bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2675: 18F-FDG-PET/CT imaging analyses for liver metastases of human colon cancer xenografts in NOG mice.
Authors: Chijiwa Tsuyoshi , Kawai Kenji , Tsukada Hideo , Suemizu Hiroshi , Nakamura Masato
Published: August 2014

Colorectal cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. Liver metastases of colorectal cancer significantly affect the fates of patients. To further understand the pathological aspects of distant metastases, we have established liver metastasis models of human colon cancer xenografts in immune-deficient NOD/Shi-scid/IL-2Rγnull (NOG) mice. 18F-fluorodeoxyglucose positron emission tomography / x-ray computed tomography (18F-FDG-PET/CT) is useful in the clinical evaluation of various human cancers. While PET analysis was established to evaluate subcutaneous lesions of human cancer xenografts in mice, its application to internal lesions including liver metastases is still being developed. We applied PET approaches for the evaluation of in vivo metastatic lesions in the internal organs of living small experimental animals. In this study, we analyzed in vivo liver metastases of human colon cancer cell lines in NOG mice using PET imaging. Experimental liver metastases were established by intrasplenic injection of human colon cancer cell line HCT116 (1.0 x 105 or 1.0 x 106 cells/mouse). 18F-FDG-PET/CT scans were performed 2 weeks after xeno-transplantation. After PET/CT scans, histopathological examinations were also performed to precisely confirm lesions. 18F-FDG-PET/CT imaging clearly demonstrated multiple hot spots on livers in spite of disadvantages in the contrast between tumor soft tissues and markedly swollen livers. PET/CT analysis also disclosed increased standardized uptake values (SUV) of FDG in metastatic foci of NOG mice (control, SUVmean 0.450 ± 0.033, SUVmax 0.635 ± 0.017; 1.0 x 105 cells, 0.853 ± 0.087, 1.254 ± 0.237; 1.0 x 106 cells, 1.211 ± 0.108, 1.701 ± 0.158). There were significant differences in FDG uptakes between the three groups (ANOVA, p=0.017 in SUVmean, p=0.044 in SUVmax, n=2). We clearly and quantitatively detected liver metastasis images in NOG mice by 18F-FDG-PET/CT in vivo. Immunohistochemical examinations confirmed more metastatic foci and more severe hepatomegaly with 1.0 x 106 than 1.0 x 105 of HTC116 cell inoculations. PET/CT analysis of human cancer xenografts is a new and reliable system to evaluate metastatic lesions in internal organs. In conclusion, this model system is useful for analyzing metastatic mechanisms and developing new therapeutic approaches for the liver metastatic lesions of human cancer.

Citation Format: Tsuyoshi Chijiwa, Kenji Kawai, Hideo Tsukada, Hiroshi Suemizu, Masato Nakamura. 18F-FDG-PET/CT imaging analyses for liver metastases of human colon cancer xenografts in NOG mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2013-2675
Abstract 2676: Measuring extracellular pH within in vivo tumors using acidoCEST MRI.
Authors: Chen Liu Qi , Howison Christine , Jeffery Justin , Robey Ian , Pagel Mark
Published: August 2014

INTRODUCTION. Extracelluar pH (pHe) is a hallmark for tumor microenvironment. We have developed a non-invasive MRI method that can accurately measure pHe to assess tumor acidosis, termed “acidoCEST MRI”. This method measures a ratio of the Chemical Exchange Saturation Transfer (CEST) effects of Iopromide, a FDA approved contrast agent for CT. Iopromide has two amides that generate different CEST effects, and a ratio of these CEST effects is correlated with pH over a range of 6.0-7.2 pH units, with a precision of 0.06 pH units, and in a manner that is independent of the agent's concentration and the sample's T1 relaxation time. We have applied acidoCEST MRI to monitor the effects of bicarbonate treatment on tumors.

METHOD. A CEST-FISP pulse sequence (2.8 μT, 5 sec, 90 Hz), with 54 saturation frequencies (+10 to -10 ppm) was used to acquire an acidoCEST image in 4.7 min on 7T MRI scanner. Each CEST spectrum was fitted to a sum of three Lorentzian line shapes (Matlab R2012B).

A mouse model of MDA-MB-231 mammary carcinoma subcutaneous flank tumor was treated with 200 mM of bicarbonate ad libitum. acidoCEST MRI was performed 1 day before and 1 day after introducing bicarbonate treatment. A bolus of 200 μL of 976 mM iopromide was injected i.v., followed by an infusion of 150 μL/hour of iopromide. Six series of acidoCEST spectroscopic images were acquired for 28 min.

RESULTS: We were able to measure an increased in pH in tumor before and after bicarbonate therapy. As shown in the table, only half of the mice studied showed a significant increase in pH after bicarbonate treatment. This is because the limit of quantification of acidoCEST using Iopromide as contrast agent was pH 7.20.

Discussion: acidoCEST MRI using Iopromide as contrast agent can measure pH of the tumor with good precision and can be applied to monitor pH-modulating therapy such as bicarbonate treatment. Our method has strong clinical translation potential because it uses low saturation powers, incomplete saturation, and a clinically approved contrast agent.

Summary of acidoCEST measurements on mammary carcinoma tumors before and after bicarbonate treatment Mouse before bicarbonate treatment after bicarbonate treatment #1 pH 6.50 ± 0.09 pH 7.15 ± 0.14 (p < 0.01) #2 pH 6.49 ± 0.09 pH 7.20 ± 0.00 (p < 0.01) #3 pH 6.53 ± 0.10 pH 7.14 ± 0.14 (p < 0.01) #4 pH 6.91 ± 0.11 pH 7.10 ± 0.13 (p < 0.02) #5 pH 7.13 ± 0.12 pH 7.16 ± 0.12 #6 pH 7.02 ± 0.13 pH 7.13 ± 0.10 #7 (necrotic tumor) pH 7.20 ± 0.00 pH 7.19 ± 0.06 #8 (necrotic tumor) pH 7.17 ± 0.09 pH 7.17 ± 0.10

Citation Format: Liu Qi Chen, Christine Howison, Justin Jeffery, Ian Robey, Mark Pagel. Measuring extracellular pH within in vivo tumors using acidoCEST MRI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2676. doi:10.1158/1538-7445.AM2013-2676
Abstract 2680: Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT.
Authors: Tuomela Johanna M. , Seppänen Jani , Haavisto Laura , Fosrback Sarita , Vahlberg Tero , Solin Olof , Haaparanta Merja
Published: August 2014

Treatment of pancreatic adenocarcinoma is difficult because the location of the lesion is often unknown. 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA) is used for imaging of neuroendocrine tumours, which take up and decarboxylate amine precursors. [18F]FDOPA use in diagnostic pancreatic imaging has increased, since the exocrine pancreas synthesises dopamine. We studied the uptake of [18F]FDOPA in healthy and diseased pancreas. Mice bearing orthotopic BxPC3 pancreatic adenocarcinoma and sham-operated controls were pre-treated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or combinations of these inhibitors. Mice were injected with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) or [18F]FDOPA and scanned via positron emission tomography/computed tomography. The absolute [18F]FDOPA uptake was determined from selected tissues using a well counter. The intratumoural biodistribution pattern of [18F]FDOPA was recorded autoradiographically. The main FDOPA metabolites present in pancreatic samples were determined with high-performance liquid chromatography. [18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in healthy pancreas and significantly lower in tumour-bearing pancreas, independent of pre-treatment. [18F]FDOPA uptake in pancreas was lowest with vehicle pre-treatment and highest with pre-treatment with the inhibitor of AADC. When mice received COMT+MAO-A inhibitors, uptake was high in healthy pancreas but low in tumour-bearing pancreas. Unequal uptake may be due to blockade of metabolism and increased availability of [18F]fluorodopamine due to the employed enzyme inhibitors. Combined use of [18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Pre-treatment with COMT+MAO-A inhibitors improved the differentiation of pancreas from surrounding tissue and healthy pancreas from tumour.

Citation Format: Johanna M. Tuomela, Jani Seppänen, Laura Haavisto, Sarita Fosrback, Tero Vahlberg, Olof Solin, Merja Haaparanta. Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2680. doi:10.1158/1538-7445.AM2013-2680
Abstract 2682: Imaging prostate cancer in an orthotopic prostate model using a 68Ga-Bombesin (BB2r) radiopharmaceutical.
Authors: Richmond Kelsey , Rold Tammy , Szczodroski Ashley , Ma Lixin , Cai Quanyu , Hoffman Timothy
Published: August 2014

Introduction: There continues to remain a significant need to develop molecular imaging agents for the diagnosis and staging of prostate cancer. The association of the bombesin receptor subtype 2 (BB2r) and prostate cancer is well known and several groups including our own have developed unique radiopharmaceutical targeting strategies for the delivery of BB2r agonists and antagonists. Earlier work from our group utilizing BB2r agonists and antagonists labeled with 99mTc and 111In demonstrated selective targeting of simple flank xenograft models of prostate cancer. We are now presenting a preclinical evaluation of 68Ga-BB2r PET imaging using an orthotopic prostate SCID mouse model.

Methods: The BB2r antagonist, DOTA-4-Amino-Carboxymethyl-Piperidine-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, (Eur J Nucl Med Mol Imaging 38(1):97, 2011) was synthesized commercially. 68Ga was obtained from a commercially available Ge-68/Ga-68 generator. The peptide (40ug) was radiolabeled with 10-20mCi (270-740MBq) of 68Ga using an automated radiosynthesis system. Reverse phase HPLC (C-18) was employed for quality control analysis using a combination of radiometric and UV analysis. Orthotopic prostate xenografted male SCID mice were administered ∼0.3mCi (11.1 MBq) of 68Ga labeled peptide and combined PET/CT imaging was performed 4 hours following injection. Each mouse received 100uL of Visipaque® before imaging commenced. Concurrent 7 T MRI data was obtained for anatomic verification of prostate tumor involvement. Pathologic confirmation of tumor involvement within the prostate bed was obtained.

Results: The 68Ga BB2r antagonists could be readily prepared in high radiochemical yield and purity as verified by HPLC. Orthotopic prostate tumor xenografts were clearly visualized using PET/CT imaging. The predominant renal clearance of the tracer presented challenges in differentiating active tumor involvement within the prostate bed from bladder contents. These challenges could be partially addressed by employing concurrent CT contrast agent administration as an aid in accurately defining bladder contents and balder wall delineation.

Conclusions: These results demonstrate that 68Ga BB2r antagonist PET/CT imaging holds the potential to accurately differentiate BB2r positive prostate tumor growth within the prostate bed as a potential screening and diagnostic tool.

Citation Format: Kelsey Richmond, Tammy Rold, Ashley Szczodroski, Lixin Ma, Quanyu Cai, Timothy Hoffman. Imaging prostate cancer in an orthotopic prostate model using a 68Ga-Bombesin (BB2r) radiopharmaceutical. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2013-2682
Abstract 2686: RSK2/CREB/Fascin-1 signaling is important for tumor metastasis.
Authors: Li Dan , Jin Lingtao , Alesi Gina , Kim Young Mee , Wang Dongsheng , Lee Benjamin H , Taunton Jack , Magliocca Kelly R , Chen Zhuo Georgia , Kang Sumin
Published: August 2014

Metastasis is the leading cause of death in patients with breast, lung or head and neck cancers. However, the molecular mechanisms underlying metastases in these cancers remain unclear. We found that the p90 ribosomal S6 kinase 2 (RSK2)-cAMP response element-binding (CREB) pathway is commonly activated in diverse metastatic human cancer cells, leading to upregulation of a CREB transcription target Fascin-1. We also observed that the protein expression patterns of RSK2 and Fascin-1 correlate in primary human tumor tissue samples from HNSCC patients. Moreover, knockdown of RSK2 disrupts filopodia formation and bundling in highly invasive cancer cells, leading to attenuated cancer cell invasion in vitro and tumor metastasis in vivo, while expression of Fascin-1 significantly rescues these phenotypes. Furthermore, targeting RSK2 by a small molecule RSK inhibitor FMK-MEA effectively attenuated the invasive and metastatic potential of cancer cells in vitro and in vivo, respectively. Together, our findings for the first time link the RSK2-CREB signaling to filopodia formation and bundling by upregulating Fascin-1, providing a pro-invasive and pro-metastatic advantage to human cancers; protein effectors of RSK2-CREB-Fascin-1 pathway represents promising biomarkers and therapeutic targets in clinical prognosis and treatment of metastatic human cancers.

Citation Format: Dan Li, Lingtao Jin, Gina Alesi, Young Mee Kim, Dongsheng Wang, Benjamin H Lee, Jack Taunton, Kelly R Magliocca, Zhuo Georgia Chen, Sumin Kang. RSK2/CREB/Fascin-1 signaling is important for tumor metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2686. doi:10.1158/1538-7445.AM2013-2686
Abstract 2689: Drivers of cancer cell invasion and metastasis.
Authors: Sharif Ghada M. , Schmidt Marcel O. , Shuptrine Casey , Weiner Louis M. , Hu Zhang-Zhi , Riegel Anna T. , Wellstein Anton
Published: August 2014

Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, evasion at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive and metastatic phenotype. Different human and mouse cancer cell lines were transduced with lentiviral-based, genome-wide or kinome-targeted shRNA libraries. Cancer cell populations of different invasive and metastatic phenotype were then selected for their loss- or gain-of-function respectively in endothelial invasion in vitro or by tumor growth and metastasis selection in vivo. After several rounds of selection, the respective shRNA targeted genes were identified from the selected cancer cell populations or from clonal cell lines. Eighteen different genes targeted by two to four distinct shRNAs each were identified in the in vitro selection for endothelial attachment and invasion. The function of the genes as well as the clinical significance appear to explain their phenotypic selection.

Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Louis M. Weiner, Zhang-Zhi Hu, Anna T. Riegel, Anton Wellstein. Drivers of cancer cell invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2013-2689
Abstract 2690: The role of musashi-1 in tumor progression in non-small cell lung cancer.
Authors: Boumber Yanis , Kudinov Alexander , Liu Xin , Ahn Young-Ho , Rodriguez Jaime , Wistuba Ignacio , Gibbons Don , Kurie Jonathan
Published: August 2014

We have previously developed an in vivo mouse model of metastatic lung adenocarcinoma by combining a mutant p53 allele (p53R175HΔG) with a KrasG12D allele (KrasLA1). From the tumors arising in KrasLA1/+; p53R172HΔG/+ mice, we have established lung adenocarcinoma cell lines (KP cells) with high (344SQ) or low (393P) metastatic potential. The metastatic potential of KP cells is governed by Zeb-1 and the microRNA-200 family, which interact through a double-negative feedback loop. Given that metastatic KP cells exhibit the capacity to form polarized epithelial spheres in Matrigel and undergo epithelial-to-mesenchymal transition (EMT) in response to extracellular cues, we hypothesized that stem cell factors serve as drivers of tumor cell invasion and metastasis in this model. We examined the expression of 13 stem cell factors in metastatic and non-metastatic KP cell lines using qPCR and found that Musashi-1 (Msi-1) was more highly expressed in metastatic KP cells, which was confirmed by western analysis. Msi1 is an RNA-binding protein that plays a central role during nervous system development, stem cell maintenance, and tumorigenesis. High levels of Msi1 have been reported in several malignancies including brain tumors, colon and breast cancer, and associate with poor survival in patients with breast and colon cancer. We have used a panel of human lung cancer cell lines and found Msi-1 protein to be expressed in lung adenocarcinoma and squamous carcinoma cell lines. Further analysis revealed that Msi-1 expression is up-regulated by Zeb-1 and down-regulated by miR-200, raising the possibility that Msi-1 functions as a downstream mediator of the Zeb-1/miR-200 axis, which we are currently investigating by carrying out Msi-1 loss-of-function and gain-of-function experiments and by analyzing human tissue microarrays to determine whether Msi-1 has prognostic value in patients. Our data suggests that Msi-1 is a candidate driver of tumor progression in NSCLC.

Citation Format: Yanis Boumber, Alexander Kudinov, Xin Liu, Young-Ho Ahn, Jaime Rodriguez, Ignacio Wistuba, Don Gibbons, Jonathan Kurie. The role of musashi-1 in tumor progression in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2013-2690
Abstract 2702: WAVE3 regulates NFκB signaling and sensitizes cancer cells to apoptosis and cell death driven by TNFα.
Authors: Davuluri Gangarao , Sossey-Alaoui Khalid , Plow Edward
Published: August 2014

ABSTRACT

WAVE3, a member of actin nucleation proteins promotes cancer invasion and metastasis, in part through regulation of expression and activity of key matrix metalloproteinases (MMPs) MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potentially important role of WAVE3 in this pathway. Mechanistically, we found that loss of WAVE3 in cancer cells leads to significant inhibition of TNFα-induced NFκB activation as a result of de-stabilization of the IκBα-NFκB complex. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity by stabilizing the IκBα-NFκB complex. We used both pharmacological and genetic manipulations of NFκB effector molecules and identified IKKβ but not IKKα as the primary upstream target for WAVE3-mediated regulation of NFκB activity. We also showed that loss of WAVE3 resulted in a significant inhibition of TNFα-mediated MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to TNFα-driven apoptosis and cell death. Finally, we showed that either the basic rich (BR) or the proline rich (PR) domain of WAVE3 is sufficient to modulate NFkB signaling. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the stabilization of NFκB complex that promotes the metastatic phenotype of cancer cells. Therefore, therapeutic targeting of WAVE3 may prove beneficial for preventing or reducing the metastatic burden in cancer patients.

Citation Format: Gangarao Davuluri, Khalid Sossey-Alaoui, Edward Plow. WAVE3 regulates NFκB signaling and sensitizes cancer cells to apoptosis and cell death driven by TNFα. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2013-2702
Abstract 2704: Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells.
Authors: Behrens Alice , Jousheghany Fariba , Yao-Borengasser Aiwei , Kieber-Emmons Thomas , Monzavi-Karbassi Behjatolah
Published: August 2014

Introduction. We have previously demonstrated that the expression levels of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness (basal-like, triple-negative and mesenchymal phenotype) of human breast cancer. The present study was performed to establish stably transfected cell lines and study the functional role of CHST11 in human breast cancer cells. Methods. MCF7 cells were transfected with a plasmid vector containing the CHST11 coding sequence. Stable transfectants, MCF-7-CHST11, were established. Flow cytometry and real-time PCR were used for expression analysis. Metastatic phenotype of cells was characterized through conduction of proliferation and migration assays. Morphology and growth pattern of MCF-7-CHST11 cells were monitored and compared to parental MCF-7 cells. Results. We observed that MCF-7-CHST11 reacted strongly with anti-chondroitin sulfate A (CS-A) antibody and recombinant P-selectin. CS-A is the immediate product of CHST11 activity. CHST11 expression significantly affected cell proliferation as MCF-7-CHST11 cells grew much faster. Migration assay using transwell chamber showed high migratory potential for MCF-7-CHST11 cells as compared to the parental MCF-7 cells. The significant migratory advantage was not proliferation dependent. The CHST11expression affected the morphology of cells and cell spreading behavior consistent with more efficient anchorage independent growth. Colony formation in soft agar, Invasion and adhesion assays are being performed at the present time. We are planning more experiments to study mechanisms involved. Conclusions. The data confirm that the CHST11 products on the cell surface can serve as P-selectin ligands. The results indicate that the CHST11 expression can affect metastatic phenotype of cancer cells by promoting cellular proliferation and migratory potential.

Citation Format: Alice Behrens, Fariba Jousheghany, Aiwei Yao-Borengasser, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2704. doi:10.1158/1538-7445.AM2013-2704
Abstract 2705: Novel functions of the homeoprotein SIX2 in mediating anchorage independence and metastasis in breast cancer.
Authors: Wang Chu-An , Jedlicka Paul , Zaberezhnyy Vadym , Tan Aik-Choon , Ford Heide
Published: August 2014

Homeobox genes encode for transcription factors that are master regulators of embryogenesis, and their misexpression has been implicated in multiple cancers. The role of the homeoprotein Six2 in developing kidney has been well demonstrated; however, its role in cancer progression is largely unknown. Here, we demonstrate, for the first time, that Six2 is causally involved in mammary tumor progression. When Six2 is knocked down (KD) in the 66cl4 mammary carcinoma cells, lung metastasis is significantly decreased compared to control KD; however, Six2 KD conferred no significant effect on growth of the primary tumor or on tumor-associated angiogenesis/lymphangiogenesis, in contrast to its closely related family member, Six1, which has been implicated in all the aforementioned properties; suggesting that Six2 may participate in later stages of the metastatic cascade. Expression of Six2 in the 4TO7 mammary carcinoma cell line (a cell line that is syngeneic with 66cl4, but expresses very low levels of endogenous Six2) led to changes in cell morphology, increased growth in soft agar, increased resistance to anoikis, and significantly enhanced lung metastasis in Balb/c mice. To determine the mechanism by which Six2 mediates metastasis, microarray analysis was performed on 4TO7-control and Six2 expressing cells. Interestingly, genes which have been implicated in lung metastasis (MMP, ANGPTL4, VCAM1) are significantly up-regulated in Six2 overexpressing 4TO7 cells; while the epithelial marker, E-Cadherin, is dramatically decreased. Finally, analysis of SIX2 expression from public microarray datasets indicates that SIX2 is increased in breast cancers compared to normal breast tissue. In addition, high expression of SIX2 correlates with poor prognosis (distant metastasis free survival, overall survival and relapse free survival) in 1881 human breast tumors examined using the GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database, and upon further investigation we found that SIX2 expression is particularly associated with poor prognosis in luminal A, normal-like and ER-positive breast tumors. Together, our studies define a novel role of Six2 in breast cancer metastasis.

Citation Format: Chu-An Wang, Paul Jedlicka, Vadym Zaberezhnyy, Aik-Choon Tan, Heide Ford. Novel functions of the homeoprotein SIX2 in mediating anchorage independence and metastasis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2705. doi:10.1158/1538-7445.AM2013-2705
Abstract 2710: Pulmonary inflammation associated with Chitinase-3-like-1 protein (CHI3L1) expression accelerates breast cancer metastasis to the lung.
Authors: Libreros Stephania , Garcia-Areas Ramon , Carrio Roberto , Iragavarapu-Charyulu Vijaya
Published: August 2014

Metastasis of a primary tumor to distant organs is the principal cause of mortality in patients with breast cancer. Recently, it was reported that asthmatic patients have a greater propensity to develop distant metastasis to the lung. Using a ragweed pulmonary inflammation model, we found that asthmatic mice implanted with 4T1 mammary tumors have a 5-fold increase in formation of metastatic foci in their lungs compared to non-asthmatic 4T1 mammary tumor-bearing mice. Further, asthmatic tumor-bearing mice showed accelerated tumor growth and shorter disease-free survival. We have previously reported that Chitinase-3 like-1 protein (CHI3L1) expression is increased in mammary tumor-bearing mice and that it induces production of prometastatic mediators, CCL2, CXCL2 and MMP-9 by macrophages. Asthmatic patients have been reported to exhibit elevated levels of CHI3L1 in their lungs and circulation while in breast cancer patients these levels are further elevated. We found that the levels of CHI3L1 are higher in circulation and lungs of asthmatic mammary tumor-bearers compared to mice with either asthma or tumor alone. It is unknown if exposure to high levels of CHI3L1 previous to tumorigenesis accelerates metastasis. We hypothesize that CHI3L1-induced pulmonary inflammation generates the proper environment for recruiting circulating breast cancer cells as well as proinflammatory leukocytes (M2 macrophages and myeloid derived suppressors), resulting in increased rate of metastasis to the lung. Treatment of 4T1 asthmatic mammary tumor-bearing mice with chitin microparticles, the ligand of CHI3L1, resulted in decreased levels of metastasis to the lung. These studies provide an understanding of the role of CHI3L1 and existing inflammation in accelerating metastasis, the major cause of death in breast cancer patients.

Citation Format: Stephania Libreros, Ramon Garcia-Areas, Roberto Carrio, Vijaya Iragavarapu-Charyulu. Pulmonary inflammation associated with Chitinase-3-like-1 protein (CHI3L1) expression accelerates breast cancer metastasis to the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2710. doi:10.1158/1538-7445.AM2013-2710
Abstract 2714: Expression and functional analysis of LGR4 in normal and cancer cells of human colon.
Authors: Yi Jing , Xiong Wei , Gong Xing , Liu Qingyun
Published: August 2014

LGR4 (leucine-rich repeat containing G protein-coupled receptor 4) and its two closely related homologues LGR5 and LGR6 have been shown to function as receptors of R-spondins to potentiate Wnt signaling. The RSPO/LGR4-6 ligand receptor system has essential functions in embryonic development, cell proliferation and survival of adult stem cells. Particularly, LGR4 is required for the survival of intestinal stem cells and the maintenance of gut epithelium while LGR5 is specifically expressed in crypt stem cells. Wnt signaling is frequently dysregulated in various types of cancer, especially in colorectal cancers of which 90% of cases are mutated within this pathway. Most recently, ∼10% of colon cancers were found to contain RSPO gene fusions, but not APC or β-catenin alterations, as an alternative mechanism of Wnt signaling activation. However, expression pattern and function of LGR4 in normal and cancer cells of human colon are largely unknown. The underlying receptor mediating the carcinogenic functions of RSPO gene fusion in colorectal cancers is also unidentified. We developed and validated a panel of LGR4 antibodies that can detect either native or denatured LGR4 with high specificity and sensitivity. Immunohistochemistry analyses were carried out in multiple matched pairs of normal and cancer tissues of human colon. In normal colon LGR4 is expressed most abundantly in epithelial cells located at the bottom of the crypts, consistent with expression data from the mouse intestine. In colon cancer, level of LGR4 is dramatically increased when compared to that of the crypt cells of the matched normal tissues. Next, we used an LGR4 antibody to isolate LGR4-high and LGR4-low cells from a colorectal cancer cell line, and compared the cell populations in rate of proliferation, migration, invasion in vitro and tumor formation and growth in xenografts. Data from these expression and functional analyses will be presented and discussed.

Citation Format: Jing Yi, Wei Xiong, Xing Gong, Qingyun Liu. Expression and functional analysis of LGR4 in normal and cancer cells of human colon. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2013-2714
Abstract 2715: Establishment of patient primary colorectal tumor xenograft models for test of anticancer agents.
Authors: Zhang Ning , Xu Chunping , Li Wen Wei , Zhou Wen , Liu Yong , He Fang , Zhou Rui , Liu Changnian
Published: August 2014

BACKGROUND Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But tumor cell lines have a relatively low transplantability, which resulted in a limited number of tumor models available for selection right models to test novel anticancer agents. Recently, we have developed a large number of patients’ primary colorectal tumor xenograft models by transplanting human fresh colorectal tumor tissues into nude mice, which have been employed for preclinical test of standard care of drugs and novel anticancer agents for their chemosensitivity screening. METHODS The fresh colorectal tumor samples were collected from local hospitals. The tumor fragments of 2-3 mm were subcutaneously implanted in the flanks of nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 0). The standard care of drugs as controls included 5-FU, irinotecan, oxaliplatin, Erbitux, and Avastin. The histopathology and gene sequencing of the established primary tumor models were analyzed and compared with patients’ original tumors. RESULTS A total of 180 patients’ colorectal tumor samples were implanted into nude mice; and 93 patient tumor derived models have been established with a tumor taking rates of 52% for the first passage. The tumor taking rates were higher in the later passages ranged from approximately 80-100%. The therapeutic efficacy of the test drugs was consistent with their clinical findings. The patients’ primary colorectal tumor xenografts from 5 passages retained a similarity in architecture, histopathological morphology, and genomic mutation status to the patients’ original tumors. CONCLUSIONS The patient primary tumor model system can provide a larger number of models for selection of right models to test novel agents in preclinical setting based on their anticancer mechanism. The primary tumor models retain a similarity in histology and genomic mutation status to their counterpart patients’ original tumors and may predict more relevant clinical response and higher correlation with clinical findings than use of traditional xenograft models established from long-term cultured cancer cell lines. Especially, they have advantages for test of target-oriented therapeutics in new drugs development programs.

Citation Format: Ning Zhang, Chunping Xu, Wen Wei Li, Wen Zhou, Yong Liu, Fang He, Rui Zhou, Changnian Liu. Establishment of patient primary colorectal tumor xenograft models for test of anticancer agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2013-2715
Abstract 2720: Newly established Barrett's adenocarcinoma cell line (TYAE-1).
Authors: Shimada Yutaka , Moriyama Makoto , Okumura Tomoyuki , Sekine Shinichi , Sawada Shigeaki , Matsui Koshi , Hojo Shozo , Shibuya Kazuto , Yoshioka Isaku , Yoshida Toru , Nagata Takuya , Tsukada Kazuhiro
Published: August 2014

Although 10 esophageal adenocarcinoma cell lines have been reported, only 5 were Barrett's adenocarcinoma cell lines. Furthermore, there was only one mouse-inoculated esophageal adenocarcinoma cell line. In this paper, we present a newly established Barrett's esophageal adenocarcinoma cell line (TYAE-1).

Material and Method: The Barrett's esophageal adenocarcinoma cells were obtained from the resected tumor of a patient (74 years-old male). The resected tumor was confirmed as well-differentiated intraepithelial Barrett's adenocarcinoma without lymphnode metastasis. The tumor was too small (T1a) for a primary culture, so we transplanted the tumor in the back of a nude mouse.

Results: One month later, the tumor grew rapidly and was able to be re-transplanted to other mice. Up to the writing of this report, the tumor (TYAE-1m) was able to be re-transplanted 10 times every month. The histology of the transplanted tumor was slightly different from the primary tumor. The majority of the tumor was cribriform type and there were a few tubular-trabecular type cells. The expression of beta-Catenin was cytoplasmic in the cribriform part and membranous in the tubular part. Both cell types did express VEGF slightly however they did not express HER-2. We also started to perform in vitro cultures from the mice transplanted tumor and established TYAE-1c cell. TYAE-1c cells were able to be transplanted in nude mice and transplanted tumor showed the same histological features as TYAE-1m. TYAE-1m and TYAE-1c cells were sensitive to Gemcitabine treatment. On the other hand, both tumors did not show sensitivity for Docetaxel treatment.

In conclusion: Although the tumor was small and had no invasive characteristics by clinicopathological examination, this tumor has aggressive behavior. This in vitro and in vivo Barrett's adenocarcinoma model may useful for understanding the behavior of Barrett's adenocarcinoma.

Citation Format: Yutaka Shimada, Makoto Moriyama, Tomoyuki Okumura, Shinichi Sekine, Shigeaki Sawada, Koshi Matsui, Shozo Hojo, Kazuto Shibuya, Isaku Yoshioka, Toru Yoshida, Takuya Nagata, Kazuhiro Tsukada. Newly established Barrett's adenocarcinoma cell line (TYAE-1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2013-2720
Abstract 2723: The chorio-allantoicmembrane (CAM) model for testing HOX gene antagonism in pancreatic cancer.
Authors: Gray Sophie , Metcalf Stephen , Boxall Angela , Middleton Gary , Bagwan Izhar , Pandha Hardev , Morgan Richard
Published: August 2014

Background

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid cancers with a 5-year survival rate approaching a dismal 5%. New therapeutic targets need to be identified in order to increase survival rates.

HOX genes are a family of 39 transcription factors involved in embryonic development and which have been found to be dys-regulated in PDAC. HOX gene function can be antagonised by HXR9, a peptide that competitively inhibits binding of HOX proteins to their co-factor PBX. We established that the CAM model recapitulates human PDAC, including desmoplasia, and is highly suitable for the evaluation of HXR9 in vivo.

Experimental procedure

Four PDAC-derived cell lines (AsPc-1, BxPc-3, PaCa-3 and PancTu1) were treated in vitro with HXR9. Ic50s were determined using both MTS and LDH assays. FACS analysis was used to determine mechanisms of cell death. Cell lines were subsequently implanted in the CAM using Marigel™ on embryonic day (EDD) 10. Tumour growth curves were established and tumours were resected and subsequently stained with

PDAC-specific cell surface markers. CAM-tumours were treated with intra-tumoral injection of HXR9 on EDD 16.

Results

PDAC-derived cell lines were sensitive to HXR9 in vitro with IC50s ranging from 13-45μM. Annexin V-PE analysis showed apoptosis was induced in HXR9 treated cells. PDAC was established in the CAM from cell lines and showed remarkable similarity to human disease including desmoplasia and phonotypic markers. Intra-tumoral treatment of CAM-PDAC showed evidence of tumour regression and apoptosis.

Conclusions

The CAM model is a simple and robust assay for modelling human PDAC and recapitulates architecture including desmoplasia. The CAM assay is suitable for testing the efficacy of HXR9. Targeting HOX gene dys-regulation is a novel approach for the treatment of PDAC.

Citation Format: Sophie Gray, Stephen Metcalf, Angela Boxall, Gary Middleton, Izhar Bagwan, Hardev Pandha, Richard Morgan. The chorio-allantoicmembrane (CAM) model for testing HOX gene antagonism in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2723. doi:10.1158/1538-7445.AM2013-2723
Abstract 2724: Prometastatic variants of mouse gastric cancer cells demonstrated the resistance to cytotoxic chemotherapy.
Authors: Park Jun Won , Kim Heung Tae , Green Jeffrey E. , Kim Hark Kyun , Jang Seok Hun
Published: August 2014

We have established a primary cultured cell line from glandular stomach of a genetically-engineered mouse that spontaneously developed adenocarcinomas in stomach and intestine using cre-mediated deletion of floxed tumor suppressor alleles in epithelial tissue of gastrointestinal tract. To derive prometastatic variant, high-Epcam+ subpopulation was obtained using FACS from this cell line. These cells were injected subcutaneously into severe combined immunodeficient (SCID) mice, and allowed to grow until the mouse showed evidence of lung metastases. The metastatic foci were re-implanted subcutaneously into a new SCID mouse. When the mouse showed lung metastases, prometastatic variant was cultured from the metastatic foci. Compared with parental cells, prometastatic variant cells showed enhanced tumorigenicity, as measured by a decreased latent period and higher take rate, and faster invivo growth as well as higher metastatic potential. Moreover, these cells readily grew orthotopically in syngeneic mice by 100% take rates. This cell line was also successfully used for testing novel immunotherapeutic agents. Importantly,this cell demonstrated increased invitro IC50 for cytotoxic chemotherapeutic agent including 5-FU, as compared with parental cells. Thus,stem-like characteristics enriched in prometastatic variants of mouse gastric cancer cells may induce the resistance to cytotoxic chemotherapy, supporting the cancer stem cell hypothesis for drug resistance in GI cancers. (supported by the Proteogenomic Research Program through the National Research Foundation of Korea funded by the Korean Ministry of Education, Science and Technology; and by Korean National Cancer Center Grant 121005).

Citation Format: Jun Won Park, Heung Tae Kim, Jeffrey E. Green, Hark Kyun Kim, Seok Hun Jang. Prometastatic variants of mouse gastric cancer cells demonstrated the resistance to cytotoxic chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2724. doi:10.1158/1538-7445.AM2013-2724

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2728: Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice.
Authors: Zeineldin Maged , McGuinness William , Blagg Brian , Rajewski Roger , Neufeld Kristi L.
Published: August 2014

Mutation of the tumor suppressor gene Adenomatous Polyposis Coli (APC) is considered an initiating event in the development of most intestinal tumors. Although much effort has been spent determining functions of the APC protein, to date little is known about the mechanisms that regulate cellular APC levels. Here we report that in cultured cells, induction of a heat-shock response, via heat or compounds such as the HSP90 inhibitor 17-AAG, resulted in increased levels of APC. A novel non-toxic small molecule that we developed, KN1, also induced a heat-shock response and led to increased APC levels in both cultured cells and mice. To investigate the effect of heat-shock response induction and elevation of Apc level in intestinal tumorigenesis, we performed a series of experiments treating mice with either KN1 or 17-AAG. We tested these compounds on two mouse models with different germline Apc mutations, ApcMin/+ and Apc1322T/+. In both cases, a moderate dose of either drug did not change tumor burden, but surprisingly altered the distribution of intestinal polyps. In a third mouse model, colonic tumors were induced via administration of the mutagen azoxymethane (AOM) and colon irritant dextran sodium sulfate (DSS) rather than by germline Apc mutation. In AOM-DSS-treated mice, KN1 reduced tumor incidence, multiplicity, and size. Moreover, KN1-treated mice lost significantly less weight and had smaller spleens than vehicle-treated mice, suggesting KN1 may also suppress colitis. We conclude that induction of the heat-shock response affects intestinal tumorigenesis in germline Apc-mutant and colitis-induced tumor models.

Citation Format: Maged Zeineldin, William McGuinness, Brian Blagg, Roger Rajewski, Kristi L. Neufeld. Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2013-2728
Abstract 2729: A genetically engineered mouse model of de novo pancreatic carcinogenesis .
Authors: Yamaguchi Takashi , Ikehara Sanae K. , Ikehara Yuzuru
Published: August 2014

Multistep models of PDAC development converge on both abnormalities in KRAS and E2F activation, which are part of the Rb/E2F pathway, and dysfunction of p53, which is associated with the MDM2/p53 pathway. Recent progress in the understanding of the protective roles of the Rb/E2F and MDM/p53 pathways against carcinogenesis showed that cross talk and complementary actions of these pathways are present in many types of cancers. In this study, we addressed directly questions regarding the collaborative roles of the Rb/E2F and MDM2/p53 pathways in suppressing the progression of multistep PDAC carcinogenesis by generating TKC (loxP-βgeo-STOP-loxPtemperature-sensitive SV40 T antigen (tsTAg)), loxP-STOP-loxP-KrasG12D, and Pdx1-Cre) mice which exhibit impairment of both pathways and K-rasG12D expression in pancreatic epithelial cells. The PDAC induced in TKC mice exhibited rapid development and absence of inflammation or PanIN formation, which occurred by de novo carcinogenesis. The tumors resembled human PDAC at the histological and molecular level. In in vitro study, we established PDAC cell lines from TKC mice and immortalized pancreatic ductal cell lines from TC mice, Cytokeratin 19 expression with which was applied to select cells as pancreatic ductal cell differentiation. In collagen gel 3D culture, PDAC cells formed atypical gland structures the same as in subcutaneous transplanted tumor in nude mice, while the Cytokeratin 19 positive immortalized ductal cell lines from TC mice formed ductal structures similar to either intralobular or interloblar pancreatic ducts. The results of the analysis of the highlighted molecular mechanisms suggest that the synthetic interaction among mutated Kras, uncontrolled E2f activation, and dysfunction of p53 results in de novo pancreatic carcinogenesis. This model represents an important tool to explore molecular targets for diagnostics and cancer therapy for PDAC via de novo pancreatic carcinogenesis.This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and New Energy and Industrial Technology Development Organization (NEDO) of Japan, and supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Citation Format: Takashi Yamaguchi, Sanae K. Ikehara, Yuzuru Ikehara. A genetically engineered mouse model of de novo pancreatic carcinogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2013-2729