bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1531: Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal metastasis.
Authors: Qian Bin-Zhi , Lang Richard A. , Pollard Jeffrey W.
Published: August 2014

Macrophages are abundantly found in the tumor microenvironment enhance malignancy. At distal metastatic sites a distinct population of metastasis associated macrophages (MAMs) promotes tumor cell extravasation, seeding and persistent growth. Compared with lung resident macrophages, MAMs associated with breast cancer lung metastasis in pre-clinical models express high levels of cell surface FMS-like tyrosine kinase 1 (Flt1). Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth of both human and murine breast tumor cell. This was confirmed using a genetic model of Flt1 tyrosine kinase domain (TK) ablation. Using bone marrow transplantation techniques, we further showed that this metastasis inhibitory effect was due to the lack of FLT1 signaling in hematopoietic cells. Furthermore, macrophage specific ablation of the tyrosine kinase domain of Flt1 by crossing Flt1 flox/flox mice with macrophage restricted Cre mice (Csf1r-Cre) also significantly inhibited tumor cell metastatic potential. Since Flt1 is not expressed by other hematopoietic cells and FLT1 inhibition did not affect the recruitment of MAMs, it indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. FLT1 regulated genes were identified by comparing gene expression profiles using microarray techniques of FACS sorted MAMs from mice treated with control and anti-FLT1 inhibitory antibody. Bioinformatics analysis showed that inflammatory response genes were highly enriched in these genes. Together, our studies indicated that FLT1 signaling is required for the activation of metastasis associated macrophages and regulates a set of inflammatory genes that promote breast tumor distal metastasis.

Citation Format: Bin-Zhi Qian, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal 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 1531. doi:10.1158/1538-7445.AM2013-1531
Abstract 1534: CD163-expressing macrophages constitute a distinct malignancy-promoting phenotype in melanoma facilitating tumor development, progression, and metastasis: Implications for novel anticancer therapy.
Authors: Maniecki Maciej B. , Etzerodt Anders , Damsky William E. , Rafique Aisha , Gonzalez David , Dyrskjøt Lars , Møller Holger J. , Bosenberg Marcus W. , Moestrup Søren K.
Published: August 2014

Tumor-infiltrating macrophages are recruited to the tumor microenvironment and believed to play a key role in tumor development, progression, and metastasis by producing growth factors and promoting neoangiogenesis. Here, we show that CD163-expressing macrophages express a distinct gene expression signature capable of facilitating tumor development and progression. This supports our earlier findings showing significant correlation between CD163+ macrophage density, tumor grade, and poor prognosis in patients with melanoma. Selective eradication of CD163-expressing tumor-associated macrophages through receptor-mediated delivery of nanoparticles encapsulating the cytotoxic agent doxorubicin significantly inhibited tumor formation and prolonged survival in an immune-competent conditional mouse model of BrafV600E-induced, Pten-deficient metastatic melanoma. High-resolution dynamic intravital imaging showed specific targeting of a subpopulation of tumor-infiltrating macrophages expressing the scavenger receptor CD163, including Tie2-expressing macrophages recently identified as being highly proangiogenic.

In conclusion, our findings identify CD163-expressing macrophages as a distinct malignancy-promoting phenotype and indicate a novel therapeutic target in cancer.

Citation Format: Maciej B. Maniecki, Anders Etzerodt, William E. Damsky Jr., Aisha Rafique, David Gonzalez, Lars Dyrskjøt, Holger J. Møller, Marcus W. Bosenberg, Søren K. Moestrup. CD163-expressing macrophages constitute a distinct malignancy-promoting phenotype in melanoma facilitating tumor development, progression, and metastasis: Implications for novel anticancer therapy. [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 1534. doi:10.1158/1538-7445.AM2013-1534
Abstract 1536: Neurropilin-1 high monocytes play an important role in trastuzumab mediated antibody-dependent cellular cytotoxicity in HER2 positive breast cancer cell lines.
Authors: Kawaguchi Kosuke , Suzuki Eiji , Sugie Tomoharu , Sato Fumiaki , Toi Masakazu
Published: August 2014

Purpose

Neuropilin-1 (NRP-1) was initially characterized as a neuronal receptor for specific secreted members of the semaphorin family involved in axon repulsion. However, it was recently reported to play an important role in the immune system. This study aimed to investigate the role of NRP-1 expression on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cell line SKBR3.

Methods

Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were prepared using Lymphoprep. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD14 and allophycocyanin (APC)-conjugated anti-NRP-1 antibodies, and an NRP-1high and NRP-1low monocyte subset was sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay using SKBR3 as target cells and trastuzumab as an antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. A CD107a degranulation assay was also used to determine the role of NRP-1 expression on monocytes in ADCC. PBMCs were co-cultured with target SKBR3 cells and trastuzumab for 2 h, stained with APC-conjugated anti-NRP-1 and PE-Cy5-conjugated anti-CD107a antibodies, and analyzed by flow cytometry.

Results

The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted NRP-1 high monocytes (12%) than in NRP-1 low monocytes (4%). Interestingly, microscopic findings showed that most NRP-1 high monocytes covered trastuzumab-treated target cancer cells (showing higher cellular cytotoxicity) while NRP-1 low monocytes did not cover the target cells but changed their morphology from a round to a dendritic shape (showing less cellular cytotoxicity) during ADCC. Moreover, the degranulation assay showed a higher expression of CD107a in NRP-1 high monocytes (17.4%) than in NRP-1 low monocytes (1.16%). Mean fluorescence intensity of CD107a in NRP-1 high and NRP-1 low monocytes was 92.6 and 26.3, respectively.

Conclusions

These data suggest that NRP-1 high monocytes exhibit higher trastuzumab-mediated ADCC than NRP-1 low monocytes in HER2-positive breast cancer cell lines. It may therefore be possible to differentiate HER2-positive breast cancer patients for whom trastuzumab will be less effective by testing for monocyte NRP-1 expression levels.

Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Tomoharu Sugie, Fumiaki Sato, Masakazu Toi. Neurropilin-1 high monocytes play an important role in trastuzumab mediated antibody-dependent cellular cytotoxicity in HER2 positive breast cancer cell lines. [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 1536. doi:10.1158/1538-7445.AM2013-1536
Abstract 1537: Characterization of tumor-infiltrating macrophages in humanized mouse model of prostate cancer.
Authors: Ryabov Vladimir , Kim David , Saito Rikka , Alexander Richard B. , Klyushnenkova Elena
Published: August 2014

Macrophage infiltration is observed in many types of human and mouse malignant tumors. Though some studies reported anti-tumor activity of tumor-associated macrophages (TAM) the majority of reports revealed contribution of TAM to tumor growth, invasion and metastasis. The phenotype and functions of TAM in prostate cancer are not well known. However, their abundance in the tumor suggests an important role in the course of malignant progression. In our novel humanized mouse model of prostate cancer, the expression of human HLA-DR*1501 (DR2b) transgene drastically impairs immune response to prostate tumor and induces excessive accumulation of TAM, which predominate in tumor tissue. In parallel, substantial accumulation of CD4+CD25+FOXP3+ T cells is observed in the tumor mass. The initial gene expression profiling of TAM in DR2b+ mice revealed their mixed M1/M2 phenotype since these cells expressed typical M2-associated genes TGFβ, IL10, arginase I, stabilin-1 and M1-associated genes IL1β, TNFα and IL6. Moreover, TAM strongly expressed chemokines CCL2, CCL3, CCL4, CCL5 and CXCL10, which are capable to recruit myeloid, NK and T cells as well as immune checkpoint receptor PDL-1. Interestingly, compared to DR2b- mice, TAM from DR2b+ animals had reduced expression of important NK and T cell survival factor IL15. Immunohistochemical investigation of the tumor tissue revealed that CD68+ TAM co-localized with T cells on the periphery of the tumor and expressed M2-associated marker stabilin-1. Moreover, T cells were found exclusively on the tumor periphery, whereas TAM were observed inside tumor mass. Altogether, present data suggest that TAM in DR2b model of prostate cancer have potential to recruit various immune cells to the tumor mass. However, elevated expression of immunosuppressive factors TGFβ, IL10, arginase I, PDL-1 and close association between TAM and T cells on tumor periphery may impair T cell activation and induce generation of Tregs.

Citation Format: Vladimir Ryabov, David Kim, Rikka Saito, Richard B. Alexander, Elena Klyushnenkova. Characterization of tumor-infiltrating macrophages in humanized mouse model of prostate 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 1537. doi:10.1158/1538-7445.AM2013-1537
Abstract 1540: TNFRs shed by tumor cells inhibit the migration of macrophages.
Authors: Rego Stephen , Helms Rachel , De Piante Alexander , Kidiyoor Amritha , Lance Amanda , Mukherjee Pinku , Dréau Didier
Published: August 2014

Breast tumor cells transform their microenvironment through the expression of pro-tumor molecules critical to their progression and metastasis. Among the targets of tumor cell secretions are macrophages, which play an essential role in tumor progression. Although macrophages possess tumor cell killing abilities, the tumor-associated macrophages (TAMs) display primarily pro-tumor functions including increasing tumor angiogenesis and promoting tumor cell invasion. Breast tumor cells shed multiple membrane-bound proteins including their tumor necrosis factor (TNF) receptors through TACE/ADAM17 activities. The release of sTNFR1/2 serves a decoy sequestering TNF and preventing TNF-driven apoptosis of tumor cells. Additionally, tumor derived sTNFR1/2 also modulate TNF effects on macrophages including their migration. Here, we investigated whether sTNFRs were shed by cells present within the breast tumor microenvironment and determined their effects on macrophage migration in response to TNF. TNF and sTNFRs concentrations were measured in murine mammary tumor and normal cells including monocytes/macrophages. In addition, the migration of murine monocytes toward TNF in the presence tumor derived soluble factors (TDSFs) was determined by transwell assays. The effects of TDSFs obtained with and without the sheddase inhibitor TAPI-0 treatment on monocyte/macrophage migration were also assessed. No TNF was detected in the TDSFs of tumor cells or secretions from normal epithelial cells. In contrast, stroma cells especially macrophages expressed high levels of TNF (p<0.001). sTNFR2 concentrations were variable in TDSFs and significantly decreased in TDSFs obtained following TAPI-0 treatment (p<0.05). Macrophages migrated toward TNF in a dose-dependent manner (p<0.05). While the addition of TDSFs prevented the macrophage migration toward TNF, the use of TDSFs collected following TAPI-0 treatment had no effects of the macrophage migration toward TNF (p<0.05). These results support the modulatory role of sTNFRs secreted by tumor cells in macrophage responses to TNF.

[This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)].

Citation Format: Stephen Rego, Rachel Helms, Alexander De Piante, Amritha Kidiyoor, Amanda Lance, Pinku Mukherjee, Didier Dréau. TNFRs shed by tumor cells inhibit the migration of macrophages. [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 1540. doi:10.1158/1538-7445.AM2013-1540
Abstract 1541: M2 macrophages co-cultured with prostate cancer cells promote osteoblast differentiation mediated by BMP-2 and BMP-7.
Authors: Wang Lihui , Huang Xin , Wang Jianhua , Lu Yi
Published: August 2014

Prostate cancers frequently metastasize to bone and cause predominantly osteoblastic lesions. When tumor cells metastasize to bone, they interact with the bone microenvironment. Bidirectional interactions between prostate cancer cells and bone microenvironment increase expression of cytokines, chemokines and adhesion molecules. These factors can be produced by tumor cells as well as bone marrow cells including osteoclasts, osteoblasts/stromal cells and macrophages. It has been demonstrated that M2 macropahge enhanced tumor growth, survival and metastasis. However, whether M2 macropahges play roles in prostate cancer-induced osteoblastic lesions, specifically in osteoblast differentiation is unknown. We initially isolated M2 macrophages from primary murine bone marrow cells culturing with IL-4 and IL-13. Then M2 macrophages were co-cultured with or without prostate cancer C4-2B or LNCaP cells at ratio 5:1. Then the conditioned media (CM) from M2 co-cultures were collected. We incubated murine bone marrow cells or MC3T3 E1 cells with different concentrations of the CM and performed Von Kossa Staining. We found that the co-cultured CM, but not the CM collected from either macrophages or prostate cancer cells, significantly induced bone marrow cells or MC3T3 E1 mineralization in vitro. This induction was partially blocked by anti-BMP-2 and anti-BMP-7 neutralizing antibodies. We further observed that the co-cultured CM significantly induced alkaline phosphatase (ALP) and osteocalcin (OCN) production. Finally, we determined the cytokine productions from the co-cultured CM by using ELISA. The production of BMP-2 and BMP-7 were detected. The conditioned media collected from M1 macrophage were used as corresponding and negative controls throughout the whole study. We concluded that M2 macrophages co-cultured with prostate cancer cells promote osteoblast differentiation mediated by BMP-2 and BMP-7. Supported by NSF projects 81171993 and 81272415; NSF key project 81130046; 973 Grant (2009CB918902); Guangxi projects 201201ZD004 and 2012GXNSFCB53004.

Citation Format: Lihui Wang, Xin Huang, Jianhua Wang, Yi Lu. M2 macrophages co-cultured with prostate cancer cells promote osteoblast differentiation mediated by BMP-2 and BMP-7. [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 1541. doi:10.1158/1538-7445.AM2013-1541
Abstract 1549: Diet-induced obesity increases tumor levels of MCP-1, macrophage infiltration and microvessel density in a murine model of breast cancer.
Authors: Ngan Emily , Pacilli Alessandra , Marshall Eva , Cutlip Kirstie , Coad James , Ammer Amanda , Vona-Davis Linda
Published: August 2014

Epidemiological studies indicate that obesity is associated with poor outcomes in breast cancer. In our previous work, we demonstrated that a chronic consumption of a high fat diet increases solid tumor growth and metastasis in a spontaneous mammary cancer mouse model. In addition to higher tumor burdens and rates of metastases, mice fed a high fat diet showed elevated levels of adipocyte derived monocyte chemoattractant protein-1 (MCP-1) in the blood. We hypothesized that diet-induced obesity promotes mammary carcinogenesis by inducing an inflammatory and tumor-supportive microenvironment. We further compared chow fed lean MMTV(PyMT) mice with those fed 45.0% (w/w) high fat (HF) to investigate the impact of body adiposity and dietary nutrient overload on levels of MCP-1 in tissue, tumor associated macrophages (TAMs) and for the degree of microvessel density. We analyzed early and late carcinoma stage tumors together with adipose tissue from mice fed HF and control diets in order to identify possible sources of MCP-1 production. MCP-1 levels in tumors of mice differed significantly by cancer stage and diet, with late stage tumors showing more MCP-1 (P<0.05). Moreover, MCP-1 production was elevated in adipose tissue of obese mice compared to lean mice. Tumors and adjacent adipose were stained with CD68 and the numbers of TAMs were quantified. Significantly more TAMs (P<0.05) were found within tumor beds and more crown-like structures were evident in adipose tissue from obese mice at both early and late stage carcinoma. Tumor beds also showed increased microvessel density when stained with CD31. In summary, diet-induced obesity increased levels of MCP-1 in tumors and adipose with more macrophage infiltration and greater vascular density evident in mammary tumor beds. Taken together, these findings suggest that high body adiposity caused by diet-induced changes promote an inflammatory and tumor-supportive microenvironment in breast cancer. NIH P20 RR016440, P30 R032138/GM103488, and P20 RR016477

Citation Format: Emily Ngan, Alessandra Pacilli, Eva Marshall, Kirstie Cutlip, James Coad, Amanda Ammer, Linda Vona-Davis. Diet-induced obesity increases tumor levels of MCP-1, macrophage infiltration and microvessel density in a murine model of 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 1549. doi:10.1158/1538-7445.AM2013-1549
Abstract 1551: Irradiation enhances tumor-homing ability of monocytes as cellular carrier.
Authors: Yu Ching-Fang , Woo Christopher W. , Lo Shao-Hua , Yen Chia-Yi , Hong Ji-Hong , Chiang Chi-Shiun
Published: August 2014

Tumor-homing ability of monocytes renders them as potential cellular delivery system for alternative cancer therapy, but the migration ability is reduced after reagents are up-taken. Approaches that can enhance monocyte tumor homing and migration abilities will facilitate the clinical value of using monocytes as cellular carrier. Previous studies have shown that irradiation (IR) could promote macrophage aggregation into the hypoxic regions. To investigate whether IR could enhance the infiltration of bone marrow-derived monocytes (BMDMs) into tumors, the infiltration of BMDMs prepared from GFP-transgenic mice were examined by fluorescence microscopy in murine prostate adenocarinoma TRAMP-C1 model. The results showed that IR did not increase the initial infiltration number of BMDMs, but enhanced the retention of monocytes within IR-treated tumors up to 2 weeks. This study also showed that BMDMs were able to uptake various imaging or therapeutic agents, but their mobility was decreased as the load was increased. However, when BMDMs were differentiated in IR-treated tumor condition medium (IR-CM), the nanoparticles-loaded migration loss was recovered in both in vitro and in vivo models. The prolong retention of monocytes within irradiated tumor tissues and the ability of IR-CM to enhance the migration ability of cargo-loaded BMDMs suggest that pre-conditioned monocytes could be a potential cellular carrier for target therapy following conventional RT. (This study is supported by NHRI-EX101-10132BI and NSC101-2627-M-007-001 grants)

Citation Format: Ching-Fang Yu, Christopher W. Woo, Shao-Hua Lo, Chia-Yi Yen, Ji-Hong Hong, Chi-Shiun Chiang. Irradiation enhances tumor-homing ability of monocytes as cellular carrier. [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 1551. doi:10.1158/1538-7445.AM2013-1551
Abstract 1555: Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance.
Authors: Gary Joy , Xu Jinfei , Simmons John , Kovalchuk Alexander , Dubois Wendy , Testa Joseph , Mock Beverly
Published: August 2014

The AKT/PI3K/mTOR signaling pathway is hyperactivated in cancers, including both solid tumors and hematologic malignancies. This hyperactivation is often due to an overexpression or mutation in PI3K, which is upstream of AKT, or a down regulation of PTEN, an inhibitor of the PI3K pathway. mTOR is a central downstream target of this pathway and is a critical modulator of cell cycle, size, proliferation and metabolism. Resistance to mTOR inhibition has been reported in many tumors as well as in certain cell lines, resulting in decreased efficacy of cancer therapy involving this class of inhibitor. In these studies, mice (Mtortm1.1Lgm) with constitutive reduction in mTOR protein level were crossed to mice (Lck-MyrAkt2) that spontaneously develop thymic lymphomas due to constitutive AKT activation to simulate mTOR inhibitor treatment of thymic lymphoma development. The Lck-MyrAkt2 mice spontaneously develop thymic lymphomas at 10-20 weeks of age, and frequently harbor a 14;15 translocation that leads to the apposition of the TCRalpha and Myc genes. Tumor formation was significantly delayed in the mTOR knockdown x Lck-MyrAkt2 cohort, and the mice had a median survival of 181 days compared to 88 days in mice with normal mTOR expression. A similar t(14;15) translocation frequency was observed in thymic lymphomas of mice from both groups. To evaluate potential mTOR resistance mechanisms, gene expression profiling (GEP) of tumors arising from both groups of mice was performed. Molecular and functional enrichment analysis of the genes differentially expressed in the tumors arising despite mTOR inhibition was performed. Increased expression of an oncogenic transcription factor was observed, and is the subject of on-going investigation.

Citation Format: Joy Gary, Jinfei Xu, John Simmons, Alexander Kovalchuk, Wendy Dubois, Joseph Testa, Beverly Mock. Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance. [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 1555. doi:10.1158/1538-7445.AM2013-1555
Abstract 1561: An evaluation of the use of genetically modified mouse models in human cancer risk assessment.
Authors: Eastmond David A. , Vulimiri Suryanarayana V. , French John E. , Sonawane Babasaheb
Published: August 2014

The development of short-term or accelerated cancer bioassays using genetically modified (GM) animals that exhibit high sensitivity to chemically induced cancers and that act through mode(s) of action relevant to humans continues to be an important focus of research. However, to date only a few GM mouse models such as the Trp53+/−, the Tg.AC and the rasH2 models have effectively been evaluated to substitute for the two-year rodent cancer bioassays. Whether these models provide an acceptable replacement for the conventional rodent cancer bioassay for assessing human cancer risks is the subject of ongoing debate. The objective of this study was to evaluate the current status of the use of GM mice for accelerated cancer bioassays in assessing the potential human health risks associated with exposure to carcinogenic agents. We compared the published data from the GM bioassays with results obtained using the National Toxicology Program's conventional 2-year mouse cancer bioassay presently used in human cancer risk assessment. The results demonstrate a moderate success for these GM models to distinguish carcinogens from non-carcinogens. Although the GM models are less efficient in detecting rodent carcinogens, they are more consistent in identifying non-carcinogens. Our analysis also shows that there continue to be concerns about the assay design and protocols used for the GM models, including issues related to sample size, study duration, genetic instability, and reproducibility. Additional issues of concern include specific toxicity pathway-dependent effects, and differences in the potential carcinogenic mechanisms operating in GM and non-GM animals, topics that still need to be investigated. In addition, the dose-responses of the GM models can vary substantially from those seen in conventional cancer bioassays. Overall, we conclude that the existing GM mouse models are of value for hazard identification, but are of limited use for dose-response analysis with currently used study designs. Hence, until these models are thoroughly validated using more robust study designs and the dose-response issues adequately addressed, caution should be exercised when using the current GM models to assess the carcinogenic risks of chemicals to humans.

Disclaimer: The views expressed in this abstract are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency or the National Institute of Environmental Health Sciences.

Citation Format: David A. Eastmond, Suryanarayana V. Vulimiri, John E. French, Babasaheb Sonawane. An evaluation of the use of genetically modified mouse models in human cancer risk assessment. [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 1561. doi:10.1158/1538-7445.AM2013-1561
Abstract 1562: Choosing the right preclinical model for cancer immunotherapy: The “SyngenOmic” toolbox..
Authors: Harper James A. , Prime John , Herath Athula , Howell Jane , Papaspyridonos Marianna , Popple Amy , Anderton Judith , Morrow Michelle , Lewis Arthur , Henley Liz , McCourt Matthew , Sainson Richard
Published: August 2014

Recent FDA approvals of ipilimumab and sipuleucel-T for the treatment of metastatic melanoma and castrate-resistant prostate cancer respectively have validated the approach to modulate the immune system for the clinical treatment of cancer. MedImmune Oncology, has an extensive pipeline and expertise in investigational Immune Mediated Therapy in Cancer (IMT-C) drugs (e.g. tremelimumab and MEDI4736), and is heavily invested in this novel era of cancer therapeutics. Pre-clinical assessment of the validity of potential IMT-C drugs can be enabled by the use of syngeneic tumours established in immuno-competent animals. MedImmune's pharmacology team has lead efforts to develop such models, to a point that a panel of 15 different validated syngeneic tumour types is available in house for studies to progress projects. The intention of selecting relevant models and of minimizing animal experimentation, reduces the number of models tested for each project, and allows for detailed genetic and proteomic characterization of treatment dependent effects within our models. Utilising both in vitro and in vivo approaches, we have generated transcriptomic and genomic data for our cell lines, the resulting implanted tumours and for the relevant lymphatic organs (draining lymph node and spleen). Using key proteomic/cell markers through IHC and FACS analysis, we are also evaluating the immune profile/status of our tumour bearing animals in parallel. Finally, in order to establish the disease relevance and prognostic value of our models we are analyzing our cell panel for the presence of somatic mutations in 50 genes previously implicated in human cancers. Once completed, the resulting dataset will hopefully help pre-clinical scientists to refine their in vivo plans and deliver more clinically-relevant pharmacology packages for the progression of cancer immunotherapy drug candidates.

Citation Format: James A. Harper, John Prime, Athula Herath, Jane Howell, Marianna Papaspyridonos, Amy Popple, Judith Anderton, Michelle Morrow, Arthur Lewis, Liz Henley, Matthew McCourt, Richard Sainson. Choosing the right preclinical model for cancer immunotherapy: The “SyngenOmic” toolbox. [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 1562. doi:10.1158/1538-7445.AM2013-1562
Abstract 1565: Significance of elemental content and distribution profiles in assessing established cell lines as surrogates for cancer tissues from two different organs.
Authors: Farah Ibrahim O. , Arslan Zikri , Ayensu Wellington K.
Published: August 2014

Studies have shown that defective metal homeostasis is very essential to the development of the cancer phenotype in many cancers including lung and liver carcinomas. Low Zn levels as well as overexpression of metalothioneins were implicated in the development and progression of various cancers. Wider elemental profiles that relate cancer and normal phenotypes with regards to metal homeostasis were not well elucidated in the literature. To this end, established cell lines are currently used as accepted models for studying cancer but the level of their representation of actual cancer tissues was not clear. This study is attempting to assess the relevance of cell lines currently is use as surrogates for cancer and establish their relationship to actual normal and cancer tissues from humans. We hypothesize that elemental content and distribution profiles in cancer and normal tissue are significantly different from those possessed by established cell lines. We used Inductivity Coupled Plasma (ICP-Mass Spectroscopy) for lung and liver samples obtained from normal and cancer human cadaver tissues (US Biomax) and cell models of lung and liver carcinomas; the A549 and HepG2 cell lines (ATCC), to analyze for elements and test the hypothesis. Samples prepared using standardized digestion procedures were loaded onto the ICP-MS equipment to test for a profile consisting of 12 elements, namely, Zn, Cu, Fe, Mg, Mn, Ca, Al, Co, Ba, Cr, Ni and Se. Analysis of data showed significant variations in elemental content and distribution profiles between the cell models and actual tissues consistent with the hypothesis. It is concluded that elemental homeostasis is essential for normal tissue function and that shifts in their distribution and content are essential in determining the use of cell models as surrogates for studying cancer. These results are promising and warrant further studies to confirm the relevance of cell line models in relation to their use as screening tools for examining targeted cancer therapeutics.

Citation Format: Ibrahim O. Farah, Zikri Arslan, Wellington K. Ayensu. Significance of elemental content and distribution profiles in assessing established cell lines as surrogates for cancer tissues from two different organs. [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 1565. doi:10.1158/1538-7445.AM2013-1565
Abstract 1570: Novel strategy for eradicating lymph node metastasis of early-stage colorectal cancers using telomerase-dependent replicating adenoviral agent.
Authors: Kikuchi Satoru , Kishimoto Hiroyuki , Tazawa Hiroshi , Hashimoto Yuuri , Uno Futoshi , Nishizaki Masahiko , Kagawa Shunsuke , Urata Yasuo , Hoffman Robert M. , Fujiwara Toshiyoshi
Published: August 2014

One of the biggest challenges in endoscopic therapeutic approach for early colorectal cancer with submucosal invasion is how to treat regional lymph nodes (LNs) possibly metastasized. The rate of LN metastasis in submucosal invasive colorectal cancer are estimated approximately 10%. Since endoscopic treatment can't clean LNs outside the bowel physically, open or laparoscopic surgical treatment is still gold standard for submucosal invasive colorectal cancer. We previously reported that in an orthotopic rectal tumor mouse model, a telomerase-dependent tumor killing adenoviral agent (OBP-301) spreads to the regional LNs, selectively replicates in cancer cells in metastasized LNs, and inhibits LN metastasis, when administered into the primary advanced rectal tumor. In this study we evaluated the effectiveness of novel less invasive treatment against submucosal invasive colorectal cancer, using local tumor resection such as endoscopic submucosal dissection (ESD) and OBP-301. HCT-116-GFP human colon cancer cells were orthotopically implanted into the submucosal layer of nude mouse rectum with resultant submucosal invasive early rectal tumor. LN metastasis was observed in 78.5% of mice as early as 7 days after cancer cell inoculation, and the rectal tumor of mice with LN metastasis got locally excised with prior peritumoral injection of OBP-301 solution, mimicking ESD technique. Seven days after tumor resection, treatment of OBP-301 showed a significant inhibition in LN metastasis status compared to control group. Moreover, LN metastatic recurrence was not detected in OBP-301 treated group even 4 weeks after tumor resection. The combination of ESD and OBP-301 has promising potential to take place of the conventional surgical approach in treating submucosal invasive colorectal cancer.

Citation Format: Satoru Kikuchi, Hiroyuki Kishimoto, Hiroshi Tazawa, Yuuri Hashimoto, Futoshi Uno, Masahiko Nishizaki, Shunsuke Kagawa, Yasuo Urata, Robert M. Hoffman, Toshiyoshi Fujiwara. Novel strategy for eradicating lymph node metastasis of early-stage colorectal cancers using telomerase-dependent replicating adenoviral agent. [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 1570. doi:10.1158/1538-7445.AM2013-1570
Abstract 1571: Tumor cell panels: New tools in genomic era.
Authors: White Chris , Volpe Lysa-Anne , Chen Luping , Bal Anupreet , Jackson Michael , Foulke John , Tian Fang
Published: August 2014

Increased understanding of cancer genome is affecting every corner of cancer research. Although human tumor cell lines have been used as essential tools for decades, there are only a few cell line panels have been developed for drug screening. The most widely used NCI60 human tumor cell line panel was composed by selecting cell lines to represent nine subtypes of cancer. The rising of targeted therapeutics has led to new generation anticancer agents. However, it's difficult to predict clinical response to these agents because of the genetic diversity and genomic heterogeneity in human cancers. Therefore, there is a gap between the new knowledge of cancer genome and the cell line based platforms for both basic and translational research.

Here, we show that new generation tumor cell panels are filling the gap. The panels were generated by selecting authenticated cell lines derived from variant cancer types, and annotated with genetic alteration information generated by large scale sequencing projects such as the Catalog of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE). There are more than 25 tumor cell line panels, which include hundreds of cell lines, have been developed in this initiative. They cover 18 most common cancer types, such as lung, breast, colon, pancreatic cancer and so on. To capture the genetic diversity of cancer, each panel includes cell lines with varying gene mutation complexity. To further facilitate targeted drug discovery, the molecular signature tumor cell line panels focus on individual driver genes, critical protein kinases, transcription factors and cell signaling pathways. Those panels have been analyzed to verify gene mutation, gene expression, protein expression and bio-functions.

Therefore, the new generation tumor cell panels provide powerful tools to accelerate the discoveries in basic cancer research, compound screening, biomarker selection, pathway analysis, and targeted therapeutic development.

Citation Format: Chris White, Lysa-Anne Volpe, Luping Chen, Anupreet Bal, Michael Jackson, John Foulke, Fang Tian. Tumor cell panels: New tools in genomic era. [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 1571. doi:10.1158/1538-7445.AM2013-1571
Abstract 1572: Influence of apc and med12 mutations on embryonic liver development in zebrafish: a potential model for investigating the role of Mediator in Wnt/β-catenin-dependent cancers.
Authors: Lescott Chamille J. , Glasgow Eric
Published: August 2014

Mutations in adenomatus polyposis coli (APC) result in overactive Wnt signaling and have been linked to colon cancer and hepatoblastoma in humans. Therefore, the Wnt pathway is an attractive target for developing cancer therapeutics. APC directs β-catenin degradation; thus, APC loss results in β-catenin accumulation and an increase in β-catenin-induced transcription. Heterozygous apc mutant zebrafish present enlarged livers at 3 days post-fertilization, and as adults they are predisposed to tumor development. Recently, the Mediator co-activator complex component, Med12, was linked to Wnt signaling. Without med12, canonical Wnt/β-catenin signaling is significantly impaired. We hypothesized that heterozygous med12 mutants will have smaller livers due to a decrease in Wnt activity. Moreover, a reduction in Wnt/β-catenin signaling due to lower levels of Med12 in double heterozygous med12;apc mutant embryos will be sufficient to rescue the large liver phenotype observed in heterozygous apc mutant embryos. In situ hybridization with lfabp probe confirmed the increased liver size in apchu745/+ embryos and showed smaller livers in med12y82/+ embryos. Importantly, the med12y82/+ mutation rescued the large liver phenotype of apchu745/+ embryos. The data suggest that med12 and apc genetically interact to regulate liver development. This interaction between med12 and apc is also likely to also play a role in the development and progression of Wnt/β-catenin-dependent cancers. Therefore, rescue of liver hyperplasia in apc mutants may serve as a model for developing anti-cancer drugs targeting WNT/β-catenin signal transduction through its interaction with MED12.

Citation Format: Chamille J. Lescott, Eric Glasgow. Influence of apc and med12 mutations on embryonic liver development in zebrafish: a potential model for investigating the role of Mediator in Wnt/β-catenin-dependent cancers. [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 1572. doi:10.1158/1538-7445.AM2013-1572

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1574: The Hippo pathway regulates liver development and tumorigenesis in zebrafish.
Authors: Cox Andrew G. , Beltz Sebastian , Evason Kimberley , Stainier Didier Y. , North Trista E. , Goessling Wolfram
Published: August 2014

The Hippo pathway is a novel signaling cascade that regulates cell proliferation, organ size and tumorigenesis in mammals. In mice, deregulation of the Hippo pathway in the liver has been shown to potently induce tumor formation. However, the molecular mechanism by which the Hippo pathway regulates hepatic growth is not well understood. In this study, we have examined liver development in NF2-/- mutant zebrafish, which exhibit systemic activation of Yap, and found that liver size was increased by 5 days post fertilization (dpf). Histological evaluation of the NF2-/- mutant zebrafish embryos revealed that the enlarged liver was composed of an extended biliary network. In order to examine the cell-autonomous effects of Yap activation in hepatocytes, we generated Tg(fabp10a:YapS87A) fish and found that liver size increased dramatically by 5 dpf. In contrast to the NF2-/- mutant, histological and FACS analysis revealed that the enlarged liver in the Tg(fabp10a:YapS87A) embryos was composed of a greater number of hepatocytes. Chemical exposure to the γ-secretase inhibitor DAPT or erlotinib rescued the hepatomegaly phenotype in 5 dpf Tg(fabp10a:YapS87A) embryos, indicating dependence on Notch and EGFR signaling pathways. To gain greater insight into how these developmental studies relate to tumorigenesis, we compared NF2+/- mutants and Tg(fabp10a:YapS87A) adults and found that both lines were predisposed to liver tumor formation, developing predominantly intrahepatic cholangiocarcinomas. Together, these studies reveal an evolutionarily conserved role of the Hippo pathway at the interface between liver development and tumorigenesis in zebrafish.

Citation Format: Andrew G. Cox, Sebastian Beltz, Kimberley Evason, Didier Y. Stainier, Trista E. North, Wolfram Goessling. The Hippo pathway regulates liver development and tumorigenesis in zebrafish. [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 1574. doi:10.1158/1538-7445.AM2013-1574
Abstract 1575: Zebrafish embryos as a model for studying the role of estrogen receptor β in cancer metastasis.
Authors: Hao Ruixin , Thomas Christoforos , Dey Prasenjit , Ström Anders , McCollum Catherine W. , Nikolos Fotis , Bondesson Maria , Gustafsson Jan-Ake
Published: August 2014

Estrogen receptor β (ERβ) has been reported to impede cancer metastasis while estrogen receptor α (ERα) can promote cancer metastasis. Several splice variants of ERβ exist in humans and they may have different functions in different tissues, but the role of these differently spliced isoforms in cancer metastasis has not been well studied. Various model systems such as xenografting of cancer cells in mice and dogs have been used to study cancer metastasis. However, difficulties in monitoring living cancer cell metastasis in large non-transparent animals have been a limitation in these studies. Zebrafish embryos are transparent and their immune system is not fully developed until several days after post fertilization. These features make zebrafish a useful model for xenografting cancer cells and monitoring their migration. In addition, the large number of offspring and small size of the embryos make the zebrafish model amendable for high throughput screening and anti-metastasis drug discovery. In our studies, we co-microinjected breast cancer cell lines bearing ERβ constructs or control constructs, expressing fluorescent proteins of different colors, into zebrafish embryos at 48 hours post fertilization (hpf). After 2 hours recovery, fish embryos with cells circulating in the blood system were removed. Embryos that contained cells only in the original injection sites were incubated at 32°C to monitor the cancer cell migration daily using a fluorescence microscope. We found that ERβ expressing breast cancer cells were less migratory than the control cells and they remained at the original injection site to a large extent. We also examined the metastasis of prostate cancer cell lines expressing ERβ splice variants in our zebrafish xenograft model and found that cells with different ERβ splice variants exhibited different migration patterns. In conclusion, zebrafish embryos work as an effective model for studying the role of ERβ and its splice variants for cancer metastasis of diverse cancer cell lines.

Citation Format: Ruixin Hao, Christoforos Thomas, Prasenjit Dey, Anders Ström, Catherine W. McCollum, Fotis Nikolos, Maria Bondesson, Jan-Ake Gustafsson. Zebrafish embryos as a model for studying the role of estrogen receptor β in cancer 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 1575. doi:10.1158/1538-7445.AM2013-1575
Abstract 1578: Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype.
Authors: Metelo Ana , Xiang Li , Baker Rania , Kamentsky Lee , Carpenter Anne , Peterson Randall
Published: August 2014

Hypoxia Inducible Factor (HIF) target genes are involved in several cellular processes including angiogenesis, erythropoiesis, metabolism and stem cell proliferation. The von Hippel-Lindau (VHL) tumor suppressor protein targets HIF for degradation in conditions of normal oxygen tension. Loss-of-VHL leads to constitutive expression of HIF. We have discovered small molecules that repress HIF-2α translation by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5’-UTR of HIF-2α mRNA and showed that these small molecules specifically down-regulate HIF-2α expression in vitro.

To validate the HIF-2α inhibitors in vivo, first we tested their efficacy to inhibit acute hif induction in wild-type (wt) zebrafish embryos challenged with the hypoxia mimetic DMOG. Treatment of wt embryos with 100μM of DMOG up-regulated the expression of zebrafish HIF-target genes, such as vascular endothelial growth factor (vegf), erythropoietin (epo) and prolyl-hydroxylase 3 (phd3). Pre-treatment of embryos with the small molecule HIF-2α inhibitor 76 (10nM) significantly decreased the DMOG-induced up-regulation of HIF target genes and resulted in decrease of DMOG-induced erythrocytosis and blood vessel sprouting.

Furthermore, we tested the efficacy of this HIF-2α inhibitor in a vhl zebrafish model (homozygougous mutants harbouring inactivating vhl mutations), which recapitulates aspects of human VHL disease (erythrocytosis, abnormal vascular proliferation in sites such as retina and brain, early lethality). Treatment with inhibitor 76 (10nM, 100nM and 1μM) down regulated the expression of the HIF-target genes phd3, epo and vegf in a dose response manner and rescued the erythrocytosis and sprouting phenotype of vhl embryos. To image and quantify the blood vessel abnormalities of vhl embryos, we generated a Fli-GFP::vhl+/- line and showed that compound 76 improves the abnormal connections between the intersegmental vessels of the Fli-GFP::vhl embryo tail, as well as their human hemangioblastoma-resembling brain vessel proliferation. Finally, we showed that treatment of vhl embryos with compound 76 rescues early lethality.

Our data demonstrate that HIF-2α inhibitor 76 is active in vivo at low concentrations and encourage further development and preclinical testing of this HIF-2α inhibitor for treatment of VHL-associated lesions, such as renal cell carcinoma, hemangioblastoma and neuroendocrine tumors.

Citation Format: Ana Metelo, Li Xiang, Rania Baker, Lee Kamentsky, Anne Carpenter, Randall Peterson. Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype. [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 1578. doi:10.1158/1538-7445.AM2013-1578