bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5270: Evaluation of Cell-able spheroid culture system for culturing patient derived primary tumor cells
Authors: Praveen Kesavan Nair , Streiner Nicole , Vo Martin , Anderes Kenna , Yokota Koichi , Ikeya Takeshi
Published: August 2012

Tumor cell microenvironment has significant impact on growth kinetics, cell signaling and response to drug treatments. 3D models are more biologically relevant models compared to 2D models and have gained preference among researchers and drug developers. It is well recognized that primary tumor cell cultures grown in 2D monolayer quickly lose the ability to proliferate while 3D culture conditions on extracellular matrix allows for spheroid formation and proliferation. Cell-able, a novel micropatterned plate has cell adhesion areas in the well bottom where the size and shape of these areas are tightly controlled. Micro-fabrication of the well bottom enables rapid cell attachment and spheroid formation. Cell-able plates are potentially an alternative to culturing cells in 3D without extracellular matrix. We have evaluated spheroid formation, morphology, duration of viability, histopathology and expression of markers in patient-derived primary tumor cells from different tumor types when cultured on Cell-able plates compared to Cultrex extracellular matrix (ECM) coated plates. In addition we have utilized Cell-able plates to grow patient-derived tumor cells and evaluate the anti-proliferative responses to cytotoxic and targeted agents using high content imaging coupled with immunofluorescence characterization enabling subpopulation analysis within the spheroids. Our results indicate that patient-derived tumor cells from multiple indications as well as tumor cell lines can be cultured on Cell-able plates as spheroids that resemble the micro architecture of tumors and are therefore a suitable 3D model. The results of high content imaging analysis suggest that Cell-able plates could be used as a valuable tool in in vitro predictive assay models in oncology drug development.

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 5270. doi:1538-7445.AM2012-5270
Abstract 5275: Biglycan is a specific marker and an autocrine angiogenic factor of tumor endothelial cells
Authors: Yamamoto Kazuyuki , Ohga Noritaka , Hida Yasuhiro , Maishi Nako , Kawamoto Taisuke , Kitayama Kazuko , Akiyama Kosuke , Osawa Takahiro , Kondoh Miyako , Kaga Kichizo , Hirano Satoshi , Shinohara Nobuo , Shindoh Masanobu , Hida Kyoko
Published: August 2012

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels are morphologically different from their normal counterparts. We isolated tumor endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. Biglycan is overexpressed in inflammation and fibrosis. However, there has been no report about the expression or the function of biglycan in TEC. In this study, we investigated the expression and the role of biglycan in TECs. Real-time PCR, western blotting and immunocytochemistry revealed higher biglycan expression levels in TECs than in NECs. Furthermore, we confirmed that biglycan was secreted from TECs. Biglycan knockdown inhibited cell migration and tube formation in TECs. TLR2 and TLR4 are the biglycan receptors. TLR2 and TLR4 blocking antibodies suppressed biglycan mediated cell migration and tube formation. We isolated TECs from human renal cell carcinoma tissue and NECs from normal renal tissue in the same patients. TECs and NECs were obtained from six patients. Real-time RT-PCR revealed that the biglycan expression levels were significantly higher in four of the six TEC samples than in the corresponding NEC samples. Furthermore, immunostaining revealed strong biglycan expression in vivo in several human tumor vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. Biglycan is an autocrine angiogenic factor stimulating tumor endothelial cell migration and tube formation. These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5275. doi:1538-7445.AM2012-5275
Abstract 5290: Heme oxygenase 1 (HO-1): An auspicious target for state of the art anti-angiogenic therapeutic strategies and new insights in prostate carcinogenesis
Authors: Gueron Geraldine , Ferrando Mercedes , Elguero Belen , Giudice Jimena , Salles Angeles , Labanca Estefania , Jaworsky Felipe , Colombo Lucas , Meiss Roberto , Navone Nora , Siervi Adriana De , Vazquez Elba
Published: August 2012

Prostate cancer (PCa) is a leading cause of death among males. Angiogenesis is critical during tumor initiation and progression. The arrival of anti-angiogenic agents as cancer treatments joining the ranks of surgery, chemotherapy and radiotherapy has been a source of renewed hope. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage. It has become increasingly clear that in addition to its effect on blocking proliferation, invasion and migration, other mechanisms including a direct effect on angiogenic factors may account for its anti-tumoral role in PCa. To further assess its properties, we investigated its potentiality to modulate PCa associated-angiogenesis. In the present study, we identified in PCa cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α, KDR and α5β1 integrin. Our results indicated also that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Furthermore, using immunofluorescence and structured illumination microscopy we visualized NFκB retention in cytoplasm and demonstrated a higher rate of co-localization with HO-1 under HO-1 over-expression. These observations correlated with repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, induced accumulation of IκB and decreased IKK mRNA levels under HO-1 modulation. These evidences strongly suggest that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports an unprecedented role of HO-1, challenging the angiogenic-switch in prostate carcinogenesis outlining a rationale for its development as an anticancer target in PCa.

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 5290. doi:1538-7445.AM2012-5290
Abstract 5383: Evaluation of immune response in preclinical settings
Authors: Mignard Caroline , Bichat Francis , Serin Guillaume , Duchamp Olivier , Mirjolet Jean-François
Published: August 2012

Among all new strategies against cancer, the treatments with vaccines, biologics, antibodies, antibody fragments, and other immunological modulators are promising. All of them aim to be more targeted, more effective, and less toxic than other therapies. In order to be developed and accurately evaluated, these immunological related therapies need appropriated preclinical models and relevant immunological readouts. Most of these immunological readouts should be analyzed in vitro, in vivo and ex vivo. A panel of tools from rodents to the bench was evaluated toward the modulation of the immune system by new therapies. All these protocols were developed and validated properly to evaluate these new immunology related cancer therapies. In immunocompetent mice, the immune cells were studied for their cell surface activation markers detection, induction of proliferative phenotype, antigen-specific T lymphocyte detection, secretion of soluble mediators such as cytokines … using FACS phenotyping, cytometric bead assay (CBA) or Luminex multiplex technologies and ELISPOT. In immunodeficient mice, the reconstitution of the mature human immune system with PBMC or naïve human immune system using hematopoietic stem cells were used to evaluate the modulation of those immune cells by therapies through human cytokine release, onset of graft versus host disease. Among other examples, characterization of antibody function (Fab and Fc mediated activities), quantification of the existence and function of antigen-specific T cell clones, phenotyping study of immune cells for activation markers as well as multiplex assays to understand the cytokines network, differentiation of hematopoietic cells by colony formation unit will be described. All of them will be presented in the context either of rodent syngenic model or humanized mouse model. All results, integrative data and examples will be presented in the poster.

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 5383. doi:1538-7445.AM2012-5383
Abstract 5390: Anti-IL10-R1 monoclonal antibody with concomitant bacillus calmette-Guérin (BCG) prevents metastatic bladder cancer in an in vivo mouse model
Authors: Askeland Eric , Newton Mark , Bockholt Nathan , Knudson Matthew , Henning Jonathan , Wang Xu , Askeland Ryan , Weady Peter , Smith George , Eisenbraun Michael , Fraser James , O'Donnell Michael , Luo Yi
Published: August 2012

Introduction: Induction of Th1 immunity is required for effective intravesical BCG immunotherapy of bladder cancer. IL-10 down-regulates the Th1 response and is associated with BCG failure. We previously demonstrated that blocking IL-10 receptor (IL-10R) by systemic administration of anti-IL-10R1 mAb enhanced intravesical BCG (living Pasteur strain) treatment of bladder cancer in an orthotopic mouse model. Here we investigated the effect of anti-IL-10R1 mAb on clinically used BCG (lyophilized TICE strain) for induction of anti-bladder cancer immunity. Methods: Splenocytes were incubated with BCG alone or plus control IgG or ant-IL-10R1 mAb for 24 hours, followed by ELISA analysis of IFN-γ production. Bladder RNA was extracted after 6 treatments (twice weekly) with intravesical (i.b.) BCG plus intraperitoneal (i.p.) control IgG or anti-IL-10R1 mAb, followed by qPCR analysis of IFN-γ mRNA expression. Three groups of 20 mice were inoculated with luciferase-expressing MB49 bladder cancer cells and treated with i.b. PBS plus i.p. PBS (Group 1), i.b. BCG plus i.p. control IgG (Group 2), or i.b. BCG plus i.p. anti-IL-10R1 mAb (Group 3) 2x/wk for 6 total treatments. Mice were monitored weekly using IVIS luminescence and followed for 76 days. A bioluminesence cutoff of 5X104 p/s was used to establish the presence of bladder cancer. At animal death bladders were collected, weighed and processed for histological analysis. In suspected cases of metastatic disease the affected organs were also collected for histological analysis. Results: BCG plus anti-IL-10R1 mAb induced increased IFN-γ production by splenocytes in a dose-dependent manner. BCG plus anti-IL-10R1 mAb substantially increased IFN-γ mRNA. Eleven, 17 and 9 mice from Groups 1, 2 and 3, respectively, developed bladder cancer. One mouse in Group 2 and 2 mice in Group 3 showed cancer regression. 36%, 53% and 0% of Groups 1, 2 and 3 developed metastatic bladder cancer, respectively (p=0.020). While bladder weights for the groups were different, the differences did not reach statistical significance. Conclusions: Anti-IL-10R1 mAb enhances BCG-induced Th1 immune responses both in vitro and in vivo. Intravesical BCG plus anti-IL-10R1 mAb shows statistical significance in preventing metastasis of bladder cancer in an orthotopic bladder tumor mouse model. Anti-IL-10R1 mAb may prove useful in clinical practice for the prevention of metastatic bladder cancer in high-risk 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 5390. doi:1538-7445.AM2012-5390
Abstract 5399: S100A9 drives MDSC migration and contributes to tumor progression by inhibiting the immune response
Authors: Condamine Thomas , Cheng Pingyan , Youn Je-In , Ortiz Myrna L. , Vogl Thomas , Roth Johannes , Gabrilovich Dmitry I.
Published: August 2012

S100A9 is a pro-inflammatory molecule, secreted by myeloid cells, which has been reported to recruit leukocytes to the sites of inflammation or tumors. We have previously found that up-regulation of S100A9 expression in myeloid progenitor cells in cancer resulted in inhibition of DC differentiation and promotion of expansion of myeloid-derived suppressor cells (MDSC). It remained unclear how S100A9 could regulate MDSC expansion and function. In this study we evaluated the effects of secreted (extracellular) vs intracellular S100A9 on MDSC. Recombinant S100A9 or S100A8 proteins separately or as heterodimers did not promote MDSC expansion from hematopoietic progenitor cells (HPC), or affect their suppressive activity. However, recombinant proteins had a strong chemotactic effect on MDSC. Interestingly, this effect on immature myeloid cells isolated from naïve tumor-free mice was substantially lower. To study intracellular activity of S100A9 we used S100A9 transgenic (Tg) and knockout (KO) mice. Mice were injected with several different weakly and strongly immunogeneic tumors. As expected Tg mice had increased expansion of MDSC and KO mice had substantially reduced number of these cells. Different levels of MDSC did not affect the growth of weakly immunogeneic tumors. In contrast, overexpression of S100A9 in mice lead to significantly accelerated growth of immunogenic tumors. Lack of S100A9 resulted in fast rejection of immunogeneic tumors. Our data indicate that extracellular and intracellular S100A9 had different signalling in myeloid cells and apparently affect different aspects of their function. S100A9 over-expression, in vivo, inhibits the immune response, most likely by promoting MDSC accumulation and/or suppressive activity. Our data suggest that targeting of S100A9 in cancer can be an attractive therapeutic option.

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 5399. doi:1538-7445.AM2012-5399
Abstract 5466: Butyrate effect on expression of quinone oxidoreductase (NQO1) and cytochrome P450 CYP1A1 enzymes
Authors: Tesfamichael Aron B.
Published: August 2012

Butyrate is a small chain fatic acid formed by bacterial fermentation of plant foods in guts. It has been shown to induce Quinone Oxidoreductase (NQO1) enzyme in tumor cell lines, which may contribute to the detoxification of dietary carcinogens with out any effect to the expression of carcinogen activation enzymes, such as cytochrome P450 CPY1A1 and epoxide hydrolase. The risk from chemical carcinogens and environmental toxins is dependent on the metabolic balance between bioactivation and detoxification enzymes. Cancer chemoprevention therefore may be achieved through alterations in the expression of these enzymes. There is substantial evidence that chemoprevention comprises multiple intervention methods to impede, arrest, or reverse carcinogenesis at various stages. Transcriptional regulation of activation and detoxification enzymes competing for the carcinogen substrate is strongly affected by intracellular oxidation reduction potential change. Treatment of cells with butyrate causes a change in intracellular redox potential which results in transcriptional regulation of activation and detoxification enzymes. Indicators of redox potential may include the concentration ratio of glutathione (GSH): glutathione disulfide (GSSG), cysteine (CYH): cystine (CYSS) and other redox couples. Treatment of human liver HepG2 cells with sodium butyrate (5mM, 48-72Hr) results in an oxidation of both GSH: GSSG and CYH: CYSS to approximately the same extent up on calculation based on Nernst equation of concentrations determined by HPLC analysis. The present study shows that both the steady-state redox potential of the cysteine/cystine couple (Eh = -155 mV) and GSH/GSSG couple (Eh = -255 mV) in cells are sufficiently oxidized (>40 mV) up on treatment of cells with butyrate. The GSH oxidation, in response to 5mM butyrate at 48 -72 Hr for HepG2 cells were associated with an approximately 3-4-fold increase in mRNA for NQO1 as measured by quantitative PCR and 40-85 fold increase as a luciferase measure for relative change in a of transcriptional activation of NQO1 as comparing to the control respectively. However, CYP1A1 expression was not significantly different from controls. Changes in redox potential may directly release transcriptional proteins from within the cytoplasm for traslocation to the nucleus or alter their affinity for transcription response elements. These possibilities are under investigation using nuclear extracts of HepG2 and HT29 cells treated to alter intracellular redox potential. Surface-enhanced laser desorption/ionization (SELDI) allows rapid identification and quantitation of changes in transcription proteins in response to intracellular redox potential change.

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 5466. doi:1538-7445.AM2012-5466
Abstract 5470: Genotoxic effect and epigenetic alterations induced by the environmental carcinogen dibenzo[a, l]pyrene in oral tissues of mice
Authors: Zhang Shang-Min , Sun Yuan-Wan , Chen Kun-Ming , Aliaga Cesar , Gowdahalli Krishnegowda , Amin Shantu , El-Bayoumy Karam
Published: August 2012

Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this environmental carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral carcinogenesis.

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 5470. doi:1538-7445.AM2012-5470
Abstract 5486: Reliability of biomarkers of inflammation from repeated measures in healthy individuals
Authors: Navarro Sandi L. , Brasky Theodore M. , Song Xiaoling , Wang C.Y. , Kristal Alan R. , Kratz Mario , White Emily , Lampe Johanna W.
Published: August 2012

Biomarkers of low-grade systemic inflammation are used to study the associations of inflammation with chronic diseases, including cancers. However, little is known about the reliability (i.e., intraindividual variability) of most of these measures. Fasting (12-hour) serum samples, which were collected at the end of ≥3 week washout periods in a 4-diet crossover feeding trial, were used to measure the inflammatory markers hsCRP, IL-6, TNF-α, IL-8 and sTNFRI and II. Participants included 32 men and 31 women (5 men and 2 women were missing from analyses of IL-8, TNF-α, and sTNFR I and II), aged 20-40, who were free of factors known to influence inflammation, e.g., chronic disease, medication use, heavy alcohol use, smoking and obesity (body mass index >30 kg/m2). Intraclass correlations (ICC) were estimated using random effects analysis of variance, across all 4 time points (∼5 weeks apart). For TNF-α and sTNFRI & II, the ICCs were very high (ICC= 0.92, 95% CI: 0.89 - 0.96; 0.92, 95% CI: 0.88 - 0.95; and 0.90, 95% CI: 0.85 - 0.94, respectively). The ICCs for IL-8 and hsCRP were 0.73 (95% CI: 0.63-0.83 and 0.68 (95% CI: 0.58-0.78), respectively. The ICC for IL-6 was considerably lower, (ICC = 0.48, 95% CI: 0.36-0.62). With the exception of IL-6, reliability of all inflammatory markers in our panel over 4 time points was high, suggesting that a single measure accurately captures the variability within an individual and is a good measure for use in epidemiologic studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5486. doi:1538-7445.AM2012-5486
Abstract 5526: Lymphangiogenesis in regional lymph nodes is an independent prognostic marker in rectal cancer patients after neoadjuvant treatment
Authors: Jakob Christiane , Aust Daniela E. , Baretton Gustavo B. , Datta Kaustubh , Muders Michael H.
Published: August 2012

One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. To evaluate the significance of lymph node lymphangiogenesis, we studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive surgery. Regional lymph node lymph vessels were detected by immunohistochemistry for podoplanin (D2-40). Our results show that the presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Indeed, our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal 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 5526. doi:1538-7445.AM2012-5526
Abstract 5531: Lumican as a novel differential diagnostic maker for Bowen's disease and actinic keratosis
Authors: Takayama Ryoko , Ishiwata Toshiyuki , Ansai Shin-ichi , Yamamoto Tetsushi , Matsuda Yoko , Kawana Seiji , Naito Zenya
Published: August 2012

BACKGROUND: Lumican is a member of the small leucine-rich proteoglycan (SLRP) family and regulates the assembly and diameter of collagen fibrils in the extracellular matrix in human tissue. Mice homozygous for a null mutation in lumican display corneal opacification and skin laxity. Recent studies have shown that lumican is expressed in various types of malignant tumors including breast, colorectal, and pancreatic cancer, and osteosarcoma and malignant melanoma, and that it plays an important role in the growth of tumors. However, little has been studied about the expression pattern and role of lumican in non-melanoma skin cancer. In this study, we examined the expression levels of lumican in in situ lesions of squamous cell carcinoma (SCC) of the skin, actinic keraotsis and Bowen's disease, to discover whether lumican can be used as a novel differential diagnostic marker for such skin tumors, particularly for bowenoid actinic keratosis and Bowen's disease in sun-exposed areas of the body. Bowenoid actinic keratosis and Bowen's disease are often difficult to distinguish in both the clinical setting and commonly prepared pathological specimens. Furthermore, invasive SCC arising in actinic keratosis is considered low-risk clinically, while Bowen's disease with invasion is considered high-risk. Thus, it is important to distinguish between actinic keratosis and Bowen's disease, and reliable differential markers for these diseases are required.METHOD: Twenty-nine cases of actinic keratosis, including 4 of the bowenoid sub-type, and 37 cases of Bowen's disease, including 6 in sun-exposed areas of the body, were selected from among patients undergoing surgical resection at the Department of Dermatology at the Nippon Medical School from 2006 to 2010. We evaluated lumican expression and mRNA in these cases using immunohistochemistry and in situ hybridization analysis, respectively. RESULTS: Immunohistochemical analysis indicated that Lumican was expressed in 34 of 37 patients with Bowen's disease (91.8%), but was not detected in any of the cases of actinic keratosis. In situ hybridization analysis revealed that lumican mRNA was expressed in the cytoplasm of immunohistochemically lumican-positive tumor cells in Bowen's disease. CONCLUSION: These findings suggest that lumican plays an important role in the pathogenesis of Bowen's disease and will be a useful marker for the differential diagnosis between Bowen's disease and actinic keratosis, particularly for bowenoid actinic keratosis and Bowen's disease occurring in sun-exposed areas of the body.

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 5531. doi:1538-7445.AM2012-5531
Abstract 5541: Molecular diagnostic tests to augment cytomorphologic diagnosis of lung cancer
Authors: Yeo Jiyoun , Crawford Erin L. , Zyrek-Betts Jill , Khuder Sadik A. , Austermiller Bradley , Willey James C.
Published: August 2012

Morphologic analysis of cytology samples obtained by transthoracic needle aspiration (TTNA) is one of the primary methods for diagnosing bronchogenic carcinoma. However, across multiple studies the false negative rate for cytomorphological analysis ranges from 0.2-0.3, and among those diagnosed with lung cancer, the false positive rate for diagnosing the sub-class of small cell carcinoma averages 0.09. These false results lead to additional invasive diagnostic studies and delay treatment. In an effort to augment accuracy and utility of cytopathologic evaluation of TTNA lung samples, we measured the previously reported c-myc x E2F-1/p21WAF1/CIP1 (p21 hereafter) gene expression index in cDNA samples from 78 non-malignant lung tissues and 57 lung cancer tissues. Using the optimal cut-off value, this test correctly classified 72/78 non-malignant and 50/57 malignant samples for a correct classification rate of 90% (95% CI 83.6% - 94.3%), sensitivity of 88%, and specificity of 92%. A CDKN2C/FOSL1 test for distinguishing non-small cell lung cancer (NSCLC) from small cell lung cancer (SCLC) had a PPV of 100% with 63% sensitivity. In order to optimize the robustness and utility of these tests, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of internal standard competitive template (CT) molecules using two-color flurometric analysis on real-time platform. For each gene, the native template was quantified with a sequence-specific FAM-labeled probe and the CT was quantified with a sequence-specific Quasar670 labeled probe. Results for each gene thus far demonstrate excellent linearity (R2 > 0.99, slope 1.0 +/- 0.05), relative accuracy (variance < 0.2), signal-to-analyte response (1.0 +/- 0.05) and precision (CV < 30%) over six orders of magnitude, and reliable detection of as few as 10 molecules. Assessing both the c-myc x E2F-1/p21 index and the CDKN2C/FOSL1 test promises to augment cytomorphologic diagnosis of bronchogenic carcinoma in TTNA samples biopsy samples.

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 5541. doi:1538-7445.AM2012-5541
Abstract 5548: High copy number of the MET gene predicts resistance to EGFR-TKI in non-small cell lung cancer patients.
Authors: Noro Rintaro , Seike Masahiro , Soeno Chie , Matsuda Kuniko , Sugano Teppei , Nishijima Nobuhiko , Toyokawa Masaru , Kitamura Kazuhiro , Kosaihira Seiji , Minegishi Yuji , Yoshimura Akinobu , Kubota Kaoru , Gemma Akihiko
Published: August 2012

Background Non-small cell lung cancer (NSCLC) patients with EGFR gene mutations have shown a dramatic responses to EGFR tyrosine kinase inhibitor (EGFR-TKI). However, it is recognized that, clinically, drug resistance eventually emerges and this limits the mean duration of response. Although mechanisms of acquired resistance to EGFR-TKI, such as T790M secondary mutation and MET amplification, have recently been described, other mechanisms of resistant to EGFR-TKI should be identified to broaden the therapeutic strategy for NSCLC in patients with EGFR gene mutations. Patients and Methods We evaluated 26 lung tumor samples from NSCLC patients who had received EGFR-TKI treatment from 2009 to 2011 at Nippon Medical School Hospital. Status of copy numbers and amplification of the MET gene were examined by fluorescence in situ hybridization (FISH) and estimated by high throughout automated image analysis. Results High copy number (>4 copies/cell) was observed in 6 patients (27%) and amplification (MET/CEP7 ≥2.0) was seen in 0 of 26 NSCLC patients. MET gene copy number status was not correlated with EGFR gene mutation, gender, histology, or smoking history. In 2 of the 6 cases with high copy number, high copy numbers were found at both the pre- and post-treatment stages. Among the 6 patients with high copy number, 5 patients achieved partial response (PR) and one patient showed stable disease (SD) with EGFR-TKI therapy. However, the 6 patients with high copy number had statistically significantly shorter progression-free survival (PFS) than the 20 patients with low copy numbers. (p=0.048, log-rank test). The median PFS of patients with High copy number and low copy numbers was 9.5 months and 24.4 months, respectively. Conclusion Detection of high copy number of the MET gene by FISH may be useful for predicting response to EGFR-TKI. The correlation between MET gene copy number and response to EGFR-TKI should be further evaluated using larger sample sizes.

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 5548. doi:1538-7445.AM2012-5548
Abstract 5680: Bortezomib-induced quiescence of multiple myeloma cells is associated with changes in the expression of p21(CIP/KIP) and the H3K27-methyltransferase EZH2
Authors: Adomako Alfred , Aguirre-Ghiso Julio A.
Published: August 2012

Bortezomib-induced quiescence of multiple myeloma cells is associated with changes in the expression of p21CIP/KIP and the H3K27-methyltransferase EZH2. Most multiple myeloma (MM) patients respond positively to the proteasome inhibitor (PI) bortezomib (Velcade). However, there are high rates of relapse after variable remission periods. It is still unclear whether these periods of remission are due to residual cells that after bortezomib treatment enter dormancy or simply due to a small residual population that slowly proliferate until patients becoming symptomatic. It is therefore important to determine whether minimal residual disease (MRD) can be rendered quiescent by bortezomib. We had shown that in MM cells, bortezomib induced ER-stress and quiescence. However, the mechanism for the induction and maintenance of this quiescent state is unclear. We hypothesized that the prolonged quiescence might involve prototypical cell cycle inhibitors and that epigenetic mechanisms sustain the growth arrest. Quantification of viable cells using immunofluorescence microscopy showed a 6-fold increase in p21 protein after a 24-hour pulse treatment with bortezomib followed by washout. p21 and p27 were also persistently induced 2-fold and 1.5-fold respectively along with the downregulation of CDK6 3 days after the drug washout. This suggests that quiescence onset is triggered by cell cycle inhibitor induction that block the G1 to S transition. This correlated with the previously reported reduction in Rb phosphorylation in bortezomib-surviving MM cells. When we tested the levels of the histone H3 methyl-transferase EZH2 and H3K27me3, these were strongly downregulated in the quiescent MM cells. EZH2 depletion was previously linked to p21-mediated senescence in melanoma. Our results suggest that changes in histone methylation and cell cycle inhibitor induction may be a mechanism regulating bortezomib-induced quiescence. Further studies will validate whether these mechanisms are active in patients by analyzing clinical samples from MM 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 5680. doi:1538-7445.AM2012-5680
Abstract 5697: Multistage vectored nanotherapeutics of breast cancer lung metastasis
Authors: Shen Haifa
Published: August 2012

Breast cancer metastasis to remote organs is the major cause of cancer death. Although significant progresses have been made on survival of patients with Stage I-III breast cancer, the situation with breast cancer metastasis is discouraging. Nanotechnology has played an important role in the fight against breast cancer in the last decade. The first nano-liposomal formulation of doxorubicin (Dox), Doxil, was FDA approved in 1994, and is currently used to treat multiple cancer types including recurrent and refractory breast cancers. More nano-formulated drugs have since been approved. These nanotherapeutics work based on the enhanced permeability and retention (EPR) effect, and still cause severe toxicity to the body, however. To overcome these defects of the first generation of nanotherapeutics, we have developed a silicon-based multistage vector (MSV) system with the aim to specifically delivery drugs to cancer tissues with the least toxicity to the normal organs. We have demonstrated highly reproducible therapeutic efficacy with this system to deliver the conventional chemotherapy drugs and siRNA oligos in nanoparticles. We have generated mouse lung metastasis models with the human breast cancer cell line MDA-MB-231 which was engineered with a luciferase gene, and used multiple doxorubicin formulations to treat the tumor mice. These included free doxorubicin (3 mg/kg weekly), Doxil (6 mg/kg biweekly), a doxorubicin prodrug (proDox, 6 mg/kg biweekly), and proDox in MSV (MSV/proDox, 6 mg/kg biweekly). Tumor growth in the lung was monitored by bioluminescence with a Xenogen IVIS200 optical in vivo imaging system. As expected, treatment with free Dox caused significant weight loss indicating drug toxicity, most likely to the heart. Tumor growth in the free Dox treatment group halted initially, but resumed two weeks later. The tumor cells in these animals might have developed resistance to Dox during the treatment. Doxil treatment also triggered weight loss initially. This correlates with reduced the cardiac side effect of the drug observed in clinic comparing to free Dox. Neither proDox nor MSV/proDox caused weight loss. Fifty percent of the untreated mice died of lung metastasis by 11 weeks. The free Dox- and proDox-treated mice extended life by 2 weeks. Half of Doxil-treated mice died by 15 weeks, when all MSV/proDox-treated mice have survived. These results indicate that we have developed a powerful system for tumor-specific delivery of therapeutic in the treatment of breast cancer metastasis.

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 5697. doi:1538-7445.AM2012-5697
Abstract 570: Breast cancer chemopreventive activity of a novel amide derivative of sulindac involves inhibition of PDE5, activation of PKG, and attenuation of Wnt/β-catenin signaling
Authors: Tinsley Heather N. , Gary Bernard D. , Whitt Jason , Zhang Wei , Chen Xi , Keeton Adam B. , Grubbs Clinton J. , Grizzle William E. , Piazza Gary A.
Published: August 2012

The nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX2) inhibitors demonstrate promising chemopreventive activity against a number of cancer types, including breast cancer. Unfortunately, potentially fatal toxicities associated with COX inhibition have precluded the development of these drugs for such indications. However, some studies suggest that their anticancer activity may be due to COX-independent mechanisms, suggesting the ability to target such mechanisms for the development of potentially safer and more effective chemopreventive agents. Here we describe a novel amide derivative of the NSAID sulindac sulfide (SS), termed sulindac sulfide amide (SSA). SSA lacked the ability to inhibit either COX1 or COX2 but demonstrated as much as a twenty-fold increase in potency to inhibit growth and induce apoptosis of breast tumor cells compared to SS. Moreover, SSA was selective for tumor cells, having no measurable effect on apoptosis of nontumorigenic mammary epithelial cells. As we have shown previously with SS, SSA inhibited the cGMP specific phosphodiesterase 5 (PDE5) enzyme. This resulted in accumulation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) at concentrations comparable to those that induced breast tumor cell apoptosis. Mirroring the effects on PKG, SSA also resulted in decreased Wnt/β-catenin signaling, including decreased expression of the Tcf/Lef controlled proteins, cyclin D1 and survivin. Furthermore, SSA caused a 50% reduction in the incidence and a 30% reduction in the multiplicity of mammary tumors using the MNU model of mammary carcinogenesis. Interestingly, mammary tumors induced by MNU displayed high levels of PDE5 expression. These findings further demonstrate the COX-independent nature of the anticancer activity of certain NSAIDs, provide additional evidence that PDE5 may serve as an effective target for breast cancer chemoprevention, and reveal the utility of chemically modifying the NSAIDs to remove COX inhibitory activity while enhancing anticancer activity in order to produce safer and more effective chemopreventive 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 570. doi:1538-7445.AM2012-570
Abstract 5705: Orally delivery alginic acid-coated chitosan nanoparticles loaded with legumain DNA vaccine protect against murine breast cancer
Authors: Liu Ze , Liu Shu , Xiang Rong , Tan Xiaoyue
Published: August 2012

Legumain DNA vaccine has been proven to be a potential treatment approach to protect against breast cancer. However, absence of safe and efficient oral delivery system highly restricts its clinical application. Here, we aim to develop a novel oral delivery system for legumain DNA vaccine with better biocompatibility and efficiency. We constructed alginic acid-coated chitosan NPs (A.C.NPs) and tested its anti-acid characteristics in vitro. DNA agrose electrophoresis data showed that compared to chitosan NPs (C.NPs), A.C.NPs significantly protected DNA from degradation in the condition of pH=1.5. Furthermore, size distribution anlysis showed that when pH<3.0, A.C.NPs aggregated to form the micrometer scale complex and dispersed to nanoparticle sized about 314 nm with the increased pH value. To visualize DNA expression, mice were treated by gavage with the A.C.NPs carrying EGFP expression plasmid. Significant EGFP expression can be detected in the intestinal peryer's patch of mice. Immunoflurescence staining showed that overlapping existed in EGFP and F4/80 (CD11b) positive cells. Full length legumain plasmids were loaded into different delivery systems including chitosan NPs (CL), Salmonella typhi (SL), A.C.NPs (ACL). PBS and A.C.NPs loaded with empty plasmid was also set as control groups (n=5 each group). Oral vaccination was performed in murine orthotropic 4T1 breast cancer model. Animals were scarified when the average tumor size in the PBS group reached 500 mm3. Our data showed that tumor volume was significantly smaller in ACL and SL groups compared to other groups. Further, we isolated splenocytes from mice and co-cultured with 4T1 cells pre-stimulated with hypoxia, which intrigued the translocation of legumain from cytoplasm to membrane. FACS assay showed that active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) in ACL group increased 4.5 folds compared to PBS group and 2.3 folds compared to CL group respectively. Our study suggests that chitosan NPs modified with alginic acid could be a potential safe and efficient tool for oral delivery plasmid DNA. Alginic acid-coated chitosan NPs loaded with legumain DNA vaccine can effectively improve auto-immune response and protect against breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5705. doi:1538-7445.AM2012-5705
Abstract 5708: Interstitial protein delivery evaluated in multicellular layers model
Authors: Kim Soo-Yeon , Kim Kwang-Suck , Kim Jung Ho , Kim Tae Hyung , Choi Jong Hoon , Lee Kang Choon , Park Ki Dong , Lee Seung-Jin , Na Kun , Kuh Hyo-Jeong
Published: August 2012

The limited efficacy of protein drugs is related to their poor distribution in tumor tissue. We examined interstitial delivery of four model proteins of different molecular size and bioaffinity as formulated of non-formulated forms in multicellular layers (MCL) of human cancer cells. Model proteins were tumor necrosis factor-related apoptosis-including ligand (TRAIL), cetuximab, RNase A, and IgG. MCLs were cultured in Transwell inserts, exposed to drugs, then cryo-sectioned for image acquisition using fluorescence microscopy (fluorescent dye-labeled TRAIL, RNase A, IgG) or Immunohistochemistry (cetuximab). TRAIL and cetuximab showed partial penetration into MCLs, whereas RNase A and IgG showed little penetration. At 10-fold higher dose, a significant increase in penetration was observed for IgG only, while cetuximab showed an intense accumulation limited to the front layers. PEGylated TRAIL and a heparin-Pluronic nanogel formulation of RNase A showed significantly improved penetration that was attributed to increased stability and extracellular matrix binding, respectively. IgG penetration was significantly enhanced with PTX pretreatment as a penetration enhancer. MCL culture was successfully used for the evaluation of protein movement in the tumor interstitum. Four proteins showed limited interstitial penetration in MCL cultures. Bioaffinity, rather than molecular size, seems to have a positive effect on tissue penetration, although strong binding affinity may lead to sequestration in the front layers. Nanoformulations, such as PEGylation and heparin-Pluronic (HP) nanogel, or penetration enhancers are potential strategies to increase interstitial delivery of anticancer biologics.

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 5708. doi:1538-7445.AM2012-5708