bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5138: Expression and circulating levels of VEGFC/VEGFD and their receptor VEGFR2, R3 in renal cell cancer: Relationship with clinicopathological parameters
Authors: de La Motte Rouge Thibault , Mouawad Roger , Comperat Eva , Thery Jean-Christophe , Vignot Stephane , Roupret Morgan , Spano Jean-Philippe , Khayat David
Published: June 2012

Background: Vascular endothelial growth factors C, VEGF-D and VEGFR-2, R-3, are overexpressed in different malignancies and associated with lymph node metastasis and poor prognosis. In renal cell cancer (RCC) lymphatic tumor spread exists but data focusing on lymphangiogenesis are rare. Since the VEGF-C/VEGF-D/VEGFR-2, R-3 axis appears to be the signaling pathway for tumor-induced lymphangiogenesis and an attractive target for therapeutic intervention; we analyzed the expression and the presence of the soluble forms in 30 non treated metastatic RCC patients and results were correlated with clinicopathological parameters. Methods: Tumor and sera from 30 metastatic renal cell cancer patients (20 clear cell (ccRCC)&10 papillary (pRCC) were included in this study. The expressions of VEGFR-2,R-3 &VEGF-C,-D expressions on tumor were evaluated by immunohistochemestry. Using ELISA assays, soluble vegfr-2–3 &sVEGF-C,-D were measured in sera of RCC patients in comparison to 20 healthy controls. Results: In overall patients, a high expression of VEGF-C,-D and their receptors were observed in more than 55% of the patients as compared to the negative control. Regarding circulating VEGF-C,-D, R2, R-3 they were variable in all samples from either patients or healthy donors. Only median sVEGF- R3 level was significantly higher (p=0.005) in RCC patients as compared to healthy donors. VEGF-C and VEGF-D expression was significantly correlated (r’=0.43, p=0.02) with each other but not with the expression of its receptors. An inverse correlation between Flt-4 expression & its soluble form was noted (r=−0.33 p=0.040). The expression of VEGF-C, VECF-D and sVEGFR-3 were significantly higher in pRCC than ccRCC (p=0.02, 0.01 and 0.035 respectively). The expression of VEGFR-2, VEGFR-3 were not different between the subgroups (p=0.11). Furthermore, no correlation with clinicopathological parameters was shown in either overall patients or in the two subgroups. Conclusions: we showed that in RCC the lymphangiogenic factors are expressed or present as soluble form, a different expression pattern in ccRCC and pRCC existe. Therefore, further studies are necessary to determine if lymphangiogenesis can play a role as a prognostic tool or a target for therapeutic intervention in renal cell carcinoma.

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 5138. doi:10.1158/1538-7445.AM2011-5138
Abstract 5139: Thymidine phosphorylase expression correlate with lymphangiogenesis and angiogenesis in metastatic gastric cancer
Authors: Shin You Keun , Jiang Xianglan , Lee Hye Ryung , Rha Sun Young , Rho Jae Kyung , Noh Sung Hoon , Chung Hyun Cheol , Jeung Hei-Cheul
Published: June 2012

Thymidine Phosphorylase (TP) (platelet-derived endothelial cell growth factor, PD-ECGF) is known as a proangiogenic factor. High TP expression in the primary tumor has been thought as a risk factor for hepatic and lymph node metastasis in patients with gastric adenocarcinoma. However, the molecular basis for induction of invasion and lymph node metastasis by TP and its therapeutic implication is largely unknown.

First, as a pilot study, the (lymph)angiogenic activity (D2-40, LYVE-1, VEGFC, VEGFR3) and TP expressions were evaluated in resected tumor samples. Sixty patients of gastric cancer with metastatic lymph nodes and in 46 patients without lymph node metastasis were included. With immunohistochemical study, we demonstrated that tumoral and stromal TP expressions were moderately correlated (Pearson correlation = 0.455). TP expression was also correlated with depth of invasion and nodal stage. TP-positive tumors had also increased lymphovascular invasion compared with TP-negative tumors (P = 0.044), and node positive tumor had higher density of lymphatic vessels, and it is correlated with TP expression (P = 0.023).

Then, we have shown that nude mice injected intraperitoneally with TP-overexpressing MKN-45 cells (MKN-45/TP) had decreased survival times compared with those injected with control cells (MKN-45/CV). MKN-45/TP tumors were more angiogenic (positive for CD31) and lymphangiogenic (positive for D2-40 and LYVE-1), and they were more invasive than MKN-45/CV tumors. A specific TP inhibitor, TPI, partially extended the survival period of TP tumor bearing mice, and its angiogenic activity was significantly reduced. All these findings imply that TP expression may be related to lymphangiogenesis in gastric cancer, and it could be further investigated as therapeutic targets for anti-metastatic therapy.

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 5139. doi:10.1158/1538-7445.AM2011-5139
Abstract 5142: VEGFA gene amplification and protein expression in breast cancer
Authors: Andreozzi Mariacarla , Schneider Sandra , Zlobec Inti , Tapia Coya , Tornillo Luigi , Eppengerger Serenella , Terracciano Luigi , Ruiz Christian
Published: June 2012

Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression.

Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers.

Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p<0.0001).

Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies.

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 5142. doi:10.1158/1538-7445.AM2011-5142
Abstract 5147: Tumor angiogenesis-promoting activity of a hypoxia-inducible cytokine, growth differentiation factor-15 (GDF-15)
Authors: Lee Jaeseob , Lee Hansoo , Choi Jeongsuk , Park Iha , Lee Jeong-Hyung , Kim Young-Myeong
Published: June 2012

Angiogenesis, growth of new blood vessels from pre-existing capillary blood vessels, is a marker of cancer and various ischemic and inflammatory diseases. Tumor growth depends on new blood vessel formation because tumor cells require oxygen and nutrients for their survival and proliferation. GDF-15, also known as MIC-1, NAG-1 and PTGF-beta, is a divergent member of the TGF-beta super family. GDF-15, like other TGF-beta family members, has shown janus-faced function in tumor development: pro-apoptotic and anti-tumorigenic vs. tumorigenic activity. Previously, we and others found that hypoxia-condition induced GDF-15 expression in various cancer cell types, leading to increased secretion from the cancer cells. Here, we present the tumor-promoting activity of GDF-15, along with pro-angiogenic activity. In in vivo mouse xenograft model using human melanoma cells, we found that tumors from GDF-15-expressing melanoma cells show higher degree of vessel formation with larger tumor volume than control tumors. Blood vessel inducing activity of GDF-15 was also observed when using human umbilical vein endothelial cells (HUVECs). GDF-15 treatment induced tube formation of HUVECs. Moreover, treatment of GDF-15 enhanced the growth and migration of HUVECs. Blood vessel inducing activity of GDF-15 was further confirmed by CAM and aortic ring sprouting assays, intravital microscopic analysis and matrigel plug assay. Furthermore, GDF-15 transgenic mice showed increased blood vessel development than wild type mice while lymphatic vessel development was no different. Interestingly, although angiogenic activity of GDF-15 was comparable to that of VEGF, its inflammatory activity was less significant than VEGF. Taken together, we show that GDF-15 functions as a potential angiogenic factor for tumor development with seemingly little inflammatory activity.

Key words: Cytokine, GDF-15, Tumor, Angiogenesis, Inflammation

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 5147. doi:10.1158/1538-7445.AM2011-5147
Abstract 5158: Plastic tumor cells engaged in vasculogenic mimicry produce PDGF-B and attract structural support
Authors: Paulis Yvette WJ , van den Oord Joost J. , Tjan-Heijnen Vivianne CG , Griffioen Arjan W.
Published: June 2012

In developmental angiogenesis, pericytes are recruited by PDGF-B-expressing endothelial cells to remodel, stabilize and support the immature blood vessel. In a process called vasculogenic mimicry (VM) highly aggressive tumor cells gain the unique ability to dedifferentiate into multiple cellular phenotypes, and obtain endothelial-like characteristics. This process can lead to the formation of vasculogenic-like matrix-embedded networks, i.e. vascular-like structures, contributing to circulation. The presence of VM is associated with increased tumor malignancy. Interestingly, also the expression of PDGF in tumor cells has been demonstrated to be associated with an increased grade of malignancy. As plastic, highly aggressive tumor cells form vasculogenic-like structures resembling immature vessels, we hypothesize that plastic tumor cells engaged in VM also functionally resemble endothelial cells by seeking structural support.

We demonstrate that plastic, aggressive melanoma cells transcribe and secrete PDGF-B, while in poorly aggressive melanoma cells, PDGF-B is absent. Myofibroblasts, acquired from the human vena saphena, cultured in the presence of plastic tumor cell conditioned medium display an activated phenotype. Additionally, in vitro analysis of 3D-culture on Matrigel demonstrated the functional contribution of myofibroblasts to plastic tumor cells as they structurally support the VM-characteristic networks formed. Immunohistochemical analysis of a series of cutaneous melanoma tissues showed a gradient PDGF-B staining towards the outer surface of the nest structures, i.e. the tumor cells aligning the VM-characteristic matrix networks. Consistent with the above described data, we observed staining for alpha-smooth muscle actin (α-SMA), a pericyte-specific marker, within the VM-characteristic matrix networks, thereby revealing the presence of pericytes inside these networks.

Together our results demonstrate that highly aggressive, VM-positive tumor cells do not solely resemble endothelial cells phenotypically, but also actively behave like endothelial cells by producing PDGF-B and attracting structural support to their immature structures. The current findings suggest that targeting of pericytes may have an unexpected anti-tumor activity through affecting vasculogenic mimicry.

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 5158. doi:10.1158/1538-7445.AM2011-5158
Abstract 5185: Evaluation of biomarker candidate genes for prostate cancer metastasis
Authors: Reyes Niradiz , Bettin Alfonso , Rebollo Juan , Tiwari Raj , Geliebter Jan
Published: June 2012

Prostate cancer is the most frequently diagnosed malignancy in men and although effective treatment is possible for early stage disease, advanced prostate cancer has a very poor prognosis. Thus, identification of biomarkers that can accurately and reliably diagnose prostate cancer in its early stages is urgently needed. MatLyLu and Dunning G are rat prostate cancer cell lines which have different metastatic capabilities. Using microarray analysis we previously identified a set of metastasis-associated genes that are over-expressed in MatLyLu. Genes found to be differentially expressed include several previously associated with the metastatic process, but not previously reported in prostate cancer. We also have found differential expression of genes that code for several proteoglycans involved in epithelial cell-extracellular matrix interaction, cell adhesion molecules, genes related to motility, growth factor receptors, and tumor suppressor genes. We are currently validating the expression of these candidate genes in human prostate cancer cell lines with different metastatic abilities (LNCaP, PC3, PWR-1E) and in clinical samples of prostate cancer at different disease stages using quantitative real time PCR (qRT-PCR) and immunohistochemistry. A better understanding of the molecular events leading to the metastatic prostate cancer phenotype will help to further identify genes or gene-products involved in this process, which may prove useful as biomarkers for diagnosis and/or prognosis of the disease.

Project code: 110745921483. Supported by Colciencias Grant # RC No.462-2008

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 5185. doi:10.1158/1538-7445.AM2011-5185
Abstract 5188: Lrig1, a cell surface negative regulator of ErbB1-4, marks both proliferative and quiescent intestinal stem cells and acts as a tumor suppressor
Authors: Wang Yang , Powell Anne E. , Li Yina , Davenport James R. , Franklin Jeffrey , Haigis Kevin M. , Coffey Robert J.
Published: June 2012

Whether proliferative and quiescent intestinal stem cells coexist in normal and cancerous tissue is controversial. Due to heightened transcript expression in the intestinal epithelial stem cell compartment, detection in quiescent hair follicle stem cells and frequent loss in breast cancer, we hypothesized that Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would mark a novel population of intestinal stem cells and may be important for intestinal homeostasis and in cancer. To study this, we generated Lrig1-CreERT2/+; R26R-LacZ mice to study the role of Lrig1. One day after Lrig1-CreERT2/+; R26R-LacZ mice received a single injection of tamoxifen, 1-2 cells were labeled in the progenitor zone. Three months after injection, 10% of colonic crypts were entirely labeled and 8% of crypts contained singly labeled, non-proliferative, Lgr5-negative cells at the crypt-base. In a regenerative response to gut injury, these singly labeled cells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the ability of these quiescent stem cells to become activated. Loss of Lrig1 resulted in heightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcinoma, supporting a functional role for Lrig1 in the maintenance of intestinal epithelial homeostasis and its ability to act as a tumor suppressor. In addition, driving stochastic loss of Apc in Lrig1-expressing cells results in multiple, large distal colonic tumors and less frequent, smaller intestinal tumors, which are common features of human familial polyposis. In summary, we show that Lrig1, a cell surface negative regulator of ErbB1-4, marks both proliferative and quiescent intestinal stem cells and acts as a tumor suppressor.

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 5188. doi:10.1158/1538-7445.AM2011-5188
Abstract 520: Local changes in tissue microenvironment regulate mammary tumor progression
Authors: Krause Silva , Tobin Heather , Brock Amy , Ingber Donald E.
Published: June 2012

Breast cancer is one of the most prominent cancers in women with approximately 40,000 women dying each year of the disease in the United States alone. Although cancer has a strong genetic basis, the tissue microenvironment also plays a significant role in tumor formation and progression. To explore this process in greater detail, we analyzed tissue structure, cellular biochemistry and gene expression change during tumor progression in a previously developed FVB C3(1)-SV40Tag transgenic mouse model in which the females spontaneously develop mammary tumors that undergo changes in cell tissue morphology that closely mimic those observed during human breast cancer progression. Histological characterization of breast tissues isolated from 8, 12, 16 and 20 week old female mice confirmed that this model is extremely robust with normal glandular morphology being observed at 8 weeks, ductal carcinoma in situ (DCIS) morphology appearing at 12 weeks and 16 weeks and large tumors with metastatic lesions by 20 weeks of age. More careful analysis revealed that tumor formation did not scale directly with expression of SV40 large T antigen that is the drive gene in this transgenic model, and instead appeared to be more closely related to variations in the tissue microenvironment. This was supported by the observation that the number of myoepithelial cells and expression levels of known breast cancer differentiation markers, including estrogen and progesterone receptors, varied among ducts within tissues at each time point even though the majority of epithelial cells in these ducts expressed T antigen. Gene microarray analysis of mammary tissues from these time points revealed consistent changes in genome-wide expression profiles, and several interesting candidates are currently being analyzed regarding their potential involvement in early microenvironmental changes that might drive the tumor progression. In summary, our preliminary results showed that despite expression of constitutively active large T antigen in the breast epithelial cells of these transgenic mice, we observe heterogeneous tumor formation within mammary tissues. These local variations in tumor progression may be related to regional changes in stromal and extracellular matrix components that are currently being analyzed by immunohistochemical analysis. Our long-term goal is to leverage the information we uncover through this effort to design differentiation therapies that suppress tumor cell growth, induce differentiation and hence, reverse breast cancer progression.

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 520. doi:10.1158/1538-7445.AM2011-520
Abstract 5200: Effect of cancer-associated fibroblast on the progression of gastric cancer stem cells
Authors: Hasegawa Tsuyoshi , Yashiro Masakazu , Matsuoka Junko , Kashiwagi Shinichiro , Fuyuhiro Yuhiko , Sakurai Katsunobu , Sakurai Katsunobu , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Yamada Nobuya , Yamada Nobuya , Maeda Kiyoshi , Ohira Masaichi , Ishikawa Tetsuro , Hirakawa Kosei
Published: June 2012

Introduction: Cancer stem cells (CSCs) are thought to possess the ability to initiate tumor growth and sustain self-renewal, which explains the tumorigenicity and the maintenance of bulk. There is an increasing evidence of the importance of the host microenvironment in conditioning the CSCs status, such as the stemness and the tumor initiation. Recently, cancer-associated fibroblasts in the cancer microenvironment have been suggested to be associated with the tumor progression. Then, the aim of this study is to clarify the relation between CSCs and cancer-associated fibroblasts.

Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblasts, CaF-37, were used. Side population (SP) cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechst 33342, and were named as OCUM-12/SP. OCUM-12/SP cells or OCUM-12 cells were cultured with or without CaF-37 fibroblasts. The percentage of SP cells was evaluated by flowcytometry, and the growth of cancer cells was examined by cell number count. In vivo tumorigenicity of OCUM-12/SP cells was evaluated by subcutaneous inoculation in the presence or absence of CaF-37 fibroblasts.

Results: The percentage of SP fraction of OCUM-12/SP cells was increased in the presence of CaF-37 fibroblasts. In contrast, CaF-37 fibroblasts significantly decreased the proliferation of OCUM-12/SP cells, while that of OCUM-12 cells was significantly increased by CaF-37 cells. The inoculation of 3×105, 1×105, and 3×104 OCUM-12/SP cells with 1×106 CaF-37 fibroblasts resulted in tumor formation in 5 (100%) of 5 mice, 4 (80%) of mice, 5 (100%) of 5 mice, respectively. However, the inoculation of 3×105, 1×105, and 3×104 OCUM-12/SP cells alone results in poor tumor formation at 3 (60%) of 5 mice, 0 (0%) of 5 mice, and 0 (0%) of 5 mice, respectively.Conclusion: Cancer-associated fibroblast might sustain the CSCs phenotype of stemness, and might increase the tumor initiation.

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 5200. doi:10.1158/1538-7445.AM2011-5200
Abstract 5203: Schlafen-3 decreases cancer stem cell marker expression and autocrine/ juxtacrine signaling in FOLFOX-resistant colon cancer cells
Authors: Oh Phil-Sun , Patel Vaishali B. , Sanders Matthew A. , Kanwar Shailender S. , Yu Yingjie , Nautiyal Jyoti , Patel Bhaumik B. , Majumdar Adhip P. N.
Published: June 2012

We have previously demonstrated that expression of the novel gene schlafen (Slfn)-3 correlates with intestinal epithelial cell differentiation. The current investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-Fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 and HT-29 cells resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 (ALDH1). This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α (TGF-α) in both FOLFOX-resistant cell lines but stimulated apoptosis. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSCs enriched FOLFOX-resistant colon cancer cells including reduction in tumorosphere/colonosphere formation, drug transporter activity and autocrine stimulation of proliferation. Thus, Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.

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 5203. doi:10.1158/1538-7445.AM2011-5203
Abstract 5206: Nanog family members are essential for spherogenicity and metastasis in colorectal carcinoma
Authors: Zhang Jingyu , Thorgeirsson Snorri S. , Jessup J. Milburn
Published: June 2012

Liver is the most common site for colorectal cancer (CRC) distant metastasis and hepatic metastasis is one of the main causes of death in CRC patients. Inhibition of liver metastases in patients with CRC may increase the survival of patients. Our postulate is that genes involved in early embryogenesis, Nanog, Oct4 and SOX2, may be reactivated and control the malignant properties of metastasis and resistance to chemo- and radiotherapy. Nanog is a family of 11 pseudogenes with the original parent gene on chromosome 12. In order to identify which gene product is expressed in human CRC, we developed a novel endonuclease restriction enzyme assay, which was used in six human CRC cell lines. Monolayer cultures of CRC cells express Nanog, NanogP8, and NanogP7 but spheroids cultured in serum-free medium only express NanogP8 which was confirmed by direct sequencing. In addition, two potential DNA methylation regions are detected in the promoters of Nanog and NanogP8 by sodium bisulfite sequencing of genomic DNA in hES and CRC CloneA cells. In hES cells, the methylation status of Nanog and NanogP8 was about 30% and 60%, respectively. In contrast, in CloneA cells, it was 65% for Nanog and about 40% for NanogP8 and lower for CX-1 CRC cells. These results suggest that NanogP8 is re-activated in CRC through promoter demethylation in CRC spheroids that are enriched for putative stem cells. Overexpression of NanogP8 in CloneA and CX-1 significantly increased the spherogenicity of single CRC cells. In addition, lentiviral shRNA to Nanog inhibited spherogenicity in vitro and tumorigenicity in vivo whereas shRNA to OCT4 and SOX2 actually increased spherogenicity and tumorigenicity. Lentiviral shRNA inhibition of Nanog prevented liver colonies after intrasplenic injection of CX-1 cells whereas 45% of NOD/SCID mice developed experimental metastases after injection of the untreated CX-1 cells or CX-1 cells treated with negative control shRNA. As a result, targeting Nanog and its family members expressed in CRC may block the progression of CRC.

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 5206. doi:10.1158/1538-7445.AM2011-5206
Abstract 5207: FGFR inhibitor regulates CD133 expression in FGFR2-amplified gastric cancer cells
Authors: Matsumoto Kazuko , Arao Tokuzo , Furuta Kazuyuki , Nagai Tomoyuki , Kudo Kanae , Kaneda Hiroyasu , Tamura Daisuke , Aomatsu Keiichi , Sakai Kazuko , Fujita Yoshihiko , Kimura Hideharu , Yamada Yasuhide , Nishio Kazuto
Published: June 2012

The underlying mechanism regulating the expression of the cancer stem cell / tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. We recently have reported that the expression of CD133 in cancer cells could be regulated though the inhibition of signal transduction by molecular-targeted drugs (Matsumoto K. et al. Cancer Res. 2009).

Here, we screened the expression changes of CD133 using wide variety of molecular-targeted drugs in FGFR2-amplified and CD133-overexpressing gastric cancer cell lines. CD133 expression was remarkably upregulated by FGFR inhibitor PD173074 exposure in these cells. Further analysis revealed that PD173074 upregulated the CD133 expression both mRNA and protein levels in dose- and time-dependent manners. In addition, the expression change was correlated with the mRNA expression levels of stem cell factors including OCT3/4, SOX2 and NANOG. As expected, PD173074 exhibited a potent growth inhibitory effect on FGFR2-amplified gastric cancer cells in vitro. These results suggest that the mechanism regulating the expression of CD133 is involved in FGFR2 signaling and also in addicted signaling pathway for cancer cells in FGFR2-amplified gastric cancer cells.

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 5207. doi:10.1158/1538-7445.AM2011-5207
Abstract 5210: Over-expression of cancer stem cell markers predicted poor prognosis in colorectal cancer
Authors: Ozawa Mayumi , Ichikawa Yasushi , Oshima Takashi , Kondo Akihisa , Nakazawa Kenichi , Miyata Hidetoshi , Zheng Yunwen , Shimozawa Manabu , Akaike Makoto , Suwa Hirokazu , Tatsumi Kenji , Watanabe Kazuteru , Ota Mitsuyoshi , Fujii Shoichi , Endo Itaru , Taniguchi Hideki
Published: June 2012

Introduction: Previous reports have demonstrated that CD133+CD44+ cells might be putative colorectal cancer stem cells (CSCs). Intestinal stem cell marker is already identified as LGR5 in mice, and it is also reportedly in human. However the correlation of CSCs and normal stem cell are unclear. The stem-cell-specific loss of APC (adenomatous polyposis coli) results in progressively growing neoplasia. Therefore, LGR5 may play a crucial role in the carcinogenesis and tumorigenesis. In this study, we investigated the possibility of LGR5 as shared common markers by CSCs and normal stem cells, furthermore to address the correlation among LGR5, CD44 and CD133.

Material and Methods: We used surgical specimens of cancer tissue to digest it into single cell suspension. The cells were immuno-stained for flow cytometory, the antigenic profiles of CD133 and CD44 were analyzed. Furthermore, the fractionated cells were sorted and transplanted into NOD/SCID mice. The xenograft tumors were analyzed by flow cytometory again or quantitative real-time PCR (qRT-PCR). And more, total RNA isolated from colorectal cancer and adjacent normal mucosa in 202 patients with untreated colorectal cancer was prepared and complementary DNA (cDNA) was synthesized from total RNA. qRT-PCR was performed to observe the expressions of LGR5, CD44 and CD133. Results: In vivo subcutaneous transplantation showed that as few as 100 CD133+CD44+ cells from primary tumor tissue had tumorigenicity in NOD/SCID mice, where in this cell fraction LGR5 was higher positive compared with other fractions. In xenograft, proportion of LGR5, CD133 and CD44 were expanded as compared with primary cancer and adjacent normal mucosa. In 202 clinical specimens, LGR5, CD44 or CD133 was expressed strongly compared with normal mucosa (p<0.0001). However, with any single marker was not enough to predict the prognosis. Instead, combined with CD44 or CD133, the group of LGR5 over-expression showed significantly low survival rate (p<0.022).

Conclusion: LGR5+, CD133+ or CD44+ cells expressed the characters of cancer stem cells in xenograft tumors as well as in primary colorectal cancers. Importantly, LGR5 is a potentially critical factor determining prognosis in clinics.

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 5210. doi:10.1158/1538-7445.AM2011-5210
Abstract 5222: Metabolomic patterns of tumors and metastases of human colon cancer xenografts
Authors: Stein Ulrike S. , Grötzinger Carsten , Detjen Katharina , Eckardt Änne , Lisec Jan , Willmitzer Lothar
Published: June 2012

Prognostic assessment of colon cancer metastasis and intervention in metastatic disease represent critical clinical challenges. Metabolomic platforms now offer the potential for a systematic survey of the metabolic profile of tumor tissues, which may allow to link key metabolites to distinct aspects of tumor biology such as metastatic capacity. Here we addressed these issues by testing the hypothesis that metastatic disease is characterized by distinct metabolites. In preparation for subsequent analyses of human tumor samples from existing tissue collections, here we employed human colon cancer lines harboring metastatic capacities to induce cancer xenografts in mice. In a first set of experiments using human colon xenografts, and applying GC-MS based metabolomics technology, we characterized the metabolite patterns of the primary tumors and of liver metastases, as well as of the mouse spleens (organ of transplantation) and the mouse livers (main target organ of distant metastasis). Subjecting the metabolite data to a Principal Component Analysis resulted in the separation of the samples into four clusters discriminating on the one hand mouse from human tumor xenograft tissues. Furthermore, mouse spleen was easily separated from mouse liver. More importantly, we identified clearly distinct metabolomic clusters comprising the primary tumor samples vs. the liver metastasis although originating from the same human colon cancer cell line. These different metabolic profiles of primary tumors and metastases were highly reproducible between independent experiments and were conserved in primary tumors or metastases of different size, suggesting they offer a representation of distinct biological features, rather than reflect tumor cell mass or common features of the tumor cell microenvironment. In addition, defined metabolites were identified that are characteristic for particular mouse organs (spleen vs. liver), and for discrimination of mouse tissues vs. human xenograft tumors. Moreover, metabolites were identified that allow discrimination of the primary tumor vs. its corresponding liver metastasis, although both tissues originate from the same human colon cancer cells. We also addressed methodological topics, such as the influence of time until sample cryo-asservation, time of storage, size and amount of the tissue samples, and reproducibility in independent in vivo experiments, on the metabolomic patterns and specific metabolites. In summary, we identified metabolomic patterns as well as defined metabolites that allow discrimination of the primary tumor vs. its corresponding liver metastasis of colon cancer xenografts in mice.

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 5222. doi:10.1158/1538-7445.AM2011-5222
Abstract 5229: Single, pre-labeled metastatic melanoma cells can be detected in the mouse brain using MRI
Authors: Daphu Inderjit K. , Wendelbo Ingvild , Immervoll Heike , Sykova Eva , Jendelova Pavla , Babic Michel , Bjerkvig Rolf , Thorsen Frits
Published: June 2012

In metastatic melanoma, brain metastases are clinically diagnosed in 40-60% of patients during the disease, but can be found in up to 90% at autopsy. The prognosis for these patients is poor, due to ineffective treatments and development of multiple brain metastasis. There is a limited knowledge about the biological mechanisms behind this development of multiple metastasis in the brain, which necessitates the development of clinically more relevant animal models.

The purpose of this study was to develop a multiple brain metastasis animal model which closely reflects the clinical setting, and specifically to determine the number of single melanoma cells able to arrest in the animal brain.

The H1 brain metastatic melanoma cell line was established from a patient melanoma brain metastases obtained during surgery. The H1 cells were pre-labeled with superparamagnetic iron oxide particles (Fedex; Babic et al, Bioconjugate Chem 2008) at a concentration of 10 μg/ml for 24 hrs. The viability of labeled cells was studied in vitro using cell proliferation assays, and Scanning/Transmission Electron Microscopy (SEM/TEM). One million pre-labeled tumor cells were then injected into the left ventricle of the heart in nod scid mice. 24 hrs after injections, T1 weighted (T1w), T2 weighted

(T2w) and T2* weighted (T2*w) MR imaging were performed. In addition, T1w and T2w MR images were obtained 5 weeks after injections. We also transfected the H1 melanoma cells with luciferase, and monitored the animals for up to 6 weeks using bioluminescence imaging (OI).

Pre-labeling of melanoma cells with Fedex did not affect cell proliferation. The SEM and TEM studies showed iron oxide particle attachment to the tumor cell membrane, as well as localisation of the labelling agents within vesicles in the cytoplasm. By encapsulating pre-labeled tumor cells at various concentrations in soft agar, we were able to detect less than 50 cell/μl using T2 mapping MRI. By pre-labeling the melanoma cells with Fedex, we were able to detect single tumour cells in the animal brains. Imaging studies (OI, MR) 4-6 weeks after injections confirmed multiple metastatic spread to the brain.

In conclusion, we have developed a melanoma metastasis model in immunodeficient mice, which reflects the metastatic tumor spread seen in patients. By using pre-labeling with Fedex, we were able to detect single tumour cells in the brain.

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 5229. doi:10.1158/1538-7445.AM2011-5229
Abstract 5238: Lung adenocarcinoma harboring EGFR L858R mutation has increased invasive ability through up-regulation of CXCR4
Authors: Chang Tzu-Hua , Yang Hsiao-Yin , Tsai Meng-Feng , Su Kang-Yi , Yang Pan-Chyr , Shih Jin-Yuan
Published: June 2012

Our previous study disclosed EGFR L858R mutation rate of lung adenocarcinoma with malignant pleural effusions of is higher than that of surgically resected lung adenocarcinoma (Wu et al, Eur Respir J 2008). The molecular mechanism is not clear. The chemokine receptor CXCR4 has recently been shown to mediate the movement of malignant cancer cells to specific organs. Stable clones of wild type and mutant L858R EGFR from lung cancer cell line H1299 were obtained from Dr. Chen (Chen YR et al Oncogene 2006). The Invasive ability of L858R EGFR H1299 cells was higher than that of wild type EGFR H1299 cell. L858R EGFR H1299 cell had higher CXCR4 expression, which was required for L858R EGFR-mediated invasion in vitro. Up-regulation of CXCR4 in L858R EGFR H1299 cell promoted invasion via increased activity of ERK. These results provide a plausible mechanism for L855R EGFR-mediated lung tumor metastasis and establish a functional link between mutant EGFR and CXCR4 signaling pathways.

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 5238. doi:10.1158/1538-7445.AM2011-5238
Abstract 5239: Licoricidin present in licorice root extract inhibits metastatic capacity of 4T1 mouse mammary carcinoma cells in vitro and in vivo systems
Authors: Park So Young , Kwon Sujin , Lim Soon Sung , Kim Jin Kyu , Kim Jong-Sang , Lee Choonghwan , Park Jung Han Yoon
Published: June 2012

Licorice root extracts have previously been shown to exhibit anti-carcinogenic activities. However, these extracts harbor considerable quantities of glycyrrhizin, which is known to exert anticarcinogenic activities and also to induce severe hypokalemia and hypertension. In our previous experiments, licoricidin was identified as an antimetastatic active component of a licorice root extract, which contains undetectable amounts of glycyrrhizin. In this study, we evaluated the effects of the licoricidin on the metastatic characteristics of 4T1 murine mammary carcinoma cells. When 4T1 cells were cultured on a transwell-filter in an in vitro model, licoricidin inhibited cell migration in a dose-dependent manner. Gelatin zymography and Western blot analysis showed that licoricidin significantly suppressed the secretion and activation of the matrix metalloproteinase (MMP)-2 and MMP-9. The secretion of tissue inhibitor of metalloproteinase-1 was reduced in licoricidin-treated cells. Licoricidin reduced the protein levels of vascular cell adhesion molecule but did not change intercellular adhesion molecule. To assess the effects of licoricidin on lung metastasis in vivo, we injected 4T1 cells (5×104 cells) into the inguinal mammary fat pads of syngeneic, immunocompetent BALB/c mice. Starting 1 week after 4T1 cell injection, the mice were given daily intraperitoneal injection of licoricidin (2 or 4 mg/kg body weight/day) for 21 days. Licoricidin treatment did not affect solid tumor growth but significantly reduced the number and volume of pulmonary tumor nodules. The expression of interleukin (IL)-1ra, IL-16, interferon γ-inducible protein (IP)-10, chemokine (C-X-C motif) ligand (CXCL)-9, MMP-3, -8, -9, osteopontin, pentraxin-3, stromal cell-derived factor (SDF)-1, and plasminogen activator inhibitor (PAI)-1 was increased in the lung tissues of mice injected with 4T1 cells, which was suppressed by the repeated injections of licoricidin. By contrast, the expression of angiopoietin-1, insulin-like growth factor binding proteins (IGFBP)-9 and IGFBP-10 was decreased in lung tissues of mice injected with 4T1 cells and was increased by the licoricidin treatment. In the present study, using both in vivo and in vitro systems, we have demonstrated that licoricidin profoundly inhibits the metastatic capacity of 4T1 mammary carcinoma cells, which may be mediated via the regulation of several metalloproteases, adhesion molecules, cytokines, and chemokines.

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 5239. doi:10.1158/1538-7445.AM2011-5239
Abstract 5247: Expression and functional role of CCR9 in lung cancer
Authors: Singh Rajesh , Lillard James W. , Grizzle William E. , Singh Shailesh
Published: June 2012

Lung cancer is leading cause of cancer related deaths worldwide in both men and women. Metastasis is responsible for most lung cancer related deaths; therefore, a better understanding of the metastatic processes and therapies designed to prevent the spread of cancer cells are greatly needed. The specific mechanisms that promote metastases have not been fully elucidated. Among all known chemokine receptors involved in cancer progression, most cancer cells including lung cancer express CXCR4. Furthermore, involvement of CXCR4 and CCR7 in breast cancer and CXCR4, CCR9 and CX3CR1 in prostate cancer progression further indicates that multiple chemokine receptors are involved in dictating the multi-step process of metastasis. For the first time, we show that lung cancer cells and tissue form Non Small Cell Lung Carcinoma (NSCLC) express CCR9, which is the receptor for CCL25. CCR9 was highly expressed by NSCLC cell lines as compared to Small Cell Lung Carcinoma (SCLC) cell lines at mRNA and protein levels. Furthermore, we have shown that CCR9-CCL25 interaction plays a crucial role in lung cancer cell migration. It addition to these, serum levels of CCL25 was significantly elevated in lung cancer patents compared to normal healthy donor. The mechanism underlying these clinically and biologically important finding need to be further explored. Increased serum CCL25 in lung cancer patients with metastatic disease required further validation as a potential therapeutic target and/or diagnostic marker for lung 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 5247. doi:10.1158/1538-7445.AM2011-5247
Abstract 525: Functional screen of paracrine signals in breast carcinoma fibroblasts
Authors: Su Gui , Beebe David J. , Friedl Andreas
Published: June 2012

Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors. However, our knowledge about the identity of paracrine mediators operating in individual patients remains incomplete. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. Collectively, neutralizing antibodies to fibroblast growth factor 2, heparin-binding epidermal growth factor, SDF1 and TGF- β1 and the enzymes heparanase-1 and MT1-MMP significantly abrogated CAF-stimulated T47D cell growth, although, a considerable degree of inter-individual heterogeneity was observed. CAF from low-grade but not high-grade carcinomas required IGF1 and TGF- β1 to stimulate carcinoma growth. CAF from estrogen or progesterone receptor negative carcinomas depended on HB-EGF activity for carcinoma growth. Paradoxically, blocking of MT1-MMP stimulated T47D cell growth in co-culture with NF. The results were largely replicated by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the paracrine mediators examined in this study. In summary, we characterize the paracrine signaling network of primary breast cancer fibroblasts with inhibitor screens. These findings have significant implications for the design of stroma-targeted therapies.

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 525. doi:10.1158/1538-7445.AM2011-525