bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4187: A novel genome-wide assay identifies chromatin domains involved in stem cell behavior
Authors: Okino Steven T. , Kong Michelle , Wang Yan
Published: August 2012

The activity of DNA regulatory elements are often controlled by chromatin structure. Active regulatory elements are located within “open” or “accessible” chromatin. If a regulatory element is in a “closed” or “inaccessible” chromatin environment it is functionally silenced. We find that when permeabilized cells are treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin, can be readily isolated. These small DNA fragments, when analyzed by next-generation sequencing, provide a global map of active genomic regulatory domains. We used this technology to identify chromatin domains involved in stem cell behavior using NTera2 cells as a model system. By comparing differentiated and undifferentiated NTera2 cells we identified more than 500 genomic regions that are accessible only in undifferentiated cells. We then used these results, together with ENCODE data analyzing human embryonic stem cells and human cancer cells, to identify genomic regulatory regions that are active only in stem cells. Importantly, many of these regions are proximal to genes that are associated with cell “stemness” (NANOG and LIN28 are examples). In addition, several uncharacterized genes that have RNA expression characteristics similar to NANOG and LIN28 were identified. Interestingly, several stem-cell specific active regulatory domains were found adjacent to previously uncharacterized, stem-cell specific non-coding RNA transcript regions. These findings demonstrate that this approach can serve as a new discovery tool to identify chromatin regions that control stem cell behavior. In addition, chromatin regions associated with other biological or pathological states could also be identified using this approach.

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 4187. doi:1538-7445.AM2012-4187
Abstract 4203: Analysis of the association of Notch pathway genes with ID4 and Ikaros gene expression in leukemia
Authors: Dalay Nejat , Gurbuz Orkun
Published: August 2012

The Notch signal pathway plays an important role in several cellular processes in a variety of tissues. In the hematopoiesis Notch expression is involved in various aspects of cellular regulation and aberrant Notch signaling components have been associated with malignant transformation. Hes1 (hairy/enhancer of split 1) gene is a Notch target gene coding for a transcriptional repressor that is necessary to maintain progenitor cell populations. HES1 can bind promoter regions of the cyclin-dependent kinase inhibitors and repress their expression. Hes-1 expression can be stimulated by growth factors The Hey family of transcriptional repressors are canonical direct targets of Notch and function as downstream effectors of Notch signaling. Expression of HES1 and HEY proteins can vary in different cell types suggesting an additional mechanism of transcription regulation. Ikaros is a member of the Kruppel transcription factor family. Expression of Ikaros down-regulates expression of the Notch target genes. Deregulation of Ikaros and Notch signalling cooperate to promote leukemogenesis, providing evidence that they function in converging pathways. The ID4 (inhibitor of differentiation 4) gene is a member of the family of ID helix-loop-helix proteins that function as dominant negative regulators of basic transcription factors. In certain cancer types downregulation of the ID4 gene has been reported resulting in low or no expression of the ID4 protein. In the current study the expression of the Hes1, Hey1, Ikaros and ID4 genes was investigated in 110 patients with acute and chronic leukemias and 32 healthy controls by real time PCR using the ΔΔCt method. Both Hes1 and Hey1 expression levels were significantly increased in the patients in all disease groups. ID4 expression was significantly decreased in % 86.4 of the patients. Expression of these genes was strongly associated. Expression of the Ikaros gene decreased only in the subgroup of patients with CML while it increased in patients with acute leukemias and CLL. Our data suggest that the Notch pathway genes Hes1 and Hey1 are activated in leukemias and may contribute to the disease development while the ID4 gene is supressed, possibly by methylation.

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 4203. doi:1538-7445.AM2012-4203
Abstract 4204: Gene expression changes of DNA repair genes in lymphocytes of breast cancer patients
Authors: Perez-Mayoral Julyann , Matta Jaime L. , Dutil Julie
Published: August 2012

Breast cancer is the most commonly diagnosed cancer among women in Puerto Rico. Previous studies have shown that dysregulation of DNA repair genes expression is important for carcinogenesis. However, most of the studies have focused on measuring DNA repair gene expression levels in tumor samples. Our preliminary studies had shown that women with decreased DNA repair capacity (DRC) are at increased risk of breast cancer. We hypothesized that changes in DRC levels may be partly explained by changes in gene expression of DNA repair genes. Our objective was to assess the contribution of the expression levels of 43 DNA repair and damage response genes in lymphocytes of breast cancer cases and controls of Puerto Rican origin. We measured the gene expression profile of 43 DNA repair genes in lymphocytes of 6 breast cancer cases with low DRC and 10 controls with high DRC using a custom SuperArray (SABiosciences). Of the 43 genes analyzed through the SuperArray, XPC, CETN2 and CCNH showed the largest fold-changes. To validate the results, we used a TaqMan-based gene expression assay in lymphocytes of 30 breast cancer cases and 30 controls. Our results showed that breast cancer cases had a 3.4x less XPC expression (p=0.002) and 2.2x less CCNH expression (p=0.01) than controls. Validation of the CETN2 SuperArray results is underway. In conclusion, we found that the XPC gene and CCNH were expressed less in breast cancer cases than in controls. Understanding the gene expression changes that occur prior to tumor development is an important step towards enhancing cancer prevention and detection strategies.

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 4204. doi:1538-7445.AM2012-4204
Abstract 4206: Identification of transcriptional regulatory motifs that control gene expression in premenopausal women with previous history of breast cancer
Authors: Cano Oscar D. , Gökmen-Polar Yesim , Chirayu Goswami , Li Lang , Sirimalle Usha , Gu Xiaoping , Nakshatri Harikrishna , Badve Sunil
Published: August 2012

Background: Age is an important risk factor for Breast Cancer. Older women have a 50 times greater risk for breast cancer as compared to younger women (under the age of 30 years). Aging is associated with activation of specific transcription factor networks. We hypothesized that premature activation of these programs may predispose to cancer. The aims of this study were 1) to identify active transcription factor networks controlling the expression of specific cancer-related genes in the normal breast and 2) to analyze whether premature activation of this network is associated with neoplastic transformation. Methods: Fresh frozen normal breast tissue of women, under the age of 50 years, with and without history of breast cancer was obtained (ncancer=8, nnon cancer=4). Total RNA was extracted, cDNA annealed and Whole-genome DASL assay (Illumina Corp) was conducted to identify genes that correlated with cancer outcome in premenopausal women. Differential expression analysis was performed using Partek® software program to identify the up or down regulated genes in women with cancer compared to those without. Transcription factors affecting the regulation of genes in patients with cancer were predicted using MotifModeler program (Liu et al). The resulting data was confirmed using qPCR analysis. Results: Whole genome DASL analysis showed differential expression of 34 genes (p-value <0.01). MotifModeler analysis predicted 18 transcription factors as major regulators of gene expression in premenopausal women with previous history of breast cancer. JUNB and FOS were the most significantly altered in this comparison. qPCR pre-validated the over expression of JUNB and FOS in association with cancer development. Further validation studies in an independent cohort are ongoing using Formalin-fixed Paraffin-embedded (FFPE) samples. Conclusion: Gene expression analysis identified transcriptional features of cancer outcome. Differential expression of the JUNB and FOS family of proteins, which are part of AP-1 transcription factor complex, may be correlated with neoplastic transformation in premenopausal women. Breast Cancer Program of the IU Simon Cancer Center funded this project.

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 4206. doi:1538-7445.AM2012-4206
Abstract 4210: Association of the 15q25 and 5p15 lung cancer susceptibility regions with gene expression in lung tumor tissue
Authors: Fehringer Gordon , Liu Geoffrey , Pintilie Melania , Sykes Jenna , Liu Ni , Cheng Dangxiao , Chen Zhuo , Seymour Lesley , Der Sandy D. , Shepherd Frances A. , Tsao Ming-Sound , Hung Rayjean J.
Published: August 2012

Lung cancer is one of the most commonly diagnosed cancers worldwide, with 1.6 million new cases reported each year and ranks first in annual global cancer deaths. Genome-wide association studies have identified two independent lung cancer susceptibility loci at chromosome 15q25 and one locus at 5p15. We examined the association of genetic variants in these lung cancer susceptibility regions with gene expression in lung tumor tissue in an effort to elucidate carcinogenic mechanisms by which these variants influence lung cancer risk. We used data from two independent studies: the JBR.10 clinical trial (131 NSCLC patients) and a patient sample from the University Health Network (UHN) in Toronto (181 NSCLC patients). We genotyped seven 15q25 and five 5p15 variants and examined their association with the expression profiles of genes in the corresponding regions measured by Affymetrix HG-U133A. The minor allele (C) of a variant representing one of the two loci at 15q25 (rs2036534) was associated with increased expression of the IREB2 (iron-responsive element binding protein 2) gene in both studies (JBR.10 P=0.042; UHN P=0.002). An FDRα0.05 in the UHN sample increased our confidence in this association. The association appears to be more prominent among patients with lung adenocarcinoma. We did not detect an association between the genotype and expression profile for the other 15q25 locus (represented by rs16969968 and rs1051730). Previous studies suggest that 15q25 variants are associated with risk through an effect on smoking behaviour, indicating risk is modulated through activity of nicotinic acetylcholine receptor genes present in this region. Our results indicate there is a second mechanism through which variants at 15q25 influence risk, operating through modulation of IREB2 gene expression. This finding may have implications for future research on lung cancer chemoprevention strategies.

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 4210. doi:1538-7445.AM2012-4210
Abstract 4215: Integrin-dependent mechanisms for sensitizing melanomas to chemotherapy
Authors: Bays Jennifer L. , Palma Melissa , DeMali Kris A.
Published: August 2012

BRAF is mutated in 50-70% of human melanomas, resulting constitutive activation of the MEK-ERK1/2 signal transduction pathway and resistance to apoptosis. Agents that target the oncogenic BRAF signaling pathway are revolutionizing the treatment of metastatic melanoma. However, it is already evident that there is both intrinsic and acquired resistance to this class of compounds suggesting that combinatorial therapies may be needed to combat melanoma resistance. We recently described a novel compound, denoted vinculin activating peptide (VAP) that engages defective integrins with the extracellular matrix and consequently sensitizes melanomas to chemotherapy. VAP is relatively large and the minimal sequences required for the sensitization of melanomas to chemotherapy are not known. In this study, we investigated the smallest domains of VAP that are required for its chemosensitizing effects. Here, we report that VAP contains three small domains, termed vinculin binding sites (VBSs) which are sufficient to bind the integrin-associated protein, vinculin and sensitize melanoma cells to chemotherapeutics. Out of the three VBSs within VAP, VBS3 exhibits stronger binding to vinculin and increases cell-matrix adhesion and focal adhesion number to a greater extent. Furthermore, VBS3 enhances melanoma sensitivity to chemotherapy more than VBS1 or VBS2. The residues of VBS3 required for the sensitization of melanomas have been investigated and suggest that divergent residues are important for chemosensitization. Collectively these data suggest that small molecule mimetics of VBS3 would be an ideal therapy to overcome the resistance of metastatic melanomas to chemotherapeutic agents.

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 4215. doi:1538-7445.AM2012-4215
Abstract 4217: Engaging CD151 inhibits cell migration and metastasis through a novel mechanism involving the cell adhesion molecule ALCAM/CD166
Authors: Palmer Trenis D. , Mazzocca Antonio , Hansen Amanda , Arnold Shanna , Jones-Paris Celestial , Yamada Masashi , Sekiguchi Kiyotoshi , Zijlstra Andries
Published: August 2012

We recently demonstrated that actively promoting the formation of cell surface complexes by engaging the tetraspanin CD151 with a monoclonal antibody (mAb 1A5) inhibits metastasis by immobilizing the tumor cells. Tetraspanins influence cellular behavior via the regulation of associated partner molecules such as integrins, cell adhesion molecules, and members of the immunoglobulin superfamily. Although CD151 is able to associate with and regulate the activity of laminin-binding integrins we demonstrate that CD151 does not associate with α3α1 in the presence of mAb 1A5. Using a 2-color flow cytometric approach we mapped the domains of CD151 required for antigen recognition by mAb 1A5 and determined that 1A5 maps to the integrin 194QRD196 motif of CD151, using this information we further performed CD151 co-immunoprecipitations and demonstrated that 1A5 recognizes CD151 not associated with α3α1.To determine the identity of the protein(s) associated with the antibody-engaged CD151 and also possibly responsible for the anti-migratory, anti-metastatic phenotype we used a proteomic approach. Using tandem mass spectrometry on CD151-associated proteins co-immunoprecipitated by mAb 1A5, we detected in excess of 200 proteins. This strategy identified several putative partners of CD151. Further analysis of the CD151 associated proteins led to our discovery of the novel CD151-associated protein ALCAM/CD166, a member of the immunoglobulin superfamily. Using an RNAi-mediated knockdown approach we demonstrate that ALCAM/CD166 is required for the CD151 complex to regulate tumor cell mobility in vitro. Conversely, CD151 is required for ALCAM to control motility. Spontaneous metastasis assays using the avian chorioallantoic membrane and subcutaneous murine xenografts demonstrate thatALCAM/CD166 is indeed required for the engaged tetraspanin to inhibit tumor cell dissemination in vivo. Further mechanistic analysis of this complex revealed that the CD151/ALCAM protein complex promotes activation of the small GTPase Rap1 via activated protein kinase C. Collectively, we present data, which demonstrate that, the ALCAM-containing CD151 complex can control metastasis. This novel complex reveals how cell-cell interactions can control migration through the regulation of cell-matrix adhesions. These results also demonstrate mechanistically that tetraspanins can integrate signaling between physically distinct molecular functions and therefore behave as a molecular clutch that controls matrix adhesion in response to cell-cell interactions.

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 4217. doi:1538-7445.AM2012-4217
Abstract 4225: Annexin A4 induces chemoresistance for multiple drugs in ovarian clear cell carcinoma
Authors: Morimoto Akiko , Enomoto Takayuki , Serada Satoshi , Matsuzaki Shinya , Yokoyama Takuhei , Kimura Toshihiro , Ueda Yutaka , Yoshino Kiyoshi , Fujita Masami , Fujimoto Minoru , Kimura Tadashi , Naka Tetsuji
Published: August 2012

BACKGROUND: Platinum drugs play the key role for postoperative adjuvant therapy and treatment in progress and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtypes of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. However, it remains unclear whether targeting Anx A4 is effective for improving the sensitization against chemotherapy, like platinum drugs. The aim of this study was to evaluate the role of Anx A4 in chemoresistance in ovarian CCC. MATERIAL AND METHODS: RMG-I cell lines were used to establish the stable knockdown of Anx A4. Plasmids encoding short hairpin RNAs (shRNAs) targeting Anx A4 were transfected. Chemoresistance was assessed by IC50 value in various drugs. RESULTS: Compared with control cells, knockdown of Anx A4 gene expression in RMG-I cells induced chemosentisitzation around 2-fold to cisplatin (IC50: 11.2 μM to 5.8 μM, P<0.01) and Carboplatin (IC50: 130.4 μM to 63.3 μM, P<0.01). Unexpectedly, knockdown of Anx A4 also improved the chemosentisitzation of epirubicin (IC50: 256.7 nM to 180.3 nM, P<0.01) and Paclitaxel (IC50: 5.5 nM to 2.9 nM, P<0.01). In consistent with the improved chemosensitivity, drugs induced p53 was markedly increased by knockdown of Anx A4 in RMG-I cells. In xenograft models, significant chemosensitization toward cisplatin was demonstrated by knockdown of Anx A4 in RMG-I cells. CONCLUSIONS: Inhibition of Anx A4 induced chemosensitization in ovarian CCC cells. Anx A4 blockade might be a novel therapeutic target of chemoresistance in patients with ovarian CCC.

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 4225. doi:1538-7445.AM2012-4225
Abstract 4228: Aryl hydrocarbon receptor nuclear translocator is associated with cisplatin resistance in cancer cells
Authors: Chen Ben-Kuen , Kalpana Sriram , Chang Wen-Chang
Published: August 2012

The (ARNT) belongs to the basic-helix-loop-helix (bHLH) transcription factors containing Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α, ARNT can also form homodimers. Our previous study had shown that ARNT is a new factor involved in EGF regulated COX-2 gene expression under normoxia involving tumorigenesis in cervical cancer tissues. Resistence to cisplatin is often observed in cervical cancer therapy. Here we showed that ARNT expression is critical for cisplatin resistance. Specifically, we showed that in HeLa cervical cancer lines, cisplatin decreased ARNT expression level. Over-expression of ARNT rendered HeLa cells resistant to cisplatin. In addition, inhibition of ARNT by small interfering RNA silencing increased cisplatin induced cell death by activation of caspase 3 and sensitized drug resistant cells to cisplatin. Using ARNT deficient and ARNT fully expressed cell lines showed that induction of apoptosis by cisplatin correlates with ARNT. Importantly down regulation of ARNT decreased drug efflux pump protein MDR1 expression, suggesting that MDR1 gene expression depends on ARNT activity. In conclusion, ARNT mediates the efflux of cisplatin and has a functional role in cervical cancer cell sensitivity to cisplatin. In a xenograft analysis of SCID mice, cisplatin also efficiently inhibited ARNT-deficient tumor formation. These results suggest that targeting ARNT could overcome cisplatin resistance in human cervical 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 4228. doi:1538-7445.AM2012-4228
Abstract 4231: Immunohistochemical analysis of claudins expression in penile squamous cell carcinoma
Authors: Bulgarelli Adriana , Lourenço Silvia Vanessa , Coutinho-Camillo Cláudia Malheiros , Guimarães Gustavo Cardoso , Soares Fernando Augusto , Vassallo José
Published: August 2012

Background: Claudins are tight junction proteins that are critical for the sealing of cellular sheets and controlling paracellular ion flux. Members of the claudin family have been demonstrated to play an important role in carcinogenesis and tumor progression. Penile squamous cell carcinoma (SCC) is a major health problem in Brazil, corresponding to 2,1% of all brazilian men tumors and 5-year survival rate drops dramatically in patients with lymph node metastasis. Objective: The aim of this study was to determine the protein expression pattern of claudins in penile SCC and correlate it with the clinicopathological features of penile cancer. Method: Tissue microarray (TMA) comprising 101 cases of penile SCC were immunohistochemically stained for claudin-1, -3, -4, -5 and -7, with which TMA core showing more than 10% of positive membranous staining cells. Results: Claudin-1, -3, -5 and -7 were downregulated in 55%, 68%, 62% and 88% of penile SCC samples, respectively. Claudin-4 was overexpressed in 58% of the tumor samples. Strong correlation was found between downregulation of claudin-1 and -3 (p< 0.001). Absence of claudin-1, -3 and -5 was associated with urethral infiltration (p=0.03 and p=0.02 and p=0.019, respectively). Loss of claudin -1 and -3 was also associated with perineural invasion (p=0.05 and p=0.02, respectively). Conclusion: Our data provides the first evidence that claudins expression was decreased in areas of invasion and infiltration and could be related to evolution and prognosis of these tumours.

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 4231. doi:1538-7445.AM2012-4231
Abstract 4234: A genome-wide RNAi screen implicates histone deacetylases as a therapeutic target for osteosarcoma
Authors: Perry Jennifer A. , Orkin Stuart
Published: August 2012

Osteosarcoma, the most common primary malignancy of bone, is an aggressive cancer occurring predominantly in children and adolescents. Studies of familial cancer syndromes and sporadic cases have strongly implicated both p53 and pRb in osteosarcoma tumorigenesis. Thus, our lab created a fully penetrant mouse model of osteosarcoma based on the osteoblast-specific deletion of p53 and pRb. This model faithfully recapitulates the defining features of human OS, including pathology, metastatic behavior, and cytogenetic complexity and shares a transcriptional profile with the human disease. Using a genome-wide shRNA screen, we have identified gene products whose inhibition synergizes with p53 and pRb deletion to kill osteosarcoma cells. This screen identified genes that are required for proliferation and survival of osteosarcoma cell lines in an unbiased fashion. The screen identified a number of druggable gene products and pathways, including members of the HDAC family of histone deacetylases. Follow-up studies with selective chemical HDAC inhibitors and shRNAs against individual HDACs demonstrate that osteosarcoma cells are vulnerable to HDAC1, 2, 3, 5 and 7 inhibition. Selective inhibition of HDACs has emerged as a promising avenue toward cancer therapy, therefore we look to understand if and how HDAC inhibition can be used as a therapeutic approach to treating osteosarcoma.

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 4234. doi:1538-7445.AM2012-4234
Abstract 4236: Comparison of optical imaging approaches in 2D and 3D in disseminated mouse tumor models
Authors: Baugher Meridith , Castorena Jeremy , Baranski Jeni , Shaw Darren , Leopold Dick , Trachet Erin , Lister Deanne , McConville Patrick
Published: August 2012

Introduction: Optical imaging allows staging and tracking of multiple tumors in disseminated tumor models and to this end, bioluminescence (BLI) imaging has been the most common approach. While BLI is cost effective and quantitative, it requires luciferase-engineered tumor lines, which have the disadvantages of time and uncertainty associated with transfection, as well as potential phenotype change in the engineered line, compared with the parent. Exogenous fluorescence probes preferentially activated by tumors provide a similar efficient optical imaging means to tumor burden tracking in these models, directly in any tumor lines of interest. This methods also allows 3D imaging through new tomographic optical imaging technologies. In this work, BLI and activated fluorescent probe imaging were used in a luciferase-expressing Raji-luc human Burkitt's lymphoma model and an M-NFS-60 mouse leukemia model. 2D fluorescence reflectance imaging was also compared to 3D tomographic imaging to determine additional utility of the 3D imaging approach. Methods: Raji-luc lymphoma cells were transfected to express luciferase using a lentiviral vector transfection method. Raji-luc cells (1x107 cells in 100µl PBS) were injected IV in CB-17 mice on Day 0. In CD-1 mice, M-NFS-60 leukemia cells (1x107 cells in 100µl PBS) were injected IP. Bioluminescence scans were used to determine the distribution of light throughout the body immediately following implantation. In vivo and ex vivo bioluminescence imaging was used to localize tumor signals and determine incidence and tumor growth. Both cathepsin- (ProSense) and MMP- (MMPSense) activated fluorescent probes were injected IV 24h prior to fluorescence reflectance and fluorescence molecular tomographic imaging. Results and Discussion: Bioluminescence imaging of intravenously injected of Raji-luc cells from one to four weeks after inoculation showed a high incidence of tumor growth at sites including hind limbs, spine, and brain in addition to lung and to a lesser extent, the axillary lymph nodes and sternum. Fluorescence imaging with tumor activated probes provided a measure of disseminated tumor burden, but had the disadvantage of normal tissue background, compared with BLI where there was inherently no normal tissue background. Fluorescence molecular tomographic imaging provided a quantitative advantage over 2D fluorescence reflectance. Activated fluorescent probe imaging provides a viable means for imaging in disseminated tumor models without the need for engineered cell lines.

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 4236. doi:1538-7445.AM2012-4236
Abstract 4241: Glioma spheroids obtained via ultrasonic aspiration are viable and express stem cell markers: a new tissue resource for glioma research
Authors: Jensen Stine S. , Aaberg-Jessen Charlotte , Jabbari Javad , Andersen Claus , Schrøder Henrik D. , Kristensen Bjarne W.
Published: August 2012

Ultrasonic aspirators allow safe, rapid and accurate removal of brain tumors. However, the tissue fragments removed are used surprisingly little in research. The aim of this study was to investigate whether such tissue fragments could be cultured as organotypic multicellular spheroids and whether these spheroids could be used in cancer stem cell research. Biopsy-derived spheroids have earlier been shown to represent an important alternative to commercially available cell lines, but access to biopsy tissue is often limited. Tissue fragments obtained by ultrasonic aspiration from 10 glioblastomas and tumor biopsy tissue from seven of these tumors were collected and cultured in serum-containing and serum-free medium. After culturing, the fragments formed spheroids, which were prepared for histological staining. For comparison, the corresponding fixed non-cultured patient biopsies were also included in the study. Hematoxylin eosin staining showed that viable glioma spheroids were obtained from both ultrasonic and biopsy tissue using both serum-containing and serum-free medium. Independent of the type of tissue used for the spheroids, the immunohistochemical expression of GFAP decreased, whereas the expression of VWF and the Ki-67 proliferation index increased in spheroids cultured in serum-free versus serum-containing medium. Independent of the type of medium, both ultrasonic and biopsy tissue expressed significantly higher levels of the investigated stem cell markers CD133, Nestin and Bmi-1 compared to the non-cultured patient biopsies. In conclusion, we found that is was easy to obtain viable glioma spheroids from ultrasonic tissue fragments. Although differences in expression of the different markers were found, the overall expression was preserved, thereby suggesting ultrasonic tissue fragments as a new tissue resource for glioma research.

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 4241. doi:1538-7445.AM2012-4241
Abstract 4245: Establishment of patients’ primary ovarian cancer xenograft models for test of anticancer agents
Authors: Liu Changnian , Li Yan , Yue Tianfu , Li Wenwei , Zhou Wen , Liu Yong , He Fang , Zhou Rui , Bai Chang
Published: August 2012

BACKGROUND Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But tumor cell lines have a relatively low transplantability, which resulted in a limited number of tumor models available for test of novel anticancer agents. Recently, we have developed a large number of patients’ primary ovarian tumor xenograft models by transplanting human fresh ovarian tumor tissues into nude mice, which have been employed for preclinical test of clinically used drugs and novel anticancer agents for their chemosensitivity screening. METHODS The fresh ovarian tumor samples were collected from local hospitals. The tumor fragments of 2-3 mm were subcutaneously implanted in the flanks of nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). The clinically used drugs included cisplatin, carboplatin, paclitaxel, and docetaxel. The histology and gene sequence of the established primary tumor models were analyzed and compared with patients’ original tumors. RESULTS A total of 76 patients’ ovarian tumor samples were implanted into nude mice; and 35 primary tumor models have been established with a tumor taking rates of 47% for the first passage. The tumor taking rates were higher in the later passages ranged from approximately 70-100%. The therapeutic efficacy of the test anticancer drugs was consistent with their clinical findings. The patients’ primary ovarian tumor xenografts from all 5 passages retained a similarity in architecture, histopathological morphology, and genomic mutation status to their patients’ original tumors. CONCLUSIONS The patient primary tumor model system can provide a larger number of models for selection of right models for preclinical testing novel agents based on their anticancer mechanism. The primary tumor models retain a similarity in histology and genomic mutation status to their patients’ original tumors. They may predict more relevant clinical response rate and higher correlation with clinical findings than use of traditional xenograft models established from long-term cultured cancer cell lines. Especially, they have advantages for test of target-oriented therapeutics in new drugs development programs.

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 4245. doi:1538-7445.AM2012-4245
Abstract 4247: Comparison of two-dimensional verses three-dimensional breast cancer models in drug discovery
Authors: Lovitt Carrie J. , Avery Vicky M.
Published: August 2012

Two-dimensional (2D) monolayer cancer cell culture is generally used in most drug discovery screening programmes to identify novel anti-cancer compounds. However, three-dimensional (3D) cell culture more closely represents the in vivo tumor environment thus providing a more accurate measure of compound activity. Development of an automated, reproducible and miniaturised 3D culture model for drug discovery in breast cancer would be highly advantageous. Thus we have developed and optimised a miniaturised in vitro 3D culture model in which breast cancer cells interact with reconstituted basement membrane from MatrigelTM. To determine the effectiveness of our 3D breast cancer model for use in future drug discovery, chemotherapeutic drugs commonly used in breast cancer therapy were tested and differences in drug activity were compared to 2D cell culture models. Once validated, this 3D culture model will be used to evaluate and further characterise novel compounds. The results presented describe the development and reproducibility of our 3D culture model and its 2D culture model counterpart. This includes the linear relationship between fluorescence intensity and cell number for measurement of metabolic activity as an indicator of cell viability when using the indicator dye Resazurin; determination of an adequate signal window and the reproducible average mean sizes of spheroids present under assay conditions. We report that there is significantly more drug resistance in our 3D cell culture model compared to our 2D culture model. This is particularly evident in the presence of taxanes and anthracyclines. Results from this study also show that the degree of drug resistance in 3D is breast cancer cell line dependent for each of the chemotherapeutic drugs studied and not necessarily correlated to the profile observed in 2D. Image based analysis demonstrated a distinct correlation of 3D cell culture morphology to drug dose, where disassociation of spheroids indicates drug effectiveness and intact spheroids indicates ineffectiveness of drug activity. Further research is underway to determine the mechanism for the drug resistance in 3D culture, which may be important in providing information on resistance mechanisms and potential drug targets. Miniaturisation of 3D culture for breast cancer cell lines allows a higher throughput of novel compound testing in a more cost effective, biologically relevant model. Our work highlights the importance of using 3D cell culture in the early stages of drug discovery in evaluating compound activity.

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 4247. doi:1538-7445.AM2012-4247
Abstract 4248: A high-fat diet induces an inflammatory tumor microenvironment in MNU-induced estrogen receptor positive mammary tumors
Authors: Zhao Shuangping , Frasor Jonna
Published: August 2012

The link between obesity and breast cancer has received much attention because it is associated with an increased risk of breast cancer, an increased incidence of recurrence, higher death rates, and reduced sensitivity to anti-hormonal therapy in post-menopausal women. In addition, obesity is associated with a chronic, low grade inflammatory status. Surprisingly, obesity can have different effects in pre-menopausal women where patient outcome is actually better in obese than lean women. This study was conducted to create an animal model to examine the effects of high-fat (HF) diet on estrogen-dependent mammary tumors in cycling rats. Sprague-Dawley rats were injected with N-Methyl-N-Nitrosourea (MNU) at 6 weeks of age to induce mammary tumors. The next day they received HF diet (45% kcal from fat) or normal fat (NF) diet (10% fat) (n=6-7) and animals were kept on these diets for an average of 15 weeks. Ninety-six percent of rats developed mammary tumors and all the tumors were ER positive. The tumor volume, tumor weight and tumor growth rate were not different between the HF or NF diet groups. Also, the HF diet did not influence the body weight, liver weight and adipose mass weight. However, the expression of pro-inflammatory cytokines, such as TNFa and IL-1b, was significantly up-regulated in tumors of the HF diet group compared to control. Macrophage infiltration was also increased in the tumors of the HF diet group. There was also a tendency of increased NFkB DNA binding activity in the HF diet group. Moreover, macrophage infiltration was significantly correlated with the amount of proinflammatory cytokine gene expression in the HF but not the NF diet group. In agreement with clinical data, cycling rats on the HF diet responded better to tamoxifen than did rats on the NF diet, with a greater reduction in tumor size over 20 days of treatment. Taken together, these results indicate that a HF diet induces a pro-inflammatory tumor microenvironment without causing obesity in cycling rats and that these tumors may be more responsive to endocrine therapy. Our findings also provide a model for future studies into the mechanism by which high fat diet may affect tumor growth and response to therapy in premenopausal women.

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 4248. doi:1538-7445.AM2012-4248
Abstract 4368: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses
Authors: Das Suvendu , Sarrou Eliana , Cassella Melanie , Feirt Nikki , Skobe Mihaela
Published: August 2012

The lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively, by the flow of lymph. Here, we demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node. In vitro, tumor migration to lymphatic endothelium (LECs) was inhibited by blocking CCR8 or CCL1. Recombinant CCL1 promoted migration of CCR8+ tumor cells. Pro-inflammatory mediators TNF-α, IL-1α and LPS increased CCL1 production by LECs as well as tumor cell migration to LECs. Blocking studies showed that CCL1 is a key molecule mediating tumor cell chemotaxis to inflamed lymphatic endothelium. In mouse and human tissues CCL1 protein was detected in lymph node lymphatic sinuses, but not in the peripheral lymphatics. In addition, CCR8 was strongly expressed by human malignant melanoma. In a mouse model, blocking CCR8 function decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1/CCR8 in metastasis and lymph node LECs as a critical check-point for entry of metastases into the lymph nodes.

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 4368. doi:1538-7445.AM2012-4368
Abstract 4375: Sunitinib alone and in combination with SN-38 is active against anaplastic thyroid cancer
Authors: Bocci Guido , Desidero Teresa Di , Fioravanti Anna , Orlandi Paola , Canu Bastianina , Danesi Romano
Published: August 2012

Background: Anaplastic thyroid cancer (ATC) is the most aggressive thyroid gland malignancy. Recent experimental evidences suggest a rationale for the use of multitarget tyrosine kinase inhibitors (TKIs). Sunitinib showed evidences of activity in a phase I trial in different tumors, including thyroid cancers. TKIs are usually more effective in combination with standard chemotherapy. Among chemotherapeutic drugs, preclinical studies using irinotecan seem to be very promising in different types of thyroid carcinomas. The aim of this study is to determine the activity of sunitinib alone and in combination with irinotecan on endothelial and ATC cells. METHODS: Proliferation and apoptotic assays were performed on human dermal microvascular endothelial cells (HMVEC-d) and ATC (8305C, FB3) cell lines exposed to sunitinib (SU) and SN-38, the active metabolite of irinotecan, for 72h. The synergism was determined with the method by Chou and quantified by the combination index (CI), where CI < 1, CI = 1, and CI > 1 indicates synergism, additive effect, and antagonism, respectively. Cell-based phospho-VEGFR-2 assay was performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 and CDK2 gene expression were performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA. 8305C xenografts in nude mice were treated with sunitinib (50 mg/kg/day) and tumour volumes were measured. RESULTS: A strong antiproliferative and pro-apoptotic activities were determined by SU on both endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2 levels significantly decreased after SU treatments in activated endothelial cells; ERK1/2 and Akt phosphorylation was significantly inhibited by SU in endothelial and cancer cells, although at lower concentrations in the endothelial ones. Moreover, SU treatment greatly inhibited the expression of the cyclin D1 and CDK2 genes in both endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration. In vivo administration of sunitinib was effective and determined a significant tumor regression. CONCLUSIONS: SU demonstrated a highly significant antiproliferative and proapoptotic activity, for activated endothelial and ATC cells. Moreover, the simultaneous combination of sunitinib and irinotecan determined a high synergism on endothelial and ATC cells, suggesting a possible translation of this schedule into the clinics.

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 4375. doi:1538-7445.AM2012-4375
Abstract 4405: Immune modulation and Treg suppression with metronomic cyclophosphamide enhances the anti-tumor effect of a therapeutic peptide vaccine in a murine model
Authors: Weir Genevieve , Stanford Marianne , Karkada Mohan , Berinstein Neil , Mansour Marc
Published: August 2012

In order to be optimally effective therapeutic cancer vaccines must be combined with immune modulatory approaches, particularly in advanced cancer where several different pathways of cancer induced immune suppression may be active. We have developed a potent cancer vaccine enhancement formulation named DepoVaxTM (DPX) that is particularly well suited for the delivery of peptide antigens. DPX is a novel vaccine platform comprised of liposomes in oil that is formulated with a TLR adjuvant, universal T-helper peptide and specific tumor peptide antigens. We have shown that DPX generates better immune responses than Montanide based emulsion formulations. A major obstacle to any cancer vaccine, however, is tumor induced immune suppression. Regulatory T cell (Treg) mediated immune suppression is an important mechanism of tumor immune evasion in many types of cancer including ovarian cancer. We sought to determine if combining a DPX-based peptide vaccine approach with immune modulation in the form of Treg suppression could enhance the anti-tumor effect of the vaccine. Metronomic cyclophosphamide (mCPA) treatment has been reported to selectively sustain reduced regulatory T cell numbers and activity. Using a murine tumor model (C3) we tested the benefit of combining mCPA and DPX vaccination for controlling well established subcutaneous solid tumors in vivo. We found that neither mCPA or DPX treatments alone were sufficient to eradicate advanced C3 tumors. However, combining mCPA with DPX vaccination provided effective tumor control. We further characterized the immune response by isolating and phenotyping tumor infiltrating lymphocytes and testing their immunogenicity by IFN-γ ELISPOT assay. Our results indicate that the DPX vaccine can increase the levels of antigen-specific CD8+ T cells and mCPA treatment reduces the CD4+FoxP3+ T regulatory cells. We conclude that immune responses generated by the DPX vaccine were more effective at controlling advanced tumors together with reduced Treg-mediated immune suppression. We have designed and initiated a phase I/II clinical trial to test a DPX vaccine targeting Survivin (DPX-Survivac), in combination with mCPA in ovarian cancer patients.

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 4405. doi:1538-7445.AM2012-4405