Primary Lines Research Articles

Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by fibroblastic foci and progressive scarring of the pulmonary parenchyma. IPF fibroblasts display increased proliferation and enhanced migration and invasion, analogous to cancer cells. This transformation-like phenotype of fibroblasts plays an important role in the development of pulmonary fibrosis, but the mechanism for this is not well understood.MethodsIn this study, we compared gene expression profiles in fibrotic lung tissues from IPF patients and normal lung tissues from patients with primary spontaneous pneumothorax using a cDNA microarray to examine the mechanisms involved in the pathogenesis of IPF. In a cDNA microarray, we found that USP13 was decreased in lung tissues from patients with IPF, which was further confirmed by results from immunohistochemistry and western blot assays. Then, we used RNA interference in MRC-5 cells to inhibit USP13 and evaluated its effects by western blot, real-time RT-PCR, bromodeoxyuridine incorporation, and transwell assays. We also used co-immunoprecipitation and immunofluorescence staining to identify the correlation between USP13 and PTEN in IPF.ResultsUSP13 expression levels were markedly reduced in fibroblastic foci and primary IPF fibroblast lines. The depletion of USP13 resulted in the transformation of fibroblasts into an aggressive phenotype with enhanced proliferative, migratory, and invasive capacities. Additionally, USP13 interacted with PTEN and mediated PTEN ubiquitination and degradation in lung fibroblasts.ConclusionsDown-regulation of USP13 mediates PTEN protein loss and fibroblast phenotypic change, and thereby plays a crucial role in IPF pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0286-3) contains supplementary material, which is available to authorized users.

OBSERVED VARIABILITY OF THE SOLAR Mg II h SPECTRAL LINE

The Mg II h&k doublet are two of the primary spectral lines observed by the Sun-pointing Interface Region Imaging Spectrograph (IRIS). These lines are tracers of the magnetic and thermal environment that spans from the photosphere to the upper chromosphere. We use a double gaussian model to fit the Mg II h profile for a full-Sun mosaic dataset taken 24-Aug-2014. We use the ensemble of high-quality profile fits to conduct a statistical study on the variability of the line profile as it relates the magnetic structure, dynamics, and center-to-limb viewing angle. The average internetwork profile contains a deeply reversed core and is weakly asymmetric at h2. In the internetwork, we find a strong correlation between h3 wavelength and profile asymmetry as well h1 width and h2 width. The average reversal depth of the h3 core is inversely related to the magnetic field. Plage and sunspots exhibit many profiles which do not contain a reversal. These profiles also occur infrequently in the internetwork. We see indications of magnetically aligned structures in plage and network in statistics associated with the line core, but these structures are not clear or extended in the internetwork. The center-to-limb variations are compared with predictions of semi-empirical model atmospheres. We measure a pronounced limb darkening in the line core which is not predicted by the model. The aim of this work is to provide a comprehensive measurement baseline and preliminary analysis on the observed structure and formation of the Mg II profiles observed by IRIS.

Endothelial cells induce cancer stem cell features in differentiated glioblastoma cells via bFGF.

BackgroundGlioblastoma multiforme (GBM) is a rapidly growing malignant brain tumor, which has been reported to be organized in a hierarchical fashion with cancer stem cells (CSCs) at the apex. Recent studies demonstrate that this hierarchy does not follow a one-way route but can be reverted with more differentiated cells giving rise to cells possessing CSC features. We investigated the role of tumor microvascular endothelial cells (tMVECs) in reverting differentiated glioblastoma cells to CSC-like cells.MethodsWe made use of primary GBM lines and tMVECs. To ensure differentiation, CSC-enriched cultures were forced into differentiation using several stimuli and cultures consisting solely of differentiated cells were obtained by sorting on the oligodendrocyte marker O4. Reversion to the CSC state was assessed phenotypically by CSC marker expression and functionally by evaluating clonogenic and multilineage differentiation potential.ResultsConditioned medium of tMVECs was able to replenish the CSC pool by phenotypically and functionally reverting differentiated GBM cells to the CSC state. Basic fibroblast growth factor (bFGF), secreted by tMVECs, recapitulated the effects of the conditioned medium in inducing re-expression of CSC markers and increasing neurosphere formation ability of differentiated GBM cells.ConclusionsOur findings demonstrate that the CSC-based hierarchy displays a high level of plasticity showing that differentiated GBM cells can acquire CSC features when placed in the right environment. These results point to the need to intersect the elaborate network of tMVECs and GBM CSCs for efficient elimination of GBM CSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0420-3) contains supplementary material, which is available to authorized users.

WNT/β-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation.

Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both naïve and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the naïve state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4.

Abstract POSTER-TECH-1105: Assessment of ovarian cancer spheroid attachment and invasion of mesothelial cells in real time to profile the molecular signature of the invasive interface

Abstract Background and Aims: Ovarian cancer is the most common cause of death from gynaecological cancer worldwide (1). Often called the “silent killer”, the high mortality rate is associated with the late stage in which the disease is diagnosed (2). Unlike other solid cancers, ovarian cancer cells rarely spread via the vasculature, instead shed from the primary tumor and disseminate within the peritoneal fluid or ascites (2). The formation of spheroids within the non-adherent peritoneal environment is a barrier to effective treatment, due to the enhanced ability of spheroids to survive chemotherapies and seed distal metastases (3-5). Invading ovarian cancer cells interact with mesothelial cells lining the peritoneal surface, attach to, and invade the underlying basement membrane to establish secondary lesions (6). The molecular events at the interface between invading ovarian cancer and peritoneal cells at the onset of invasion have been poorly studied, existing methods capture events after invasion is well-underway. Therefore, the regulatory signals which govern the initiation of a metastatic lesion are poorly understood. We aimed: i) to develop an innovative method to co-culture ascites-derived primary ovarian cancer cells to measure invasion through peritoneal mesothelial cell layers in real time (7); ii) identify the unique protein expression profile at the ovarian cancer spheroid-peritoneal interface during early invasion using proteomics. Methods: Real Time Cell Analysis (RTCA) technology (xCELLigence, ACEA) was adapted to establish a three-dimensional co-culture model of the tumor microenvironment of the peritoneum (7). RTCA wells were coated with Matrigel (basement membrane matrix) and a confluent monolayer of human mesothelial cells. Multicellular spheroids were generated from primary ovarian cancer lines (high-grade serous carcinoma, leiomysarcoma, adenocarcinoma, carcinosarcoma) or benign ascites derived cells cultured under non-adherent conditions. Spheroids were added to the RTCA plate and real time measurements were conducted to determine the precise period when invasion commenced. Parallel cultures were histologically-preserved at invasion initiation and processed for assessment by Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging. Results: We assessed invasion in a panel of ovarian cancer samples, benign ascites cells and the mesothelial cell line. Continuous real time measurements revealed that individual ovarian cancer samples exhibited differences in the timing of the onset of invasion. High-grade serous carcinoma samples initiated invasion rapidly after addition, with some samples detected in under 30 min, a time period not captured by traditional Transwell assays. The benign and mesothelial cells themselves did not invade. MALDI imaging allowed for the identification of peptides expressed at the tumor-mesothelial interface during early invasion. These molecules represent novel molecules involved in the initiation of the metastatic process and are the subject of further characterisation. Conclusion: This method represents a high-throughput quantitative analysis of ovarian cancer spheroid invasion of mesothelial and ECM barriers. The study has provided an unprecedented insight into the molecular mechanisms and molecules involved in the initiation of ovarian cancer invasion of the mesothelium, identifying new therapeutic targets.

Abstract 3223: A true orthotopic ovarian cancer patient-derived xenograft (PDX) model

Abstract Introduction: Patient derived xenografts (PDXs) show promise as models to study cancer as they recapitulate the principle characteristics of the patients’ original tumor such as histology, mutational status, DNA copy number, gene expression patterns, and clinical behavior while remaining biologically stable through passaged in mice. We used an orthotopic model transplanting tumor directly to the fallopian tube/ovary in order to accurately study the native environment and the progression of the ovarian cancer (OVCA). Experimental Design: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week old mice. Tumor growth was followed over time. Tumors were evaluated by IHC. Alu II probe staining was performed to evaluate human stroma content. RPPA and targeted DNA sequencing are in-process. Drug studies are in progress. Primary tumor lines are being generated. Results: To date, we have transplanted tumor from 18 primary, 2 interval, and 5 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 85% success rate in generating tumors in mouse passage 1 (MP1). The median time for tumors to reach ∼2 cm is 3 months when tumors are then passaged again (MP2/MP3). We have generated 8 BRCA 1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrates human cells in the stroma. We have generated 8 primary tumor cell lines from original tumors/PDXs. Conclusions: We conclude that although technically more challenging, the orthotopic transplantation technique is feasible in generating ovarian cancer PDXs that most closely resemble the natural environment for ovarian cancer progression. Citation Format: Hongmei Cui, Yingyan He, Clemens Krepler, Janos Tanyi, Mark A. Morgan, Robert A. Burger, Sarah Kim, Emily Ko, Tan Ince, Meenhard Herlyn, Fiona Simpkins. A true orthotopic ovarian cancer patient-derived xenograft (PDX) model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2015-3223

Abstract 5177: Heterogeneity of pancreatic ductal adenocarcinoma visualized

Abstract The outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal. Factors that contribute to the lethality of this disease include its intrinsic resistance to therapeutics, an aggressive growth pattern, and a large degree of both inter- as well as intratumor heterogeneity. All these features have been attributed (at least in part) to the presence and function of a population of pluripotent cells called cancer stem cells. Despite the alleged importance of these cells for clinical outcome, the methods that have been used to study these cells in PDAC are troubled by some serious caveats. Defining important quantitative parameters for these cells in an unbiased, marker-agnostic, way is called for and we aim to achieve this by unbiased tracing methods in patient derived xenografts. A panel of PDX-derived lines generated in our institute was labeled using the LeGO (Lentiviral Gene Ontology; Weber et al. 2012 Nat. Prot) system to enable tracking of single cell clones by color. The system utilizes three vectors coding for red, green, and blue fluorophores much like a television uses these colors to generate an almost infinite range of colors in the visible range. Nuances in color and intensity are generated by the many possible combinations of fluorophores, the different integration numbers of the genes coding for them, and the variations in expression levels determined by for instance the integration sites. The end result is a population of cells in which each cell is endowed with a unique color. This will allow detection of the offspring of such a cell by identifying clones within a structural unit in for instance a tumor, or in a culture dish, of similar color. The ability to give rise to such a structure is then, depending on the experimental context and outcome, considered a proxy for stem cell function. Several primary lines have proven amenable to LeGO-marking. These lines showed stable color patterns during culturing in vitro as analyzed by continuous FACS measurements, and tumors derived from marked cells did not show a difference to umarked tumors as assessed by gross histology and growth rate. We are currently testing the ability of these cells to faithfully occupy and mark structural units in PDAC, much like intestinal stem cells would in a lineage tracing experiment in the gut, and derive numbers for stem cell number as well as replacement rate. Experiments are currently being performed to assess the system's potential to measure parameters associated with stemness, and possibly also differentiation status of the original tumor, as the cells grow in vitro. (Radio)chemotherapy regimens are included to formally address the purported resistance of cancer stem cells to these treatments without having to rely on an a priori chosen marker. Furthermore, more detailed comparison should reveal use of LeGO-derived data to measure the intra- and intertumor heterogeneity in PDAC tumor biology in our panel of PDXs. Citation Format: Veronique Veenstra, Helene Damhofer, Tom van Leusden, Jan Kessler, Jan Paul Medema, Hanneke van Laarhoven, Louis Vermeulen, Maarten Bijlsma. Heterogeneity of pancreatic ductal adenocarcinoma visualized. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2015-5177

Abstract 4027: Aggressive breast cancer associated fibroblasts communicate with cancer cells via microRNAs and promote an aggressive breast cancer phenotype

Abstract Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells CAFs are known to promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival, and response to hormone therapy in ER+ patients. Breast tumors from the TCGA and METABRIC breast cancer datasets classified as high hMAPK-miRNA exhibit signficant stromal and immune cell infiltrate, suggesting that these cell types may contribute to the hMAPK-miRNA signature. We have established primary breast CAF lines, from different breast cancer subtypes, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability, and have found that the CAFs differentially express several hMAPK-microRNAs compared to the DTs. Results: To investigate the role of specific expression of hMAPK-miRNAs secreted by CAFs, CM was isolated from CAFs from “aggressive” tumors (TNBC), from “indolent” tumors (ER+, luminal A), and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors showed an overexpression of various secreted hMAPK-microRNAs into the CM than do HMFs or CAFs from indolent tumors. Several hMAPK microRNAs were secreted by “aggressive” CAFs compared to “indolent” CAFs or HMFs, including miR221/222. The secreted miR221/222 could down-regulate both ER and TRPS1 (a negative regulator of EMT), known targets of miR221/222, in MCF-7 breast cancer cells. The hMAPK-regulated miR155 is also secreted by “aggressive” CAFs, and regulates Myeloid derived suppressor cell induction To determine if the secreted miRNA differences exhibited by the different CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Conclusion: Together, these data suggest that CAFs from aggressive tumors supports the aggressive breast tumor phenotype by repressing ER, facilitating EMT and helping tumor cells escape immune surveillance. Citation Format: Sanket Shah, Zheng Ao, Philip Miller, Ram Dattar, Marc Lippman, Dorraya El-Ashry. Aggressive breast cancer associated fibroblasts communicate with cancer cells via microRNAs and promote an aggressive breast cancer phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2015-4027

Abstract 1718: The importance of clinically relevant rapid autopsy specimens and LuCaP patient-derived xenograft models to interrogate the heterogeneous and evolving treatment resistance of castration-resistant prostate cancer

Abstract Recent advances in our understanding of metastatic castration-resistant prostate cancer (mCRPC) biology have led to the approval and use of multiple novel drugs, including abiraterone, enzalutamide, cabazitaxel, sipuleucel-T and radium-223. Although these agents all provide improvement in survival, resistance always develops after an initial response. To understand the biology of responses and resistance, clinically relevant specimens and preclinical models are needed. To address this critical need, our objectives are: 1) to collect samples of metastases prior to and after treatment for evaluation of potential biomarkers of responses and resistance and 2) to generate patient-derived xenografts (PDXs) that are representative of these novel phenotypes of advanced mCRPC. A Rapid Autopsy Program (RAP) was established at the University of Washington to study the lethal phenotype of mCRPC. Through the RAP we have collected more than 3000 tissue specimens, including bone and visceral metastases from over 110 patients who died of mCRPC. The samples were processed for paraffin embedding and frozen for future histological and molecular analyses. All samples are tracked in a relational database and cross-linked to clinical and pathologic data. To provide clinically relevant preclinical models for studies of late stage advanced prostate cancer, we also implant tissues acquired during the RAP into immunocompromised mice in order to establish novel CRPC PDXs. At present we have 28 primary PDXs (the LuCaP series), and 10 castration-resistant sublines of the primary lines. The LuCaP models have been extensively characterized by light microscopy, immunohistochemistry, expression arrays, RNASeq, IHC, exome sequencing and other methodologies. We have also determined responses of these PDXs to castration and docetaxel. To address the need for models representing the current tumor phenotypes that exist in patients who develop resistance to secondary androgen-targeting therapy, we have also evaluated responses to abiraterone and enzalutamide, and are experimentally developing abiraterone- and enzalutamide-resistant LuCaP PDXs. In addition, we are continuing to generate novel PDXs using abiraterone- and enzalutamide-resistant patient samples, and have four models that were established from abiraterone-resistant tumors. In summary, we have established a large biorepository of mCRPC specimens and PDXs. Moreover, due to the constant evolution of the advanced PCa phenotype we continue to acquire mCRPC specimens and establish novel PDXs that are refractory to the most recent treatments which allow us to investigate biology of late stage mCRPC and to provide valuable clinical specimens and LuCaP PDXs to the PCa research community. Citation Format: Colm Morrissey, Ming H. Lam, Tia S. Higano, Lawrence D. True, Martine Roudier, Robert B. Montgomery, Peter S. Nelson, Paul H. Lange, Evan Y. Yu, Robert L. Vessella, Eva Corey. The importance of clinically relevant rapid autopsy specimens and LuCaP patient-derived xenograft models to interrogate the heterogeneous and evolving treatment resistance of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1718. doi:10.1158/1538-7445.AM2015-1718

Pseudomonas aeruginosa Quorum Sensing Molecule N-(3-Oxododecanoyl)-L-Homoserine-Lactone Induces HLA-G Expression in Human Immune Cells.

HLA-G is a nonclassical class I human leukocyte antigen (HLA) involved in mechanisms of immune tolerance. The objective of this study was to determine whether N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL), a quorum sensing molecule produced by Pseudomonas aeruginosa, could modify HLA-G expression to control the host immune response. We evaluated the ability of 3O-C12-HSL to induce HLA-G expression in primary immune cells, monocytes (U937 and THP1), and T-cell lines (Jurkat) in vitro and analyzed the cellular pathway responsible for HLA-G expression. We studied the HLA-G promoter with a luciferase assay and interleukin-10 (IL-10) and p38/CREB signaling with enzyme-linked immunosorbent assay and immunofluorescence, respectively. We observed that 3O-C12-HSL is able to induce HLA-G expression in human monocytes and T cells. We showed that the induction of HLA-G by 3O-C12-HSL is p38/CREB and IL-10 dependent. 3O-C12-HSL treatment is able to arrest only the U937 cell cycle, possibly due to the peculiar expression of the ILT2 receptor in the U937 cell line. Our observations suggest HLA-G as a mechanism to create a protected niche for the bacterial reservoir, similar to the role of HLA-G molecules during viral infections.

A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas.

Meningiomas are the most common primary intracranial adult tumor. All Neurofibromatosis 2 (NF2)-associated meningiomas and ~60% of sporadic meningiomas show loss of NF2 tumor suppressor protein. There are no effective medical therapies for progressive and recurrent meningiomas. Our previous work demonstrated aberrant activation of mTORC1 signaling that led to ongoing clinical trials with rapamycin analogs for NF2 and sporadic meningioma patients. Here we performed a high-throughput kinome screen to identify kinases responsible for mTORC1 pathway activation in NF2-deficient meningioma cells. Among the emerging top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-deficient meningioma cells, inhibition of SGK1 rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also leads to attenuated mTORC1 but not mTORC2 signaling, suggesting that mTORC2/SGK1 and Rac1/PAK1 pathways are independently responsible for mTORC1 activation in NF2-deficient meningiomas. Using CRISPR-Cas9 genome editing, we generated isogenic human arachnoidal cell lines (ACs), the origin cell type for meningiomas, expressing or lacking NF2. NF2-null CRISPR ACs recapitulate the signaling of NF2-deficient meningioma cells. Interestingly, we observe increased SGK1 transcription and protein expression in NF2-CRISPR ACs and in primary NF2-negative meningioma lines. Moreover, we demonstrate that the dual mTORC1/mTORC2 inhibitor, AZD2014 is superior to rapamycin and PAK inhibitor FRAX597 in blocking proliferation of meningioma cells. Importantly, AZD2014 is currently in use in several clinical trials of cancer. Therefore, we believe that AZD2014 may provide therapeutic advantage over rapalogs for recurrent and progressive meningiomas.

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

73. Effects of the Innate Immune System on Oncolytic Measles Virotherapy

Vaccine strain derived Measles Virus is currently being tested in clinical trials for the treatment of several tumor types. Clinical response to MV therapy can vary, despite similar stages of disease, pre-existing antibody titers and MV entry receptor expression among treated patients. By using Next-Gen Sequencing in tumor samples deriving from ovarian cancer (OvCa) and glioblastoma (GBM) patients who were treated with MV in clinical trials, we observed that expression of interferon-stimulated genes (ISG), such as IFI44, MX1, MX2, RSAD2, and IFI27, correlated with permissiveness to MV infection and clinical outcome. Therefore, we hypothesized that elevated baseline levels of ISG expression directly confers resistance to MV infection in GBM and OvCa patients, and can possibly serve as predictive markers of response to MV therapy. To test this hypothesis we have assessed the baseline ISG expression in 27 primary patient-derived GBM xenograft lines. We found that 4 of the 27 lines have elevated baseline levels of ISG expression. Four primary GBM lines with low baseline levels of ISG expression were infected with MV-NIS (MOI 1 and 0.1). Upon infection, these cells were effectively killed and produced high titers of virus (>104 TCID50/ml by 48hr). In contrast, three primary GBM lines with high baseline levels of ISG expression were resistant to cell killing and produced significantly less virus upon infection with MV-NIS (MOI 1 and 0.1). ISG expression is regulated by the ISGF-3 complex via Stat-1/-2 and Jak-1 activation. Treatment with 3μm of the FDA approved Jak1/2 inhibitor ruxolitinib (Jakafi) for 48hr prior to MV infection (MOI 1 and 0.1) significantly increased MV production and cell killing in the high ISG expressing primary GBM39 line. The observed Jakafi effect is mediated via decreased ISG expression both at baseline and in response to MV infection. Experiments assessing the use of Jakafi in combination with MV in vivo are ongoing. In order to explore an association between ISG expression and MV restriction in OvCa patients, we assessed baseline ISG expression in 118 primary patient-derived ovarian xenograft lines. We found that 57 of the 118 samples have elevated baseline levels of ISG expression. MV therapy in an OvCa xenograft with low baseline levels of ISG expression led to significant prolongation of survival. Ongoing in vivo work in orthotopic ovarian models with variable ISG levels is assessing the association with efficacy of MV therapy. These experiments can lead to elucidation of the role baseline ISG expression plays in controlling response to MV therapy, which could be critical for patient selection and future clinical trial planning. Funding by: Brain SPORE (P50 CA108961), R01 CA154348 and Ovarian SPORE (P50 CA136393)

Abstract P4-04-15: Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression

Abstract Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.

Analyzing the theoretical capacity of railway networks with a radial-backbone topology

In this work we propose a mechanism to optimize the capacity of the main corridor within a railway network with a radial-backbone or X-tree structure. The radial-backbone (or X-tree) structure is composed of two types of lines: the primary lines that travel exclusively on the common backbone (main corridor) and radial lines which, starting from the common backbone, branch out to individual locations. We define possible line configurations as binary strings and propose operators on them for their analysis, yielding an effective algorithm for generating an optimal design and train frequencies. We test our algorithm on real data for the high speed line Madrid–Seville. A frequency plan consistent with the optimal capacity is then proposed in order to eliminate the number of transfers between lines as well as to minimize the network fleet size, determining the minimum number of vehicles needed to serve all travel demand at maximum occupancy.

Hyaluronan expression in primary and secondary brain tumors.

Collectively, primary and secondary brain tumors represent a major public health challenge. Glioblastoma (GBM) is the most common primary brain tumor in adults and is associated with a dismal 5-year survival of only 10%. Breast cancer causes secondary tumors; it occurs in 200,000 patients yearly and 30% of these individuals develop brain metastases which also lead to a very poor prognosis. GBM and primary breast tumors are known to express hyaluronan (HA) which may serve as a therapeutic target. For the present study we had two aims: (I) to identify suitable preclinical models for HA in GBM by examining HA expression in human GBM cell lines implanted orthotopically in mice; and (II) to determine whether breast cancer brain metastases in human patients express HA similar to the primary tumor. Forty human surgical samples of primary breast tumors and breast cancer brain metastases were processed and stained for HA. Athymic nu/nu mice were orthotopically implanted with one of 15 GBM lines and after tumors were established, quantitative immunohistochemistry determined whether. HA was expressed. All GBM cell lines and patient-derived orthotopic tumors did express HA, with 3 primary human lines expressing the highest staining intensity, above that of normal brain. All 40 human primary breast tumors and brain metastases examined also contained HA, though staining intensity was highly variable. Our data support the use of specific patient-derived GBM cell lines in nu/nu mice for preclinical studies on HA-targeting therapies. Additionally, our research provides a basis for the assessment of HA expression and HA-targeting therapeutic agents for the treatment of breast cancer brain metastases.

Neratinib, an irreversible pan-ErbB receptor inhibitor, is highly effective against primary cervical cancer cell lines harboring HER2/neu gene mutations

(OS) and progression-free survival (PFS) were estimated using the Kaplan–Meier method. The associations of smoking and of smoking + treatment (i.e., bevacizumab vs. no bevacizumab) with PFS and OS were evaluated with the Cox proportional hazards model. Results: Smoking questionnaires were available for analysis in 99% of study participants. Two hundred eighty-six women (64%) were active or former smokers. In the entire study population, this group had significantly worse OS than never smokers (14.2 vs. 16.8 months, HR 1.29, 95% CI 1.03–1.61, P= 0.026). Smokers who received chemotherapy plus bevacizumab (n= 148, 52%) experienced significantly longer PFS (median 7.9 vs. 5.7 months, log-rank P b 0.01) and significantly longer OS (median 16.7 vs. 12.0 months, log rank P= 0.027) than smokers who received chemotherapy alone (n= 138, 48%). Overall response rate among smokers was 49.3% (chemotherapy plus bevacizumab) vs. 29% (chemotherapy alone) (P= 0.002). The median number of cycles of therapy was 6 for smokers and nonsmokers (interquartile range, 4–10, smokers vs. 2–9, nonsmokers) (P=ns). Smokers receiving bevacizumab experienced the following adverse events: grade 2+ fistula (16.2%), grade 3+ venous thromboembolism (9.5%), grade 2+hypertension (27%), and grade 2+proteinuria (2.7%). Cervical cancer was the reported cause of death in 74.5% of smokers. Conclusions: Smoking is common and associated with a 29% increased risk of death among women with advanced or recurrent cervical cancer. The survival benefits conferred by antiangiogenesis therapy are sustained, even among smokers in this population. Smokers with CxCA should be informed of these results and efforts at smoking cessation strongly encouraged.

Appropriateness of using patient-derived xenograft models for pharmacologic evaluation of novel therapies for esophageal/gastro-esophageal junction cancers.

The high morbidity and mortality of patients with esophageal (E) and gastro-esophageal junction (GEJ) cancers, warrants new pre-clinical models for drug testing. The utility of primary tumor xenografts (PTXGs) as pre-clinical models was assessed. Clinicopathological, immunohistochemical markers (p53, p16, Ki-67, Her-2/neu and EGFR), and global mRNA abundance profiles were evaluated to determine selection biases of samples implanted or engrafted, compared with the underlying population. Nine primary E/GEJ adenocarcinoma xenograft lines were further characterized for the spectrum and stability of gene/protein expression over passages. Seven primary esophageal adenocarcinoma xenograft lines were treated with individual or combination chemotherapy. Tumors that were implanted (n=55) in NOD/SCID mice had features suggestive of more aggressive biology than tumors that were never implanted (n=32). Of those implanted, 21/55 engrafted; engraftment was associated with poorly differentiated tumors (p=0.04) and older patients (p=0.01). Expression of immunohistochemical markers were similar between patient sample and corresponding xenograft. mRNA differences observed between patient tumors and first passage xenografts were largely due to loss of human stroma in xenografts. mRNA patterns of early vs late passage xenografts and of small vs large tumors of the same passage were similar. Complete resistance was present in 2/7 xenografts while the remaining tumors showed varying degrees of sensitivity, that remained constant across passages. Because of their ability to recapitulate primary tumor characteristics during engraftment and across serial passaging, PTXGs can be useful clinical systems for assessment of drug sensitivity of human E/GEJ cancers.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma

AbstractWe studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)–DLBCL compared with activated B-cell (ABC)–DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the “gold standard” GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

The effect of valproic acid in combination with irradiation and temozolomide on primary human glioblastoma cells

Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival of less than 2 years. While there have not been any novel anti-GBM therapeutics approved for many years, there has been the gradual accumulation of clinical data suggesting that the widely used anti-convulsant agent, valproic acid (VPA) may significantly prolong survival in GBM patients. This pre-clinical study aimed to determine the potential clinical utility of VPA in the treatment of GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination with temozolomide and irradiation. At clinically achievable concentrations, VPA was shown to be effective as a monotherapy agent in the five primary lines tested. VPA was then used as a sensitizing agent to in vitro radiation and showed significant augmentation of in vitro irradiation therapy. In addition, when VPA, radiation and temozolomide were combined an additive, rather than synergistic effect was noted. Gene expression profiling demonstrated close clustering of triple treated cells with VPA mono-treated cells while untreated cells clustered closer with TMZ-irradiation dual treated cells. These microarray data suggest a dominant role of VPA at the gene expression level when combining these different treatment options. Moreover, in an in vivo tumor transplantation model, we were able to demonstrate an increase in animal survival when cells were pre-treated with irradiation-VPA and when triple treated. These findings provide a significant rationale for the investigation of VPA in the treatment of GBM patients.