bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4046: Kinetic quantification of caspase-3/7 mediated apoptosis using live-cell imaging
Authors: Appledorn Daniel M. , Nelson Thomas , Dale Tim , Alcantara Susana , Artymovich Katherine , Endsley Eric W. , Trezise Derek J. , Groppi Vince E.
Published: August 2012

Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that typically results in the activation of executioner caspase-3. Numerous in vitro assays have been designed to measure the activation of caspase-3. The majority of these assays utilize reagent substrates that incorporate the DEVD motif which is recognized by both activated caspases 3 and 7. Such assays result in single, user-defined time point measurements of caspase-3/7 activity and typically require multiple wash steps or cell lifting prior to data collection; potentially resulting in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions. In this study, we optimized an assay system incorporating a bi-functional reagent containing the DEVD motif linked to a DNA dye for use on the IncuCyte FLR imaging system. When added to cell culture growth medium, this relatively inert, non-fluorescent molecule crosses the cell membrane where it is available to be a substrate for activated caspase-3/7. The reaction product is a green fluorescent DNA dye that labels the nuclei of cells undergoing apoptosis. Our data show that this strategy can quantitatively measure the kinetic activation of caspase-3/7 through extrinsic stimulation of human tumor derived MDA-MB-231 breast adenocarcinoma cells, HeLa cervical adenocarcinoma cells, A549 lung epithelial carcinoma cells, and HT 1080 fibrosarcoma cells following treatment with TNFα and cycloheximide. Moreover, we exemplify the use of this strategy in 96-well plate screening protocols using both HT 1080 and MDA-MB-231 cells to measure the induction of caspase-3/7 activity in the presence of staurosporine, a well-known general protein kinase inhibitor and inducer of caspase-3/7 mediated apoptosis with Z’ values α0.6. We provide evidence that this stable, kinetic, mix-and-read assay can provide both quantitative and qualitative assessments of caspase-3/7 activity using fluorescent images and high-definition phase contrast images, respectively.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4046. doi:1538-7445.AM2012-4046
Abstract 4049: Early prediction of response to targeted drugs treatment in malignant lymphoma and sarcoma using in vivo proliferation marker 18F-FLT
Authors: Li Zhoulei , Graf Nicolas , Herrmann Ken , Pirsig Sabine , Schwaiger Markus , Scheidhauer Klemens , Buck Andreas , Keller Ulrich , Dechow Tobias
Published: August 2012

Purpose: Last year we have shown that 18F-FLT is superior surrogate marker than 18F-FDG for very early response assessment to therapy in malignant Lymphoma. In this study, we use FLT-PET to predict the response of ewing sarcoma to different cancer therapy and investigate its relation to cancer cell death, especially apoptosis. Methods: FLT and/ or FDG PET were performed prior and early in each therapeutic course on SCID mice bearing with various tumor cells. Ewing sarcoma A673 was applied for investigation of the response to sorafenib treatment or Radiotherapy. GFP labelled Lymphoma cells (Myc449, Myc665) with or without transportation of Bcl2 gene was employed for the observation of cell death especially apoptosis after the treatment with aura kinase inhibitor or chemotherapy. Tumor-to-background ratios (TBR) of FLT-PET were compared to that of FDG-PET. PET findings were correlated with optical Imaging, histopathology and in vitro data comprising cellular tracer uptake, cell cycle related protein expression, cell cycle distribution and viability assessment. Results: In vitro study shows that sarcoma A673 cells were sensitive to all therapy. For in vivo studies, tumor volume of sorafenib treated animals showed modest increase of 0.1cm3 (range 0.02cm3 to 0.2cm3, SD=0.09) within the first week as opposed to a 15-fold increase in control mice. Median TBR of FLT-PET decreased significantly to 30.1% compared to baseline as early as 5d after therapy (range 11.3-53.5%, n=12, p=0.0004). On the other side, TBR in FDG-PET also showed the similar tendency (median TBR 74.6%, range 28.1%-131.8%, n=12, p=0.02). But the reduction of TBR in FLT was significantly stronger than that in FDG (p<0.001). In vitro study of lymphoma cells with Bcl2 transduction showed a strong anti- cell death ability, which will be served for the link between FLT PET and cell death. Conclusion: Compared with FDG-PET, FLT-PET is able to predict the response in the early therapeutic course and has a high specificity for the detection of ewing sarcoma. Continuing studies are intensively carried out to investigate FLT and FDG PET in relation to therapy-induced cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4049. doi:1538-7445.AM2012-4049
Abstract 4051: A mouse model of head and neck cancer with infrared-based molecular imaging
Authors: Mouchli Anas , Han Ziying , Zheng Jun , Lajud Shayanne A. , O'Malley Bert W. , Li Daqing
Published: August 2012

Introduction Head and neck cancer (HNC) is notoriously difficult to treat and the five-year survival rate has improved very little over the past thirty years. Head and neck squamous cell carcinomas (HNSCC) mouse models are invaluable tools for furthering mechanistic HNC research. Molecular imaging (MI) is a promising new modality that may improve current HNC mouse models. Visible florescent proteins (VFPs) have been widely used with animal models for tumor detection using MI. However, the low emission wavelength of VFP results in its overlapping with the auto-fluorescence of many tissues and causes insufficient penetration into deeper tissues, preventing the application of VFP to track local and metastatic tumor progressions. The recent development of infrared-fluorescent protein (IFP) with Biliverdin as a chromophore has greatly advanced in vivo molecular imaging of deep tissues in animals. Hypothesis The ultimate goal of this study is to develop a novel orthotopic mouse model of HNC with regional spreading and distant metastasis tumors that closely mimics human HNC and can be accurately detected by a noninvasive IFP-based molecular imaging (MI) system. Methods A stable human HNC cell line expressing IFP was established. This stable cell line was used for generating a mouse model with HNC. The tumor formation and tumor progression with this model were accurately monitored by a MI system. Results The IFP signal was clearly detected in our mouse model of HNC. Scanned imaging demonstrated a progression pattern of tumor growth in this model. The tumor can be detected earlier with molecular imaging than by conventional external caliper measurement. Unlike surgical measurement, the tumor can be quantified without disturbing the tumor environment. More important, our data suggest that local-regional metastasis tumors can be detected with this model. Conclusion This novel animal model represents an orthotopic human HNC model. The IFP signals penetrate tissues well and can be detected in the deep tissues with this model. Thus, our model has the potential to be used for monitoring tumor spreading and metastasis of advanced stage HNC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4051. doi:1538-7445.AM2012-4051
Abstract 4052: Highlighting intratumoral heterogeneity and peritumoral areas in human melanoma biopsies by infrared spectral microimaging
Authors: Sebiskveradze David , Gobinet Cyril , CARDOT-LECCIA Nathalie , ORTONNE Jean-Paul , MANFAIT Michel , JEANNESSON Pierre , PIOT Olivier
Published: August 2012

Infrared (IR) spectral microimaging is an efficient label-free optical method to analyze biological samples and to provide information about their intrinsic biochemical composition. Recent studies have shown its potential to detect and characterize cancerous tissues in their early stages, independently of visual morphology. IR microimaging appears more informative than conventional histology and could thus be developed as a sensitive, non-destructive and objective diagnostic tool in clinical oncology. The discrimination between tumoral and surrounding tissues relies on highlighting subtle IR spectral differences. An innovative fuzzy clustering based algorithm was thus developed in order to introduce the notion of nuance into the clustering of IR image pixels and to automatically determine the optimal clustering parameters, particularly the number of clusters. IR spectral imaging has been directly acquired on formalin-fixed paraffin-embedded tissue sections of human cutaneous melanomas with different degrees of invasiveness (n=10 patients). After the application of the fuzzy algorithm, the reconstructed color-coded clustering images allow recovering different histological structures automatically, particularly to precisely localize tumoral areas and their normal counterparts. However, more than reproducing classical histology, our algorithm can give access to additional information about the tissular structures. The notion of nuance introduced by fuzzy clustering is valuable for the pixels located at the interface between tumoral tissue and its microenvironment. Heterogeneous transitional areas between tumor and marginal normal tissue are identified, what is not possible on hematoxylin-eosin stained section or by conventional clustering. In addition, this automatic approach of IR data processing permits to highlight the interconnectivity between heterogeneous intratumoral areas and between tumoral and peritumoral structures. These preliminary results show significant potential for probing tumor progression and for early determination of tumor aggressiveness in cutaneous melanomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4052. doi:1538-7445.AM2012-4052
Abstract 4056: Gaussia luciferase complementation demonstrates that secreted CXCL12 (SDF-1) forms dimer under physiologic conditions
Authors: Ray Paramita , Lewin Sarah A. , Mihalko Laura Anne , Lesher-Perez Sasha Cai , Takayama Shuichi , Luker Kathryn E. , Luker Gary D.
Published: August 2012

Chemokine CXCL12 signaling through receptors CXCR4 and/or CXCR7 promotes tumor growth and metastasis in more than 20 different human malignancies, establishing CXCL12 as a promising therapeutic target in cancer. Under physiologic conditions, many chemokines form homodimers, which may activate signaling pathways distinct from those regulated by monomers of the same chemokine. Prior studies with recombinant, mutant forms of CXCL12 engineered to form either monomers or dimers show that monomeric and dimeric CXCL12 preferentially activate different downstream signaling molecules. However, the extent to which CXCL12 forms homodimers under physiologic conditions and resultant effects on CXCR4 and CXCR7 signaling remain to be determined. To analyze dimerization of CXCL12 secreted by mammalian cells, we used bioluminescence imaging of CXCL12 fused to either full length or complementation fragments of Gaussia luciferase (GL). Using column chromatography and GL protein fragment complementation, we demonstrated that CXCL12 is secreted from mammalian cells as both monomers and homodimers, and the homodimer remains stable in the extracellular environment. In 3D spheroid cultures, we also established that CXCL12 secreted from two independent cell populations can form homodimers in the extracellular space. Relative to dimeric CXCL12, monomeric CXCL12 preferentially signaled via CXCR4 to Gαi proteins to activate AKT and suppress intracellular cAMP. By comparison, dimeric CXCL12 was more potent at recruiting α-arrestin 2 to CXCR4 and regulating chemotaxis CXCR4+ breast cancer cells. In cell-based assays and an orthotopic tumor xenograft model of human breast cancer, we also found that CXCR7 preferentially sequestered monomeric CXCL12 and had minimal accumulation of the dimer. From these studies, we conclude that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions, and monomeric versus homodimeric CXCL12 regulate distinct signaling pathways. These results inform ongoing efforts to target CXCL12 and its receptors for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4056. doi:1538-7445.AM2012-4056
Abstract 4058: Multispectral imaging of mismatch repair proteins in colorectal cancers and tumor cells isolated from blood
Authors: Zhou Lanlan , Yang Zhaohai , Baker Maria , Dicker David T. , El-Deiry Wafik S.
Published: August 2012

DNA mismatch repair (MMR) is a system for recognizing and repairing erroneous insertion, deletion and misincorporation of bases that can arise during DNA replication and recombination, or some forms of DNA damage. MMR is required for maintaining genomic stability and is highly conserved from prokaryotes to eukaryotes. MMR defects are found in about 15% of sporadic colorectal cancer and in more than 90% of hereditary non-polyposis colorectal cancer (HNPCC)-associated tumors. Detection of defective MMR through analysis of loss of MMR protein immunostaining in colorectal carcinomas stratifies the risk of having an inherited cancer predisposition syndrome and identifies patients who might benefit from subsequent genetic testing and counseling. Multispectral imaging of colorectal cancer (CRC) HCT116, HT29, SW480 and SW620 cells cultured in chambered slides with multiplexed different color quantum dots (Qdots) conjugated MMR proteins MLH1, MSH2, MSH6 and PMS2 shows that HCT116 cells harbor defective MLH1 and PMS2, which is consistent with the finding from conventional single protein immunofluorescence staining. Multispectral imaging of CRC xenografted tumor sections with Qdot-conjugated antibodies to MMR proteins demonstrates that the human colorectal HCT116 tumor xenograft has MLH1 and PMS2 defects, consistent with individual protein immunohistochemical staining. To explore the application of multispectral imaging of MMRs in circulating tumor cells (CTCs), we spiked CRC cells into human blood and recovered CRC cells with the FDA-approved Veridex CellSearch system. Multispectral imaging of recovered CRC cells displayed similar MMR expression spectrum to conventional single protein immunofluorescence staining. The methodology may provide an adjunctive screening tool for detection of MMR defects in CTCs from patients with advanced disease. The utility of this novel assay for detection of MMR gene expression abnormalities will be further examined in the colorectal cancer clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4058. doi:1538-7445.AM2012-4058
Abstract 4063: Image-guided fluorescent endomicroscopy of glioblastoma with SVV-GFP viral labeling
Authors: Wong Kelvin , Alvarado Miguel Valdivia y , Huang Yulun , Zhao Xiumei , Wong Stephen , Li Xiao-nan
Published: August 2012

We propose a novel method to improve detection of infiltrative microscopic GBM tumors during surgery, by labeling the infiltrative tumor cells in vivo by a single intravenous injection of a highly specific tumor seeking fluorescence virus, Seneca Valley Virus (SVV-GFP). The feasibility of this approach was demonstrated in a permissive pediatric GBM (IC-2305GBM) and an adult GBM (IC-A46GBM) orthotopic xenograft mouse model. Both models were derived from primary tissue of GBM from patients to preservethe histopathological and immunophenotypical characteristics of the original tumors. After establishing the xenograft tumors, SVV-GFP was injected intravenously at a dose of 5x1014 viral particles/kg. The animals were imaged at day 3 after SVV-GFP virus injection. During the imaging procedure, the animals were euthanized through anesthesia overdose and a small window was immediately drilled in the skull to introduce a 1mm outer diameter fiberoptic fluorescence probe through the tumor. Fluorescent labeled cells were recorded as a real-time video sequence along the track of the probe. Image quantification starts with low frequency temporal drift correction using Fast Fourier Transform based lowpass filtering. Air image frames (before probe insertion) were used as a zero reference and the intensity range of the video is shifted accordingly. A normal tissue fluorescence image was then estimated using a median statistics of all image frames acquired in normal brain tissue. This image was used to correct for lower detection sensitivity near the probe edge or any field inhomogeneity. The video sequence was then normalized to the normal tissue image to create a TBR map. By definition, air is used as reference with TBR=0 and normal tissue is at TBR=1. A cutoff of TBR>1.1 is used and tumor cells exceeding that threshold were classified as GFP-positive tumor cells. Fractional area occupied by the virus-labeled microscopic tumors were computed with the TBR exceeding 1.1 within the field of view. A low fractional area (<0.01%) was found in normal brain tissue using this method, demonstrating that our method is robust. To summarize, we demonstrated an innovative approach for microscopic tumor detection in glioblastoma that is two to three orders of magnitude smaller than those currently detected with in vivo imaging modalities.This method can potentially be applied to detect microscopic tumors in metastatic retinoblastoma, medulloblastoma, endocrine tumors and small cell lung cancer which are shown to be permissive to infection of this virus.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4063. doi:1538-7445.AM2012-4063
Abstract 4068: A novel microfluidic platform for isolation of circulating tumor cells intended to facilitate downstream biomarker analyses
Authors: Conant Carolyn G. , Nevill Tanner , Ionescu-Zanetti Cristian , Schwartz Micahel
Published: August 2012

Circulating tumor cells (CTCs) inherently hold important biomarker information potentially pertinent to cancer patient therapy and prognosis. Many existent technologies capture CTCs inside of a ‘chip’. Because of the difficulties in efficiently removing very small numbers of CTCs from ‘chips’, these technologies have limited potential for downstream (off-chip) analysis opportunities. We have developed a novel microfluidic system for high purity CTC isolation and analysis specifically for post-capture CTC use. Using our device, analytical studies were performed with cell lines (HT29, SKBR3, PC3) to demonstrate recovery and purity capabilities. We isolated CTCs with high purity (from 1.5- 50%) and high capture efficiency (up to 96%) in volumes as low as 5 µl. Clinical samples from stage IV carcinoma patients of several different pathologies were processed and benchmarked against the predicate system. We found, in all cases, CTCs could be identified even when not identified on the predicate system. Further clinical samples indicated in some cases CTCs could be detected as early as stage II. A technique designed to identify somatic mutations in the KRAS oncogene was employed successfully to characterize samples isolated on the device from colon carcinoma patients. Fluorescence in situ hybridization (FISH) was used to characterize Her-2 and Top2A gene status in CTCs isolated from breast carcinoma patients. The use of this device is likely extensible to a wide variety of molecular biology and biochemistry-based biomarker studies which previously have been precluded due to insufficient CTC sample quality.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4068. doi:1538-7445.AM2012-4068
Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation
Authors: Ball Corbie , Ramireddy Bommireddy , Keenan Michael , Stern Stephen , Azhar Mohamad , Gard Constance , Besselsen David G. , Doetschman Thomas
Published: August 2012

Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+/+ mice and ran Real Time-PCR or Western Blot for different Toll-like Receptor Pathway components. Results: qRT-PCR showed that Smad3−/-− mice have a significantly lower quantity of Bacteroides sp; the introduction of Helicobacter hepaticus results in a significant increase in B. distasonis and a significant decrease in B. thetaiotaomicron in Smad3−/− mice. These data suggest that loss of SMAD3 and the addition of Helicobacter hepaticus alters the colonic microflora. Previous studies illustrated the importance of epithelial signaling in microbial induced inflammation. To examine if Smad3 is altering the Toll-like receptor pathway we examined RT-PCR of the cecum and found a significant increase in Tlr4, Irak4, Cd14, Myd88, Nfkb, Cox2, and Nos2 mRNA in the Smad3−/−− mice compared to Smad3+/+ mice. Western blots confirmed these data by showing that cecal tissue from Smad3−/− mice has increased IRAK4 and pNF-κB. Conclusion: Smad3−/− mice show heightened Toll-like receptor pathway activity which leads to a hyper-inflammatory response of the mucosa to endogenous microbes. Further, loss of SMAD3 results in dysbiosis of the gut microbiota. These findings suggest that SMAD3 plays a role in microbial-host homeostasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 407. doi:1538-7445.AM2012-407
Abstract 4073: Comprehensive cell health analysis on a novel multi-parametric cell analyzer
Authors: Gillis Katherine , Khan Asima , Clor Julie , Tyagarajan Kamala
Published: August 2012

The study of anti-cancer compounds and cancerous cell lines requires access to cell health information that includes parameters such as cellular vitality, cell cycle and apoptosis and provides a complete picture of cell health. Traditionally, expensive and complicated instrumentation has been required for comprehensive cell health analysis. Here we present data from a novel, low-cost, easy to use, compact, high performance instrument, the Muse Cell Analyzer which provides multi-dimensional cell health information. The instrument utilizes a combination of highly simplified mix and read assay protocols with a touch-screen interface which greatly simplifies data acquisition and analysis. The objective of this study was to evaluate performance of Muse Count and Viability Assays when compared to traditional predicate methods including hemacytometers and automated image based systems. The mix and read format of the Muse™ Count & Viability assay provides quantitative detection of multiple suspension cell lines (e.g. Jurkats, Hl60s, K562s) and adherent cell lines (e.g. CHOs, PC3s, MCF-7s) across a wide range of concentrations and viabilities. Comparison of count and viability data from multiple platforms indicate that count and viability information on a vast variety of cancer cell lines can be robustly and accurately obtained on the Muse Cell Analyzer and the system depicts good linearity over a wide concentration range. Superior precision was obtained on the Muse Cell Analyzer for a variety of sample types with %CV ranging from 1-8% compared to %CV of 5-20% for predicate methods on the same samples. Cytotoxicity effects of anti-cancer compounds such as staurosporine, gambogic acid etc.on multiple cell types could be reliabily obtained on the instrument platform. The mix and read format of the Annexin V & Dead Cell assay and simplified Cell Cycle protocol on the instrument further allows for the easy and reliable study of impacts of anti-cancer compounds. The combination of three critical assays for cell count and viability, apoptosis and cell cycle status on a simple low cost instrument makes the Muse Cell Analyzer a versatile and accurate solution for comprehensive cell health analysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4073. doi:1538-7445.AM2012-4073
Abstract 408: Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells
Authors: Bhaskara Vasantha Kumar , Mohanam Indra , Rao Jasti S. , Mohanam Sanjeeva
Published: August 2012

Neuroblastoma is the most common extracranial pediatric solid tumor that is derived from the developing sympathetic nervous system and results from the improper differentiation of neural crest cells. Neuroblastomas show a tremendous clinical heterogeneity ranging from benign ganglioneuromas to highly aggressive immature tumors. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. Intermittent hypoxia (IH) which is characterized by cyclic periods of hypoxia and reoxygenation, occurs in tumor cells that are dependent on tumor blood vessels having intermittent perfusion fluctuations in blood flow. The occurrence of IH episodes varies significantly in rapidly growing malignant tumors. Bone is one of the target organs of metastasis in advanced neuroblastoma. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by the osteoclast. The present study focuses on how IH-conditioned neuroblastoma cells modulate the differentiation of osteoclasts. IH-conditioned cells were derived by exposing tumor cells to 10 repeated cycles of hypoxia followed by reoxygenation. In order to assess the role of HIF-1α overexpression in osteolysis, we stably transfected human neuroblastoma cells with an expression vector containing a HIF-1α cDNA. Additionally, cells were stably transfected with a plasmid expressing HIF-1α shRNA or luciferase shRNA and selected in neomycin. The expression of HIF-1α protein was analyzed by western blotting in IH-conditioned and stable transfectants of neuroblastoma cells. Changes in gene expression were determined by real-time PCR for IL-8, MCP-1α, PTHrP, CXCR-4 and RANKL and the expression of osteoclastogenic factors was found increased in IH-conditioned and HIF-1α protein overexpressing cells. Conditioned medium collected from IH-conditioned and HIF-1α cDNA overexpressing neuroblastoma cells has shown enhanced osteoclast formation capabilities in the tartrate-resistant acid phosphatase (TRAP) assay. IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells secrete increased amounts of VEGF protein, which has been found to promote osteoclast activity. Calcium-sensing receptor (CaR) plays a pivotal role in osteoclast differentiation. We found enhanced expression of CaR in RAW 264.7 (osteoclast precursors) cells treated with conditioned medium collected from IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells compared with the control. Thus, IH was found to enhance osteolytic capabilities of neuroblastoma cells in vitro in part through HIF-1α protein stabilization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 408. doi:1538-7445.AM2012-408
Abstract 4090: Epigenetic profiling identifies differentially methylated loci in metastatic vs. non-metastatic primary squamous and basal cell skin cancers
Authors: Colacino Justin A. , Tang Alice , Moyer Jeffrey , Rozek Laura
Published: August 2012

Nonmelanoma skin cancer is the most commonly diagnosed cancer in the United States. Over 2 million new cases of these cancers are diagnosed each year, and incidence rates are increasing worldwide. Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin are generally considered less aggressive than melanomas, which is reflected in their lower relative mortality rates. Despite this, a clinically relevant subgroup of both of these cancers will metastasize and develop an aggressive disease phenotype. In order to characterize the molecular pathways that may be involved in the development of metastatic disease in these common cancers, we measured DNA methylation profiles at 1505 CpG sites associated with 807 related to cancer using the Illumina Goldengate Methylation Cancer Panel BeadArray in BCCs and SCCs isolated from patients treated at the University of Michigan. DNA methylation was measured in metastatic (n=9) and non-metastatic (n=19) primary SCCs and in metastatic (n=3) and non-metastatic (n=9) primary BCCs. Due to the known divergent mechanisms of carcinogenesis of basal and squamous cell carcinomas, separate analyses were conducted for these tumor types. Unsupervised hierarchical clustering was used to identify overall differences in DNA methylation patterns between metastatic and non-metastatic disease. Gene specific analyses using linear modeling coupled with an empirical Bayes method of variance estimation were conducted to identify if methylation at individual CpG sites is associated with metastatic potential. SCCs were not found to cluster based on metastasis, however, metastatic BCCs were found to cluster together, suggesting that metastatic BCCs may share common mechanisms of pathogenesis. In gene specific analyses, FRZB was found to be statistically significantly hypermethylated in metastatic compared to non-metastatic primary SCCs (median methylation: 54.2% vs. 4.7%; p=3.8x10-5). Two sites in MYCL2 were found to be significantly hypomethylated in metastatic vs. non-metastatic BCCs (3.7% and 4.0% vs. 77.9% and 53.5%; p=1.87x10-6 and 3.69x10-5 respectively). The results from this pilot study give insight into the molecular mechanism of metastasis for nonmelanoma skin cancers and identify the divergent characteristics of BCCs and SCCs. The differentially methylation regions identified in this study may be useful epigenetic biomarkers of metastatic potential for nonmelanoma skin cancers to be examined in an extended patient population in future studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4090. doi:1538-7445.AM2012-4090
Abstract 410: NLRP3 is involved in tumor development by regulating immune function
Authors: Le-Bitoux Marie-Aude , Stamenkovic Ivan
Published: August 2012

The NLRP3 inflammasome is a multiprotein cytoplasmic complex that mediates the processing of pro-inflammatory interleukin-1β (IL-1β), IL-18 and possibly IL-33, via the activation of Caspase-1. While NLRP3 has been widely described in the pathogenesis of inflammatory diseases, its role in tumor-host interactions, where inflammation is known to be a major player, has yet to be fully elucidated. We observed that DMBA carcinogen treatment that induced large and numerous papillomas in the skin of wt mice induced barely visible lesions in NLRP3 deficient counterparts. Similarly IV injection of B16F10 melanoma cells resulted in multiple lung metastases in wt mice but virtually no metastatic growth in NLRP3-deficient animals. The NLRP3 inflammasome thus appears to be a key helper for tumor cell growth and survival at both primary and secondary sites. Bone marrow transplantation revealed that leukocyte-derived NLRP3 plays a key role in supporting tumor cell survival prompting us to compare the immune cell distribution and function in NLRP3 deficient mice to those in wt littermates, in both the presence and absence of tumor growth. Flow cytometry analysis of immune cell populations in these mice allowed us to link NRLP3 to the recruitment, adaptation and ability of the immune system to target tumor cells. We observed that compared to wt animals, NLRP3 KO mice had fewer CD3+CD4+ T cells, NK cells and F4/80+CD11c+ myeloid cells but more CD11b+Gr1+ cells, B lymphocytes and F4/80+CD206+ macrophages of the M2 phenotype in the spleen. The increased numbers of myeloid derived suppressor cells (MDSC) and M2 macrophages in mice that displayed decreased tumor growth was counterintuitive but was associated with functional impairment that could explain decreased permissiveness of the microenvironment for tumor growth. Our observations provide a framework to investigate the mechanistic implication of NLRP3 in the relationship between tumor cells and their microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 410. doi:1538-7445.AM2012-410
Abstract 4102: Histone deacetylase inhibitor, vorinostat, decreases transcription of survivin and is an enhancer of gemcitabine therapy for resistant pancreatic tumors
Authors: Green Leila J. , Koshkina Nadezhda V. , Curley Steven A.
Published: August 2012

Introduction: Overall 5-year survival rates of patients with pancreatic cancer remains below 15% due to delay in diagnosis and chemotherapeutic resistance. Combinational therapy has become the mainstay of treatment in patients who have metastatic pancreatic cancer. We hypothesize that 1) vorinostat (suberoylanilide hydoxamic acid), a histone deacetylase inhibitor, will improve efficacy of gemcitabine in gemcitabine-resistant pancreatic tumor cells; and 2) vorinostat works through decreasing the transcription inhibitors of apoptosis proteins (IAPs). Methods: Six different human pancreatic cell lines were treated with various levels of vorinostat with or without gemcitabine. Viability of tumor cells was determined via MTT assay and cytoplasmic levels of IAPs, including survivin, were analyzed via western blot assay. Real-time RT-PCR was performed on cell lysates to determine changes in survivin gene expression. Results: Combinations of vorinostat with gemcitabine significantly inhibited pancreatic cancer cells growth in vitro when compared with either treatment alone (p<0.05). Screening of IAPs expression in tumor cells after exposure to vorinostat revealed variation of their expression in pancreatic tumor cells except survivin, whose expression declined consistently in all cells after vorinostat treatment. Overall cytoplasmic levels of survivin were seen to decline across all cell lines treated. Real-time RT-PCR showed a decrease in survivin gene expression in all cell lines tested as compared to control. Conclusion: We conclude that 1) vorinostat increases the efficacy of gemcitabine on pancreatic cells; and 2) decline of survivin protein levels in after exposure to vorinostat is due to a decrease in gene transcription and indicates that survivin plays an important role in improving the chemotherapeutic effect of gemcitabine. We are currently conducting in vivo experiments to verify our findings using orthotopic pancreatic tumor animal models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4102. doi:1538-7445.AM2012-4102
Abstract 4107: Possible role of autophagy inhibition in hypoxia-induced chemoresistance of pancreatic cancer cells
Authors: Ko Yoon Ho , Won Hye Sung , Jeon Eun Kyoung , Hong Young Seon , Cho Young-Seok
Published: August 2012

BACKGROUND: Autophagy is a catabolic process and provides metabolic support for the cell by degradation of intracellular macromolecules. Various types of stress, including hypoxia, activate autophagy. Recent studies have suggested that hypoxia has been shown to associate with resistance to chemotherapy and radiotherapy and hence poor prognosis in pancreatic cancer. This study investigated the role of autophagy in the treatment of pancreatic cancer with gemcitabine under hypoxic condition. METHODS: To evaluate the role of autophagy inhibition in hypoxia-induced chemoresistance, BxPC-3 human pancreatic cancer cell line was used under normoxic and hypoxic conditions. We evaluated the extent of LC3-II, as an autophagosome marker, by Western blotting to measure the hypoxia- or chemotherapy-induced autophagy. We then examined the effects of gemcitabine on induction of apoptosis under normoxic and hypoxic conditions. Finally, to determine the effect of 3-MA, a known inhibitor of autophagy, on overcoming hypoxia-induced chemoresistance, the MTS assay and flow cytometry were performed.RESULTS: Compared with normoxia, gemcitabine-induced cell death was significantly decreased under hypoxia, as a result of the reduced apoptosis. Western blotting analysis demonstrated that LC3-II was increased under hypoxia, compared with normoxia. However, we found that 3-MA can enhance the growth inhibition and apoptotic effect of gemcitabine, even under hypoxia. These findings mean that autophagy mediates the chemoresistance under hypoxia. Conclusions: Activated autophagy plays a role in hypoxia-induced chemoresistance of pancreatic cancer cells. These findings may have important implications for future therapeutic strategies using gemcitabine against pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4107. doi:1538-7445.AM2012-4107
Abstract 411: PI3 Kinase gamma control of Arginase-1 expression promotes tumor immunosuppression
Authors: Schmid Michael C. , Foubert Philppe , Varner Judith A.
Published: August 2012

Tumor inflammation promotes angiogenesis, immunosuppression, and tumor growth. Tumor associated myeloid cells, which include incompletely differentiated myeloid derived suppressor cells, macrophages and granulocytes, establish an immunosuppressive microenvironment in tumors through the expression of Arginase-1, which depletes the tumor microenvironment of arginine, thereby promoting T cell death and NK cell inhibition. Suppression of Arginase-1 expression and function could alter the course of cancer therapy by stimulating anti-tumor immunity to work in concert with anti-tumor vaccine or chemotherapeutic agents targeting tumor cell proliferation and survival. We found that myeloid cells express Arginase-1 in response to IL-6 stimulation in a Jak2/Stat3 and PI3Kinase gamma/mTORc1 dependent manner by promoting the activation of the transcriptional enhancer C/EBPbeta. Pharmacological or genetic blockade of Jak2, Stat3, PI3Kgamma, mTOR or C/EBPbeta suppressed expression of Arginase-1. Suppression of PI3Kgamma or Arginase-1 expression blocked myeloid cell induced death of T cells in vitro. PI3Kgamma inhibition blocked Arginase-1 expression in vivo, thereby increasing the number of CD8+ T cells in tumors, stimulating T cell-mediated cytotoxicity of tumor cells in vivo and suppressing growth and metastasis of implanted and spontaneous tumors. These studies reveal that suppression of Arginase-1 expression with PI3Kgamma or Jak2/Stat3 inhibitors can serve as an effective therapeutic strategy in oncology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 411. doi:1538-7445.AM2012-411
Abstract 4110: Measurement of autophagy in sensitive versus resistant breast cancer cells
Authors: Shajahan Ayesha N. , Olson Rory , Webb Mathew , Samoy Jennifer , Zielinski John , Mullenix Michael , Clarke Robert
Published: August 2012

In 2011, there will be almost 192,000 newly diagnosed cases of invasive breast cancer in the U.S. with estrogen receptor positive ≤ positive (ER+) tumors. For these patients, endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), is the least toxic and most effective means to manage their hormone-dependent breast cancer. Tamoxifen (TAM) produces a 26% proportional reduction in mortality, however, advanced ER+ breast cancer largely remains an incurable disease and resistance to endocrine therapy remains a significant clinical problem. We and others have shown that antiestrogen resistant breast cancer cells have higher level of basal autophagy, a process that allows the cells to survive stress by digesting its own organelles. However, the precise role of this sustained autophagy in antiestrogen resistance is unclear and the measurement of autophagy flux, the dynamic measurement of passage of substrates through the autophagic pathway, is essential to determine levels of autophagy in a cell. In this study, we investigate the rate of autophagosome formation and degradation in antiestrogen sensitive versus antiestrogen resistant breast cancer cells. Autophagosome formation was measured using autophagosome specific fluorescent dye incorporation (Cyto-IDTM). Progression of autophagy was indicated by the degradation SQSTM1/p62 and NBR1 in cell lysates as measured by colorimentric enzyme immunoassays (ELISAs). The ELISAs were validated to provide biologically relevant sensitivity, dilutional linearity, reproducibility and recovery in native samples. Cells further progressing into apoptosis and cell death were identified by fluorescence using a combination of yellow emitting dye labeled Annexin V and a red emitting viability dye (GFP-Certified™Apoptosis/Necrosis detection kit). We hypothesize that combination of sustained autophagosome formation and inhibition of degradation are crucial in promoting pro-survival autophagy in antiestrogen resistant breast cancer cells. We show that while autophagosome formation and concurrent degradation of SQSTM1/p62 and NBR1 leads to cell death in sensitive cells, resistant cells have higher levels of autophagosomes, lower levels of degradation of SQSTM1/p62 and NBR1 and decreased cell death. Our data will highlight the importance of determining autophagy flux in pro-death versus pro-survival autophagy in drug resistance in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4110. doi:1538-7445.AM2012-4110
Abstract 4116: Mammary adipocyte-specific metabolic alterations are associated with paracrine effects on mammary tumorigenesis
Authors: Volden Paul A. , Wonder Erin L. , Skor Maxwell N. , Carmean Christopher M. , Ye Honggang , Kocherginsky Masha , McClintock Martha K. , Brady Matthew J. , Conzen Suzanne D.
Published: August 2012

In rodent models of breast cancer, chronic social stress from isolated housing is associated with increased mammary tumor growth. Interestingly, in several diverse mouse strains, social isolation is also associated with increased expression of the key metabolic genes Acaca, Hk2 and Acly in the adipocyte fraction of the mammary gland. We hypothesized that these gene expression changes would be associated with increased mammary fat glucose metabolism, lipid synthesis, and secretion of paracrine growth promoting adipokines. We found that increased metabolic gene expression in mammary fat did indeed increase both lipid metabolism and leptin secretion from the mammary adipocytes of isolated mice. Surprisingly, visceral fat metabolism and systemic metabolic parameters were unchanged in social isolates, suggesting an effect of social stress that is specific to the mammary fat depot. When mouse mammary cancer cells were treated with culture media derived from the incubation of isolated versus group-housed mouse mammary adipose tissue, the mammary fat secretome of social isolates resulted in significantly increased cancer cell proliferation compared to mammary fat from grouped mice. Current experiments are designed to elucidate the metabolic factors promoting tumor cell proliferation in the mammary fat derived from the stressed animals. We propose a model wherein social isolation and the ensuing heightened neuroendocrine response significantly alters mammary adipocyte metabolism and associated adipokine secretion, thereby potentiating estrogen-independent mammary tumor growth. These studies have implications for understanding the environmental, endocrine, and molecular risk factors associated with estrogen receptor-negative breast cancer. DOD W81XWH-11-1-014901, NIH/NIDDK T32 DK087703, NIH R01CA148814

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4116. doi:1538-7445.AM2012-4116