showing that ethanol alone cured the weak L1758 [+PSI] variant might explain why there is such a low frequency of [+PSI] in wild-type strains [+PSI] variants that spontaneously form in yeast. Unexpectedly, we also observed that it was not necessary to overexpress Hsp104 to cure the L1758  cells overexpressing Hsp104 in 10% ethanol. The images showed a progressive loss of foci and a complete loss of foci in some cells without cell division. These results are not consistent with the asymmetric segregation model for curing since the seeds did not form large aggregates and were freely mobile with no anchoring to a subcellular structure. The loss of foci independent of cell division is consistent with our previous observation that Hsp104 causes a loss of prion seeds due to the trimming activity of Hsp104, which ultimately produces curing when all of the seeds are eliminated by trimming [We also imaged GFP-labeldSup35 foci in [trimming . +PSI] is heat shock [+PSI] by asymmetric segregation of the prion seeds, a suggestion that was supported by their finding that deleting proteins which were essential for polarisome formation markedly inhibited curing. However, the findings of the laboratories differ in that the Chernoff laboratory reported that the daughter cells were preferentially cured relative to the mother cells whereas the Serio laboratory reported the opposite; i.e., that the mother cells were preferentially cured relative to the daughter cells. Furthermore, the Chernoff laboratory proposed that the asymmetric segregation of the prion seeds was due to the sequestering of the aggregated prion seeds in the mother cell, whereas the Serio laboratory proposed that the asymmetric segregation was due to the presence of higher levels of Hsp104 in the mother cells than in the daughter cells. Despite the differences between these laboratories, their results make clear that heat shock causes [+PSI] curing by a very different mechanism than ethanol stress. Therefore, one cannot generalize that because heat shock causes [+PSI] curing by the mechanism of asymmetric segregation of the prion seeds, this is also the case for other conditions where Hsp104 is overexpressed; e.g., ethanol stress. Rather, our results clearly show that ethanol stress causes [+PSI] curing by the dissolution mechanism; i.e., curing is caused by the trimming activity of the highly expressed Hsp104. Another stress that cures  by Hsp104 overexpression was also dependent on the N-terminal domain of Hsp104, indicating that this domain functions in the trimming of the prion seeds by Hsp104  yeast were counted as [+PSI]. A minimum of three colonies were averaged for each experiment. Yeasts were grown at 30 °C on synthetic defined medium  with complete supplement mixture or the appropriate amino acid dropout for selection and maintenance of the plasmid (Sunrise Scientific). Synthetic galactose  medium contains both 2% galactose and 2% raffinose in place of glucose. ½ YPD solid medium used in the plating assays contains 0.5% yeast extract, 2% peptone, and 2% glucose. Cultures were always maintained in active growing conditions (ODCells were kept in the logarithmic phase (OD 600 < 0.6) for 24 h prior to experiment and were grown in a 30 °C incubator. At the start of the experiment, cells were diluted to an OD 600 of 0.20. To inhibit cell division, we used 15 µg/mL of nocodazole , 5 µg/mL alpha-factor  or 0.2 M hydroxyurea (Sigma). To arrest with ethanol, 100% ethanol was added to the cells to make a final concentration of 10% ethanol. Yeast growth was also monitored by measuring the absorbance of the samples at 600 nm using the Synergy H1 microplate reader (Biotek). Yeast in a 48-well tissue culture plates (Sarstedt) were maintained with constant shaking at 30 °C. Measurements were obtained at 10 min intervals for 24 h. +PSI] yeasts were kept in the logarithmic phase (OD 600 < 0.6) for 24 h prior to experiment and were grown at 30 °C. For staining, fluorescent disodium 28 brightener (Sigma) was added to the cells at a final concentration of 0.005% for 5 min [L1758 [or 5 min . After 52 chambered coverslips (Cellvis).Fluorescence images were obtained on the Zeiss LSM 880 Airyscan confocal microscope equipped with a 63× 1.4 NA objective. Z-stack confocal images were taken for all cells. The yeasts were imaged in 8-well 25-mmCell lysis and Western blotting were performed as described previously  The foll


Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models.Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances.In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture. However, this assumption has been disputed by a growing body of research, in which genetic instability has been explicitly characterized in various cell lines during in vitro culture.TP53, PTEN, and KRAS.Genomic instability, one of the enabling characteristics of cancer,In a research setting, CIN is often a consequence of CRISPR‐Cas9 gene editing. One of the major concerns associated with the use of CRISPR‐Cas9 is the potential for unintended off‐target effects.CIN poses a significant challenge to cancer research because the genetic and phenotypic heterogeneity that arises due to CIN in cell lines can compromise the accuracy and reproducibility of experiments. Additionally, the genetic changes that occur in cell lines over time may not accurately reflect the genetic alterations present in the original tumor. While background controls are often included in published studies, they may not fully capture the indirect effects of CIN. For instance, Chromosome segregation errors during mitosis can produce unstable micronuclei that rupture and release genomic DNA into the cytosol.This review will provide an overview of the fundamental mechanisms of CIN, highlight the implications of CIN in fundamental research, and discuss how CIN can be monitored and controlled during cell culture.2Chromosome stability relies on the correct separation of duplicated chromosomes to daughter cells during mitosis.Over the past two decades, extensive work has been dedicated to understanding the molecular principles underlying CIN. To date, numerous processes that contribute to CIN have been identified, including defects in kinetochore‐microtubule (k‐MT) attachment, telomere maintenance, DNA damage response (DDR), mitotic checkpoints and cell cycle regulation. While these mechanisms have varied prevalence across different cell types and were assumed to be independent, they are intrinsically connected and often interact with each other.3Chromosome segregation is the fundamental process of cell division; fidelity in this process during mitosis is crucial for genomic stability. Correct bipolar attachment is a prerequisite for proper chromosome segregation, which demands that kinetochores on every sister chromatid pair attach to microtubules emanating from opposite centrosomes Figure . There a4Another mechanism that has been proposed to induce CIN is defective telomere maintenance. Telomeres are regions of repetitive DNA sequences found at the ends of chromosomes that protect the genetic material from degradation or fusion with other chromosomes.5Chromosomal DNA is constantly damaged by internal and external factors, such as ionizing radiation, ultraviolet radiation, and reactive oxygen species.A novel connection between DDR and CIN was discovered by Bakhoum et al.6The mitotic checkpoint, also known as the spindle assembly checkpoint (SAC), is a critical surveillance mechanism that maintains the correct chromosome number during mitosis.The key components of the SAC include Mitotic arrest deficiency 1 (Mad1), Mad2, budding uninhibited by benzimidazoles 1 (Bub1), Bub3, BubR1, and Cell division cycle protein 20 homolog (Cdc20).7The mechanism of cell cycle disturbances is a broad topic because it is implicated in many of the mechanisms mentioned above. There are many cell‐cycle regulators that play an important role in CIN, such as cyclin E, p53 and forkhead box protein M1 (FoxM1).8Established cell lines have played an essential part in biomedical research as an inexhaustible source of biological material. However, researchers have generally found considerable CIN in many different cell lines, and questions have been raised about the comparability of cell lines to the original cells from which they were derived. Previous studies have shown that cell cultures have the potential to undergo major karyotypic changes over time due to CIN, which results in the constant emergence of variants and subsequent heterogeneity within an assumed monoclonal cell line.Vargas‐Rondón9The source of the cell lines should be carefully selected. Numerous academic or commercial organizations have established cell culture banks, such as American type culture collection (ATCC), Cellbank Australia, and Coriell cell repository.Frozen stocks of continuous cell lines should be produced at specific time points, so researchers can regularly return to these stocks to minimize the passage number or repeat experiments. Also, when a cell line is not used, it is preferable to return it to a cryopreserved bank to avoid unnecessary long‐term cell culture and passaging. When working with cell lines, it is recommended that primary tests be conducted to specify a passage number range that is appropriate for maintaining consistent cell functions that is, only a limited number of passages are performed before returning to another batch of the same stock. Importantly, since there is no easy way to determine how many times a cell culture has been passaged, every passaging must be properly recorded. When thawed from the freezer the passage number continues and is added to the number at freezing.2 levels. Additionally, good laboratory practices are also critical to prevent contamination. Mycoplasma infection is a common contamination issue that can cause CIN by releasing enzymes that break down DNA, leading to mutations and chromosomal aberrations.The growth environment of cell lines is a crucial factor that can significantly impact the stability of the cells. Even slight variations in the culture conditions can lead to significant changes in the characteristics of the cells. Frequent passages and transfers between laboratories can expose cells to varying environments, potentially leading to the selection of clones better adapted to the new conditions.Observing cell morphology under an inverted microscope is considered the simplest way to assess the quality and stability of cells. In general, cell lines with high CIN levels often undergo morphological changes and display heterogeneous cellular morphology.Cell line authentication and characterization should be performed regularly using suitable methods, and baselines should be established to check for unacceptable changes throughout experiments. If certain cellular products or functions are being investigated, these characteristics should also be monitored to determine whether and how they change over time in culture. Currently, short tandem repeat (STR) profiling is widely used as a standard method for authenticating human cell lines because it is inexpensive and easy to interpret.Cell line instability has been a long‐standing issue highlighting concerns about data validity and reproducibility. While it is impossible to prevent a cell line from evolving in culture, researchers should take actions to improve the reliability and reproducibility of cell‐based research. Here we set out some recommendations for researchers to monitor and control CIN during cell culture.10As discussed in this review, CIN is a complex and multifaceted phenomenon that is caused by many different mechanisms and is regulated by hundreds of genes. Further research will help identify the central drivers and suppressors of CIN, as well as their interactions. Ultimately, a better understanding of CIN will enable us to prevent its associated issues during cell culture.Current research on CIN is challenging due to the lack of a standardized definition and measurement criteria for CIN. Many studies based their estimates of CIN levels on their own data, and there is a lack of consensus about how to measure the level of CIN. Such ambiguity makes it difficult to interpret data and compare the results of different studies. CIN is defined as an increase in the rate of gain or loss of whole or fractions of chromosomes.Not only do we need to clarify the definition of CIN, but we also need the appropriate techniques to measure it. The broad range of chromosomal abnormalities makes it extremely difficult to quantify CIN. Apart from common structural aberrations like translocations, duplications, deletions, and inversions, there are many unclassified aberrations that cannot be easily detected or quantified. One such chromosomal aberration is chromothripsis, a process of rapid and massive genome re‐organization that results in highly rearranged chromosomes.As discussed earlier, CIN is, by definition, the rate of gain or loss of fractional or whole chromosomes in a population of cells. Hence, the measurement of CIN requires monitoring of chromosomal changes over time. Such time‐based measurements are feasible on cell cultures but impossible on fixed tissue. Alternatively, CIN can be more readily assessed by measuring cell‐to‐cell variability at a single time point.Understanding the consequences of chromosomal instability (CIN) in cancer cell lines is important for cancer research in several ways. The presence of CIN can be used as a diagnostic and prognostic marker for cancer and help identify subtypes of cancer that are more likely to have a poor prognosis or be resistant to treatment. Bold et al.CIN is a key driver of cancer development and progression. Understanding the mechanisms and consequences of CIN can help identify potential preventive strategies, such as lifestyle changes or targeted interventions to reduce the risk of cancer. Understanding the consequences of CIN in different cancer cell lines can help guide the development of personalized treatment strategies that consider the specific molecular characteristics of an individual's cancer. For example, Martín et al.To conclude, cell lines remain a powerful tool for biomedical research, but the high level of CIN during culture and the variation across cell line strains must be well considered in experimental design and data interpretation. The scientific community need to work together to increase engagement and awareness. Organizations can formulate and refine the guidelines for the handling of cell lines. We, as responsible researchers, should take practical steps to address the issues arising from unstable cell lines and ensure the validity of our research.Zichen He: Investigation (lead); writing – original draft (lead). Andrew David Wilson: Formal analysis (supporting); project administration (supporting); supervision (supporting); writing – original draft (supporting). Fenella Jean Rich: Investigation (supporting); resources (supporting). Diane Kenwright: Investigation (supporting); resources . Aaron John Stevens: Supervision (supporting); writing – review and editing (supporting). Yee Syuen Low: Investigation (supporting); project administration (supporting); resources (supporting). Michelle Thunders: Conceptualization ; funding acquisition ; project administration ; resources ; supervision (lead); writing – review and editing .The authors have stated explicitly that there are no conflicts of interest in connection with this article.This is a review article and did not require consent or ethical approval.


Salvia officinalis L.) collected along the Adriatic coast of Croatia. Scanning electron microscopy (SEM) was used to examine the morphology of the stem and leaf surfaces. Essential oil excretory glands were detected on both the leaves and stem surfaces. The essential oils were isolated by hydrodistillation, and their chemical composition was determined by gas chromatography and mass spectrometry (GC-MS). Sage essential oils were mixtures of terpene compounds, among which the most common were: α- and β-thujone, camphor, and 1,8-cineol. Cytotoxic activity was tested using MTS assay on multiple cell lines: normal and immortalized fibroblasts (HF77FA and HDF-Tert), immortalized lung line (BEAS-2B), and breast adenocarcinoma (MDA-MB-231). The growth of treated cells was determined relative to control conditions without treatment. The immortalized lung line was the least resistant to the activity of the essential oils, whereas immortalized fibroblasts were the most resistant. Statistical analysis has connected the cytotoxic effect and chemical composition of the studied essential oils. To the best of our knowledge, this work is the first testing of the cytotoxic activity of S. officinalis EO’s on the BEAS-2B, HF77FA, and HDF-Tert cell lines. The presented data on essential oil chemical composition and cytotoxic effect on 4 types of human cells supports pharmacotherapeutic potential this plant is known to have.In our study, we investigated the chemical composition and cytotoxic activity of essential oils isolated from Dalmatian sage ( Lamiaceae family  vs. normalized response—Variable slope in-built equation provided in the software. Calculated IC50 values are expressed as mean ± standard deviation. Data distribution was determined by performing Shapiro-Wilk normality test. Data were normally distributed (s tested . To inteS. officinalis using SEM, following two protocols that preserve the natural structure of the tissue surface. We also analyzed the chemical composition of seven S. officinalis EOs using GC/MS and assessed cell viability using MTS assay at various doses of EOs on healthy and cancerous cells.In this study, we investigated the stem and leaf tissue structures of S. officinalis EOs showed that they contain varying percentages of common chemical constituents: α- and β-thujone, camphor, and 1,8-cineole. The biological effects of sage EOs on cell viability were tested on different healthy and cancerous cells. The results showed that skin and immortalized cells were more resistant to sage EO, while the BEAS-2B cell line was the least resistant compared to the other cell lines. Using one cancerous and three healthy cell lines, we investigated the cytotoxic effect of our EOs to get results that simulate physiological conditions. Our results showed that two fibroblast and cancer-derived cell lines were less susceptible to S. officinalis EO than non-cancer cell lines derived from lung tissue. Healthy cell lines were more resistant to cytotoxic agents than cancer cell lines.The chemical analysis of seven S. officinalis EOs are related to the differences in chemical composition.The statistical analysis and correlation of the chemical composition of EO and the tested cell viability have shown that the different cytotoxic activities of S. officinalis EOs on BEAS-2B, HF77FA, and HDF-Tert cell lines. The data presented on the chemical composition of the EOs and the cytotoxic effect on 4 types of human cells support the pharmacotherapeutic potential that this plant is known to possess. High IC50 values of EOs tested on healthy human dermal fibroblasts give more arguments to the traditional use of S. officinalis EOs for skin treatments and cosmetic purposes. Further investigation of biological activities of mixtures of isolated chemical constituents and/or combinations of chemical isolated constituents, present in S. officinalis essential oils, could provide a more in depth understanding of the pharmacological properties of this plant.To the best of our knowledge, this is the first study on the cytotoxic activity of


The Human Pluripotent Stem Cell Registry established a database of clinical studies using human pluripotent stem cells (PSCs) as starting material for cell therapies. Since 2018, we have observed a switch toward human induced pluripotent stem cells (iPSCs) from human embryonic stem cells. However, rather than using iPSCs for personalized medicines, allogeneic approaches dominate. Most treatments target ophthalmopathies, and genetically modified iPSCs are used to generate tailored cells. We observe a lack of standardization and transparency about the PSCs lines used, characterization of the PSC-derived cells, and the preclinical models and assays applied to show efficacy and safety. https://hpscreg.eu/clinical_studies). Persistent lack of standardization and transparency about the source cell lines, characterization of the therapeutic product, applied preclinical models, and efficacy and safety assays are mounting obstacles to clinical study development and reproducibility.In this perspective, Kobold et al. identify 109 clinical studies using pluripotent stem cells performed in 14 countries targeting 44 diseases by analyzing data from hPSCreg ( For efficient progress in the field at this early stage, it is therefore important to have access to information on ongoing clinical studies, transparency about cell sources, derived cells as a final product, relevant efficacy and safety assays, and outcomes. Because this information is difficult to obtain, a clinical study database dedicated to the use of human PSCs as starting material for cell therapies has been established . It is pOur data confirm a strong trend toward studies using human induced PSCs (iPSCs), while human embryonic stem cells (ESCs) continue to be used in a significant portion of roughly 50% of all studies. While the majority of studies up to 2017 utilized ESCs, since 2018, most studies were based on hiPSC-derived products. There is a remarkable drop of clinical studies initiated in 2022 compared with the previous two periods, which may reflect the impact of coronavirus disease 2019 (COVID-19).https://hpscreg.eu) provides a resource for available iPSC and ESC lines and certifies their identity, ethical provenance, and biological properties, among other related information. Together with its clinical study database, a resource linking information on PSC lines and clinical trials is available at hPSCreg. However, most clinical trials using iPSCs do not publicly disclose the PSC lines used or register these lines in hPSCreg. In fact, we are only able to trace the source PSC cell lines to 11 ESC lines and 1 iPSC line, of which 10 and 0 lines, respectively, are registered in hPSCreg . The name of each clinical trial sponsor is a required data element in the ICTRP standards for clinical trial registries who wish to be included in the ICTRP registry network ). In most cases (102 of 109 studies), the sponsor can be identified from the clinical study registry record. Within the last 5 years, industry has sponsored more clinical studies since the first PSC-derived cell therapy study was started in 2010 ;  in 2010 . IndustrThere are clear differences between countries. While in the United States, the number of industry-sponsored trials outweighs academically/government-sponsored trials  and are strongly represented even before 2018, Japan and China have only within the last 5 years started trials with industry sponsors, and the bulk of their trials remains predominantly academically/government sponsored as a result of strong national funding programs . Only a Within the last decade, the number of PSC-derived cell types used for therapies has grown from 3 to 22 cell types, encompassing 44 target indications/diseases . The mosclinicaltrials.gov: NCT02302157).The multiplicity of studies employing a single cell type also reflects different clinical study phases of the same cell product  or clinical application of the cells to diseases where cell degeneration is a common denominator. Another important additional consideration is clinical testing of any medical devices that have been developed to deliver the cell product to the appropriate site in the patient . For exawww.hpscreg.eu/browse/trials). For these 31 publications, 5 include preclinical work, 1 reports only preclinical data, 5 include manufacturing protocols and cell quality assessment parameters, and 30 report clinical study design and outcomes. Reports on termination, withdrawal, or failure of clinical studies are not published and, unfortunately, only in a few cases recorded in public registries. One could assume that those 17 studies that published outcome results are completed, and, thus, that, of the 109 recorded clinical studies, at most 93 are currently active.Disease targets for PSC-based therapies have broadened moderately . While 1Study duration, as defined by time between study start and study, is available for 90 of 109 studies and varies between 1 and 19 years . The time frame from start to completion may serve as a surrogate indicator for follow-up duration. No correlation was found between the duration of study, national regulatory framework, year when the study started, or disease category.https://hpscreg.eu; The sourced database hosted by hPSCreg uses a combination of search tools to gather information on worldwide clinical studies using PSCs as source material. The data are manually curated before being entered into the clinical study database and made publicly accessible at the hPSCreg portal (https://icmje.org/recommendations/). Moreover, information on cells used  is only occasionally available. Furthermore, although employed in hPSCreg, standardized ontologies, taxonomies, or terms to describe cell- or disease-specific data are rarely used in other clinical trial registries. Access to the data, comparability, and searchability require further use of standard terms and taxonomies; for example, for therapeutic cell type, target clinical feature or disease, or experimental procedures.Public clinical study registries, resources, and trial publications often do not use the same standards for reporting, which makes comparisons of data across the field and understanding potential causes of trial failure difficult. The hPSCreg database orients its reporting requirements on the WHO data fields to ease cross-linking of information, adhering to the International Committee of Medical Journal Editors (ICMJE) recommendations  encourages sharing of output data and study-related publications, albeit on a voluntary basis. For example, the ICMJE introduced a requirement for authors to report how individual patient data (IPD) will be shared when publishing clinical trial results . Relevant resources are now emerging, such as the European Union (EU)-funded COST (Cooperation in Science & Technology) innovation network “Generation of Human Induced Pluripotent Stem Cells From Haplo-Selected Cord Blood Samples” ; however, haplotyped iPSC lines have not yet been broadly exploited for clinical studies , the United States (Regenerative Medicine Advanced Therapy [RMAT] designation), and now in the EU (Priority Medicines [PRIME]) to streamline the process for approval of cell therapies . These trends confirm an enormous imbalance of global resources and likely will perpetuate unequal access to therapeutic benefits, which, of course, is not restricted to advanced therapies . Measureherapies . FurtherThe number of PSC-based cell clinical studies has rapidly increased in the last 5 years. In the next decade, we expect to see more strategic collaborations within industry as big pharma seeks to acquire the expertise of smaller companies to enter the cell therapy arena. At the same time, smaller companies forge strategic alliances with one another through licensing or collaboration agreements . MergersS.K., collection and assembly of data, data analysis, and generation of figures; N.B., implementation of the database and collection and assembly of data; G.S. and A.K., conception, design, and writing of the manuscript; S.C.M., writing and revision of the manuscript; N.M., data analysis and interpretation, manuscript writing, and generation of figures and tables.


Here, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines (C57BL6 genetic background): Panc02 , and two cell lines originated from PDACs developed in two different KPC  mouse models. Our study (i) characterized the ability of a widely used attenuated oncolytic vesicular stomatitis virus VSV-ΔM51-GFP to infect, replicate in, and kill mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-ΔM51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-ΔM51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. The exome analysis demonstrated that more VSV-ΔM51-GFP-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3.Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies demonstrated that human PDAC cell lines are highly variable in their permissiveness to OVs. Mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during cancer therapies. Mouse PDAC cell lines showed high divergence in their permissiveness to oncolytic vesicular stomatitis virus (VSV), which negatively correlated with their abilities to mount innate antiviral responses. Also, we discovered that more VSV-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. Our study provides essential information about three model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against different PDACs in immunocompetent mice.Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies using various human PDAC cell lines demonstrated that they are highly variable in their permissiveness to OVs. In this study, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines, which are widely used for  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that accounts for approximately 95% of pancreatic cancers and is the fourth cause of cancer-related deaths in the USA. The 5-year survival rate for PDAC patients has remained around 10%, while survival rates for other cancers have significantly improved .Mononegavirales, family Rhabdoviridae, genus Vesiculovirus) and a promising OV (Oncolytic virus (OV) therapy is a promising anticancer approach that utilizes replication-competent viruses that preferentially infect, replicate in, and kill cancer cells , 4. Vesiising OV –7. VSVising OV . The panising OV . The oncising OV –12, beising OV .in vivo PDAC mouse model systems fail to recapitulate all key characteristics of human PDAC disease   –16. Ime, etc.) . This ise, etc.) –20. MoIn this study, we examined three different allograftable mouse PDAC cell lines. Two of these cell lines originated from genetically engineered mouse models (GEMMs) of PDAC. GEMMs are created by introducing specific gene mutations in oncogenes and/or tumor suppressor genes that are central in human PDAC, effectively recapitulating PDAC in the mouse. The most robust and well-described PDAC GEMM is the KPC mouse, which is characterized by mutations in the Kras and Trp53, both of which are driven by a pancreas-specific Cre recombinase (via the Pdx1 promoter) which is expressed in all cells of the pancreas from early stages in development . ImportaIn this study, we examined phenotypically and genotypically three mouse PDAC cell lines (two KPCs and one non-KPC). Our study (i) characterized the ability of a widely used attenuated oncolytic virus VSV-ΔM51-GFP to infect, replicate in, and kill these mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-ΔM51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-ΔM51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. Our study provides essential data about three allograftable model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against phenotypically different PDACs in immunocompetent mice.2 atmosphere at 37°C. For all experiments, cells were kept for no more than 15 passages. All described experiments were approved by the University of North Carolina at Charlotte Institutional Biosafety Committee (IBC).The recombinant virus VSV-ΔM51-GFP was previously described , in whic2, then read using a multiwell plate reader at 450 nm. This was repeated every 24 h for 7 days. Cell doubling time (TD) was calculated using the equation TD  =  (0.693t)/ln (Nt/N0) where t =  time difference in h during log phase, Nt =  absorbance value at time t, and N0 =  absorbance value at the initial time.1,000 cells per well in DMEM with 10% FBS were seeded into 96-well plates. Cells were given 24 h to adhere. After each 24-h period, WST-8  cell viability reagent was added to each well for 4 h at 37°C in 5% CO2 for the duration of the experiment. Virus-encoded GFP fluorescence was measured periodically over a 72-h time course using a fluorescence multiwell plate reader. GFP fluorescence was measured at 485/535 nm.Virus titers were calculated using standard plaque assays on BHK-21 cells in 12 or 24-well plates. For virus replication kinetics experiments, cells were seeded into 96-well plates and were given 24 h to adhere. Virus dilutions were prepared in DMEM with 0% FBS. Cells were washed with phosphate-buffered saline (PBS), followed by the addition of the virus for 1 h at 37°C. Virus-containing medium was then aspirated and fresh DMEM with 5% FBS was added back to cells and incubated at 37°C in 5% CO2, then read using a multiwell plate reader at 450 nm. Results are expressed as fold change compared with mock treatment.In a 96-well plate layout, cells were seeded at 90% confluence and were given 24 h to adhere. Cells were then washed once with PBS and mock-infected, or infected at either multiplicity of infection (MOI) 1, 0.1, 0.01, 0.001, or 0.0001. After 1-h incubation at 37°C, the virus was removed and a fresh medium containing 5% FBS was added to each well. At 70 h p.i., WST-8  was added to each well for 4 h at 37°C in 5% COTwelve-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with VSV-ΔM51-GFP dilutions or mock-infected (control) for 24, 72, and 120 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I-0710) in DMEM with 5% FBS. After 24, 72, or 120 h, formalin was added to fix cells for 4 h. After fixation, agarose was removed and cells were stained with crystal violet . For the plaque assay figures, each row represents one 12-well plate that was cut and stitched to view the highest amount of virus to the least amount of virus from left to right.Cells were seeded into 12-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and cells were washed once with PBS. Cell lines were either mock-treated or infected with VSV-ΔM51-GFP at MOIs of 0.1 and 0.001 either based on virus titer on BHK-21 cells or based on virus titer on each cell line, in medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, the medium was removed and fresh medium with 0% FBS was added to each well. Cells were lysed and total protein was collected 24 h after infection using buffer as described previously . Total pCells were seeded into 12-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and washed once with PBS. VSV-ΔM51-GFP was added at MOIs of 1 and 0.1 (based on each cell line) in a medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, fresh medium was added to each well containing 0% FBS. After 24 h, cell supernatants (for cytokine array) and lysates (for western blot) were collected and stored at −80°C. Supernatants were then sent to EVE Technologies Corp for Mouse Cytokine/Chemokine 44-Plex Discovery Assay Array (MD44).50 values were calculated using GraphPad Prism 9.3.1.Cells were seeded into 96-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and cells were washed once with PBS. The virus was then added at MOI of 0.01 in medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, the medium was removed and fresh medium (0% FBS) containing mouse IFN alpha  was added at 2,500, 500, 100, 20, 4, or 0 units/mL. Virus-encoded GFP fluorescence was measured at 485/535 nm periodically over a 73-h time course using a fluorescence multiwell plate reader. ICSix-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with sixfold serial dilutions of VSV-ΔM51-GFP or VSV-Mwt-GFP from 1E−4 to 7.7E−8 or mock-infected for 72 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I- 0710) in DMEM with 5% FBS. After 72 h, formalin was added to fix cells for 3 h. After fixation, agarose was removed and cells were stained with crystal violet .Twelve-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with threefold serial dilutions of VSV-ΔM51-GFP or VSV-Mwt-GFP from 1E−4 to 2E−9 or mock-infected for 72 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I-0710) in DMEM with 5% FBS. After 24 h, virus-driven GFP focus forming units (FFUs) were counted using a fluorescent microscope. The results shown are relative to the titer on the KPC-Luc-4580 cell line.Cells were seeded into 12-well plates at 95% co0nfluence and were given 24 h to adhere. The medium was removed, and cells were washed with PBS. Cells were placed on ice approximately 5 min before virus infection to cool cells. Virus in DMEM with 0% FBS was added to cells on ice, and cells were incubated for 1 h at 4°C. After incubation, the virus-containing medium was aspirated, and cells were washed three times with cold PBS to remove any unbound virus. Samples were then either collected for cell lysates immediately (to examine attachment) or incubated for an additional 7 h at 37°C (to examine replication) and then cell lysates were collected. Total VSV protein was analyzed by western blot as described above. Membranes were blocked in 5% nonfat milk in TBS-T. Membranes were then incubated with a 1:5,000 dilution of rabbit polyclonal anti-VSV antibodies (raised against VSV virions) in TBS-T with 5% nonfat milk, followed by a 1:10,000 dilution of antirabbit secondary antibodies. To verify the total protein in each sample, loaded membranes were stained with Coomassie blue.50 values were calculated using GraphPad Prism 9.3.1.For gemcitabine and 5-FU determination, cells were seeded into 96-well plates for approximately 50% confluency in a medium supplemented with 10% FBS. The next day, cells were treated with serial dilutions of either gemcitabine  or 5-FU . The serial dilution ranges for gemcitabine and 5-FU were 1,000–0.008 µM and 300–0.006 µM, respectively. A WST-8  cell viability assay was performed 72 h later. ICEach cell line was grown to 70% confluence in a T-75 flask (Greiner #658170) and sent to the CytoGenomics Core Laboratory at the University of Minnesota. Adherent cells were harvested with colcemid arrest, treated with 0.75 M KCl hypotonic solution, and fixation with 3:1 methanol:acetic acid. The resulting cells were spread onto glass slides according to standard cytogenetic protocols. A spectral karyotyping (SKY) slide was processed according to the manufacturer’s protocol [Applied Spectral Imaging (ASI)]. SKY uses a unique combination of five fluorescent dyes to paint all 24 chromosomes. Seven metaphase cells per sample were examined by SKY using the Olympus BX61 microscope with DAPI and SKY fluorescence filter sets. G-band and SKY metaphase cells were imaged and karyotyped using ASI software. The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH Grant #P30 CA077598.6 cells), trypsinized, pelleted, frozen, and sent to Azenta US  for DNA extraction, library preparations, sequencing reactions, and bioinformatic analysis. DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Genomic DNA samples were quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Enrichment probes were designed against the region of interest and synthesized through Twist Biosciences—Twist Mouse Comprehensive Panel . Library preparation was performed according to the manufacturer’s guidelines. Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up, end-repaired, and adenylated at the 3′ends. Adapters were ligated to the DNA fragments, and adapter-ligated DNA fragments were enriched with limited cycle PCR. Adapter-ligated DNA fragments were validated using Agilent TapeStation (Agilent Technologies) and quantified using Qubit 2.0 Fluorometer. Adapter-ligated DNA fragments were hybridized with biotinylated baits. The hybrid DNAs were captured by streptavidin-coated binding beads. After washing, the captured DNAs were amplified and indexed with Illumina indexing primers. Post-captured DNA libraries were validated using Agilent TapeStation (Agilent) and quantified using Qubit 2.0 Fluorometer and Real-Time PCR (KAPA Biosystems). Libraries were sequenced 2 × 150 on an Illumina HiSeq instrument. For data analysis, sequencing adapters and low-quality bases in raw reads were trimmed using Trimmomatic 0.39. The reads were then aligned to the GRCm37 reference genome using Sentieon 202112.01. Aligned sequences were then sorted and PCR/Optical duplicates were marked, producing BAM files. Somatic SNVs and small INDELs were called by using Sentieon 202112 . The VCF files generated by the pipeline were then normalized  using bcftools 1.13. Overlapped transcripts were identified for each variant and the effects of the variants on the transcripts were predicted by Ensembl Variant Effect Predictor v104. The most severe impact was selected for each variant and they are used for downstream cohort analysis. The impact of the variants was also classified based on MAF document specifications. The exome data were deposited to the NCBI GenBank Sequence Read Archive (SRA) database under BioProject PRJNA944630. Exome analysis of three murine pancreatic cancer cell lines was deposited under the following accessions: PANC02-Luc , KPC-Luc-4580 , and KPC-Luc-A .Each cell line was grown to 90% confluence  were performed using GraphPad Prism 9.3.1 software. Tests used are indicated in the legends of the figures.in vivo.In this study, we examined three clinically relevant allograftable mouse PDAC cell lines: PANC02-Luc, KPC-Luc-A, and KPC-Luc-4580. These cell lines originated from C57BL6 mice that had three different genetic backgrounds in the pancreas. The PANC02 cell line  is one of the most widely used mouse PDAC cell lines, and it was generated by chemically inducing PDAC in mice via the implantation of 3-methyl-cholantrene (3-MC) (a carcinogen) into the pancreas of genetically unaltered C57BL6 mice  Fig. 1AFig. 1A. TD) of these three cell lines  cell lines, which may contribute to the observed difference in growth kinetics. The KPC-Luc-4580 cells displayed the fasted growth with a doubling time of 21.23 days, and KPC-Luc-A cells were intermittent with a TD of 24.03 days. Although the KPC cell lines were generated similarly, differences in the overall history of each cell line  may contribute to the differences in growth kinetics.First, we compared growth kinetics and determined the doubling time . In addition, BHK-21, a baby hamster kidney cell line, was used as a reference cell line for comparison as it is highly permissive to VSV and widely used for VSV amplification and plaque assays. Also, our previous studies demonstrated that there is a wide range of permissiveness of human PDAC cells to OV therapy, from highly permissive to highly resistant, and the success of OV therapy greatly depends on permissiveness of human PDAC cell lines to OVs –20, 33To further examine the ability of VSV to replicate in these mouse PDAC cell lines, we analyzed VSV-driven GFP fluorescence using a panel of human and mouse PDAC cell lines and BHK-21 cells. Cells were seeded in 96-well plates and either mock-infected or infected with VSV-ΔM51-GFP at an MOI of 10, 1, 0.1, 0.01, 0.001, 0.0001, or 0.00001 (based virus titer on BHK-21 cells). As shown in To examine the effect of VSV infection on cell viability, cells were infected with VSV-ΔM51-GFP at different MOIs  for 70 h, followed by a cell viability assay . In geneIn general, our data indicate that VSV-ΔM51-GFP differs in its ability to replicate in, spread, and kill each of the three mouse PDAC cell lines. Our data show that PANC02-Luc cells are the overall most resistant to VSV-ΔM51-GFP, followed by KPC-Luc-A and KPC-Luc-4580.The differences in VSV-ΔM51-GFP replication, spread, and cell lysis observed between the mouse PDAC cell lines suggest potentially varied levels of antiviral signaling. Our previous studies have established that the capacities for type I IFN antiviral signaling between human PDAC cell lines vary dramatically and are strongly correlated with resistance to VSV and other OVs , 18–2050) for mIFNα, the amount of mIFNα needed to result in 50% inhibition of virus replication  (50 = 5.37 U/mL), followed by PANC02-Luc (IC50 = 33.1 U/mL), KPC-Luc-A (IC50 = 61.6 U/mL), and KPC 4580 (IC50 = 354.8 U/mL). These data support our hypothesis that resistance of mouse KPC cells to VSV is at least in part due to their abilities to mount antiviral responses. In agreement with this hypothesis, we see that PANC02-Luc cells have both the greatest sensitivity to mIFNα and the greatest overall resistance to VSV-ΔM51-GFP among tested mouse PDAC cell lines.To further study the role of antiviral signaling in mouse PDAC cell lines, we examined the antiviral effect of mouse IFNα (mIFNα) on VSV-ΔM51-GFP replication in each cell line. We also used the reference human PDAC cell line, SUIT-2, as this cell line is extensively used in our lab and exhibits an intermediate level of type I IFN antiviral signaling. Importantly, human cells are known to be also sensitive to mIFNα , 46. Oure cells) . The anaAs mentioned above, the functional innate immune responses require not only the ability of cells to respond to IFN but to produce and secrete IFNs and other antiviral cytokines. We hypothesized that cell lines would differ in their ability to produce and secrete such cytokines. To test this hypothesis, cells were either mock-treated or infected with VSV-ΔM51-GFP at MOI 1 and 0.1 (based on virus titer on each cell line). At 24 h p.i., cell supernatants were collected and analyzed using the Mouse Cytokine/Chemokine 44-Plex Discovery Assay Array (MD44), which includes critical antiviral and proinflammatory cytokines known to control viral infections. Out of the 44 different cytokines that were measured, we show in Our data suggest that the observed differences in permissiveness of mouse PDAC cell lines to VSV-ΔM51-GFP are likely due to the higher levels of antiviral restriction factors (constitutive and/or induced) present in more resistant cell lines. This hypothesis is consistent with our previous studies demonstrating that the ability of cells to mount functional type I IFN antiviral responses is a major determinant of resistance of human PDAC cell lines to VSV-ΔM51-GFP , 20. HowTo compare VSV-ΔM51-GFP and VSV-Mwt-GFP, we first tested the abilities of each virus to initiate infections  and spreWe previously showed that, while abnormal or residual type I IFN antiviral activities play a major role in resistance of some human PDAC cell lines to VSV-ΔM51-GFP, some human PDAC cell lines also show inefficient attachment of VSV . Our dat50 for each drug on each cell line that was used as a measure of drug resistance. We found no positive correlation between the level of resistance to VSV and resistance to either gemcitabine or 5-FU. In sharp contrast, PANC02-Luc cells, which are the most resistant mouse PDAC cell line among our three to VSV, demonstrated the lowest IC50 for both gemcitabine and 5-FU, at 28.8 and 2,880 nM, respectively. The IC50 values for gemcitabine and 5-FU in KPC-Luc-A cells were 131.8 and 12,020 nM. Interestingly, the mouse PDAC cell line most permissive to VSV among our three, KPC-Luc-4580, demonstrated the greatest resistance to gemcitabine and 5-FU with IC50 values of 199.5 and 48,900 nM. These data reveal that the level of chemoresistance in mouse PDAC cell lines does not correlate with the efficacy of VSV-based OV-therapy and, promisingly, suggest that chemoresistant tumors can be good targets for OV therapy.We have recently shown that some human PDAC cell lines, which acquire resistance to chemotherapeutical drugs, can also simultaneously develop resistance to OV therapy . In addiin vivo, to our knowledge, they have never been tested in detail for overall chromosomal abnormalities or all genomic mutations in protein-coding sequences. To examine the karyotype of these cells, each cell line was grown to 70% confluence and sent to the Cytogenomics Core Laboratory at the University of Minnesota for SKY. We found that each cell line contained numerous chromosomal aberrations , and two KPC cell lines originated from PDACs developed in different modified KPC mice encoding major driver mutations KrasG12D and Trp53R172H in the pancreas. We found that the three mouse PDAC cell lines were genotypically and phenotypically distinct and showed very different permissiveness to OV-based therapy and, therefore, can serve as promising cell lines to use and address intertumoral heterogeneity 50 values for each cell line represent the relative ability of each cell line to respond to IFN. Between the three tested mouse cell lines, PANC02-Luc displayed the lowest mIFNα IC50 (33.1 U/mL), versus the highest IC50 in KPC-Luc-4580 cells (354.8 U/mL), and intermediate IC50 of 61.6 U/mL for KPC-Luc-A. This means that it takes almost 11 times the amount of mIFNα to bring KPC-Luc-4580 cells to the same level of type I IFN antiviral signaling as PANC02-Luc cells. We further highlight the differential levels of type I (and III) IFN antiviral signaling between these cell lines in We found that each of the three mouse PDAC cell lines exhibited varying levels of permissiveness to VSV infection and VSV-mediated cell killing . We also2/M cell cycle phase are transcriptionally repressed, leading to a decreased ability of cells to mount antiviral responses  to VSV could be also explained by the presence of KrasG12D driver mutation in these 2 KPC cell lines, while it is absent in PANC02-Luc . Severalther OVs –63. Fother OVs . Additiother OVs . Therefother OVs , which mInterestingly, we found that KPC-Luc-A and KPC-Luc-4580 cell lines, even though isolated from PDAC tumors generated from cells that had the same driver mutations, exhibited differential responses to VSV infection. Multiple possible reasons for the differences in phenotypes and genotypes between KPC-Lu-A and KPC-Luc-4580 include: (i) different environmental factors and selective pressures during tumor development in mice; (ii) different selective pressures throughout cell culture adaptation, and (iii) random genetic changes. To our knowledge, the only difference in the original engineering design between KPC-Luc-A and KPC-Luc-4580 cell lines was the method by which each cell line was engineered to express luciferase . In KPC-Whatever the reason for the phenotypic and genotypic differences between the tested KPC cell lines, these differences help contribute to their clinical translatability, as they likely better represent characteristic heterogeneity among and within PDAC patients.50 for both gemcitabine and 5-FU, and the least resistant cell line to VSV (KPC-Luc-4580) had the highest IC50 for gemcitabine and 5-FU. These data suggest that even if PDAC in patients is inherently chemoresistant, VSV-based OV therapy remains a viable treatment option.As VSV-based OV therapy becomes a more commonly tested therapeutic option in clinical trials , it is necessary to better understand how the effectiveness of VSV-based OV therapy is affected by the chemoresistance status of cancer cells. It has been observed in our previous studies and others , 57, 66 The ideal PDAC mouse model system should address both the intertumoral heterogeneity and the ability to detect and evaluate innate and adaptive immune responses against both tumor and OV. In In conclusion, our study provides essential data about three allograftable model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against phenotypically and genotypically distinct PDACs in immunocompetent mice. This study will be useful for ongoing and future studies in the field of PDAC therapeutics. Although this study focused on VSV as the oncolytic agent, alternative OVs can be tested using the model cell lines characterized in this study.


Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na+,K+-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces  PNPLAs via destabilization of a direct PNPLA7 ligand apolipoprotein-E  or TPP . Advanced MEM, DMEM, OptiMEM, GlutaMAX, MEM vitamin solution, fetal bovine serum (FBS), Fungizone (250 µg/mL of amphotericin B), gentamicin (10 mg/mL), trypsin-EDTA, Pierce Enhanced Chemiluminescence (ECL) Western Blotting substrate, High Capacity cDNA Reverse Transcription Kit, TaqMan Universal Master Mix with UNG and TaqMan gene expression assays were purchased from Thermo Fisher Scientific . 4-12% Criterion Bis-Tris polyacrylamide gels, XT MES electrophoresis buffer, goat anti-mouse IgG-horseradish peroxidase conjugate, anti-rabbit IgG-horseradish peroxidase conjugate, PCR plates and PCR plate sealing films were purchased from Bio-Rad . Amersham ECL Full-Range Rainbow Molecular Weight Marker was purchased from . Polyvinylidene fluoride (PVDF) membrane, forskolin, dibutyryl-cAMP (db-cAMP), bovine serum albumin (BSA), and all other reagents were purchased from Merck/Sigma-Aldrich . Insulin (Actrapid) was obtained from NovoNordisk (Denmark). E.Z.N.A HP Total RNA Kit was purchased from Omega Bio-Tek . Dexamethasone was purchased from KRKA (Slovenia).2.2semitendinosus muscle obtained during routine orthopaedic surgery as surgical waste, with informed written consent signed by the participants as described  FBS, 1% (v/v) MEM vitamin solution, 0.3% (v/v) Fungizone and 0.15% (v/v) gentamicin). Experiments were performed on myotubes after 7-10 days of differentiation.Human skeletal muscle cells were prepared from samples of the escribed –32. One 2HPO4, 1.8 mM KH2PO4, pH=7.4) and switched to serum- and insulin-free DMEM (without antibiotics and antimycotics), which contained 0.5 g/L, 1 g/L, or 4.5 g/L glucose. Treatments with insulin (0.1 µg/mL and/or 10 µg/mL), dexamethasone (1 µM), forskolin (5 µM in DMSO), db-cAMP (200 µM) and/or vehicle lasted for 16 h. At the end of experiment myotube samples were lyzed and collected for the subsequent mRNA and protein analysis.Experiments were performed in serum-free DMEM. To remove FBS and insulin, which is present in Advanced MEM, myotubes were washed with warm PBS  with the following sequence: CEVGYQHGRTVFDIWGRSGVLEKMLRDQQGPSKKPASAVLTCPNASFTDLAEIVSRIEPAKPAMVDDESDYQTEYEEELLDVPRDAYADFQSTSAQQGSDLEDESSLRHRHPSLAFPKLSE. Briefly, primary antibody against PNPLA7 was incubated with the excess of PNPLA7 peptide antigen used for immunization. Incubation mixture contained 10 μL of PNPLA7 antibody (0.4 μg/mL), 1.1 μL of PNPLA7 PrEST antigen (0.8 μg/mL) and 29 μL of primary antibody buffer with 1 M urea. The mixture was incubated for 3.5 h at 22°C and constant shaking (300 rpm). After incubation, the mixture was diluted with the primary antibody buffer (containing 1 M urea) to the final volume of 5000 μL, the final antibody dilution thus reaching 1:500. The solution containing the neutralized PNPLA7 antibody solution was subsequently used for immunoblotting. To detect PNPLA7, homogenates of human semitendinosus muscle or human myotubes were used. Tissue homogenates, prepared in Laemmli buffer, were collected from our previous study  Antigen  were seeded into 6-well plates and cultured overnight in Advanced MEM supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX and 1% (v/v) MEM vitamin solution, without antibiotics. The transfection was performed using Lipofectamine 2000 reagent (Thermo Fisher Scientific) and siRNA according to the manufacturer’s protocol. As a negative control, non-targeting scrambled RNA (siSCR) was used. Lipofectamine and siRNA were separately diluted in Opti-MEM and combined just before the addition to the cell culture medium. Final concentration of siRNA against PNPLA7  and scrambled siRNA  in the medium was 10 nM. Final concentration of Lipofectamine was 2 ‰(v/v). After 24 h of incubation in the presence of the siRNA, the cell medium was replaced with the complete growth medium. Analyses of mRNA and protein levels were performed 48 and 72 h after the beginning of siRNA treatment, respectively.2.6® Universal Master Mix II with UNG and TaqMan gene expression assays (PNPLA7 (Hs00295012_m1), IL-6 (Hs00174131_m1), acetyl-CoA carboxylase 1 , acetyl-CoA carboxylase 2 , β-actin , 18S rRNA (Hs99999901_s1)) in a 96-well plate. β-actin (ACTB) and 18S rRNA were used as reference genes (endogenous controls). Results were calculated as gene expression ratios: (1+Ereference)Ct,reference/(1+Etarget gene)Ct,target gene where E is the PCR efficiency and Ct is the threshold cycle. Since two reference genes were used for normalization, geometric mean of both expression ratios was calculated at the last step, while the efficiency of PCR was analyzed with the LinRegPCR software  on Epoch™ spectrophotometer . RNA extracted from cell lysates was reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription Kit and was performed in PTC-100 Programmable Thermal Controler (MJ Research Inc). qPCR was performed on Quant Studio 3  using TaqMansoftware , 35, as  Hs001741_m1, acet2.7Immunoblotting was performed as described –32. Brie2.8post hoc test. The difference between the groups was considered statistically significant when p ≤ 0.05.Data are presented as means with the standard error of the mean (± SEM). In all experiments, n refers to the number of donors (primary cultures obtained from different donors). Statistical analysis was performed with Microsoft Excel (Microsoft Office 2016) and GraphPad Prism 6 (GraphPad Software). Statistical significance was determined using t-test or a two-way ANOVA with Tukey 33.1Pnpla7 mRNA in mouse skeletal muscle was determined using Northern blot . Taken together, these results indicated that primary human skeletal muscle cells express PNPLA7 protein and that the ~150 kDa band, obtained with HPA009130 could be used for its quantification.As an additional control, the antigen sequence of HPA009130 was tested for possible alignments with other proteins using BLAST , which r3.2+,K+-ATPase (NKA aka sodium pump), whose function is known to be affected by lysophosphatidylcholine and membrane lipid environment , which by producing malonyl-CoA catalyzes the first step of de novo synthesis of fatty acids, and the ACC2 isoform , which by producing malonyl-CoA inhibits carnitine palmitoyltransferase and β-oxidation, were similar in control and knock-down cells  AMPK (at Thr172) was significantly reduced (The AMPK pathway was assessed by measuring the expression and phosphorylation of AMPK substrate acetyl-CoA carboxylase (ACC), which plays a major role in fatty acid metabolism by catalyzing the conversion of acetyl-CoA to malonyl-CoA. The abundance of the total ACC was reduced in PNPLA7 knock-down cells   of STAT3. The expression of IL6 mRNA (6 (IL-6) , and lowpression . To dete effects , 39, 40,Collectively, these results suggested that deficiency of PNPLA7 selectively affects various aspects of myoblast function, thereby indirectly indicating a role for PNPLA7 in human skeletal muscle.3.3Pnpla7 mRNA levels in mouse skeletal muscle , a well-characterized down-stream target of PKA , a cell-permeable analogue of cAMP, at low (0.5 g/L ≈ 2.78 mM), physiological (1 g/L), or high (4.5 g/L) glucose concentrations. The 4PNPLA7 mRNA in skeletal muscle is dependent on hormonal or metabolic status as well as that PNPLA7 deficiency leads to myopathy, highlighting the functional significance of PNPLA7 in skeletal muscle physiology. However, little is known about the expression and regulation of PNPLA7 in human skeletal muscle. Our preliminary results suggested that human myotubes might express PNPLA7 protein  by mTORC1, p70S6K phosphorylates ribosomal protein S6, thus promoting protein translation , 42. WhiPnpla7 mRNA levels in skeletal muscle  was not sufficient to observe a decrease in PNPLA7 protein levels. Interestingly, insulin did not alter PNPLA7 mRNA levels at low (0.5 g/L) or high (4.5 g/L) glucose, showing that glucose modulated its expression. Consistent with this view, the protein abundance of PNPLA7 in cultured myotubes inversely correlated with glucose concentrations. Taken together with previously published work  modulates expression of La cells . Further pathway . It woul pathway , 45, 46,r PNPLA7 , there ir PNPLA7 , 10.PNPLA7 did not affect expression level of IL6 mRNA or phosphorylation of STAT3, indicating PNPLA7 is not important for IL-6 signalling in human skeletal muscle cells. Conversely, in Pnpla7 knock-out mice the concentrations of IL-6 in plasma were increased   . While tPNPLA7 depends on the interpretation of the immunoblotting data, which might be affected by non-specific binding of the primary antibody HPA009130. To avoid technical artefacts, we performed antibody validation experiments, which included antibody neutralization and gene silencing of PNPLA7. Antibody neutralization experiments indicated that both the ~150 kDa and ~225 kDa likely reflect specific binding of HPA009130. Taken together with the predicted molecular weight of PNPLA7 (~145.7 kDa) and gene silencing experiments, which resulted in marked suppression of the ~150 kDa band, it is highly likely that the ~150 kDa band represents PNPLA7. Conversely, the ~225 kDa band, which was not detectable during incubation with PrEST-treated HPA009130, was not affected by gene silencing, suggesting it does not represent a protein encoded by the PNPLA7 gene. The discrepancy could be explained if a dimer or another PNPLA7-containing complex has a longer half-life than a PNPLA7 monomer, meaning that incubation was too short to observe a significant decrease in the ~225 kDa immunoreactivity. However, while patatin, isolated from Solanum tuberosum, is expressed as a dimer . Full statement: The primary human skeletal muscle cell cultures were prepared from the samples of semitendinosus muscle obtained during routine orthopaedic surgery as surgical waste, with informed written consent signed by the participants as described –31. PrepKM and AM-L performed experiments. MK, AM-L, and KM analyzed the data and prepared the first draft of the manuscript. TM planned experiments and analyzed the data. SP analyzed the data, edited, redrafted, and finalized the manuscript. SP and MK provided funds, planned and supervised the experiments. SP, KM, and MK conceptualized the study. All authors contributed to the article and approved the submitted version.


As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds , previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines  and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL. The proArtemis, a DNA nuclease in the metallo-β-lactamase family, is an enzyme that is specifically responsible for opening these DNA hairpins . TherefoOne strategy that we have been pursuing for targeting ALL cells takes advantage of the fact that over 90% of ALL patient cell lines express the RAG complex . TherefoThis rationale was supported here by examining the expression level data for Artemis in primary and relapse human ALL patients. Based on this rationale, a high throughput screen was done using a proprietary library to evaluate inhibition of the Artemis catalytic domain and its nuclease activity (to be reported elsewhere). Following hit confirmation and medicinal chemistry optimization to improve potency and selectivity, four compounds were assessed for inhibition of primary and relapse ALL cells.Bone marrow (BM) and peripheral blood (PB) samples from ALL patients were acquired in compliance with the Institutional Review Board regulations of each institution. Informed consent for cell banking was obtained from all human subjects. Leukemia cells were processed and cultured as previously described . Human sNormal mature human B cell lines, immortalized lymphoblastoid cell lines 3301015 and 5680001, were generated using EBV-transformation of peripheral blood from patients with no hematopoietic abnormalities.BCR-ABL1 or hypodiploidy) were excluded from the study (http://www.R-project.org/).Clinical and gene expression microarray data from 207 high-risk B-precursor ALL patients from the COG Clinical Trial P9906 were obtained from the GEO database (GSE11877) . The pathe study . CryopreD binding affinity to Artemis from 20 to 150 nM. The commercially available DNA-PK inhibitor (AZD7648)  was evaluated in parallel as a positive control. Proliferation was determined by measuring the green fluorescence intensity per image (9 images were captured per well) by Incucyte every 8 h for 3 days. Raw values were normalized to the zero-time DMSO control. On day 3, viability of the treated cells was determined by annexin V/DAPI staining via flow cytometry.Lentivirally green fluorescence protein (GFP) labeled patient-derived B-ALL cells  or mature B cell lines 3301015 and 5680001 were plated on irradiated murine stromal OP9 cells and treated with four compounds optimized for Artemis inhibition  . These compounds ranged in Kλex = 485 nm, λem = 520 nm) with 1-min reads for 40 min. Compounds were tested in 10-point concentration (10 different concentrations with 3-fold dilutions) responses using the slopes of the enzyme readouts to determine percent inhibition which was fit to a 4-parameter logistic curve to calculate the IC50 of each compound. We have previously published the purity of the protein preparations  on the 3’ end. The assay buffer used was 25 mM MOPS, pH 7.5, 10 mM MgCl2, 10 mM KCl, 10 μM ATP, 1 mM DTT, 0.05% (w/v) CHAPS with 5 nM FL-Artemis, 5 nM DNA-PKcs, and 200 nM DNA FAM hairpin substrate. The fluorescence signal was monitored kinetically on a Perkin Elmer Envision microplate reader with FITC filters  and Artemislow expressing cases . Artemishigh leukemias were associated with inferior outcome with lower overall survival and relapse-free survival. This supported the rationale for targeting Artemis.To determine the role of Artemis in ALL, expression of Artemis mRNA (DCLRE1C) in 207 ALL patients uniformly treated with the COG P9906 clinical trial  was corrA high throughput screen of Artemis active site inhibitors using a proprietary library to assess for inhibition of the Artemis catalytic domain and its nuclease activity identified four small molecule inhibitors . The med2+ conditions is entirely inactive without DNA-PKcs  were transduced with GFP and 100,000 cells were plated in 24-well plates seeded with irradiated OP9 cells. Increasing green fluorescence intensity is indicative of proliferation as measured by Incucyte every 8 h. Although the GFP expression varied between cell lines , the norOur studies indicate that the 827171 compound substantially inhibits the proliferation of primary patient B-ALL lines, and it is comparable to the AZD7648 compound . The advThe effects of 827171 on viability, in contrast to proliferation, are among the mildest of the compounds evaluated. This likely reflects that DSBs in nondividing cells may not cause death immediately or at all. In contrast, an unrepaired DSB in proliferating cells represents more of a challenge. This increased effect of DSBs on proliferating cells may be because of a much higher probability of losing large chromosome fragments. Such a loss is because only the portion of the chromosome with the centromere is retained at the metaphase plate during cell division, and the chromosomal fragment lacking a centromere portion is easily lost. In addition, inhibition of Artemis may interfere with the ability of cells to exit from S phase .The large majority of acute lymphoblastic leukemias (ALL) produce the RAG enzyme complex encoded by the RAG genes . The RAGHere, we evaluated four of the Artemis inhibitors generated from a high-throughput screen and medicinal chemistry effort. These were assessed in three primary human ALL samples, with human mature B cell lines used for comparison. For comparison with the Artemis inhibitors, we used a known DNA-PKcs inhibitor, AZD7648, because DNA-PKcs is a kinase which regulates Artemis activity, and this compound has been evaluated in clinical trials for utility in a variety of non-lymphoid human malignancies .We found that the 827171-compound reduced proliferation of the three B-ALL lines at 1, 5, and 10 µM without affecting the mature B-cell control lines. In contrast, the DNA-PKcs inhibitor, AZD7648, showed little inhibition of the B-ALL lines, and instead suppressed the growth of the mature B cell control lines. These findings are encouraging for future work to make derivatives of the 827171 compounds. Importantly, the DNA-PKcs inhibitor AZD7648 decreased the proliferation less than 827171 in these same lines. This suggests that inhibition of hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis, namely, DNA-PKcs. This may be because Artemis can be activated by DNA ligase IV, even when DNA-PKcs is not present . TherefoWhile the 827171 compound shows promise for inhibition of the active site of Artemis, the brief study here also raises the interest in parallel strategies for inhibiting Artemis. Future work may take advantage of the recently described cryo-EM structure for the basal state of the Artemis:DNA-PKcs complex to develop inhibitors that prevent Artemis from being activated . Combina


After this article  was publDisregarding the erroneous bioinformatic analysis results about HNF-4α expression in the GSE84402 dataset, the article’s main conclusion relies on data obtained in cell line experiments. There are concerns about the validity of the control cell line used in comparisons of HCC cell line vs. control cell line expression: as is noted in the Discussion, the control cell line used is of embryonic hepatic origin and as such may not serve as an appropriate control for experiments using adult HCC cell lines. Furthermore, the study did not include loss of function studies or studies in native tissue samples to support claims about a role of HNF-4α in HCC proliferation.PLOS ONE Editors concluded that the article’s conclusions are not adequately supported when disregarding the erroneous bioinformatic analysis results and considering the methodological concerns discussed above. Therefore, the PLOS ONE Editors retract this article.The All authors did not comment on the retraction decision.