Severe acute and chronic graft versus host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Historically, cord blood and matched sibling transplantation has been associated with the lowest rates of GVHD. Newer methods have modified the lymphocyte components to minimize alloimmunity, including: anti-thymocyte globulin, post-transplant cyclophosphamide, alpha/beta T cell depletion, and abatacept. These agents have shown promise in reducing severe GVHD, however, can be associated with increased risks of relapse, graft failure, infections, and delayed immune reconstitution. Nonetheless, these GVHD prophylaxis strategies have permitted expansion of donor sources, especially critical for those of non-Caucasian decent who previously lacked transplant options. This review will focus on the biologic mechanisms driving GVHD, the method by which each agent impacts these activated pathways, and the clinical consequences of these modern prophylaxis approaches. In addition, emerging novel targeted strategies will be described. These GVHD prophylaxis approaches have revolutionized our ability to increase access to transplant and have provided important insights into the biology of GVHD and immune reconstitution. Acute GVHD is thought to primarily be caused by the recognition of self (or host) antigens by donor T cells. Tissue damage is a key component to initiate aGVHD and often begins prior to the introduction of the graft. This tissue injury is due to inflammation associated with the HCT preparative regimen but may also have occurred as part the underlying disease  or its treatment . Myeloablative and total body irradiation-based regimens associated with greater tissue inflammation have been linked to higher rates of aGVHD , 2. Notay milieu . This pry milieu , 9. Thesy milieu . aGVHD hy milieu  and Th2 y milieu , 12. Addy milieu . Murine y milieu –16, thouy milieu –22. In s). Tissue injury may be pre-existing, due to pre-transplant preparative regimens such as in aGVHD, or due to second insults, induced by later infections and the impact on the microbiome, or local injury e.g. sun-exposure  which will be discussed. High resolution HLA typing for unrelated donors is recommended to include HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DPB1 loci with HLA-DQB1 and DRB3/4/5 optional . Some stvia ex vivo and in vivo lymphocyte selection, depleting functional malevolent lymphocytes to achieve long term durable tolerance. The potential risks of this approach that curtails the infused lymphocyte activity are increased relapse rates, graft failure, or infections . The application of the GVHD-free, relapse free, survival proportion has enhanced our ability to compare these GVHD-reduction approaches, including these key risks in a single outcome measure, and thus included in the discussions below where possible. However, it is important to note, that the risks of GVHD and relapse may be different in terms of long-term outcomes, so there remains value in reporting rates of relapse and acute and chronic GVHD separately. Herein, we describe the published data for each method of GVHD reduction and then discuss the controversies in the field between the disparate donor and prophylaxis regimen options.Other strategies to reduce acute and/or chronic GVHD have focused on preparative regimens and graft manipulation. In addition to the use of immature thymus-derived T cells in cord blood, these have largely included either prioritizing genetically HLA-matched donors and/or graft engineering in vivo, one study linked reduced cGVHD to lower naïve helper T cells after ATG exposure, and suggested that this was a primary mechanism of action (Anti-thymocyte globulin (ATG) is composed of antibodies to T cells generated in a horse or rabbit and thus work by antibody mediated clearance of T cells. ATG is typically added to a standard backbone of methotrexate and calcineurin inhibitor though rare studies include other agents . The equine preparation was generated against human thymus cells. Rabbit ATG is generated by inoculating rabbits with either a human T cell leukemia cell line (ATG-F) or human thymocytes (ATG-T). Because these antibodies are developed in response to different T cell populations, it is not surprising that the polyclonal antibody composition differs. While ATG-F and ATG-T both react to CD2, CD8, CD11a, CD107a, ATG-F has a higher response to CD28 and ATG-T has greater activity against CD3 and CD4 . Data haf action . The impf action –66. ThusStudies have consistently shown reduction of acute and chronic GVHD after ATG exposure. Most studies are performed using peripheral blood, many randomized to controls, and most include matched related and unrelated donors –76. ThesCyclophosphamide is an alkylating agent that causes DNA strand breakage and replication stress in dividing cells. Its use in the post-transplant setting to prevent GVHD was pioneered at Johns Hopkins University. It has been posited that post-transplant cyclophosphamide (PTCy) works through the preferential killing of alloreactive donor T cells followed by peripheral tolerance induction through clonal deletion and Treg suppression and lastly by central deletion of alloreactive T cell precursors in the thymus . HoweverPTCy was first evaluated in non-myeloablative conditioning regimens for haploidentical transplants utilizing bone marrow grafts , 80. A dPTCy has also been evaluated in the matched related and matched and mismatched unrelated donor setting , 91–93. With the success of PTCy in the haploidentical and unrelated donor setting, there has been an increasing debate over whether a matched unrelated or haploidentical donor would be preferred given the lower cost and increased accessibility with a related donor. A recent CIBMTR analysis compared haploidentical and matched unrelated donor (MUD) transplants in which both groups received PTCy GVHD prophylaxis . In the The majority of the initial trials evaluating PTCy utilized bone marrow grafts but more recently PBSC grafts have been used. There have been no randomized prospective studies comparing graft sources however multiple retrospective studies have been performed with mixed results –97. WhilDelayed immune reconstitution and associated increased risk of infections has been observed with haploidentical transplant with PTCy –101. A rPTCy has been less extensively studied in non-malignant disease and mostly limited to smaller studies. Low rates of GVHD and NRM have been observed however graft failure rates have been high with nonmyeloablative regimens –108. ImpAn additional consideration when using PTCy is the toxicities associated with cyclophosphamide. Cardiotoxicity is a known side effect of cyclophosphamide and tends to occur within the first few weeks following administration , 110. A In summary, PTCy has been evaluated in the haploidentical, matched-related, and the matched and mismatched unrelated donor setting and is associated with low rates of aGVHD and cGVHD. While PTCy has been associated with delayed immune reconstitution and increased risk of viral infections, the nonrelapse mortality rates are low. The impact on relapse and EFS is variable among studies but is improved in myeloablative and more HLA-matched transplants.For T cells to become activated they require both antigen presentation through T cell Receptor (TCR) engagement of the MHC on APCs and costimulation through multiple different costimulatory pathways . The besBased on encouraging data from an initial pilot study, one large prospective, multi-center, phase II, randomized, placebo-controlled, consortium study has been conducted to use abatacept to prevent aGVHD . Abatacein vivo approaches to reduce GVHD, ex vivo approaches utilize graft engineering to modify the product prior to infusion. CD34+ positive selection was one of the first such approaches, which the goal of a product highly enriched for CD34+ stem cells with minimal CD3+ lymphocytes, administered on a backbone of ATG  without subsequent pharmacologic immunosuppression. The CD34+ selection strategy has largely been used in adult recipients of matched related or unrelated donors for the treatment of hematologic malignancy. CD34+ cell doses are a median of 6-8e6/kg with T cell doses infused of 2-6e6/kg  as part of the transplant regimen, with the former aimed to decrease the risks of graft rejection and GVHD and the latter to decrease EBV reactivation. Most of these protocols do not employ post-transplant pharmacologic GVHD prophylaxis, testing whether the graft engineering approach is sufficient to avert alloimmunity alone. This αßTB depletion approach was developed to increase donor accessibility with haplo-identical transplantation, removing the cells most likely to induce GVHD, while preserving graft-vs-leukemia and anti-infectious immunity through preservation of natural killer and gamma-delta T cells. Thus, most but not all the studies use this graft engineering approach with related HLA-mismatched donors. This newer therapy has largely been studied in children, comprising 95% of the nearly 700 total patients reported in the literature, and a still small cohort compared to other alternative donor approaches  obtained proaches –141. MorαßTB depletion removes the T cells most closely linked to acute and chronic GVHD and depletes the B cells that can contribute to chronic GVHD, the rates of these complications were anticipated to be lower than that observed after matched donor HCTs despite the increased HLA-mismatch. Overall, this has been observed with median rates of grade II-IV aGVHD of 18% (ranging from 11-28%), and cGVHD of 8% (range 0-30%), with 0-21% experiencing extensive cGVHD  have yet to be established. As expected with αßTB cell depletion, the rates of viral reactivation were higher than matched transplant approaches. Those reporting viral reactivations reported consistent rates of 40-65% of patients experiencing reactivations, with 20-65% reactivating CMV, 0-44% EBV, up to 57% adenovirus, 21% HHV6, and 23% BK virus , the incidence was higher in nonmalignant recipients, ranging from 4-30%, and appears higher than the rates of graft failure rates reported in a large CIBTMR study of similar pediatric populations  was first identified as a viable alternative donor source in 1989 , permittOne advantage of UCB transplant has been its success even in the setting of HLA disparity. Due to lower immunogenicity, UCB transplant requires less stringent HLA-matching . Greater HLA disparity has been associated with increased rates of graft failure and GVHD , 156–158Large studies evaluating single and double UCB transplant have reported median rates of grade II-IV aGVHD of 38% (ranging from 24-45%), 17% grade III-IV aGVHD (9-25%) and 27% cGVHD 14-53%) with the range of relapse, EFS, and OS being 12-45%, 28-70%, and 31-75% respectively -53% with, 161–170In summary, UCB transplant is associated with tolerable rates of grade II-IV aGVHD and reduced rates of cGVHD while permitting greater HLA disparity, though mismatched donors have been associated with higher GVHD and NRM. Delayed immune reconstitution and higher infection rates and their effect on NRM are a concern with UCB transplant while the graft versus leukemia effect appears to be preserved.in vivo depletion with Obinutuzumab (CD20 blockade) NCT02867384, ustekinumab (IL-12 and IL-23 blockade) NCT04572815, tildrakizumab (IL-23 blockade) NCT04112810, or BAFF inhibition with belimumab NCT03207958, or the addition of medications that modulate the immune system, e.g. proteasome inhibition with Ixazomib NCT03225417, histone deacetylase inhibition with vorinostat NCT03842696, JAK1 inhibition with itacitiniab NCT04859946, or the addition of alpha-1-antitrypsin NCT03805789.The prevention of graft versus host disease after allogeneic hematopoietic cell transplantation is a rapidly evolving field. In addition to the strategies listed above, many are currently in development and/or clinical trials. A clinicaltrials.gov search yielded over 50 studies currently active, including engineered grafts that supplement immunomodulatory cells e.g. Treg cells (“Orca-T”) NCT03802695 or interferon gamma-primed mesenchymal stromal cells NCT04328714, While aGVHD is a risk factor for cGVHD, not all regimens that reduce one reduce the other. The early data for naïve T cell depletion does not appear to impact rates of aGVHD, especially as this was tested in patients with matched sibling donors and thus those at the lowest risk for GVHD. That said, if one develops highly treatable aGVHD with low rates of cGVHD and relapse, would this be the method of choice for patients with very high-risk hematologic malignancies and a matched sibling donor? Notably, this was tested in young adults and evaluation in pediatrics is underway but unknown. In addition, there is no data for nonmalignant indications, and for these patients neither aGVHD nor cGVHD provides benefit . ATG may reduce both but is this needed for all matched sibling transplants? This may be an especially critical query for young children who incur low rates of chronic GVHD and may have aggressive malignant disease with higher relapse rates. Abatacept has also been tested in children and adults with matched and mismatched unrelated donors and has reduced the rates of severe aGVHD in those with high-risk hematologic malignancies but with the same query for 8/8 matched transplants. Similarly, should CD34 selection be chosen for these patients even if it reduces the rates of acute and chronic GVHD? Should the age of the recipient and/or donor influence the choice of additional prophylaxis? Perhaps, one should consider late teens to older adults a population to consider for these trials in the future?αßTB depletion, which reduced cGVHD without aGHVD, with similar risks of graft failure and viral reactivations though as yet to be studied in adults. Additionally, cord blood reduces the risk of cGVHD, especially in matched settings, while still incurring a risk of aGVHD but potentially preserving GVL. Abatacept, ATG, cord blood, αßTB depletion, and PTCy markedly increase the donor pool to include mismatched donors without severe acute and/or chronic GVHD, critical as many HCT recipients lack matched donors. Collectively, these data support a personalized approach to the risks of acute and chronic GVHD, including thoughtful evaluation of the risk of relapse, the risk of severe acute or chronic GVHD , the risk of nonengraftment, and the risks of severe infections.While this remains an ongoing question in the matched transplant donor setting, historical data clearly justify the use of additional GVHD prophylaxis for alternative donors (<8/8 match), in which unacceptable rates of acute and/or chronic GVHD have been demonstrated. For those without an 8/8 matched donor, the choices typically include a 7/8 matched unrelated donor, a haplo-identical donor, and cord blood transplantation. For 7/8 matched unrelated donors, abatacept maintained good antileukemia control, with event free survival rates matching matched cohorts, albeit with some increase in cGVHD. The dose of abatacept is now being extended to evaluate if both acute and chronic GVHD may be averted by this method. However, on balance, good leukemia control may be more valuable with high-risk diseases and merit a higher risk of cGVHD, and this approach did extend the donor pool for many patients by including mismatched unrelated donors. ATG impacted aGVHD and cGVHD in this setting as well, though some studies suggested an impact on graft versus leukemia effects. Alternatively, in the haplo-identical setting, PTCy reduced aGVHD and cGVHD, albeit with some increased in viral reactivations in both approaches and some studies showing higher relapse and nonengraftment. Similarly, in children, the haplo-identical platform has included It is noteworthy that there is large variability in the number of pediatric and adult patients in whom each approach has been tested. The largest data exists for ATG, cord, and PTCy with each of these including some prospective comparison studies with less data for the other (often newer) approaches. In addition, a limitation to comparing approaches is that not all studies report acute and chronic GVHD incidence, infections, graft failure, relapse rates, and GRFS. As we move forward, it would be helpful for each trial to present complete data on these variables to aid in precision medicine of GVHD prophylaxis and to prioritize prospective comparison trials.αßTB depletion, and cord blood may be potentially associated with increased risks of nonengraftment compared to other approaches, these approaches might be deprioritized in these patients. Further, one might also consider the impact of residual ATG on these patients, adopting a pharmacokinetic approach to mitigate the effect on donor T cells that can aid in engraftment. Alternatively, there may be other ways to mitigate this risk, such as testing for donor specific antibodies and using this for donor selection, agents that could enhance the stromal function, or targeting higher stem cell doses either by increased volume at collection or by CD34 stem cell expansion strategies.Certain indications for transplant incur higher risks of nonengraftment, due to either decreased fertile stromal milieu  or activated host T cell populations . When choosing GVHD prophylaxis regimens for these patients, the risks of nonengraftment should be prioritized. Given that PTCy, The regimen that best preserves the graft versus leukemia effect remains to be determined. In part, this is because the key lymphocytes and tumor milieu that mediate GVL remain elusive. Data support that functional lymphocytes are key to maintain GVL . Thus, Gvia pharmacologic interventions  or via adoptive transfer of donor lymphocytes. These donor lymphocytes infusions have historically been unmanipulated, though newer approaches include removal of CD45RA naïve T cells, or manipulation to expand T cells specific for tumor antigens. Collectively, these considerations suggest that these factors should be carefully considered as the preparative and GVHD prophylaxis regimens are developed, including optimization of initial disease reduction, consideration of the effects of graft versus tumor effect on the GVHD prophylaxis chosen, and the post-transplant options for maintenance of remission.It is also important to consider other factors that alter the risk of relapse. Reduced preparative regimens increase relapse risk, though are necessary for those with diminished organ function or advanced age. Alternatively, some approaches rely upon total body irradiation which may increase the anti-leukemia benefit by disease reduction in sites with diminished blood flow or high residence in the marrow stromal niches. An increased risk of relapse after HCT may be mitigated by post-transplant disease reduction approaches as well, Many patients who undergo transplant for nonmalignant diseases have increased risk for infections , non-engraftment, or increased risk for particular organ impairments . Those who enter HCT with active viral infections in the setting of pre-existing T cell dysfunction may benefit from optimizing early T cell function to avert life-threatening viral disease. In this case, abatacept and ATG may be better than PTCy, CD34+ selection, αßTB depleted haplo, or cord blood in which the viral reactivations may be higher. Alternatively, other supportive care approaches may also be employed to mitigate this risk. Letermovir is now approved for prophylaxis of CMV in adult HCT recipients at high risk of reactivation and thus diminish this risk. Similarly, the planned use of T cells specific for particular viruses (through selective expansion or cytokine capture) may also enable more donor choices through decreasing these viral risks.αßTB depletion, permitting expedited HCT with mismatched related donor, both accessible and likely motivated to proceed.In contrast, those with sickle cell disease who are less likely to have matched donors may benefit from PTCy or abatacept to increase the donor pool without increasing adverse effects (to the kidney and lungs). Some of these patients with nonmalignant disease may require rapid transplantation which may prioritize the PTCy or It is also notable that several groups are developing preparative regimens without cytotoxic chemotherapy. This may shift the landscape for these patients, potentially mitigating aGVHD risk, and permitting prioritization of reduction of cGVHD for these patients.Finally, there are a dearth of data regarding GVHD prophylaxis to treat nonmalignancy. This is surprising as this population may be the ideal population to test various prophylaxis options. Relapse is not a competing risk and estimates suggest ~25% of patients will develop cGVHD .The approaches to prevent GVHD, including donor sources, have varying degrees of impact on immune reconstitution. Delayed immune reconstitution is linked to increased infectious complications and associated mortality. Some patients have baseline infectious risk factors necessitating a more rapid immune recovery, e.g., neutrophil recovery for active fungal disease or high-risk bacterial infections or T cell recovery for a history of mycobacterial infections or viral reactivations or active infections. Cord blood has been associated with delayed T cell reconstitution and infections compared to adult donors, attributed to the absence of memory cells to effect viral immunity. Additionally, PBSC grafts have been found to have significantly quicker neutrophil recovery than BM grafts. PTCy has also been linked to delayed T cell reconstitution and increased risk of viral infections, while abatacept showed similar immune reconstitution to controls.Full T cell reconstitution requires two pathways for recovery. Initially, the infused donor T cells will increase number but not diversity through peripheral expansion. These initial T cells can provide GVL and viral immunosurveillance. Full recovery of diverse T cell reconstitution requires thymopoiesis, an option for children and young adults after hematopoietic cell transplantation. Little is known about the thymic recovery of the different GVHD prophylaxis approaches. However, minimizing the risk of GVHD overall should diminish one risk of thymus impairment, that of direct T cell attack on the thymus tissue. Including studies of thymus-derived T cells, diversity of T cell receptor, evidence of T cell function , would be beneficial to evaluate the overall effect of each approach on lymphoid immune reconstitution.In conclusion, the approach to acute and chronic GVHD prophylaxis should be personalized, factoring in the age of the recipient , the preexisting conditions and disease indication, the intensity of the conditioning regimen, the risk of, and opportunities to mitigate infections or disease relapse/recurrence, and the risks of nonengraftment and delayed immune reconstitution. Recently, there have been many new approaches that have both diminished the rates of severe acute and/or chronic GVHD, that have increased both the available donor pool and the options for personalized GVHD prophylaxis regimens. Comparisons of alternative donor approaches would potentially be valuable for particular disease indications across the lifetime spectrum of the recipient, to assist practitioners in these weighty decisions. In addition, the optimal GVHD prophylaxis is influenced by other aspects of the HCT, e.g., preparative regimen intensity, enhanced supportive care for the diagnosis and treatment of infections, which may reprioritize the prophylaxis regimens for particular HCT recipients.BW and KW designed, wrote, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malignant pleural mesothelioma (MPM) is a disease of the pleura related to asbestos exposure. Despite the advancements in new therapeutic frontiers, it has a dismal prognosis and very limited treatment options. To find novel weapons in the care of MPM, we undertook a drug-repurposing approach that consists of evaluating existing drugs already approved for other human diseases. We screened 1170 drugs, and we observed that cephalomannine, a taxane; ouabain, a cardiac glycoside; thonzonium bromide, an antifungal surfactant; and emetine, an emetic alkaloid, had marked activity against immortalized and patient-derived primary MPM cell lines. These compounds were shown to be promising, and they will be evaluated in further studies, both in vitro and in vivo. We believe that drug repurposing is a valuable strategy to facilitate and accelerate the definition of novel treatment options for the management of MPM.The lack of effective therapies remains one of the main challenges for malignant pleural mesothelioma (MPM). In this perspective, drug repositioning could accelerate the identification of novel treatments. We screened 1170 FDA-approved drugs on a SV40-immortalized mesothelial (MeT-5A) and five MPM  cell lines. Biological assays were carried out for 41 drugs, showing the highest cytotoxicity and for whom there were a complete lack of published literature in MPM. Cytotoxicity and caspase activation were evaluated with commercially available kits and cell proliferation was assayed using MTT assay and by clonogenic activity with standard protocols. Moreover, the five most effective drugs were further evaluated on patient-derived primary MPM cell lines. The most active molecules were cephalomannine, ouabain, alexidine, thonzonium bromide, and emetine. Except for alexidine, these drugs inhibited the clonogenic ability and caspase activation in all cancer lines tested. The proliferation was inhibited also on an extended panel of cell lines, including primary MPM cells. Thus, we suggest that cephalomannine, ouabain, thonzonium bromide, and emetine could represent novel candidates to be repurposed for improving the arsenal of therapeutic weapons in the fight against MPM. Malignant pleural mesothelioma (MPM) is a rare tumour of the pleura mainly caused by a past exposure to asbestos and often diagnosed in advanced stages . As firs2 in Medium 199 , supplemented with 2 mmol/L l-glutamine (Euroclone S.p.A.), 20 ng/mL hEGF , 100 U/mL penicillin, 10% FBS (Euroclone S.p.A.), 100 μg/mL streptomycin (Euroclone S.p.A.), and 0.4 μg/mL hydrocortisone (Sigma-Aldrich). The local Ethic Committee approved the study .All the materials and methods have been published in detail on a previous manuscript . Here, w® Luminescent Cell Viability Assay . For the preliminary screening, all compounds are tested initially at a single dose of 10 μM, and only at a later stage, a selection of them will progress to the full five-dose assay. This one-dose screen methodological approach has already been used successfully before [® Luminescent Cell Viability Assay, as reported previously [The FDA-approved drug library was composed of 1170 entities  . Cell viy before ,17,18,19eviously . Three i® Life Science , whereas thonzonium bromide and pralatrexate were purchased from MedChemExpress .Individual chemicals, i.e., albendazole, alexidine, azelastine, bosutinib, carmofur, cladribine, digoxigenin, dronedarone, emetine, miconazole, pentamidine, pitavastatin, sertraline, solifenacin, sulcanozole, tadalafil, terfenadine, thioridazine, trifluorothymidine, and toremifene were purchased from Cayman Chemical Company . Additionally, 10-deacetylbaccatin, allyl-thiourea, aminoacridine, bazedoxifene, cephalomannine, cetrimonium bromide, ciclopirox, clomifene, cytarabine, fenbendazole, ouabain, oxethazaine, penfluridol, pyrithione zinc, tioconazole, thioguanine, and vinorelbine were purchased from Sigma-Aldrich. Flubendazole was purchased from LKT Laboratories, Inc. , and gemcitabine and niclosamide were purchased from AdipoGen® 3/7 Assay (Promega) was used for detecting the activation of caspases 3/7, a marker of apoptosis. The assay was carried out according to the manufacturer’s instruction and as reported in the previous study [® Omega microplate reader . Three independent experiments with three technical replicates each were carried out.Drugs showing a dose-response activity on the previous assay were further studied for proliferation with MTT or SRB assays, both described in detail in the previous manuscript . For CFAus study . Lumines2, and growth was monitored daily and recorded using a light microscope . Then, after excluding aggregates of outliers showing extra-large dimensions or unusual shapes, frankly isolated spheroids were analysed at the microscope for their size or morphology. The size was measured as area occupied on the surface of the visual field by counting (with the software ImageJ) the total number of pixels covered by spheroids. Then, the average spheroid area in each well was calculated as a ratio of the total pixels shared by the total number of spheroids  and expressed as “spheroid average size” (arbitrary unit). Alternatively, we used the grade of damage to spheroids, seen as sloughing of outer layers. The average damage in each well was measured as the ratio of the “number of damaged spheroids/total number of spheroids” and expressed as “morphological score” (arbitrary unit). Both methods of counting were carried out by two independent readers blinded for the state of spheroids. Data were reported as mean ± standard error of the mean (SEM) of two experiments, each performed in duplicate.Among our panel of cells, we firstly assessed that only MeT-5A, Mero-25, IST-Mes2, MSTO-211H, HMC7, MMP1, and MMP4 were able to form spheroids. Cells were seeded in Nunclon™ Sphera™ 96-Well microplates , allowing spheroids to aggregate for 48 h before treatments with either controls or drugs. TB and CM were used at concentrations of 0.2, 1, and 5 μM, whereas OB and EM at concentrations of 0.025, 0.05, and 0.1 μM. All treatments lasted for 72 h. Spheroids were kept at 37 °C and 5% CO50s was carried out with the software GraphPad Prism 7 . To rank drugs according to the overall activity, the −Log10 of the IC50s summed across all the cell lines was calculated.Similar to what was reported in , the valp-value of 0.05 was employed as the statistical threshold of significance.For cell proliferation, the ODs obtained from three independent experiments  were analysed with the multivariate analysis of variance (MANOVA) with interaction . Within this type of analysis, the differences between control and treatments at each dose were obtained through the Sidak multiple comparison test. CFA, caspase activation, and spheroids’ formation assays were evaluated with the one-way analysis of variance and the Dunnett’s multiple comparisons test. The statistical analyses were carried out with GraphPad Prism 7 and with Statgraphics Centurion XVI . A ® assay, and it was performed on MeT-5A  and five permanent MPM cell lines. The re-evaluation allowed us to identify 90 cytotoxic drugs active in at least four out of five MPM cell lines and to a various extent, also in the MeT-5A cell line. The results of these 90 molecules are reported in In the present study we re-evaluated the results of our recent screening of 1170 FDA-approved drugs . It was According to the published literature, 56 of the 90 drugs were never assayed in MPM models (either in vitro or in vivo). Thus, we selected 38 of these drugs and evaluated whether their cytotoxicity could be validated in a dose-dependent manner  in the MeT-5A, Mero-14, Mero-25, IST-Mes2, NCI-H28, and MSTO-211H cell lines. As a control, we also employed four of the thirty-four positive molecules previously published as being cytotoxic in MPM cells: cytarabine, gemcitabine, pralatrexate, and vinorelbine.50s are reported in 50s were added together from across all cell lines and their −Log10 were calculated. Thus, the overall activity of these compounds and their rankings are represented in The complete set of dose–response curves used for the calculation of the ICAmong the compounds cytotoxic at the lowest doses, there were several molecules known to be active on MPM, such as pralatrexate, gemcitabine, cytarabine, and vinorelbine. However, in the present work, we deepen the investigation on cephalomannine , ouabain , alexidine , thonzonium bromide , and emetine , as they showed the strongest cytotoxicity compared to the other drugs. In any case, the remaining molecules are still of interest, and they could be the objects of future studies. The dose–response curves for CM, OB, AX, TB, and EM are shown in (a) CM. CM caused decreased proliferation, statistically significant in all cell lines at 72 h after the treatment (p < 0.0001) (p < 0.0001) ((b) OB. A statistically significant decrease of proliferation was observed in all cell lines analyzed, starting from 48 h after the treatment (p < 0.0001). (c) AX. Treatment with AX at 0.1 μM did not elicit significant changes in any cell line under investigation, neither in the proliferation ((d) TB. Upon treatment of 1 μM TB, we observed an inhibition of the growth of MPM cell lines following 72 h of culturing post treatment (p < 0.0001) (p < 0.0001) ((e) EM. Treatment with EM caused a statistically significant reduction of the proliferation of Mero-14, IST-Mes2, and MSTO-211H at 72 h (p < 0.0001) (p < 0.01). In addition, EM prevented all the cell lines from forming colonies (p < 0.0001)  . However 0.0001) . Finally 0.0001) . (b) OB.reatment . OB alsoreatment . Figure feration  nor in tferation  nor in tferation . For thireatment . MeT-5A reatment . This direatment . In addi 0.0001) . Finally 0.0001) . (e) EM. 0.0001) . EM also 0.0001) .The toxicity of the selected molecules was further assayed by using the same doses on a different panel of MPM cell lines of epithelioid origin , two non-malignant cell lines , and three primary cell lines derived from MPM patients .After 72 h of treatment, a statistically significant reduction in cell viability caused by the drugs analysed was confirmed in almost all the MPM cell lines, except for CM in Mero-41 and TB in Mero-95  [Candida albicans where it can inhibit the vacuolar “V” ATPase [Psychotria ipecacuanha plant. It disturbs DNA, RNA, and protein synthesis [Strophantus gratus plant. Based on its positive ionotropic activity occurring via the inhibition of Na/K-ATPase, OB is used in the treatment of congestive heart failure and cardiac arrhythmias [The goal of the present study was the identification of novel drugs active against the malignant behavior of MPM cells with a drug-repurposing approach. Thus, 1170 FDA-approved drugs were screened on one non-MPM (MeT-5A) and five MPM  cell lines, and CM, OB, AX, TB, and EM were among the most active. The activity was further confirmed for all the molecules, except for AX, in other in vitro systems. We are aware that the preliminary screening performed at a single dose of 10 μM could poses a bias in the selection of the best active molecules. In fact, some drugs could be effective against MPM at higher doses or eliciting mechanisms not detected through the set of assays employed in this paper . Moreover, since a single-dose assay could be less informative about the toxic activity of a drug than a full-range dose assay, some promising candidates may have been missed. However, the FDA-approved drug library employed in this work has been successfully used at this concentration previously ,19, and nadensis ,23. The nadensis ,25. Its nadensis . TB is a strains , Cooperi” ATPase ,30. Here” ATPase  showed, ” ATPase ,33. EM i” ATPase  extracteynthesis ,36,37, aynthesis . OB is ahythmias ,40. Howehythmias .Drug repurposing could represent a valuable strategy, especially when alternatives are not available for the cure of unmet diseases, such as MPM. Theoretically, CM, OB, and TB should be further investigated with in vivo studies followed by clinical trials. Despite the reduced costs, the clinical research of repurposed drugs could still be challenging, as further studies need to be carried out on formulations, doses, posology, and toxicity profiles. For example, TB has been approved for topic uses, but its effects when administered through other modalities is not known. Moreover, it would be very important to add knowledge on the interactions with other chemotherapeutic agents or in the context of immuno-therapy, a type of research that is very time demanding and resource consuming. Since immuno-therapy was shown to be less effective in MPM than in other tumors , novel cIn conclusion, the present study allowed the identification of several molecules active against MPM cell lines never previously reported with such an activity. However, it should not be forgotten that there were more molecules placed lower in the rank that appeared potentially interesting and worthy of an evaluation in future experiments. Here, CM, OB, TB, and EM showed marked activity on MPM cells, warranting their evaluation in future in vitro and in vivo experiments, also to ascertain their underlying mechanisms of action. Once additional evidence on their effectiveness is proven, it could be foreseen as an application in a potential combined therapy.


NOTCH1 are drivers of T-cell type acute lymphoblastic leukemia/lymphoma. The γ-secretase inhibitor (GSI), which suppresses the function of NOTCH1, is expected to be a molecular-targeted agent. NOTCH1 is also expressed in other malignant neoplasms. We aimed to determine the function of NOTCH1 expression and the effects of GSI on adult T-cell leukemia/lymphoma (ATL) caused by long-term human T-cell leukemia virus type I (HTLV-1) infection.Activated mutations in We analyzed the expression of NOTCH1 in six ATL- and HTLV-1-infected cell lines and investigated the influence of activated NOTCH1  together with GSI on cell proliferation.NOTCH1 mutations were undetectable in six ATL- and HTLV-1-infected cell lines, regardless of abundant NOTCH1 expression. Moreover, GSI did not suppress the growth of ATL cell lines.Activated NOTCH1 found in ATL- and HTLV-1-infected cell lines was undetectable after incubation with GSI, regardless of Tax expression (HTLV-1-coded protein). Whole-exome sequencing revealed that activated NOTCH1-mutation-negative ATL cell proliferation.These findings suggested that NOTCH1 protein is constitutively activated but is likely a passenger during The online version contains supplementary material available at 10.1186/s12885-022-10003-w. NOTCH1 was discovered by analyzing the DNA flanking the breakpoints of a recurrent t  chromosomal translocation, which juxtaposes truncated NOTCH1 with T-cell receptor β, directing the overexpression of N-terminally truncated polypeptides, in < 1% T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) -S-phenyl-glycine t-butyl ester  was dissolved in 10 mM dimethyl sulfoxide (DMSO) stock solution.Cell lines were cultured for 96 h with the indicated concentrations of DAPT or DMSO. Cell viability was then determined using MTT assays .We created libraries using Ion AmpliSeq™ Exome technology (Thermo Fisher Scientific Inc.) and then genotyped amplicons by whole-exome sequencing (WES) using an Ion Proton™ platform  as described by the manufacturer. Sequences were aligned against a reference genome (GRCh37/hg19) using TMAP (Thermo Fisher Scientific Inc.), and then genotyped variants were confirmed by Sanger sequencing.p < 0.05 were considered statistically significant.Data are shown as means ± standard error (SE). Statistical significance was determined using Student t-tests. Values with Expression of NOTCH1 protein is excessive in ATL and HTLV-1-infected T-cell lines.Figure HTLV-1-Tax protein did not affect NOTCH1 protein expression in ATL and HTLV-1- infected T-cell lines.Tax mRNA, S1TcTax05, and S1TcTax10 [We analyzed NOTCH1 protein expression in the Tax-negative S1T-cell line and a clone expressing 1TcTax10 . The expActivated NOTCH1 dissipation was affected by GSI.NOTCH1 rearrangement t  [NOTCH1 mutation  generated via GS-cleavage and labeled using a TM/RAM domain antibody was detected in ATL- and HTLV-1-infected T-cell lines, except for the Su9T01 cell line Fig. . Activat3;q11.2)  than in Growth of ATL and HTLV-1-infected T-cell lines was not suppressed by GSI.NOTCH1 was constitutively activated but probably acted as a passenger in the proliferation of ATL and HTLV-1-infected T-cell lines without NOTCH1 mutation . Activated NOTCH1 mutations, especially in the PEST domain and inactivated FBXW7 mutations, were undetectable in six ATL- and HTLV-1-infected cell lines , in HD-MAR and HT-1 T-ALL cell lines, both of which are dependent on the NOTCH1 signaling pathway for proliferation. GSI reduced proliferation in t -harboring HD-MAR and HT-1 cells, where NOTCH1 is truncated inside the HD under transcriptional control of the T-cell receptor alpha variable [NOTCH1 mutation, needs to be developed to provide a better view of the role of NOTCH1 in ATL pathogenesis. Another reason for the discrepancy is that we could not discount the possibility that additional mutations that occurred during culture for several years might have resulted in NOTCH1 signaling becoming dispensable for growth in vitro. Furthermore, we examined high-concentration of DAPT (up to 10 μM) for 96 h, but it may be the case that the actual effect on the proliferation of ATL/HTLV-1-infected T-cell lines only occurs after long-term incubation.Pancewicz et el. reported that 30% of ATLs have PEST domain single-substitution mutations and showed that the GSI inhibition of NOTCH1 signaling reduces tumor cell proliferation and tumor formation in mice engrafted with ATL. They concluded that NOTCH1 signaling could result in cellular proliferation in ATL . Althougvariable . The weavariable . Howeverhttps://www.ncbi.nlm.nih.gov/clinvar/). Our data suggested that NOTCH1 p.Arg879Gln could be a loss-of-function mutation, and the entity described herein provides a model cell line for topics of clinical and scientific interest.We detected NOTCH1 p.Arg879Gln in Su9T01 cells Table . This vaProgression might be less dependent on the NOTCH1 signaling pathway in ATL cells than in T-ALL.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70–75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers. Pluripotent stem cells are capable of unlimited proliferation and self-renewal. The application of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), is intended to make a revolutionary breakthrough in the field of regenerative medicine, the creation of new drugs, the in vitro modeling of human diseases, and cell bank development ,2,3,4. IUnder in vitro conditions, hPSCs grow in the form of monolayer multicellular colonies with characteristic morphological features. The use of hPSCs in clinic requires a high level of quality control; a homogeneous, clonal composition of the cell population is one of the necessary conditions for their safe application. Various molecular methods are used to assess the state of undifferentiated, healthy colonies. However, the use of invasive assessment methods does not allow these cells to be further used in clinical practice. The noninvasive assessment of cell culture quality is currently not quantitative and can often be biased. It may depend on the experience of the investigator or subtle morphological differences between cell lines and clones. The various existing commercial high-content/high-throughput image acquisition systems not always can be applicable for the morphological analysis of hPSC colonies, since multicellular colonies are formed by close-packed small cells (10–16 μm in diameter), many parameters of which may be overlooked during automatic image analyses ,8,9,10. Clonality is another important characteristic of hPSC colonies. The clonal origin of hiPSC colonies guarantees their further safe use in the clinic, since the heterogeneous composition of the colony is directly related to adaptation in culture leading to the occurrence of chromosomal aberrations . The losMany studies focused on studying the genetic, epigenetic, molecular, and signaling pathways that control the induction and maintenance of pluripotency, as well as the mechanisms of hPSC self-renewal. At the same time, how a stem cell colony is formed, how its growth and cell behavior are regulated, and how this is related to the morphological and functional parameters remain poorly understood. It is important to emphasize that the parametric analysis of the morphological characteristics of various hiPSC clones is in good agreement with the gene expression data; thus, it is a reliable indicator of their quality ,17. ThesIn this study, we extracted and quantitatively analyzed seven morphological parameters of cells and colonies from three hPSC lines and linked these data to their clonality, pluripotency status, and differentiation ability towards three germ layers. We identified specific morphological parameters as the most informative ones in terms of variance between lines and different morphological features and used these parameters to train classification models of colony phenotypes. Finally, we linked the data of the morphological phenotype to the pluripotency status, reflecting the expression of pluripotency marker genes and differentiation markers, and further linked it to create the morphological “portrait” of the colony with a good phenotype. Our results indicated the fundamental possibility of constructing a morphological “portrait” of a colony informative for the automatic identification of the phenotype and link this portrait to the expression of pluripotency genes.The routine culture of hPSCs presents some unique challenges when compared with culturing other human cell types. It is well accepted that healthy and high-quality hPSCs should exhibit the following characteristics: relatively round shapes; tightly packed cells with a high nucleus: cytoplasm ratio, where the nucleus practically inhabits the entire cell; prominent nucleoli; colony centers becoming very dense; and a well-defined edge .We extracted and analyzed seven parameters  characteParameters Area and Perimeter describe the size of the cell’s/colony’s interior and boundary, respectively, while the Minor axis and Feret’s diameters represent the level of elongation. Shape factor quantifies the circularity of cells/colonies, thus representing a measure of their compactness. This parameter, also known as isoperimetric quotient, varies between 0 and 1, where 1 corresponds to the circularity of a circle and smaller values are associated with less circular and compact shapes. One of the indicators of the good quality of hPSCs is the ability for compactness as the colony reaches 72 h of culture. We quantified the compact packing of cells within colonies via the area of the free intercellular space (AIS), with larger values of this parameter being associated with less compact packing. Therefore, AIS represents an additional measure of compactness for colonies.At first, we investigated how the morphological parameters of cells and colonies varied across lines, passages, and growing times. The colony growth process led to a gradual increase over time in most parameters, except Shape factor . The H9 p > 0.05) in any parameter. These data probably point to the possibility to access the quality of the culture at the early time points (24–48 h) and to terminate it in the case of the sign of spontaneous differentiation.The analysis also showed that the variance in the morphological parameters of colonies from the same cell line came mostly from the time point changes rather than from the difference between various passages. For example, the comparison of control hiPSC colonies taken from the middle passage range (passages 21–28) and the late one (passages 39 and 56), fixed at the same time range (24–48 h) for each passage, did not show a significant difference  as a control parameter .p < 0.05). A closer comparison of the groups demonstrated that different lines were characterized by different morphological parameters sensitive to phenotypic separation (p < 0.05) in mean values between colonies with “bad” and “good” phenotypes. The same was true for hiPSC line CaSR, except for the fact that Perimeter only showed a trend towards statistical significance (p = 0.07). Therefore, these two lines exhibited similar sets of colony morphological parameters that could be considered as informative for assessing colony phenotypes. On the other hand, no colony parameter for hiPSC line AD3 showed a significant difference in mean values for different phenotypes  difference between the groups of good and bad phenotypes for hiPSC line AD3. Cells in hiPSC line AD3 colonies with the good phenotype on average had smaller mean values of Area, Perimeter, Minor axis, and both Feret’s diameters then cells with the bad phenotype, which could have been related to the more compact cells in good colonies. They were also more regular in form in the good colonies, which manifested as higher mean Shape factor for the good phenotype as compared with the bad one. Cells from the H9 line also demonstrated a statistically significant difference between phenotypes in the mean values of Shape factor, but in contrast to hiPSC line AD3, this change was associated with a lower average value of this parameter in the good colonies. This could probably indicate differences in colony growth patterns between these cell lines. We did not observe a statistically significant difference in the mean values of cell morphological parameters for hiPSC line CaSR . The resulting cross-validation accuracy of this model was 70 ± 6%, indicating that the phenotype could be predicted with good precision when the parameters were considered in combination.To find out which parameters were more informative in the context of such classification, we trained a set of classification models on the same data using all possible subsets of colony parameters as predictors, with the subset length ranging from two to seven parameters. As expected, the accuracy of these models grew almost monotonically as more parameters were added . We thenWe combined a given number of the best parameters from Applying the minimal classification model to the whole data sample led to a phenotype prediction accuracy of 77% . Bad andNext, we performed a similar analysis of the phenotype classification models based on cellular morphological parameters as predictors. Preliminary calculations showed that Feret’s D and Minimal Feret’s D contributed to the model accuracy in a measure comparable to the contribution of Minor axis, so we excluded these two parameters from consideration to simplify model training. A trained classification model including all other cell parameters (“full model”) produced a cross-validation accuracy of phenotype prediction equal to 69 ± 3%, which was less accurate than when colonial parameters were used as predictors.Classification models based on all possible subsets of the four cellular parameters showed that on average, three parameters were sufficient to reach the highest possible accuracy . The anaApplying the minimal model based on the cellular parameters to the whole data sample led to a phenotype prediction accuracy of 69% . Bad andDNMTB3, SALL4, IGFR1, CD9, DPPA4, OCT4, REX1, NANOG, SOX2, and KLF4 genes) in colonies from the three cell lines with different clonality statuses and phenotypes. By comparing the quantitative gene expression levels between all cell lines, we found that hiPSC line AD3 was mostly characterized by an elevated expression as compared with H9, while some markers that differentiated hiPSC line CaSR from H9 tended to be underexpressed in hiPSC line CaSR , SALL4 (hiPSC line AD3), SOX2 (both hiPSC lines), DNMTB3 (in H9 and hiPSC line AD3), IGFR1 (H9), KLF4 (hiPSC line AD3), DPPA4 (H9), and OCT4 (hiPSC line CaSR). The following genes were overexpressed in nonclonal colonies compared with clonal ones: SOX2 (H9), OCT4 (hiPSC line AD3), SALL4 (H9 and hiPSC line CaSR), KLF4 (H9), DNMTB3 (hiPSC line CaSR), NANOG (H9), IGFR1 (hiPSC line CaSR), CD9 (hiPSC line CaSR), and DPPA4 (hiPSC line CaSR). The fact that the sign of change in expression (over- or underexpression) of many genes varied across the lines pointed to the existence of line-dependent mechanisms that control pluripotency, in agreement with previous publications , where “data” is a list of data values in the form: list of parameter values for a colony/cell → phenotype. With this form of ValidationSet option, the validation set was selected automatically from “data” during the model training. As a control, we performed computational experiments on classification on the cellular data with the manual separation of the training, validation, and testing data sets and obtained the same model performance , and Minor axis and Perimeter (from the cell data). The classification models demonstrated lower performance on hiPSC line CaSR data. To find out the link between pluripotency and clonality in different lines, we examined pluripotency marker expression in hPSC colonies with good and bad morphological phenotypes of clonal and nonclonal (bulk) expansion. We observed significant changes in the expression levels of the pluripotency marker genes in a line- and clonality-dependent manner. Among them,


Coessentiality networks derived from CRISPR screens in cell lines provide a powerful framework for identifying functional modules in the cell and for inferring the roles of uncharacterized genes. However, these networks integrate signal across all underlying data and can mask strong interactions that occur in only a subset of the cell lines analyzed.IGF1R-PIK3CA, and illustrate the implications for drug targeting. We further demonstrate how context-dependent functional interactions can elucidate lineage-specific gene function, as illustrated by the maturation of proreceptors IGF1R and MET by proteases FURIN and CPD.Here, we decipher dynamic functional interactions by identifying significant cellular contexts, primarily by oncogenic mutation, lineage, and tumor type, and discovering coessentiality relationships that depend on these contexts. We recapitulate well-known gene-context interactions such as oncogene-mutation, paralog buffering, and tissue-specific essential genes, show how mutation rewires known signal transduction pathways, including RAS/RAF and This approach advances our understanding of context-dependent interactions and how they can be gleaned from these data. We provide an online resource to explore these context-dependent interactions at diffnet.hart-lab.org.The online version contains supplementary material available at 10.1186/s13059-022-02712-z. ESR1-FOXA1 pathway in ER+ breast cancer cell lines and the BRAFV600E module in melanoma cells  matrix of quantile normalized BFs, and n is the number of cell lines associated with the “context.” Positive dPCC represents a gain of interaction (GOI) because the higher PCCall depends on the presence of cells with the feature of interest, and negative dPCC represents loss of interaction (LOI) because excluding cells associated with the feature of interest increases the PCC.where PCCobs) for each gene pair/feature combination empirically. Briefly, we determined n, the number of cell lines associated with the feature. Then we randomly removed n cell lines from the set of all 808 cell lines and calculated the all-by-all PCC matrix for all pairs of genes, PCCrand. For each gene pair, we measured dPCCrand = PCCall − PCCrand. We repeated this process 1000× and compared the observed dPCCobs to the bootstrapped distribution of dPCCrand to calculate a two-sided empirical P-value that dPCCobs is higher or lower than random expectation. In general, our sampled null distributions of dPCCrand are not well estimated by classical distributions, and our estimate of P-value is therefore limited to a discrete distribution in steps of 0.001. We repeated this bootstrap resampling to generate null distributions of dPCCrand for every gene pair for each of the 393 contexts identified by the logistic regression model . See “calc_diff_coess_pval.py” for code and details.We evaluated the statistical significance of the observed dPCC , LN-18 (CRL-2610), and U-118 MG (HTB-15) were sourced from the American Type Culture Collection (ATCC). Cell line identities were confirmed by STR fingerprinting by M.D. Anderson Cancer Center’s Cytogenetic and Cell Authentication Core (Promega Powerplex 16 High Sensitivity Assay). All cell lines were routinely tested for mycoplasma contamination using cells cultured in non-antibiotic medium .2. Cell lines were cultured in HEPES modified Dulbecco’s modified Eagle’s medium (Sigma D6171) supplemented with 10% FBS (Sigma), 1 mM sodium pyruvate (Gibco), 2 mM L-alanyl-L-glutamine dipeptide (Gibco), 1× penicillin-streptomycin (Gibco), and 100 μg/mL Normocin (InvivoGen).All cell lines were grown at 37 °C in humidified incubators at 5.0% COLentivirus was produced by the University of Michigan Vector Core. Virus stocks were not titered in advance. All transductions were performed in a 6-well plate format across multiple plates with a range of virus volumes and 8 μg/mL polybrene (EMD Millipore). The pool with the most optimal transduction efficiency was expanded and collected for western blotting.5 LN-18 per well, 6 × 105 U-118 MG per well, 6 × 105 M059K per well.Non-transduced cells were eliminated via selection with 2 μg/mL puromycin dihydrochloride (Gibco). Selection was maintained until all non-transduced control cells reached 0% viability. Once selection with puromycin was complete, three replicates were seeded in a 6-well plate format using the following amounts of viable cells: 4 × 10Cells were washed with PBS and harvested in RIPA lysis buffer  supplemented with Halt Protease Inhibitor Cocktail . Twenty micrograms of cell lysate was denatured in Laemmli SDS-Sample Buffer  and loaded on a precast 4–15% Mini-Protean TGX Stain-Free gel (Bio-Rad). After electrophoresis and transfer, the PVDF membrane (Bio-Rad) was blocked in 5% nonfat milk with TBST (Bio-Rad) at room temperature for 1 h and incubated with primary antibody overnight. MET  antibody was purchased from cell signaling technology. CPD  and GAPDH  antibodies were purchased from Thermo Fisher Scientific. The membranes were then washed in TBST three times, followed by incubation with goat anti-rabbit/Mouse secondary antibodies  for 1 h at room temperature. After washing with TBST, the membranes were developed by films. All western blots were repeated twice. Image quantification was performed in ImageJ.Additional file 1: Figure S1. Regression model. Figure S2. Screenshot of Diffnet web service at https://diffnet.hart-lab.org. Figure S3. Selected regression model features. Figure S4. Raw Western blots from Fig. Additional file 2. Tab-delimited table of regression coefficients, with 2918 genomic and lineage features used to predict background-specific essentiality of 2987 genes.Additional file 3. Tab-delimited table of top 2644 hits from differential PCC analysis.Additional file 4. Quantitative data from Western blot experiments.Additional file 5. Review history.


They provide a key tool with which to study cell toxicity and responses to environmental stressors on a cellular level in wild bird species of interest.The validation of the use of primary cell lines from non-lethal matrixes of feathers and blood of nestlings of a wild bird species, the tawny owl  is described. Tawny Owl Feather Fibroblast (TOFF) cells and peripheral blood mononuclear cells (PBMCs) were isolated and cultured from the pulp of the secondary wing feathers and whole blood respectively from free-living tawny owl nestlings. Cell growth was registered up until 48 h for both the PBMC cells and the TOFFs. The validation of these primary cell lines in free-living birds has the potential to advance the assessment of immunotoxicological effects in wildlife  Utilising in vitro tools such as primary and secondary cell lines can make it possible to study the cellular responses to stressors in wildlife via a non-destructive manner. For example, in vitro studies on blood primary cell lines of peripheral blood mononuclear cells (PBMCs) have shown to be good substitutes for exposure experiments in live animals since they represent an attractive tissue source in molecular and immunologic studies  cell lines that have lost the true characteristics of the original tissue from which they are isolated. Serial passaging is known to cause genotypic and phenotypic variation in cell lines. Variation can often be so far from that of the original tissue to where they do not adequately mimic the in vitro for many validated downstream applications from personalised medicine to veterinary medicine, and wildlife research. However, taking blood samples from live animals can be logistically challenging and requires specialised expertise in the handling and extracting of blood. The amount of blood that can be taken from an individual to minimise adverse outcomes is also limited, especially in smaller species. Other non-destructive matrixes could then be explored for example skin biopsies from free-swimming humpback whales  and one non-adherent cell line (PBMCs). They were chosen because they are both: 1) easily accessed without using lethal methods; 2) represent cells with immunological importance (which will be the focus of our downstream applications). Overall, a standardised tool to assess immunotoxicology in free-living birds is lacking, yet very timely given the increase in epidemics such as avian influenzas  in central Norway. A nest box with three tawny owl nestlings was visited on the 26th of May 2021, 3 weeks after hatching. Blood samples (approx. 1–1.5 ml) were taken from the wing vein from each of the three nestlings using a pre-heparinized sterile 2 ml BD PlastiPak syringe with a 0.6 × 25 mm BD Microlance 3 23G no.16 and transferred to a heparinized tube. One secondary wing feather was taken from each of the nestlings and stored in zip bags. Samples were transported to the laboratory facilities within 1 h of collection. Samples were collected as part of a larger ongoing project .2 incubator at 38°C and 5% CO2 gas for 30 days.All the following steps were completed within 24 h of feather sampling. Feather samples from each individual was stored in separate plastic bags in the cooler or fridge (4°C) from sampling until the culturing was performed. The calamus was separated from the rest of the wing feather  using a g for 10 min for samples that were treated within 12 h, or 20 min for samples that were treated within 12–24 h, with the break on. The supernatant with the PBMCs containing the white blood cells (WBCs) was poured off into a new 15 ml tube. The WBC suspension was then washed with the same amount of DPBS with 2% FBS and centrifuged at 300 g for 10 min. The supernatant was discarded, and the pellet was resuspended. The cells resuspension was added into 10 ml Dulbecco’s modified Eagle’s medium  added 5% foetal calf serum  and 1% of Pen-Strep, 100 U/ml Penicillin and 100 μg/ml Streptomycin (cell culture media) in T-25 flasks. A subset of cells was then plated out into 96 well plates to allow for viewing under the microscope. Cells were incubated in a CO2 incubator at 38°C and 5% CO2 gas for 30 days.All the following steps were performed with blood samples stored within 24 h after blood sampling. The blood samples were stored in a dark area at room temperature. Approximately 2 ml of whole blood was transferred into a 15 ml sterile tube. The same amount of Dulbecco’s phosphate-buffered-saline with 2% foetal bovine serum  was also transferred to the tube. The blood/DPBS mixture was then added to a separation tube (SepMate™ -15 (IVD), Stemcell Technologies) that contained 4.5 ml density gradient media  and 80 µl Lymphocyte Enrichment Cocktail  to enrich lymphocytes from the whole blood samples. The separation tube was then centrifuged at 1200 Cells in the 96 well plates were checked daily and photographed weekly. Cell culture media was added to the cell cultures approximately once a week. Photos were taken either with an apple iPhone through the lens of a Leica DM1000/DM3000 microscope or on the EVOS XL Core image software.Growth of PBMCs and TOFFs were registered in the 96 well plates after 5 days  and blood (PBMCs) from a free-living raptor species, the tawny owl for this purpose. Fibroblasts and PBMCs have key immunological properties , therefoin vitro studies can be further expanded into wildlife, instead of model species. This non-lethal method of a primary cultures creates possibilities of expanding the number of species and individuals to look for pollutant effects and investigate biological processes rather than the sacrificing of one individual to immortalize a secondary cell line; which are not as biological relevant as primary cell lines. Which in turn, create possibilities of making an environment of cell exposure for specific species that are suffering from pollution exposure in the wild. By using cell lines from wildlife species of interest, the results have the potential to have less inter-species bias (both between different wild bird species and between domesticated vs wild bird species).Historically, cell lines have been based on domestic species like the domestic chicken  or quail (Coturnix coturnix) . By valiin vitro experiments. TOFF cells can be used for infection/exposure studies with pollutants and pathogens of choice  on a cellular level of any wild bird species of interest. For further studies, it is also possible to increase the levels of pollutants in the in-vitro studies compared to the current environmental concentrations to simulate how future contamination levels will affect the immune response.The next step in this procedure will be to utilise Tawny Owl PBMCs and TOFFs for immunotoxicology studies by exposing the cells to environmentally relevant levels of pollutants . PBMCs can be used for classic lymphocyte proliferation assays, which makes it feasible to measure toxicity of the cells. The assay discovers the compound’s ability to block or cause biological activity without having toxic effects on cells , which mf choice ). Previof choice . These svia pollution levels. However, care must be taken when interpreting the results on cell lines because they cannot completely replace or reflect the whole complexity of the physiological processes that takes place in an entire organism (Current anthropogenic threats makes raptors more vulnerable to toxicants as they are susceptible to bioaccumulate high levels of metals and other pollutants . Furtherorganism . However


ABQ-1-17) as anti-CML agents. In the current work, ABQ-3 was taken to the National Cancer Institute (NCI) for screening to determine its in vitro antiproliferative effects against a large panel of human tumor cell lines at five doses. ABQ-3 revealed significant growth inhibition against HCT-116 CRC and MCF-7 breast cancer cells with 2.00 µM and 2.35 µM GI50 values, respectively. The MTT test also showed that ABQ-3 possessed anticancer effects towards HCT-116 and MCF-7 cells with IC50 values of 5.22 ± 2.41 μM and 7.46 ± 2.76 μM, respectively. Further experiments indicated that ABQ-3 induced apoptosis in both cell lines, and molecular docking studies explicitly suggested that ABQ-3 exhibited DNA binding in a similar fashion to previously reported compounds. Based on in silico pharmacokinetic prediction, ABQ-3 might display drug-like features enabling this compound to become a lead molecule for future studies.Colorectal cancer (CRC), breast cancer, and chronic myeloid leukemia (CML) are life-threatening malignancies worldwide. Although potent therapeutic and screening strategies have been developed so far, these cancer types are still major public health problems. Therefore, the exploration of more potent and selective new agents is urgently required for the treatment of these cancers. Quinones represent one of the most important structures in anticancer drug discovery. We have previously identified a series of quinone-based compounds ( Colorectal cancer (CRC) is a very malignant and prevalent tumor worldwide. Age, local inflammatory changes, genetic components, and a substantial number of environmental and lifestyle factors account for important risks of CRC development. The great majority of patients with CRC are generally diagnosed at late stages with metastases requiring the administration of radiation therapy and chemotherapy as the leading therapeutic strategies for controlling the disease. Although these therapeutic options have evolved to targeted therapy and immunotherapy, the prognosis of CRC has never reached to the desired rate beyond a delay of disease progression so far. The complexity of the underlying mechanisms of the disease and the development of resistance are the major challenges for the treatment failure ,4,5,6,7.On the other hand, breast cancer is the most prevalent and second most deadly cancer among women across the world. It is heterogenous in nature, containing various subtypes with specific properties. Age, heredity, and steroid hormones are major risk factors in breast cancer development. In spite of traditional treatment strategies, including surgery, chemotherapy, and radiotherapy along with hormonotherapy and targeted therapy options to improve overall survival in breast cancer patients, drug resistance is the major impediment to achieving effective anticancer treatment ,10,11,12Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of chromosomal abnormality leading to the aberrant Abelson oncogene (Abl)-breakpoint cluster region (Bcr) gene fusion, which encodes for the Bcr-Abl tyrosine kinase. Tyrosine kinase inhibitors such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib are the mainstay of CML treatment. However, due to the resistance and toxicity problems emerging during treatment with these agents, new treatment approaches to be efficient in CML treatment are required ,14,15.Apoptosis, known as programmed cell death, maintains the elimination of large numbers of cells that are no longer needed without causing any harm to the developing organism ,17. NecrIt has also gained great importance in developing new, more effective, and selective drugs that precisely target/block the changes that cause cancer growth and proliferation. Natural products are associated with the development of novel therapeutic products for cancer treatment; new natural product analogs revealing great chemical diversity and high potential biological activities have become the apple of the eye of scientists ,24,25,26para position revealed the highest inhibitory capacity. The biological potency decreased in these structures with the extension of the bulky alkyl tail in the alkoxy group , illustrated in The 1,4-quinone moiety is a prominent source of biologically active substances, as shown in In order to enhance biological activity, we additionally studied the lead molecules to determine further activity against HCT-116 CRC and MCF7 breast cancer cells. Moreover, the most effective anticancer compound was subjected to further mechanistic experiments, such as the in vitro determination of levels of apoptosis in both cell lines and the in silico analysis of the DNA binding mode. Several pharmacokinetic determinants of this compound were also anticipated in silico.ABQ-3, NCI: D-827196/1, ABQ-11, NCI: D-827197/1, and ABQ-12, NCI: D-827198/1) towards 60 cancer cell lines [ABQ-3 (2-chloro-3-((4-methoxyphenyl)amino)-5,6-dimethyl-1,4-benzoquinone) was appointed as a lead PQ analog because of its significant selective anticancer potential compared to ABQ-11 and ABQ-12 for five-dose in vitro anticancer activity assessment in the range of 0.01–100 µM.In our previous study, PQ analogs  were docll lines  at a sinll lines ,43. Afte50 (growth inhibitory activity), TGI (cytostatic activity), and LC50 (cytotoxic activity)) [ABQ-3. The GI50 is an indicative concentration with a 50% growth inhibition, whereas TGI refers to the total growth inhibitory activity, and LC50 is an indicative concentration in which 50% of cancer cells died. These parameters were calculated for each cell line from the log concentration versus % growth inhibition curves on 60 human cancer cell lines to create dose–response curves [In the current work, GItivity))  were usee curves ,45.ABQ-3 showed high anticancer effects against all leukemia cell lines with GI50 values of around 2.50 µM. ABQ-3 revealed sensitivity towards all leukemia cell lines in concordance with our previous encouraging results that the IC50 values of ABQ-3 against K562, Jurkat, and MT-2 cells were found as 0.82 ± 0.07 µM, 1.51 ± 0.29 µM and 5.41 ± 0.95 µM, respectively. ABQ-3 also exerted low cytotoxicity against the healthy cell line  with the values of 1.79, and 1.40 µM GI50, respectively. HCT-116 (CRC) had a value of 2.00 µM GI50, LOX IMVI, and UACC-257 (melanoma) had values of 1.79 and 1.86 µM GI50, IGROV1, OVCAR-3, OVCAR-4, OVCAR-8, and NCI/ADR-RES (ovarian cancer) had GI50 values in the range of 1.71–2.12 µM, and MCF-7, MDA-MB-231/ATCC, T-47D, and MDA-MB-468 (breast cancer) had GI50 values in the range of 1.29–2.35 µM. TGI values lower than 20.00 µM were determined with most of the mentioned cell types. Finally, ABQ-3 demonstrated a potent lethal function towards the majority of cancer cell lines in the range of 5.60–26.60 µM LC50 values. All the dose–response curves of ABQ-3 against the 60 human cancer cell lines are outlined in On the other hand, EKVX, HOP-92 (NSCLC), HCT-116, SW-620 (CRC), LOX IMVI, MDA-MB-435, and UACC-257 (melanoma), IGROV1, OVCAR-3, OVCAR-4, OVCAR-8, and NCI/ADR-RES (ovarian cancer), MCF-7, MDA-MB-231/ATCC, T-47D, and MDA-MB-468 (breast cancer) were also found susceptible to ABQ-3 . This anABQ-3 displayed significant sensitivity towards HCT-116 and MCF-7 cell lines based on NCI GI50, TGI, and LC50 parameters. In addition, CRC and breast cancer are important research platforms that we have been working on for a long time. Therefore, we further examined the anticancer effects of ABQ-3 on these two cell lines via an MTT -2,5-diphenyltetrazolium bromide) assay at five dose concentrations  in comparison with cisplatin. Cisplatin was selected as a control because cisplatin has been used for the treatment of various cancers such as CRC and breast cancer [t cancer ,48,49,50ABQ-3 inhibited the cell viability of HCT-116 and MCF-7 with notable IC50 values of 5.22 ± 2.41 μM and 7.46 ± 2.76 μM compared to cisplatin   . The perreatment .ABQ-3 displayed notable anticancer effects on CRC and breast cancer cells, we further searched for its apoptotic effects in HCT-116 and MCF-7 cells using the annexin V/ethidium homodimer III staining assay, which was detected by a fluorescence microscope representing apoptosis, necrosis, or late apoptosis/necrosis with green, yellow and red staining, respectively (ABQ-3 was found to possess similar apoptotic behavior in HCT-116 cells (61.80%) with cisplatin (62.30%). In addition, ABQ-3 showed 22.70% late apoptotic/necrotic and 15.50% necrotic activity in HCT-116 cells when compared with cisplatin   B. The di-7 cells B.ABQ-3 in comparison with ABQ-11 and ABQ-12 in the minor groove of DNA  and −6.610 kcal/mol (PDB ID: 2GB9) indicated that ABQ-3 was able to bind to DNA with a higher binding potential compared to ABQ-11 and ABQ-12   using MDs: 2GWA . The docd ABQ-12 . ABQ-3 pctively) A,B.ABQ-3 were estimated by projecting compounds on the QikProp algorithm [ABQ-3 on cytochrome P450 (CYP) enzymes, the bioavailability, passive gastrointestinal absorption, and brain penetration of ABQ-3 were in silico anticipated with the help of the SwissADME web service [ABQ-3 served a drug-like character that crucial pharmacokinetic determinants, including the octanol/water partition coefficient (QPlogPo/w), aqueous solubility (QPlogS), human serum albumin binding (QPlogKhsa), and brain/blood partition coefficient (QPlogBB) were coherent within specified ranges (ABQ-3 exhibited an excellent % of absorption (100%) and followed Lipinski’s rule of five and Jorgensen’s rule of three without any violation.The pharmacokinetic features of lgorithm . Moreove service ,59. ABQ-d ranges . ABQ-3 eABQ-3 based on the SwissADME tool , implying its high penetration through the BBB. ABQ-3 was also detected with a red dot, explaining that it was not a substrate for P-glycoprotein [The demonstration of physicochemical determinants of DME tool  indicategg model  refers toprotein ,60,61.There are many gaps to fill in the treatment of CRC and breast cancer, albeit to the advancements in the screening programs and in the therapeutic options related to prognostic biomarkers. The discovery of new potential anticancer agents to be effective in both cancers may hold the key to the enhancement of treatment responses for patients with CRC and breast cancer. Several lines of evidence have documented that quinone-based compounds, in particular PQ analogs, stand out as promising candidates for anticancer drug discovery ,63.1  parameters of  enzymes ,73,74.ABQ-3, ABQ-11, and ABQ-12 were carried out previously. Their structures were characterized by spectral analysis previously [The synthetic experiments of eviously .The PQs were investigated for their growth inhibitory activity by the NCI  protocol at a 10 µM concentration in DMSO for 60 cancer cell lines. Compounds were added to the microtiter culture plates and incubated for 48 h at 37 °C. Sulforhodamine B (SRB) was applied for end-point detection. The percentage of the growth of the exposed cells was measured in comparison to the nonexposed control cells, and the findings of each tested compound were calculated ,44,74.ABQ-3) for a higher testing level to determine GI50, TGI, and LC50 for each cell line after generating a dose–response curve, including concentrations of 0.01, 0.1, 1, 10, and 100 µM for ABQ-3 [Serial 5 × 10-fold dilution was carried out from an initial DMSO stock solution before incubation at each individual concentration. The Developmental Therapeutics Program (DTP)-NCI screened the most effective compound  at a 4 × 104 cells/mL concentration for 48 h (the optimum cell number was quantified and linked with an earlier study) [ABQ-3 was prepared in DMSO  and cisplatin in DMF  at concentrations in the range of 0.1–10 mM and further was diluted with fresh culture medium. The concentration of DMSO and DMF in the final culture medium was 1% without affecting the cell viability [HCT-116 cells ) and MCF-7 cells  were incubated in Dulbecco’s modified Eagle’s medium (DMEM)  and RPMI 1640 , respectively. All media  was supplemented with 10% fetal bovine serum (FBS)  and 89 μg/mL streptomycin  at 37 °C, 5% COiability ,40.ABQ-3 and cisplatin on cell viability. HCT-116 and MCF-7 cell lines were subjected to ABQ-3 and cisplatin at 1, 3, 10, 30, and 100 μM concentrations at 37 °C for 48 h before being stained with an MTT solution and further incubated for 4 h. After the removal of supernatants, 100 μL DMSO and DMF (for cisplatin) were added to each well. For the analysis of the absorbance of the solution, a plate reader Infinite M1000  was used. All experiments were applied with three repeats, and IC50 values were identified as the drug concentrations, which decreased the absorbance to 50% of the control values.MTT  assay was used as previously explained ,40 for tABQ-3 and cisplatin at an IC50 concentration for 12 h before the cell death detection kit  was carried out with some modifications to the manufacturer’s protocol [HCT-116 and MCF-7 cells were incubated with protocol . Both ceprotocol .p < 0.05, ** p < 0.01, and *** p < 0.001. GraphPad Prism7  was used for the determination of the IC50 values.All findings were exhibited as means ± SD. Data were screened using a one-way analysis of variance, and diversity was accepted at * ABQ-3, ABQ-11, and ABQ-12 were also prepared using the LigPrep module of Maestro. Ultimately, Glide/XP docking protocols were administered for all these ligands [Initially, DNA was downloaded from RSCB (PDB IDs: 2GWA  and 2GB9 ligands ,53,54,55ABQ-3.The QikProp  and SwisABQ-3 was selected for NCI-60 during in vitro screening at five doses towards a huge panel of cancer cells, including HCT-116 and MCF-7 cell lines. ABQ-3 displayed 2.00 µM GI50, 4.37 µM TGI, and 9.55 µM LC50 values against HCT-116 cells, whereas these values were detected as 2.35 µM, 6.05 µM and 30.60 µM, respectively, against MCF-7 cells, demonstrating the growth inhibitory, cytostatic, and cytotoxic effects of ABQ-3 on these cell lines. Based on MTT screening, this compound also showed significant cytotoxicity against HCT-116 and MCF-7 cells. Furthermore, the cell death assay demonstrated that ABQ-3 enhanced apoptotic activity in HCT-116 and MCF-7 cell lines when compared with cisplatin. Molecular docking studies suggested the strong DNA binding of ABQ-3. In silico ADME prediction indicated that ABQ-3 afforded positive drug-likeness values, thus, making this titled compound a potentially good and orally active anticancer agent for future studies.The quinone-based compound


This study aimed to establish a mathematical survival model database containing cell-specific coefficients from human digestive system cells exposed to electroporation pulses (EPs).A total of 20 types of human digestive system cell lines were selected to investigate the effect of EPs on cell viability. Cell viability was measured after exposure to various pulse settings, and a cell survival model was established using the Peleg–Fermi model. Next, the cell-specific coefficients of each cell line were determined.Cell viability tended to decrease when exposed to stronger electric field strength (EFS), longer pulse duration, and more pulse number, but the decreasing tendency varied among different cell lines. When exposed to a lower EFS , only a slight decrease in cell viability occurred. All cell lines showed a similar tendency: the extent of electrical injury (EI) increased with the increase in pulse number and duration. However, there existed differences in heat sensitivity among organs.This database can be used for the application of electroporation-based treatment (EBT) in the digestive system to predict cell survival and tissue injury distribution during the treatment. In termury (TI) , 4. As Eury (TI) .Although the mechanism of cell electroporation is still controversial, it is well-established that the electric field distribution plays a major role in the biological outcome of EPs, which is primarily determined by the pulse parameter, electrode configuration, environmental conditions, and tissue properties , 6. Therin vitro experiment data and mathematical modeling, Dermol and Miklavčič compared the goodness of fit and applicable situation among different mathematical models of electroporation, confirming Peleg-Fermi model is suitable for treatment planning of electrochemotherapy and IRE  exposed to EPs was established by in vitro. Detailed information on the cell lines is provided in 2 humidified incubator  at 37°C.A total of 20 types of human digestive system cell lines, including 17 cancer cell lines and three normal cell lines, were selected to investigate the effect of EPs on cell viability 4 cells/ml. The cell suspension (500 μl) was added to a 2 mm gap cuvette . A square wave pulse generator  was used to generate a burst of EPs. The detailed protocol of the pulse setting for each procedure is summarized in Before exposure to EPs, the cells were washed with phosphate-buffered saline solution, harvested by trypsin, and resuspended in DMEM culture medium to a concentration of 8 × 10After exposure to EPs, the cell suspension was re-collected immediately, dispensed into 96-well plates, and incubated for 24 h. Cell viability was assessed using the cell counting kit (CCK)-8 commercial kit  following the manufacturer's instructions. The optical density (OD) value at a wavelength of 450 nm was measured using an automatic microplate reader . Cell viability was calculated using the following equation:Aexp, Ablan, and Acon are the mean OD values of the experimental, blank medium, and control groups, respectively. For each parameter setting of each cell line, the experiment was repeated 10 times. All data were analyzed using GraphPad .where The equation for the Peleg–Fermi model was as following:S is the cell viability after EP; E is the EFS; Ec(N) and Ac(N) are two functions that depend on the pulse number (N) and cell-specific coefficients, where Ec(N) represents the EFS required for the half lethality of cells, and Ac(N) is the constant of the cell survival curve slope under specific pulse parameters; and E0, A0, k1, and k2 are the coefficients related to the pulse duration, frequency, and cell type. R2 was calculated as statistical measure for the goodness of fit, which indicates the correlation between the calculated and practically determined value. The value of Ec(N) and Ac(N) was calculated based on Equation (2) as first step. Then the value of the coefficients was calculated subsequently based on the results of the first step and Equations (3) and (4), respectively. Thus, two R2 values was acquired in step 2 because E0 and k1 were fitted separately from A0 and k2 based on different equation.where in vitro study were not suitable for curve fitting. To solve this problem, the modified Peleg–Fermi model and power function model was used instead  and t0 = 1 ms.where Ec(N) (N = 100) were estimated using Equation (3) for each cell line.The survival model curve fitting and calculation of the coefficients were referenced from Goldberg et al. , and werEc(N), the field at which 50% of a population of cells, as the threshold for subsequent numerical analysis.In Pelge-Fermi equation, with the increase of the pulse number, even very weak electric filed will induce electric injury, which is inconsistent with the reality. Therefore, a threshold is needed to limit the calculation for IRE. Here, we defined To evaluate the efficacy of the mathematical model for IRE, a numerical analysis was performed using COMSOL Multiphysics  using the finite element method in accordance with our previous study . To simpA time-dependent study was performed to investigate the accumulative electrical and thermal effects of EP on the tissue. Specifically, the pulse parameter was set to 2,000 V in pulse voltage, 100 μs in pulse duration, and 1 Hz in frequency. A total of 100 pulses were used for EI analysis. To fully evaluate the thermal effect of IRE and heat sensitivity of each organ, 200 pulses  were set for the analysis of TI.Ec(N) for each cell line.Because the results from the Peleg–Fermi model and Arrhenius model depicted survival rate, the threshold of irreversible injury for the probability of EI and TI was set as 0.5, implying that if the probability of EI or TI was >0.5, it could be treated as an irreversible injury. The effective electric field was defined as the threshold of EFS for electroporation that was set as the corresponding value of To quantitatively evaluate the model from a certain cell line, the relative area ratio of the probability of EI and the probability of TI were calculated using Equations (7) and (8):SEI is the area of EI, STI is the area of TI, and Stotal is the total area of the tissue domain. All data were analyzed and compared using GraphPad . As the calculation of EI is based on the specific coefficients obtained from the Peleg–Fermi model at the cellular level, the corresponding SEI can be acquired for each of the 20 cell lines. Since the calculation of TI is based on the Arrhenius equation at the organ level, STI can only be obtained for each organ.where Statistical analysis was performed using GraphPad. The results are expressed as mean ± SD.The viability of each cell line exposed to EPs under various pulse durations, pulse numbers, and EFS was determined. Ec, Ac, and Ec(100) of the 20 cell lines under different pulse durations. Ec and Ac for each cell line with the increase in pulse number under a pulse duration of 100 μs. The data of Ec and Ac under pulse durations of 25, 50, and 75 μs are available in the online database. Moreover, the results of power function model of all cell lines are available in the online database as well  values were in the range of 800–1,000 V/cm, and for a pulse duration of 100 μs, most of the Ec(100) values were between 400 and 600 V/cm. For most of the cell lines, the value of Ec(100) decreased with increasing pulse duration. When treated with EPs with a 100 μs pulse duration, the Ec(100) value varied from 459 to 684 V/cm for esophageal cells, 85 to 536 V/cm for gastric cells, 316 to 632 V/cm for colon cells, 401 to 665 V/cm for hepatic cells, 174 to 485 V/cm for biliary cells, and 532 to 713 V/cm for pancreatic cells.For the 25 μs pulse duration, most of the SEI and STI of each cell line from the digestive system are shown in SEI and STI for specific pulse numbers can be found in the online database  and cholangiocarcinoma cell lines (HCCC-9810 and QBC-939) showed higher slopes and maximum values of the SEI curve than the other cell lines. In contrast, the viability of cell lines from the esophagus and pancreas showed a relatively lower inhibition rate when exposed to EPs  of all cell lines was calculated and compared in this study. For most cell lines, the value of Ec(100) was approximately 500–700 V/cm, which is generally considered as the threshold for electroporation  and 60 days . They also found that harmless, self-limiting adverse effect and lasting tissue regeneration occurred during the 60 days follow-up . Recentlin vitro cell experiments that cannot completely reflect the effect of EPs at the tissue level. However, there is still no qualified method for directly evaluating cell activity under EP in vivo. This study can assess the biological effect of EPs on the survival of the digestive system to a large extent, laying a theoretical foundation for future studies. Second, the Peleg–Fermi model contains the EFS, pulse number, and pulse duration without frequency; however, pulse frequency may have a considerable effect on both EIs and TIs. This model may not be suitable for high-frequency EPs. Third, the EI and TI were calculated via the Peleg–Fermi equation and Arrhenius equation; however, there may exist an electrical-thermal coupling effect during electroporation, which needs to be further investigated for a more accurate model. Additionally, normal tissue properties were used to establish the numerical analysis model due to the lack of data from the tumor; therefore, the efficacy of this model needs to be further verified by in vivo studies in the future.This study has some limitations. First, the cell survival model was established based on in vitro cell experiments and in silico analysis. The efficacy of the database was evaluated by numerical analysis of both EIs and TIs of the digestive system. This database can be used for basic research, computational simulation, medical device design, and treatment planning that can predict cell survival and tissue injury distribution after exposure to EPs during EBT.In this study, a mathematical model database for cell survival containing cell-specific coefficients of 20 human digestive cell lines was established based on The original contributions presented in the study are included in the article/XH and NZ designed the study, performed the related experiment, and wrote and edited the manuscript. YZ, ZL, JZ, XC, and ZQ researched and analyzed the data, helped in writing, and reviewed the manuscript. QL, YW, LM, TH, and YLi helped to perform the related experiments. FR and YLv designed the study and revised the manuscript. All authors have approved the final manuscript.This study was supported by National Natural Science Foundation of China (grant no. 81727802), the Fundamental Research Funds for Central Universities (grant no. XJH012020022), the Scientific Development Funding of the First Affiliated Hospital of Xi'an Jiaotong University (2020QN-08), and the Basic Natural Science Research Project of Shaanxi Province (2021JQ-401). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.