This pilot study examined radiobiological responses to irradiation and subsequent regeneration in two HNC cell lines (HPV positive and negative). A novel approach was taken to develop generational cultures of HNC cell lines, UM-SCC-1 (HPV negative) and UM-SCC-47 (HPV positive). MTT assays were used to determine surviving metabolic activity as a function of dose following 6 MV X-ray irradiation. Parallel cultures surviving 4 Gy irradiation  were re-cultured and passaged to develop subsequent generations which were re-irradiated and analysed for generational change in radiation response. Second and 3rd generations of UM-SCC-1 showed decreasing metabolic activity with dose but little difference was evident in surviving fractions between these generations. Significantly lower metabolic activity in the 3rd generation at <6 Gy, compared to the 2nd generation, showed UM-SCC-47 becoming progressively more radiosensitive. HPV positive UM-SCC-47 showed generational progression in radiosensitisation not seen in the HPV negative UM-SCC-1.Head and neck cancers (HNCs) are aggressive epithelial tumours frequently treated using radiation. HNC biology shows distinctions dependent on the oncologic involvement of the human papilloma virus (HPV). Clinically, HPV positive HNCs respond better to radiotherapy but few  Aetiological factors linked to the incidence of HNSCC are carcinogens such as tobacco, alcohol and betel quid. In developed countries, an increasing incidence of oropharyngeal HNSCC results from infection with the human papilloma virus (HPV). The prevalence of this form of HNSCC has resulted in the status classification of HNSCC as HPV positive or negative4. The global incidence for HNSCC is around 680,000 new cases annually and the survival rate of around 50% has changed little over the last few decades7. Although primary recurrence of HNSCC is the most frequent cause of mortality, distant metastasis is also an important cause of cancer specific death9. HNSCCs are often invasive cancers, involving surrounding vital tissue which may preclude surgical resection of the entire tumour. A multidisciplinary treatment approach is frequently required in which radiotherapy plays a prominent role10. Prescribed radiation doses, with intent to cure, are delivered in fractionated schedules, typically over 6–7 weeks. Complicating the prospect of tumour control are the responsive changes seen among cancer cells to radiation that can alter the parametrics by which the therapy has been prescribed12. Radiation induced changes in radiosensitivity can result quickly between the first and last treatment fractions13.Head and neck cancers are typically aggressive epithelial tumours arising in the mucosae lining the mouth, nose, pharynx and larynx. More than 90% of these cancers are squamous cell carcinoma (HNSCC)14. Treatment recommendations for HNSCC however, generally do not distinguish between the HPV statuses and this may partially result from a current lack of explanation for the difference seen in clinical outcomes16. Understanding radiobiological responsiveness of cancer cells treated at therapeutic dose levels, and radiation induced changes with fractionation, is critical in the application of radiotherapy for tumour control and in the minimisation of patient morbidities. Investigating the observed differences in treatment outcomes, based on the HPV status of disease, is clinically relevant and may help to identify features of the HPV negative disease that make it more refractory.Clinical studies show HNSCC responds differently to radiotherapy, dependent on HPV status, such that radiotherapy exerts better tumour control in HPV positive patients than in the HPV negative patients. Consequently, a HPV positive status is taken to be a favourable prognostic indicator17. This identified ‘generational’ change in the metabolic survival of each cell line after exposure to a range of X-ray doses using standard MTT assay18. Generational changes in metabolic activity were also compared between cell lines of different HPV status. This approach results in the development of multiple concurrent cultures of each cell line and this study was undertaken as a pilot to test significance in change of response after subsequent irradiations and to inform future work.This study investigated specific radiobiological responses, as a function of dose, in 2 HNSCC cell lines re-cultured following repeated X-ray irradiation. A novel approach was taken whereby cell line cultures were re-assayed for metabolic activity in surviving fractions following previous radiation exposures and passage19. Original non-irradiated cultures were considered the 1st generation of each cell line. Cell cultures irradiated once with 4 Gy X-ray irradiation, then re-cultured and passaged, became the 2nd generation. Where 2nd generation cultures were re-irradiated with 4 Gy, the re-cultured cells became the 3rd generation  and UM-SCC-47 (HPV positive) were compared. Radiation dose to cell lines was measured as absorbed dose in gray (Gy), defined as the energy in joules deposited in mass by kilogramsMTT assays were used in this study to assess resulting cell viabilities by metabolic activity in surviving populations after irradiation. MTT assays were performed as both cell lines proved to have low viability when seeded at low density for clonogenic assays and MTT assays facilitate quicker and higher throughput of sampling. Determinations of cell metabolic activity, as a function of dose, were compared between generations within cell lines and between cell lines by generation.20. Cell cultures were grown in T75 flasks using RPMI 1640 cell growth medium (Sigma-Aldrich® Darmstadt DE) supplemented with 10% foetal calf serum (FCS), 10 mM HEPES, 12.5 μg/ml penicillin and 16 μg/ml gentamycin. Cultures were grown in monolayer while incubated in humidified atmosphere at 37 °C with 5% CO2 and passaged after reaching exponential growth prior to confluency. Both cell lines were tested for the presence of mycoplasma  and found to be negative.Two head and neck squamous cell carcinoma cell lines were purchased from Merck Millipore (Darmstadt Germany) for this study. 1) UM-SCC-1 is a HPV negative cell line isolated from a recurrent tumour in the floor of mouth of a 73-year-old male. 2) UM-SCC-47 is a HPV positive cell line derived from a primary squamous cell carcinoma of the lateral tongue. Both cell lines originated from the laboratory of Dr. Thomas Carey, University of Michigan21 and the radiation dose output checked on the day of irradiation with Daily QA 3™ device  before each radiation treatment. To achieve an electronic equilibrium at the cell monolayer, flasks were placed on top of a 13 mm slab of solid water  with the table top at the level of the isocentre  X-ray beam from a Varian 600 C/D linear accelerator . The linear accelerator was calibrated using IAEA TRS398 protocol22. Sham irradiations of T75 flask cultures were performed as a control.To investigate generational differences in metabolic activity as a function of dose, 3 generations of UM-SCC-47 were exposed to 1.0, 2.0, 4.0 and 6.0 Gy. As the UM-SCC-1 cells were more radioresistant, 3 generations of this cell line were exposed to 1.0, 2.0, 4.0, 6.0 and 8.0 Gy23. Crystal solvents such as isopropanol or DMSO (Dimethyl sulfoxide) are used to homogenise the purple colouration. Spectrometry reading is automated by the use of plate readers which measure resulting optical density from formazan development by the absorbance of monochromatic light, typically around 570 nm. Absorbance values for each plate are indicative of the surviving metabolic activity for that culture. MTT assays are commonly used to measure cell viability post treatment (e.g. post irradiation) as they are comparatively quick and allow for high throughput analysis23.MTT is a colourimetric non-clonogenic assay that measures cell viability in a culture by its metabolic activity. Briefly, cell cultures are stained with a yellow tetrazolium substrate -2,5-diphenyltetrazolium bromide) (MTT). Viable cells metabolise the substrate by reduction with NADH (Nicotinamide adenine dinucleotide) producing purple, water insoluble, formazan crystals. Dead cells do not reduce tetrazolium and thus the accumulation of purple colouration is proportional to metabolic activity and surviving cell viability® Darmstadt DE). As MTT assays show a non-linear relationship between cell numbers and absorbance at high plating numbers, populations of 1.5 × 104 cells per well (n = 10) were plated to be within linear function24. Staining and analysis was performed at 3 days post irradiation to allow sufficient time for apoptosis following irradiation. For each generation of both cell lines, 2 × 96 well plates were used. Five wells were seeded for each radiation dose in each plate, giving 10 wells of cells per radiation dose, plus control. Wells surrounding those seeded for culture were dosed with 150 ml PBS for humidification. The 96 well plates were incubated for 72 hours prior to staining with MTT, tetrazolium salts  (Sigma-Aldrich®) and before cell populations reached confluency. At 72 hours, the cell medium was removed from the wells of adherent cells. 100 µL of 0.2 mg/ml tetrazolium salts was added to each well and incubated for 2 hours at 37 °C in 5% CO2 before checking formazan crystal formation by microscopy. The supernatant was removed from the wells and formazan crystals were dissolved by the addition of 200 µL isopropanol to each well to achieve homogenous optical density after 30 minutes. An additional column of empty wells received 200 µL of isopropanol to be read as blanks by microplate reader.To assay metabolic activity post irradiation, cells were trypsinised and centrifuged at 350 g for 5 minutes  then resuspended and counted by haemocytometer before replating in 96 well plates . The optical density from blue-purple colouration in each well was measured as absorbance by the attenuation of 570 nm light. Absorbance was read in 10 wells for each dose (2 × 96 well plates) and for blanks where wells contained isopropanol only. Mean blank values for each plate were subtracted from absorbance values for each well.Absorbency data were analysed using Prism 7.01 . Resulting absorbance values from 10-fold analysis of each dose were plotted as the ratio of mean absorbance to mean of control with standard error of the mean. Significance of results for intergenerational difference within cell lines were calculated by two-way ANOVA using Prism 7.01 and multiple comparisons within groups was performed using Tukey’s test. Significance of results between cell lines was calculated by multiple t test. Results of p < 0.05 were considered significant .Supporting data is available from the corresponding author upon reasonable request.st generation than the control by way of radiation induced MTT effect, up to 4 Gy, which is explained further in the discussion. At higher doses, the 1st generation continued to show greater metabolic activity than the latter two generations. The 2nd and 3rd generations show decreasing metabolic activity at >2 Gy, as shown in Fig. nd and 3rd generations across the full range of radiation doses.MTT assay results for UM-SCC-1 showed greater metabolic activity in the 1st generation. Metabolic activity higher than the control was seen across the dose range up to 6 Gy in the 1st generation and up to 2 Gy in the 2nd generation. An increasingly significant reduction in metabolic activity was observed however, in the 2nd generation, against the 1st, at 4 and 6 Gy. The 3rd generation of UM-SCC-47 showed even further reduction in metabolic activity by dose against the first 2 generations up to 4 Gy  causing membrane disruption resulting from lipid peroxidation3. It was noted in this study that this effect was most pronounced in the 1st generations of both UM-SCC-1 and UM-SCC-47 and was seen only at lower doses in the 2nd generations. Optical densities from MTT assay, greater than the controls, may possibly demonstrate greater radiobiological effect on cell membrane in the previously unirradiated cells, at this dose range, than cellular lethality and loss of metabolic function26.Changes in generational responses in the HPV negative UM-SCC-1 are only evident between the 124. In this investigation MTT assays were used as both UM-SCC-1 and UM-SCC-47 showed poor plating efficiencies and proved unsuitable for clonogenic assay. It is important to note that the MTT assay does not specifically measure clonogen survival but rather the viability of all cells in a culture irrespective of differentiation status. MTT assays do however have the advantage of not requiring colony formation at low plating densities and, being a semi-automated process, enable high volume throughput and easier repeat experimentation.Although clonogenic assays remain the gold standard for determining radiosensitivity by surviving fractions from irradiated cells, it has the drawbacks of lengthy incubation times and difficulties arise with non-colony forming cell linesrd generation of UM-SCC-47, demonstrated depressed metabolic activity post irradiation, after passage and re-culture following 2 previous exposures of 4 Gy, compared to 1st and 2nd generations. The observed changes in cellular responsiveness to dose indicate increasing radiosensitivity with subsequent irradiation at 4 Gy. Curiously, the 3rd generation of UM-SCC-47 also showed and elevated response at 6 Gy which matched that of the 2nd generation.Metabolic activity results showed significant alterations in surviving function, for dose, between generations within each cell line. The 3st and 2nd generations, possibly due to the radiation induced MTT effect in the 1st generation. Unlike UM-SCC-47, there was no further increase in radiosensitivity with the 3rd generation of UM-SCC-1. This resulted in significant differences observed in radiosensitivity between the 2 cell lines in the 3rd generation. Here the HPV positive cell line showed a continuing increase in radiation sensitivity in contrast to the HPV negative cell line. This is of interest given better clinical outcomes observed for HPV positive HNSCC and illustrates a distinction between the cell lines that may be related to their HPV status. An investigation by Kimple et al.27 of 4 HPV positive and 4 HPV negative cell lines (including UM-SCC-1 and 47) found all HPV positive cell lines more radiosensitive than the HPV negative lines. Radiosensitivity was assessed by the surviving fraction of cells after a 2 Gy exposure (SF2). The mean SF2 for the HPV positive status cell lines was 22% compared to the negative status SF2 of 59%. On a molecular basis, studies examining differences in radiosensitivity between HPV positive and negative HNSCC, investigated the development and decay of the γH2AX molecule where γH2AX form detectable foci at DNA double strand breaks (DSB) resulting from ionising radiation. The extent to which these foci are resolved is indicative of cellular DNA repair28. HPV positive cell lines have demonstrated significantly greater levels of γH2AX foci, persisting after 24 hours, than HPV negative cell lines, suggestive of poorer DNA repair and consequently greater radiosensitivity. Elevated γH2AX foci were also found to correlate with G2 arrest and share an inverse relationship with cell survival29. While differential mechanisms of radiosensitivity between HPV positive and negative aetiologies remain largely unclear, such investigations by cell line, however small, can direct future work.Significant change was only observed in UM-SCC-1 between the 115.The radiobiological differences in HNSCC cell lines, according to HPV status, demonstrated in our study and others, are consistent with the better clinical outcomes observed for HPV positive HNSCC. These results should encourage further work to identify the mechanisms underlying the observed differences in radiobiological responsiveness between HPV positive and negative HNSCC cell lines. This information will be important because clinical trials are currently underway to investigate reductions in the intensity of therapeutic regimens employing radiotherapy in HPV positive HNSCCIn this study, we have used a novel generational approach to demonstrate altered cellular responsiveness to irradiation in the two HNSCC cell lines investigated, after repeated X-ray exposures. Our findings of altered to radio-responsiveness by subsequent exposures and re-culturing of cell lines show generational increases in radiosensitivity. Although increasing radiosensitivity in UM-SCC-1 cells was not apparent after the 2nd generation, it was observed to continue for the HPV positive UM-SCC-47 cells. Unlike dose fractionation schedules applied clinically, our data do not indicate that repeated irradiation of HNSCC cell lines resulted in increased radioresistance. These results however must be viewed in light of their limitations where the surviving cultures, examined after 3 days, are irrespective of the clonogenic population which has greater bearing on post-radiotherapy tumour re-growth. To this end, clonogenic assays are required in future work to examine the generational behaviour of clonogens in response to subsequent irradiations. The conclusions of this study, in respect of differences between HPV positive and HPV negative cell lines, are limited by the small representation of cell lines. Future work requires extended representation by greater number of cell lines of each HPV status to determine radiobiological behaviour common to each status. Additionally, the development of further generations of each cell line needs to be undertaken to show more extensively radiobiological change in cell lines following fractionated exposures. Ultimately, the generational technique developed and observations of significant differences in radiobiological responses in this work need to be investigated and validated using tumour biopsies taken from tumours of different origins and stages.


The overall 5-year survival rate of lung cancer is about 15% even with therapeutic drugs like tyrosine kinase inhibitors. Ideal models are urgently needed for exploring mechanisms and finding new drugs. Patient-derived xenografts (PDX) models and primary cells are both used to screen therapeutic regimens for cancer. However, PDX models and primary cells from the same patient are difficult to establish. Their consistency to the original tumor tissue is not well studied.EGFR and K-RAS mutation detection and gefitinib sensitive assay were performed to evaluate the characteristic of established PDX models. Immunofluorescence analysis, anchorage-independent cell growth, Western blotting and gefitinib sensitive assay were performed to assay the characteristic of established primary cell lines. The whole-exome sequencing was used to compare the characteristic of the patient’s tumor tissue, established PDX and primary cell line.31 lung cancer patient tissues were procured to establish the lung cancer PDX models and primary cell lines. Tumor growth measurements, histological and immunohistochemistry analysis, Western blotting, EGFR mutation PDX model and primary cell line (LG1) were sensitive to gefitinib treatment. The expression of CK8/18, TTF1 and NapsinA in LG1 and LG50 primary cells were also positive. And the activated signal pathways were activated in LG1 and LG50 primary cell lines. Furthermore, the gene mutation in PDX tumor tissues and primary cell line (LG50) was consistent with the mutation in LG50 patient’s tumor tissues.Twenty-one lung cancer PDX models  and ten primary cell lines  were established from patients’ tumor tissues. The histology and pathological immunohistochemistry of PDX xenografts are consistent with the patients’ tumor samples. Various signal pathways were activated in different PDX models (n = 5) and primary cell lines (n = 2). These data suggested that established lung cancer PDX models and primary cell lines reserved mostly molecular characteristics of primary lung cancer and could provide a new tool to further understand the mechanisms and explore new therapeutic strategies. Lung cancer is the leading cause of cancer-related deaths worldwide –4. Lung Here, we established and characterized lung cancer patient-derived xenografts and established primary cell lines from patients’ tissues. We evaluated the pathological characteristic of the PDX models and primary cell lines. The differences between PDX models and the original tumors were also investigated. We found both the PDX models and primary cell lines mostly reserved molecular characteristics and heterogeneity of original cancer tissues, even drug sensitivity. Therefore, these PDX models and primary cell lines provide a platform for further understanding the molecular mechanisms and therapeutic screening, regimen for lung patient from cell to the animal level.31 lung cancer patients had undergone the surgical procedures at the First Affiliated Hospital of Zhengzhou University  and lung cancer tissues were obtained from August 2014 to October 2015. All patients did not receive both chemotherapy and radiotherapy and followed written informed consent before surgery. All research protocols were approved by the research ethics committee of Zhengzhou University. Tissue histology was analyzed and confirmed by two pathologists. Lung cancer specimens were harvested from the periphery of whole tumor tissues to maintain high activity and low necrotic part. All tissues were transported to the lab in transport media (FBS-free PRMI DMEM with penicillin and streptomycin).6–8 weeks old female CB17/severe combined Immunodeficiency (SCID) mice of 18 ~ 20 g average body weight  were used in the studies. 4–5 mice were kept in a pathogen-free environment with light controlled rooms (12 h cycles) and provided with food and water adlibitum.3. PDX models were used for study when the tumors were passaged for three generations. All animal studies were performed according to guidelines approved by the Zhengzhou University Institutional Animal Care and Use Committee.Tissue samples were placed in Petri dishes containing PBS with penicillin and streptomycin. The tissue was divided into four parts . The detailed method of implanting into SCID mice was described in our previous works . In brie2/2, where V was tumor volume, LD was longest tumor diameter, and SD was the shortest tumor diameter. Tumor growth curves were plotted as tumor volume.The tumors were measured with a vernier caliper twice a week. Tumor volume was calculated using the formula V = LD × (SD)One part of tumor tissue was embedded into paraffin for histopathologic examination and immunohistochemistry analysis. All the slides were stained with Harris Hematoxylin after dewaxing 5 μm thick sections with dimethylbenzene and were evaluated by two pathologists. Tissue sections were incubated with CK5/6, P63, P40, or CK8/18, TTF1, NapsinA antibodies  overnight at 4 °C. HRP-IgG secondary antibody was used at 37 °C for 15 min and detected by the diaminobenzidine (DAB) reactions. All slides were observed and measured by Olympus microscope (Japan).The PDX tissues were ground in liquid nitrogen and then were lysed. Next, protein was extracted by centrifugation for 30 min at 12,000 rpm. Protein (50 μg) was separated in 10% SDS-PAGE gel, and transferred to PVDF membrane at 90 V for 2 h. The membrane was blocked with 5% no-fat milk for 60 min. Later, membrane was incubated with mTOR, p-mTOR (Ser2481), STAT1, p-STAT1 (Tyr701), STAT3, p-STAT3 (Tyr705), AKT1, p-AKT (Ser473), ERK, p-ERK (Thr202/Tyr204) antibodies  overnight at 4 °C. All antibodies were used at 1:1000. The STAT3 antibody was from mouse, other antibodies were from rabbit. The fluorescent secondary antibody was incubated at room temperature for 1.5 h. The PVDF membranes were scanned by Odyssey and analyzed by Image Studio Ver 2.1.EGFR)  and K-RAS (exons 2 and 3) were screened by the mutant-enriched liquid chip polymerase chain reaction method.Lung cancer patients’ tissues and xenograft tissues from PDX models were pathologically reviewed to ensure that tumor cells were more than 80% and that no significant tumor necrosis had occurred. Genomic DNA was extracted from each sample using Puregene Cell and Tissue Kit . The quantity and quality of DNA samples were measured by Nanodrop ND-1000 UV/VIS spectrophotometer . DNA fragment integrity was confirmed by electrophoresis using 1% agarose gel. The concentration of DNA samples was normalized to 20 ng/μl and stored at − 20 °C. ‘Hotspot’ mutations in epidermal growth factor receptor  and gefitinib (100 mg/kg). Body weight and tumor size measurements were performed twice a week.After establishing PDX models, we chose LG1 and LG50 PDX xenografts for gefitinib treatment. Mice were divided into two groups (10 mice per group) which were vehicle control and gefitinib treatment group. Once the tumor volumes reached approximately 25 mm3 per well) for proliferation were seeded into 96-well plates. After overnight incubation, cells were treated with different concentrations of geftinib  and incubated for 24, 48, 72, or 96 h. CCK8 Solution  was then added and cells were incubated for another 1.5–2 h. Absorbance was measured at 450 nm.LG1 and LG50 primary cells  at the concentration of 200 U/ml and digested for 2–4 h at 37 °C. The enzymatic digestion was stopped when most of the tissues became cell suspensions. Following washing in RPMI-DMEM and centrifuging at 300g for 5 min, cells were transferred into standard tissue culture coated flasks  and cultured in the Defined Keratinocyte-Serum Free Medium (DK-SFM) supplemented with l-glutamine , EGF 20 ng/ml, basic-FGF 10 ng/ml , 2% B27 , penicillin and streptomycin, and amphotericin B . All primary cells were cultured at 37 °C in a humidified incubator with 5% CO2. Culture medium was changed every 2–3 days. Cells were passaged after 80–90% confluence.The fresh lung cancer tissues were minced into small pieces less than 1 mm5 primary cells were seeded in 24-well plate which was placed a sterilized glass slide in every well, incubated for 24 h, and fixed with 4% paraformaldehyde for 30 min. CK8/18, TTF1, NapsinA antibodies  was incubated at 4 °C overnight, and then FITC-conjugated rabbit IgG antibody was used. Cells with green fluorescent signals in the nucleus and cytoplasm were counted as positive expression cells. The In Cell 6000 Analyzer (GE) was used for detecting the fluorescence.2.5 × 102 for 2–3 weeks, and cell colony numbers were counted by In Cell 6000 Analyzer (GE).A total of 8000 primary cells were suspended in 0.33% basal medium eagle agar supplemented with 10% FBS. The cells were maintained at 37 °C, 5% COhttp://snpeff.sourceforge.net/SnpEff_manual.html) was applied to perform a series of annotations for variants. The final variants and annotation results were used in the advanced downstream analysis.We used the Illumina HiSeq to perform whole-exome sequencing of LG50 patient tissue, LG50 PDX xenografts, and LG50 primary cell line. All genomic variations including SNPs and In-Dels were detected by the software, such as HaplotypeCaller of GATK (v3.3.0). After that, the hard-filtering method was applied to get high-confident variant calls. Then the SnpEff tool . All the patients did not have any apparent distant metastases before surgery and had not been previously treated. In these samples, there are 10 adenocarcinoma samples, 1 adenosquamous carcinoma, 1 mucoepidermoid carcinoma, 1 neuroendocrine and SCLC, 1 non-small cell lung carcinomas (NSCLC), 1 SCLC, 16 squamous carcinomas. There are 2 well-differentiated samples, 4 moderately-well, 12 moderate, 7 moderately-poor and 6 poor differentiated samples. According to lymph node metastasis, 8 of 11 samples with lymph node metastasis had been established, and 13 of 20 samples without lymph node metastasis had been established. All patients’ clinical data were shown in Table 31 tumor specimens were transplanted to SCID mice and 21 xenografts were established  was lowest in LG22. p-p70S6K (Ser424) in LG17  and INsertion/DELetion (InDel) were analyzed among LG50 patient tissue, LG50 primary cells, and PDX tumor tissue. The summary statistics of SNPs was shown in Table 3 on day 168 and 154 for LG50 and LG33, respectively.Identification and characterization of tumor molecular changes and genetics are pivotal for screening targeted drugs and precise treatments , 16, 17.Different pathological type of cancer have different indexes in clinical. P40, P63 and CK5/6 were specific clinical diagnosis indexes for squamous carcinoma. CK8/18, TTF1 and NapsinA were specific clinical diagnosis indexes for adenocarcinoma. Our results found lung squamous carcinoma indexes and lung adenocarcinoma indexes still maintained in the third passage of the PDX models. These data indicated that our PDX models reflected the characteristics of patients’ samples. Different signaling pathways such as AKT/mTOR pathway, STAT1, and STAT3 pathway, ERK pathway played a critical role in the progression of lung cancer –22. TargEGFR plays an essential role in regulating cell proliferation and apoptosis of lung adenocarcinoma. EGFR mutation and K-Ras mutation are regarded as mutation initiator in lung cancer patients [EGFR in 17% of NSCLC patients is more frequent than in 5% of SCLC [EGFR mutation and 1 K-Ras mutation. We assessed the EGFR inhibitor sensitivity of these models. PDX model with EGFR L858R mutation was sensitive to gefitinib. However, PDX model with EGFR wild-type was resistant to gefitinib (Fig. Genetic and epigenetic abnormalities of primary cancer influence the processes of invasion, metastasis and drug resistance , 25, 26.patients , 28. Mut of SCLC . In the The primary cell lines have advantages over conventional cell lines which may lose the diversity of tumor cells. Therefore, we established the primary cell lines from the patients’ lung cancer tissues. We checked the molecular characteristics of these primary cell lines. CK8/18, TTF1, and NapsinA were mostly located in the cytoplasm and cell nucleus of the LG1 and LG50 primary cells Fig. . The LG1We established lung cancer PDX models and paired primary cell lines. The established PDX models and primary cell lines have uttermostly reserved molecular characteristics, heterogeneity, and drug sensitivity of patients’ tumor. These PDX models and primary cell lines provide a useful tool for understanding the molecular mechanisms and screening new compounds for lung cancer therapy.


Leukemia/lymphoma cell lines have been critical in the investigation of the pathogenesis and therapy of hematological malignancies. While human LL cell lines have generally been found to recapitulate the primary tumors from which they were derived, appropriate characterization including cytogenetic and transcriptional assessment is crucial for assessing their clinical predictive value.In the following study, five canine LL cell lines, CLBL-1, Ema, TL-1 (Nody-1), UL-1, and 3132, were characterized using extensive immunophenotyping, karyotypic analysis, oligonucleotide array comparative genomic hybridization (oaCGH), and gene expression profiling. Genome-wide DNA copy number data from the cell lines were also directly compared with 299 primary canine round cell tumors to determine whether the cell lines represent primary tumors, and, if so, what subtype each most closely resembled.Based on integrated analyses, CLBL-1 was classified as B-cell lymphoma, Ema and TL-1 as T-cell lymphoma, and UL-1 as T-cell acute lymphoblastic leukemia. 3132, originally classified as a B-cell lymphoma, was reclassified as a histiocytic sarcoma based on characteristic cytogenomic properties. In combination, these data begin to elucidate the clinical predictive value of these cell lines which will enhance the appropriate selection of in vitro models for future studies of canine hematological malignancies.The online version of this article (doi:10.1186/s12917-016-0836-z) contains supplementary material, which is available to authorized users. Hematological diseases in humans are widely heterogeneous including numerous molecular subtypes with wide ranging prognoses and therapeutic responses. Oncogenesis is well understood for some subtypes, while the molecular changes leading to other subtypes remain unknown . A similAlthough the use of cell lines provides numerous advantages, including ease of handling and manipulation, high homogeneity, provision of a continuous source of sample material, and accessibility to the scientific community , detaileCell line characterization minimally requires that a comprehensive set of immunophenotyping and cytogenetic data have been published . HoweverSpontaneously occurring lymphoid malignancies in dogs share the same histopathological and clinical features of their human couterparts, in addition to evolutionarily conserved chromosome aberrations and mutations, indicating shared pathogenesis across species –9. ThereWe present a comprehensive characterization of five canine LL cell lines starting with an extended panel of immunophenotyping. High resolution oligonucleotide array comparative genomic hybridization (oaCGH) was performed to assess genome-wide copy number status, and multicolor fluorescence in situ hybridization (FISH) analysis was used to further identify copy number imbalances and structural changes in karyotype architecture. Transcription status of each cell line was investigated using high-density array based gene expression profiling (GEP) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Additionally, genome wide copy number data of each cell line were compared with data from primary canine round cell tumors to further confirm their classification and relevance as in vitro preclinical models of lymphoid neoplasia for canine and comparative medicine.2 in RPMI-1640 culture medium  supplemented with 10 % fetal bovine serum , 2 mM Glutamax , and 100 μg/ml Primocin  and tested negative using a PCR Mycoplasma test kit .Five previously established canine LL cell lines with varying levels of initial characterization were included in this study: CLBL-1   was completed as previously described , 28 to a7cells were removed from the same culture flask at the same time point for isolation of DNA and RNA and preparation of metaphase chromosome preparations to ensure consistency in downstream analyses. DNA was isolated using the DNeasy Blood and Tissue kit  and manufacturer’s protocol, and quantity and quality of DNA were evaluated using spectrophotometry (260/280 > 1.8) and gel electrophoresis. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) and manufacturer’s protocol, and assessed using the 2100 Bioanalyzer RNA 6000 Nano Kit  to confirm an RNA integrity number (RIN) >9.0. Metaphase chromosome preparations were prepared from each cell line as previously described [Aliquots of 1 × 10escribed  using cooaCGH was completed as previously described using a 180,000 feature canine oligonucleotide array (Agilent Technologies) with repeat-masked 60mer oligonucleotides spaced ~13 kb across the genome . An equihttp://genome.ucsc.edu/) and the NCBI gene database (http://www.ncbi.nlm.nih.gov/gene). Genes previously associated with cancer were based on those reported in the Cancer Gene Census (http://cancer.sanger.ac.uk/cosmic/census) [Scan data were processed using Feature Extraction v.10.10 software (Agilent Technologies) and imported into Nexus Copy Number v7.5 . Raw data were evaluated to identify and exclude probes displaying non-uniform hybridization or signal saturation, and copy number calls were made using Biodiscovery’s FASST2 segmentation algorithm. Copy number calls were based on a minimum of three consecutive probes per segment, and mean log2 cell line:reference thresholds of +/− 0.2 were used to define gain and loss, respectively. Genes within the defined intervals were identified using the UCSC canine genome browser  .Further statistical analyses using the Feature Extraction data were performed using R . The sigProcessed ratio is the centered and normalized ratio of the Agilent processed fluorescent signals. Segmentation was performed across all chromosomes using circular binary segmentation [Where, entation . Data weentation , 106 lymwww.chori.org). Initially, two clones (326 K03 and 330E21), previously determined to hybridize to the centromeric regions of canine autosomes [FISH of all cells lines was performed as previously described  to evaluutosomes , were flBCL11B, IGH, VEGFA, CCNC, FOXO3A, CDKN2A, MYC, KIT, CDK6, EZH2, MYCBP2, FLT3, PTEN, HEY1, E2F5, NFKB2, ERG, MLLT2, CD83, and DEK  located in a region of CNA in at least one of the five cells lines, (2) displayed differential expression between cell lines based on the GEP, and (3) have been associated with human and/or canine lymphoid malignancies in prior studies , 45, 46. located Canis familiaris mRNA transcripts and over 20,000 non-redundant predicted genes. Additionally, total RNA was isolated as described from lymph nodes harvested from six healthy mixed breed dogs that showed no evidence of lymphoid neoplasia at necropsy [Total RNA from each cell line was used to perform gene expression profiling (GEP) as described elsewhere , 47 usinnecropsy . Microarnecropsy  and signnecropsy , 50.One ug of total RNA from each cell line and two normal lymph node controls was used to perform qRT-PCR to validate GEP as previously described . qRT-PCRMYC, KIT, FLT3, PTEN, and RPL32 (Table T) was performed as described previously using normal lymph node as the baseline for comparisons. RPL32 was used as the reference gene based on its stable expression across all samples in GEP analysis and previous identification as a suitable qRT-PCR reference gene for canine lymphoid neoplasia [Primers were designed using NCBI Primer-BLAST as previously described  for MYC,eoplasia .Table 2PIGH gene. Ema was positive for Thy-1, a T-cell marker. TL-1 cells were negative for most antigens, however weak expression of MHCII was observed, which may indicate a T-cell phenotype as MHCII is expected to have high intensity on B cells and antigen presenting cells, with much weaker expression observed on T cells [TCRG gene , further supporting a mature T-cell phenotype for both cell lines. UL-1 expressed most antigens including CD34, which indicates an immature precursor phenotype. Positive expression for CD3, CD4, CD5, CD8α, CD8β, CD11d, and Thy-1 all support a T-cell phenotype. UL-1 is also positive for CD14, which is normally a marker for myeloid cells; however, expression is also demonstrated in B and T cell precursors but not mature cells [TCRG indicated a monoclonal product further supporting T-cell phenotype. 3132 is most likely of dendritc/histiocytic origin based on the positive expression of CD1a and CD11c, which is characteristic of histiocytic diseases [TCRG.Flow cytometry data are presented in Table  T cells . TL-1 anre cells , furtherdiseases  and a codiseases , 11. AddChromosome enumeration and centromere localization via FISH of metaphase chromosomes was used to assess the gross karyotypic architecture of each cell line. The normal dog karyotype includes 38 pairs of acrocentric autosomes, a large sub-metacentric X chromosome and a small metacentric Y chromosome . EnumeraoaCGH analysis revealed varying levels of genomic imbalance in each of the cell lines with detected CNAs ranging in size from 19.5 Mb to entire chromosomes Fig. . Large dFISH analysis was completed for 20 genes relevant to lymphoid neoplasia to verify oaCGH data, enumerate the level of imbalance at each locus, and investigate any structural aberrations associated with the selected genes of interest. The 20 genes were divided into four panels of five for multicolor FISH analysis. Each of the four FISH panels is identified in the insets of Fig. Canis familiaris 13 (CFA 13) joined by the centromere resulting in the formation of a metacentric chromosome  in the majority of cells (>87 %). UL-1 had more genes gained, with three copies of BCL11B, IGH, VEGFA, CCNC, FOXO3A, FLT3, and NFKB2 in the vast majority (>93 %) of cells. Additionally, 50 % of cells gained an extra copy of CDK6. FISH analysis of 3132 showed copy number changes at 19/20 loci, many of which were strikingly heterogeneous. MYC, KIT, and CD83 each have proportions of cells with normal copy number and gains of 3, 4, and >4 copies. Losses of ERG and EZH2 were identified in all or nearly all 3132 cells, with proportions (10–23 %) of cells displaying homozygous deletion.A summary of the frequency of CNA at each of the 20 investigated loci is presented in Fig. GEP data were filtered to remove probe sets with limited variation (standard deviation <2) across the five cell lines and six non-neoplastic lymph nodes resulting in 1153 probe sets used for subsequent analyses. Unsupervised clustering resulted in immediate branching of 3132 from the other 10 samples. The remaining samples were then separated into two discrete groups including the four cell lines, and the six control lymph nodes Fig. . Normal Fold change analysis was also completed by comparing each cell line with the mean expression of the non-neoplastic lymph nodes. Fold changes of known cancer related genes and the transcripts with the 50 largest absolute fold changes (up or down) in each cell line are listed in Additional file MYC, KIT, FLT3, and PTEN . Fold changes in MYC and KIT are more variable across the cell lines and have minimal correlation with copy number changes observed. CLBL-1 and Ema had slight downregulation of MYC, while the other three showed modest upregulation. KIT was largely downregulated in CLBL-1, Ema, and 3132 (two of which had copy number gains), and upregulated 8-fold in TL-1 in which the loci was copy number neutral.qRT-PCR was performed to verify GEP findings and analyze the relationship between copy number change and expression change in four well known cancer associated genes including TEN Fig. . HomozygFinally, we compared the five cell lines with genome wide copy number data generated in our laboratory from 299 primary round cell tumors via clustering analysis to determine whether the cell lines represented primary tumors, and, if so, what subtype each most closely resembled  for CD11a and CD18 and negative for CD3. Similarly, TL-1 was originally positive for CD3, CD18, CD45, CD45RA, and MHC11 while our immunophenotyping indicated no expression of CD3 and CD18. The slight differences in the immunophenotyping data may be due to factors that can influence flow cytometry data including, the amount of antibody used, the call threshold of positive versus negative, instrumentation, and other reagents. Alternatively, it’s possible that the loss or gain of antigens on the cell surface occurred while in culture. Both Ema and TL-1 were previously T-cell PARR positive, which is consistent with our PARR data. Overall, the changes observed in immunophenotype did not impact the overall interpretation of the classification of both these cell lines as mature T-cell lymphoid neoplasia.CDKN2A in oaCGH analysis, which was further confirmed to be a homozygous loss of the gene loci in FISH analysis. CDKN2A loss occurs in >55 % of T-cell lymphomas (T-LSA) and <2 % of other subtypes of canine leukemia and lymphoma [Both Ema and TL-1 were found to have a copy number loss in the region containing lymphoma , 34. Emalymphoma . Ema andUL-1 immunophenotyping at the time of establishment was positive for CD8α, CD18, CD45, CD45RA. We found additional positive expression of CD3, CD4, CD5, CD14, CD21, CD34, Thy-1, and MHC-II. All were detected at an intermediate level therefore, as discussed earlier, differences in methods and data analysis could have resulted in previous negative findings. UL-1 was also T-cell PARR positive which agrees with our PARR findings. UL-1 was previously classified as a T cell lymphoma; however, based on expression of CD34, a surface glycoprotein expressed on hematopoietic stem cells, we suggest it is more representative of a T-cell acute leukemia (T-cell ALL). In veterinary medicine, CD34 expression can be used to suggest an acute leukemia since it is less commonly expressed in malignancies of more mature cells, such as lymphomas and chronic lymphocytic leukemias , 56. Sev3132 immunophenotyping in our laboratory, in conjunction with the original description of the cells  suggests this cell line is representative of a disseminated histiocytic sarcoma (HS). No immunophentypic or genomic analysis of this cell line has been previously reported, although it was later described as a B-cell lymphoma based on detectable surface immunoglobulins , and it KRAS, NRAS, PIK3R5, PLCG2, TGFBR2, TNFAIP3, TRADD, BCL2A1, CAMK2D, NFATC2 [BUB1B, PRKCD, CD83, CXCL13, CD36, IL8, IL2, CD40LG, LCK, LTBR, and TNFSF11 [KIT  [ZAP70 downregulation (decreased 10 fold) [Dysregulation of several genes previously associated with canine diffuse large B-cell lymphoma in the NF-kB signaling and B-cell receptor signaling pathways shared conserved expression in CLBL-1 including the following: , NFATC2 , BUB1B,  TNFSF11 . SimilarqRT-PCR)  and ZAP710 fold) .KIT expression is decreased in canine T-cell LSA [SYK expression is downregulated in canine T cell malignancies [MMP9 and TIMP1 expression is significantly upregulated in canine T-cell lymphomas [FLT3, and increased FLT3 expression has been previously identified in canine a subset of acute lymphocytic leukemias and the GL-1 cell line [VEGFA was also found in UL-1 and has been previously reported in canine ALL [GTSF1, LUM, and PYPH and downregulation of CLEC12A and CD9 in primary canine histiocytic sarcomas [There are less reported data regarding gene expression changes in other canine hematopoietic malignancies. cell LSA , and wasgnancies  and was ymphomas , and wasnine ALL . Finallysarcomas , 64 wereCross contamination of cell lines is a common issue in the scientific community with ~19 % of human LL cell lines being cross contaminated . This prIn summary, we present the comprehensive immunophenotypic and genomic characterization of 5 canine LL cell lines that confirms their cell of origin in 3 cell lines  and refutes their cell of origin in 2 cell lines (UL-1 and 3132). These data provide valuable information that can be used to select cell lines for preclinical in vitro studies based on the presence or absence of particular immunophenotypic and/or genomic characteristics of interest, which will enhance their clinical predictive value. The generation of integrated molecular profiling of cell lines and comparison with primary tumors will allow further exploration into their biology and clinical utility in veterinary medicine and contribute to comparative and translational studies of hematopoietic malignancies in dogs and humans.


Streptococcus pneumoniae and Listeria monocytogenes, cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology.Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens,  Streptococcus pneumoniae  also prefers to reside [Clostridium perfringens causes disease [Cholesterol-dependent cytolysins (CDCs) represent a broad group of protein toxins that originate from Gram-positive bacteria and contribute substantially to the pathogenicity of their bacterial carriers . Toxins o reside , and per disease . NeverthListeria monocytogenes and S. pneumoniae produce debilitating inflammations of brain meninges, with pneumococcus being considered the leading cause of meningitis in adults [PLY and LLO are two of the most extensively studied members of the group and share some pathogenic similarities, such as the affinity of their carriers to the brain. n adults ,9. The rn adults . Many non adults , astrocyn adults ,13, actin adults , and pron adults . AlthougL. monocytogenes is an important foodborne pathogen that has a significant impact on both public health and the economy worldwide [L. monocytogenes has the potential to cause serious and life-threatening diseases, preferentially in immunocompromised patients, such as septicemia, meningoencephalitis, meningitis, or abortion in pregnant women.Much less is known about the neurotoxic potential of LLO, and most information has been obtained from indirect analyses of pathologic brain materials of patients with listerial meningitis and encephalitis . L. monoorldwide . AlthougL. monocytogenes and cell-to-cell spread [L. monocytogenes meningitis and encephalitis. Thus, we have performed an extensive analysis of LLO neurotoxicity both in primary glial cultures and in acute brain slices, focusing on dendritic and astrocyte changes.LLO is studied largely in the context of its role in facilitating pathogen internalization, intracellular phagosome disruption, release of l spread . LLO is l spread . Its dirl spread . Thus, dFirst, we examined the cytotoxicity of recombinant LLO. We compared LLO with PLY, a well-studied neurotoxin from the same group. At physiological pH, our recombinant PLY demonstrated hemolytic activity of 20,000 hemolytic units (HU)/mg, whereas the activity of the recombinant LLO was 4000 HU/mg in human erythrocyte lytic test (not shown). In a lactate dehydrogenase (LDH) release assay in primary glial cells, LLO was sub-lytic  a2/5% CO2) . We prepared acute brain slices from C57Bl/6 mice, postnatal day (PD) 10–14, and incubated them for 5 h with 2 HU/mL and 4 HU/mL LLO. At these concentrations, only varicosity  formation increased significantly  . In the A major effect of PLY in brains is astrocyte cell shrinkage and shape changes, which can lead to glial remodeling in brain neocortex. LLO was capable of inducing astrocyte cell shrinkage and shape remodeling in non-permeabilized primary astrocytes at concentrations as low as 2 HU/mL a. Membraglia limitans at the neocortical surface, which is considered a barrier against toxic substances from the CSF. Surprisingly, we did not find any apparent disruption of the astrocyte layer or substantial cell shape changes in the slices, despite the evidence of cell shape remodeling in culture  to 2 HU/mL LLO for 5 h and analyzed the anti-GFAP  immunostaining of the astrocytes of  culture a–c. In t culture d.Our experiments demonstrated a distinct neurotoxic profile of LLO that is associated with dendritic varicosity formation in an NMDA-dependent manner and astrocyte cell shape changes in culture. In contrast to PLY, the other typical neurotoxin of the CDC group, LLO demonstrated a delayed cell permeabilization profile, no manifestations of dendritic spine collapse (indicative of diminished synaptotoxic effect), and morphologically significant astrocyte remodeling in brain tissue at equivalent lytic amounts. L. monocytogenes in juvenile meningitis and encephalitis [Members of the CDC group have attracted great attention, mostly due to their unique mechanism of membrane penetration, which are observed among members of the MACPF group as well . Cellulaphalitis . In our study, LLO demonstrated a permeabilization profile in primary glial cells that was slower than that of PLY with a comparable lytic capacity . SeveralDendritic varicosity formation (swelling of the dendrites that are mostly NMDA glutamate receptor-dependent). A similar phenomenon is observed in ischemic brain damage . StudiesDendritic spine collapse, which represents a morphological substrate of synaptic loss . DendritIt has been shown that at non-lytic concentrations in brain tissue, PLY initiates two major types of neuropathological changes in the neocortex :DendritL. monocytogenes, this effect would be enhanced [In our study, LLO demonstrated only partial resemblance to PLY; it initiated NMDA-dependent dendritic varicosity formation in a similar manner but surprisingly did not induce the collapse of dendritic spines. The occurrence of dendritic varicosities represents a typical and early pathologic finding in ischemic brain damage and often precedes dendritic spine collapse ,30. Appaenhanced . L. monocytogenes in the brain tissue. In contrast to listeria, S. pneumoniae releases PLY in the course of meningitis and other relevant pathologies, extracellularly [LLO is the only member of the CDC toxin group, which is produced by an intracellular pathogen. Nevertheless, some of the toxin is released extracellularly as well. The pathogenic role of intra-/extracellularly released LLO on host cell physiology differs . Intracellularly .Listerial meningitis accounts for 8% of all meningitis cases, and its diagnosis represents a major clinical challenge. The pathogen is confirmed in the CSF of only a third of all patients, and in most cases, empirical therapy is applied . A specithe magnitude and mechanical strength of cell shape changes differ from toxin to toxin;the time frame of cell shape changes by LLO may be slower compared to PLY, thus allowing more effective tissue adaptation; continuous exposure of tissue to LLO in normal pH conditions may lead to toxin inactivation; orinhibitory cellular metabolites specifically inhibit LLO. Another finding of the current work is the ability of LLO, similar to PLY, to initiate non-lytic astrocytic cell shape changes in culture. This indicates that cytoskeletal reorganization and cell shape remodeling represent a feature of other members of the CDC group toxins (apart from PLY) and are associated with early effects on the membrane due to prepore or pore formation by these toxins. In the last years, ample evidence has accumulated that supports the massive cytoskeletal reorganization capacity of PLY at sub-lytic concentrations, which consists of actin remodeling and bundling of microtubules ,37,38. WS. pneumoniae and L. monocytogenes is the presence of two distinct phospholipases produced by the latter bacterium that may act in concert with LLO to exacerbate changes in cell function. Furthermore, the phosphatidylcholine-specific phospholipase C from L. monocytogenes is an important virulence factor in murine cerebral listeriosis [In the current study, only the effects of recombinant purified toxins on cells and tissues were analyzed. All bacterial diseases, however, represent complex host/pathogen interactions in which a plethora of bacterial and host defense factors playing pathogenic roles. For example, an obvious difference between teriosis . Our worL. innocua 6a strain as described previously [2PO4, pH 6.2). The non-absorbed fraction was centrifuged and desalted by transferring through a super loop to a HiPrepTM26/10 desalting column . Loading buffer  was used to elute the desalted fraction. This fraction was subsequently filtered through a 0.22 μm filter and loaded onto a Resource S column  previously equilibrated with 50 mM NaH2PO4, pH 6.2. The pure toxin eluted reproducibly from the column at 0.21 to 0.28 M NaCl using elution buffer . Protein desalting and purification processes were carried out using the high performance chromatography system ÄKTA explorer and UNICORNTM control system .LPS-free LLO was expressed and purified from the wild type eviously . ShortlyEscherichia coli BL-21 cells  and purified via metal affinity chromatography as described previously [Wild-type PLY was expressed in eviously . The pureviously . Briefly2 cell culture flasks , the cells were harvested. Culture treatment with PLY and LLO was performed in serum-free medium.Primary mouse astrocytes were prepared from the cortices of newborn C57BL/6 mice  3–5) as mixed cultures with microglia in Dulbecco’s modified Eagle’s medium (high glutamate) . The growth medium was supplemented with 10% heat-inactivated fetal calf serum (FCS)  and 1% penicillin/streptomycin (Gibco). Fourteen days after seeding in 75 cm2, 5% CO2) at 4 °C. The slices were allowed to adapt in carbogenated Basal Medium of Eagle (Gibco) with 1% penicillin/streptavidin and 1% glucose at 37 °C for 1 h before being challenged with PLY or with LLO in the 5% CO2-buffered medium (pH = 7.3). In these acute slices, cell lysis never exceeded 7% within 12 h.Acute brain slices were prepared from infant (PD 10–14) C57Bl/6 mice via decapitation and vibratome sectioning  in artificial CSF continuously oxygenized with carbogen gas . Cells were incubated in Leibowitz medium (Gibco) with stable pH outside a COeviously . PrimaryLactate dehydrogenase test  was performed according to manufacturer´s instructions. Shortly, LDH released from lysed cells was detected by conversion of a tetrazolium salt into a red formazan product for 30 min with subsequent absorption detection at 560 nm on a Tecan Photometer . Acute mouse brain slices were fixed in 1.5% paraformaldehyde  in PBS (pH = 7.3) for 30 min and processed either for immunohistochemistry or for neuron-specific DiI staining ,42. BrieFor the immunohistochemical experiments, acute Vibratome slices were prepared, treated, and fixed in 2% formalin solution. After overnight permeabilization in 1% Triton X-100 in PBS, the slices were blocked in 4% BSA for 1 h and incubated overnight with anti-GFAP rabbit antibody  and with secondary antibody goat anti-rabbit tagged with Cy3 . All samples were preserved with ProLong antifade reagent (Life Technologies).All samples were analyzed on an Olympus Cell^M imaging fluorescent system using 10× and 20× dry objective or a 60× oil immersion objective, or on a Leica SP5 laser-scanning microscope /Zeiss LSM 880 with Airyscan  using 63× oil immersion objectives and optical zoom between 4 and 8. Image processing and analysis were performed using the ImageJ software .U-tests (comparing two groups differing in one parameter) or one-way ANOVA with a Bonferroni post-test (comparing three or more groups differing in one parameter). Permeabilization kinetics analysis was performed by non-linear regression analysis, utilizing one phase exponential association. Statistical analysis was performed using GraphPad Prism 4.02 for Windows . The statistical tests included Mann-Whitney


Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/β-catenin pathway was activated through direct interaction of p-ERK and GSK3β. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3β seems to be a mechanism for increase of ITF-2.Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected  B-RAF-mutated melanoma and K-RAS/B-RAF-mutated colorectal cancer) [B-RAFV600E or B-RAFV600K mutations, as detected by an FDA-approved test (https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm481951.pdf). AZD6244 (Selumetinib) is a non-ATP competitive inhibitor and is highly specific for MEK1/2. Multiple phase 2 studies of this drug have been conducted in patients with a wide range of cancers [Intracellular growth-promoting signals are transduced through the activation of the small G protein RAS, which activates RAF and then activates mitogen-activated protein kinase (MAPK) kinase (MEK), leading to the phosphorylation and activation of MAPK . MEK1 and MEK2 show a unique specificity for ERK1/2 and the ERKs have wide spectrum substrates, many of which play a role in promoting cell proliferation and survival [ cancer) . PD09805 cancer)  and seveB-RAF gene represent the most important predictive biomarker for sensitivity to MEK inhibitors [B-RAF mutant tumors were not uniformly responsive to MEK inhibitors although tumors with these mutations seemed to be more sensitive. [RAS mutations or ERK activation correlate poorly with the sensitivity of cells to MEK inhibitors [Although recent studies have demonstrated impressive antitumor activities of MEK inhibitors, many tumors show intrinsic resistance of these agents. Reliable biomarkers of susceptibility or resistance to MEK inhibitors are thus needed to allow tailoring of individualized treatments with these compounds and to reduce the risk of unnecessary drug toxicities. It has been suggested that activating mutations in the hibitors , 7. Howensitive. , 9 Furthnsitive. . Other bhibitors , 12.The NCI-60 is a set of 60 human cancer cell lines derived from diverse tissues, including brain, blood and bone marrow, breast, colon, kidney, lung, ovary, prostate, and skin. These cell lines have now been subjected to a battery of experiments including extensive pharmacological characterization analyses via treatment with over 100,000 chemical compounds, chromosome karyotyping, and gene expression analysis using various DNA microarray platforms . The curImmunoglobulin Transcription Factor-2 (ITF-2) is one of several specific target genes of T-cell factor-dependent transcription upon translocation of β-catenin to the nucleus [The proto-oncogene β-catenin has been linked to the pathogenesis of hepatocellular carcinoma, colorectal carcinoma, lung cancer, malignant breast tumors, and leukemia through the Wnt-signal transduction pathway  Immunogl nucleus .In our current study we aimed to screen for novel response predictive markers for the AZD6244, MEK inhibitor by analyzing published microarray data from AZD6244 sensitivity profiling of the NCI-60 cell lines and also aimed to validate the candidate markers in both primary and acquired resistance models.50 ≤ 0.5 μM) and 6 resistant (IC50 > 5 μM) cell lines . One resistant and 5 sensitive cell lines harbored a B-RAF mutation; and 1 resistant and 1 sensitive line contained a K-RAS mutation. One resistant cell line had an N-RAS mutation  gene because it showed the strongest upregulation in resistant lines and previous studies have suggested that this gene is a downstream target of the Wnt/β-catenin/T-cell factor pathway [Following public microarray data acquisition and analysis, a total of 62 differentially expressed genes (DEGs) were selected from 12 cell lines  . Of thes pathway .ITF-2 in AZD6244 resistant cancer cell lines were found to be significantly higher than in cell lines sensitive to this inhibitor    . The apoTF-2 #2)  and incrTF-2 #2) .These results suggest that the ITF-2 suppression could enhance the susceptibility to AZD6244 in melanoma cells with primary or acquired resistance.Because ITF-2 is known as a downstream target of Wnt signaling pathway, we examined the pathway in AZD6244 sensitive cell line (M14) and resistant cell lines (M14/AZD-3 and LOX-IMVI). Translocation of dephosphorylated β-catenin to the nucleus and accumulation of phosphorylated Ser9 of GSK3β in cytosol fraction were found in AZD6244 resistant cell lines (M14/AZD-3 and LOX-IMVI), but not in AZD6244 sensitive cell line, M14 Figure . The samWe assumed that p-ERK might directly interact with GSK3β and we performed GST pull-down assay using pGEX-GSK3β and Glutathione-Sepharose 4B (GST) beads. GSK3β fused GST bead was interacted with phosphorylated p90RSK (Tyr573) and ERK (T202/Y204) in M14 but the interaction did not activate phosphorylation of GSK3β (S9). In contrast, the bead was interacted with phosphorylated ERK (T202/Y204) and phosphorylation of GSK3β (S9) was induced in both resistant cell lines without p90RSK Figure . The finMEK1 mutations, [ITF-2 might be a novel biomarker of cellular resistance to MEK inhibitors because the mRNA and protein levels of this gene in AZD6244 resistant cell lines were significantly elevated and suppression of ITF-2 by siRNA enhanced the susceptibility of AZD6244 resistant cells to this inhibitor. These results were observed with both primary AZD6244 resistance cell line (LOX-IMVI) and acquired resistance cell line (M14/AZD), which was established as a model for acquired resistance using a sensitive cell line harboring B-RAF mutation. Our study results might provide a clue to prevent or overcome the development of acquired resistance to MEK inhibitors, which is a common and serious problem in real clinical settings.Drug resistance, either primary or acquired, is one of the most challenging aspects of anticancer agents. A critical component of cancer drug development is the identification of biomarkers that can be used to predict resistance to these agents. Biomarker identification is also important for minimizing patient exposure to potentially toxic agents for little clinical benefit and to develop rational approaches for overcoming drug resistance. Many studies have been under taken to gain an improved understanding of the mechanisms of cancer drug resistance and have identified factors that seemed to be associated with resistance to MEK inhibitors,  includintations,  over-exptations,  aberranttations,  concurretations, , 25 or ttations,  and an ‘tations, . In our ITF-2 gene  is located on human chromosome 18q21, consists of 20 exons (exons 1 and 20 are noncoding), and spans 360 kb. ITF-2 belongs to the class A subfamily of basic helix-loop-helix (bHLH) transcriptional regulators [T-cell transcription factor 4 on human chromosome 10q25.3, which was previously termed TCF4, but is now designated as TCF7L2. The ITF-2 gene encodes two splice variants, ITF-2A and ITF-2B. ITF-2A is a truncated splice variant with a low transcriptional potency. ITF-2B produces a larger protein, which is ubiquitously expressed and dimerizes with other classes of bHLH proteins to regulate tissue-specific gene expression through E-box sites in various cell types including myocytes, osteoblasts, B and T lymphocytes, and neuronal cells [TCF7L2 pathway, which is frequently disrupted in human cancers [c-MYC and cyclin D1, ITF-2 functions as an oncogene when deregulated in human colon cancer or ovarian cancer, [ITF-2 may play a role in early gastric carcinoma progression or in the colonic adenoma to carcinoma transition [ITF-2 gene in AZD6244 resistant cells  and a MEK inhibitor (AZD6244) was reported, and genetic or pharmacological inhibition of the PI3K/AKT/mTOR pathway was found to partially or completely reverse this resistance [B-RAF or RAS mutations, and also to delay the onset of acquired resistance and in some cases overcome acquired resistance to AZD6244 [BCR-ABL in nilotinib-resistant chronic myeloid leukemia (CML) cells and nilotinib acts synergistically with MEK inhibitors to kill drug-resistant CML cells both in vitro and in vivo [In vitro studies using leukemia cell lines have reported that MEK inhibitors enhance the cytotoxic effects of cytarabine or doxorubicin [ITF-2 siRNA induced cell death in AZD6244 resistant cell lines, although the magnitude of enhanced susceptibility to AZD6244 was not adequate for clinical application, probably due to the only partial suppression of ITF-2 expression by siRNA. Wnt/ β-catenin pathway inhibitors might suppress ITF-2 more effectively and may provide a novel combination approach for MEK inhibitors.MEK inhibitors have generally shown limited effectiveness as monotherapeutic agents and combination regimens are a possible treatment approach to overcome MEK inhibitor resistance. Indeed, simultaneously inhibition of the MAPK and PI3K/AKT/mTOR pathways has been proposed for melanoma . Moreovee tumors . In anotsistance . Further AZD6244 . The MAP in vivo . The ava in vivo . It has orubicin , 44. Simorubicin  and biliorubicin  colon, [orubicin , 48 lungorubicin . When MEorubicin , 48. In ITF-2 gene as a novel resistance biomarker for an MEK inhibitor, AZD6244. The mRNA and protein levels of ITF-2 were found to be significantly elevated in cell lines with primary or acquired resistance to AZD6244 and a targeted silencing of ITF-2 by siRNA significantly enhanced the susceptibility of primary resistant cells (LOX-IMVI) and acquired resistant cells (M14/AZD-3) to AZD6244. Activation of Wnt signaling pathway through direct interaction of p-ERK and GSK3β seems to be a mechanism for increase of ITF-2.In summary, we have identified the 2 in RPMI-1640 medium containing 5% or 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin . Information regarding the mutation status of the B-RAF, K-RAS, and N-RAS genes of each cell line was obtained from the Wellcome Trust Sanger institute website .The NCI-60 cell lines were purchased from the Jackson Laboratory  and were cultured at 37°C in 5% COin vitro. M14 cells were continuously exposed to gradually increasing concentration of AZD6244 up to 500 nM. An AZD6244 resistant subclone was isolated using limiting dilution method and AZD6244 resistant cell lines  could be established. In most experiments, M14/AZD-3 was used.Human melanoma M14 cell line was sensitive to AZD6244 ™ X Light luminescence plate reader . The concentration of AZD6244 required for 50% growth inhibition was scored as the half maximal inhibitory concentration (IC50) values.Cell viability was assessed by the luminescent-based CellTiter-Glo system  and Cell counting kit -SK  in accordance with the manufacturer's instructions. Briefly, cells were plated at 1,000–3,000 per well in a 96-well opaque plate and were incubated in complete growth medium. Cells were treated with various concentrations of MEK1/2 inhibitor (AZD6244), which was synthesized by Chemizon  and prepared as a 50 mmol/L stock solution in DMSO. After 48 h, cell viability was determined by measuring luminescent signals with a VICTORwww.ncbi.nlm.nih.gov/geo). The chip data were analyzed with RNA-eXpress (www.rnaexpress.org) using its default analysis settings and a quintile normalization method. Analyses of preprocessed data were performed using BRB-ArrayTools (linus.nci.nih.gov/BRB-ArrayTools.html). A two-sample t-test was performed between sensitive and resistant groups at P < 0.005 to select for DEGs between the two groups.To identify candidate genes associated with resistant or sensitive responses to AZD6244 genome-wide gene expression profiling data from Affymetrix HG-U133A chips were downloaded from the Gene Expression Omnibus . All PCR amplifications were performed with HotStar HiFidelity Polymerase kit  following the manufacturer's instructions.  methods . The seq® Reagent (Invitrogen) and converted into cDNA using a QuantiTect Reverse Transcription Kit . The transcript levels were quantified via the SYBR Green real time PCR method using LightCycler® 480 SYBR Green I Master Mix (Roche Applied Science) on a Roche-480 system using primer sets  and siITF-2 #2 . The level of off-target effects was assessed by western blot analysis using an anti-ITF-2 mAb in M14/AZD-3 and both ITF-2 siRNA's significantly reduced ITF-2 expression levels . Cells were then incubated at 37°C for 24 h.The siRNA was used for targeted silencing of n levels . A non-tCells were lysed with lysis buffer  and protein samples (50 μg) were separated by SDS-PAGE and were blotted onto polyvinylidene difluoride membranes . After blocking with 5% (w/v) non-fat dry milk powder for 1 h, the membranes were incubated with primary antibodies overnight at 4°C, then with secondary antibody conjugated with horseradish peroxidase . Specific antigen-antibody complexes were detected by enhanced chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate . Specific antibodies were as follows: ITF-2 , phospho-beta-catenin (p-beta-catenin), beta-catenin, phospho-ERK (p-ERK), ERK, phospho-GSK3 beta (Y216 and S9), GSK-3 alpha and beta, phospho-90RSK (Y573), 90RSK, PARP, cleaved caspase-3 , dephospho-beta-catenin (de-p-beta-catenin) (Enzo bioscience Inc. France), KRAS, B-RAF, phosphor-MEK, MEK, and beta-actin .® Nuclear and cytoplasmic extraction reagents  according to the manufacturer's protocol. For comparison, one equivalent of cytoplasmic proteins and two equivalents of nuclear proteins were analyzed side-by-side by western blot analysis with different antibodies.To isolate cytoplasmic and nuclear fractions, cells were incubated using NE-PER5) grown on sterile glass coverslips were transfected with either non-targeting control siRNA or ITF-2 siRNA. These transfected cells were then incubated with 1:50 anti-ITF-2  at room temperature for 1 h after fixation with 4% paraformaldehyde and permeabilization with triton X-100. The cells were then incubated with secondary anti-goat antibody conjugated with fluorescein isothiocyanate  and counterstained with a 4 μg/mL DAPI solution. After the coverslips were mounted with fluorescence mounting medium , cells were viewed using a fluorescence microscope .Cells (2 × 106) were treated with AZD6244 for 24 h, washed twice with cold PBS, and resuspended in 1× Binding Buffer at a concentration of 1 × 106 cells/mL. A 100 μL aliquot of this solution (1 × 105 cells) was then transferred to a 5 mL culture tube to which 5 μL of FITC Annexin V was added. These cells were incubated for 15 min at room temperature in the dark before assaying with an Annexin V–FITC Apoptosis Detection Kit I , in accordance with the manufacturer's protocol. The percentage of apoptotic cells was analyzed by flow cytometry within 1 h .Cells (1 × 10Cell death after siRNA transfection with or without AZD6244 treatment was assessed using Live-Dead Cell Staining Kit (Enzo Life Sciences). This kit utilizes a cell permeable green fluorescent dye to stain live cells. Dead cells were stained by propidium iodide, a red fluorescent dye, which is actively pumped out of the cytoplasm in viable cells. Stained cells were visualized by fluorescence microscopy .Escherichia coli BL21(DE3) cells  by transformation and the cells were cultured in 5 ml LB medium at 37°C to the mid-log phase. Protein expression was induced by 1mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG). After culturing for 3 h, cells were pelleted by centrifugation and then suspended in 100 μL of a lysis buffer . Recombinant GST-GSK3β was purified using Glutathione- Sepharose 4B beads . Protein lysates of M14, M14/AZD-3, and LOX-IMVI cell lines were respectively combined with 50 μL pre-cleaned Glutathione- Sepharose 4B beads and the mixture was incubated under shaking for 16 h at 4°C. The incubated beads were washed three times with lysis buffer . After washing, 2× SDS-PAGE loading buffer was added to the washed beads and proteins were extracted from the beads by boiled for 5 min. Proteins were analyzed by 10% SDS-PAGE and western blot analysis.pGEX-GSK3β  was introduced into t test, while those in relative expression of endogenous ITF-2 mRNA were evaluated by the Mann-Whitney U test. Death rates of two cell lines in assessment of Live-Dead cell staining were compared by a Fisher's exact test. Statistical analyses were performed using SPSS version 21.0 software. For all analyses, the P values were 2-tailed, and P < 0.05 was considered as statistically significant.Statistical differences in cell viability, presented as mean ± standard error of the mean (SEM), were evaluated by the Student


The prognosis for patients with disseminated lung cancer is poor and current treatments have limited survival benefit as resistance often occurs, and is often associated with significant toxicity. A possible strategy to improve treatment and evade chemoresistance may be to find new combinations of drugs. The aim of this study was to analyze the potential of combining proteasome inhibitors (PIs) with chemotherapeutic drugs used in the routine treatment for lung cancer patients.The median-effect method was applied to the Fluorometric Microculture Cytotoxicity Assay (FMCA) to evaluate effects of combining two different PIs (bortezomib and b-AP15) with clinically used chemotherapeutic drugs representing different mechanisms of action  in two lung cancer cell lines (one sensitive and one resistant). Proteasome inhibition in combination with cisplatin, gemcitabine or vinorelbine had synergistic effects in at least one of the tested cell lines. Furthermore, the effect of gefitinib appeared strongly potentiated by the PI in the least resistant lung cancer cell line, although the level of synergy could not be determined with the median-effect method.Combining PIs with cisplatin, gefitinib, gemcitabine or vinorelbine show potential as new combination chemotherapy for the treatment of lung cancer.The online version of this article (10.1186/s13104-017-2842-z) contains supplementary material, which is available to authorized users. Lung cancer (LC) is causing most cancer-related deaths in the world and is responsible for 1.2 million new cases per year . CurrentThe ubiquitin–proteasome system is responsible for degradation of proteins by tagging them with ubiquitin which leads to recognition by the proteasome complex and degradation of the proteins into small peptides. The ubiquitin–proteasome system is important for maintenance of cell homeostasis which is critical for normal function and survival of all cells. Dysregulation of protein degradation has been shown to play an important role in the growth and survival of tumors  and protThere is still a lack of knowledge of the optimal combinations of PIs and chemotherapeutics, their synergistic effects and potential use in resistance to chemotherapy in LC. The present work aims to address these issues with two PIs targeting the 19S or the 20S proteasome in combination with commonly used chemotherapeutics in LC representing different mechanisms of action.l-Glutamine  and cells were maintained in 37 °C humidified air with 5% CO2. The cell lines were tested for mycoplasma during the project with MycoAlert™ Mycoplasma Detection Kit (Lonza).The NSCLC squamous cell carcinoma cell lines U-1752 and NCI-H157, the NSCLC large cell carcinoma cell line U-1810, the NSCLC adenocarcinoma cell line NCI-H23 and the SCLC cell lines U-1906-L and U-2020 were obtained from American Type Culture Collection (ATCC) and cultivated in RPMI-1640 Medium  supplemented with 10% FBS (Sigma-Aldrich) and 50 values were determined from nonlinear regression of the dose–response relationships, with the GraphPad Prism software .The Fluorometric Microculture Cytotoxicity Assay (FMCA) was used as previously described  to inves50-values determined from single drug experiments with the FMCA  and high (> 97.5%) SI-values were excluded from the calculations as recommended by Chou [Combinations of the chemotherapeutic drugs and the PIs were evaluated by applying the median effect method, by Chou and Talalay , to the A Tables , 2. For  by Chou . CombinaAll drugs and drug combinations were tested in duplicates and each FMCA experiment was repeated at least two times.50-values varied between the drugs and the cell lines, and are presented in Table 50 values are presented in Additional file The FMCA method was used to investigate the single agent activity of the chemotherapeutic drugs cisplatin, gefitinib, gemcitabine and vinorelbine and the PIs bortezomib and b-AP15 in six LC cell lines. The obtained IC50) and the most sensitive  cell line in the panel . The outcomes of combining the PIs bortezomib and b-AP15 with each of the four chemotherapeutic drugs were evaluated with the median effect method [50 values > 50 µM) the combination with either b-AP15 or bortezomib had close to 100% effect in the least gefitinib-resistant cell line U-2020 . Resistance to gefitinib is strongly connected with the mutation status of EGFR [50 value below median), whereas NCI-H23 was the second most resistant cell line and U-1752 the most resistant cell line (IC50 > 310 µM) to gefitinib, indicating that other factors than EGFR mutation status may also influence the sensitivity to gefitinib. Anyhow, gefitinib combined with PIs had close to 100% effect even at the lowest tested concentrations (0.25 × IC50 for each drug) in the least resistant cell line U-2020. Due to the high effect at all tested concentrations for gefitinib the level of synergy could not be determined with the Calcusyn Software [All cell lines were highly resistant to gefitinib in vitro (all IC of EGFR . To our  of EGFR –25. NCI-Software . Hence, Comparing the combination effects of the PIs showed that they had similar effects. However, bortezomib combined with cisplatin, gemcitabine and vinorelbine had more effects in the resistant cells compared with the more sensitive cells, in regard to each therapeutic drug. This could not be seen for b-AP15. Since proteasomal inhibition causes proteotoxic stress with an increase in free radicals which increases the effect of DNA damaging agents, especially in resistant cells, this indicates that b-AP15 may induce lower levels of free radicals than bortezomib and that bortezomib may be a more effective second-line treatment than b-AP15 for LC patients who have become resistant to DNA damaging agents.There are several preclinical and clinical studies indicating that the drug combinations tested in our study have potential for the treatment of LC patients. Bortezomib potentiates the effect of cisplatin in different cancer cell types , 27 and Since drug resistance is a major clinical problem in LC, as well as in other tumor types, there may be a rationale to concomitantly combine treatment with PIs with other chemotherapeutic agents upfront in order to prevent or delay resistance from occurring. Our study also demonstrates the synergistic action when combining the drugs concomitantly and would argue against using PIs and chemotherapeutics in sequence. This concomitant approach is also supported by results from other studies in vitro and in vivo. Although, further studies evaluating the potential of increasing the synergistic effects by sequential administration would be interesting.It is possible that combinations of more than two of the agents that were used in this study may have even stronger anti-tumor effects than combinations with two agents. For example, a Children’s Oncology Group clinical trial investigated if bortezomib increased the efficacy of ifosfamide and vinorelbine in paediatric Hodgkin lymphoma and demonstrated a promising response rate with the triple combination . BortezoPIs combined with chemotherapeutic agents used in routine treatment for LC patients have synergistic effects in LC cells in vitro. Clinical trials have indicated that these combinations are safe for cancer patients and may provide benefit for LC patients. However, further in vitro and in vivo studies of these combinations are needed to establish the optimal combination strategies and to confirm its efficacy and safety in larger clinical trials with LC patients.Additional file 1. SIs for each replicate of the FMCA experiments of single drugs. SI is defined as the fraction of living cells in a drug treated sample compared with an untreated sample. Each replicate is a mean of the duplicates in each experiment.Additional file 2. Effect levels for each replicate of the FMCA experiments of the drug combinations. Effect is defined as 1 minus the fraction of living cells in a drug treated sample compared with an untreated sample. Each replicate is a mean of the duplicates in each experiment.


Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2800 transgenic lines for use with the split-GAL4 intersectional method.The ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The  Drosophila melanogaster . In these lines (referred to below as generation 1 GAL4 lines), expression of the transcription factor GAL4 is driven by a 2–3-kb segment of genomic DNA that contains one or more transcriptional enhancer sequences. While many lines show expression in a small fraction (< 1%) of neurons in the adult brain, only ∼1 in 1000 appears to drive expression in a single cell type  and verified by EcoRI restriction digestion. The HL9 (Ddc), Tdc2 (Tyrosine decarboxylase 2), TH (ple), and Trh (Tryptophan hydroxylase) hemidrivers are described in Lines were constructed essentially as described by Sp-1/CyO; TM2/TM6B  landing site (Drosophila Stock Center (http://flystocks.bio.indiana.edu). The expression patterns driven by the parent generation 1 GAL4 and lexA lines are available at www.janelia.org/gal4-gen1. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.5987542.The hemidriver lines shown in Supplemental Material, Table S1 have been deposited in the Bloomington www.janelia.org/gal4-gen1) to identify enhancers that drive expression in the desired cell type. Once a list of enhancers has been identified, AD and DBD hemidrivers that use those enhancers can be selected from the lines in Table S1 and crossed using a cross scheme such as that shown in The first step in generating a split-GAL4 line for a given cell type is screening the images of generation 1 GAL4 lines , and these images can serve as a useful guide for the expression pattern of the enhancer inserted at that landing site. Examples of three such comparisons are shown in Because expression patterns vary with insertion site, the hemidriver expression pattern may differ from that of the generation 1 GAL4 line with the same enhancer because the AD hemidrivers are inserted into attP40 and the generation 1 lines are inserted in attP2. Different insertion sites for the AD and DBD hemidrivers were used to avoid transvection  and to mA successful intersection requires the identification of at least two generation 1 lines that express in the cell type of interest; for some cell types, the lines in Table S1 will be insufficient for this purpose. In this case, there is little choice but to expand the set of hemidriver lines. For example, we have been able to obtain successful intersection in such cases by also incorporating the set of hemidrivers described in Once combinations of hemidrivers that yield successful intersections have been identified in the screening process, we generally construct stable split-GAL4 lines that carry a combination of the two hemidrivers, using the cross scheme shown in Using the combined hemidriver collections described in this paper and Drosophila [for example, We anticipate rapid progress on electron microscopic level connectomics in