bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5233: Imaging breast cancer xenograft using multienergy modality and target-specific molecular agents
Authors: Wang Wei , Qiu Xiuchun , Carmeron Arlin G. , Zhang Siyuan , Yu Dihua , Mawad Michel E. , Shi Ke
Published: February 2011

Interleukin-11 (IL-11) and its receptor (IL-11R) involve in many normal and abnormal biological progress. Studies strongly suggest that interleukin-11 (IL-11) plays an important role in early bone metastases. We synthesized an IL-11Rα homing peptide identified from in vivo patient screening with a peptide phage display and to conjugate it to both an NIR dye for optical imaging and a chelator for nuclear imaging. Cell binding and internalization of the imaging agent was evaluated using NIR confocal microscopy and human breast cancer MDA-MB-231 cell lines. Specificity was demonstrated by comparing with free dye and blocking with the unlabeled recombinant IL-11. Tumor cell growth, vasculature formation and IL-11 agent binding to the tumor were demonstrated in vivo using luciferase positive tumor xenograft model and the injection of both vasculature and IL-11 agents. Tissue autoradiography and H&E staining of the same tissue sections were conducted to verify imaging data. Biodistribution was analyzed by percentage injection dose per gram tissue. Confocal microscopy demonstrated that the target specific imaging agent accumulated on the cell membrane and internalized into cell while free dye had significant lower cell binding than the IL-11 agent. Pre-incubated by the presence of IL-11 protein significantly decreased the agent binding to the cell. Multienergy in vivo imaging revealed the tumor growth, vasculature formation and IL-11 agent binding on the tumor as early as 4 days after the tumor cell inoculation. Overlaid the autoradiography tissue image with H&E staining results demonstrated the heterogeneous distribution of the imaging agent inside tumor mass. Furthermore, the images showed the IL-11 agent only bound to the viable tumor cells. These results suggest a potential imaging agent for study IL-11Rα expression at noninvasive whole body level as well as organ, tissue and cellular levels.

*WW, XQ: These authors contributed equally to this work.

Grant support: DoD W81XWH-08-1-0489, Department of Defense, Department of Radiology research fund, Curtis Hankamer Basic Research Fund and the L.E. and Josephine S. Gordy Memorial Cancer Research Fund, Dan L. Duncan Cancer Center Scholar, American Society for Clinical Oncology and Hope Street Kids Foundation, and National Science Foundation of China grants (30471988, 30973409, and 30873027).

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5233.
Abstract 5247: Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines
Authors: Schiapparelli Paula , Enguita Monica , Balbuena Jana , Fan Xing , Lazcoz Paula , Castresana Javier S.
Published: February 2011

Neuroblastoma is the most common extracranial solid tumor of childhood. Cellular heterogeneity is a hallmark of this embryonal malignancy, as distinct neural crest lineages can be found within the same tumor sample.

The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 12 neuroblastoma cell lines. RT-PCR and flow citometry were performed in order to analyze different kinds of “stemness genes” such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). Also, glial and neuronal markers like NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Interestingly, we found that neuroblastoma cell lines showed a particular pattern of expression suggesting the presence of a subpopulation of immature stem like cancer cells.

Epigenetic changes regarding the CD133 (Prominin-1) promoter gene were also analyzed. This protein has been extensively used to enrich putative cancer propagating stem-like cell populations of different kinds of solid tumors. We found unusual DNA methylation, as 7 of the 12 neuroblastoma cell lines analysed featured a half-methylated state. An increase of RNA and protein levels of CD133 was achieved following demethylation assays using 5-aza-2′-deoxycytidine.

Because cancer stem cells are believed responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Also, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5247.
Abstract 5256: The identification of fatty acid synthase (FASN) as a candidate oncogene and therapeutic target in neuroblastoma
Authors: Mayes Patrick A. , Diskin Sharon , Attiyeh Edward F. , Toporovskaya Yana , Maris John M.
Published: February 2011

Neuroblastoma is a common and lethal pediatric cancer. More than half of patients are at high-risk for relapse and die despite intensive therapy; thus presenting an urgent need for new, rationally designed drugs. To address the need for new therapeutic targets we sought to identify metabolic genes that are critical to neuroblastoma growth and survival. We restricted our analysis to a set of 12 metabolic genes that can be targeted therapeutically and had previously been implicated in other cancers. Analysis of 188 primary human neuroblastomas genotyped on the Illumina HumanHap SNP Array identified high-level, unbalanced gain at the FASN locus (17q25) in 23% of tumors. Analysis of FASN mRNA expression in 99 of these primary neuroblastomas using the IlluminaHT Expression Array identified significant increases in FASN expression restricted to high-risk neuroblastomas with amplification of the MYCN oncogene (P=0.0001). Within the subset of high-risk tumors with MYCN amplification, those that harbored FASN DNA copy number gains showed greater FASN mRNA expression than those with two copy FASN DNA. We next tested siRNAs targeted to the set of 12 metabolic genes in a panel of 12 neuroblastoma cell lines. We found that siRNA inhibition of fatty acid synthase (FASN) and hexokinase 2 (HK2) resulted in significant growth inhibition in 9/12 and 6/12 cell lines, respectively, whereas the others showed no appreciable cytotoxicity. Inhibition of FASN resulted in significant growth inhibition and apoptosis in 8/8 neuroblastoma cell lines with MYCN amplification, suggesting a synthetic lethal effect of FASN inhibition in neuroblastomas with MYCN amplification. Taken together, an integrated genomic and functional analytic approach has identified fatty acid synthase, a key enzymatic mediator of de novo fatty acid metabolism, as a candidate oncogene and therapeutic target in neuroblastoma, especially those harboring MYCN amplification. Ongoing analyses of pharmacologic inhibitors of FASN in neuroblastoma cell line animal models, including impact on global lipidomic profiles will be reported.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5256.
Abstract 526: Tetraspanin CD81/TAPA-1 up-regulates MT1-MMP involved in melanoma cell motility through Akt-dependent Sp1 activation signaling pathways
Authors: Hong In-Kee , Wang Sun-Ju , Lee Moon-Sung , Park Iha , Kim Young-Myeong , Lee Hansoo
Published: February 2011

Tetraspanin CD81 is a member of transmembrane 4 superfamily proteins that have four putative membrane-spanning domains, short C- and N-terminal cytoplasmic domains, two extracellular loops and small intracellular loop. Tetraspanin CD81 is involved in cell adhesion, cell proliferation, motility and metastasis. In this study, to reveal the role of CD81 in tumor invasion, we examined the effect of CD81 overexpression on invasive phenotype in human melanoma cells. We also investigated the intracellular signaling pathways mediated by CD81 involved in these invasive phenotypes. For this study, we generated MelJuSo human melanoma cell lines stably expressing CD81. Cell invasion and motility of the CD81-transfected cells was enhanced significantly as compared to control transfectant cells lacking CD81 expression. In addition, MT1-MMP expression is up-regulated in CD81 transfectant cells as compared to control transfectant cells. MT1-MMP knockdown in CD81 transfectant cells abolished the increase of cell motility by CD81 expression, suggesting the role of MT1-MMP in CD81-induced cell motility. Through the studies using chemical inhibitors and siRNAs for gene knockdown, we found that Akt/PI3K signaling pathway is involved in CD81-mediated MT1-MMP expression. The enhancement of cell motility by CD81 expression was abolished by knockdown of Akt. Finally, we found the involvement of Sp1 transcription factor in the induction of MT1-MMP expression mediated by CD81. Sp1 binding site in MT1-MMP gene promoter was indispensable for the induction of MT1-MMP expression by CD81. Electrophoretic mobility shift assay confirmed the involvement of Sp1 in MT1-MMP expression by CD81. Taken together, these results suggest that tetraspanin CD81 stimulates melanoma cell invasion and motility by up-regulating MT1-MMP expression through the activation of AKT signaling pathways and Sp1 transcription factor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 526.
Abstract 5262: Resistance to fenretinide in neuroblastoma is associated with increased expression of sphingosine kinase and sphingosine-1-phosphate receptors and targeting sphingosine kinase reverses resistance
Authors: Ghent Matthew V. , Kim Youngleem , Jakimenko Ana , Reynolds C Patrick
Published: February 2011

The synthetic retinoid fenretinide, N-(4-hydroxyphenyl) retinamide (4-HPR), is selectively cytotoxic to cancer cells via increases in dihydroceramide levels and generation of reactive oxygen species and has clinical activity against recurrent neuroblastoma (J Clin Oncol 27: 15s abstr 10009, 2009). Metabolism of ceramides into various sphingolipid species, including sphingosine, and phosphorylation of dihydrosphingosine and sphingosine by sphingosine kinase 1 (SPHK1) are anti-apoptotic and thought to counter cytotoxicity mediated by ceramides. Multistep selection for resistance to 4-HPR was performed with a 4-HPR sensitive neuroblastoma cell line, SMS-KCNR (IC99=4.6 uM), resulting in the 4-HPR resistant line, KCNR-FR (IC99=19.9 uM). We generated a TaqMan low-density array (TLDA) that measures expression of 42 genes involved in ceramide synthesis and metabolism by quantitative RT-PCR. Genes overexpressed in KCNR-FR relative to SMS-KCNR included ceramide desaturase 2 (14.1-fold), sphingosine-1-phosphate receptors S1PR2, 3, and 4 (2.4, 1.6, and 1.3-fold, respectively), and SPHK1 (5.2-fold). These data point toward 4-HPR resistance being mediated by sphingosine-1-phosphate or dihydrosphingosine-1-phosphate, both of which are generated via phosphorylation of sphingosine by SPHK1. SPHK1-specific RNAi knockdown decreased SPHK1 mRNA expression (36% ± 0.007 of scrambled RNAi, p<0.05) and significantly increased apoptosis (35.8 ± 1.7%; p<0.05) in KCNR-FR treated with 4-HPR relative to scrambled RNAi controls (8.8 ± 6.5%; p<0.05). Safingol (L-threo-dihydrosphingosine), a putative sphingosine kinase inhibitor in phase I trials, partially reversed KCNR-FR resistance to 4-HPR (Combination Index = 0.56). Thus, selection for resistance to 4-HPR in neuroblastoma was associated with increased expression of multiple components of the anti-apoptotic sphingosine kinase signaling pathway, and such resistance can be partially overcome by targeting sphingosine kinase.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5262.
Abstract 5264: Are the methylation of MGMT (O6-methylguanine-DNA methyltransferase) and the DNA mismatch repair (MMR) mechanism frequently involved in pediatric cancers?
Authors: Nguyen Aurelia , LEGRAIN Michele , Neuville Agnès , Pencreach Erwan , Guerin Eric , Lutz Patrick , Gaub Marie Pierre , Guenot Dominique , Entz-Werlé Natacha
Published: February 2011

In pediatric oncology more and more protocols are including now temozolomide (TMZ) as a drug in the first line treatment or at relapse time. MGMT activity is known to circumvent the toxicity of alkylating agents such as TMZ. The defect of MMR mechanism is involved also as a cellular way for cancers to resist to TMZ. Only few studies have been already done in the pediatric field focusing on the study of these markers of TMZ resistance. Based on the new therapeutic indications of this drug in pediatric cancers, we performed a study in 100 malignant pediatric tumors from various origins to evaluate the MGMT methylation and the MMR mechanism.

Material and methods: 12 high grade gliomas (HGG), 13 medulloblastomas (MB), 15 osteosarcomas (OS), 15 Ewing sarcomas (EWS), 15 rhabdomyosarcomas (RMS), 15 NBs and 15 acute lymphoblastic leukemias (ALLs) were included retrospectively in the study. DNA was extracted from tumors at diagnosis. In all tumors, the methylated status of MGMT was analyzed by methylation specific PCR (MS-PCR). Simultaneously, its expression was studied by immunohistochemistry. To analyze the MMR status in each tumor, an allelotyping method was performed in these tumors by using the NIH recommended microsatellites.

Results: Out of these 100 samples, 3 tumors (2 HGGs and 1 MB) were presenting a MSI (microsatellite instability), witness of a MMR deficiency. Only 5 tumors (2 EWSs, 2 HGGs and 1 MB) had a methylated MGMT promoter. One HGG was presenting both abnormalities at the same time. The immunohistochemical results were concordant with MS-PCR observations. Concerning the tumors treated with TMZ (12 HGGs, 4 MBs, 2 EWSs and 4 NBs), we do not observed any differences on survival between the unmethylated and methylated tumors.

Discussion: Methylation of MGMT promoter and MMR deficiency are rare in pediatric tumors and therefore seem not to be the major mechanisms involved in the sensitivity to TMZ. Our results suggest that in pediatric tumors, other mechanisms for TMZ sensitivity or resistance should be activated.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5264.
Abstract 5281: The effect of RF through JAG/Notch pathway in tumorigenesis, angiogenesis and migration
Authors: Kim Mi Hwa , You Ho Jin
Published: February 2011

RF has known as multifunctional protein with the two of domains, redox and repair domains. Although RF is highly expressed in a lot of cancer cell lines, its function has not been exactly reported. The Jagged1/Notch proteins participate in a variety of cellular processes, such as cell fates specification, proliferation, differentiation, apoptosis, adhesion, epithelial-mesenchymal transition, migration, and angiogenesis. In the present study, Jagged1 and Notch-1 are overexpressed in RF-transfected fibroblast cell(GM00637/RF) and several human cancer cell lines. Through DNA microarray, we show that JAGGED1/Notch1 expression is up-regulated by RF. In promoter study, RF regulates the expression of JAGGED1 by binding Egr-1 on JAGGED1 promoter. Also, immunofluorescence confocal microscopy shows Notch active form(Notch intracellular domain) is in the nucleus. Furthermore, down-regulation of RF or JAG by RNA interference reduces the activities of focal adhesion molecules(vinculin, pFAK) and MMP-2 which have known to be involved in cell migration and invasion in cancer. In nude mouse model, RF or JAG siRNA significantly reduces tumor size and angiogenesis. We suggest that RF may act as a potential regulator in tumorigenesis, angiogenesis and migration through JAGGED1/Notch pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5281.
Abstract 53: Krebs cycle substrate-level phosphorylation drives metastatic cancer cells
Authors: Shelton Laura M. , Strelko Cheryl L. , Roberts Mary F. , Seyfried Thomas N.
Published: February 2011

Many metastatic cancers are derived from compromised respiration in tissue cells of myeloid origin (e.g., macrophages), which use glutamine as a major energy substrate. Although impaired respiration can render tumor cells dependent on glycolysis for energy in the presence of oxygen (Warburg effect), it has not been determined if glutamine alone could provide enough energy (ATP) through substrate level phosphorylation in the TCA (Krebs) cycle to maintain tumor cell viability. Using a bioluminescent-based ATP assay, we determined the viability of metastatic mouse VM-M3 tumor cells grown in serum free medium in the presence of glucose alone (25 mM), glutamine alone (4 mM), or in glucose + glutamine. The VM-M3 cells could not survive on glucose alone, but could survive in glutamine alone indicating an absolute requirement for glutamine in these metastatic tumor cells. Glucose + glutamine acted synergistically in providing growth and viability. Lactic acid production was 10-fold lower in the cells grown in glutamine alone than in glucose alone. Glutamine could also maintain viability in the absence of glucose and in the presence of the F1 ATPase inhibitor oligomycin. Glutamine could not maintain viability in the presence of the TCA cycle enzyme inhibitor, 3-Nitropropionic acid. NMR analysis indicated that glutamine metabolism via the TCA cycle was active resulting in accumulation of both succinate and aspartate. The data indicate that glutamine can provide ATP for viability in the metastatic VM-M3 cells through Krebs cycle phosphorylation in the absence of energy from either glycolysis or oxidative phosphorylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 53.
Abstract 530: Evaluation of in vitro invasion of primary vs recurrent glioblastoma multiforme cells using organotypic rodent brain slice culture
Authors: Rossi Marco , Tarantino Antonio , Roncarati Renza , Salerno Massimiliano , Giordano Cinzia , Tunici Patrizia , Bakker Annette
Published: February 2011

Glioblastoma multiforme (GBM) is the most common primary malignant tumor of the adult central nervous system. The highly lethal nature of this tumor partly derives from the acquisition of an invasive phenotype, which allows the tumor cells to infiltrate the surrounding brain tissues. In fact the tumor cells, leading to the recurrence of the primary neoplasm highly contribute to the lack of success in eradicating this disease.

In order to measure the migrating ability of GBM cells and the potential difference in infiltration capacity of primary versus recurrent GBM cells, we developed and validated an organotypic rodent brain slice assay and visualized the fluorescently labeled cells using confocal laser scanning microscopy. The invasion capacity has been quantified applying an innovative image analysis method using a panel of primary and recurrent glioma cell lines derived from patient biopsies and the commercially available GBM cell lines, DBTRG-05MG (recurrent) and T98G (primary).

For validation purposes, cytochalasin D, a potent inhibitor of actin polymerization and hence migration and invasion, was used. The results revealed clear differences between primary and recurrent GBM cells: They differ both in their migrating capacity on the surface of the brain slice as well as by their ability to invade into the brain slice.

A small population of recurrent GBM cells seems to acquire high infiltrative capacity allowing the invasion of brain tissue to greater distance than the primary GBM cells. Higher invasiveness may be an important characteristic of recurrent tumours, adding to our understanding of their worse clinical evolution and responsiveness to therapy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 530.
Abstract 5301: MUC16-Siglec-9 binding leads to adhesion between ovarian tumor cells and specific subsets of NK cells, B cells, and monocytes
Authors: Patankar Manish S. , Belisle Jennifer A. , Horibata Sachi , Felder Mildred , Gubbels Jennifer A.A. , Connor Joseph P.
Published: February 2011

MUC16 is a cell surface mucin expressed at high levels by epithelial ovarian cancer cells. Following proteolytic cleavage, cell surface MUC16 (csMUC16) is shed in the extracellular milieu and is detected in the serum of cancer patients as the tumor marker CA125. csMUC16 acts as an adhesion molecule and facilitates peritoneal metastasis of ovarian tumors. Both sMUC16 and csMUC16 also protect cancer cells from cytotoxic responses of natural killer (NK) cells. In a previous study, we demonstrated that sMUC16 binds to a specific subset of NK cells. Here, we demonstrate that in addition to NK cells, sMUC16 also binds to B cells and monocytes isolated from the peripheral circulation and the peritoneal fluid. The I-type lectin, Siglec-9, is identified as the sMUC16 receptor on these immune cells. The inhibitory receptor Siglec-9 is expressed on approximately 30-40% of CD16pos/CD56dim NK cells, 20-30% of B cells and >95% of monocytes. sMUC16 binds to the majority of the Siglec-9pos NK cells, B cells and monocytes. sMUC16 is released from the immune cells following neuraminidase treatment. While sMUC16 binds to Siglec-9, it has no affinity for another I-type lectin Siglec-7. Experiments with Siglec-9 transfected Jurkat cells and monocytes isolated from healthy donors demonstrate that immune cells can bind to ovarian tumor cells via Siglec-9-csMUC16 interaction. Siglec-9 is an inhibitory receptor that attenuates T cell and NK cell function. Our studies indicate that sMUC16/csMUC16 may each serve as Siglec-9 ligands and mediate inhibition of anti-tumor immune responses.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5301.
Abstract 5307: Indomethacin reverts tumor mediated immune suppression in a murine lung adenocarcinoma
Authors: Blidner Ada G. , Salatino Mariana , Karas Romina A. , Jasnis Maria A. , Rabinovich Gabriel A. , Diament Miriam J. , Klein Slobodanka M.
Published: February 2011

Tumor-induced immune suppression is widespread in patients and experimental animals with malignant tumors and is likely to be a significant impediment to immunotherapy. Multiple mechanisms are thought to facilitate this suppression state, being Myeloid-Derived Suppressor Cells (MDSC) and T regulatory cells (Tregs) major contributors. Our aim was to analyze immune parameters indicative of immune suppression in BALB/C mice bearing LP07 (COX+) lung tumor (TBM) during tumor growth and the effect of a Non Steroidal Anti-inflammatory drug (NSAID), Indomethacin, which is a non-specific COX inhibitor, on these parameters. We determined 1) the effect of Indomethacin on LP07 tumor growth; 2) the content of MDSC and Treg cells in lymphoid organs (spleen, tumor-draining lymph nodes-TDLN-) and in the tumors at different times by FACS; 3) arginase activity in lung, spleen, liver and tumor measured as µg Urea/mg protein; 4) specific Delayed Type Hypersensitivity (DTH) response with formalinized LP07 cells, by the foot pad swelling assay. Results: Indomethacin treatment significantly inhibited tumor growth. The percentage of MDSC (CD11b+ Gr1+) augmented significantly in the spleen of TBM in advanced stages of tumor progression (33 days): Normal: 2,6%; TBM 10 days: 2,4%; TBM 20 days: 5,6%; TBM 33 days: 8,3%, p<0,05 vs control. This rise was prevented by Indomethacin treatment (4,6%). Intratumoral and TDLN CD4+CD25+Foxp3+ Tregs frequency augmented during tumor growth, peaking at day 20. Interestingly, Indomethacin treatment decreased Treg cell and MDSC induction (p<0.05) in TDLN. Arginase activity increased in all analyzed tissues of TBM compared to those of control mice. Furthermore arginase activity increase in the spleen was in line with MDSC induction. Specific DTH response decreased in late stages of tumor progression, while Indomethacin treatment reversed this inhibition (TBM: 0,06 ± 0,045 mm; Indo:0,14 ± 0,038 mm). Conclusion: In our model, the increment in MDSC and Tregs frequency during tumor progression correlates with specific immunesuppression demonstrated by DTH responses and increased arginase activity. We suggest that Indomethacin treatment prevented this immunesuppression by modulating COX activity in the tumor microenvironment, which is known to activate MDSC and differentiate Treg cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5307.
Abstract 5310: mTOR knock-down mice exhibit altered T and B cell activation, differentiation and immune response
Authors: Zhang Shuling , Readinger Julie , Roth Laura , Pruitt Maggie , Robinson Richard , DuBois Wendy , Bliskovsky Val , Zhang Ke , Wu Julie , Tessarollo Lino , Schwartzberg Pam , Mock Beverly A.
Published: February 2011

Mammalian TOR (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. Because disruption of the mouse mTOR gene leads to early postimplantation lethality, insights into in vivo function of mTOR have been limited. In this study, we generated a mouse knock-down model, which recapitulates treatment with mTOR inhibitors. Neo insertion interrupts transcription, leading to reduced mTOR expression. The homozygous knock-down mice exhibit reductions in body, organ, and cell size. T cell development in the thymus appears normal, however, T cell receptor (TCR)-induced phosphorylation of AKT was reduced in CD4+ and CD8+ T cells. Spleens are disproportionately small relative to overall body weight. The number of CD4+ and CD8+ T cells is reduced in the spleen. Cell proliferation was decreased in splenic T and B cells. Both TCR-mediated proliferation in T cells and LPS-induced proliferation in B cells were reduced. Splenic T cells show reductions in migration to TECK and SDF, and increased FoxP3 expression. Splenic B cell populations had more mature cells, with fewer transitional cells and markedly reduced marginal zone and follicular B cells. Phosphorylation of AKT was increased in B cells after LPS stimulation, similar to what has been reported in tumor cells exposed to long-term rapamycin treatment. B220+ B cells were decreased in bone marrow and alterations were seen in pre-B cell maturation. We investigated the immunologic consequences of immunization with NP-CGG, and knock-down mice show decreases in NP-specific Ig responses and defects in producing primary germinal center reactions. These data suggest that mTOR plays a role in the maturation and differentiation of immune cell lineages, as well as in the regulation of humoral immunity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5310.
Abstract 5316: Inflammation-induced myeloid-derived suppressor cells have enhanced resistance to apoptosis
Authors: Chornoguz Olesya Y. , Grmail Lydia , Sinha Pratima , Artemenko Konstantin , Zubarev Roman , Ostrand-Rosenberg Suzanne
Published: February 2011

The purpose of this study is to deduce the influence of inflammation on apoptosis of Myeloid-Derived Suppressor cells. Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression. MDSC induced in heightened inflammatory environments with high levels of IL-1β and/or IL-6 accumulate more rapidly, are more potent suppressors of T cell activation, produce more IL-10, and have a greater ability to down-regulate macrophage production of IL-12, as compared to MDSC that develop in environments with lower levels of these cytokines. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory vs. less inflammatory settings. Proteomic pathway analysis identified Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by down-regulating Fas-mediated MDSC apoptosis. Using cytochrome C and activated caspase-3 and caspase-8 as indicators of apoptosis, confocal microscopy and flow cytometry demonstrated that inflammation-induced MDSC have enhanced resistance to Fas-mediated apoptosis. These results suggest that an inflammatory environment protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings. (supported by NIH R01CA84232 and RO1CA115880).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5316.
Abstract 5328: Expansion of CD11b+CD33lowCD15high immunosuppressive myeloid cell population in patients with bladder carcinoma
Authors: Kusmartsev Sergei A. , Eruslanov Evgeniy , McCullers Molly , Rosser Charles , Daurkin Irina , Vincent Charles , Willis Daniel , Vieweg Johannes
Published: February 2011

Recent studies highlighted the immunoregulatory role of CD11b myeloid cells in cancer-associated immune non-responsiveness. Increased presence of myeloid-derived suppressor cells (MDSC) is one of the major factors responsible for immune suppression in tumor hosts. Here we describe the expanded CD11b myeloid cell subset in peripheral blood obtained from patients with bladder cancer. Based on expression of CD33 and CD15 myeloid cells markers, the circulating CD11b-positive cells in patients with bladder cancer could be subdivided into two major subpopulations: CD33lowCD15high and CD33highCD15low. First subpopulation (CD11b+CD33lowCD15high cells) has granulocyte-type morphology, express G-CSF receptor (CD114) and secrete elevated amounts of pro-inflammatory cytokines and chemokines such as G-CSF, MCP-1 MIP-1alpha, MIP-1beta. Second major myeloid cell subset from cancer patients (CD11b+CD33high CD15lowcells) displays a monocyte-type cell morphology and co-express CD14, M-CSF receptor (CD115), CCR2 and HLA-DR. We show here that proportion of granulocytic CD33lowCD15high cells is highly elevated in cancer patients as compared to healthy individuals. Patients with bladder cancer but not healthy volunteers show weak T cell proliferative response after in vitro stimulation. We demonstrate that granulocyte-type CD33lowCD15high cell population is involved in T cell hyporesponsiveness, since specific depletion of this cell subset restored the T cell proliferation. Obtained results suggest that fraction of PBMC in patients with bladder cancer patients contains markedly elevated proportion of CD11b+CD33lowCD15high granulocyte-type myeloid cell population which contributes to cancer-associated inflammation and tumor-induced immune suppression.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5328.
Abstract 533: Critical role of MMP-9 dimers in cell migration: design of inhibitory peptides
Authors: Dufour Antoine , Sampson Nicole S. , Kuscu Cem , Zucker Stanley , Cao Jian
Published: February 2011

Non-proteolytic activities of matrix metalloproteinases (MMPs) have recently been shown to impact cell migration, but the precise mechanism remains to be understood. We previously demonstrated that the hemopexin (PEX) domain of MMP-9 is a prerequisite for enhanced cell migration. Using a biochemical approach, we now report that dimerization of MMP-9 through the PEX domain is necessary for MMP-9-enhanced cell migration. Following a series of substitution mutations within the MMP-9 PEX domain, two critical motifs in the outermost strands of the first and fourth blades of MMP-9 PEX domain were found to be involved in MMP-9 homo- and hetero- dimer formations. Peptides mimicking these motifs efficiently blocked MMP-9 dimer formations and inhibited MMP-9-mediated cell migration, but not membrane type 1- MMP (MT1-MMP)-induced cell migration. In addition, these peptides demonstrate inhibitory effects on migration of human fibrosarcoma HT1080 cells, which express high level of endogenous MMP-9. Using a shRNA approach, CD44 was found to be a critical molecule in MMP-9-mediated cell migration. Furthermore, a novel axis involving an MMP-9-CD44-EGFR signaling pathway in cell migration was identified using antibody microarray analysis and specific receptor tyrosine kinase inhibitors. In conclusion, we dissected the mechanism of proMMP-9-enhanced cell migration and developed structure-based inhibitory peptides specific targeting of MMP-9-mediated cell migration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 533.
Abstract 5335: The antigen target of IMGN901, CD56, is expressed at significant levels in merkel cell carcinoma (MCC)
Authors: Carrigan Christina N. , Xu Shanqin , Zhao Yiwei , O'Keefe Jessica , Qin Albert , O'Leary James , Payne Gillian , Lutz Robert
Published: February 2011

Background: IMGN901 consists of a CD56-binding monoclonal antibody, huN901, with a maytansinoid agent, DM1, attached via a stable disulfide linker, SPP. The CD56 antigen, also known as neural cell adhesion molecule (NCAM), is present in a number of malignancies including Merkel cell carcinoma (MCC), small cell lung carcinoma and other neuroendocrine tumors, ovarian cancers and multiple myeloma. Once bound to CD56, IMGN901 is internalized into a cancer cell and its DM1 is released to cause cell death via inhibition of tubulin polymerization. IMGN901 is currently in Phase I testing for the treatment of CD56-positive multiple myeloma and CD56-positive solid tumors. Six patients with MCC have received IMGN901, used as a single agent, in one of the Phase I trials underway. Clinical benefit was observed in 3 of these 6 patients: one complete response (CR), one partial response (PR) and one stable disease (SD).

Objective: The goal of this study was to assess the potential utility of IMGN901 as a treatment for MCC by identifying the range of CD56 antigen expression levels in MCC.

Methods: A calibrated immunohistochemical (IHC) staining method for CD56 was developed. CD56 staining conditions were established where 4 formalin fixed paraffin embedded (FFPE) MCC samples showed differential staining. FFPE cell pellet controls prepared from human tumor cell lines were next identified which showed CD56 staining in the same range as the MCC samples. The CD56 antigen level for each tumor cell line was subsequently quantified by flow cytometry. The calibrated IHC staining method was then applied to positive cell pellet controls and an extended panel of MCC tissues. For each resulting tissue section, the extent and intensity of immunostaining was evaluated and the CD56 expression level was estimated by comparing to the cell pellet controls.

Results: The majority of samples from the MCC panel exhibited heterogeneous or homogeneous staining with significant levels of CD56 expression. Tissues from primary biopsy sites and from metastases showed comparable levels of CD56 expression. To date, biopsy samples have been acquired from 3 of the 6 MCC patients who have received IMGN901 (two IMGN901 non-responders and one responder) and these all showed high and homogeneous levels of CD56 expression.

Conclusion: This IHC study indicates that the majority of MCC patient tissues express significant levels of CD56, supporting the development of the CD56-targeting compound, IMGN901, for this indication.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5335.
Abstract 5336: Change in pattern of relapse following anti-angiogenic therapy in high grade gliomas
Authors: Narayana Ashwatha , Kunnakkat Saroj , Medabalmi Praveen , Golfinos John , Parker Erik , Knopp Edmond , Zagzag David , Gruber Deborah , Gruber Michael L.
Published: February 2011

Purpose:. Local recurrence (LR) accounts for >90% at the time of relapse in high grade glioma (HGG) following conventional therapy. Anti-angiogenic therapy with bevacizumab has shown impressive radiological and clinical responses in HGG. However, an apparent increase in diffuse invasive relapse (DIR) following therapy necessitated a detailed analysis of pattern of recurrence following therapy.

Methods: One hundred and sixty five consecutive patients with HGG, either newly diagnosed (n=75) or recurrent (n= 90) received therapy with bevacizumab at 10 mg/kg every two weeks along with conventional chemotherapy ± involved field radiotherapy. The treatment was continued till the time of disease progression or the patient developed dose limiting toxicity. Treatment evaluation was done with neurological examination and magnetic resonance imaging at base line and every 8 weeks subsequently. Pattern of recurrence and time to relapse were the primary aims of the study. DIR was defined as involvement of multiple lobes ± crossing the midline. Survival was calculated from the start of bevacizumab therapy.

Results: With a mean follow-up of 6.8 months (range 1-58), 100 of the 165 patients (60.6%) have relapsed with 76 as DIR and 70 have died. The median progression free survival (PFS) and overall survival (OS) were 5 and 9 months respectively. Of the 62 patients who failed initially as LR, 38 (61.3%) patients subsequently developed DIR. 38/75 (50.7%) patients with newly diagnosed and 38/90 (42.2%) of recurrent HGG failed ultimately as DIR. All 9 patients who presented with multicentric HGG relapsed as DIR. The median OS for the patients who failed as DIR was 9 months Vs 6 months for patients with LR alone (p=0.06). While the hazard risk for LR remained linear over time, the risk of DR increased exponentially with time (R2 = 0.933). Biopsies done on eight of these patients at the time of recurrence showed decreased expression of Olig-2 in 6, increased expression of SDF-1 in 5 and a variable expression of CXCR-4 in 6 patients. No change in expression of HIF-1, p53, PDGF or CD163 was seen.

Conclusion: Invasion remains a dominant process in HGG as demonstrated by both imaging and pathology. Angiogenic blockade that results in increased invasiveness of HGG is worrisome and needs to be addressed in future clinical trials.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5336.
Abstract 5342: Phage display selelction on tumor histological specimens
Authors: sun yujing , Shukla Girja , Pero Stephanie , Peletskaya Elena , Krag David
Published: February 2011

A method was developed to obtain phage displayed ligands that bind to a select population of cells in histological specimens of freshly harvested solid human cancers. It combines phage display panning with laser capture microdissection (LCM). This method allows selection of phage ligands bound to subpopulations of specific cells contained in tumor tissue on histological sections. Naïve phage scFv library was incubated directly on a histological section of human breast cancer that was snap frozen immediately after surgical resection. Tumor stromal cells were captured by LCM and bound phages were recovered by bacterial infection. Individual phage clones selected after three rounds of panning were evaluated for their binding ability by immunofluorescence staining on tumor tissue from the same patient. Based on DNA analysis, 20% of 3rd output clones represented a single clone. This clone selectively bound tumor stroma in the breast cancer specimen and did not bind the malignant cell population. This clone, expressed as a soluble scFv, showed no significant binding to normal tissues, 13 other breast cancers or 4 colon cancer samples. This method is applicable for selection of ligands to virtually any portion of a histological specimen amenable to LCM. This may speed the process of generating ligands to any subset of cells or noncellular feature present on histological specimens.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5342.
Abstract 5354: HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of PLK4, a master regulator of centriole biogenesis
Authors: Korzeniewski Nina , Duensing Anette , Duensing Stefan
Published: February 2011

Human papillomavirus (HPV)-associated neoplasms exhibit abnormal centrosome and centriole numbers that can cause mitotic defects, chromosome missegregation and aneuploidy. Centrosomes are the major microtubule-organizing centers in most mammalian cells and orchestrate the formation of mitotic spindle poles. The centrosome consists of a pair of centrioles that normally duplicate precisely once prior to mitosis. The HPV-16 E7 oncoprotein has been found to rapidly stimulate centriole overduplication within a single cell division cycle, thereby increasing the risk for cell division errors. We discovered that this activity of the HPV-16 E7 oncoprotein involves the concurrent formation of multiple daughter centrioles at single maternal centrioles (centriole multiplication). We found that deregulated cyclin E/cyclin dependent kinase 2 (CDK2) activity, as occurs following HPV-16 E7 oncoprotein expression, leads to the aberrant recruitment of polo-like kinase 4 (PLK4) to maternal centrioles, which is subsequently degraded by the SCF ubiqutin ligase complex. These and several other results underscore that the level of PLK4 at maternal centrioles is rate-limiting for centriole multiplication. Hence, we asked whether the HPV-16 E7 oncoprotein can alter PLK4 expression and/or localization. We found aberrant PLK4 protein expression at maternal centrioles in primary human keratinocytes engineered to stably express HPV-16 E7. Real-time quantitative RT-PCR revealed an increase in PLK4 transcription in keratinocytes stably expressing HPV-16 E7. The ability of HPV-16 E7 to upregulate PLK4 mRNA was found to depend on its ability to degrade the retinoblastoma (pRb) protein, suggesting a role of E2F-mediated gene transcription in deregulation of PLK4. Collectively, these results highlight the critical role of PLK4 as a regulator of centriole biogenesis and identify PLK4 as a novel target for small molecules to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5354.
Abstract 535A: The tyrosine kinase Src co-operates with the ERM family member ezrin to promote Met activation and ECM degradation in a breast epithelial cell model
Authors: Hoskin Victoria , Organ Shawna , Furmaniak-Kazmierczak Emilia , Pilon Jenna , Meens Jalna , Cote Graham , Elliott Bruce
Published: February 2011

Over-expression and aberrant activation of the Met proto-oncogene frequently occurs in invasive breast cancer, and is linked with poor patient prognosis. We and others have shown that the tyrosine kinase Src mediates cell-adhesion dependent activation of Met in the absence of ligand (JCB 107:1168-81, 2009). We have also shown that Src acts co-operatively with the membrane cytoskeletal crosslinker ezrin to disrupt cell-cell contacts, and to increase cell spreading and scattering, all characteristics of epithelial-to-mesenchymal transition (EMT) (JCB 92:16-28, 2004). We therefore examined the role of Src and ezrin in Met activation and invasion of breast epithelial cells. As a model, we used the HC11 breast epithelial cell line expressing various combinations of activated Src and ezrin mutants, as well as a dominant negative ezrin mutant. In contrast to activated Src, which promotes the phosphorylation and hence activation of Met and cell spreading, activated ezrin alone induces the formation of numerous filopodia-like structures, but not Met phosphorylation or cell spreading. Co-activation of Src and ezrin produces a combination of both cell spreading and filopodia-like structures, and strongly increases Met phosphorylation over levels resulting from activated Src alone. Interestingly, Src/ezrin co-operativity dramatically increases tyrosine-phosphorylation of cortactin and MMP2 expression, resulting in the formation of invadopodia and extracellular matrix degradation, a hallmark feature of invading cancer cells. However expression of a dominant negative ezrin domain (aa1-309) abolishes Src and ezrin-induced morphological phenotypes, as well as Src-dependent Met phosphorylation and invadopodia formation, suggesting an important requirement for ezrin in these processes. These findings implicate the Src/ezrin axis in regulating adhesion-dependent Met activation and invadopodia formation, and may provide important clues toward treatment strategies for invasive breast tumors which exploit this signaling pathway (Supported by CBCRA and CIHR).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 535A.