Graft versus host disease (GVHD) is the major non-relapse complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, GVHD occurs in roughly half of patients following this therapy and can induce severe life-threatening side effects and premature mortality. The pathophysiology of GVHD is driven by alloreactive donor T cells that induce a proinflammatory environment to cause pathological damage in the skin, gastrointestinal (GI) tract, lung, and liver during the acute phase of this disease. Recent work has demonstrated that the GI tract is a pivotal target organ and a primary driver of morbidity and mortality in patients. Prevention of this complication has therefore emerged as an important goal of prophylaxis strategies given the primacy of this tissue site in GVHD pathophysiology. In this review, we summarize foundational pre-clinical studies that have been conducted in animal models to prevent GI tract GVHD and examine the efficacy of these approaches upon subsequent translation into the clinic. Specifically, we focus on therapies designed to block inflammatory cytokine pathways, inhibit cellular trafficking of alloreactive donor T cells to the GI tract, and reconstitute impaired regulatory networks for the prevention of GVHD in the GI tract. Graft versus host disease (GVHD) is the major non-relapse cause of morbidity and mortality occurring after allogenic hematopoietic stem cell transplantation (HSCT) , 2. GVHDCorticosteroids have long been first line therapy for patients with acute GVHD in the GI tract as they function to inhibit inflammatory pathways and cytokine production , 10. HowAmelioration of this complication in humans is therefore dependent upon increasing our understanding of the pathophysiology of GI GVHD. To unravel pathophysiologic mechanisms by which this disease is propagated and devise potentially translatable clinical strategies, animal models, primarily using mice, have been employed to examine how dysregulation of the immune system occurs in this setting , 13–16. H17 cells (Interleukin-1 (IL-1) was the first interleukin to be described and exhibits a myriad of functions that are critical for inflammation. IL-1, along with 10 other members, comprise the IL-1 super family since they possess a highly conserved gene structure and are primarily clustered in a 400kb region of human chromosome 2 , 18. Whi17 cells . The rol17 cells .McCarthy et al. first demonstrated that IL-1 could be a critical modulator of acute GVHD in murine studies. IL-1α was observed to be elevated in the skin of mice with GVHD and inhibition of IL-1 signaling with an IL-1R antagonist reduced GVHD mortality without impairing engraftment . SubsequMore recently, Park and colleagues evaluated the mechanism for how IL-1 blockade alleviates GVHD severity . They deBased on preclinical data indicating that IL-1 inhibition could be beneficial for GVHD, Antin et al. tested whether treatment with a recombinant human IL-1 receptor antagonist could be beneficial for the prevention of acute GVHD in a phase I/II trial. They observed stage-specific improvements of GVHD in the skin, liver and particularly the GI tract (82% of patients) as well as demonstrated that the treatment was safe . AnotherTNF-α, a pleiotropic inflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and cancer also plays a role in acute GVHD. While TNFα is primarily produced by macrophages and monocytes during acute inflammation, context-dependent insults can induce TNFα to be released by lymphocytes, endothelial cells and other cell types as well. TNFα signaling occurs through 2 receptors; TNFR1, which is ubiquitously expressed and promotes inflammation and tissue damage, and TNFR2, which is restricted to a few cell types and responsible for homeostatic functions .One of the first pre-clinical studies to evaluate the role of TNFα in GVHD was performed in 1987. Piguet et al. utilized a semi-allogenic murine model to assess whether administration of anti-TNFα antibodies eight days after GVHD induction could be beneficial. This therapeutic approach limited skin epidermal necrosis, reduced gut epithelial cell damage, and decreased mortality . The autEarly clinical studies helped to define the kinetics of TNFα production following GVHD. This work demonstrated that systemic TNFα levels were increased during the conditioning phase  as well Administration of infliximab, a chimeric IgG1 monoclonal antibody which binds to soluble and transmembrane human TNF-α, has been given to patients for GVHD treatment with some success , but theH17 cells and plays an integral role in skewing naïve T cells towards proinflammatory phenotypes limiting regulatory T cell (Treg) differentiation. Several studies have identified a role for IL-6 and members of the IL-6 superfamily  in contributing to autoimmune disorders, cancer, and GVHD  and reduce acute GVHD. They concluded that donor cytotoxic T cells utilize CCR5 to enter the gut and that the PP is an essential site for initiating GVHD . Conversph nodes . Collectin vitro as well as performed a phase 1/2 study on 38 high-risk patients who received standard immune suppression along with maraviroc as GVHD prophylaxis , also known as α4β7 integrin, is responsible for homing into gut-associated lymphoid tissue. When expressed on T lymphocytes, α4β7 integrin licenses cells to bind to mucosal addressin cell adhesion molecule (MAdCAM), which is chiefly expressed on high endothelial venules of mucosal lymphoid organs as well as intestinal lamina propria . Given t−/− T cells resulted in significantly reduced GVHD-induced mortality compared to wild type T cells which was attributed to delayed homing to the intestines and liver  compared to patients with skin GVHD (1.3%) and no GVHD (1.0%). α4β7 was not differentially expressed on any CD4+ or CD8+ T cell subsets that were analyzed. Therefore, this study highlights the importance of α4β7 expression on CD8+ T cells particularly for propagating human GVHD symptoms within the gut.In clinical studies, Chen et al. examined the peripheral blood of patients with symptoms of acute GVHD before treatment . The colVedolizumab, a monoclonal antibody that binds to α4β7, has been approved for treatment of ulcerative colitis and Crohn’s disease and, more recently, has been examined as a treatment for steroid refractory GI GVHD with variable results , 88. DanThese data demonstrate that delayed α4β7 inhibition has limited efficacy for the treatment of acute GVHD patients who are steroid refractory. This could be due to the fact that there are multiple trafficking mechanisms employed by allogeneic T cells that contribute to lower GI tract damage, or that pathological damage facilitated by T cell entry into the gut occurs rapidly after transplant and may be dispensable at later time points. Cumulatively, these data suggest that earlier intervention may be necessary and that vedolizumab may be better suited as a preventive therapy rather than as steroid-refractory secondary treatment for gut-associated acute GVHD. To that end, there is currently a trial evaluating the efficacy of vedolizumab for acute GVHD prophylaxis (NCT03657160). This trial will assess the effect of vedolizumab on decreasing the incidence of GI-acute GVHD and acute GVHD-induced mortality 6 months after transplant. Results from this study will help to determine whether administration of vedolizumab could be appropriate for GI GVHD prevention.+ T cells are immunosuppressive lymphocytes that express high levels of the IL-2 receptor alpha-chain CD25, as well as the fork-head box transcription factor, Foxp3 . This trial is designed to co-culture recipient dendritic cells and donor Tregs prior to allogeneic stem cell transplantation to determine whether the incidence of acute GVHD is reduced.Macmillan performed a phase I study to determine the safety and efficacy of induced Tregs (iTregs) on GVHD prophylaxis in adults with high-risk malignancy . They rein vitro (α1-antitrypsin (A1AT), is a protease inhibitor produced by the liver and can inactivate serine proteases produced by myeloid cells and suppress their ability to produce pro-inflammatory cytokines. Pre-clinical studies have been performed which have revealed unique mechanisms for how A1AT influences acute GVHD. Marcondes et al., 2011 showed that A1AT could mediate protection by first demonstrating that it suppresses IL-32 and T cell proliferation in vitro . UtilizaWith respect to GVHD prevention, Gergoudis et al. performed a biomarker-guided preemptive study examining whether administration of A1AT could reduce the incidence of GVHD in patients deemed to be at high risk for steroid-resistant complications . Thirty Histone deacetylases (HDACs) play a key role in regulating gene transcription by acting as transcriptional repressors to remove acetyl groups and promote chromatin condensation . HDAC inex vivo with HDAC inhibitors before transplant was sufficient to protect mice from GVHD. These findings illustrate the prominent role HDACs play in regulating DC function to aggravate intestinal damage associated with GVHD. Another group corroborated the benefits of HDAC inhibition for GVHD by elaborating on its mechanism for protection of GVHD mice  could improve GVHD-induced morbidity and mortality in an MHC-mismatched murine model of the disease . They deVHD mice . Leng etThe pre-clinical success observed with HDAC inhibition laid the foundation for testing whether this therapy could be beneficial for GVHD prophylaxis. Choi et al. performed a phase I/II trial to evaluate whether the HDAC inhibitor, vorinostat, could reduce the incidence of GVHD if administered 10 days before transplantation until day 100 in patients with high-risk hematological malignant disease who received stem cell grafts from matched related donors after reduced intensity conditioning . They foA second HDAC inhibitor, panobinostat, was recently evaluated both in a phase I trial for GVHD treatment  and in aProteasomes are large catalytic protein complexes that cleave and degrade misfolded, damaged or erroneous proteins into peptides . They cain vitro and protect mice from acute GVHD in vivo without adversely affecting donor reconstitution when administered at the time of transplant. Subsequently, Vodanovic-Janovic et al. evaluated whether administration of bortezomib could protect mice from GVHD  in 2003  and was rom GVHD . They reSun et al. corroborated these findings and demonstrated that prolonged bortezomib administration increased serum levels of TNFα and IFNγ in multiple murine models of GVHD and led to early mortality . The detBased on preclinical results indicating a protective role of bortezomib administration for acute GVHD, Koreth and colleagues conducted a phase I/II trial to test whether a short course of bortezomib could be an applicable intervention for GVHD prophylaxis following transplantation from HLA-mismatched unrelated donors for patients with hematologic malignancies . Of the The GI tract is the major site of morbidity and mortality associated with the development of acute GVHD. Unfortunately, a significant percentage of patients fail to respond to first line therapy with corticosteroids and require second line therapy for steroid refractory disease. Outcomes for these patients is significantly worse and a substantial proportion of these patients ultimately do not respond to salvage therapy. In addition, patients who develop GI GVHD often require hospitalization and are at risk for secondary infections due to compromised epithelial barrier integrity, which adversely impacts quality of life and can also result in premature fatality. Consequently, prevention of this complication, particularly within the GI tract, is critically vital to improve overall treatment outcomes and should be a primary goal of GVHD prophylaxis strategies.To that end, preclinical studies have identified that inhibition of inflammatory cytokine pathways, blockade of gut homing molecules that are expressed on the surface of alloreactive donor T cells, and reconstitution of regulatory pathways as potential therapeutic strategies that have shown promise and led to translation in human clinical trials. Unfortunately, many of these strategies, while promising in animal studies, have not translated well into the clinic. Reasons for this are not entirely clear but are likely multifactorial and related to limitations of mouse models that do not fully replicate the complexity of human allogeneic stem cell transplantation with respect to recipient age, stem cell source, conditioning regimen intensity, and MHC disparity which all impact GVHD severity. In other cases, some of these approaches have only recently entered clinical trials for GVHD prophylaxis  so the verdict is still out on whether they will be efficacious for prevention of GI tract GVHD. To date, blockade of IL-6 signaling, administration of Treg infusions, and histone deacetylase inhibition have reported clinical outcomes in which there appears to be a reduction in GI tract GVHD; however, definitive data are still lacking with these approaches. Thus, additional investigations are required to clearly identify effective prophylactic strategies that will ameliorate toxicity to this important tissue site, and secondarily lead to an improvement in overall transplant outcomes.AR and WD wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by grants from the National Institutes of Health .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.


E. coli) and Gram-positive Staphylococcus aureus (S. aureus), but CDFs exhibited better antibacterial performance, and S. aureus could be completely inhibited with the minimum inhibitory concentration of 30 μg/mL. This reveals CDFs magnify both the stability and antibacterial activity, which would be more promising for practical applications.Carbon dots (CDs) have been widely used as antimicrobials due to their active surface, but some CDs suffer instability. Therefore, the relative applications such as the antibacterial activity may not be reliable for long-term use. Herein, we synthesize CDs with blue fluorescence by a hydrothermal process. Thereafter, polyethylenimine was applied for the assembly of CDs into CDs-based frameworks (CDFs). The CDFs exhibited quenched fluorescence but showed more stable properties based on the scanning electron microscope and zeta potential investigations. Both CDs and CDFs show antibacterial activity toward Gram-negative Escherichia coli (The online version contains supplementary material available at 10.1186/s11671-021-03582-3. Bacterial infections show a serious threat to human lives, and the development of effective medicines to disinfect bacteria is in great demand . VariousE. coli) and Gram-positive Staphylococcus aureus (S. aureus) compared to CDs, indicating their broad-spectrum antibacterial performance, though many CDs only eradicated Gram-positive bacteria [To meet the demand for practical applications, the antimicrobials should have the following characteristics: (a) Excellent stability remains unchanged for a certain time in an ambient environment; (b) Excellent biocompatibility and low toxicity: (c) high antibacterial activity. The larger nanomaterials tend to be more stable, but they might have relatively weaker antibacterial activity due to the smaller active surface area. Considering both the weakness of small and large nanomaterials, we report the assembly of the small CDs into large CDFs by simply adding polyethyleneimine (PEI) Fig. . CDs werbacteria . In addibacteria . This woX-ray surface photoelectron spectra (XPS) were recorded on an ESCALAB250Xi X-ray surface photoelectron spectroscopy (XPS) instrument. The transmission electron microscope (TEM) was performed by a JEM-2100 microscope operating at 200 kV. The fluorescence of the materials was obtained using the F97 fluorescence spectrometer. The FDA/PI staining of the bacterial cells was recorded in tapping mode with a Leica DFC450C microscope. Fluorescence lifetime was measured on a time-correlated single-photon counting (TCSPC) system using a Nanolog spectrofluorometer . Ultraviolet–visible spectroscopy (UV–vis) spectra are obtained from the UV-1600 instrument. The bacterial imaging was observed by a confocal microscope (Olympus FLUOVIEW FV1000 c). All the reagents were of analytical grades. The deionized water was used through the experiments. Cell counting kit-8 was obtained from Beyotime Biotechnology.L- cysteine (1.0 g) was dissolved in 10.0 mL of deionized water and mixed well. Then, the pH of the solution was adjusted to 9.0 with 1.0 M NaOH. The solution was transferred to a hydrothermal reactor and heated at 160 °C for 24 h. After the solution was cooled to room temperature, the resulting solution was subjected to dialysis using a dialysis bag (MW 7000 cut off) for one day. The as-obtained CDs were used for the following characterizations and experiments. For the preparation of CDFs, 40 µL of 1% PEI was added to 1 mL of the CDs. The mixture was allowed to stay for 1 h. The product was purified by dialysis using the same method as the purification of CDs. For HR-TEM characterization, the samples were concentrated into a small volume, transferred to a silica gel column, and eluted with methanol and dichloromethane to obtain the further purified products.PC12 cells were seeded in 96-well plates for 12 h and then were incubated with CDs and CDFs with different concentrations. The number of viable cells was investigated using a Cell Counting Kit-8 assay (CCK-8). 3--2, 5-diphenyltetrazolium bromide (MTT) (5 mg/mL in PBS) was added at 1/10 culture volume, and the cells were returned to the incubator. After that the supernatants were discarded and 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The crystals were dissolved by shaking the plates for 10 min. The absorbance at 490 nm was measured using the Microreader (Varioskan LUX Multimode Reader). Blank control wells were included for all the absorbance measurements.E. coli and S. aureus were incubated in the absence and presence of CDs and CDFs at 37 °C with 250 rpm shake. Growth of the bacterial cells in Lysogeny broth (LB) culture was measured by the Microreader at 600 nm wavelength (OD600). LB medium was used as the blank control. The OD600 stands for the cell density, and the relative cell viability was calculated based on the comparison between the cultured bacterial cells in the presence of the materials to the control group . The live/dead bacteria are evaluated by the FDA/PI staining protocol [protocol .Intracellular ROS production was measured in bacterial cells before and after the treatment by CDs and CDFs for 12 h, with a 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe following the instructions. The fluorescence was detected at an emission wavelength of 525 nm with excitation at 488 nm.Figure Zeta-potential investigations were used to measure the degree of electrostatic repulsion of CDs and CDFs between adjacent charged particles in the dispersed system Fig. . It can The fluorescence emission spectra were used to monitor the assembly process of CDs to CDFs Fig. . As the For evaluation of the safety of the materials, the toxicity of the CDs and CDFs to PC12 cells is investigated. Assays of MTT were conducted to investigate the influence of the materials on cell viabilities Fig. . After iS. aureus and E. coli cells. Especially, the viability of the S. aureus cells was almost 0 when larger than 30 µg/mL of the CDFs were used. Similarly, the E. coli cells were disinfected by both CDs and CDFs. CDs showed multiple charges ,HR-TEM (b), and AFM  of the as-obtained CDs; Figure S2: SEM of CDscharacterized at four random times; Figure S3: (a) UV-vis, (b) the fluorescenceexcitation and emission spectra of the CDs; Figure S4: XPS survey (a) and FTIR ofthe as-obtained CDs; Table S1: Lifetime of the CDs and CDFs.


Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Treatment options for UM include radiotherapy, thermotherapy and tumor resection. Electrochemotherapy (ECT) is a new therapeutic modality for local tumor control in various cancer entities. The current study assesses the radiosensitizing effect of concomitant ECT with bleomycin and irradiation on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines. The evaluation of the radiosensitizing effect of ECT as a drug delivery system was based on the changes in the spheroid growth, the cell viability as well as the cytotoxic long-term effect of the combined treatment. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses in comparison to cell lines originating from previously irratiated tumors. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors. Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines  and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses. BRCA1-associated protein (BAP1) tumor predisposition syndrome [Uveal melanoma (UM) is the most common primary intraocular tumor in adults and has an incidence of approximately 5.1 per million per year in the USA and of 1.3–8.6 per million per year in Europe, following a north-to-south decreasing gradient from a maximum of 8 per million per year in Denmark and Norway to a minimum of 2 per million per year in the Mediterranean region [syndrome ,5. The mBRAF), neuroblastoma RAS viral oncogene homolog (NRAS) and neurofibromatosis type 1 (NF1) are not common in a UM, which are characterized by different oncogenic genes or mutations with a loss of function [GNAQ/GNA11), which appear in a mutually exclusive manner and occur in approximately 90% of posterior UMs [SF3B1), eukaryotic translation initiation factor 1A, X-linked (EIF1AX) and telomerase reverse transcriptase promoter (TERTp) have also been associated with UMs. A direct lineage between cytogenetic alterations and prognosis has been described and M3, 8q+ 6p+ and 1p- can consequently be used as biomarkers of prognostic importance [A UM has distinct molecular features compared with other melanoma subtypes. The cutaneous melanoma-associated mutations such as v-raf murine sarcoma viral oncogene homolog B1 (function . By nextrior UMs . Other mportance .The primary treatment options for a UM include radiotherapy (brachytherapy or teletherapy), transpupillary thermotherapy and tumor resection  ,2. The oElectrochemotherapy (ECT) is a nonthermal treatment modality that combines the application of electric pulses to the tumor tissue with chemotherapeutic agents ,10. ECT Moreover, ECT has been reported as a radiosensitizing agent for radiation therapy, postulating that a single session before irradiation can significantly enhance the tumor response. The combination of electrochemotherapy preceding irradiation leads to an increased radioresponse with an enhancement factor of up to 4.6 using a radiomimetic (bleomycin) or a radiosensitizing (cisplatin) drug ,18,19,20Our study examines the radiosensitizing effect of ECT with various drug concentrations as well as alternating pulse and radiation settings in 3D spheroids using two established UM cell lines  and two primary UM cell lines . Not only the spheroid growth but also the cell viability, cell survival and the long-term cytotoxic effect of the tumor spheroids were assessed in order to determine the efficacy of concurrent irradiation and ECT in a primary UM.Spheroids were generated from two UM cell lines, UM92.1 or Mel270, and from two uveal primary melanoma (UPM) cells lines, UPMD2 or UPMM3. The characteristics of the cell lines are summarized in The different tumor spheroids may reflect heterogeneous uveal melanomas and were therefore subjected to radiation and/or ECT on day 7. The short-time cytotoxic effect on the spheroid growth of different UM and UPM spheroids was observed five days after treatment (at day 12). Overall, radiation had little effect on spheroid morphology whereas the ECT dose dependently changed the spheroid size and appearance in a cell line-specific manner . In termOverall, ECT with both bleomycin dosages of 1 or 2.5 µg/mL were effective in the size reduction of spheroids and radiation prior to ECT had little additional effects on the spheroid size. Strikingly, the treatments led to a reduction in the spheroid size not only compared with untreated spheroids at day 12 but also compared with untreated spheroids at day 7 indicating the shrinking of the spheroids below the initial size . The cell viability of UM spheroids or UPM spheroids were differently affected by the treatments . UM spheIn contrast, the UPM spheroid cell viability was reduced already by radiation. Strikingly, ECT on its own as well as in combination with radiation dramatically reduced the spheroid cell viability . Basically, UM or UPM spheroids displayed a different ability of cell out-growth and proliferation in a long-time culture. The untreated UM spheroid cells formed a confluent and homogeneous cell monolayer  while UPM spheroid cells remained as a subconfluent and inhomogeneous monolayer . The respective cell density was reflected by Crystal Violet (CV) staining of the cell nuclei/DNA .The uveal melanoma cell line UM92.1 and uveal primary melanoma cell lines UMPD2 and UPMM3 were provided by Dr. M. Zeschnigk  ,27,28,29The cell cultures were trypsinized and the resultant cell suspension was mixed with an equal amount of 4% trypan blue stain . The number of living and dead cells was determined by trypan exclusion using a TC20 automated cell counter .3 living cells in round bottom 96-well ultra-low attachment plates  in 100 µL of the respective cell culture medium. The medium was refreshed two times per week. The spheroid cultures were incubated in a humified incubator  for the indicated time of period. Compact spheroids could be generated on day 4 of the culture.The spheroids were generated by seeding 5 × 10n = 5 each condition) were imaged at day 4, 7 and 12 using a Zeiss Primovert bright-field microscope at 4 × magnification. The images were recorded with a Zeiss Axiocam 105 and ZENcore software and analyzed by using image processing software Fiji ImageJ 1.53c . The spheroid size was determined by calculating the cross-sectional area of the spheroids (µm2). The spheroid compactness was determined by calculating the optical density of the spheroid area . In order to determine the living/dead cells, the spheroids (n = 8–16 in triplicate) were trypsinized and the resulting cell suspension was subjected to a cell viability assay as described above.The spheroids (The spheroid cultures were irradiated and/or treated with electrochemotherapy (ECT). In terms of combination therapy, the spheroid cultures were first irradiated and additionally treated with ECT within one hour after radiation. As controls, the spheroid cultures remained untreated.60Co source X-ray irradiator  at 300 kV, 10 mA and a dose rate of 0.9 Gy/min.Radiation: spheroid cultures were irradiated in a 100 µL medium in multi-wells with a dose of 5 or 20 Gy by using a ECT: the spheroids were treated with a 100 µL medium containing 1.0 µg/mL or 2.5 µg/mL bleomycin and electroporated by using two parallel aluminum electrodes 4 mm apart. Eight pulses  of a 1000 V/cm or 750 V/cm pulse strength were applied by a voltage pulse generator . As controls, further samples were treated with bleomycin or electroporation only. After 24 h of treatment, the spheroids were washed with the medium and incubated in a fresh medium for the indicated period of time.n = 3–8 each condition) were fixed in buffered formalin (Histofix). Formalin-fixed paraffin embedded sections were cut at a thickness of 3 µm. The Ki67 immunohistochemistry was performed on a Ventana BenchMark Ultra system  using clone 30-9, a monoclonal mouse IgG antibody . An immunohistochemistry analysis was performed with the following protocol: pretreatment cell conditioning 1, 95 °C, 32 min.; incubation 36 °C, 16 min. The visualization was conducted using an OptiView DAB System . Thereafter, the slides were scanned using a Leica DM4000B microscope, Leica DFC290 camera and analyzed using the Leica Suite version 2.8.1 . The slides were reviewed by a board-certified pathologist (ST).The spheroids (The spheroid cell viability was assessed using the CellTiter-Glo 3D cell viability assay (Promega) according to the manufacturer’s instructions. Equal amounts of CellTiter-Glo and spheroid cultures were mixed by pipetting up and down for 30 s to enable a complete lysis of the spheroid cells and the release of ATP. The mixture was transferred to white opaque-walled multi-well plates (Nunc Micro well). After five minutes of incubation on a shaker (750 rpm) and a further 30 min of incubation, the luminescence was recorded using a reader FluostarOmega . The ATP luminescence was given in RLU (relative light units).n = 12 each condition) were individually transferred to 24-well plate dishes. The spheroids were allowed to attach to the uncoated flat plastic bottom to enable the out-growth of spheroid cells. After eight weeks in the culture, the cell confluence was determined by bright-field microscopy. The adherent cells were fixed with formalin for 5 min and the cell nuclei/DNA were stained with 0.05% Crystal Violet for 30 min. The absorbance of Crystal Violet (CV) was measured at OD 540 nm  using a FluostarOmega  reader.Five days after treatment (at day 12), the spheroid cultures . A value of This study presented the positive radiosensitizing effect and the long-term results of concomitant irradiation and ECT with bleomycin in primary UM 3D spheroids with or without monosomy 3. The results indicated that uveal melanoma cell lines could be radiosensitized after one ECT treatment, making a clinical application practicable. The different levels of radiosensitivity between the various UM cell lines could be attributed to the tumor origin and prior treatments as well as to the intrinsic tumor characteristics and genetics. As shown for the Mel270 and UM92.1 cell lines, the combined treatment with electrochemotherapy and tumor irradiation could be used to treat radioresistant tumors and larger tumor nodules, which would not achieve a satisfactory tumor control with other single-modality treatments. Further studies are necessary for the examination of the time setting and the interval between ECT and irradiation as well as of the effect of repeated ECT sessions and a fractionated irradiation regimen in uveal melanoma tumors.


Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established. Based on levels of the estrogen, progesterone and HER2 receptors, breast cancer can be divided in different subtypes. The triple-negative subtype (TNBC) lacking the expression of these three hormone receptors accounts for 15–20% of all tumors2 and is the most aggressive subtype, often leading to metastases4. Despite the efforts, there is still no targeted therapy for TNBC available5. A major reason for this lack in clinical translation may be the use of two-dimensional in vitro experiments that do poorly represent the three-dimensional (3D) tissue physiology observed in human cancer patients. To increase translation from in vitro findings to a clinical setting, different 3D culture systems are now explored, such as organoid cultures, patient-derived xenograft models, reprogrammed stem cell like models, tumor-on-a-chip and 3D cultures of immortalized breast cancer cell lines6. While the majority of breast cancer drug screening studies in the last decade have still been performed in 2D12, there is an increasing number of drug screens performed in more complex models such as patient-derived organoids14, tumor-on-a-chip15 and patient-derived xenograft16 models. Although these complex models better represent human physiology and should increase clinical translation19, drawbacks of these models include reduced reproducibility21, increasing costs, inconvenient maintenance, difficulties in expanding them and generating genetic modifications, making these models less suitable for high-throughput screening22. Next to the already widely studied phenotypic changes between different culturing models23 and phenotypic classification of different tumor subtypes6, transcriptomic and proteomic analyses can contribute to the understanding of the differences between established in vitro models and help to determine the most suitable model in terms of both clinical translation, costs and efficiency.Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women with an estimated 40,610 deaths in the United States in 2017Here, we performed RNA-sequencing of 14 breast cancer cell lines cultured on a 2D plastic substrate as well as in a 3D matrigel-collagen environment. In this 3D model, cells spontaneously form spheroid-like structures exhibiting cell–cell as well as cell–extracellular matrix interactions, thereby changing their cell polarity and shape. We unraveled the transcriptomic differences linked to the invasive phenotype of basal B (or claudin-low) TNBC compared to basal A and luminal breast cancer and uncovered a spectrum of genes higher expressed in 2D cultures that were related to metastatic progression in breast cancer patients. Since the transcriptomic correlation of in vitro cultured cell models to patient tumor tissue was highly subtype and pathway dependent, we established a bioinformatics tool that can be used in future studies to select the most suitable cell type and culture conditions for the pathway of interest. Altogether, this study unraveled the transcriptomic variance between different breast cancer in vitro models and provides an important database that can contribute to selection of the most effective and relevant drug candidates for the treatment of TNBC.28 with selected cell lines representing the different BC subtypes ). These cell line subtypes were validated in our RNA sequencing dataset; hierarchical clustering based on RNA expression of previously published luminal and basal markers clearly separated the different subtypes  cells, we performed RNA sequencing of 52 human breast cancer cell lines cultured on 2D tissue culture plastic and 14 cell lines cultured in a 3D matrigel-collagen environment   tumors) derived from The Cancer Genome Atlas (TCGA). Since we observed clear differences in culture-specific gene expression changes for the different subtypes, we performed these analyses for the DEGs obtained from the paired analysis for all cell lines, basal A and basal B specific DEGs Fig. A-B. Sinc42. The respective targets of these various inhibitors were significantly lower expressed in 3D cultures  and showed that the sensitivity to EGFR inhibitors such as gefitinib is determined by the variation rather than the mean EGFR expression; PDX models bearing heterogeneous expression of EGFR were more sensitive towards gefitinib46. EGFR-high subpopulations were characterized by growth inhibition upon gefitinib treatment and enhanced expression of mesenchymal-stem cell markers46. Interestingly, the same mesenchymal-stem cell markers were also more highly expressed in non-invasive EGFR-inhibitor sensitive cells in both 2D  uncover the differential pathway expression linked to subtype-specific morphological changes and (2) identify the most suitable model for future drug screens. Consistent with previous comparisons of 2D and 3D cultures28. Also needs to be noted that the MDA-MB-231 cell line that was classified as basal B and has been used in this context in many studies, demonstrated to be a poor basal cell line representative in a recent study48. These contrasting phenotypes between basal A and basal B cells are likely related to differences in expression of a small subset of extracellular matrix interacting and cell–cell adhesion genes. Thus, the basal B cells upregulated genes involved in collagen biosynthesis and breakdown of the extracellular matrix in 3D cultures, while these genes were downregulated in basal A cells. Moreover, the higher basal levels of E-cadherin in the basal A subtype could contribute to the less invasive phenotype observed in 3D cultures. The differences in cell line invasiveness and related pathway regulation for basal subtypes could be specific to the selected culture environment. Future studies using a 3D environment without the presence of an external extracellular matrix such as ultra-low attachment plates or spinner vessels would reveal the effects of the matrix composition on pathway regulation and cell phenotype.We observed that cell lines of the basal A subtype showed a clear non-invasive spheroid morphology in 3D. In contrast, cell lines of the basal B subtype demonstrated a distinct invasive phenotype; an exception for the basal B phenotype was the SUM149PT, that shows ambiguous classification and has also sometimes been classified as basal A36. Next, proteasomal inhibition could disrupt essential cellular pathways resulting in cancer cell death 50. To investigate whether transcriptomic differences change drug sensitivity and define the optimal in vitro model for future drug screening; we systematically treated cell lines in both 2D and 3D cultures with specific inhibitors against CDKs, aurora kinases and the proteasome. Whereas previous studies mainly identified reduced sensitivity towards established drugs  in 3D cultures in mostly HER2 positive cell lines53, no change in sensitivity for CDKs, aurora kinases and proteasomal factors comparing 2D and 3D cultures was observed with our experimental setup. While the drugs that we tested were not evaluated in these studies, the discrepancies can also be explained by the differences in cell lines used and culture conditions. Additionally, we cannot exclude that limited differential sensitivity between 2 and 3D for the selected drug targets is related to limited changes of the target protein levels. For example, EGFR expression per se does not explain EGFR inhibitor sensitivity, and that EGFR inhibitors are effective in blocking EGFR auto-phosphorylation, without impacting on downstream signaling54. Hence, the TNBC proliferative and survival signaling wiring is complex and multiple pathways can contribute to overcome kinase inhibitor efficacy. Regardless, our data do indicate that across a broader panel of TNBC cell lines, typically 2D screening for the candidate drug targets will be as effective as 3D screening in identifying drug responses. To determine the optimal future screening model to assess efficacy of new targeted therapies, a thorough understanding of the overall clinical translation of the test system for specific cancer types, including TNBC, will be essential.CDKs, aurora kinases and proteasomal factors that were higher expressed in 2D, showed genuine higher expression levels in more aggressive breast tumors and cell lines, and were positively related to metastatic disease in breast cancer patients. Several aurora kinase inhibitors and Cell cycle-related CDK inhibitors have been developed of which some show promising results in clinical trials and are almost facing towards the clinicFor basal BC cell lines the transcriptome of 3D cultures demonstrated increased similarity towards human patient tumor transcriptomes, which was mainly caused by elevated levels of genes involved in extracellular matrix interactions. On the contrary, the transcriptome profile of luminal cell lines was already highly comparable to the patient tumor transcriptome in 2D conditions, but did not improve in 3D settings. Importantly, similarity of transcript levels was highly pathway and subtype dependent. Next to the pathway of interest, the ideal in vitro model might change depending on the BC subtype of interest. In general, pathways in triple-negative patient tumors are better correlated to basal cell lines than the same pathways in ER-positive tumors.Altogether, we can conclude that our systematic transcriptome analysis uncovered major differences between 2 and 3D models thereby increasing the global understanding of these in vitro test systems. Defining ‘the best in vitro model’ with efficient clinical translation is yet difficult and dependent on both the pathway and subtype of interest, although in general 3D cultures correlated better to human patient tumors. To support such a selection, we established an in silico web tool in to allow (1) differential expression of genes between 2 and 3D BC cell lines, (2) collect gene expression levels in different breast cancer subtypes (cell lines as well as patient tumor data) and (3) calculate correlation between different cell lines/culture conditions with human patient tumors (10.5281/zenodo.4560297).57. These models that are closer to the patient situation have higher costs and cannot yet be applied in similar throughput as 2D plastic models, but are recently widely exploited for their application in drug screening61. Future systematic comparisons of the transcriptome and proteome of all these test systems in comparison with large BC patient transcriptome and proteome datasets, and their response to candidate drug targeted therapeutic approaches will be essential to provide clarity about the most optimal pre-clinical in vitro test systems.We performed a systematic transcriptomic comparison between 2 and 3D BC cell lines. Although clear transcriptomic differences were identified between 2 and 3D culturing systems, additional proteomic analysis would add a layer of information that could help to validate the transcriptomic findings and more intensively discriminate between the different models. In this study, we did not include other in vitro cell models that are of relevance for BC anticancer therapy development, including BC tumor organoids, short-term patient-derived xenograft cell cultures, stem cell reprogramming and tumors-on-a-chip2 incubator. For 2D cultures, cells were grown on plastic tissue culture plates (Corning). For 3D cultures, cells were grown in a gel-mixture containing 6 mg/ml matrigel , 0.5 mg/ml collagen , 3.7 g/L NaHCO3 and 0.05 M HEPES.All cell lines were purchased from ATCC. Cells were grown in RPMI-1640 medium  supplemented with 10% FBS , 25 IU/ml penicillin and 25 µg/ml streptomycin (full RPMI) (ThermoFisher Scientific) at 37 °C in a humidified 5% COFor 2D cultures, cells were plated in a 6-well tissue culture plate  in full RPMI. RNA was collected 2 days after plating at 70% confluency using RNeasy plus mini kit (Qiagen) according to the manufacturer’s protocol.g), the upper aqueous phase was collected and RNA was precipitated by mixing with 600 μl isopropanol. Samples were incubated for 10 min at room temperature, followed by 15 min centrifugation at 4 °C  and washing with 1.2 ml 75% v/v ethanol. Samples were centrifuged for 5 min at 4 °C (7500 × g). Then the supernatant was removed and the pellet was air-dried and dissolved in 30 μl RNase free water.For 3D cultures, cells were plated in a 24-wells tissue culture plate . Wells were pre-coated with 200 μl matrigel  and cells were plated in a total volume of 400 μl. The following densities were used for the different cell lines: MDA-MB-231 (200 cells/μl), Hs578T (200 cells/μl), BT549 (200 cells/μl), SUM149PT (250 cells/μl), HCC1143 (250 cells/μl), HCC1806 (250 cells/μl), HCC1954 (250 cells/μl) and MDA-MB-468 (250 cells/μl). After 30 min incubation at 37 °C, 1200 μl full RPMI was added. RNA was collected 7 days after plating using the QIAzol method to reduce matrigel pollution. RPMI was removed, 1.2 ml QIAzol (Qiagen) was added per well and cells were lysed by repetitive pipetting. Cells were incubated for 5 min at room temperature and 240 μl chloroform was added, followed by 2–3 min incubation at room temperature. Samples were centrifuged for 15 min at 4 °C  developed by Dr. Richard Simon and the BRB-ArrayTools Development Team and R (v3.2.2). Genes with p-value < 0.01 were considered significant. Upon acceptance, RNA sequencing data will be available in the Sequence Read Archive.DNA libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit and sequenced according to the Illumina TruSeq v3 protocol on an Illumina HiSeq2500 sequencer. 100 base pair paired-end reads were generated and alignment was performed using the HiSat2 aligner (version 2.2.0.4) against the human GRCh38 reference genome. Gene expression was quantified using the HTseq-count software (version 0.6.1) based on the ENSEMBL gene annotation for GRCH38 (release 84). Count data was normalized using the DESeq2 package63 previously described by our group64.For 2D drug screening, cells were plated in a 96-well tissue culture plate  using the following cell densities: HCC1143 (8000 cells/well), HCC1806 , HCC1954 (8000 cells/well), MDA-MB-468 , Hs578T , MDA-MB-231 (5000 cells/well), SUM149PT (8000 cells/well) and BT549 . Drug treatment was performed 16 h after plating and 4 days after treatment, cells were fixed for sulforhodamine B (SRB) colorimetric proliferation assayFor 3D drug screening, cells were plated in a 384-well µclear plate  using a CyBi Selma 96/60 robotic liquid dispenser . The same gel composition and cell densities were used as in the RNA isolation procedure. Cells were plated in a total volume of 14.5 μl matrigel-collagen mix. After 30 min incubation at 37 °C, 44.5 μl full RPMI was added. Drug treatment was performed 3 days after plating and 4 days after treatment, cells were fixed and stained in 0.05 µM Phalloidin Rhodamin (Sigma Aldrich), 0.4 µg/ml Hoechst 33258 (Fisher Biotech), 0.1% Triton X-100 Triton (Sigma Aldrich) and 3.7% formaldehyde (Sigma Aldrich) in PBS O/N at 4 °C. Plates were washed with PBS and imaged using ImageXpress Micro XLS system .Both in 2D and 3D cultures, cells were treated with 6 concentrations of the following inhibitors: MG132, bortezomib, flavopiridol HCl, dinaciclib, palbociclib, milciclib, aurora kinase inhibitor I, TAK-901, gefitinib and erlotinib . DMSO was used as drug vehicle control.SRB absorbance values were used to analyze 2D cell line proliferation. To analyze the 3D cultures the acquired image stacks  were processed in OcellO Ominer 3D image analysis platform. The software processes the DAPI channel to measure the number of nuclei and the shapes of the nuclei in each image stack and integrates this information with the shape and number of tumor cell clusters detected in the TRITC channel. The software was used to process the total phalloidin area as measurement of proliferation.Proliferation was normalized to the absorbance (2D) or phalloidin area (3D) before treatment (set at 0%) and DMSO treatment (set at 100%). A nonlinear sigmoidal dose–response (with variable slope) curve was used to fit the data and retrieve the IC50 values using Graphpad Prism 7.00.For 2D immunofluorescence staining, cells were plated in a 96-well μclear plate (Greiner). Cells were fixed and permeabilized 72 h after plating by incubation with 1% formaldehyde and 0.1% Triton X-100 in PBS and blocked with 0.5% w/v BSA in PBS. Cells were incubated with 1:10,000 Hoechst 33258 combined with 1:2000 rhodamine-phalloidin  for 1 h at room temperature. Cells were imaged with a Nikon Eclipse Ti microscope and 20 × oil objective.65 using KEGG and Reactome databases. Ranked gene set enrichment analysis (GSEA) was performed on the full ranked gene lists66. T-sne plots were generated using the tsne R package. Hierarchical clusterings were generated using the pheatmap R package, based on complete linkage and Euclidean distance or correlations as described in the figure legend.Over-representation analysis was performed using Consensus PathDB67 after the new release in January 2017. Normalized reads were log2 transformed before further analyses were performed. For calculating spearman correlations between the TCGA and cell line RNA sequencing data, log2 normalized data were used for both datasets. Microarray data of primary tumors of 867 untreated patients (MA-867 dataset) used to calculate hazard ratios was previously published and publicly available . Raw .cel files were downloaded, processed with fRMA and batch effects were corrected using ComBat.Normalized total gene expression data from The Cancer Genome Atlas (TCGA) were obtained by using the TCGA Assembler R packageThis article does not contain any studies with human participants or animals performed by any of the authors.Supplementary Figure Legends.Supplementary Figures.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.


Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs.Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the ‘The Cancer Genome Atlas’.A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs . Western blotting confirmed regulation of key resistance-associated proteins . GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines.High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.The online version contains supplementary material available at 10.1007/s00345-022-03936-1. Testicular germ cell tumours (GCTs) are the most common malignant solid neoplasms in men between the age of 15 and 40 with an increasing incidence seen over the last 4 decades . NSGCT cIn this study, we compared cisplatin-resistant and cisplatin-sensitive cell lineages of pluripotent NTERA-2 and NCCIT as well as the nullipotent 2102EP cells  , 14 by hl-glutamine . The incubation temperature was 37 °C in a humid atmosphere with 5% carbon dioxide in the air.The two human GCT cell lines NTERA-2 (CRL 1973) and NCCIT (CRL 2073) were supplied by ATCC, USA. 2102EP cells were a kind gift from PD Dr. Dr. Friedemann Honecker. All cell lines were cultured in HEPES-buffered RPMI-1640  supplemented with fetal calf serum , penicillin , streptomycin  and For all parental GCT cell lines , isogenic cisplatin-resistant sublines  were established through repeated cisplatin-exposure to increasing sublethal cisplatin concentrations (0.01–0.5 µg/ml) over a time period of 9–12 months, as described previously . Cisplatl-arginine-13C6 (Arg + 6) and 0.275 mM l-lysine-2D4 (Lys + 4) or equimolar amounts of l-arginine-13C615N4 (Arg + 10) and 0.275 mM l-lysine-13C6,15N2-Lys (Lys + 8)  for at least ten cell cycles. Labelled cells were lysed in 0.5% Nonidet P-40 buffer containing 50 mM Tris/HCl, pH 7.8, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 0.2% lauryl maltoside and protease inhibitors . After isolation, the protein amounts were determined by a colorimetric BCA assay. Equal amounts of SILAC-labelled proteins , were mixed and separated by SDS-PAGE . Proteins were visualized with Coomassie brilliant blue stain and each lane cut into 11 equidistant slices irrespective of staining. Gel slices were reduced with 10 mM DTT for 55 min at 56 °C, alkylated with 55 mM IAA for 20 min at 26 °C and digested with modified trypsin  overnight at 37 °C. Resulting peptides were concentrated on a C18 precolumn  for 5 min at a flow rate of 10 μl/min and separated on a C18 capillary column  at a flow rate of 300 nl/min, with a gradient of 5–35% acetonitrile 0.1% formic acid over 90 min using a Proxeon nano LC coupled to a Q Exactive mass spectrometer . MS conditions were as follows: spray voltage, 1.8 kV; heated capillary temperature, 270 °C; and normalized collision-energy (NCE) 28. The mass spectrometer was operated in data-dependent acquisition mode. Survey MS spectra were acquired in the Orbitrap (m/z 350–1600) with a resolution setting of 70,000 at m/z 200, a target fill value of 1e6 and a maximum fill time of 60 ms. The 15 most intense ions were sequentially isolated for HCD MS/MS fragmentation and detection at a resolution setting of 17,500, a target fill value of 2e5 and a maximum fill time of 60 ms. Raw data were analysed with MaxQuant software version 1.3.0.5  against the UniprotKB human reference proteome revision 02–2017 containing 92,928 sequence entries. Up to two missed cleavages of trypsin were allowed. Oxidized methionine, N-terminal protein acetylation and the respective isotope-labelled arginine and lysine residues were searched as variable modifications and cysteine carbamidomethylation as fixed modification. The false discovery rates at the protein and peptide levels were both set to 1%, respectively. Missing values in individual experiments are denoted by ‘Not a Number (NaN)’. No further data imputation or similar was used.We performed protein expression profiling by mass spectrometry in combination with stable isotope labelling by amino acids in cell culture (SILAC) as described before –18. NTER2 ratios of quantification to the intensity, Log10 values were calculated for intensities of each mean value. Afterwards, significance B analysis using a Wilcoxon-Mann–Whitney test with a Benjamini–Hochberg FDR < 5% was performed by Perseus software to calculate the p values of outlier proteins from the 1/1 ratio [10 intensity was plotted against Log2 ratios of quantified resistant/parental ratios. Proteins were coloured according to their p values where blue colour means > 0.05, red color 0.05–0.01, yellow between 0.01 and 0.001 and light green coloured proteins mean that they have p values less than 0.001.Perseus Software version 1.5.2.6  was used for statistical evaluation. The mean of the biological replicates was calculated for each cell line . In order to compare Log/1 ratio . Log10 i® CA-630) with protease inhibitors. Protein concentration was quantified by the Bio-Rad DC Protein Assay . The Western blot analyses were performed using the following primary antibody dilutions: monoclonal rabbit anti-CBS , polyclonal rabbit anti-LDHA , monoclonal rabbit anti-ANXA1 , monoclonal rabbit anti-CTH  monoclonal mouse anti FDXR . Polyclonal immunoglobulins/HRP secondary antibodies  were used for the detection of primary antibodies. Membranes were developed using the ECL system .Cell lines were lysed in RIPA buffer  [p values < 0.05 and FDR q < 25% as suggested by GSEA. For visualization in a bar graph p- and q-values were − log2 transformed. Enrichment plots were saved from the GSEA reports.Functional annotations were predicted using gene set enrichment analysis (GSEA) software downloaded from the home page (dex.jsp) , 22. Loghttps://string-db.org) [https://david.ncifcrf.gov/home.jsp) [https://www.cbioportal.org/) [The STRING algorithm was used to predict protein–protein interaction by confidence using standard settings (-db.org) . The DAVome.jsp) . The proal.org/) , 26.2 SILAC ratios were seen in all three cell lines  and NADPH-adrenodoxin oxidoreductase (FDXR) were increased in all three resistant cell lines . In each cisplatin-resistant cell line individually, we found a heterogenous set of processes affected. The analysis of NTERA-2-R detected ‘Interferon alpha and gamma’ signalling and ‘epithelial to mesenchymal transition’ (EMT) as mainly enriched gene sets . Two sets related to ‘Myc targeting’ and ‘DNA repair’ were significantly affected in NCCIT-R cell line . Furthermore, GSEA revealed seven significantly affected gene sets including ‘P53 signalling’, ‘hypoxia’, ‘fatty acid metabolism’, ‘glycolysis’, ‘late estrogen response’, ‘oxidative phosphorylation’ and ‘IL2 STAT5 signalling’ for 2102EP-R . The combined analysis of the three cell lines highlighted ‘DNA repair’, ‘oxidative phosphorylation’ and ‘early estrogen response’ as the three most prominently enriched gene sets  of all genes coding for proteins altered in each cell line individually or commonly. GSEA determines, if a defined set of genes shows a statistically significant difference in enrichment between two biological states (cisplatin resistance vs. sensitivity) , 22. We First, the DAVID Gene ontology tool was used to predict molecular functions of all proteins altered in the resistant cell lines . Proteins increased in amount in the resistant cells compared with the parental cell lines were involved in oxidoreductase activity, acetylation and metal binding were among others commonly deregulated in all three cell lines. Next, the STRING algorithm was used to predict protein–protein interactions of the enriched proteins . Proteins involved in the molecular processes found by the DAVID analysis were highlighted by colour. In contrast, proteins decreased in amount in resistant cell lines compared with the parental cell lines were involved in phosphatidyl-inositol binding and DNA methyltransferase activity .IFITM3 (enriched in 2102EP-R) harboring a deep deletion in 2.1% of samples, GDF3 being amplified in 7% (enriched in NCCIT-R) and STK38L (enriched in NT2/D1-R) being amplified in 8% as most affected genes by mutation.Furthermore, using cBioPortal, we screened the TCGA GCT tissue cohort for mutational footprints and expression of all genes coding for affected proteins in resistant cell lines individually . In general, 149 GCT (seminoma and nonseminoma) samples were included. The majority of the samples were highly aneuploid, harbored a 12gain, were mainly in the typical age of 14–44 years and were isolated from testis. Indeed, expression of analyzed genes was mainly associated with a nonseminomatous cell character . Nevertheless, expression of a subset of genes could also be related to seminomas. Mutations in the analyzed genes were overall rare, with Cisplatin resistance in GCTs rarely occurs in primary diseases, but in up to 15% of metastatic diseases with poor prognosis for the affected patient . Differe2S) [2S production might activate the intrinsic apoptotic pathway via release of mitochondrial cytochrom C [S-transferase (GST) [Cystathionine beta-synthase (CBS) was significantly decreased in amount in all three cisplatin-resistant cell lines in comparison to their cisplatin-sensitive parental lineage. CBS is an enzyme that regulates homocysteine metabolism and catalyzes the formation of cystathionine . Further2S) . CBS is 2S) , 31. Its2S) . For exa2S)  as well 2S) , colorec2S)  and pros2S)  have shoochrom C . Accordiochrom C , testicuse (GST) . These f2S. It plays a key role in the bodies H2S production [2S in metastatic prostate cancer [Another enzyme of the cysteine metabolism, namely cystathionine gamma lyase (CTH), showed decreased levels in two of the three resistant cell lines (NTERA-2-R and NCCIT-R). CTH catalyzes the breakdown of cystathionine to cysteine, alpha-ketobutyrate and Hoduction , 30. Itse cancer , similare cancer , 39. InhThe Annexin A1 (ANXA1) levels were significantly increased in resistant NTERA-2 and NCCIT cell lines. ANXA1 is a calcium-dependent phospholipid binding protein that belongs to the Annexin superfamily –42. ANXAIn addition, the protein levels of L-lactate dehydrogenase A chain (LDHA) were elevated in NTERA-2-R and 2102EP-R cells. LDHA plays an important role in anaerobic glycolysis , 49. In Furthermore, a significantly increase of COL1A1 and COL1A2 levels was detected in cisplatin-resistant NTERA-2 cell lines in comparison with the parental cell line, with decrease in resistant NCCIT cell line. Both proteins resemble extracellular matrix proteins for that increased levels have been detected in stressed cells . They haGSEA revealed that DNA repair is the only significant gene set deregulated in all three cell lines, which has been considered for cisplatin resistance in GCT before . In NTERDAVID Gene Ontology and STRING analyses showed a deregulation of proteins involved in oxidoreductase activity, acetylation and metal binding. The upregulation of NADPH-adrenodoxin oxidoreductase (FDXR) in resistant cell lines could be validated by Western blot. Oxidoreductase activity, acetylation and metal binding have been described in connection to p53 –57. A reScreening of the TCGA GCT cohort demonstrated that most genes/proteins found increased in the resistant situation, show already basal expression in GCT tissues (mostly nonseminomas). Thus, rather an overshooting/upregulation of expression than an induction of gene expression is associated with acquisition of therapy resistance. The mutational burden was overall low, nevertheless some genes were frequently mutated in GCT tissues, putatively affecting their molecular function. During development of cisplatin resistance, these mutated genes might be induced as well, further contributing to cisplatin resistance by their altered function or increased expression turnover.In summary, high resolution mass spectrometry in combination with SILAC quantification is a powerful tool to detect differences of protein levels in cisplatin-resistant and cisplatin-sensitive cell lines. We detected 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. The findings of mass spectrometry could be validated by Western blot analysis. With this study, we therefore provide a large proteomic resource in vitro library for studying proteomic alterations contributing to acquired cisplatin resistance in testicular germ cell tumour cell lines. The detected and analysed proteins need further investigations to unravel their putative role in cisplatin resistance of GCT and to determine possible new treatment approaches.Supplementary file1 (PPTX 277 KB) Supplementary Figure 1: Distribution of proteins in all cell lines. This figure shows the normal distribution of proteins from SILAC analysis in all cisplatin-resistant and cisplatin-sensitive cell lines.Supplementary file2 (XLSX 695 KB)Supplementary file3 (PPTX 1041 KB) Supplementary Figure 2: GSEA analysis of all three GCT cell lines separated. (a) The top ten affected gene sets of NTERA-2 represented as the − log2 of the NOM q-value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of NT2: (b) INTERFERON ALPHA RESPONSE , (c) INTERFERON GAMMA RESPONSE  and (d) MESENCHYMAL TRANSITION . (e) The top ten affected gene sets of NCCIT represented as the − log2 of the NOM q value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of NCCIT: (f) MYC TARGETS_V1 , (g) DNA REPAIR  and (h) MYC TARGETS V2 . (i) The ten top affected gene sets of 2102EP represented as the − log2 of the NOM q value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of 2102EP: (j) P53 PATHWAY , (k) HYPOXIA , (l) FATTY ACID METABOLISM , (m) GLYCOLYSIS , (n) ESTROGEN RESPONSE_LATE , (o) OXIDATIVE PHOSPHORYLATION  and (p) IL2 STAT5 SIGNALLING Supplementary file4 (PPTX 10786 KB) Supplementary Figure 3: Summary of the results of STRING analysis and DAVID annotation.  Increased proteins in NTERA-2-R cell lines: green – phosphoprotein, blue – acetylation, yellow – oxidoreductase, red – PcG protein complex.  Increased proteins in NCCIT-R cell lines: blue – oxidoreductase, red – mitochondrial biogenesis, green – cholesterol biosynthesis, yellow – lipid biosynthesis, pink – glutamine family amino acid biosynthetic process.  Increased proteins in 2102EP-R cell lines: blue – RNA N6-mehtyladenosine methyltransferase complex, red – glycogen storage disease.  Decreased proteins in NTERA-2-R cell lines: red – metabolism, green – phosphatidylinositol binding.  Downregulated proteins in NCCIT-R cell lines: red – metabolism, biosynthesis of amino acids, green – phosphatidylinositol binding.  Decreased proteins in 2102EP-R cell lines: green – Ubl conjugation, red – DNA methyltransferase activity, blue – microRNAs in cancer, yellow – kinase, pink – signal recognition particleSupplementary file5 (PDF 1232 KB) Comparison of data to TCGA GCT cohortBelow is the link to the electronic supplementary material.


WT  and hSOD1G93A  cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with most cases being sporadic; however, 5–10% are familial .G93A) and wild-type  cell lines [3H]L-tryptophan was lower in the MT than in the control cell lines L-arginine uptake under neuroinflammatory conditions.Our study aimed to evaluate the transport characteristics of arginine in ALS cell lines and to investigate nNOS expression. Using quantitative real-time polymerase chain reaction (qPCR) analysis, we further sought to investigate nNOS regulation after treating NSC-34 cell lines with arginine and citrulline. In addition, we also investigated the effect of [3H]L-arginine was purchased from PerkinElmer . Other reagent-grade chemicals and drugs were purchased from Sigma Aldrich .A total of 45.1 Ci/mmol of L-arginine uptake study in NSC-34 cells was conducted according to previously described methods [3H]L-arginine  to each well, cell lines were incubated for 5 min at 37 °C. Next, ice-cold ECF buffer was used to terminate [3H]L-arginine uptake. Thereafter, 750 µL of 1 N NaOH was used to solubilize the cells overnight at room temperature. Radioactivity was measured using a liquid scintillation counter . The cell-to-medium ratio (µL/mg protein) was calculated as follows: [3H] radioactivity (dpm/µL) in the sample per milligram of cellular protein (dpm/mg protein) L-arginine at 0–1 mM was calculated using the Michaelis–Menten constant (Km) and maximum uptake rate (Vmax) as shown in the following equation 1:max1 and Vmax2 are the maximum uptake rates for high- and low-affinity processes, respectively; Km1 and Km2 are the Michaelis–Menten constants for the high- and low-affinity processes, respectively; and Kns1 and Kns2 are first-order constants for the non-saturable components in the high- and low-affinity processes, respectively.For saturation kinetics, the initial uptake of L-Arginine uptake was found to be concentration-dependent in NSC-34 cell lines; however, its uptake in the MT cell lines was lower than that in the WT cell lines  and maximum uptake rate (Vmax) were fitted to the Equation (1), and at the high-affinity site, the affinity (Km) was 23-fold lower and the capacity (Vmax) was approximately 40-fold higher in the MT cell lines than in the WT cell lines. However, at the low-affinity site, both affinity and capacity in the MT cell lines did not markedly differ from those in the WT cell lines , a system y+ specific inhibitor, significantly inhibited the uptake in both cell lines, showing sensitivity to system y+. N-methylmaleimide (NMM), which is system y+L inhibitor, also showed significant inhibition in both cell lines. However, 2-aminobicyclo--heptane-2-carboxylic acid (BCH), a system Bo,+ inhibitor, showed no inhibition in either cell line  strongly inhibited [ell line .3H]L-arginine transport was scrutinized in NSC-34 cell lines. Quinidine, an antiarrhythmic drug, significantly inhibited [3H]L-arginine uptake at a concentration of 2 mM. Notably, [3H]L-arginine uptake was inhibited by more than 50% in the presence of gabapentin in both cell lines, whereas verapamil showed 68 and 73% inhibition in MT and WT cell lines, respectively. Clonidine and donepezil did not inhibit [3H]L-arginine uptake in either cell lines  in the presence or absence of 500 µM lysine, quinidine, and gabapentin. In the case of lysine, the two lines of the plot intersected at the ordinate, indicating that lysine competitively inhibited L-arginine a,b.Quinidine showed non-competitive inhibition in the WT cell lines, with a Ki value of 2.2 mM, and competitive inhibition in the MT cell lines, with a Ki value of 0.64 mM c,d. In aTo assess the pattern of nNOS levels, we measured mRNA expression using qPCR in WT and MT cell lines, with or without the addition of arginine and citrulline. nNOS mRNA expression was lower in the MT cell lines than in the control; however, nNOS relative gene expression significantly increased in MT cell lines treated with arginine and citrulline, in comparison to untreated MT cell lines. In the WT cell lines, there was no statistical difference in the mRNA expression between the untreated and arginine-treated cells. In contrast, citrulline treatment resulted in a significant increase in nNOS expression compared with that in untreated WT cell lines .3H]L-arginine uptake upon preincubation of NSC-34 cell lines with 100 µM H2O2 are shown in Changes in [2O2 significantly decreased [3H]L-arginine uptake in both cell lines compared with that in control. Additionally, 10 mM of arginine and citrulline increased [3H]L-arginine transport rate in the MT cell lines. The Slc7a1 mRNA expression was markedly decreased in the presence of H2O2 alone in the MT cell lines; however, in addition to co-treatment with H2O2 and arginine, co-treatment with H2O2 and citrulline also resulted in a significant increase in the [3H]L-arginine uptake rate. These results suggest the restorative and neuroprotective roles of arginine and citrulline in ALS cell lines.Hwt) and SOD1 (G93A) gene mutation and are widely used for the study of ALS disease mechanisms and also to discover the therapeutically effective drug regimen in motor neurodegeneration L-Arginine uptake occurs in a Na+-independent manner, ruling out the idea of a Na+-dependent transport system such as system B0,+ in NSC-34 cell lines. In addition, [3H]L-arginine uptake was voltage-sensitive  and polyamines and is involved in various biochemical pathways, such as protein synthesis and the urea cycle .For the neration ,35. Argill lines , suggestll lines . [3H]L-Aensitive , consist+, b0,+, B0,+, and y+L. Previous studies have also shown that system y+ is an arginine-specific transporter associated with several cell types, including neurons L-arginine uptake was significantly inhibited by typical CAT-1 substrates, including arginine, lysine, histidine, and ornithine, in both cell lines  [+ and our data revealed significant inhibition of [3H]L-arginine transport due to NEM in both cell lines, leading to the hypothesis that system y+ was primarily involved in arginine transport in these cell lines  . NEM, isll lines . However3H]L-arginine uptake in ALS model cell lines. Earlier studies have shown the effectiveness of quinidine combined with dextromethorphan in treating pseudobulbar affect, a consequence of neurological damage caused by ALS, AD, and other traumatic brain injuries, overall the combination has shown to improve the quality of life of ALS patients [3H]L-arginine uptake in ALS model cell lines (3H]L-arginine was competitively inhibited by lysine, which is also a substrate of CAT-1, suggesting that the high-affinity uptake of arginine and lysine both shared the same transport process which is system y+. Therefore, showed competitive inhibition in both cell lines. Similar results have been reported previously on arginine, in which it was competitively inhibited by ornithine, another CAT-1 substrate L-arginine uptake was markedly reduced in the presence of H2O2 in NSC-34 cell lines, which might be attributed to the reduced expression of the transporters involved in arginine transport in ALS cell lines. However, arginine and citrulline pretreatment significantly increased arginine uptake in the MT cell lines, indicating that arginine and citrulline restored arginine uptake by restoring the expression of transporters. Our qPCR analysis revealed a similar pattern of results (2O2 and was recovered by lysine. L-a results . Previou lysine. . Overall lysine. , and out lysine. .+ in NSC-34 cell lines and that two saturable transport processes were involved in both NSC-34 cell lines. Additionally, arginine exhibited regulatory effects, restoring nNOS expression in the disease model cell lines, and a neuroprotective effect against the oxidizing stress-inducing agent H2O2. Therefore, by understanding the transport system involved in arginine transport using motor neuron models, such as NSC-34 cell lines, L-arginine may act as a potential candidate for improving the pathological conditions in ALS and other neurodegenerative diseases.In conclusion, our study revealed that arginine transport was mainly mediated by system y


The compound 1a was highly anti-proliferative and reached IC50 values in the nanomolar concentration range. 1a was superior to the known anti-tumorally active curcuminoid EF24 (2) and its known N-ethyl-piperidone analog 1b in all tested tumor cell lines. Furthermore, 1a induced a noticeable increase of intracellular reactive oxygen species in HT-29 colon adenocarcinoma cells, which possibly leads to a distinct increase in sub-G1 cells, as assessed by cell cycle analysis. A considerable activation of the executioner-caspases 3 and 7 as well as nuclei fragmentation, cell rounding, and membrane protrusions suggest the triggering of an apoptotic mechanism. Yet another effect was the re-organization of the actin cytoskeleton shown by the formation of stress fibers and actin aggregation. 1a also caused cell death in the adherently cultured glioblastoma cell lines U251 and Mz54. We furthermore observed that 1a strongly suppressed the stem cell properties of glioma stem-like cell lines including one primary line, highlighting the potential therapeutic relevance of this new compound.A new and readily available pentafluorothiophenyl-substituted N-methyl-piperidone curcuminoid  Though displaying promising anticancer activities, its potential as a drug candidate is limited by its rapid metabolic degradation, its low water solubility and its insufficient bioavailability both in plasma and in tissues. , 540 (42), 513 (41), 512 (41), 414 (11), 310 (22), 296 (23), 270 (45), 242 (8), 143 (7), 115 (13), 43 (14); HRMS (ESI) C20H18F10NOS2 [M + H]+ calcd. 542.06709, found 542.06612. 1-Methyl-4-piperidone  was dissolved in MeOH (5 mL) and 4-(pentafluorothio)-benzaldehyde  was added. NaOH  and HVbl (ACC-149) cervix carcinoma, MCF-7Topo (ACC-115) breast carcinoma, HT-29 (ACC-299), HCT-116 (ACC-581), DLD-1 (ACC-278), SW-480 (CCL-228) colon carcinoma, were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and 1% antibiotic-antimycotic at 37 °C, 5% CO2, and 95% humidity. To keep MCF-7Topo and KB-V1Vbl cells resistant, the maximum-tolerated dose of topotecan or vinblastine were respectively added to the cell culture medium 24 h after every passage. The cells were used in passages between 40 and 47 (518A2), 102 and 110 (Panc-1), 223 and 229 (KB-V1Vbl), 41 and 46 (MCF-7), 34 and 51 (HT-29), 67 and 73 (HCT-116), 51 and 56 (DLD-1), as well as 43 and 48 (SW-480). U251   A A2) can  3 and 7 , suggestd 7  from the characteristic cortical filaments (shown by the untreated control) to cell rounding and actin clustering.  which effectively blocks proliferation and stemness in GSCs -4-piperidinone (EF24), which evokes promising antitumour properties such as cell cycle arrest, apoptosis induction, and inhibition of HIF-α and NF-κB. ,42,43,44ctivity.  Due to tuminoids . Comparioids [1a . 1a effectively reduces the viability of numerous cancer cell lines derived from multiple cancer entities. Similarly, the lead compound Curcumin (3) has been shown to be an effective anticancer agent across several cancer cell lines as well as cancer models in vitro and in vivo. [1a exerts comparable pleiotropic effects by acting on similar processes than Curcumin, albeit at much lower concentrations. As such, we observed potent inhibition of angiogenesis, induction of ROS, cell cycle inhibition and an increase of effector caspases 3 and 7. Similarly, Curcumin has been shown to inhibit angiogenesis for example in models of pancreatic cancer [We could previously demonstrate that Curcumin effectively targets GBM cells both in vitro and in vivo ,47 and win vivo.  Based onc cancer  and breac cancer . Furtherc cancer  Addition1a not only decreases viability of GSCs, but also reduces their stem-cell frequency using sub-lethal drug concentrations indicating that 1a has the potential to target the stem-like phenotype of cancer cells. In fact, it is well-established that GBM cells can obtain a stem-like phenotype in vivo and that these cells are largely responsible for treatment resistance and finally disease recurrence. [Importantly, we next focused on a more complex model system by employing three GSC spheroid cell lines, which are known to more closely mimic the original tumor and as such are a better preclinical model system . We coulurrence.  Althoughurrence.  Similarlurrence. ,54 Thus,1a was prepared in one step and tested against a panel of tumor cell lines. Our working hypothesis was that 1a is a superior anticancer drug compared to 1b and 2 and our primary endpoint of analyses was after assessment of this anticancer activity across several cancer cell lines derived from multiple cancer entities. The compound showed a notable higher anti-proliferative activity than close congeners such as EF24, Curcumin, and 1b. Cell death and apoptosis induction by 1a occurred at lower doses than by 1b. This was further corroborated by a strong increase in ROS production and the Sub-G1 phase of the cell cycle of HT-29 adenocarcinoma by 1a could be determined. Also, the effect on the actin cytoskeleton in treated cells was clearly improved compared to 1b. Additionally, 1a showed similar growth-inhibiting efficacy when tested against several GSC cultures, including the primary culture 17/02, which is derived from a heavily pre-treated GBM patient. 1a was also able to effectively inhibit stemness in all three cultures in low nanomolar doses. Hence, 1a appears to be an optimized anti-tumor curcuminoid and further investigation of this promising compound is warranted.A new Curcumin derivative


Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors. Many forms of cancer have developed mechanisms to prevent mitochondrial apoptosis by altering the equilibrium between pro- and antiapoptotic proteins2. Expression levels and functional importance of individual antiapoptotic proteins are considered to be different in the different types of tumors1.Apoptosis or programmed cell death is a process which eliminates old cells or cells that are otherwise no longer needed or detrimental. Decisive for the fate of the individual cell is the interaction between pro- and antiapoptotic members of the BCL2 family4. Mimicking proapoptotic BH3-only proteins, BH3 mimetics antagonize the function of antiapoptotic BCL2 family proteins, providing a new opportunity for treatment of these diseases4. Antagonizing BCL2, venetoclax was the first BH3 mimetic approved for CLL and then—in combination with hypomethylating drugs—also for AML5. Various new drugs targeting BCL2 family members are currently evaluated in clinical trials1.The aberrant expression of BCL2 and of other members of the BCL2 family is well-described in follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and in acute myeloid leukemia (AML)6.Aim of our study was to identify hematologic entities not yet in the focus of broad interest, but with a conspicuous expression pattern of antiapoptotic BCL2 family members, indicating a potential for the application of BH3 mimetics. To delineate disease-specific expression patterns of antiapoptotic genes in hematologic tumors, we screened cell lines from the leukemia-lymphoma 100 (LL-100) panel. In 2019, we had developed this panel of cell lines covering 22 entities of leukemia and lymphoma malignancies as novel platform for the development of targeted therapies9. BH3 mimetics targeting MCL1 are promising options for the therapy of both diseases8. We describe the effects of the MCL1 inhibitor AZD-5991 on growth and apoptosis in ALCL and PEL cell lines. Furthermore, we discuss under which circumstances the combination of AZD-5991 with other BCL2 family inhibitors might be of clinical benefit. Inhibitors against the antiapoptotic AKT pathway are an alternative for combination treatment with AZD-5991.RNA-seq analysis of the LL-100 panel showed that cell lines derived from two rare lymphomas, anaplastic large cell lymphoma (ALCL) and from primary effusion lymphoma (PEL) stood out by high expression of MCL1. MCL1 had already been described as essential mediator for the survival of multiple myeloma (MM) and of AML11. Deregulation of BCL2 in hematopoietic tumors could be traced back to the translocation t, amplification or hypomethylation of the gene2.Overexpression of antiapoptotic BCL2 family genes promotes survival of cells in hematological malignancies12. To find potential targets of BH3 mimetics, we examined the expression of BCL2 family genes in the LL-100 cell lines panel. Remarkably, all ALCL and PEL cell lines showed an MCL1high/BCL2low expression pattern  cells15.MCL1 is the dominant antiapoptotic BCL2 family member in MM, more critical than BCL2 and BCLXLMCL1 gene is located on chromosome 1 (q21.3), in a region which is often amplified in human cancer16. According to our quantitative genomic PCR, 6/7 ALCL cell lines and 6/8 PEL cell lines carry gains of MCL1 (Suppl. Table MCL1 locus (9n), suggesting that focal gain can play a role in the aberrant overexpression of MCL1  are emerging agents for the treatment of BCL2 dependent tumors. Different compounds are in clinical trials, with the BCL2 inhibitor Venetoclax (ABT-199) being the first BH3 mimetic approved by the FDA for CLLpos/BCL2neg) and RI-1 (MCL1low/ BCL2pos) , AZD-5991 efficiently inhibited growth of the MCL1pos cell line (U-2946)  and of one BCL2/BCLXL inhibitor (ABT-263) on the diffuse large B cell lymphoma (DLBCL) cell lines U-2946 . Therefore, we assessed BCLXL as candidate antiapoptotic mediator in ALCL.ABT-263 inhibits not only BCL2, but also BCLXL and BCL-W. Therefore, expression of the latter two genes might explain responsiveness of a BCL2pos/BCLXLpos cell line L-82  RNA oligonucleotides was verified by qRT-PCR  and ABT-263 (300 nM) yielded synergistic antiproliferative and proapoptotic effects when compared to the effects of each inhibitor alone  expressed BCLXL   encoding the NPM-ALK fusion gene.MCL1 had been described as the main antiapoptotic mediator in ALCLhigh), but not in L-82 (BCL2A1low)  to test whether this drug would repress MCL1 in our cell lines. We speculated that a double hit against MCL1, reducing expression (BEZ-235) and inhibiting its function (AZD-5991), might create cooperative effects.The rationale for these experiments lies in earlier studies reporting that apoptosis in ALCL and DLBCL models was induced by downregulation of MCL1 following inhibition of the AKT/mTOR pathway29  resulted in a dramatic drop of MCL1  and induced apoptosis (12 h) in a dose-dependent manner  induced expression of several dozen genes in cell lines L-82 and SU-DHL-1 . Cell lines were authenticated by DNA profiling and cytogenetics. Detailed references and cultivation protocols have been described previously6. RNA-seq data were deposited at ArrayExpress E-MTAB-7721. For the heatmap visualization 0.1 was added to normalized expression levels prior to log2 transformation.Total RNA was extracted via miRNeasy Mini Kit  including DNase digestion. Library preparation and sequencing were commissioned to GATC Biotech . RNA expression analysis was performed as described previously4 cells (in 50 µl) were seeded in triplicate in 96-well flat bottom microtiter plates. Effectors were added as 2 × concentrated solutions in a 50 µl volume. Growth and apoptosis were determined with the IncuCyte S3 Live-Cell analysis System . We used the Caspase 3/7 reagent (Essen Bioscience #4704) to detect apoptotic cells.About 2 × 1039. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA were obtained from Qiagen. The siRNA (80 pmol) was transfected into 1 × 106 cells by electroporation using an EPI-2500 impulse generator  at 350 V for 10 ms. Treated cells were harvested after 24 h or 48 h.Transfection with siRNA oligonucleotides was performed as described previouslyBAX (Hs00180269_m1), BCL2 (Hs00153350_m1), BCL2A1 (Hs00187845_m1), BCLW (Hs00187848_m1), BCLXL (Hs00236329_m1), CDKN1A (Hs00355783_m1), MCL1 (Hs03043899_m1), MDM2 (Hs01066930_m1) and NOXA1 (Hs00611456_g1) using TBP as endogenous control. DNA was isolated with the High Pure PCR Template Preparation Kit . Quantitative genomic PCR was performed with SYBR-green reagents (Applied Biosystems). Primers and PCR conditions for genomic PCR are shown in Table RNA was prepared using the RNeasy Mini kit (Qiagen). For mRNA quantification, reverse transcription was performed using the SuperScript II reverse transcriptase kit . PCR was performed on a 7500 Applied Biosystems . TaqMan probes (Applied Biosystems) were used to quantify human 40. Anti AKT (#4691), pAKT (Ser473) (#4060), BAD (#9239), pBAD (Ser136) (#4366), BCL2 (#4223), BCL2A1 (#14093), BCL-W (#2724), BCLXL (#2764), pGSK3B (Ser9) (#9323), MCL1 (#39224), pMCL1 (Ser159/Thr163) (#4579), S6 (#2317) and pS6 (Ser235/236) (#2211) antibodies were purchased from Cell Signaling . Anti GAPDH (ab8245) antibody was from Abcam . Specific bands on nitrocellulose membranes were visualized with the biotin/streptavidin–horseradish peroxidase system  in combination with the “Renaissance Western Blot Chemoluminescence Reagent” protocol .Samples were prepared as described previouslyCell lines L-82 and SU-DHL-1 were stimulated with BEZ-235 . RNA was isolated using the RNeasy Mini kit (Qiagen). Quality and integrity of total RNA was controlled on Agilent Technologies 2100 Bioanalyzer . For biotin labelling 2–10 ng of total RNA were used according to GeneChip Pico Kit (Affymetrix). Then, 5.5 µg of biotinylated cDNA were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls  as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix Clariom S (400 Format) for 17 h at 45 °C. Hybridization was done for 16 h at conditions recommended by the manufacturer. Clariom S chips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix GCS 3000. Image analysis was done by Affymetrix GeneChip Command Console Software (AGCC) and Affymetrix Expression Console Software. Clariom S arrays were analyzed via R/Bioconducor using the package oligo including multi-array average (RMA) background correction, quantile normalization and log2 transformation, whereas the annotation file was retrieved from Thermofisher . Genes were filtered to at least log2FC < -1 in control versus 6 h after BEZ-235 treatment. The data were deposited with ArrayExpress under E-TAB-10903.Supplementary Information.