bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 511: Adipocyte-derived fibroblasts contribute to the desmoplastic reaction in breast cancer: A new link between breast cancer and obesity
Authors: Bochet Ludivine , Dauvillier Stéphanie , Dirat Béatrice , Dabek Marta , Gonidec Sophie Le , Mari Pierre-Olivier , Escourrou Ghislaine , Valet Philippe , Muller Catherine
Published: June 2012

Now, it is widely established that malignant tumors, not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Accordingly, one of the features of many carcinomas, particularly breast, is the presence of a dense collagenous stroma, the so-called desmoplastic response, comprising fibroblast-like cells which play a crucial role in tumor progression. Peritumoral fibroblasts are composed of several subpopulations that are morphologically undistinguishable and their origins remain debated. Our results suggest that a part of this fibroblast could arise from the “reorientation” of mature adipocytes, highly represented in breast tumour microenvironment. Using an original 2D coculture system, we show that mature adipocytes cocultivated with breast tumour cells for 3 to 8 days lose their lipid content as well as their adipocyte differentiation markers (HSL, Adipsin, Adiponectin, C/EBPα, PPARγ, Resistin). This process is associated with intense morphological and functional changes leading to an activated fibroblast phenotype (exhibition of actin stress fibers, increase of focal adhesion sites). In this way, we have identified for the first time a new fibroblastic population called Adipocyte Derived Fibroblast (ADF). Interestingly, the reorientation occur in vivo since the injection of far red fluorescent tumour cells into a mice fatpad containing GFP-expressing adipocytes induce the apparition of GFP-fibroblast-like cells that interdigitate with tumour cells. Moreover, our results show that the ADF phenotype depends on the reactivation of the Wnt/β-catenin pathway. Finally, ADFs exhibit an increase of migratory and invasive abilities, with enhanced profibrotic secretions (fibronectin and collagen 1) suggesting that these cells could be key players in desmoplastic reaction. Ongoing experiments are performed to assess the presence of ADFs in breast human tumors. Our results might provide an explanation for the poor prognosis observed in localized breast cancer in obese women, since the nature of the desmoplastic reaction and the secretion pattern of ADF might be profoundly altered in this physiopathogical condition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2011-511
Abstract 5118: Comparative study on feature selection methods in ovarian cancer diagnosis using chemometrics tools
Authors: Yoon Seongkyu , Lee Hae Woo , Liu Jay , Na Young Jung
Published: June 2012

Ovarian cancer has the poorest prognosis of the gynecological cancers. 75% of the patients are diagnosed with stage III-IV on their diagnosis. The 5 year survival rate of ovarian cancer patients whose disease was diagnosed at stage III or IV is less than 20%, but that of the patients diagnosed with stage I is higher than 90%. The early detection and diagnosis of cancer is one of the major application areas in omics technology and has received tremendous attention in last few years.

The main focus of this study is aimed at the investigation of robust formulations for the feature selection algorithms which are utilized during the statistical analysis of omics profiles in diagnosing ovarian cancer. The common difficulty in the feature selection for cancer-specific biomarker discovery is the inconsistency of the feature sets selected among different samples or by different selection methods. This issue is particularly crucial in biological applications where subsequent biomedical analysis of selected features requires relatively considerable time and costs. In this study, we analyzed the reproducibility of different feature selection algorithms usually employed in chemometrics and investigated their stability to provide valuable insight for designing proper feature selection algorithms in the domain of cancer-specific biomarker discovery. Two data sets of MALDI-TOF-MS (proteomics) and LC/TOF-MS (metabolomics) spectra measured from serum samples to diagnose ovarian cancer were analyzed. To evaluate the feature selection methods in terms of their consistency, we systematically quantified a measure of similarity (similarity≤1) for the outcome of each method by running each selection procedures repetitively.

The results show that, in general, the multivariate feature selection methods outperformed the univariate methods, in that they selected more consistent features regardless of training set perturbations. The results clearly show that some feature selection methods can suffer severely from the irreproducibility issue and this might deflate the potential benefits of omics technology for cancer diagnosis. A possible remedy might be multi-objective formulation of the feature selection problem by simultaneously optimizing stability and accuracy of classification models, and this will be investigated further in future research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2011-5118
Abstract 5130: Neuropilin-1 expression in breast, colorectal and lung cancer
Authors: Jubb Adrian M. , Strickland Laura A. , Chen Daniel S. , Schmidt Maike , Koeppen Hartmut
Published: June 2012

Neuropilin-1 is a coreceptor for vascular endothelial growth factor, which is expressed by endothelial cells and tumor cells. Preclinical data suggest that targeting neuropilin-1 would directly inhibit tumor cell growth and invasion, in addition to suppressing angiogenesis with indirect suppression of tumor growth. A humanized monoclonal antibody to neuropilin-1 is currently being evaluated in a phase one clinical trial of patients with solid malignancies. The aim of this study was to characterize the in situ expression of neuropilin-1 in normal and neoplastic human tissues.

A monoclonal antibody to human neuropilin-1 was generated and validated for immunohistochemistry by comparison with isotopic in situ hybridization data, use of formalin-fixed human embryonic kidney (HEK)-293 cell pellets transfected with neuropilin-1, western blotting and quantitative reverse transcriptase (RT)-PCR on microdissected pure cell populations. Neuropilin-1 expression was assessed in whole sections of 65 primary breast carcinomas, 95 primary colorectal adenocarcinomas and 80 primary non-small cell lung carcinomas. An additional 50 breast, colorectal and lung metastases were also evaluated.

The neuropilin-1 monoclonal antibody showed appropriate reactivity by western blotting and in formalin-fixed cell pellet controls, with co-localization of protein (immunohistochemistry) and mRNA (in situ hybridization and laser-capture microdissection followed by quantitative RT-PCR). Immunoreactivity for neuropilin-1 was seen in endothelium from normal tissues adjacent cancer and 98 to 100% of primary and secondary carcinomas. Tumor cell expression of neuropilin-1 was also observed in 34% of primary non-small cell lung carcinomas and 6% of primary breast carcinomas, but no colorectal adenocarcinomas.

This is the first comprehensive analysis of neuropilin-1 protein expression in neoplastic human tissue with validated reagents. The assay will inform the likely activity and toxicity of agents that target neuropilin-1 and warrants evaluation as a biomarker of anti-neuropilin-1 activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2011-5130
Abstract 514: A novel dual color mouse model for studying the tumor microenvironment of pancreatic adenocarcinoma
Authors: Snyder Cynthia S. , Kaushal Sharmeela , Hardamon Chanae R. , Tran Cao Hop S. , Menen Rhiana S. , Metildi Cristina A. , Lowy Andrew M. , Hoffman Robert M. , Bouvet Michael
Published: June 2012

We have developed a novel, dual color, immunocompetent mouse model for studying the pancreatic tumor microenvironment using fluorescence imaging. To develop this model, we used mice engineered to constitutively express a conditional tdTomato fluorescent transgene that converts to the expression of EGFP following Cre recombinase-mediated intramolecular rearrangement of the transgene. The reporter transgene is driven by a strong, ubiquitous promoter from a well-characterized genomic locus. Both tdTomato and EGFP fluorescent proteins are membrane-targeted, allowing distinction of single cells and cell processes within the microenvironmental setting. For our studies of the pancreatic microenvironment, we used PdxCre (Pdx-1-Cre) transgenic mice. PdxCre mice express Cre recombinase within the developing pancreas during embryogenesis. To generate mice in which the pancreatic parenchyma, expressing membrane-targeted green fluorescence (mG), is highlighted against the red fluorescent (mT) backlight of stromal and non-pancreatic tissues, we crossed PdxCre transgenic mice with conditional fluorescent (mTmG) indicator mice. Both lines are on a mixed C57Bl/6 × Sv129 background, backcrossed several generations onto C57Bl/6. We used PDA4964 pancreatic adenocarcinoma cells, derived from genetically engineered triple transgenic (LSL-KrasG12D/+;LSL-Trp53R172H/+; PdxCre) mutant mice, to generate orthotopic tumors in the pancreata of PdxCre+, mTmG+ dual transgenic, dual color mice. Using a dosage of 1 × 106 tumor cells per mouse, half of the mice developed tumors within 4-6 weeks. Ex vivo imaging of orthotopic tumor-bearing mice at necropsy revealed large pancreatic tumors comprised of non-fluorescent tumor cells supported and nourished by red fluorescent host stromal and vascular cells. The expanding tumor masses distorted and displaced the adjacent green fluorescent host pancreatic parenchyma. Upon microscopic examination of the tumor-host interface, cell borders of individual host pancreatic parenchymal and stromal cells were clearly outlined. Our preliminary findings indicate that this novel, dual color, immunocompetent mouse model will be valuable for studying the pancreatic tumor microenvironment using fluorescence imaging.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2011-514
Abstract 5140: Development and evaluation of a liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants in non-small cell lung cancer (NSCLC)
Authors: Parisi Kleo , Markou Athina , Tsaroucha Emily , Kaklamanis Loukas , Lianidou Evi S.
Published: June 2012

Introduction. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key regulator of angiogenesis. The aim of our study was to develop and validate a novel liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants, based on the Luminex technology and evaluate it in NSCLC.

Materials and methods. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of VEGF splice variants VEGF121, VEGF165 and VEGF189. We included PBGD as a reference gene, in order to evaluate sample RNA quality. The developed assay is based on liquid bead array technology (Luminex® xMAP® and xTAG®). The assay is based on the following procedure: total RNA isolation from samples, cDNA synthesis, multiplex PCR, treatment with ExoSAP-IT® reagent, target specific primer extension (TSPE), biotinylation of PCR products, hybridization of the biotinylated products to xMAP microspheres, addition of detection reagent (Streptavidin- phycoerythrin) and measurement in the Luminex analyzer. Before processing to patients samples an extensive optimization of every step of the method was performed in terms of analytical sensitivity, specificity, repeatability and reproducibility. The clinical performance of the assay was evaluated in 20 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissue samples originating from patients with NSCLC. The results were compared with RT-qPCR using our previously developed methodology for VEGF splice variants, (Zygalaki et al, Clin Chem, 2007).

Results. When using the tumor cell line MCF-7 the developed method can detect down to 10 cells, is highly specific and is characterized by satisfactory repeatability and reproducibility for each VEGF splice variant. Comparison between the developed liquid bead array for VEGF splice variants and RT-qPCR has shown an accordance of 16/20 pairs (80%) for PBGD, 16/20 for VEGF121 (80%), 18/20 for VEGF165 (90%) and 17/20 for VEGF189 (85%).

Conclusions. The developed liquid bead array can be used to detect VEGF splice variants simultaneously in clinical samples with high specificity and sensitivity. The method can be extended to analyze additional gene targets, while the use of an internal control could further enable quantification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5140. doi:10.1158/1538-7445.AM2011-5140
Abstract 5144: Oligonucleotide-based inhibition of the pro-angiogenic activity of Hepatoma-derived growth factor related protein 3 in neuroblastoma
Authors: Pastorino Fabio , Dewilde Sarah , Loi Monica , Tosti Sara , Ricci Laura , Cilli Michele , Ribatti Domenico , Sapio Roberto , Bucci Enrico , Barone Domenico , Ponzoni Mirco
Published: June 2012

Hepatoma-derived growth factor related protein 3 (HDGFRP3) belongs to the HDGF polypeptide family. Expression of HDGF is a prognostic factor for NSCLC, pancreatic and gastrointestinal tumors, and silencing of HDGF suppressed cell proliferation, migration and invasion of squamous lung cancer cells. Here we investigated the expression of HDGFRP3 in various tumor cell lines by quantitative PCR and western blot analysis. HDGFRP3 is highly expressed and secreted from neuroblastoma (NB) cell lines. The presence of high levels of HDGFRP3 was confirmed in two animal models of human NB. Since HDGFRP3 shares a conserved Pro-Trp-Trp-Pro motif (PWWP) domain with its family members, we assessed whether it could promote proliferation, migration and angiogenesis in analogy to HDGF. Recombinant HDGFRP3 inhibited migration of endothelial cells in a modified Boyden chamber experiment, while cell survival was not affected. Nevertheless, the protein promoted angiogenesis in vitro, as assessed through vascular sprouting and tubulogenesis assays. Starting from the structural insight of the PWWP domain, an oligonucleotide-based inhibitor was designed and tested to block the pro-angiogenic effect of HDGFRP3. A single stranded DNA inhibited the pro-angiogenic properties of HDGFRP3 in vitro. After modifications to improve its serum stability, the cytotoxicity of the designed oligonucleotide-based molecule (BN210) was tested by an MTT cell viability assay on various NB cell lines. BN210 slightly reduced NB cells proliferation. To determine if BN210 could be used both as single anti-angiogenic agent and in combination with chemotherapy in vivo, orthotopic NB tumor-bearing mice were randomly divided in different groups of treatment: 1 mg/kg of vincristine (VCR), once a week for 4 weeks; 7-21 mg/kg of BN210, either i.p. (5 days/week) or i.v (every 2 days). Other mice were treated with a combination of VCR and BN210. NB-bearing mice treated with BN210 alone showed a partial increase in life span, compared to control mice. However, mice treated with the combination of VCR plus BN210 had statistically significant increased life span, compared to both the control and the single treatment groups, with long term survivors obtained with the combined schedule when BN210 was injected i.v. Thus, the oligonucleotide-based inhibition of the pro-angiogenic activity of HDGFRP3 can be considered as a novel strategy for combined anti-tumor therapy in NB.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5144. doi:10.1158/1538-7445.AM2011-5144
Abstract 5149: Intermedin stimulates cell growth, migration, and angiogenesis in human colorectal adenocarcinoma and hepatocellular carcinoma
Authors: Guo Xiaojia , Schmitz John , Uchio Edward , Kenney Barton , Kulkarni Sanjay , Cha Charles
Published: June 2012

Intermedin (IMD) functions as an angiogenic factor in a rat ischemic model and human endothelial cells. In our search for anti-angiogenic therapy for solid tumors, we analyzed the expression and possible role of IMD in tumor angiogenesis. IMD was expressed in human colorectal adenocarcinoma (CRC) and hepatocellular carcinoma (HCC). The IMD expression was significantly higher in stage I than stage 0 of both colon and liver tumors (stage I/0 ratio of mRNA level is 7.7 ± 0.4 and 3.2 ± 0.3, respectively). IMD was widely expressed in colon and liver tumor lines. Immunohistochemistry showed that the tumor regions were significantly more immunoreactive for IMD than adjacent benign regions of CRC and HCC specimens. Inhibition of IMD expression by RNA interference reduced cell proliferation by 29.7 ± 6.7% (n = 3, p = 0.047) and impaired cell invasion by 27.8 ± 3.6% (n = 3, p = 0.016) in a HCC cell line SK-Hep-1. Addition of IMD peptide for 5 minutes to serum starved SK-Hep-1 cells increased the phosphorylation level of p44 and p42 MAP Kinase (Erk1/2), suggesting that IMD regulates Sk-Hep-1 cell growth, at least partly, through activating Erk1/2 signaling pathway. Similarly, IMD knockdown in a CRC cell line HCT116 reduced cell growth by 35.0 ± 1.4 % (n = 3, p = 0.018). Conditioned medium from IMD siRNA-transfected SK-Hep-1 cells reduced endothelial cell tube formation by 31.6 ± 6.8%. Conversely, over-expression of IMD in CRC RKO cells increased cell growth and induced cell migration significantly. Furthermore, treatment with RKO/IMD conditioned medium increased endothelial cell tube formation by 14. 7 ± 0.8% (n=4, p = 0.049). Thus, IMD stimulated HCC and CRC cell growth, migration, and invasion, indicating a role for IMD in tumor angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5149. doi:10.1158/1538-7445.AM2011-5149
Abstract 515: Membrane type 1-matrix metalloproteinase cooperates with KrasG12D to promote pancreatic fibrosis through transforming growth factor-β activation of pancreatic stellate cells
Authors: Krantz Seth B. , Shields Mario S. , Dangi-Garimella Surabhi , Cheon Eric C. , Barron Morgan R. , Hwang Rosa F. , Rao M Sambasiva , Grippo Paul J. , Bentrem David J. , Munshi Hidayatullah G.
Published: June 2012

Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) that is required for growth and invasion in the collagen-rich microenvironment. In this report we show that compared to control mice (Kras+/MT1-MMP-) that express an activating mutation necessary for pancreatic cancer development, KrasG12D, littermate mice that express both MT1-MMP and KrasG12D (Kras+/MT1-MMP+) developed a greater number of large mucin-containing papillary lesions with a high degree of cellular atypia. These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin positive cells in the stroma, indicative of activated myofibroblasts, and a greater extent of Smad2 phosphorylation. To further understand how MT1-MMP promotes fibrosis, we established an in vitro co-culture model to examine the effect of expressing MT1-MMP in PDAC cells on stellate cell collagen deposition. Conditioned media from MT1-MMP-expressing PDAC cells grown in 3D collagen enhance Smad2 nuclear translocation, promote Smad2 phosphorylation and increase collagen production by stellate cells. Inhibiting the activity or expression of the TGF-β type I receptor in stellate cells attenuated MT1-MMP-conditioned media induced collagen expression by stellate cells. Additionally, a function-blocking anti-TGF-β antibody also inhibited MT1-MMP-induced collagen mRNA expression in stellate cells. Overall, we demonstrate that the bona fide collagenase MT1-MMP paradoxically contributes to fibrosis in vivo by increasing TGF-β signaling and that targeting MT1-MMP may help to mitigate fibrosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2011-515
Abstract 5156: Biochemical characterization of recombinant woodchuck vascular endothelium growth factor (wVEGF)
Authors: Huang Huayi , Salavaggione Oreste E. , Tennant Bud , Iyer Renuka V. , Adjei Araba A.
Published: June 2012

Vascular endothelial growth factor (VEGF) is one of the most potent and predominant angiogeneic signals. It binds receptors on blood vessels and stimulates proliferation, migration, survival and permeability of endothelial cells that form new vasculature for malignant tumors. Human hepatocellular carcinoma (HCC) is a highly VEGF-driven tumor and frequently occurs in the context of chronic hepatitis B or C virus infection. The woodchuck is a well characterized model of hepatitis B virus related HCC as it develops acute and chronic infection after neonatal infection with sera containing woodchuck hepatitis virus. This is a valuable model for translational studies of novel VEGF targeted agents as results may have great clinical relevance. We cloned a VEGF cDNA (wVEGF) from woodchuck liver tissue and transiently expressed it in COS-1 cells. A variety of parameters were measured to functionally characterize the wVEGF. Woodchuck cell lines, namely WCH17, WHK44 (woodchuck HCC), WC3 (woodchuck hepatocyte) and HepG2 cells were also characterized. The wVEGF peptide sequence was 94% identical to the human VEGFA. Two protein bands were detected in the supernatant (secreted) with molecular weights of approximately 25 and 17kDa, and a single band of 25kDa in the cell lysates. The major band observed in the cell lines was 25kD. The amount of wVEGF protein in the supernatant was 14-fold higher than in the lysate. The mRNA level in the cell lysate was significantly higher than was present in either the COS cells transfected with empty vector and also in the woodchuck and HepG2 cell lines. After 72 hours of stimulation with supernatant wVEGF, COS cells exhibited approximately 34% higher growth compared to cells in absence of wVEGF. The antiproliferative property of sunitinib, (a VEGF receptor tyrosine kinase inhibitor) on COS cells overexpressing wVEGF showed similar IC50 values for MTS assay 7.6±1.4 µM and for cell death (cell counts), 6.4±1.5 µM. In the cell lines, sensitivity to sunitinib ranked from WHK44 (4.5±1.2 µM) > WC3 (4.8±1.3 µM) >HepG2 (9.5±1.4 µM) >WCH17 (17.9±1.4 µM). In summary, the wVEGF was 94% identical in amino acid sequence to the human VEGF 189. It produced two protein bands that were associated with the supernatant after overexpressing the wVEGF in COS cells, one protein band was associated with the cell lysate. The wVEGF protein stimulated cell growth and exhibited sensitivity to the sunitinib with reduction in cell proliferation.

(Support:American Cancer Society MRSG-08-096-01-CCE, Roswell Park Alliance Grant, PI Iyer)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5156. doi:10.1158/1538-7445.AM2011-5156
Abstract 5157: A role of the FGF-pathway in the VEGF/VEGFR targeting
Authors: Tsimafeyeu Ilya , Demidov Lev , Wynn Nygel
Published: June 2012

Background: The growth of new blood vessels is regulated at multiple steps by interactions between several pro- and antiangiogenic factors. We believe that the angiogenesis induced by basic fibroblast growth factor (bFGF) is resistant to anti-VEGF/VEGFR (vascular endothelial growth factor/receptor) therapy.

Methods: The Corneal Micro Pocket Assay was performed. 70 Charles River female C57BL/6 mice (age at start day, 6 weeks) were randomized to 7 arms (10 mice in each group): 1) bFGF; 2) VEGF-A; 3) bFGF negative, VEGF-A negative; 4) bFGF and sunitinib; 5) VEGF-A and sunitinib; 6) bFGF and bevacizumab; 7) VEGF-A and bevacizumab. Doses of bFGF, VEGF-A (R&D Systems), sunitinib (Pfizer), and bevacizumab (Roche) were 200 ng, 400 ng, 10 mg/kg, 50 mg/kg per animal, respectively. Hydron pellets preparation, surgical procedure, and quantification of angiogenesis (angiogenic score) were performed as previously reported (Kenyon BM et al.). Statistical significance was determined by the Student's t test.

Results: There was no neovascularization in bFGF negative, VEGF-A negative group (mean, 0). The effect of 200 ng/pellet of bFGF (mean, 4.2; SEM, 0.05) was compared with that of 400 ng/pellet VEGF-A (mean, 4.08; SEM, 0.09), P=0.7. In bFGF-induced angiogenesis, sunitinib (mean, 3.9; SEM, 0.1; P=0.2) and bevacizumab (mean, 4.71; SEM, 0.33; P=0.85) did not impact on neovascularization in comparison with bFGF positive control. The angiogenic effect of VEGF-A was significantly inhibited by both sunitinib (mean, 0.38; SEM, 0.06; P=0.001) and bevacizumab (mean, 0.75; SEM, 0.05; P=0.001) in comparison with VEGF-A positive control. No significant differences between 2 targeted agents in bFGF and VEGF-A models were obtained.

Conclusion: Our recent findings demonstrate that anti-VEGF(R) therapy significantly impacts on VEGF-A-induced angiogenesis and not on bFGF-induced neovascularization. Further studies are needed to assess the role of FGF-pathway in resistance to VEGF(R) therapy. The study was supported by Terry Fox Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5157. doi:10.1158/1538-7445.AM2011-5157
Abstract 5159: Wnt signaling protein (Wisp2/CCN5) stimulates angiogenesis and invasion in prostate cancer
Authors: Hashimoto Yasunobu , Singh Rajendra K. , Muñoz Daniel , Lokeshwar Bal L.
Published: June 2012

Introduction: Angiogenesis and invasion are essential attributes of metastatic cancer cells. The Wnt-Induced Signaling Protein-2 (Wisp-2 /CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since chronic inflammation is thought to be significant in CaP metastasis and IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells.

Hypothesis; Wisp-2 is a down-stream effector of IL-8 induced angiogenesis and invasion, and is essential for metastatic progression of prostate cancer.

Methods: Quantitative PCR (qPCR) was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells (e.g. RWPE-1, LNCaP, LAPC-4, DU145, and PC3). Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells (HUVEC cells). Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), Matrigel invasion assay and chemotaxis assay.

Results: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ≥15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemo-invasion activity (28%) and secretion of MMP-9 (25%).

Conclusions: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential.

(Grant Support; VA MERIT Review, VA5312.01 (BLL))

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2011-5159
Abstract 5161: Cell mechanics-cytoskeleton-membrane protein transduction loop mediates brain metastasis of breast cancer cells
Authors: Zhao Hong , Nie Fang , Cui Kemi , Gong Zhongcheng , Penmnesta Siva , Mao Yong , Wang Zhiyong , Xue Zhong , Que Long , Wong Stephen
Published: June 2012

Brain metastasis is the worst stage of breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cells get through blood-brain barrier and grow in brain tissue. To provide information in this direction, we developed a microfluidic single-cell patch clamp microdevice to study the electrophysiological and biomechanical properties of human breast cancer brain metastatic cells for the first time. The results show that the brain metastatic cells possess higher membrane resistance and greater membrane stiffness compared to non-brain metastatic cells. The over-expression of HER2 and EGFR decrease the cell membrane resistance, while increase the cell stiffness, which correlates with the enhanced cell invasion through the in vitro blood-brain barrier and increased formation of brain metastatic loci in the xenograft mice. Gene expression analysis of the brain metastatic cells identifies cell membrane protein PCDH7 mediates the increased cell mechanics property functionally, and PCDH7 is also an enhancer of blood-brain barrier crossing and brain colonization for the brain metastatic cells, through the cytoskeleton rearrangement mechanism. This cell mechanics-cytoskeleton-PCDH7 protein loop mechanism may prove to be an attraction therapeutic target to block the brain metastasis of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2011-5161
Abstract 5174: HYAL4: a new player in bladder cancer progression and diagnosis
Authors: Escudero Diogo O. , Knapp Judith , Lokeshwar Soum , Manoharan Murugesan , Soloway Mark , Lokeshwar Vinata B.
Published: June 2012

BACKGROUND: Hyaluronidase (HAase) family of enzymes degrades hyaluronic acid (HA). In the human genome there are 6 HAase genes clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). While PH20 and HYAL-2 are testicular and lysosomal HAases, HYAL1 is a tumor-derived HAase. HYAL1 promotes tumor growth, invasion, and angiogenesis and is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal cells or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine.

METHODS: Using time RT-PCR, we examined the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). The levels were normalized to β-actin. HYAL1 and HYAL4 were localized in bladder tissues by immunohistochemistry using anti-HYAL1 and anti-HYAL4 antibodies, respectively. The staining was graded for intensity (0 – 3) and area of staining. HYAL4 function was analyzed by siRNA transfection of HT1376 and HT5637 cell lines.

RESULTS: HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (1.2 ± 0.6; 2.2 ± 2.4; 4.2 ± 6.6; P 0.05). cDNA cloning revealed that HYALP1 is a pseudo gene that encodes a non-functional truncated protein. HYAL1 and HYAL4 staining was significantly elevated in TBL tissues (188 ± 57; 232 ± 91) when compared to NBL tissues (68.5 ± 21.3; 93 ± 46; P < 0.001). The staining for both HAases was exclusively localized in tumor cells. When compared to HYAL3, PH20, and HYAL2 mRNA levels, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly elevated in BCa patients’ urine (P < 001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than HYAL1 mRNA levels (72.3%; 76.2%) to detect BCa. Both HAases detected high-grade BCa with 83% – 86% sensitivity. Down regulation of HYAL4 expression by siRNA decreased proliferation of HT1376 and HT5637 cells (2.3-fold) and induced apoptosis (2.6-fold).

Conclusion: This is the first study showing that, like HYAL1, HYAL4 is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression.

Support: Grant NCI/NIH R01 CA-72821-09; Bankhead Coley Cancer Research Program Sylvester Comprehensive Cancer Center and CURED

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2011-5174
Abstract 5175: Predictors of metastatic spread and progression free survival in uterine papillary serous carcinoma: A pilot study
Authors: Schuler Kevin M. , Banet Natalie , Bae-Jump Victoria , Lininger Ruth , Gehrig Paola A.
Published: June 2012

Background: Uterine Papillary Serous Carcinoma (UPSC) comprises 5-10% of all endometrial cancers but is responsible for nearly 40% of all endometrial cancer-related deaths. Progression-free survival (PFS) controlled for stage is also markedly shorter in UPSC compared to Type 1 endometrial cancers. Unlike endometrioid carcinoma, in which depth of myometrial invasion, grade, and lymphovascular space invasion (LVSI) are risk factors for extrauterine spread, UPSC often metastasizes in those with no myometrial invasion.

Objective: The objective of this study is to identify markers that may predict metastatic disease in women with non-invasive UPSC or portend a shorter progression free survival.

Materials: A retrospective chart review was performed on women diagnosed with Stage IA or Stage III/IV UPSC without myometrial invasion from 1995-2008. Examined factors include age, parity, race, weight, LVSI, CA125 levels, and tumor location within the endometrium. Tissue microarrays (TMAs) were constructed using paraffin blocked specimens and stained for ER, PR, p53, VEGF, Claudin 3, Claudin 4, Her-2/neu, Her-3, Synuclein gamma, and Maspin. Chi-square analysis, Student's t-test, and Fisher's Exact analysis were used where appropriate to detect statistically significant differences in these 2 groups with Kaplan-Meyer analysis used to determine PFS. Significance was represented at p<0.05.

Results: Thirty-two patients were identified to have UPSC without myometrial invasion, 24 (75%) comprising Stage IA and 8 (25%) having Stage III/IV disease. Pathology blocks were available for 21 patients (14 Stage I and 7 Stage III/IV). CA125 was the only factor to achieve significance (p=0.04) in predicting metastatic spread; biomarkers, either alone or in combination, were not predictive of metastatic disease. In Stage I disease, positive ER status was associated with a significantly longer PFS (p<0.001) while in Stage III/IV disease, positive Her-3 status was associated with a shorter PFS (p=0.014).

Conclusions: In addition to traditional risk factors not being predictive of metastatic disease, molecular markers that have been associated with metastatic disease and a worsening prognosis do not appear to be predictive in UPSC. However, the contribution of ER status and Her-3 needs to be further evaluated as this may offer new insight into therapeutic options for treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5175. doi:10.1158/1538-7445.AM2011-5175
Abstract 5177: Primary tumor induces lymphangiogenesis and immune suppression in sentinel lymph nodes
Authors: Chikamatsu Kazuaki , Ishii Hiroki , Sakakura Koichi , Miyata Masanori , Masuyama Keisuke
Published: June 2012

The main factor that affects the prognosis of patients with squamous cell carcinoma of the head and neck (SCCHN) is regional lymph node metastases, which usually spreads first to the sentinel lymph nodes (SLNs). Recent studies using animal models have demonstrated that tumor cells can induce lymphangiogenesis in SLNs before metastasizing. In this study, we investigated the association of SLN lymphangiogenesis with the status of primary tumors. Moreover, the immunological status of SLNs was also examined. The expression of lymphatic-specific markers, including VEGFR-3, Prox-1, and LYVE-1 in 23 metastasis-negative SLNs obtained from 10 patients with SCCHN was investigated using quantitative real-time RT-PCR. Moreover, primary tumors obtained from patients were evaluated for the expression of VEGF-A, -C, and -D by immunohistochemistry. The level of LYVE-1 expression in SLNs was significantly higher than in control lymph nodes from patients with non-cancerous diseases. Moreover, SLNs from patients with VEGF-C-positive tumor showed a significantly higher expression of VEGFR-3 than those from patients with VEGF-C-negative tumor. VEGFR-3 is expressed mainly in lymphatic endothelium but also in a subset of antigen-presenting cells including immature dendritic cells (DCs), therefore; we furthermore examined the infiltration of DCs into SLN by immunohistochemistry. Interestingly, S-100+ and CD1a+ immature DCs infiltrated significantly into SLNs compared to non-SLNs in patients with SCCHN, suggesting that predominant migration of immature DCs, which suppress T-cell activation and support regulatory T cell development, would contribute to inhibit rather than develop anti-tumor immunity. Our findings suggest that primary tumor actively induces lymphangiogenesis in SLNs prior to the onset of metastases, and where tumor-derived VEGF-C plays an important role. Moreover, active lymphangiogenesis may concomitantly lead to down-regulation of SLN immunity. Thus, both lymphangiogenesis and immune suppression in SLNs are likely the basis of its susceptibility to tumor metastases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2011-5177
Abstract 5178: Expression of CD44/CD24 and aldehyde dehydrogenase 1 in resected breast cancer: CD24-positivity predicts early relapse
Authors: Moon Yongwha , Koo Ja Seung , Han Hyunju , Park Byeong-Woo , Kim Seung Il , Park Seho , Kim Joo-Hang , Sohn Joo Hyuk
Published: June 2012

Background: CD44+/CD24- or aldehyde dehydrogenase 1 (ALDH1) has been suggested as potential markers for breast cancer stem cells, although little is known about its clinical implications. We evaluated the expression of CD44/CD24 and ALDH1 in breast cancer patients in both ends of the prognostic spectrum.

Methods: Of 819 resected breast cancer cohort with positive estrogen receptor, ‘early relapse group’ within 5 years postsurgery and ‘no relapse group’ longer than 10 years postsurgery were defined. Paraffin-embedded tumor tissues were available in 31 early relapsed patients and 68 patients from no relapse group. CD44/CD24 and ALDH1 expression were evaluated with immunohistochemical staining. Positive criteria were designated as more than 15% tumor cell membranous staining for CD44/CD24 and more than 5% tumor cell cytosolic staining for ALDH1.

Results: Early relapse group contained higher N stage and higher histologic grade than no relapse group but there were no differences in age, menopauseal status, progesterone receptor status, HER2 status, and T stage between both groups. Phenotypes of CD44/CD24 were CD44+/CD24- in one patient (1%), CD44+/CD24+ in one (1%), CD44-/CD24+ in 12 (12%), and CD44-/CD24- in 67 (68%). Four patients (4%) showed ALDH1-positivity. Due to rarity of CD44-positivity or ALDH1-positivity, staining results were simply dichotomized into CD24-positive (13%, 13/99 patients) and CD24-negative status (87%, 86/99 patients) and only the status of CD24 was further analyzed. CD24-positivity was higher in early relapse group (32%) than in no relapse group (4%). CD24-positivity was associated with negative progesterone receptor (P=0.026), higher N stage (P=0.029), and higher histologic grade (P=0.034). However, in the multivariate logistic regression adjusted by known prognostic factors, CD24-positivity was a still significant predictive factor for early relapse (hazard ratio=8.51; P=0.006).

Conclusions: The incidence of CD44+/CD24- and ALDH1-positivity which are potential breast cancer stem cell markers, were low. However, in resected breast cancer, expression of CD24 was a significant predictive factor for early relapse. CD24 is worth further investigation as a novel biomarker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2011-5178
Abstract 5192: The mobilization and recruitment of bone marrow-derived stem cells contributes to the acceleration of tumor growth after surgery
Authors: Takemoto Yoshihiro , Li Tao-Sheng , Kubo Masayuki , Ohshima Mako , Ueda Kazuhiro , Harada Eijirou , Uesugi Naomasa , Enoki Tadahiko , Hamano Kimikazu
Published: June 2012

Background. Although surgical resection of the primary tumor lesion is still the most promising strategy for cancer therapy, surgical injury may accelerate tumor growth and metastasis, negatively affecting patients’ prognosis. Previous studies have demonstrated that many factors, including the robust release of cytokines and temporal changes of immunological and inflammatory mediators may contribute to the acceleration of tumor growth and metastasis after surgery. However, the precise mechanisms are not yet fully understood. We tested the hypothesis that surgical injury induces mobilization and recruitment of bone marrow-derived stem cells, thereby enhancing angiogenesis and accelerating tumor growth.

Methods. Mice were subjected to partial gastrectomy, and non-gastrectomized mice were used as controls. Mobilization of bone marrow stem cells was monitored after surgery. Using an established tumor model in GFP+ bone marrow-transplanted chimera mice, we further investigated whether the mobilized stem cells affected tumor growth. To evaluate the influence on tumor growth of surgical injury and the inhibition of SDF-1/CXCR4 signals, we used AMD3100, a CXCR4 antagonist.

Results. Compared to control, gastrectomy significantly increased the populations of CD34+ cells (6.9±4.5% vs 3.3±0.4%, p<0.05) and CD34+/Flk-1+ cells (0.08±0.02% vs 0.05±0.01%, p<0.05) in peripheral blood 12 hours after surgery. Twelve days after surgery, the tumor volume was almost double in gastrectomized mice compared with control (580±106 mm3 vs 299±162 mm3, p<0.05). Histological analysis of tumor tissue revealed that the microvessel density and number of proliferating cells were significantly higher, but those of apoptotic cells were significantly lower, in gastrectomized mice as compared with control. Furthermore, the number of GFP+ cells found in tumor tissue was significantly larger in mice that underwent gastrectomy than in control. Some of the stained GFP+ cells were positive for CD34 and had been incorporated into microvessels. However, administration of AMD3100 significantly inhibited the recruitment of GFP+ cells and completely negated the acceleration in tumor growth after surgery (345±172 mm3, p<0.05).

Conclusion. Surgical injury induces mobilization and recruitment of bone marrow stem cells, thereby enhancing angiogenesis and accelerating tumor growth. Temporary inhibition of the SDF-1/CXCR4 signals may represent new therapeutic strategies for preventing acceleration of tumor growth after surgery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2011-5192