Treatment results for multiple myeloma and plasma cell leukemia have considerably improved, but cure remains elusive and establishing new therapeutic approaches constitutes a major unmet clinical need. We analyzed the anti-myeloma properties of the aza-anthracenedione pixantrone which has been successfully used in a phase III study for the treatment of patients with aggressive non-Hodgkin’s lymphoma as monotherapy as well as in combination regimes in vitro and in an adapted in vivo model  assay). Pixantrone significantly inhibited proliferation and metabolic activity of all investigated myeloma cell lines. Importantly, anti-myeloma effects were more pronounced in tumor cell lines than in stromal cells, mesenchymal stem cells, and peripheral blood mononuclear cells of healthy controls. Apoptosis of myeloma cell lines was observed only after a 7-day incubation period, indicating a fast cytostatic and a slower cytotoxic effect of this drug. Pixantrone reduced the viability of primary plasma cells of patients and induced downregulation of myeloma-cell growth in the CAM assay. Additionally, we demonstrate in vitro synergism between pixantrone and the histone deacetylase inhibitor panobinostat with respect to its anti-proliferative features. From these data, we conclude that systematic investigations of the clinical usefulness of pixantrone in the framework of controlled clinical trials are clearly indicated .The online version of this article (10.1007/s00277-019-03797-6) contains supplementary material, which is available to authorized users. Multiple myeloma (MM) is an incurable malignant disease of immunoglobulin and/or free light chains producing monoclonal plasma cells, clinically characterized by hypercalcemia, renal failure, anemia, and osteolytic bone disease . Considerable clinical progress has been made with respect to higher response rates, better remission-induction efficiency and depth, as well as the improvement of both progression-free and overall survival. However, cure remains an elusive therapeutic goal. Nearly all patients will sooner or later relapse and develop treatment resistance to all available drugs and treatment modalities . Median Plasma cell leukemia (PCL) is a leukemic variant of myeloma arising either de novo or from clinically pre-existent MM in mostly heavily pretreated patients and carries an even worse prognosis than does relapsed/refractory MM. Treatment options are limited, and no drug has ever been explicitly registered for this indication. Furthermore, there is no generally accepted standard of care, although most hematologists use—depending on previous treatments—combinations of MM drugs, steroids, and chemotherapy . AnthracPixantrone (PIX) is structurally similar to the anthracycline doxorubicin (Dox), but with an improved toxicity profile, especially with regard to cardiotoxicity , 12, whiHere, we investigated the anti-myeloma properties of PIX by (1) analyzing its effects on myeloma cell lines, (2) screening in vitro for anti-myeloma synergisms with putative combination partners, (3) confirming its anti-myeloma effect in a chorioallantoic membrane assay (CAM), and finally (4) generating confirmatory evidence with primary patient material.All patients gave written informed consent to the use of their biologic materials for research (EU FP7 consortium OPTATIO) and documentation of their clinical data via the Austrian Myeloma Registry (AMR) after extensive discussion of the respective procedures.Patient disposition is outlined in Table 2. The cells were serially passaged twice a week. All cell lines were authenticated by typing short tandem repeats. Mycoplasma contamination was routinely monitored and only mycoplasma-free cultures were used. Mesenchymal stem cells were purchased from PromoCell  and cultured in Mesenchymal Stem Cell Growth Medium, according to the manufacturer’s instructions.Myeloma cell lines ) and the stromal cell line HS-5  were cultured in RPMI 1640 without phenol red, supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and fetal calf serum . Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO4) were incubated with 1 μg/mL phytohemagglutinin  and appropriate concentrations of PIX for 72 h.PBMC from two patients with de-novo PCL, three patients with secondary PCL, and two patients with relapsed/refractory MM as well as four healthy volunteers were isolated by Ficoll density gradient centrifugation. To generate activated PBMC cells -2,5-diphenyltetrazolium bromide (MTT) assay  according to the manufacturer’s instructions. To exclude unspecific staining by FCS-containing medium, the optical density (OD) of the respective medium was subtracted. Metabolic activity in the absence of the compounds was set at 100%.Induction of programmed cell death for cell lines and primary cells after different time periods was determined by staining with Annexin V (Ann V)-FITC-labeled antibody and propidium iodide (PI). Samples were further analyzed by flow cytometry  and data were evaluated utilizing FlowJo software .The CAM assay used in this study was essentially performed as described previously . In detap value < 0.05 was considered statistically significant. Additive and synergistic effects of drugs were defined according to the following formula: additive mode of action: surviving fraction (SF) (compound A + compound B) = SF (A) × SF (B); synergistic mode of action: SF (A + B) < SF (A) × SF (B) [The Wilcoxon rank-sum test was used to analyze the differences between proliferation, metabolic activity, and apoptosis induction in the absence and the presence of the respective concentration of the compounds . A × SF (B) .Proliferation of myeloma cell lines was determined after 72 h of incubation with various concentrations of PIX. All cell lines tested showed reduced proliferation, and the extent was dose-dependent, starting at 0.05 μM PIX Fig. . At a PIAs PIX structurally resembles Dox, a drug still widely used for MM treatment, the anti-proliferative capacity of Dox was analyzed in parallel in selected myeloma cell lines after a 72-h incubation. As shown in Fig. Similar anti-proliferative effects of PIX were observed in a co-culture system with the stromal cell line HS-5 . Apoptosis induction, however, was observed only after a 7-day incubation  in combination with various “new” anti-myeloma compounds such as bortezomib (5 and 10 nM), panobinostat (3 nM), and lenalidomide (10 and 25 μM) and “classical” MM drugs such as Dox (10 and 50 nM), bendamustine (10 and 50 μM), and dexamethasone . The most effective combination was PIX (50 nM) and panobinostat  Fig. . This inAs already shown for primary cells, PIX (50 nM) and Dox (50 nM) revealed also synergistic effects in the myeloma cell lines suggesting additional mechanisms of these drugs. Similar strong anti-proliferative activity was observed with PIX (50 nM) and bendamustine (10 μM).PIX is approved in the EU for the treatment of aggressive non-Hodgkin’s lymphoma patients. We therefore investigated if PIX might also be effective in MM and PCL.In all cell lines tested, relatively low PIX concentrations (between 0.1 and 0.25 μM) already significantly inhibited cell proliferation. Importantly, a concentration of 0.25 μM corresponds to plasma levels achievable with standard-dose PIX in patients . This loImportantly, anti-myeloma effects were more pronounced in myeloma cell lines than in the stromal cell line HS-5, MSC, and activated PBMC of healthy controls. The results of the CAM assay further support the putative anti-tumor specificity of PIX while inducing no toxic effect on the embryo.Inhibition of metabolic activity showed a more variant pattern and may reflect the clinical and biological heterogeneity of this disease. The inhibition of metabolic activity after a 3-day culture period was accompanied by apoptosis induction of the MM cell lines after 7 days only, which, nevertheless, is in accordance with the literature .In direct comparison to Dox, higher PIX concentrations were necessary to induce similar strong effects in myeloma cell lines as well as in primary patient cells. However, the improved toxicity profile of PIX , 11 arguWe were able to demonstrate an impressive in vitro synergism between PIX and PAN with respect to their anti-proliferative features on myeloma cell lines. PAN is a potent, oral pan-deactylase inhibitor that increases acetylation of proteins involved in multiple oncogenic pathways . It has Similar strong combinatorial activity was observed with Dox (in cell lines as well as in primary cells) and bendamustine. As at least bendamustine is a good therapeutic option in myeloma therapy—especially in renal failure—another interesting option to be investigated might thus be a bendamustine-PIX combination , 28.In conclusion, PIX induced a strong and fast cytostatic effect in myeloma cell lines and induced apoptosis after a longer incubation period suggesting a putative role of PIX as part of possible combination therapies in relapsed/refractory MM and PCL patients in the framework of controlled clinical trials.Supplementary Figure 1(DOCX 18 kb)


Drug delivery systems are molecular platforms in which an active compound is packed into or loaded on a biocompatible nanoparticle. Such a solution improves the activity of the applied drug or decreases its side effects. Dendrimers are promising molecular platforms for drug delivery due to their unique properties. These macromolecules are known for their defined size, shape, and molecular weight, as well as their monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, dendrimer cytotoxicity depended on the generation, the number of surface groups, and the nature of terminal moieties . Higher cytotoxicity occurred for higher-generation dendrimers and for dendrimers with positive charges on the surface. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with polyethylene glycol (PEG), acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. Dendrimers clearly have great potential for wide utilization as drug and gene carriers. Moreover, some dendrimers have biological properties per se, being anti-fungal, anti-bacterial, or toxic to cancer cells without affecting normal cells. Therefore, intrinsic cytotoxicity is a comprehensive problem and should be considered individually depending on the potential destination of the nanoparticle.  Dendrimers, also known as starburst polymers , cascadeFirstly, dendrimers are characterized by a compact structure and a strictly predictable molecular weight (MW). They are monodisperse, polyvalent, usually globular macromolecules with a regular and highly branched three-dimensional architecture. They consist of a central core molecule surrounded by branches called dendrons, and are terminated with functional surface groups. The essential feature of these nanomolecules is their generation, defined as the number of layers attached to the core and denoted as Gn, where n can range from 0 to 12. All these properties make dendrimers excellent candidates for nanomedical applications. As compounds that are so intensively studied for their potential use in biomedicine, dendrimers should meet several criteria. Specifically, they should be non-toxic, non-immunogenic, biopermeable (possess the ability to cross biobarriers), able to stay in blood circulation until the desired effects occur, and able to target specific biological structures. Many groups of dendrimers fulfill most of these requirements; however, their utilization in biomedicine is often limited due to their high cytotoxicity . This rePoly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers are the two most commonly studied dendrimers, probably due to their broad commercial availability . However2 dendrimer through electrostatic interactions between the dendrimer amines and the carboxyl group of the drug. The complexed drug entered A549 cells much more rapidly than the pure drug, suggesting that dendrimers were able to carry the complexed drug inside cells efficiently [Boas et al. showed that thiourea-functionalized PPI dendrimers can be used as a drug delivery system. The encapsulation properties of these dendrimers are based on the formation of dense, hydrogen-bonded surface shells with solid-state characteristics. Small guest molecules captured in the interiors of the dendrimers were unable to escape even after extensive dialysis. The number of encapsulated guest molecules was directly proportional to their size and shape. Such a dendrimer box could be opened controllably by partial or total hydrolysis to release either some or all of the entrapped guest molecules . In addiiciently . Anothericiently . Interesiciently ,20. Khaniciently , while Giciently . Photodyiciently .Dendrimers can be used as therapeutic factors in neurodegenerative disorders. In vitro studies showed that dendrimers were capable of interfering with the formation of amyloid fibrillar structures typical to the onset and development of neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and prion diseases. Wasiak et al. demonstrated that cationic phosphorus dendrimers of generations 3 and 4  were able to affect β-amyloid and MAP-Tau protein aggregation processes. Assessment of the cytotoxicity of formed fibrils and intermediate products during Aβ 1–28 aggregation was performed using a neuroblastoma cell line (N2a). The results indicated that CPDs were able to reduce the toxicity of aggregated forms of Aβ 1–28 . SimilarCandida albicans. It was shown that maltose-modified PPI dendrimers displayed the highest antibacterial activity and a striking selectivity toward a Gram-positive bacterium, Staphylococcus aureus. Simultaneously, these nanomolecules exhibited insignificant toxicity toward eukaryotic cells [®, was developed by Starpharma . Preclinical development studies demonstrated that VivaGel® was 100% effective at preventing infections of primates exposed to a humanized strain of simian immunodeficiency virus (SHIV). In 2012, the company started crucial phase III clinical trials for the treatment of bacterial vaginosis. First, VivaGel®-containing condoms were approved on the Australian market. Then, the product was approved for the treatment and prevention of bacterial vaginosis (BV) and the prevention of sexually transmitted infections [Dendrimers and dendrimer complexes proved to be effective antipathogenic agents in vitro. Unmodified and maltose-modified PPI dendrimers were tested for their potential against a set of pathogenic bacteria and yeast—ic cells . Moreoveic cells . These cic cells ,34. PAMAic cells . One of ic cells . The PLLfections .Plasmodium falciparum T- and B-cell stimulatory peptides, is a good example of such a system [Many researchers aimed to use dendrimers for vaccine and immunization purposes. A multiple antigenic peptide (MAP) system, based on PLL dendrimers, was pioneered by Tam et al. in the late 1980s ,39. It ca system . Furthera system .Since the pioneering work of Lauterbur, Wiener, and Tomalia et al. , numerouMany studies focused on the utilization of amino-terminated PAMAM or PPI dendrimers as non-viral gene transfer agents, increasing the transfection efficiency ,45. DendInterestingly, dendrimers were also investigated for their use in biomedical applications as artificial proteins. Owing to their nanoscale dimensions, globular shape, and other properties, they can mimic many important proteins. For example, the size of insulin (3 nm) is comparable to the size of a PAMAM G3 dendrimer. Similarly, cytochrome C (diameter = 4 nm) and hemoglobin (diameter = 5.5 nm) correspond to the sizes of PAMAM G4 and G5, respectively. A comprehensive strategy for designing dendritic nanostructures to mimic globular proteins was described by Tomalia et. al. . They syDendrimers possess great biomedical potential and, as presented in The cytotoxicity of dendrimers depends strongly on the number and nature of functional surface groups. Cationic dendrimers often exhibit high toxicity, whereas anionic and neutral dendrimers show slight or no toxic effects. For example, poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers possessing terminal primary amines are characterized by concentration- and generation-dependent toxicity ,50, wherVibrio fischeri, Daphnia magna, Thamnocephalus platyurus, and two fish cell lines. The toxicological response correlated well with the dendrimer generation and, therefore, with the particle surface area; an increase in surface area led to an increased toxic response. Daphnia magna was found to be the most sensitive model, and the RTG-2 fish cell line was the least sensitive [Mukherjee et al. published an interesting paper, disclosing an indirect impact of PAMAM dendrimers on cell viability. The authors observed generation-dependent toxicity of PAMAM G4, G5, and G6 to human keratinocytes (HaCaT) and primary colon adenocarcinoma cells (SW480). Based on spectroscopic methods, and the measurements of the zeta size and potential of dendrimers in fetal bovine serum (FBS)-supplemented medium, it was found that dendrimers absorbed proteins from the culture media. These data suggested the indirect mechanism of PAMAM cytotoxicity resulting from the depletion of medium components . In the ensitive . Interesensitive . 2, COONa, COOH, PEO) on their in vitro behavior. Dendrimers with amino terminal groups displayed concentration- and generation-dependent hemolysis, while anionic and CSi–PEO dendrimers were neither hemolytic nor cytotoxic toward a panel of cell lines in vitro. Polyether dendrimers with carboxylate and malonate surfaces were not hemolytic at 1 h; however, after 24 h, unlike anionic PAMAM dendrimers, they were lytic [Several types of dendrimers  or diaminoethane (DAE) core, poly(ethylene oxide) (PEO)–grafted carbosilane (CSi–PEO), and polyether dendrimers) were compared regarding the influence of generation , and surface functionality  groups. Dose–response effects of both modified and unmodified PAMAM were examined on two cell lines—a fish cell line (RTG-2) and a rat hepatoma cell line (H4IIE). No toxic effects of amino-terminated PAMAM dendrimers were observed for both H4IIE and RTG-2 cells lines when the concentration was below 500 μg/mL. For surface-modified PAMAM dendrimers, higher cytotoxicity was found for the H4IIE cell line [As mentioned above, surface modification is a common way to increase the biocompatibility of dendrimers. For example, modified PAMAM dendrimers of generation G4 with 4-carbomethoxypyrrolidone surface groups (PAMAM–pyrrolidone dendrimer) showed only minor toxicity and no ability to induce apoptosis. The most important finding was the lack of influence of the PAMAM–pyrrolidone dendrimer on intracellular ROS level and mitochondrial membrane potential even at the highest tested concentration (200 µM) ,60. PAMAell line . Wen et ell line . In anotell line . The worell line . In theiell line . Janaszeell line . In anotell line . It seemThe ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Indeed, a safe alternative to the viral system used in gene therapy is a nonviral carrier, and dendrimers seem to be excellent for this role. Albertazzi et al. revealed that PAMAM dendrimers G2, G4, G6, and lipidated G4 possess similar properties to widely used cell-penetrating peptides (the arginine-rich motif derived from the HIV protein TAT). Furthermore, they are not toxic toward the HeLa cell line (1.5 μM concentration for 1 h) and, thanks to their chemical tunability, they represent an attractive option for drug and gene delivery . Also, CNavath et al. demonstrated the development of biocompatible PAMAM G4 dendrimers, enabling the attachment of drugs (indomethacin and dexamethasone) and imaging agents (dexamethasone and FITC) without using any specific linkers. This could be achieved thanks to multiple amino-acid-based orthogonal surface groups at the dendrimer periphery. Furthermore, one of the two functional handles at the periphery was used to develop in situ forming hydrogels, whereas the other handle could be used for conjugating drugs. An in vitro cytotoxicity test and a hemolysis assay showed that the heterobifunctional dendrimers were non-cytotoxic in the 100 ng/mL to 1 mg/mL concentration range . Krishna et al. reported the synthesis of several PPI dendrons and dendrimers, which were constructed by involving an ether as the linker component and an imine as a branching component. The adopted synthetic sequence allowed an alcohol, an amine, or a carboxylic acid to be installed at the peripheries. The carboxylic-acid-terminated dendrons and dendrimers were evaluated for their cytotoxic properties, and, while most dendrons and dendrimers did not exhibit any measurable cytotoxicity, even up to 100 μg/mL, the second-generation dendrimer with the benzenoid core exhibited mild toxicity at concentrations above 30 μg/mL . It was w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL–PEG copolymer was non-toxic for cells, indicating that this type of block copolymer could be used as a drug delivery carrier [Wang et al. proposed the synthesis of a star polymer composed of amphiphilic block copolymer arms. The core of the star polymer was a PAMAM dendrimer, the inner block of the arm was lipophilic poly(ε-caprolactone) (PCL), and the outer block of the arm was hydrophilic PEG. The star-PCL polymer was synthesized first via ring-opening polymerization of ε-caprolactone with a PAMAM-OH dendrimer as an initiator. The PEG polymer was then attached to the PCL terminus via an ester-forming reaction. It is known that hydrophobic dyes and drugs can be encapsulated in the micelles. A similar effect was observed for the star polymer. A loading capacity of up to 22% ( carrier .Cyprinus carpio (EPC) [50 values ranging from 0.3 to 1.6 μM. Interestingly, the complexation of the terminal ligands of 1G3 dendrimers by copper(II) metal strongly increased IC50 values in non-cancer cell lines [Lazniewska et al. studied toxic responses of two low generations of cationic phosphorus dendrimers (CPDs) of generation 2 and generation 3 toward murine embryonic hippocampal cells (mHippoE-18) and N2a cells. The obtained results showed that CPDs G3 were highly cytotoxic at concentrations above 1 μM and at 0.7 μM for mHippoE-18 cells. A significant decrease in cell viability corresponded to severe changes in cellular processes, such as massive ROS generation. Unlike other cytotoxic dendrimers, which activated the apoptotic pathway in cells, CPDs caused a breakdown of cellular processes followed by a necrotic cell death . The cytio (EPC) . It was ll lines .Escherichia coli, and the toxicity against human cells was evaluated with the human epithelial cell line A549. The silver particles, especially those prepared from dendrimers of higher generations, demonstrated a significant antibacterial effect against E. coli. No toxicity against human cells was observed for the silver particles even in case of the highest investigated silver concentration. Therefore, such a construct could offer great potential for application as an antibacterial agent with low human toxicity [Zeng et al. presented hyperbranched polyester Boltorn decorated with PEG groups, which showed potential as an efficient and specific delivery platform for drugs and imaging agents. The cytotoxicity, cellular uptake profiles, and intracellular trafficking of polymer micelles were tested in MDA-MB468 breast cancer cells. The uptake of these nanoparticles was positively correlated with time and concentration and was energy dependent. These nanoparticles were shown to be internalized into cells via clathrin- and macropinocytosis-mediated endocytosis. Moreover, they did not significantly affect the viability of the cells . Hydroxytoxicity . Feliu etoxicity . The biotoxicity . One more architecture characterized by improved biological properties is dendritic polyglycerol (dPG). Various dPG derivatives possessing neutral, cationic, and anionic charges were compared with amine- and hydroxyl-terminated PAMAM dendrimers. In the U-937 cell line, dPG with terminal hydroxyl groups, dPG sodium sulfate, dextran, and linear PEG caused no toxic effect compared to dexamethasone used as a reference standard. Similarly, amine terminal dPGs (7% and 18% amines) were also almost non-toxic . On the other hand, three polymers, namely, dPG with higher amine functionality (45% and 100%), PAMAM G4 hydroxyl and amine dendrimer, and PEI, caused higher cell toxicity than the uncharged hydroxyl dPGs, dextran, and PEG. The cell compatibility results showed that the dendritic polyglycerols were as safe as linear PEG polymer or dextran, which indicates the suitability of dPG derivatives in delivering therapeutic agents .tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Cbz) protecting groups, Boc-protected or unprotected natural amino acid residues, ethylene-diamine ligands, and/or dansyl fluorescence labels. The cytotoxicity was determined in vitro in concentration-dependent assays using the MCF-7 cell line. The internal structure of the presented dendrimers did not seem to play a profound role in cytotoxicity, despite the common view that the interior of low-generation dendrimers is accessible. The surface decoration, however, was crucial for toxic effects. Most of the examined non-charged dendrimers  were non-toxic, although they were clearly bioavailable as proven by cell uptake experiments, and all completely diaminopropionic-acid-decorated dendrimers (positively charged) were also non-toxic. These results, especially the latter case, indicated that positive charges on a dendrimer surface did not always lead to cell toxicity and that another structure/toxicity correlation might also play a role [Fuchs et al. developed a new series of dendrimers G1 and G2 possessing various surface functionalities. Terminal amine groups of these dendrimers were decorated with protons, y a role . Huang et al. also described synthesis of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. These novel nanomolecules were designed as biocompatible carriers of bioactive cargo able to cross the cell membrane and localize in targeted cell compartments. The behavior of dendrimers was comparable in two tested cell lines: NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC). The differential localization patterns of the two molecular transporters could be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations utilizing this practical approach might overcome limitations in drug delivery .Although cytotoxicity is usually the limiting factor for dendrimer application, it plays a more comprehensive role in clinical practice. Dendritic polymers chosen for the potential treatment of infectious diseases should be able to kill a pathogen while remaining harmless for a patient. Similarly, in anti-cancer therapy, dendrimers should affect abnormal cells only. This effect can be achieved with proper dose selection and by utilizing specific ligands or effects such as “enhanced permeability or retention”. This section reviews recent research on the cytotoxicity of dendrimers in the context of their potential clinical applications.Staphylococcus aureus and Staphylococcus epidermidis, Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and yeast, Candida albicans. Cytotoxicity of all tested dendrimers was checked on B14, human liver hepatocellular carcinoma (HepG2), N2a, and rat liver cell lines (BRL-3A). The obtained results indicated that unmodified PPI dendrimers and PPI dendrimers modified by 25% maltose displayed antibacterial activity and a striking selectivity toward S. aureus, at the concentration of 1 µM, which, at the same time, was harmless for the eukaryotic cell lines [S. aureus) and Gram-negative (E. coli) bacteria, as well as against a fungal pathogen . However, contrary to the PPI dendrimers, lysine-based peptide ones exhibited some level of toxicity, although most of them caused only a slight (ca. 20%) decrease in B-14 cell viability for minimum inhibitory concentrations. The study also showed that the degree of branching, steric distribution, and types of hydrophobic (aromatic) groups and cationic centers are important components of dendrimeric structure and influence both antimicrobial potency and toxicity [PPI dendrimers were investigated for their cytotoxic and antibacterial activities by Felczak et al.; original PPI G4 dendrimers and PPI G4 with a surface modified by 25% and 100% maltose  were evaluated for their antibacterial activity against Gram-positive bacteria, ll lines . Klajnertoxicity . Bacillus subtilis and S. aureus (Gram-positive), as well as E. coli and Salmonella typhi (Gram-negative). Metallodendrimers also showed anti-tumor activity against the MFC-cell line. Pt(II)-containing metallodendrimers were found to be more efficient in the induction of MFC-7 death than Pd(II)-containing metallodendrimers, and both showed lower cytotoxicity than cisplatin (standard drug) [n = 1–3), were synthesized, and the pyrenyl group of the dendritic molecules was encapsulated in the arene ruthenium metallacages. The host–guest systems inhibited the growth of both cisplatin-sensitive and -resistant cancer cells (A2780 and A2780cisR). Moreover, these water-soluble host–guest systems showed cytotoxicity toward the cisplatin-resistant cell line that was comparable to free cisplatin in non-resistant human ovarian cancer cells [Metallodendrimers  are another group of dendritic compounds. It was revealed that polyamide metallodendrimers possess anti-bacterial activity against rd drug) . Pitto-Ber cells .Thiagarajan et al. characterized the activity of a PAMAM dendrimer conjugated with an anticancer drug camptothecin (PAMAM–CPT) on human colorectal carcinoma cells (HCT-116). The conjugate was stable under physiological pH (7.4) in phosphate-buffered saline (PBS) and in growth media (with 10% FBS) with minimal release of 4% and 6% drug, respectively, at 48 h. PAMAM–CPT inhibited the proliferation of HCT-116 cells and induced cell-cycle arrest with up to 68% of cells blocked in the gap 2 (G2) phase. Thus, the PAMAM–CPT conjugate was active against colorectal cancer cells in vitro, inhibiting their growth and inducing nuclear fragmentation. However, it would be useful to test the cytotoxicity of this conjugate against normal cells . p < 0.05) increased the cytotoxicity against both cell types. The conjugate had approximately 12-fold greater permeability across both apical and basolateral cell monolayers than that of PTX alone [Surface modifications of dendrimers were shown to be a factor enhancing the cell-penetrating ability of the drug–dendrimer complex, thus contributing to the inhibitory effect on cancer cell growth. Jia et al. modified PAMAM dendrimers G5 with acryloyloxyethyl phosphorylcholine (APC), a molecule possessing biomimetic properties. The hydrophobic interior of the modified dendrimer (G5-PC) was used to incorporate the anti-cancer drug adriamycin (ADR) and the G5-PC showed sustained release behavior for ADR. Cell viability and morphology analyses on the cancerous HepG2 cell line revealed that G5-PC was characterized by much lower toxicity in comparison to the unmodified PAMAM dendrimer. Furthermore, the drug-loaded G5-PC was shown to be internalized into cancer cells and could effectively decrease their viability . Teow etTX alone . PaclitaTX alone . In another study, PEGylated PAMAM dendrimer of generation 3 was loaded with fluorouracil (5-FU) and tested in vitro on the MCF-7 cell line. The results indicated that the PAMAM dendrimer presented higher toxicity as compared to its PEGylated counterpart. However, PEG-modified PAMAM dendrimer loaded with 5-FU exhibited antiproliferative activity against the cancer cell line. An additional benefit from the encapsulation of fluorouracil in the PEGylated dendrimer was a slow release profile of the drug. Moreover, in vivo studies revealed that the tested complex exhibited a significant decrease in the volume of the tumors, which were generated by the MCF-7 cancer cells .Despite their cytotoxicity, PPI dendrimers were also widely used as effective delivery vehicles for drugs. Wang et al. decreased the cytotoxicity of PPI dendrimers by acetylation and then used them to encapsulate anticancer drugs, including methotrexate sodium, sodium deoxycholate, and doxorubicin. The results indicated that the degree of acetylation determined the level of cytotoxicity and drug-loading capacity of the dendrimer. The drug-loading capacity of acetylated PPI dendrimers increased proportionally with the degree of acetylation on the dendrimer surface. PPI dendrimers with greater than 80% acetylation did not affect the viability of MCF-7 and A549 cells. The cytotoxicities of methotrexate sodium and doxorubicin were also significantly reduced when they were complexed with acetylated PPI dendrimers with high degrees of acetylation (>80%) owing to sustained drug release from the dendrimers. The results suggested that surface acetylation can reduce the cytotoxicity and improve the anticancer drug-loading capacity of cationic dendrimers . KesharwThe use of vector molecules for the targeted delivery of antitumor drugs provides selectivity for cancer cells. Yabbarov et al. used a recombinant receptor-binding fragment of alpha-fetoprotein (rAFP3D) as a vector molecule. The receptor of alpha-fetoprotein, a tumor marker, is expressed on the surface of many tumor cells, but not in normal human tissues. The vector rAFP3D was conjugated with a PAMAM G2 dendrimer and an anticancer drug, doxorubicin (Dox). The conjugate demonstrated a high cytotoxicity against human ovarian adenocarcinoma cell lines (Dox-sensitive SKOV3 cells and Dox-resistant SKVLB cells), while having low toxicity against human peripheral blood lymphocytes . AnotherZhang et al. synthesized a saccharide-terminated PAMAM G3 dendrimer, which was then conjugated with the drug methotrexate (MTX). This construct was shown to have potential as an anticancer nanodevice for the specific targeting and killing of folate receptor (FR)-expressing tumor cells. Their results showed that G3–MTX presented enhancement in binding avidity to folate-binding protein (FBP) that was three orders of magnitude higher than a free folic acid (FA), and internalized into FR-expressing KB cells  in a dose-dependent and receptor-mediated fashion . Han et Gupta et al. added DOX (approximately 26 and 65%) to the PPI dendrimers, as well as folate-conjugated PPI (PPI–FA) dendrimers. PPI–FA–DOX exhibited the highest percentage cell-growth inhibition compared to other formulations and to the drug itself. All three formulations showed a dose-dependent inhibition of MCF-7 cells. The higher uptake in the case of PPI–FA–DOX was possibly due to the ligand-specific targeting of dendrimers due to surface conjugation of the folic acid . The published toxicity levels of all dendrimers mentioned in this paper are summarized in Although there are active compounds that are able to inhibit or cure particular diseases, they are often not effective due to weak solubility, lack of ability to cross biological barriers, poor targetability, and sensitivity to the cell environment (pH) and its molecular machinery . A solution to this problem could be “packing” the active compound into or loading it onto a biocompatible delivery platform. One of the most promising options here is represented by dendrimers.Dendrimers, which were synthesized in the 1980s, quickly attracted attention of researchers from the biomedical field, and, in the 1990s, first works concerning their biocompatibility, cytotoxicity, and potential applications appeared. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, toxic effects caused by dendrimers were dependent on the generation, the number of surface groups, and the nature of terminal moieties . A higher generation and an increased number of positive charges on the surface resulted in higher cytotoxicity. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with PEG, acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. These properties include defined size, shape, and molecular weight, as well as monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Intensive studies on dendrimers and their constant improvement resulted in their current status as systems of great potential in the biomedical field. In particular, they are developed as nanocarriers of drugs , nucleic acids in gene therapy , and imaging agents for MRI . Furthermore, some dendrimers also exhibit intrinsic biological activity, with anti-bacterial, anti-fungal, and anti-viral properties of dendritic nanomolecules previously demonstrated. Moreover, other dendrimers were reported to inhibit processes involved in neurodegenerative disorders, i.e., fibrillation and aggregation of specific proteins. Lastly, in several cases, it was demonstrated that dendrimers can be toxic to cancerous cells, while exhibiting no or low toxic effects toward normal cells. A synergistic effect of the combination of the active dendrimer and its cargo molecules (drugs) can result in a very potent therapy.The unique structure of dendrimers enables modifying the surface groups, which are largely responsible for properties of dendrimers, including cytotoxicity. However, it should be taken into account that diminishing cationic charge on the surface often reduces the therapeutic activity of dendrimers, e.g., the ability to create complexes with DNA or RNA. Therefore, partial modification is sometimes applied in order to achieve a balance between reduced toxicity and retained activity; this is frequently done via partial modification of PPI dendrimers by maltose or maltotriose .Depending on the specific medical application, a higher or lower dose of the dendrimer is needed. It allows finding the right dendrimer for a specific purpose from the large library of available dendrimers. Therefore, the toxicity of the dendrimers is not the main issue that slows down the process of commercializing dendrimers. However, this field certainly needs better consistency when testing the long-existing and new compounds. Overall, the intrinsic cytotoxicity of dendrimers can be overcome by precise chemical modifications. Their complexation with various bioactive molecules can be perfectly optimized, while being delivery-specific and safe for both the cell and the cargo. At present, dendrimers are an outstanding alternative to other drug carrier platforms by increasing the bioavailability and efficiency of transported compounds, thereby enhancing their therapeutic effects.


ATP7B. Chromosomal alterations were characterized by segmented deletions and gains leading to severely altered karyotypes. The few chromosomal changes shared among LTTs included gains involving the anti-apoptotic BCL2L1 gene and losses involving the NRF2 regulator KEAP1. Overall, the extent of genomic changes paralleled cisplatin treatment concentrations. In conclusion, bladder cancer cell lines selected for cisplatin-resistance contain abundant and characteristic drug-induced genomic changes. Cisplatin treatment may therefore generate novel tumor genomes during patient treatment.The DNA-damaging compound cisplatin is broadly employed for cancer chemotherapy. The mutagenic effects of cisplatin on cancer cell genomes are poorly studied and might even contribute to drug resistance. We have therefore analyzed mutations and chromosomal alterations in four cisplatin-resistant bladder cancer cell lines (LTTs) by whole-exome-sequencing and array-CGH. 720–7479 genes in the LTTs contained point mutations, with a characteristic mutational signature. Only 53 genes were mutated in all LTTs, including the presumed cisplatin exporter  It is particularly efficacious in testicular germ cell cancers, whereas its efficacy in other cancer types, such as ovarian or urinary bladder cancer, is often limited by inherent or acquired resistance3 caused by multifaceted mechanisms9.Cisplatin 2-d(GpG), 25% cis-Pt(NH3)2-d(ApG) 1,2-intrastrand adducts and 5–10% 1,3-intrastrand adducts12. In addition, a small, but relevant amount of interstrand crosslinks is created. Intrastrand crosslinks are repaired by nucleotide excision repair (NER) or may be bypassed by translesion synthesis during replication. Resolving interstrand crosslinks requires the crosslink-homologous repair system. The various types of DNA damage caused directly or indirectly by cisplatin may thus lead to base mutations or even double-strand breaks and chromosomal instability. Very recently, a specific mutational signature for cisplatin was deduced from treated mammary and hepatic tumor cells and likewise detected in human tumor samples treated with cisplatin13.Following activation in the cell by aquation, cisplatin forms monoadducts with guanine bases in DNA which react with adjacent purine bases to form intrastrand crosslinks, namely about 65% 14. These mechanisms have been studied in depth in many cancer types, especially in cell line models. A less explored issue is which genomic changes are elicited by cisplatin in cancer cells that acquire resistance to the DNA-damaging drug, and whether and to which extent these contribute to the various mechanisms of resistance.Many mechanisms are involved in the resistance of cancer cells to cisplatin treatment, including changes in transport and metabolism of the drug, altered DNA repair and checkpoint signaling as well as evasion of apoptosis50 values for 72 h cisplatin treatment, as determined by standard viability assays, ranged from 1.9 to 12 µM, but increased in the respective cisplatin-resistant variants to 10 to 210 µM, yielding resistance indices of 5–17 fold7. They are maintained with regular cisplatin treatment, which would be lethal for the parental cells, but from which the LTTs recover within a few days. As described in detail in previous publications, the LTTs presented with a broad spectrum of resistance mechanisms, each to various extents, prominently increased cisplatin detoxification, driven partly by activation of NRF2 signaling8, which increases glutathione biosynthesis and the MRP2 glutathione conjugate exporter, among others, and decreased apoptosis with higher expression of anti-apoptotic factors like survivin7. The LTTs are not enriched for bladder cancer stem cell-like populations, but canonical WNT signaling is partly activated and some markers of epithelial-mesenchymal transition are increased5.We have previously generated cisplatin-resistant variants, named LTTs (for long-term treated), from four different bladder cancer cell lines by repeated treatment with increasing concentrations of cisplatin. In the initial cell lines ICHere, we report on the genomic differences between the parental RT-112, J82, 253J, and T-24 cell lines and their cisplatin-resistant LTT variants, as determined by whole exome sequencing (WES), array comparative genomic hybridization (aCGH) and karyotyping. Based on this data, we addressed several questions. First, to which extent does cisplatin induce point mutations in bladder cancer cell lines? Second, do these mutations conform to the signature proposed by recent studies? Third, is there evidence for induction of chromosomal alterations by cisplatin? Fourth, are mutations and chromosomal alterations gained during initial selection or accrued gradually? Fifth, is it possible to identify genomic changes accounting for the observed mechanisms of resistance in the LTTs?et al.13, namely frequent C > A transversions preferentially in a ACC or GCC context, more frequent C > T transitions preferentially in CCC or CCT and less commonly in CCA or CCT contexts, with rarer transversions of C > G, predominantly in a GCC context and T > A, with a preceding C and any base following  ranged between 0.90 and 0.97  mutations (27.0–59% vs. 25% expected by chance), indicating no positive selection for functionally important mutations in general.15. Among these genes, mutations likely to have a functional impact  were found in NRXN2, encoding the cell adhesion protein neurexin 2, HECW1, encoding the eponymous ubiquitin ligase , FAM205A, encoding a poorly studied transmembrane protein, and ATP7B, a copper transporting ATPase.The mutations in the four LTTs affected between 720 and 7479 genes. Only 53 protein-coding genes were mutated in all four LTTs Fig. , Table 3The variant allele frequency of the new variants extended continuously to 1 in all LTTs Fig. . In factATP7B, and RT-112-LTT additionally contained mutations in ATP7A  and FAM205A , are entirely unexplored in the context of cancer and chemoresistance. NRXN2 encodes Neurexin-2, a plasma membrane and adhesion protein predominantly expressed in the brain. Most of the mutations found in NRXN2 were observed in the first three Laminin domains  or maintenance concentrations (LTTs). Knockdown of ATP7B enhanced the effect of cisplatin in all LTTs, but interestingly also in 253J and T24 parental cells  and cell adhesion molecules were overrepresented among the mutated genes. However, these attributes may rather reflect that larger genes are more likely to be hit. We therefore searched specifically for mutations in genes previously implicated in mechanisms of cisplatin resistance (abbreviated CRG here) as well as for TCGA-SMG. Among the CRG, a KEAP1 mutation previously described in RT-112-LTT8 is very likely to contribute to NRF2 activation in this cell line, as KEAP1 negatively regulates NRF2. A mutation in BACH1, albeit relatively conservative (A678G) might influence the same pathway. Mutations in three genes, CHUK (G289R), NFKB1 (nonframeshift substitution) and SIRT6 (G22A) might alter NFκB survival signaling. A different, presumably heterozygous mutation of CHUK (A42E) was also detected in 253J-LTT. Three LTT lines acquired mutations in HDAC6, a histone deacetylase involved in cell stress responses. Most interestingly, all LTTs contained missense mutations in ATP7B, and RT-112-LTT additionally contained mutations in ATP7A, which encode copper transporters thought to support cisplatin extrusion . It is therefore not clear which mutations functionally contribute to cisplatin resistance. Accordingly, an analysis using the DAVID functional annotation toolon Table .TP53 (R198H), FOXA1 (A83T in the forkhead N-domain) at high allele frequency, ERCC2 (Y16N in the helicase domain), ARID1A (G406R and R1566K) and STAG2 (G667T) at presumable heterozygote frequency. Interestingly, 253J-LTT acquired two mutations in ARID1A (R1566K and P1567A) as well, albeit at moderate allelic frequency. All TCGA-SMG mutations in T-24-LTT occurred at relatively low variant allele frequencies and none were detected in J82-LTT . In contrast, RT-112-LTT cells had a significantly higher number of chromosomes compared to RT-112 parental cells  including several new markers  and decreased on average in J82-LTT vs. J82 , likewise with new markers .To detect karyotype changes in the LTT lines, array-CGH was performed for all four cell line pairs. Since all four cell lines are aneuploid, rather than comparing each cell line against a reference genome and then calculating the differences between parental and LTT variant, DNA from each parental cell line was hybridized against that of its LTT variant Figs . The valers Fig. . The others Fig. . A slighIn the aCGH analyses, chromosomal instability presented as multiple segmental chromosomal gains and losses as well as focal deletions and amplifications, as illustrated exemplarily for RT-112-LTT in Fig. SLC6A11, SLC6A1 (within gain 3p25.3-p24.3), IKKB, POLB, SLC20A2, DKK4 (gain 8p11.23-p11.21), MRE11A, MRE11B (loss 11q14.2-q21), BCL2A1 (gain 15q24.3-q25.3), BLM (gain 15q26.1-q26.3), TOP2A, ERBB2, KRT14, STAT3, BECN1, BRCA1, und WNT3 (gain 17q12q-21.32). Many of these genes have functions relevant for cisplatin resistance, such as cell survival, DNA repair and WNT signaling9. The KEAP1 copy number in the 19p13.3-q13.33 region appeared diminished in all cell lines, including RT-112  cell lines selected for resistance to high concentrations of cisplatin, we observed a large number of mutations with distinctive characteristics by whole exome sequencing and pronounced chromosomal instability by aCGH. Since all parental cell lines can be safely considered as optimally evolved for long-term propagation in culture, mutations and chromosomal changes observed in the resistant LTTs are expected to reflect the genotoxic impact of cisplatin and potentially positive selection for resistance to the drug, but not selection for other properties like faster growth. In fact, the LTTs grow more slowly than their respective parental cellsTwo of our questions were to which extent cisplatin induced point mutations in the cell lines during selection and whether these mutations had a distinct profile. The number of point mutations, ranging from 865 to 12,500, largely paralleled the tolerated drug doses. By comparison, the parental cell lines harbor between 34,400 and 36,800 variants compared to the reference genome, meaning that between 2.3% and 34.1% mutations were additionally induced. These are minimum percentages, as a fraction of the variants in the parental cells will be germ-line variants and as we did not consider rare variants  in the LTTs. Thus, treatment with cisplatin induced a large number of mutations.20. The profiles of new mutations in the three UC LTTs with sufficient numbers of mutations fitted exactly with that defined by Boot et al. for hepatoma HepG2 and breast cancer MCF7A cells13. Thus, across three different human tumor entities, cisplatin appears to elicit the same mutation profile. Moreover, the mutation profile seen by Boot et al.13 and in our analysis is highly similar to a new mutational signature derived by Liu et al.21 from a comparison of UC tissue samples before and after treatment with cisplatin-based chemotherapy, which in turn bears strong similarities to that observed in another study on post-chemotherapy metastatic urothelial carcinomas22. Liu et al. proposed that this signature is likely caused by cisplatin21. Our results strongly support this contention. The newly defined cisplatin mutational signature resembles the cisplatin mutation profile previously described in C. elegans, S. cerevisiae, and DT-40 chicken cells25 in its predominance of C > A and C > T changes, but the profiles differ in their base contexts, suggesting differences in cisplatin mutagenicity between species. Of note, in addition to the distinctive three-base contexts, we observed further characteristics of cisplatin mutagenesis in the LTTs, such as double-base replacements and single base deletions. On a note of caution, the cisplatin mutational signature cannot be identified in J82-LTT. While this is plausibly due to the low number of mutations in this cell line, we cannot formally exclude that it presents a different mutational signature.Currently, five cancer signatures have been identified as prevalent in bladder cancer, more precisely, signatures 1, 2, 5, 10, and 13in vivo, the relative number of mutations differs strongly, as Liu et al. observed only a low percentage of new mutations (<1%)21. Plausible explanations for this difference are (i) the higher drug concentrations used on the cell lines, as supported by the positive association between cisplatin maintenance concentration and number of mutations, and (ii) the longer treatment duration in the cell lines, as supported by the large fraction of subclonal mutations in an overall clonal environment, which suggests accrual over time. Further potential factors accounting for the difference could be the use of drug combinations in patient treatment and more pronounced tumor heterogeneity in vivo. Notably, unlike Liu et al. in patient tissues, we did not observe APOBEC-related mutational signatures for the new variants in the LTTs, even though UC cell lines express APOBEC3B27.Whereas the spectrum of cisplatin-induced point mutations observed in our study in urothelial carcinoma cell lines is very similar to that observed in UC tissues following neoadjuvant chemotherapy et al. in bladder cancer tissues following chemotherapy21. In addition, detection of CNAs by aCGH in the pure tumor cell population in cell lines is likely to be more sensitive and robust than inference of CNA from exome sequencing of cancer tissues with a variable stromal content. Cisplatin is known to induce chromosomal aberrations, presumably mostly as a consequence of interstrand crosslinks. Accordingly, cisplatin sensitivity is related to the cellular capacity for interstrand crosslink and homologous recombination repair. In the bladder cancer cell lines used, interstrand crosslink repair is functional30. In the LTTs, several new variants in DNA repair genes were detected, but all occurred at relatively low allelic frequency and almost all were silent or conservative changes. Most UC cell lines, however, are deficient in DNA damage checkpoint signaling due to inactivation of p53 and RB1 function, a defining property of almost all urothelial cancers31, including those investigated by Liu et al.21. Therefore, the different extent of CNAs is unlikely to result from differences in DNA repair and checkpoint signaling between tumor tissues and cell lines. As for the number of mutations, the intensity and duration of cisplatin treatment may represent the decisive factor. The lower amount of chromosomal aberrations in 253J-LTT cells is in line with this idea. Our results therefore clearly indicate the potential of cisplatin to induce severe chromosomal aberrations, while the comparison to the tissue data suggests that their extent may depend on drug concentration and treatment duration. These dependencies deserve further investigation to better understand the clastogenic effects of cisplatin and their impact in patients.The same factors may also account for the discrepancy between the high chromosome instability detected in the LTTs and the low number of copy number alterations (CNA) reported by Liu 50s of their parental cells, but below their acquired IC50. These concentrations still induce DNA damage as evidenced by accumulation of cells in S-phase and growth arrest, from which the LTTs - unlike parental cells - recover within a few days7. Thus, during the initial selection period, more DNA damage is incurred, whereas during further propagation, cells sustain DNA damage only to an extent with which they can cope. Accordingly, any mutagenic effects of cisplatin will also be less dramatic.Both the results from WES and aCGH argue that the LTT cell lines were generated by the initial selection for a surviving clone with multiple new mutations and chromosomal alterations followed by gradual acquisition of further mutations and - albeit to a limited degree - of copy number changes. From the WES data, this conclusion is supported by the relatively high fractions of homozygous (or near homozygous) new mutations in each cell line Figs  and 3 acin vivo are more likely to be genomically heterogeneous at the start of treatment and populations emerging during treatment are oligoclonal22. In such tumors, point mutations are more easily detected than copy number changes by current deep sequencing techniques.Notably, clonal selection in the LTTs may constitute another factor explaining the higher extent of CNAs in the cell lines compared to tumor tissues. In the cell model, a single clone apparently was selected from a relatively homogeneous cell population. Cancers KEAP1 mutation promoting NRF2 activation in RT-112-LTT8, we detected mutations or copy number changes in genes related to NF-κB-signaling, DNA repair and cell survival regulation, among others. In addition, several point mutations affected large chromatin regulators like ARID1A, which are known to be relevant in UC in general and are important for transcriptional regulation of cell survival. Whether these genomic alterations are functionally important needs to be ascertained individually.Because of the high number of mutations and copy number changes in the LTTs, our last question, namely whether genomic changes may underlie the observed mechanisms of resistance in these cells, is difficult to answer. In addition to the previously reported ATP7A and ATP7B metal ion transporters. Both are capable of exporting platinum compounds and are established as contributing to cisplatin resistance16. Specifically, mutations in ATP7B have been observed in patients with metastatic bladder cancer and may be predictive for the response to cisplatin-based chemotherapy32. In the LTTs, expression of ATP7A and ATP7B is at most moderately changed7, but the present findings raise the possibility that missense mutations, especially in ATP7B, might contribute to cisplatin resistance. These mutations are located within the HMA  domains in the N-terminal metal-binding region where site-directed mutations have been demonstrated to influence cisplatin binding and ATPase activation16. Interestingly, some mutations described in the TCGA data set are also located in HMA domains. Our data thus support the assumption that mutations in ATP7B may be a significant mechanism in the development of cisplatin resistance in UC. As a further support of their functional importance this idea, we observed that siRNA-mediated knockdown of ATP7B sensitized all four LTTs to cisplatin. The precise effect of each missense mutation on cisplatin binding and extrusion would however need to be tested in appropriate models16.Cases in point are the frequent missense mutations observed in the HECW1 deserves further investigation33. Its gene product, NEDL1, is best characterized as an ubiquitin ligase for the WNT/β-catenin pathway signal transducer DVL1 and its loss of function could therefore increase the activity of the pathway. Increased activity of this pathway has previously been implicated in cisplatin resistance34 and, interestingly, we have observed increased expression of some of pathway targets in the LTTs5. NEDL1 is moreover capable of inducing apoptosis in a p53-dependent manner. The UC cell lines used in our study are all p53-mutant, but the influence of NEDL1 on apoptosis in UC cell lines should likewise be investigated in future work.In addition, the functional significance of mutations in 21, which might be used to detect the impact of cisplatin with respect to point mutations in vivo. For several reasons, including polyclonality and tumor heterogeneity, copy number changes are more difficult to detect in vivo. However, if detectable, segmental gains and losses as well as focal alterations of the sort seen in the LTTs could be indicators of cisplatin clastogenic activity in tumor and other tissues.In conclusion, our investigation demonstrates that the emergence of cisplatin-resistant UC cell lines in a continuous treatment model is accompanied by massive genomic changes, both point mutations and chromosomal alterations. These findings underline the potential of cisplatin to cause genomic changes in cancers and patients. Our data confirm a mutational signature proposed by others5.The human UC cell lines (UCCs) RT-112 and T-24 were obtained from the DSMZ , whereas 253J and J82 cell lines were kindly provided by J. Fogh . Parental UCCs and their long-term cisplatin-treated sublines were grown in DMEM GlutaMAX-I  containing 10% heat-inactivated fetal bovine serum. Long-term cisplatin-treated sublines (LTTs) were established by continuously escalating cisplatin  dosages over several months until stable resistant cell lines could be maintained at doses of 50 μM (RT-112-LTT), 3.3 μM (J82-LTT), 6.6 μM (253J-LTT), and 23 μM (T-24-LTT) as described previously5 using primers amplifying ATP7B  or the housekeeping gene SDHA  for normalisation. Relative expression was calculated by the 2−ΔΔCt method Relative cell viability was measured in quadruplicates by the CellTiter-Glo assay .The siRNA-mediated knockdown of ATP7B in UCCs was performed by transfecting 10 nmol/L siATP7B or a non-targeting control  using Lipofectamine RNAiMAX Reagent  according to the manufacturer’s protocol. RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as previously described on the Lightcycler 96 system 35.For chromosome preparation the cells were grown to near confluency, and incubated with 10 μg/ml colcemid  at 37 °C for 0.5–2 hours. Cells were harvested by using 0.05% trypsin/ 0.02% EDTA (ethylene diamine tetraacetic acid) in phosphate-buffered saline (PBS) , treated by hypotonic solution (0.56% KCl w/v) for 30 min at room temperature, and fixed in ice cold methanol/acetic acid solution . The fixed cells were dropped onto ice cold wet slides, and air dried. Giemsa banding using Trypsin and Giemsa (GTG-banding) was performed according to standard protocols. For chromosome banding analysis a digital karyotyping system  was used. A total of 20 metaphases of each cell line were analyzed. The karyotype was designated according to the International System for Human Cytogenetic NomenclatureDNA was extracted by a Blood & Cell Culture DNA Midi Kit  according to the manufacturer’s protocol. Comparative genomic hybridization (CGH) was performed using an array of 60mer DNA-oligonucleotide probes with a median overall probe spacing of 13 kb. Array CGH was done essentially according to the protocol of the manufacturer  using 1.0 μg of each cyanine 3-dUTP (Cy3) labelled tumor DNA and cyanine 5-dUTP labeled (Cy5) reference DNA tumor DNA .36, using freely available R packages from Bioconductor (bioconductor.org) and CRAN (cran.r-project.org). The R-script operated as a pipeline implementing the following steps: 1) Conversion of the reference/tumor log10-ratios as returned by the feature extraction software to tumor/reference log2-ratios. 2) Removal of improper oligo-nucleotides, flagged by the feature extraction software as having a too inhomogeneous distribution of pixel intensities throughout the spot representing the oligo (flags gIsFeatNonUnifOL = 1 or rIsFeatNonUnifOL = 1), or as being oversaturated (flags gIsSaturated = 1 or rIsSaturated = 1) or undersaturated or otherwise nonsensical (log-ratio set to zero). 3) Breakpoint detection using three different R packages: DNAcopy37, GLAD 38, and HaarSeg40. For GLAD and HaarSeg the recommended default parameters were used; for DNAcopy, the parameter “UNDO.SD” was calculated as 0.25 / derivative log ratio spread (data), adjusting the approximate minimum log-ratio difference between adjacent segments to our own log-ratio cutoff (see point 4). 4) Assignment of an aberration status  to the regions defined by the breakpoints based on the following criteria: To be called aberrant, a region had to span at least five oligos with an average log2-ratio >0.25 for duplications and <−0.25 for deletions, and it had to be supported by at least two of the three breakpoint methods used. 5) Creation of a detailed report listing the calls, their positions, sizes, log-ratios, and affected genes. For detecting recurrent aberrations, a second R-script was used to combine the results of multiple array analyses into one, reporting the relative number of samples sharing the same aberrations in textual form and as graphics (penetrance plots). For further visual inspection Agilent Workbench Software (Version 7.0) was used.Microarrays were scanned using an Agilent Technologies Scanner G2505C. Agilent’s Feature Extraction Software version 11.5.1.1 was used to extract log-ratios from the scanned images. The bioinformatic analysis was carried out with a custom-built script for the R Statistical EnvironmentTM System (Life Technologies) using Ion PI™ v2 chip with the Ion PI™ Hi-Q™ Sequencing 200 Kit according to the manufacturer’s protocol . Amplicon sequences were aligned to the human reference genome GRCh37(hg19) in the target region of the AmpliSeq™ exome using the Torrent SuiteTM software 5.2.0 with the implemented TMAP algorithm (Life Technologies). Subsequently, variants were detected using the Torrent Variant Caller Plugin v5.2.0.34 and the predefined parameter set ‘germ line low stringency’. Detailed annotations to each detected variant were collected in the databases of the NCBI Reference Sequence Database , Exome Sequencing Project (ESP6500), 1000 Genomes Project (1000 g), ClinVar (2015/06/29) and dbSNP142 using the open source software tool ANNOVAR. Further information was added by the implemented prediction tool MutationTaster. The Integrative GenomicsViewer (IGV 2.4.4) was used to visualize the read alignments and to check possible errors, for example due to homopolymeric stretches. Further information on filtering parameters and according numbers of variant is given in Supplementary Table The exome NGS (next generation sequencing) library was generated from high quality genomic DNA from RT-112, J82, 253J, and T-24 parental and LTT cell lines with the Ion AmpliSeq™ Exome RDY Kit . Amplification and adapter ligation were performed according to the manufacturer’s protocol . The library was barcoded during the ligation reaction using the Ion Xpress™ Barcode Adapter Kit (Life Technologies). Library concentration was determined by Qubit measurement (Life Technologies) and library fragment length was analyzed using the 2100 Bioanalyzer . The library was diluted to a final concentration of 8 pM. Emulsion-PCR and enrichment were performed on the Ion OneTouch™ 2 System (Life Technologies) using the Ion PI™ Hi-Q™ OT2 200 Kit following the manufacturer’s protocol . Sequencing was performed on the Ion Proton42. We calculated the relative contributions of these 96 mutated trinucleotides for each sample using the SomaticSignatures R package version 2.14.043. We closer examined the base substitutions caused by cisplatin long-term treatment in each cell line by removing mutations identical in LTT and control and calculating the relative contributions of mutations that were exclusively present in each LTT cell line. The fraction of mutations at the 96 mutated trinucleotides are represented in bar charts. Cosine similarity among the mutational profiles of the LTT lines and to those of HepG2 and MCF10A . Each of these classes can be subdivided into 16 groups by sequence context if the bases immediately 5′ and 3′ to each mutated base are considered0A Table , which w46.ATP7B, HECW1, NRXN2, and FAM205A mutations in 404 bladder urothelial carcinoma samples (TCGA) were analyzed using the cBioPortal for Cancer GenomicsSupplementary materialsSuppl Table 1Suppl Table 2Suppl Table 3Suppl Table 4Suppl Table 5Suppl Table 6Suppl Table 7Suppl Table 8Suppl Table 9Suppl Table 10Suppl Table 11Suppl Table 12Suppl Table 13Suppl Table 14Suppl Table 15Suppl Table 16Suppl Table 17Suppl Table 18Suppl Table 19Suppl Table 20


Although low-grade serous ovarian cancer (LGSC) is rare, case-fatality rates are high as most patients present with advanced disease and current cytotoxic therapies are not overly effective. Recognizing that these cancers may be driven by MAPK pathway activation, MEK inhibitors (MEKi) are being tested in clinical trials. LGSC respond to MEKi only in a subgroup of patients, so predictive biomarkers and better therapies will be needed.We evaluated a number of patient-derived LGSC cell lines, previously classified according to their MEKi sensitivity. Two cell lines were genomically compared against their matching tumors samples. MEKi-sensitive and MEKi-resistant lines were compared using whole exome sequencing and reverse phase protein array. Two treatment combinations targeting MEKi resistance markers were also evaluated using cell proliferation, cell viability, cell signaling, and drug synergism assays.KRAS mutation status, and EGFR and PKC-alpha protein expression. The biomarkers were validated in three newly developed LGSC cell lines. Sub-lethal combination of MEK and EGFR inhibition showed drug synergy and caused complete cell death in two of four MEKi-resistant cell lines tested.Low-grade serous ovarian cancer cell lines recapitulated the genomic aberrations from their matching tumor samples. We identified three potential predictive biomarkers that distinguish MEKi sensitive and resistant lines: KRAS mutations and the protein expression of EGFR and PKC-alpha should be evaluated as predictive biomarkers in patients with LGSC treated with MEKi. Combination therapy using a MEKi with EGFR inhibition may represent a promising new therapy for patients with MEKi-resistant LGSC.The online version of this article (10.1186/s12935-019-0725-1) contains supplementary material, which is available to authorized users. Each year in Canada and the United States, over 25,000 women are diagnosed with ovarian cancer , 2. Low-TP53 gene are rare (8% in LGSC versus 96% in HGSC) [KRAS (20–40%), NRAS (7–26%) and BRAF (5–33%) genes [It is now recognized that LGSC has unique clinical, pathological, and molecular characteristics compared to other types of ovarian cancers, such as the high-grade serous ovarian carcinoma (HGSC) , 8. Molein HGSC) , and thain HGSC) , 12. LGS%) genes –20. Evid%) genes  led to a%) genes . A secon%) genes . CurrentTo date, preclinical laboratory research in LGSC has been limited to tumor tissues. The low frequency and slow growth rate of these tumors have challenged the development of cell lines and animal xenograft models. In the past 5 years, our laboratory has successfully established a collection of patient-derived LGSC cell lines that are now available for pre-clinical drug testing. Previously, we evaluated the effects of four different MEKi  in eight advanced/recurrent LGSC cell lines. Our results indicated that there were substantial differences in cellular response and on-target drug efficacy between cell lines and drugs . EncouraAdvanced or recurrent LGSC samples (tumor and ascites) were obtained from the OvCaRe gynecologic tumor bank , and the John and Mary Knight Translational Ovarian Cancer Research Unit . Tumor bank protocols, cell line derivation, and all research relating to this study was approved by institutional human ethics review boards at BCCA (H14-02859), the University of British Columbia , and the University of Western Ontario (HSREB 12668E). Clinical information was extracted retrospectively from patient records. Tumor histology was confirmed by a gynecological pathologist.2. No immortalization methods were used. Doubling time of these cells ranged from 30 to 80 h, with an average of 47 h, reflecting the clinical slow growth rate of LGSC.Low-grade serous ovarian cancer patient-derived cell lines were established through continuous in vitro culture of patient material obtained through OvCaRe or the John and Mary Knight Translational Ovarian Cancer Research Unit (cell line iOvCa241) tumor banks. Cultures were established and maintained in M199:MCDB105 (1:1) media  supplemented with 10% defined fetal bovine serum  maintained at 37 °C and 5% COMicrosatellite analysis of short tandem repeats (STRs) was performed on LGSC cell lines and corresponding tumor and buffy coat samples for cell line authentication. STR analyses of 10 loci were performed by Genewiz Inc.  (Data available upon request). STR results confirm that all LGSC cell lines and buffy coat samples match to corresponding tumor samples.Whole exome sequencing (WES): Agilent SureSelect RNA Library All Exons v6 protocol was performed by Beijing Genome Institute, per manufacturer’s guidelines. Quality and quantity of post-capture libraries were assessed using an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina Hiseq 4000 (PE 100). Copy number variation (CNV) analysis: Data analysis was performed using Nexus Copy Number Discovery Edition Version 9.0 . Samples were processed using the Nexus NGS functionality (BAM ngCGH) with the FASST2 segmentation. The log ratio thresholds for single copy gain and single copy loss were set at + 0.18 and − 0.18, respectively. The log ratio thresholds for gain of 2 or more copies and for a homozygous loss were set at + 0.6 and − 1.0, respectively. Tumor sample BAM files were processed with corresponding normal tissue BAM files. Probes were normalized to median. Mutation analysis: Sequence alignment and mutation calling were performed in Partek Flow environment (© 2017 Partek Inc). Sequence reads were aligned to GRCh38/hg38 human genome build using bwa 0.7.2. Variants were called using Strelka 1.0.15 for all cell lines except for VOA-1312 (lacking buffy coat sample). VOA-1312 variant calling was performed using LoFreq 2.1.3.a. The called variants were annotated using the wAnnovar software (reference obtained from: http://jmg.bmj.com/content/49/7/433.citation-tools). Annotated calls were then filtered to show only protein-changing SNVs that were present in cell line DNA at allele frequencies (AF) greater than 0.1 and coverage higher than 16×. For VOA-1312, all calls not present in dbSNP (version 138) were retained, while of the calls that were present in dbSNP, only calls with (average heterozygocity + aveHet standard error) < 0.1 were retained. These were additionally filtered using the same criteria as for the Strelka calls.Whole genome sequencing (WGS): Genomic data from LGSC tumors T7 and T11 were obtained from the personalized oncogenomics (POG) program at the BCCA. Methodology has been previously described in detail [n detail . To summAssessment of MEKi sensitivity using trametinib  and selumetinib , were performed as previously described . Phase c2. Absorbance at 490 nm was measured using a microplate reader (BioTek Epoch SN257811). Viability for each treatment was compared to DMSO treated cells. Wells were subsequently stained with crystal violet (CV) to determine residual cells after treatment. Statistical analysis using t-test were used to compare treatment conditions and differences were considered significant at a p-value < 0.05.Cell viability was measured using MTS-Cell Titer 96R Aqueous Non-Radioactive Cell Proliferation Assay, following manufacturer recommendations  at endpoint of Incucyte™ proliferation assays. Treatment media was replaced with 100 μL of fresh media and 20 μL of MTS. Plates were incubated for 3.5 h at 37 °C in humidified 5% COErlotinib (Cat. No. S7786) were purchased from Selleck Chemicals . Dimethylsulfoxide  was purchased from Sigma-Aldrich . Cells were seeded in 96-well plates at 40–50% confluence and treated after 24 h with DMSO or a range of drug concentrations. The inhibitory concentration  was determined using crystal violet staining after 72 h drug treatment.Cell lysates were prepared according to previously published protocols , then 20Reverse-phase protein array on whole tumor and cell line lysates was performed as previously described , 29. ProPRKCA shRNA sequences and one non-target shRNA (Cat. No. TLHVU1401-5578) was purchased from transOMIC Technologies . VOA-3723 and VOA-6406 were plated at 50% confluence in 6-well tissue culture dishes 24 h prior to lentiviral transduction. 199:105 media supplemented with 1% Hyclone dFBS and polybrene  and lentivirus expressing non-targeting shRNA or PRKCA shRNA  in a total volume of 1.5 mL was added. After 24 h, cells were washed with PBS and complete media was added. Successful transduction was confirmed using confocal microscopy. After an additional 24-h recovery, transduced LGSC cells were selected and maintained using puromycin .shERWOOD-UltramiR shRNA lentiviral target gene set containing three http://www.combosyn.com). This software is based on the median-effect principle and the combination index-isobologram theorem  [Cell proliferation, viability and crystal violet results from in vitro drug testing (single drug and drug combinations) were used to assess drug synergism using CompuSyn software  . Drug doOur laboratory previously established a collection of LGSC cell lines derived from patients with advanced/recurrent disease. Preclinical evaluation of four MEKi in eight different LGSC cell lines resulted in the identification of two distinct phenotypes: MEKi-sensitive (MEKi-Se) cell lines n = 2), and MEKi-resistant (MEKi-Re) cell lines (n = 6). In this first study, MEKi drug concentrations and IC50 values were reported . Recogni, and MEKWES was performed to characterize the genomic profiles of our LGSC cell lines and primary cultures. First, we compared the copy number profiles of two of our cell cultures  with those of their associated tumor samples (from WGS data). As shown in Fig. MTAP and CDKN2A tumor suppressor genes, was found in all samples. As expected, KRAS and NRAS non-synonymous mutations were both found most frequently. Either mutation was present in 28.6% of all cell lines/cultures, and in 36.4% (KRAS) and 27.3% (NRAS) respectively when analyzed by patient (some cell lines were derived from the same patient at different times). KRAS and NRAS mutations co-existed in only one cell line (VOA-8862). Only one BRAF mutation was detected . Additionally we analyzed gene mutations and copy-number changes affecting 61 well-known MAPK-pathway genes is shown in Additional file RAS mutations, copy-number variation (CNV) findings, and MEKi sensitivity in each LGSC cell culture (n = 14) is shown in Table Results from the WES analysis in our LGSC cell lines and primary cultures n = 14) show variable levels of genomic aberration and non-synonymous mutations (NsMs), ranging from 1 to 66% total genome change and 24-111 mutation calls per cell line , while MEKi-Re cell lines were either NRAS mutant , or KRAS/NRAS wt . Of interest, the VOA-8862 cell line (mutations in both KRAS and NRAS) was found to be sensitive to all four MEKi tested. In this line, the KRAS mutation variant detected (G12D) is known to be oncogenic, while the NRAS mutation variant detected (C118Y) was not found in the COSMIC database, therefore its oncogenic potential remains unknown. We did not observe any obvious correlation between the degree of CNV in each cell line (copy number high versus low) and MEKi response.We found that all MEKi-Se cell lines  carried oncogenic mutations in To identify biomarkers of MEKi response, we compared two MEKi-Se  and six MEKi-Re  LGSC cell lines using reverse phase protein array (RPPA) analysis. To do so, lines treated with DMSO, 1 μM selumetinib, or 0.1 μM trametinib were screened using a panel of 91 validated antibodies , with and without MEK inhibition (using selumetinib or trametinib), in four MEKi-Re LGSC cell lines . IC50 values for erlotinib in these cell lines are shown in Additional file With the highest dose of erlotinib treatment alone , all four MEKi-Re cell lines continue to proliferate. Interestingly as shown in Fig. The effects of erlotinib, with and without MEKi treatment, on EGFR and MAPK signaling pathways were evaluated using WB. Levels of total and phosphorylated EGFR and ERK1/2, as well as total and cleaved PARP (c-PARP) were measured after 24 h treatment. Results from these experiments indicated that drug effects on cell signaling were cell line dependent  carried PRKCA copy-number gain and two MEKi-Se cells (VOA-9164 and VOA-8862) had PRKCA copy-number loss. To determine whether PKC-alpha protein expression played a role in mediating MEKi resistance we evaluated the effects of PKC-alpha knockdown using lentiviral shRNA, with and without selumetinib or trametinib in two MEKi-Re LGSC cell lines (VOA-6406 and VOA-3723). As shown in Fig. PRKCA shRNA resulted in a complete PKC-alpha protein knockdown in VOA-6406 cells and a partial knockdown in VOA-3723 cells by WB. Subsequent MEKi treatment (1.0 μM selumetinib or 0.1 μM trametinib) experiments showed no significant changes in cell viability when compared to non-target shRNA transduced lines  are frequently found in cancer. MAPK pathway inhibitors, such as MEK inhibitors, were developed as targeted therapeutics to potentially treat such cancers , 42. MEKMTAP and CDKN2A genes) [KRAS and NRAS genes, in agreement with results from previous studies on LGSC tumor tissues [RAS mutations were often associated with RAS copy number gain. As previously reported [In this study, we utilized genomic and proteomic techniques to molecularly characterize a collection of LGSC cell lines and primary cultures (derived from advanced/recurrent LGSC patients), and identify markers that predict response (sensitivity/resistance) to MEKi treatment in vitro. Genomic profiles of two of these cell models were compared with their corresponding tumor samples from the same patient and showed remarkably similar copy number profiles, supporting the utility of these cell models for preclinical research. Subsequent comparisons of genomic profiles from an additional twelve LGSC cell models showed frequent deletion of Chr9p (including loss of A genes) , 49 and  tissues –15. Addireported , 46, 50 KRAS in all four MEKi-Se lines which were absent in all six MEK-Re lines. Previous results from a clinical trial using selumetinib  in this study. In agreement with our results, two recent case reports on LGSC patients with remarkable and durable clinical responses (> 5 years) to MEKi therapy have reported oncogenic KRAS mutations (both G12V) in their tumors [RAS mutation status as a predictive biomarker. It is not unexpected that a single biomarker, such as KRAS mutation status, will not accurately predict responses to MEKi treatment, recognizing that LGSC harbor other MAPK-pathway gene mutations and significant MAPK copy number changes. Furthermore, KRAS copy-number amplification (described as one activating mechanism) could also play a role in mediating MEKi efficacy [Further evaluation of mutation profiles in eight LGSC cell lines with different sensitivity to MEKi treatment (two MEKi-Se and six MEKi-Re) showed oncogenic mutations in y et al. ) did notr tumors , 47. As efficacy .Using RPPA to compare MEKi-Se and MEKi-Re LGSC cell lines, we found that all MEKi-Re lines had higher levels of EGFR and PKC-alpha expression. These results were subsequently validated in three newly established LGSC cell lines. Using this approach, we also described proteomic changes specific to each MEKi tested (selumetinib or trametinib). The changes we observed may be particularly relevant when evaluating differences in drug efficacy, as MEKi may exhibit differences in MEK isoform specificity or off-target effects . InteresIn colorectal cancer, preclinical studies with BRAF inhibitors have reported adaptive feedback reactivation of MAPK signaling involving EGFR , 51. ThiKRAS mutant status. Though the treatment was tolerated, significant side effects occurred with combination therapy. If these drug treatment combinations are going to be effective in LGSC, optimal drug dosing will be required in order to minimize side effects without loss of treatment efficacy.There is a growing body of evidence supporting the use of combining a targeted therapy with other targeted agents or with traditional chemotherapeutic agents , 52. ComBRAF mutant melanomas, and combining a BRAFi and an EGFRi has improved tumor regression in BRAF mutant colorectal cancer xenografts [KRAS G12D mutation and a CUL1:BRAF fusion) were identified in the tumors of both patients [KRAS G12V [MEK1 (Q56_V60del) gene mutations experienced disease stabilization in response to this drug treatment combination [Combination therapy with BRAFi and MEKi has remarkably improved survival in the adjuvant setting for patients with nografts , 54. In patients . AdditioRAS G12V , 47 and bination .PRKCA copy number gain. PKC-alpha knockdown by itself did not affect cell proliferation in either cell line. The results of our experiments suggest that PKC-alpha protein expression appears to be a predictive biomarker but is not a therapeutic target mediating MEKi resistance.PKC-alpha expression has been implicated in chemotherapy drug resistance in some cancers , 37. To BRAF mutations, they have demonstrated little effect in the treatment of BRAF mutant colon cancers [KRAS are more common than in BRAF [Identifying molecular characteristics to predict drug sensitivity/resistance in individual patients with solid tumors has proved to be challenging. The efficacy of therapies designed to target specific mutations are known to be dependent on the cancer type. For example, while BRAF inhibitors have shown to be effective in melanomas carrying  cancers , 55, 56. in BRAF , 57, 58.KRAS mutations and low levels of EGFR and PKC-alpha protein expression. The confirmation of these results in MEKi treated LGSC tumors samples could lead to better patient selection for MEKi treatment, and further avoid unnecessary treatment and toxicities in patients unlikely to respond. Our study also suggests that a significant portion of those LGSC patients whose tumors are unresponsive to MEKi therapy may benefit from combination therapy with EGFR and MEK inhibition. As LGSC xenografts are not yet available for research, we are currently unable to validate these results in vivo. However, we are now using our LGSC patient-derived cell lines to establish xenograft models. The potential predictive value of the three molecular markers of MEKi response identified in our LGSC cell line models should be considered for further validation in clinical trials using MEKi for the treatment of LGSC.In summary, this proteogenomic study is the first to perform predictive biomarker discovery for MEKi treatment in LGSC cell lines. MEKi-Se cell lines were found to have oncogenic Additional file 1: Table S1. List of antibodies included in the RPPA analysis.Additional file 2: Figure S1. Preclinical MEKi evaluation in novel LGSC cell lines.Additional file 3: Table S2. List of all NsMs and genomic aberrations (CNV and NsMs) affecting 61 MAPK-related genes .Additional file 4: Table S3. List of proteins differentially expressed between MEKi-se and MEKi-re LGSC cell lines .Additional file 5: Table S4. EGFR information on LGSC cell lines.Additional file 6: Figure S2. Biological effects of trametinib and erlotinib drug combination in MEKi-re LGSC cell lines.Additional file 7: Table S5. Compusyn data from selumetinib/trametinib and erlotinib drug combinations in MEKi-re LGSC cell lines.Additional file 8: Figure S3. Effects of trametinib and erlotinib combination on LGSC cell lines by WB analysis.


Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes.TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC.We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes , TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives.In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 activity is not a marker for CPT sensitivity in breast cancer. Breast cancer (BC) is the most frequent form of cancer among women . The incCamptothecin (CPT) is the mother compound of an important class of drugs that specifically target the nuclear enzyme topoisomerase I (TOP1). The members of this class of compounds are currently among the most effective anti-cancer drugs for the treatment of advanced-stage gastrointestinal  malignancies, ovarian cancer, and recurrent small-cell lung cancer (SCL) –19. BiolCPT inhibits the re-ligation step of the TOP1 catalytic cycle, leading to the accumulation of cytotoxic covalent TOP1-DNA cleavage complexes (TOP1cc). The cytotoxic effect of TOP1cc is specific to the S-phase and is thought to reflect collision events between the replication machinery and TOP1cc, leading to DNA fragmentation and cell death . DecreasTOP1 gene copy number may be used as an alternative to TOP1 protein expression as a predictive biomarker for stratification of patients with CPT-responsive colorectal cancer  exhibited greater resistance to the drug, with IC50 of 0.448 ± 0.054 μM and 0.481 ± 0.060 μM, respectively  and HCC1419 (HER2 subtype)] showed high sensitivity toward CPT treatment, exhibiting an ICIn addition to TOP1 activity, the drug susceptibility of the enzyme itself can influence cellular sensitivity to CPT. The susceptibility of TOP1 toward CPT was estimated by measuring TOP1 activity in nuclear extracts prepared from the eight cell lines selected in the presence of 60 μM CPT, using the REEAD assay. Note that CPT susceptibility as measured in terms of inhibition of the (nuclear-extracted) TOP1 re-ligation activity is not dependent on TOP1 activity per se  = 0.0003). Hence, all four TNBC-like cell lines  had a high growth rate  . The aim was to investigate possible associations of cellular response to CPT with various cellular and molecular factors, including TOP1 gene is common in patients with BC and is often considered as a potential biomarker of response to treatment with TOP1 inhibitors [TOP1 was found to be amplified in two of five Luminal cell lines, one of four HER2 cell lines, and nine of 14 TNBC-like cell lines . Jandu and co-authors reported a putative association between TOP1 protein level and gene copy number in nine BC cell lines, but it was not possible to determine the significance of this presumptive correlation, because the level of TOP1 protein expression was evaluated by eye rather than by quantitative densitometry [TOP1 locus or by other aspects of cell metabolism. Current data regarding TOP1 gene status in breast tumor tissue are rather controversial. An investigation of TOP1 gene copy number (including 1033 cases) based on use of the The Cancer Genome Atlas (TCGA) portal revealed that only 2% of breast tumors exhibited increased TOP1 copy number [TOP1 amplification (> 30% of BC patients had gene copy numbers > 4). This discrepancy in the overall analysis of gene copy number may be explained by a dilution effect caused by the presence of normal stromal cells [It has been shown that a gain of the hibitors , 57, 58.l lines  Fig. 1)..TOP1 genitometry . In geneal cells .We observed a direct association between protein level and cleavage-ligation activity of TOP1 in Luminal and TNBC-like subtypes but not for the HER2 subtype. The lack of a significant correlation for HER2-like cells may be partially explained by the small number of cell lines of this subtype available for experimentation or, alternatively, by specific cellular metabolic features associated with HER2 amplification .TOP1 gene copy number, protein expression, enzyme activity, and CPT-induced cytotoxicity. This is in contrast to results obtained from cell lines originating from tumors other than breast cancer, which demonstrated a direct correlation between TOP1 gene copy number and IC50 [Given the labor-intensity of experiments involving the analysis of multiple parameters  across numerous cell lines, we selected eight cell lines that represented three main BC subtypes. A Spearman correlation test applied to all cell lines investigated showed that there was no correlation between and IC50 –62. Howeand IC50 , 46. Forand IC50 .In addition to the TOP1 activity, drug susceptibility of the enzyme itself may influence cellular sensitivity to CPT , 55. We TDP1 is an enzyme that catalyzes the repair of several types of DNA damage, including CPT-induced DNA damage , 63. Conp < 0.05) lower growth rates, when compared to their parental and DMSO controls [It is well established that when a replication fork is encountered by CPT-stabilized TOP1cc, DNA fragmentation and cell death follow. It can be assumed that rapidly dividing cells are more drug sensitive than slowly dividing cells. We therefore investigated whether the differential drug response observed among the cell lines analyzed correlated with growth rate. When dividing the cell lines into two broad categories slow growing (double time above 89 h) and fast growing (doubling time below 34 h) we identified a direct association between the rate of cell division and sensitivity to CPT. This finding is in agreement with previous studies reporting that the cell-killing mechanism of CPT is S-phase dependent , 65. Ourcontrols .It is striking to note that four of the cell lines  characterized by a high growth rate and high CPT sensitivity have low TOP1 activity. This may suggest off target effects of CPT, but is more likely explained by the fact that the cytotoxicity of CPT depend on collision of replication forks with CPT induced TOP1cc resulting in the generation of lethal DNA damage. It is possible that in very fast-growing cells even a low activity of TOP1 is sufficient to generate sufficient TOP1cc in the presence of CPT for efficient cell killing. Indeed, in cells with defect repair pathways, which is a key mark of cancer cells, even a single TOP1cc at the right place and time may kill the cells . Hence, TOP1 gene copy number is a poor indicator for CPT response in BC cell lines. More comprehensive studies are needed to confirm this hypothesis for each breast cancer subtype. Indeed, for cell lines derived from the HER2 and Luminal subtypes, our data point to a putative direct correlation between gene copy number, TOP1 activity, and cellular sensitivity to CPT, suggesting that TOP1 at either the genomic or enzymatic level may be a predictive marker for response to CPT in these BC subtypes. For all BC subtypes, there is a direct correlation between growth rate and cellular response to CPT. Collectively, our results suggest that more aggressive and quickly dividing malignant tumors may be targeted more effectively by CPT. The extremely powerful response of cell lines derived from TNBC tumors treated with CPT appears promising, suggesting that it may be worthwhile to investigate the possibility of treating patients with this subtype of BC with CPT derivatives.Taken together, our results suggest that Additional file 1: Table S1. description of the cell lines used in the study. Table S2. Shows the result of the quantifications of the TOP1 protein levels. Figure S1. Nuclear TOP1 IHC staining. Figure S2. Shows the correlation between TOP1 protein expression level and TOP1 gene copy number in cell lines of the luminal subtype. Figure S3. Shows the correlation between TOP1 protein expression level and TOP1 gene copy number in cell lines of the HER2 subtype. Figure S4. Shows the correlation between TOP1 protein expression level and TOP1 gene copy number in cell lines of the TNBC subtype. Figure S5. Shows the correlation between TOP1 activity and protein expression level in cell lines of the luminal subtype. Figure S6. Shows the correlation between TOP1 activity and protein expression level in cell lines of the TNBC subtype. Figure S7. Shows the correlation between TOP1 activity and protein expression level in cell lines of the HER2 subtype. Figure S8. Shows a graphically depiction of TOP1 susceptibility to CPT in nuclear extracts. Table S3. Summary of the results obtained for all investigated parameters.