We have tested the effects of IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on human colon, pancreatic carcinoma cell, and glioblastoma cell lines and primary cultures. Although OSI and BMS are able to inhibit IGF-1R activity at low doses, the differential effect on cell proliferation and cell-cycle phase distribution shown by both compounds probes that many effects observed are mediated by BMS off-target interactions. Using MAPKs ELISAs and phospho-RTK array analysis, we have identified several BMS regulated putative kinases able to mediate BMS off-target effects. Interestingly, molecular docking assays suggest that BMS could affect these kinases not only by blocking their ATP-binding domain, but also by means of allosteric interactions. Since BMS has an important antineoplastic effect on these poor prognosis types of cancer, these compounds could be taken in consideration for treatment independently of IGF-1R status.2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G IGF1R gene is translated into a single polypeptide precursor that is cleaved to yield an α subunit, which contains the ligand-interacting domain, and a β subunit, which contains the transmembrane and tyrosine kinase domains [IGF-1R (UniProtKB code P08069) is a tyrosine kinase receptor located in the plasma membrane, which is involved in the processes of cell growth, development, and differentiation. In addition, it exhibits a very strong antiapoptotic activity [ domains . These t domains . The rec domains . Its lig domains . Activat domains .Numerous studies have shown that IGF-1R is overexpressed in primary tumors and cancer-derived cells. This increase in IGF-1R levels reflects a reversion to more primitive, less differentiated and oncogenic states that are characterized by high concentrations of IGF-1R mRNA and IGF-binding sites . In this0/G1 phase.Currently, strategies are being developed in order to exploit IGF-1R as a therapeutic target . InhibitIn this work, we have addressed the effects of BMS-754807 and OSI-906 on cell proliferation and cell-cycle phase distribution in several human colon, and pancreatic carcinoma, and glioblastoma cell lines and primary cultures derived from glioblastoma patients. Our results show that BMS-754807 mainly acts through off-target effects exerted on other protein kinases independently of IGF-1R inhibition. Given that BMS-754807 exhibits a potent antiproliferative effects on glioblastoma, colon and pancreatic carcinoma cellular models analyzed in this work, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.Given that both compounds, BMS and OSI, have been developed as IGF-1R and IR inhibitors, we decided to study whether equivalent doses of these drugs were able to inhibit IGF-1R phosphorylation to a similar extent. With this purpose, we tested the effects of a 10 µM dose of BMS or OSI on IGF-1R phosphorylation in the human pancreatic cell lines IMIM-PC-2 and RWP-1. As shown in Next, we assessed by molecular docking assays the interaction between both inhibitors, BMS and OSI, on the IGF-1R protein structure, as shown in In order to analyze the effect of both IGF-1R inhibitors on cell lines derived from different types of human tumors, we carried out MTT cell-proliferation assays in the presence of BMS or OSI. The results shown in 1 phase, BMS elicited an arrest in the G2+M phases of the cell cycle. BMS also causes an increase  in the fraction of cells in sub-G1 phase , on glioblastoma cellular models, and determined that the molecular mechanism of cell death induced by this compound was not a caspase-dependent apoptosis . AccordiGiven that both compounds, BMS and OSI, are inhibitors of IGF-1R tyrosine kinase activity, we set to assess whether their observed differential effects on cancer cell lines were attributable to their possible ability to inhibit off-target protein kinases. With this purpose, we started by analyzing their putative effect on the activity of different MAP kinases. We performed these experiments in the pancreatic carcinoma cell line RWP-1, since both compounds inhibited their proliferation and affected their cell cycle phase distribution A,B. An EIn an attempt to identify possible differential targets for BMS and OSI, we used a human phospho-kinase array. The most significant results of these experiments are shown in D (KD = expΔG/RT) for compounds with a ΔG ≤ −10.5 kcal/mol was in the nanomolar or subnanomolar range [ar range ,24. As nThis work shows that both BMS and OSI small molecules indeed act as inhibitors of IGF-1R, as previously reported ,19,25,261 phase  and the ligand in each binding pose . InitialOther data proving that the effect of BMS is independent of IGF-1R levels arise from checking the UALCAN database  . In thisBMS-754807 exerts a strong antineoplastic effect on several cancer model cells, apparently in a way mostly independent of IGF-1R inhibition and is able to inhibit the ERK1/2 and PI3K/AKT/mTOR signaling pathways, which are crucial for tumor cells to survive. The identification of the primary targets of BMS responsible for mediating its antiproliferative effects should allow one to develop new compounds able to directly act on them with a potential antineoplastic effect and fewer side effects.The IGF-1R small molecule tyrosine kinase inhibitors BMS-754807 (PubChem 24785538) and OSI-906 (PubChem 11640390) were purchased from Selleck Chemicals . The pan-caspase inhibitor Z-VAD-FMK was obtained from Calbiochem  and used to assess caspase-dependent apoptosis.The human colorectal cancer cell lines SW480, SW620, HCT-15 and DLD-1, and pancreatic carcinoma cell lines IMIM-PC-1 (M186), IMIM-PC-2 (M220), HS766T, PANC-1 and RWP-1, were kindly donated by the Instituto Municipal de Investigaciones Médicas . The human glioblastoma cell lines U87-MG (U87), U-87ΔEGFR and T98G (T98) were obtained from the American Type Culture Collection . HGUE-GB-15, HGUE-GB-16 and HGUE-GB-17 primary GBM cell cultures were obtained from brain aspirates of patients who had been diagnosed with GBM, as previously described . These bv/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) of a penicillin and streptomycin mixture  and incubated at 37 °C in a humidified 5% (v/v) CO2 atmosphere. Primary cultures were maintained under the same conditions previously described, except that the medium was supplemented with F-12 Nutrient Mixture .All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) High Glucose  supplemented with 10%   was added at 0.25 mg−1, and after incubation for 3 h the medium was removed and 200 μL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added. After shaking at room temperature for 30 min to dissolve the formazan crystals [Cells were seeded in 96-well plates  at a density of 4000 cells per well and incubated at 37 °C with high humidity and 5% COcrystals , the absv/v) for at least 30 min at −20 °C. Thereafter, the cells were pelleted, resuspended in 0.5 mL of phosphate-buffered saline (PBS) in the presence of 0.5% Triton X-100, 25 µg mL−1 RNase A  and 25 µg mL−1 propidium iodide (Sigma Aldrich), and incubated for 30 min at room temperature in the dark. Finally, the cell cycle phase distribution was determined as a function of the cellular DNA content in a BD FACSCanto™ flow cytometer .Cells were seeded in 6-well plates , and after treatments they were harvested by trypsinization and fixed in cold ethanol , following the manufacturer’s instructions. Briefly, the cells were lysed in an appropriate volume of the cell lysis mix provided in the kit (100 µL for a 96-well plate) with shaking (300 rpm) at room temperature for 10 min. Then, 50 µL of cell lysate was added to each well of InstantOne ELISA microplates and, after the addition of 50 µL of the antibody cocktail, incubation was carried out for 1 h. Fifty µL of cell lysis mix or of the control lysate provided in the kit were used as negative and positive controls, respectively. Thereafter, three washes with 200 µL of wash buffer per well each time were carried out, and 100 µL of detection reagent was finally added. After shaking for 30 min, stop solution was added and the absorbance was read at 450 and 650 nm.RWP-1 cells were treated with BMS-754807 or OSI-906 for 6 h, and then harvested and analyzed for levels of phosphorylated tyrosine kinases using the Human Phospho-RTK Array Kit  or Human2O molecules, ions and inhibitors were removed. The addition and visualization of protein structures and the elaboration of figures were carried out using the PyMOL v.2.3 software from the Schrödinger LLC platform .To date, a number of crystal structures of the catalytic domain of protein kinases analyzed in this study have been solved and deposited in the Protein Data Bank (PDB). A complete list of all structures used in assays of molecular docking against the catalytic site of these protein kinases is included in http://ccc.umh.es/) of the Miguel Hernández University (UMH). The chemical structures of BMS-754807 and OSI-906 were retrieved from the NCBI PubChem Compound database [Molecular docking assays of ligands (BMS and OSI) against the ATP-binding site of each protein kinase catalytic domain were carried out as previously described ,24,47. Bdatabase . To seardatabase , where adatabase .t-test or the Mann–Whitney U test was used. p-values lower than 0.05 were considered statistically significant.The results are shown as the mean ± standard error of the mean (SEM) of at least three independent experiments. A descriptive statistic was performed with the GraphPad Prism version 5  calculating the mean, standard error of the mean, and standard deviation for the values. The Shapiro–Wilk statistical test was used to evaluate the normal distribution of the data, and, to analyze the association between variables, Student’s Although OSI and BMS are able to inhibit IGF-1R activity at low doses, the differential effect shown by both compounds against colon, pancreatic and glioblastoma cancer cellular models is mediated by BMS off-target effects. Since BMS has an important antineoplastic effect on these poor prognosis types of cancer, this compound could be taken into consideration for treatment independently of IGF-1R status.We have identified several kinases that can mediate the BMS off-target effects. Interestingly, our data suggest that BMS could affect these kinases not only blocking their ATP-binding domain, but also by means of allosteric interactions.The differential molecular mechanisms of cell death induction evoked by BMS and PPP (another IGF-1R inhibitor) on the same cellular models open the possibility to find a new therapeutic target for poor prognosis cancers based on these molecular mechanisms of cell death.


Although rare, pancreatic neuroendocrine tumours (pNETs) represent the second most common group of pancreatic neoplasms, and associated patient outcomes remain largely poor. Advances in diagnosis and management rely strongly on the ability to accurately model these tumours ex vivo; however, currently available models are limited. Moreover, the importance of the extracellular matrix in disease development and progression should be heavily considered when modelling the disease. This review will outline the most clinically relevant disease models of pNETs and challenges in their use, as well as recent advances and future directions in their modelling.Pancreatic neuroendocrine tumours (pNETs) are a heterogeneous group of epithelial tumours with neuroendocrine differentiation. Although rare , they are the second most common group of pancreatic neoplasms after pancreatic ductal adenocarcinoma (PDAC). pNET incidence is however on the rise and patient outcomes, although variable, have been linked with 5-year survival rates as low as 40%. Improvement of diagnostic and treatment modalities strongly relies on disease models that reconstruct the disease ex vivo. A key constraint in pNET research, however, is the absence of human pNET models that accurately capture the original tumour phenotype. In attempts to more closely mimic the disease in its native environment, three-dimensional culture models as well as in vivo models, such as genetically engineered mouse models (GEMMs), have been developed. Despite adding significant contributions to our understanding of more complex biological processes associated with the development and progression of pNETs, factors such as ethical considerations and low rates of clinical translatability limit their use. Furthermore, a role for the site-specific extracellular matrix (ECM) in disease development and progression has become clear. Advances in tissue engineering have enabled the use of tissue constructs that are designed to establish disease ex vivo within a close to native ECM that can recapitulate tumour-associated tissue remodelling. Yet, such advanced models for studying pNETs remain underdeveloped. This review summarises the most clinically relevant disease models of pNETs currently used, as well as future directions for improved modelling of the disease. Pancreatic neuroendocrine tumours (pNETs) are the second most common subtype of primary pancreatic tumours and represent around 1–2% of pancreatic neoplasms despite their relative rarity  ,4. pNETsPreclinical disease models offer platforms for studying the complex tumour biology and pathophysiology as well as the effects of different drug treatments in a controlled environment. Popular models include cell lines, patient derived xenograft cells and animal models ), while three-dimensional (3D) models have been a recent focus of attempts to better mimic the in vivo like behaviour of cancer cells and allow the study of complex molecular interactions in cancers .Surrogate models for studying diseases are pivotal yet are not easy to construct in a way that completely recapitulates the complex physiology and cell–cell interactions with both cancerous and noncancerous cells of the tumour microenvironment. Models for studying pNETs are scarce, due in part to the rarity of the disease. However, considering the rise in incidence and the often poor outcomes for patients diagnosed with advanced disease, ongoing development of such models to better mimic and reflect the biology of these tumours are warranted. In this review we summarise the most clinically relevant models of pNETs in current use.Available established pNET cell lines include four human, three mouse and two rat cell lines. Animal cell lines have mostly been used for the purpose of studying islet hormone production and their regulation mechanisms in the settings of diabetes mellitus , rather The most widely used human cell lines are BON-1 and QGP-1. The BON-1 cell line was isolated from a pancreatic serotonin-producing metastatic carcinoid present in a lymph node excised from a 28-year-old male patient , while QQGP-1 and BON-1 cells have both been used for the study of molecular processes associated with tumourigenesis  and the effects of their inhibition on disease progression. Both cell lines have been key to studies of current first-line treatments, such as everolimus  and somatostatin analogues for treating gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), as well as studies aimed at overcoming drug resistance to these using novel PI3K/mTOR pathway inhibitors ,12,13,14Additional human pNET cell lines include the CM and NT-3 lines. The CM cell line was isolated from the ascitic fluid of a patient with a primary pNET; due to the high resemblance to pancreatic β-cells , it has been used for the study of β-cell function . More reRecent studies aimed at mapping the genomic landscape of BON-1 and QGP-1 cells, however, raise certain issues with respect to their true origin and epithelial/pancreatic-endocrine phenotype.P53, TSC2 and NRAS mutations alongside aberrant activation of the mTOR/Akt pathways, while QGP-1 cells harbour mutations in P53, ATRX and SMAD4, both showing only partial resemblance to commonly occurring pNET genetic mutations [BON-1 cells bear utations . With reutations , and thautations . Luley eutations . While autations . Lastly,utations . These futations ,21,22.Two-dimensional (2D) cell culture systems do not take into account the biology and physiology of the original tissue structure. Instead, cells are typically grown in monolayers leading to polarised cell adhesion, as opposed to the three-dimensional (3D) environment of neoplasms. Consequently, gene expression patterns, the complex cell-to-cell interactions and mechanical and biochemical cell signals that occur in vivo are significantly modified ,24. PharFurther attempts to create a more realistic tumour environment have led to the popular use of orthotopic transplant models. Implantation of BON-1 and QGP-1 cells in immunocompromised mice enables their propagation as tumours that retain their neuroendocrine phenotype . An orthRecognising the above discussed shortcomings of cell lines and their questionable predictive value following modification while in long-term culture, the use of primary cells taken from biopsies obtained surgically or by endoscopic sampling has gained popularity among researchers. The culture of primary pNET cells taken from 25 tumours in bovine ECM has been described by Mohamed et al. . This nePDX models require tumour resection or sampling followed by their transplantation into animals for expansion (most commonly immunodeficient mice), and they offer the advantage of preserving the original histological and genetic tumour properties as opposed to the altered genomic landscape of established cell lines ,34. CancPDX models enable the study of cancer biology in a natural microenvironment, reflecting the tumour heterogenic nature . HoweverMouse models provide a productive platform to study pNETs as they allow simultaneous observation of small populations, which have well-characterised genotypes and a short life span, alongside the ease with which malignant lesions and their precursors can be retrieved. Moreover, advances in genetics and gene manipulation allow relatively simple induction of tumourigenesis . The rarThe RIP-Tag2 and RIP-Tag5 models are examples of genetic alteration in which the tissue-specific, transgenic expression of the oncogenic Simian virus 40 large T-antigen is directed by the rat insulin gene-2 promoter (RIP) ,44. The TAM/RIP-Bcl-xL-RIP models, transgenic stimulation and inhibition of the c-Myc oncogene and the apoptosis inhibitor Bcl-xL, respectively, promote the formation of an aggressive insulinoma subtype [RIP-Tag5 mice develop tumours at a later stage, when the host immune response to these lesions can be studied . Similar subtype .Glucagon-producing tumours can be studied using GLU-Tag2 transgenic mice which form tumours not only in the pancreas, but also in the brain . The GLU−/− model, in which glucagon receptor deletion is used to study diabetes-associated glucagon signalling and the oncogenic effects of deletion of the p53 and Rb tumour-suppressor genes in renin-expressing cells. These mice harbour renal and subcutaneous tumours together with glucagonomas [−/− mice usually remain localised, and hence the process of metastasis cannot be studied adequately in this model. To overcome this deficiency, the Gcg gfp/gfp mouse model (GCGKO), homozygous for glucagon-knockout/green fluorescent protein knock-in alleles, was developed [−/− models but also develop more invasive metastatic neuroendocrine tumours. The difference between the two models might be explained by the absence of GLP-1 expression in Gcg gfp/gfp mice, owing to its assumed role in inhibition of α-cell proliferation and subsequent hyperplasia [−/− for studying dissemination and metastasis in pNETs.Several GEMMs that were originally designed to study other disease processes but surprisingly were found to develop pNETs have been reported. These include the Gcgreveloped . Gcg gfperplasia . FurtherPrkar1afl/fl and Pdx1-CRE) is characterised by inactivation of the PRKAR1A gene, a regulator of the cAMP-dependent kinase PKA [The NfpNET GEMM . This model was developed by crossbreeding MEN-1 floxed mice (MEN-1L/L) carrying a tamoxifen-inducible Cre recombinase with mice expressing a modified oestrogen receptor [flox/flox Ptenflox/flox RIP-Cre) and MPM (MEN-1flox/flox Ptenflox/flox MIP-Cre)—characterised by loss of both MEN-1 and PTEN1 using the Cre-LoxP system. Both models developed well-differentiated insulinomas faster than control models with a single MEN-1 gene inactivation. Even though mice with a single PTEN gene loss do not develop pNETs, this gene may cooperate with MEN-1 to suppress pNET tumourigenesis. Consistent with PTEN deletion causing an aberrant activation of the mTOR pathway, treatment with the mTOR inhibitor rapamycin induced delayed tumour growth in these models.The in vivo study of MEN-1-associated pNETs is possible using MEN-1-knockout mice . The devreceptor . Wong etreceptor  subsequeThe use of 3D in vitro models has gained popularity in recent years under the assumption that these better recapitulate the complex cell-to-cell interactions that exist in tumours. Despite their reduced capture of certain features of the physiological microenvironment  that detrimentally alter cancer cell phenotypic properties ,60, a 3D3D cultures also allow control over most experimental variables compared to in vivo animal models and rely on the induction of cellular aggregate (tumour spheroid) formation that can be incorporated into or grown on top of an extracellular substitute, acting as miniature 3D models of the tumour and its immediate environment . MoreoveSpheroids are 3D culture models that are grown under nonadherent conditions, with their use going back to the early 1970s . Tumour Cellular spheroids are concentrically arranged into several layers —an exterSpheroid models of pancreatic ductal adenocarcinomas have previously demonstrated higher chemoresistance compared to conventional monolayer platforms using the MiaPaca2 and PANC-1 cell lines . The useOrganoid and spheroid cultures have gained significant popularity in recent years as disease models for the study of organ development, tumour biology, drug screening and toxicity . The terDespite their advantage over traditional monolayer cultures, spheroid and organoid cultures neglect the role of the tumour microenvironment in disease progression. The ECM is a complex network of structural and signalling protein, also consisting of proteoglycans which serve not only as a support framework in different tissues but also as significant participants in mechanical and biochemical signalling mediated by cell surface receptors ,92. ECM Noncancerous supporting cells promote cancer cell survival and tumour structure and function, and they are associated with the chemoresistance that challenges effective cancer treatment .Cancer-associated stromal cells are also able to modulate the antitumour immune response by interacting with immune cells by secreting chemokines that have an immunosuppressive as well as proapoptotic role against T-cells .The use of animal and 3D culture models has enabled researchers not only to study genetic aberrations leading to pNET formation and the efficacy of novel therapeutic agents, but also to study the close interaction between tumour cells and their microenvironment. In vitro cancer disease models have also been significantly modified to include both artificial and natural ECM components, as well as a variety of cancer-supporting cells, in attempts to study cancer development, progression and response to treatment under more realistic biological conditions.Hpa-Tg), which constitutively overexpress heparinase, and heparanase-knockout mice (−/−Hpse) [Studies looking into the close interaction between NETs and their stroma have revealed that NETs are one of the highly vascularised cancers and are 10-fold more vascularised compared to epithelial-cell-derived carcinomas . NET celHpse−/−) .Products of ECM degradation produced by proteolytic activity of enzymes such as MMPs are collectively called matrikines . ModulatDespite the overall benefit gained so far, the use of animal models raises ethical issues around subjecting animals to discomfort and death. Moreover, such models are limited in their ability to represent complex biological processes around cancer physiology, development and progression in humans due to interspecies differences in genetics and biology, as well as variability in their expression of molecular targets . AlongsiAdvances in proteomics, more specifically ‘bottom-up’ proteomics where samples are enzymatically digested into peptides prior to analysis using mass spectrometry, enable quantitative profiling of the complex in vivo ECM protein composition . DiffereTissue decellularisation can also be applied to construct platforms for in vitro 3D disease modelling of cancers within their native tissue. When designing culture models, it is not only the ability to combine known components of the ECM, such as collagens, fibronectin, proteoglycans and growth factors, that is key. The association, concentrations and ratios of ECM components play an important role in preserving its function and unique characteristics that will support cellular processes, growth and function ,119. TheNeuroendocrine tumours of the pancreas are a rare entity, yet their incidence is on the rise. The often poor outcomes associated with pNETs, however, warrant improvement in their management and therefore support further research of the disease. Disease models are pivotal in ex vivo studies of biological and molecular pathogenetic processes in cancer and have evolved over the last few decades, with technological advances allowing the development of more complex platforms.Traditional 2D monolayer cultures of established cell lines were key in preclinical drug development stages that resulted in approval of current first-line agents for treatment of pNETs such as sunitinib. Such models, however, are insufficient for studying more complex genetic characteristics and overlook the in vivo like effects of the tumour environment on disease progression and chemoresistance. Moreover, they do not align with the current shift in research toward a more personalised treatment approach. Models that consider the in vivo like cell–cell interactions and spatial arrangement, such as spheroids, allow for more realistic studies preserving to some extent oxygen and nutrient gradients that are observed in malignant masses. With respect to neuroendocrine cancer of pancreatic origin, reports of spheroid cultures have mostly focused on model characterisation while observing specific cell processes, investigating molecular signalling  and conducting in vitro drug screening.Organoids that are derived directly from patient biopsies more closely resemble the tumour of origin than 2D cell lines and maintain genetic expression and aberrations while preserving the tumour architecture yet are technically more challenging. Despite a recent trend toward organoid-based research in other cancer types, reports of NET-derived organoids are lacking. This is mostly attributed to the rarity of the disease and the technical challenges (including slow tumour growth rates) involved in culture of pNET primary cells. Since organoids have been used for the study of embryonic tissue development, the study of the role of cancer stem cells, as well as cancer-specific processes, is also likely to prove valuable for studying the development and progression of NETs. A very recent study by April-Monn et al. highlighted the potential of organoid models as a personalised drug screening approach performed on primary pNET cultures .The ability to artificially induce known associated genetic aberrations which lead to tumour formation made GEMMs an attractive platform for studying pNETs , which have been extensively linked with genetic syndromes and various signalling pathway alterations. Transgenic mouse models in which MEN-1 and PTEN gene knockouts are induced (resembling mutations identified in a subset of pNETs) are therefore extremely valuable. Animal models also enable the study of cancer cell–ECM interactions, as tumours are observed within their supporting tissue. However, the low clinical translatability rates of findings in animals could perhaps be improved by using tissue constructs obtained from surgical resections and from transplant and deceased donors. Decellularised tissue depleted of resident cells retains the ECM components that confer important features such as physiological substrate stiffness and protein signatures of the native tissue. In the context of studies involving the pancreatic endocrine system, such existing models mostly focus on tissue regeneration and islet transplantation for the treatment of diabetes. The combination of such tissue constructs with primary patient cancer cells has the potential to provide a more accurate ex vivo platform to study cancer-induced ECM remodelling.


Furthermore, colonic inflammation score and percentage of human CD8+ T cell populations in AMSC-treated mice were significantly lower than in GVHD control and BMSC-treated mice. Interestingly, gene expression and protein secretion of the PD-1 ligands were higher in AMSC than in BMSC. These findings are the first to demonstrate that AMSC exhibit marked immunosuppression and delay acute GVHD progression by preventing T cell activation and proliferation via the PD-1 pathway.Acute graft-versus-host disease (GVHD) is characterized by severe tissue damage that is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been increasingly examined for the treatment of immune-related diseases. We aimed to assess the immunosuppressive effects of human amnion-derived MSC (AMSC) in a xenogeneic GVHD NOD/Shi-scid IL2rγnull mouse model using human peripheral blood mononuclear cells (PBMC). Additionally, we used human bone marrow-derived MSC (BMSC) as comparative controls to determine differences in immunomodulatory functions depending on the MSC origin. Administration of AMSC significantly prolonged survival, and reduced human tumor necrosis factor-α (TNF-α) concentration and percentage of programmed cell death protein-1 receptor (PD-1) Donor lymphocytes attacking various host tissues can lead to cutaneous inflammation, intractable diarrhea, hepatobiliary disease, pulmonary fibrosis, and musculoskeletal injury2. However, patients without response to corticosteroid therapy have a dismal long-term prognosis, with an overall survival rate of only 5‒30%3. Anti-thymocyte globulin as second-line treatment for steroid-refractory acute GVHD have been reported to be associated with an increased incidence of infectious disease in a meta-analysis4. Recently, ruxolitinib, a selective Janus kinase (JAK1 and JAK2) inhibitor showed potential efficacy in phase 2 and 3 trials, and the Food and Drug Administration approved for use in patients 12 years of age or older who had steroid-refractory acute GVHD5. Considering drug resistance and relapse, more treatment options are desirable.In Japanese patients with grade II‒IV acute GVHD, response rates to corticosteroids, which are the first-line treatment for acute GVHD, range from 40 to 70%6. Importantly, bone marrow-derived MSC (BMSC) have been introduced as a cell-based therapy for acute GVHD7 and registered as regenerative medicine for steroid-refractory acute GVHD in Japan8. Although the cytokine-mediated effects of MSC occur primarily via immunomodulation, the underlying cellular and molecular mechanisms remain unclear.Mesenchymal stem or stromal cells (MSC) isolated from multiple tissues such as bone marrow, adipose, and perinatal tissues are recognized for their potential immunomodulatory effects9. Several recent studies reported that human amnion-derived MSC (AMSC) exhibited therapeutic efficacy in rodent disease models including colitis induced by chemicals11, heart failure induced by coronary artery ligation12, and allo-GVHD13.The advantages of utilizing amnion, a perinatal tissue normally discarded after delivery, as a stem cell source include non-invasive procurement and vast abundance of source material. Additionally, the immunomodulatory characteristics of mesenchymal cells within fetal membranes play key roles in fetal-maternal tolerance16. NOD/Shi-scid IL2rγnull (NOG) mice lack T, B, and NK cells, and have macrophages and dendritic cells with reduced functions. These mice exhibit a higher level of immunodeficiency. Ito et al. 17 reported a novel xenogeneic (xeno) GVHD model using NOG mice and human peripheral blood mononuclear cells (PBMC) in the absence of total body irradiation. Xeno-GVHD mice are suitable animal models to elucidate the immunoregulatory properties of human MSC in vivo prior to their assessment in clinical trials.Immunodeficient mice engrafted with human cells and tissues, commonly known as humanized mice, are used for medical research on various human diseases20. Identifying more effective MSC with improved immunomodulation from different sources can considerably contribute to cell therapy.Several studies have indicated that the immunosuppressive function of MSC in xeno-GVHD mice might depend on the MSC originWe elucidated the immunosuppressive effects of AMSC in a xeno-GVHD mouse model in vivo and in a mitogenic stimulation model in vitro. Additionally, we used BMSC as comparative controls to determine differences in immunomodulatory functions depending on the MSC origin.13. Thus, we first determined whether human AMSC administration had an immunosuppressive effect on mice with severe xeno-GVHD induced by human PBMC21 compared with human BMSC administration22. All GVHD induction groups started to decrease body weight at around week 2 , although the difference was not significant between AMSC and BMSC treatment in this regard  , although the difference was not significant between AMSC and BMSC treatment in this regard. Conversely, percentage of human CD8+ T cell populations was significantly lower in AMSC-treated mice than in GVHD controls and BMSC-treated mice (P < 0.05), respectively, at week 3.To evaluate the mechanism underlying acute GVHD suppression by human AMSC, we analyzed the temporal changes in human lymphocyte subpopulation in the blood of xeno-GVHD mice. In this middle-term experiment, there were no significant differences in relative body weight or survival in any of GVHD induction groups Fig. a,b. Perc23. Percentage of human PD-1+CD8+ T cell populations was significantly lower in AMSC-treated mice than in GVHD controls at weeks 2 and 3 , although the difference was not significant between AMSC and BMSC treatment in this regard.We also measured programmed cell death protein-1 receptor (PD-1), which is an activation marker of T cells and expressed in multiple GVHD target organsP < 0.05). No differences were observed in IL-10 concentrations among GVHD induction groups. Histopathologically, AMSC treatment, but not BMSC treatment, reduced the colonic inflammation score by a significant difference compared with GVHD controls (P < 0.05)  Fig. e, which 24, and T cells in human PBMC as responders. To examine whether the immunomodulatory effect of AMSC need cell–cell contact or not, we used a transwell system for indirect coculture to separate T cells and MSC. By stimulating with PHA/IL-2 related to T cell activation and proliferation, numbers of relative CD3+ T cell, and percentages of regulatory T cells +), and T cells (CD4+ or CD8+) expressing IFN-γ, TNF-α, and PD-1 populations were increased in PHA/IL-2-stimulated PBMC (No MSC) compared with in unstimulated PBMC controls  as a mitogen, IL-2 as a proliferation inducer+ T cell proliferation compared with No MSC (P < 0.01 and P < 0.05) , although it was not in PBMC indirectly cocultured with MSC    and decreased in indirect PBMC–AMSC cocultures on the contrary (P < 0.01) , whereas these were not significantly changed in PBMC–AMSC direct coculture. Similarly, concentrations of CXCL11 in direct PBMC–BMSC coculture were 1.8 times higher than in direct PBMC-AMSC coculture  Fig. a. Concen01) Fig. a. Surpri01) Fig. . Interesure Fig. b. Additiure Fig. .Figure 425 which were shown to be elevated both in vivo and in vitro. Further, we measured PD-L1 and PD-L2, well-known immune modulators secreted by BMSC27. PD-L1 and PD-L2 mRNA expressions were significantly higher in AMSC than in BMSC independent of IFN-γ/TNF-α treatment (P < 0.01)    or the middle phase (weeks 3‒5). Relative body weight started to decline at around week 3 in all GVHD induction groups Fig. a, and th05) Fig. b.Figure 1. Our data suggest that the key roles of AMSC include reduction in PD-1+CD8+ T cell population (donor T cell activation) and inhibition of colonic inflammation (target tissue destruction) and indicate that AMSC prolong survival of xeno-GVHD mice by delaying disease progression.GVHD progresses in three sequential phases: (1) antigen-presenting cells (APC) activation; (2) activation, proliferation, differentiation, and migration of donor T cells; (3) target tissue destruction10 reported that intravenous AMSC administration ameliorated the disease activity index score in rats with dextran sulfate sodium-induced colitis. Low Tnf-α mRNA expressions in the rectal tissues of these AMSC-treated rats with colitis coincide with our data showing low TNF-α protein concentrations in blood of AMSC-treated GVHD mice in vivo and mitogen-stimulated PBMC in vitro. Cooke et al.28 revealed that TNF-α mediated the development of intestinal inflammation in the early phase of allo-HSCT in mice. We predicted that AMSC attenuated TNF-α producing T cells in the early phase of GVHD in mice, thereby mitigating colonic inflammation. Additionally, other groups have recently demonstrated that the local administration of adipose tissue-derived MSC was potentially therapeutic in perianal fistulizing Crohn’s disease29. Therefore, human MSC might have common abilities to suppress gut inflammation. Kamel et al.30 demonstrated that IFN-γ secreted by donor cells were higher in the plasma of patients with acute GVHD, consistent with our observations in xeno-GVHD control mice and mitogen-stimulated PBMC controls in the absence of MSC. However, AMSC treatment did not decrease IFN-γ concentrations in vivo, contrary to that observed for TNF-α concentrations.We found that AMSC improved colonic inflammation in xeno-GVHD mice, without any changes in liver, spleen, or skin. Onishi et al.31. Ahn et al.32 reported that CD8+ T cells expressed PD-1 early after virus infection, whereas Simonetta et al.33 demonstrated that population of CD8+ T cells expressing PD-1 increased early in patients undergoing allo-HSCT. We confirmed that percentage of PD-1+CD8+ T cell population, which was increased over time in xeno-GVHD control mice , was significantly ameliorated by AMSC infusion.PD-1 is upregulated on T cells during immune activation34 indicated that MSC secrete soluble factors, including prostaglandin E2, indoleamine 2,3-dioxygenase, transforming growth factor beta 1, galectin-1, IL-6, nitric oxide, and PD-L1, to downregulate effector T cells and monocytes. We showed that AMSC and BMSC inhibited CD3+ T cell proliferation and PD-1+ T cell activation as same levels in vitro regardless of the culture conditions, indicating that contact-independent mechanisms were important.Weiss et al.35. Consistent with our results that Treg populations were more increased in direct PBMC–MSC cocultures, English et al.36 reported that cell–cell contact was more important than soluble factors based on an in vitro Treg induction assay using a transwell system. CXCL9, CXCL10, and CXCL11 are ligands of CXCR3, a T cell-specific chemokine receptor associated with chronic inflammatory diseases including hepatitis C virus infection38. Their concentrations increased in direct PBMC–BMSC coculture, but were markedly suppressed in direct PBMC–AMSC coculture. Because these chemokines induce the migration and activation of leukocytes40, AMSC might have the ability to decrease proinflammatory chemokines and lead to calm inflammation by contacting stimulated PBMC. On the other hand, concentrations of some proinflammatory cytokines and chemokines were elevated in PBMC–MSC coculture medium. Previously, MSC derived from bone marrow, umbilical cord, adipose tissue, and Wharton’s jelly were reported to increase IL-6 and IL-8 concentrations in coculture with PBMC25. These changes were similar in that they were partially due to allogeneic response of PBMC versus MSC and should be carefully monitored to avoid the promoting GVHD during clinical setting.Conversely, Treg population and concentrations of some cytokines and chemokines were changed in direct cocultures versus in indirect cocultures comprising MSC and PBMC. Typical MSC are well-known to induce Treg by several mechanisms including soluble mediator secretion, cell–cell interactions, and APC modulation+ T cells stimulated by mitogens41. Additionally, IL-10 concentrations were elevated in mitogen-stimulated PBMC directly cocultured with AMSC in vitro but not in xeno-GVHD mice in this study. Therefore, the differences between xenogeneic and allogenic response in the context of cell–cell contact warrant careful consideration.Further, concentration of anti-inflammatory cytokine IL-10 only increased in direct PBMC–AMSC coculture, but not in PBMC–BMSC coculture and indirect coculture. Because Treg populations were not different, AMSC were presumed to be involved in high functionality of Treg. Notably, FOXP3 is expressed not only in Treg but also in naïve CD426 reported that PD-L1 and PD-L2 were secreted by BMSC to dampen the AKT pathway, allowing BMSC-mediated suppression of T cell activation. The authors also described that PD-L2 had a 2‒6 fold higher affinity for PD-1 receptor than PD-L1. Further, Survival benefit in AMSC-treated xeno-GVHD mice were maintained although the number of AMSC administrations was reduced from four to three in the early phase but not in the middle phase, indicating that early-phase AMSC infusion was more effective in attenuating acute GVHD progression. Based on our results demonstrating reduction in TNF-α concentrations and percentage of PD-1+CD8+ T cell population in vivo, together with decrease in T cell proliferation and proinflammatory chemokine concentrations as well as increase in Treg population and IL-10 concentration in vitro, AMSC might have immunomodulative effect related to the inhibition of activation, proliferation, differentiation, and migration of donor T cells, eventually delaying acute GVHD progression.Expressions of PD-L1 and PD-L2 were more upregulated in AMSC than in BMSC cultured with IFN-γ and TNF-α, whereas the specific PD-L2 blockade tended to be weakened the immune functions of AMSC in vitro. Davies et al.+ T cell proliferation in xeno-GVHD mice. In common, Grégoire et al.20 reported that administration of MSC from different origin  slightly prolonged overall survival in xeno-GVHD mice. These results indicated that therapeutic effects did not remarkably differ even though the origin of MSC was different. However, compared to BMSC, AMSC have merits as stem cell source for clinical use include non-invasive procurement, vast abundance of source material and small effect of donor age42.In this study, highly similar immunosuppressive effects with no significant differences were observed between AMSC and BMSC, except for colonic inflammation and CD8We hypothesized that not only AMSC directly inhibit T cell activation and proliferation, but it also contributes to the suppression of GVHD indirectly by modulating inflammatory-related cytokines and chemokines via the PD-1 pathway. However, immune response observed in xeno-GVHD mice is far from alloreactive GVHD observed in patients, and data to determine the appropriate AMSC dose are lacking. Therefore, future studies are warranted to translate our findings into clinical application.To the best of our knowledge, we have provided the first evidence that AMSC exert marked immunosuppression in xeno-GVHD mice in vivo and in mitogen-stimulated PBMC in vitro. We estimated that the therapeutic potential of AMSC was equal to that of BMSC, but AMSC was greater than BMSC in suppressing T cell activation and proliferation. These immunosuppressive effects of AMSC might also be applicable to other immune-related diseases.12. Amniotic membranes procured following cesarean section of pregnant females were treated with collagenase and dispase 12. Human BMSC from healthy donors were purchased from AllCells . The culture conditions of AMSC and BMSC  were determined based on previous studies43. In detail, MSC were cultured in MEMα  supplemented with 1% Antibiotic–Antimycotic (Thermo Fisher Scientific) and 5% human platelet lysate44 or 10% fetal bovine serum , and incubated at 37 °C in a humidified incubator at 5% CO2. After reaching confluence, spindle-shaped cells were detached using trypsin and cryopreserved in liquid nitrogen until further experiment. AMSC from four donors and BMSC from two donors were used at passages 2‒5. MSC were characterized based on positivity (> 95%) for CD73, CD90, and CD105 and negativity (< 5%) for CD45 by flow cytometry  were transplanted intravenously via the tail vein in NOG mice on week 0. Body weight was measured twice a week. To investigate the immunosuppressive effects of MSC in xeno-GVHD mice, 8 × 106 cells/kg AMSC or BMSC were administered intravenously once a week for 3‒4 consecutive weeks. Sham control mice were administered PBS instead of PBMC and the cryopreservation vehicle (5% dimethyl sulfoxide and human serum in culture medium) instead of MSC. GVHD control mice were administered PBMC to induce GVHD and the cryopreservation vehicle instead of MSC. Moribund mice and those that survived at the study termination (week 7 in the long-term experiment and week 4 in the middle-term experiment) were sacrificed by exsanguination from the descending aorta under isoflurane inhalation anesthesia . Plasma were cryopreserved at − 80 °C. Liver, skin, colon, and spleen were fixed with 10% buffered formalin and embedded in paraffin. Moribund mice with advanced disease were recorded as deaths. All animal experiments were approved by the Animal Care and Use Committee of Kaneka Corporation (reference number: 2019-7) and performed in accordance with the relevant local and national guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.Severe immunodeficient male NOG mice aged 7‒8-weeks were purchased from In-Vivo Science . Mice were divided into groups by stratified randomization according to weight. Human PBMC were purchased from HemaCare , and 3 × 104 cells/mL in 24-well plates. After 24 h, the cells were incubated with 10 μg/mL mitomycin C (Fujifilm) for 2 h at 37 °C to inhibit cell proliferation. Next, 6 × 105 PBMC/mL were added directly or indirectly using a transwell system  at a PBMC/MSC ratio of 10:1 or 100:1. The lymphocytes in PBMC were stimulated with 4 μg/mL PHA (Fujifilm) and 100 U/mL IL-2 . Anti-human antibodies against PD-L1 and PD-L2  were used at 250 or 500 ng/mL to inhibit ligand function. PHA/IL-2-stimulated PBMC and MSC were cocultured for 3 days.MSC were seeded at a density of 0.6‒6 × 105 cells/mL in 6-well plates and stimulated with 100 U/mL IFN-γ (R&D Systems) and 100 ng/mL TNF-α (R&D Systems). After 24 h, the cells and supernatants were collected for gene expression and protein secretion analyses.MSC were seeded at 3 × 10A small amount of peripheral blood was collected once a week from mouse tail vein using heparinized capillary tubes . Blood cells obtained from survived mice were processed to obtain single-cell suspensions using High-Yield Lyse (Thermo Fisher Scientific) for preprocessing. The cells were measured by flow cytometer using Attune NxT (Thermo Fisher Scientific) or Accuri C6 . Live cells were gated to exclude dead cells based on propidium iodide (Sigma) or LIVE/DEAD fixable dead cell stains (Thermo Fisher Scientific). Monoclonal antibodies against human CD3, CD4, CD8, CD25, FOXP3, IFN-γ, TNF-α, and PD-1 were used. For intracellular staining, 2 μM monensin (Fujifilm) was added for 4 h at 37 °C and cells permeabilized using the FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Data were analyzed using FlowJo software . The antibody information is provided in Table (GAPDH), Hs99999905-m1; PD-L1, Hs00204257_m1; PD-L2, Hs00228839_m1 (Applied Biosystems). All data were expressed as the ratio of target gene and GAPDH expression.Total RNA of MSC was extracted using RNeasy Mini Kit , and reverse transcription was performed using the PrimeScript RT Reagent Kit . Quantitative real-time polymerase chain reaction was performed using QuantStudio 7 Flex with TaqMan Fast Advanced MasterMix and Taqman Gene Expression Assay . The following primers were used: glyceraldehyde 3-phosphate dehydrogenase Human PD-L1 and PD-L2 protein concentrations were determined by ELISA (Thermo Fisher Scientific and R&D Systems) according to the manufacturer’s protocols. Optical density was measured at 450 nm by Spectra Max . To measure the protein concentrations of multiple human cytokines and chemokines, LEGENDplex multibead-based immunoassays  were used according to the manufacturer’s protocols with minor modifications. Data were analyzed using LEGENDplex software (BioLegend).45 with minor modifications. Histopathological data from dead mice were basically included. But it was omitted in case of abnormal changes such as gut autolysis. Histopathological score was interpreted by a pathologist: 0 as normal and rare, 1 as focal and slight, 2 as diffuse and mild, 3 as diffuse and moderate, and 4 as diffuse and severe.Paraffin sections (4 μm) of mouse tissues were stained with hematoxylin/eosin. GVHD lesions, primarily the extent of inflammatory cell infiltration, were evaluated according to the semiquantitative grading systemU test. In vitro assays were analyzed using two-tailed, unpaired Student’s t test, or one-way analysis of variance followed by Dunnett’s test for multiple comparisons. All data were presented as mean ± standard error of the mean. Differences with a P < 0.05 were considered statistically significant.Statistical analyses were performed using Prism 7 . Survival data were plotted using the Kaplan–Meier method and analyzed by the log-rank test. The remaining results of in vivo assays were analyzed by the nonparametric, two-tailed, unpaired Mann–Whitney Supplementary Information 1.


Breast cancer (BC) is the most prevalent type of cancer in women in western countries. BC mortality has not declined despite early detection by screening, indicating the need for better informed treatment decisions. Therefore, a novel noninvasive diagnostic tool for BC would give the opportunity of subtype-specific treatment and improved prospects for the patients. Heterogeneity of BC tumor subtypes is reflected in the expression levels of enzymes in lipid metabolism. The aim of the study was to investigate whether the subtype defined by the transcriptome is reflected in the lipidome of BC cell lines. A liquid chromatography mass spectrometry (LC-MS) platform was applied to analyze the lipidome of six cell lines derived from human BC cell lines representing different BC subtypes. We identified an increased abundance of triacylglycerols (TG) ≥ C-48 with moderate or multiple unsaturation in fatty acyl chains and down-regulated ether-phosphatidylethanolamines (PE) (C-34 to C-38) in cell lines representing estrogen receptor and progesterone receptor positive tumor subtypes. In a cell line representing HER2-overexpressing tumor subtype an elevated expression of TG (≤ C-46), phosphatidylcholines (PC) and PE containing short-chained (≤ C-16) saturated or monounsaturated fatty acids were observed. Increased abundance of PC ≥ C-40 was found in cell lines of triple negative BC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in differences in the lipidome and, furthermore, potentially biologically relevant differences may exist within these defined subtypes. Breast cancer (BC) is mainly diagnosed using routine mammography and self-examination. Recent large scale retrospective studies of Norwegian, European and North American women indicated that these routine examinations had little or no impact on BC mortality , 2. The neu) is amplified. The subtypes are: I) luminal A , II) luminal B , III) HER2-overexpressing , IV) triple negative breast cancer  and, V) normal-like subtype +, furthermore, neutral loss of headgroup choline or ethanolamine were used to confirm the identity of PC, SM, and PE, respectively. Glycerolipids (GL) were identified mainly by [H+NH4]+, [M+H-H2O]+, and [M+Na]+. A combination of an in-house database containing lipid identifier (ID), estimated retention time and neutral mass, online databases e.g. LipidMaps ipidMaps , 39 and ipidMaps . A possiLipids from the classes GPL, GL, SL were investigated in this study. The lipids are annotated according to their lipid subclass; PC, LPC, PE, LPE, PG, PI, SM, DG and TG. Individual lipids are characterized by length and composition of the fatty acyls and are annotated as “lipid subclass total carbon number in fatty acyl chains: total number of double bonds” e.g. PC 30:0. Ether-PC and -PE are subdivided into plasmenyl  or plasmanyl and will be denoted with the prefix P- or O- respectively  e.g. PC RStudio  was applied for data analysis, generation of heatmap with hierarchical dendrogram (Euclidean distance and Ward's linkage method) and statistics. GraphPad Prism 5.03  was used for graphical presentation and statistical analysis. Student t-test and two-way ANOVA followed by Bonferroni correction were applied to test statistical difference between mean abundance in BC cell lines and reference cell line for individual lipids.Fig 1).In order to evaluate the lipidome of BC cell lines, a fit-for-purpose analytical method is needed for detection and identification of lipids. Here we report a UPLC-QTOF-MS method for analysis of extracted lipids from cultured cell lines. The estimated retention times of individual lipid subclass were 1.5–3.0 min for LPC and LPE, 4.5–8.5 min for PC, PE and SM, 7.0–8.5 min for DG, and 8.5–10 min for TG . Principal component (PC) 1 on the x-axis explained 29.9% of the variance while PC 2 (y-axis) explained 20.5%. When the QC samples were included, they cluster in the center of the PCA score scatter plot indicating high analytical precision and accuracy . The biological replicates from each individual cell line clustered together indicating low variance within the cell lines. Notably, BC cell lines MCF7, SK-BR-3 and MDA-MB-231 cluster furthest from each other and from the reference cell line, showing the largest difference between components in these cells. The cell lines CAMA-1 and T-47D cluster separate from these BC cell lines and closer to the reference cell line and the MDA-MB-436 cell line. The least difference based on the componential analysis seems to be between MDA-MB-436 and MCF10A.PCA modelling of 439 ion features detected in Progenesis QI shows that each cell line clustered separate from other cell lines including the reference cell line MCF10A  highlights the between-cell-lines variance. The explained variance in the predictive component was 74.5% and 8.99% in the orthogonal component, indicating that the variance between the cell lines was larger than between biological and technical replicates of MCF10A or MCF7 cell lines. The selection from the S-plot returned 76 up-regulated (red) and 98 down-regulated (blue) ion features in MCF7 cells compare with MCF10A . The ion features selected across the BC cell lines based on OPLS-DA models included 242 ion features in total and there was substantial overlap between the cell lines.An OPLS-DA model for MCF7 cell line compared to the reference cell line (MCF10A) was utilized to uncover the most reliable class discriminating variables  Figs. ScFig 4). The dendrogram showed closest similarity between the biological replicates within each cell line. The cell lines MDA-MB-231 and MDA-MB-436 representing TNBC subtype cluster together and so do cell lines T-47D and MCF7 representing luminal BC subtype. However, high similarity was found between the reference cell line (MCF 10A) and the cell line CAMA-1. These cell lines share similarities with MDA-MB-231 and MDA-MB-436 cell lines. SK-BR-3 cell line was distinguished clearly from the remaining cell lines in the dendrogram.A possible lipid ID was assigned to 67 of the 242 ion features belonging to the lipid classes PC, PE, SM, DG and TG. The normalized abundance (log transformed) of these 67 lipid features was included in a heatmap with hierarchical dendrogram (S2 Table). These are mainly C-32 to C-36 lipids, except for two C-42 SM. A group of ether-PE and -PC (C-34 to C-38) had a very low abundance  in MCF7 and T-47D compared to the other cell lines. Some of the TG grouped together and showed a high abundance in either MCF7 or SK-BR-3. Otherwise, the heatmap shows that differences between BC cell lines were related to individual lipids belonging to subclasses PC, PE, SM and DG, which will be elaborated below.In the middle of the heatmap, a group of highly abundant lipids of the subclasses SM and PC across all cell lines is seen . In SK-BR-3 cells, TG ≤ C-46 with saturated or monounsaturated FA (MUFA) in side chains  were found to be up-regulated compared with all other cell lines . MCF7 cells showed a significantly higher abundance of TG 46:1 and 46:2, and C-48 to C-56 TG . The abundance was remarkably high in TG with di- or tri-unsaturated FAs  when compared with the other cell lines . TG ≥C-50 were generally down-regulated in SK-BR-3 and in one or both of the MDA-MB-436 or MDA-MB-231 TNBC cell lines .In two BC cell lines, SK-BR-3 and MCF7, TG were found to be especially abundant  showed that the trends for the lipid subclasses PC, PE, SM and DG, were not as clear as for the TG. However, even though many of the identified lipids were present in multiple BC cell lines, a differences were found in up- or down-regulation of specific lipids between the cell lines . PC with a low number of total carbons and saturated FA or MUFA in side chains PC 28:0, PC 28:1, or PC 30:1 were up-regulated in MCF7, CAMA-1 and/or SK-BR-3, but not in MDA-MB-231 or MDA-MB-436 . A shared tendency between MCF7 and T-47D was a significant down-regulation in ether-PE . In SK-BR-3 PE P-32:1 and PE P-34:2/O-34:3 were also up-regulated, however, PE P-36:4 or O-36:5 and PE P-38:4 or O-38:5 were down-regulated . The luminal cell lines and SK-BR-3 cells shared features in significant up-regulation of specific PE . In CAMA-1 and T-47D PE 38:0 was significantly up-regulated. SM 44:1 and SM 44:2 were highly up-regulated in CAMA-1 cells, which was not the case for SM of shorter chain lengths, thus SM 32:1 was significantly down-regulated . SM 44:2 was also up-regulated in MCF7 (The heatmap ( in MCF7 . MCF7 shth quadrant and MCF7 and T-47D in 3rd quadrant). CAMA-1 was, however, more related to the reference cell line than the other luminal cell lines based on the dendrogram and in the PCA plot it was in quadrant 1 close to the reference cell line, suggesting similarities in the lipidome of these cells. Conflicting results exist in the literature regarding the PgR status of CAMA-1 which is reported to be positive or negative in different studies. Furthermore, the TP53 status is different for the three luminal cell lines with T-47D expressing mutated protein [TP53 is known to regulate lipid metabolism in cancer [The PCA scatter plot clearly defined clustering of each BC cell line indicating that the ion features detected are sufficient to distinguish between the cell lines analyzed. Clustering of the cell lines in a hierarchical dendrogram based on lipids from the subclasses PC, PE, SM, DG and TG, showed the relative similarities between the cell lines. The closest similarities were observed between cell lines MDA-MB-231 and MDA-MB-436 which are both TNBC tumor subtype. Furthermore, the luminal cell lines MCF7 and T-47D were similarly clustered in the dendrogram. These similarities found in the dendrogram are reflected in the PCA by clustering in the same quadrant (MDA-MB-231 and MDA-MB-436 in 4 protein  and TP53n cancer . This inn cancer . The cluTG stored in the cell as lipid droplets represent a reservoir of FAs ready to undergo β-oxidation. We detected significant changes in TG particularly in SK-BR-3 and MCF7 cells. The perilipins (PLIN 1–5) are generally involved in regulation of TG storage. PLIN1 suppresses the hydrolysis of TG from lipid droplets and is generally down-regulated in BC with the lowest expression in TNBC , 17. In S2 Table), indicating that the side chains were C-16:0, C-16:1, C-18:0, or C-18:0 FA which are predominantly synthesized via FASN [Fig 6). Therefore, these abundant lipids will not be considered further for distinguishing BC subtypes in this paper.In our study, the most abundant PC and PE had a composition of C-32 to C-36 saturated or moderately unsaturated  have also been reported in BC patients , 50. DG droplets , 55. Lipdroplets , 55. LPIdroplets . In combdroplets , 56, 57.droplets . PC 40:6droplets . ELOVL 1droplets . This suEther-GPL are abundant in biological membranes, they play a role as second messengers, in differentiation and storage of long-chain PUFAs, they are abundant in lipid raft microdomains and they are suggested to protect cells from reactive oxygen species . The bioin-vitro conditions. The cell lines represent different defined subtypes of breast cancer, but only one cell line was derived from a HER2-positive cancer. The comprehensive comparison of Jiang et al. of 68 breast cancer cell lines and primary breast cancer tissue revealed strong correlations particularly for mRNA expression but weaker for genomic profiles and protein expression. In a combined correlation score the T47-D, CAMA-1 and SK-BR-3 cell lines scored high, but MCF-7 and MDA-MB-231 and MDA-MB-436 had a lower score [This study is based only on cell lines. This has the advantage of using well-defined material that has also been used in other lipidomic studies, thus offering the opportunity for comparison. A major drawback is that each cell line can only reflect the individual tissue of origin and caution is needed in generalizing findings. Furthermore, these cell lines have been in culture for a long time and have adapted to er score . The coner score  and 2D cer score . The addIn this work, we describe the most significant differences in the lipidome between individual BC cell lines and a reference cell line. Firstly, we established the overall similarities in the lipidome of the cell lines based on PCA and hierarchical dendrogram. The most similar cell lines belonged to the same subtypes; the TNBC cell lines (MDA-MB-231 and MDA-MB-436) and the two luminal cell lines (T-47D and MCF7). The luminal cell line CAMA-1 did not share strong similarity with the other luminal cell lines revealing within-subtype variations in the lipidome. The cell line that differed most based on the lipidome from the other cell lines was the HER2-overexpressing, SK-BR-3 cell line. Some general trends in the lipidome may be useful for distinguishing between BC tumor subtypes in clinical samples, particularly if applied to plasma samples. Most interestingly, we suggest that ER- and PgR-positive tumor subtypes can be identified by a significantly increased abundance of TG ≥ C-48 with moderate or multiple unsaturated FA chains, which in contrast are significantly down-regulated in ER- and PgR-negative subtypes (HER2 and TNBC). Furthermore, ether-PE, especially those containing the most abundant fatty acyls C-16, C-18 and C-22:6 or C-20:4 may be down-regulated in ER- and PgR-positive subtypes and to a lesser degree in ER- and PgR-negative subtypes. Therefore, these PE could be of particular interest for distinguishing between ER- and PgR-positive and -negative subtypes. Furthermore, we suggest that the HER2-overexpressing tumor subtype is characterized by elevated levels of TG, PC and PE containing saturated FA or MUFA ≤ C-16 in the side-chains. Significantly increased abundance of PC ≥ C-40 may be useful for identification of the TNBC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in the lipidome which may be used to define further subdivision within the BC subtypes.in vitro and in vivo. The potential of lipidomics as a diagnostic tool needs to be investigated further, and the recently published study of Santoro et al. implies that this is a promising approach [in vitro cultured BC cell lines and actual BC tumor subtypes needs to be confirmed in clinical samples.The cells analyzed in this study are grown under controlled conditions. They represent good and reasonably stable models of clinical phenotypes and reflect to some extent molecular characteristics of primary tumors , 63. Theapproach . The resS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigAbundance of 439 ion features normalized to all compounds . Pareto scaling and normalization applied to data prior to modelling. Score scatter plot of principal component (PC) 1 and PC 2 after PCA modelling visualizes the differences and similarities in the ion feature profile between the individual cell lines. The white sphere in the model plot represents the Hotelling T2 with 95% confidence. Three biological replicates were analyzed three times with each dot representing one analytical sample and QC samples consisted of 11 injections throughout the analytical batch.(TIF)Click here for additional data file.S2 Figa) OPLS-DA score scatter plot of T-47D cell line compared to reference cell line the reference cell line MCF10A. b) Corresponding S-plot comparing T-47D to the reference cell line MCF10A. c) OPLS-DA score scatter plot of CAMA-1 cell line compared to reference cell line MCF10A. The black spheres represent MCF10A and green represents CAMA-1. d) Corresponding S-plot comparing CAMA-1 to the reference cell line MCF10A. e) OPLS-DA score scatter plot of SK-BR-3 cell line compared to reference cell line MCF10A. The black spheres represent MCF10A and green represents SK-BR-3. f) Corresponding S-plot comparing SK-BR-3 to the reference cell line MCF10A. Scores scatter plots highlight the between class variance in the predictive component on the x-axis (R2Xo [(TIF)Click here for additional data file.S3 Figa) OPLS-DA score scatter plot of MDA-MB-231 cell line compared to reference cell line MCF10A. The black spheres represent MCF10A and green represents MDA-MB-231. b) Corresponding S-plot comparing MDA-MB-231 to the reference cell line MCF10A. c) OPLS-DA score scatter plot of MDA-MB-436 cell line compared to reference cell line MCF10A. The black spheres represent MCF10A and green represents MDA-MB-436. d) Corresponding S-plot comparing MDA-MB-436 to the reference cell line MCF10A. Scores scatter plots highlight the between class variance in the predictive component on the x-axis (R2Xo [(TIF)Click here for additional data file.S4 FigBars represent mean abundance of three biological replicates, error bars represent SD. Normalised abundance is shown on the y-axis. Statistically significant up- or down-regulation in normalised abundance compared to MCF10A is indicated by * (p< 0.05), ** (p<0.01), *** (p<0.001), insignificant changes are unmarked.(TIF)Click here for additional data file.


NRASQ61K mutation and characterized the sensitivity of this model to MEK and TORC1/2 inhibitors. Despite the clinical diagnosis of SCLC, the majority (8/11) of cases were either of nonpulmonary origin or of mixed histology and included atypical carcinoid (n = 1), mixed non‐small‐cell lung carcinoma and SCLC (n = 4), unspecified poorly differentiated carcinoma (n = 1), or small‐cell carcinoma from different origins (n = 2). RB1 and TP53 mutations were found in four and five cases, respectively. Predicted driver mutations were detected in EGFR (n = 2), NRAS (n = 1), KRAS (n = 1), BRCA1 (n = 1), and ATM (n = 1), and one case harbored a TMPRSS2‐ERG fusion. DFCI168 (NRASQ61K) exhibited marked sensitivity to MEK inhibitors in vitro and in vivo. The combination of MEK and mTORC1/2 inhibitors synergized to prevent compensatory mTOR activation, resulting in prolonged growth inhibition in this model and in three other NRAS mutant lung cancer cell lines. SCLC in never/former light smokers is rare and is potentially a distinct disease entity comprised of oncogenic driver mutation‐harboring carcinomas morphologically and/or clinically mimicking SCLC. Comprehensive pathologic review integrated with genomic profiling is critical in refining the diagnosis and in identifying potential therapeutic options.Small‐cell lung cancer (SCLC) occurs infrequently in never/former light smokers. We sought to study this rare clinical subset through next‐generation sequencing (NGS) and by characterizing a representative patient‐derived model. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. We established a patient‐derived model from one such patient (DFCI168) harboring an  EGFR, NRAS, KRAS, BRCA1, and ATM, and one case harbored a TMPRSS2‐ERG fusion.Small‐cell lung cancer occurs infrequently in never/former light smokers. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. The majority of cases were either of nonpulmonary origin or of mixed histology. Predicted driver mutations were detected in  EGFRepidermal growth factor receptorNEneuroendocrineNSCLCnon‐small‐cell lung cancerPDXpatient‐derived xenograftsSCLCsmall‐cell lung cancer1et al., et al., et al., et al., et al., et al., Small‐cell lung cancer (SCLC) is one of the most challenging cancers to treat, with a 5‐year survival rate of 4–5%  or light former smokers  were analyzed for NRAS mutations alone by Sanger sequencing (n = 8) or comprehensively by targeted next‐generation sequencing . SW1271 was grown in Dulbecco’s modified Eagle medium  with 10% FBS. Trametinib  and stored at −80 °C. Cell lines were authenticated by single tandem repeat analysis at Michigan State University in October 2017 and tested negative for mycoplasma as determined by the Mycoplasma Plus PCR Primer Set .SW1271, H1299, H2087, H2347, H69, H82, H209, and Glc16 were purchased from American Type Culture Collection (ATCC). All cell lines except SW1271 were maintained in Roswell Park Memorial Institute (RPMI)‐1640  supplemented with 10% FBS, 100 U·mL2.3et al., A pleural effusion was obtained from a patient with SCLC. The sample was subjected to red blood cell lysis using RBC Lysis Buffer , and the cells were suspended in HITES medium . For NRAS siRNA knockdown experiments, cell viability was measured using CellTiter‐Glo luminescent assay (Promega).Inhibition of growth by targeted kinase inhibitors was evaluated by MTS assay according to the manufacturer’s instructions . Cells were plated in 96‐well plates at a density of 2000–8000 per well and treated on the following day. At 72 h after drug addition, cell viability was measured. Combination index (CI) was calculated using 2.56 cells from pleural effusion were implanted subcutaneously in 8‐week‐old female NSG mice in accordance with the guidelines approved by the DFCI Institutional Animal Care and Use Committee (IACUC) and tumor growth was monitored by caliper measurements. Once tumors grew to a size of 1 cm3, tumors were isolated and cut into pieces of ~ 2 × 2 × 2 mm and transplanted subcutaneously in additional NSG mice. Tumors were passaged for no more than five times. Samples from all passages were viably frozen in liquid nitrogen and used for further experiments. The tumor fidelity from various passages was confirmed by Hematoxylin and eosin (H&E) staining and the existence of the NRASQ61K mutation.To establish the DFCI168 PDX model, ~ 5 × 102.6in vivo studies were conducted at DFCI with the approval of the Institutional Animal Care and Use Committee in an AAALAC‐accredited vivarium. The DFCI168 tumor fragments were transplanted subcutaneously on the right flanks of 8‐week‐old female NSG mice. Tumors were allowed to establish to 219 ± 54 mm3 in size before randomization into vehicle‐treated, trametinib‐treated , torin2‐treated , and combo‐treated groups  of 8 mice per group. Trametinib was administered in 0.5% hydroxypropyl methyl cellulose with 0.2% Tween 80. Torin2 was dissolved in captisol (1 : 40) followed by dilution in water to 3 mg·mL−1. Tumor volumes were determined from caliper measurements by using the formula V = (length × width2)/2. Tumor sizes and body weights were measured twice weekly. For pharmacodynamic (PD) study, mice were treated for 2 days and tumor samples were collected at 4 h (n = 3) after the last dose. Tumor samples were analyzed by western blot.All 2.7facsdiva software .NCAM/epithelial cell adhesion molecule (EpCAM) expression in the DFCI168 cells was evaluated with anti‐human NCAM APC antibody/anti‐human EpCAM‐PE antibody or with fluorophore‐tagged isotype control . Early and late apoptotic cell death was assessed by Annexin V‐FITC/PI  or Annexin V‐PE/7AAD staining (BD Biosciences) according to the manufacturer’s protocol. The samples were analyzed with a BD LSR Fortessa flow cytometer with BD 2.84 cells per well. Two days after plating, cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilization with 0.5% Triton X‐100 for 10 min. After blocking with 10% goat serum for 1 h, the cells were stained with Phalloidin Green 488 . For vimentin staining, the following antibodies and dilutions were used: rabbit anti‐vimentin  and goat anti‐rabbit Alexa Fluor 594 . Slides were mounted with antifade mounting media containing DAPI. Images were obtained using the Nikon Eclipse 80i microscope .The DFCI168 cells were plated in 8‐well chamber slides at a concentration of 3 × 102.9NRAS mutant cell lines using DNeasy Blood & Tissue Kit  and analyzed by massively parallel sequencing using a solution‐phase Agilent SureSelect hybrid capture kit and an Illumina HiSeq 2500 sequencer. Further details were described elsewhere  .pBabe 2.11Cells were grown and treated as described and lysed with RIPA buffer with Triton X‐100 (Boston BioProducts) supplemented with protease and phosphatase inhibitors. Immunoblotting was performed according to the antibody manufacturers’ recommendations. The following antibodies were obtained from Cell Signaling Technology : Anti‐Synaptophysin (SYP), phospho‐ERK1/2 (Thr202/Tyr204), ERK1/2, phospho‐RSK (Thr359/Ser363), RSK, phospho‐S6 (Ser235/236), phospho‐S6 (Ser240/244), S6, phospho‐4EBP (Thr37/46), 4EBP, phospho‐Akt (Ser473), Akt, Hsp90, phospho‐RB (Ser780), phospho‐RB (Ser807/811), RB, Vimentin, ZEB1, Snail, and Lamin B1. Anti‐ASCL1 antibody was purchased from BD Biosciences. Anti‐tubulin antibody is purchased from Sigma‐Aldrich. Anti‐NRAS antibody is purchased from Abcam.2.12m of control siRNA pool (D‐001206‐13‐05) or with NRAS specific siRNA pool . After 48 h, cell viability was measured by CellTiter‐Glo. NRAS silencing efficiency was verified by immunoblotting.Cells were transfected with 25 n2.13RNA was extracted using TRIzol (Invitrogen) and column‐purified using RNeasy Kit (Qiagen). cDNA was generated from 1 µg of RNA using QuantiTect Reverse Transcription Kit (Qiagen). qPCR was performed and analyzed on Step One Plus Real‐time PCR System using TaqMan probes  for E‐cadherin (Hs01023894), vimentin (Hs00958111), SNAI1 (Hs00195591), TWIST (Hs00361186), ZEB1 (Hs00232783), ASCL1 (Hs00269932), and SYP (Hs00300531). GUSB was used as a housekeeping gene.33.1n = 7) or light former (n = 4) smokers with clinically diagnosed SCLC treated at DFCI from 2013 to 2018  cases (n = 7), two cases were positive for EGFR mutations , and the others harbored NRASQ61K (n = 1), BRCA1L502Afs*2 (n = 1), and ATMT1953I (n = 1) and high MET amplification (n = 1). In RB1 mutant cases (n = 4), KRASG12V (n = 1) and TMPRSS2‐ERG fusion (n = 1) were detected. An additional eight patients with SCLC (never smokers (n = 4) or light former smokers (n = 4)) with only limited DNA were specifically queried for an NRAS mutation, but no NRAS mutations were detected (data not shown).We identified 11 cases of never (EGFR mutations (case 8 and case 10) showed admixed non‐small‐cell and small‐cell features, suggestive of de novo small‐cell transformation of EGFR‐mutated adenocarcinomas. Case 1 with an ATM mutation and case 2 with a MET amplification showed features intermediate between large‐cell NE and small‐cell carcinomas. Case 7 with a NRAS mutation was verified to be a keratin‐positive small round blue cell tumor but lacked any specific features to confirm a diagnosis of SCLC. A BRCA1 mutation‐harboring case 3 was reclassified as grade 2 NE tumor/atypical carcinoid of possible pancreas or lung primary in the available clinical and radiographic context. Among the five cases with classic small‐cell carcinoma histology, case 4 with KRASG12V was suspected to be of possibly of hepatobiliary origin. Case 11 harbored a TMPRSS2‐ERG fusion, an oncogenic fusion common in prostate cancers . The patient is a 49‐year‐old male who presented with a large right hilar mass , but no RB1 mutation was detected by OncoPanel in SW1271. The NRASQ61K mutation was confirmed in both the xenograft tumor and the DFCI168 cell line by Sanger sequencing . At autopsy, this tumor was best classified as an undifferentiated carcinoma with NE differentiation by IHC, presenting clinically and being treated as SCLC.Unlike most SCLC cell lines that grow in culture as floating aggregates, DFCI168 grows in a monolayer and displays a spindle‐shaped morphology Fig. , left. W168 Fig. , right. ing Fig. . Flow cying Fig. A. Consis271 Fig. B. DFCI16271 Fig.  and S2D.271 Fig. , right, 271 Fig. , left, w3.3NRAS knockdown . Both trametinib and selumetinib showed selective potency in NRAS mutant cell lines , but not in NRAS WT SCLC cell lines   Fig. .3.4NRASQ61 mutation on MEK signaling, we tested three NRAS mutant non‐small‐cell lung cancer (NSCLC) lines  for sensitivity to MEK inhibitors. H1299 showed resistance (IC50 > 10 µm) to trametinib and selumetinib in the cell viability assay  . We also confirmed that the combination of 100 nm of trametinib and torin2 abrogates the phosphorylation of S6 in DFCI168 and H2087  Figs  and S3, nes Fig. . Importa087 Fig. .et al., We further investigated the synergistic effects of the combination of MEK and mTORC1/2 inhibitors. MTS‐based cell viability assays were performed after a 72‐h treatment with trametinib and torin2, either alone or in combination at a constant ratio of 1 : 1 Fig. . No syneTo test whether the drugs induce apoptosis, the cell lines were treated with trametinib and torin2 for 48 h and stained with either FITC‐labeled Annexin V and PI‐ or PE‐labeled Annexin V and 7AAD Fig. . The com3.6in vitro, we further evaluated the effect of trametinib in vivo using a PDX model from DFCI 168. The DFCI168 tumor‐bearing mice were treated with trametinib at 3 mg·kg−1 daily for 3 weeks. The tumor initially regressed significantly but grew back within 4 weeks after treatment was stopped, likely from the residual cells not eliminated by drug treatment . While trametinib at 3 mg·kg−1 was well‐tolerated, torin2 at 30 mg·kg−1 treatment required a drug holiday in three out of eight mice. The combination treatment resulted in > 15% body weight loss in four out of seven mice, also requiring drug holidays. To confirm on‐target inhibition of the combination treatment in this model, we conducted a 2‐day PD study  , but the similarities between SW1271 and DFCI168 raise a question whether SW1271 is in fact a true SCLC cell line. To date, SW1271 has been commonly used as a SCLC cell line in several prior studies  from a clinically diagnosed SCLC never smoker with a 68 from aEGFR mutations and ALK rearrangements, are more commonly observed in never smokers than in those with a history of smoking , but detected no RAS mutations  and found no NRAS mutations reported in the 210 SCLC cases regardless of smoking status. Considering that NRAS mutations in NSCLC are more frequent in previous/current smokers  Flow cytometric analysis of NCAM/EpCAM expression on DFCI168. (B) (left) The lysates from nuclear fractions of lung cancer cell lines were examined for RB, pRB (Ser807/811) and lamin B (loading control). (right) IHC image of DFCI168 PDX stained with RB. Scale bar 50 µm. (C) The activation of PI3K/Akt/mTOR and MEK/ERK pathways in the NRAS mutant lung cancer cell lines was assessed by western blot. (D) Immunofluorescence images of DFCI168 stained with phalloidin (green) and VIM (red). Scale bars 100 µm.Click here for additional data file.Fig. S3. H1299 and SW1271 were treated with trametinib at the indicated concentration for the indicated times. The cell extracts were immunoblotted using the indicated antibodies.Click here for additional data file.Fig. S4. The comparison of IC50 values of various inhibitors for NRAS mutant lung cancer cell lines after 72 h of treatment. The results were obtained from three independent experiments, and the bar represents the mean ± SD.Click here for additional data file.Fig. S5. DFCI168 and H2087 were treated with 100 nm of trametinib/torin2 either alone or in combination for the indicated times. The cell extracts were immunoblotted using the indicated antibodies.Click here for additional data file.Table S1. Mutations and gene amplification found by OncoPanel sequencing in 11 clinically diagnosed never/light smokers with SCLC.Click here for additional data file.