bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 32: The role of Lrig1 signaling in mammary gland development and tumorigenesis
Authors: Simion Catalina , Chen Qian J. , Wilkerson Charles L. , Rafidi Hanine , Borowsky Alexander D. , Sweeney Colleen
Published: August 2015

Lrig1 is a transmembrane leucine-rich repeat protein and a negative regulator of oncogenic receptor tyrosine kinases (RTKs) such as the ErbB family members. The Sweeney lab recently demonstrated that under-expression of Lrig1 predicts poor prognosis in breast cancer. Breast cancer patients with high Lrig1 expression show significantly longer relapse-free survival, identifying Lrig1 as a new prognostic marker. Recently, Lrig1 was found to be a tumor suppressor in intestinal tissue. It was shown that Lrig1 null mice develop ErbB receptor-dependent duodenal adenomas. Based on Lrig1's function as a negative regulator of RTKs and its reported tumor suppressor activity in intestinal epithelium, we hypothesized that Lrig1 null mice would be susceptible to mammary tumorigenesis. To characterize the function of Lrig1 in the mammary gland, we compared mammary development in Lrig1 wild type (+/+) and null (-/-) mice at regular intervals from 4 to 12 weeks. Mice were screened for evidence of hyperplasia, pre-malignant lesions or tumors by whole mount analysis across age and the intensity of specific proteins was surveyed by western blot. Organotypic growth of the mammary epithelial cells from wild type and null mice was examined in three-dimensional cell culture to examine whether any phenotypes are inherent to the epithelium. Since Lrig1 has been defined as a stem cell marker in the intestine, its role in stem cell maintenance was determined by examining the impact of Lrig1 loss on mammary stem cells. Although these studies are ongoing, early results indicate an increase in the amount of Lrig1-target proteins by western blot in the Lrig1-/- mice as opposed to the Lrig1+/+ mice mammary gland tissue. Epithelium from Lrig1-/- mice present an exaggerated branching response when stimulated with TGFα in comparison to Lrig1+/+ epithelium. Our studies also indicate that Lrig1-/- mammary glands are enriched in stem cells when compared to Lrig1+/+ mammary glands. In addition, a number of proliferative lesions were encountered in the mammary gland whole mounts of the Lrig1-/- mice, but not that of the Lrig1+/+ mice, which strongly suggests that Lrig1 plays a growth suppressor role in the mammary gland.

Citation Format: Catalina Simion, Qian J. Chen, Charles L. Wilkerson, Hanine Rafidi, Alexander D. Borowsky, Colleen Sweeney. The role of Lrig1 signaling in mammary gland development and tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2015-32
Abstract 3204: Tissue engineered models of metastatic bone disease for the study of prostate cancer cell dormancy
Authors: Liao Yanwan , Tan Jun Jie Aaron , Tamilselvan Sahaana , Chan Jerry , Ge Ruowen , Lee Lui Shiong , Chong Mark
Published: August 2015

Cellular dormancy refers to a state of quiescence during metastatic progression, in which disseminated tumor cells enter cell cycle arrest. Such dormant cells are thought to influence cancer drug sensitivity, and strategies have hence emerged to target dormant cells for novel therapeutic approaches. Mechanisms of cellular dormancy remain poorly understood, with current evidence suggesting a major role of the metastatic microenvironment in deciding cell fate. Research in this area, however, is hampered by the lack of appropriate experimental models of cancer cell dormancy. Here, we described the use of tissue engineering methods to generate an in vitro model of bone-metastatic disease. Briefly, human fetal mesenchymal stem cell (hfMSC) and human umbilical vein endothelial cell (HUVEC) were co-cultured to respectively represent the osteo-progenitor and vascular compartments, and subsequently induced to form a vascularized bone marrow model. Metastatic prostate cancer PC3 cells were then added to simulate metastatic dissemination and the co-culture was maintained for 28 days. Reporter genes were employed to facilitate live-monitoring of HUVEC and PC3 cells in the co-culture. Using this system, we observed single cell dormancy, particularly when osteogenic induction was performed. Additionally, PC3 cells were observed to exert osteo-modulatory effects on the engineered bone microenvironment. Immunostaining for cellular markers indicated low expression of proliferation marker Ki-67 and various mesenchymal markers, indicating absence of Epithelial-Mesenchymal Transition (EMT). Dormancy was functionally assessed by retrieving the PC3 cells and subsequently replating under normal culture conditions to rescue cellular proliferation. Finally, drug sensitivity assays demonstrated reduced susceptibility of dormant cells to conventional chemotherapeutic drugs. This in vitro model of prostate cancer cell dormancy in the engineered bone marrow may thus be useful for furthering our understanding of cellular dormancy, and may further provide a useful tool for drug testing.

Citation Format: Yanwan Liao, Jun Jie Aaron Tan, Sahaana Tamilselvan, Jerry Chan, Ruowen Ge, Lui Shiong Lee, Mark Chong. Tissue engineered models of metastatic bone disease for the study of prostate cancer cell dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2015-3204
Abstract 3207: The roles of cancer-associated fibroblasts in stemness maintenance of lung cancer cells
Authors: Lin Chih-An , Ho Chao-Chi , Chen Jeremy J.W.
Published: August 2015

Lung cancer is the leading cause of death worldwide including Taiwan. A lot of therapies have been developed to improve cure rate of lung cancer, however the prognosis remains dismal. Cancer cells can acquire resistance to therapies by several known or unknown mechanisms. Microenvironment is also important for acquiring resistance to corresponding treatments of cancer cells. Cancer-associated fibroblasts (CAFs) are known to support many different aspects of tumor initiation, growth and progression by secretion of growth stimulatory, pro-survival and angiogenic factors. In our studies, we compare the differences between normal fibroblasts (NFs) and CAFs which were cultured from surgical specimens of lung cancer patients. CAFs shows increasing tumorous sphere formation ability and significantly up-regulating stem cell markers such as nanog, klf-4, Sox-2, Oct4 and c-myc of cancer cells. We also found that CAFs regulate cancer stem cells (CSCs) growth through numerous paracrine factors such as IGF-II, HGF and SDF1 and by inducing expression of the corresponding receptors in CSCs, including IGF1R, IGF2R, HGFR and CXCR4. In our studies, CAFs obtain more advanced ability compared to NFs, but the mechanisms of how NFs are educated to become CAFs are still unknown, thus we trying to clarify the mechanisms by genome and epigenome-wide high-throughput analysis, including cDNA array, DNA methylation array and miRNA array.

Citation Format: Chih-An Lin, Chao-Chi Ho, Jeremy J.W. Chen. The roles of cancer-associated fibroblasts in stemness maintenance of lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2015-3207
Abstract 3210: Level of Osteoprotegerin expression is breast cancer subtype specific
Authors: Tsang Stephanie , Renaud Ashleigh , Roseman Kim , Imaizumi Yuko , Yadav Nalini , Weichhaus Michael , Connelly Linda
Published: August 2015

Osteoprotegerin (OPG) is a regulator of bone metabolism that is also produced by breast tumor cells. There is accumulating evidence that OPG has tumor-promoting effects in breast cancer. We have recently demonstrated that OPG can promote invasion and metastasis of triple negative breast cancer cells. We are now investigating the expression of OPG across major breast cancer subtypes.

We cultured 3 breast cancer cells lines from each of the major subtypes: MCF-7, ZR-75-1 and T47-D (Estrogen Receptor positive; ER+); SKBR-3, HCC1954 and HCC202 (Human Epidermal Growth Factor Receptor 2 positive; HER2+) and MDA-MB-231, MDA-MB-436 and BT-549 (triple negative). We incubated cells for 24 hours and then measured OPG mRNA expression by real time RT-PCR and secreted OPG protein in cell culture supernatant by ELISA.

The triple negative cells that we tested expressed the highest levels of OPG mRNA and protein. A much lower level of OPG expression was observed in the HER2+ cells. Contrasting results were observed with the ER+ cells. While there was no expression from the ZR-75-1 or T47-D cells after 24 hours incubation, the MCF-7 cells produced levels of OPG similar to those observed with the triple negative breast cancer cells.

We also looked at whether OPG expression levels could be altered by incubating breast cancer cells with macrophages, a cell type found in the breast tumor microenvironment. We co-cultured cells from each subtype with THP-1 human macrophages for 8 hours and extracted mRNA from the breast cancer cells or incubated the breast cancer cells for an additional 16 hours alone to measure secreted OPG protein. We found that co-culture with macrophages significantly increased OPG mRNA and protein expression in MCF-7 (ER+) and SKBR-3 (HER2+) cells but there was no increase in the MDA-MB-231 cells (triple negative).

In conclusion, while OPG was expressed at comparatively high levels in triple negative breast cancer cells, there were generally low levels or no expression in the ER+ and HER2+ cells tested. This suggests that the tumor-promoting effects of OPG may contribute to the more aggressive phenotype in triple negative cells. However OPG expression in ER+ and HER2+ cells can be stimulated through co-culture with macrophages. Thus conditions within the breast tumor microenvironment could amplify the effects of OPG in these tumor subtypes.

Citation Format: Stephanie Tsang, Ashleigh Renaud, Kim Roseman, Yuko Imaizumi, Nalini Yadav, Michael Weichhaus, Linda Connelly. Level of Osteoprotegerin expression is breast cancer subtype specific. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3210. doi:10.1158/1538-7445.AM2015-3210
Abstract 3218: Transcriptome characterization by RNA sequencing identifies molecular and clinical subgroups in high risk neuroblastoma
Authors: Zhang Shile , Wei Jun S. , Patidar Rajesh , Song Young K. , Sindiri Sivasish , Wen Xinyu , Asgharzadeh Shahab , Seeger Robert C. , Maris John M. , Guidry Auvil Jamie M. , Gerhard Daniela S. , Khan Javed
Published: August 2015

Background/objectives: Neuroblastoma (NBL) is characterized with its heterogeneous clinical and biological behavior. Despite improvement of survival rate with multimodal chemo- and immunotherapy, high mortality and morbidity is still substantial for patients with metastatic disease. Previous DNA sequencing studies characterized the genetic basis of the disease and revealed a very low somatic mutation burden and surprisingly few recurrently somatic mutated genes comparing to adult solid tumors. In order to identify the novel molecular therapeutic target and understand the biology of the high-risk neuroblastoma, we investigated gene expression profiles of tumors from a cohort of high-risk neuroblastoma patient using RNA sequencing as an integral part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project.

Methods: We performed RNA sequencing in a cohort of 150 high risk patients (97 survival and 53 death) using polyA selected mRNA. Gene and transcript isoform expression was used to correlate to the clinical parameters for this clinically well-annotated patient cohort. Whole exome sequencing (WES) data was available for 106 of the 150 patients cohort for combined analysis of DNA and RNA sequencing to identify expressed mutation.

Results: On average, we found 12.5% of the human genome, which include 63.7% of exon, 18.0% of intron, and 4.6% of intergenic regions covered by RNA-seq reads in NB samples. Preliminary gene expression analysis showed four molecular subgroups of NB tumors with distinct outcomes (log-ranked p = 0.002). Sixty-two percent of somatic mutations identified by WES was present (>10% VAF) in the RNA transcriptome when the locus was expressed (10X). We found that some of the important pathogenic somatic mutations such as TP53 p.R243W can be only detectable by integrated analysis of the sequencing data of RNA and DNA, possibly due to low tumor purity.

Future directions: We will assess the expressed mutations, fusion genes, mRNA expression, and splicing profiles to provide clinically relevant classification and offer insight into the tumor biology especially for those without detectable oncogenic driver mutation by DNA sequencing.

Citation Format: Shile Zhang, Jun S. Wei, Rajesh Patidar, Young K. Song, Sivasish Sindiri, Xinyu Wen, Shahab Asgharzadeh, Robert C. Seeger, John M. Maris, Jamie M. Guidry Auvil, Daniela S. Gerhard, Javed Khan. Transcriptome characterization by RNA sequencing identifies molecular and clinical subgroups in high risk neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3218. doi:10.1158/1538-7445.AM2015-3218
Abstract 3221: Development of patient-derived xenograft (PDX) models for gastrointestinal stromal tumors (GIST) as a pre-clinical platform for drug development
Authors: Ricono Jill , Mullins Chelsea , Nair Praveen , Mirsaidi Cyrus , Broudy Thomas
Published: August 2015

Patients with gastrointestinal stromal tumors (GIST) continue to face a poor prognosis. GISTs are a type of soft tissue sarcoma primarily affecting adults over the age of 50. They are the most common mesenchymal tumor within the GI tract. Survival rates vary greatly depending on the location when it is first diagnosed and there has been minimal progress in improving the survival rates of metastatic GISTs. It has been difficult to accurately evaluate the effectiveness of targeted drugs to develop treatment strategies due to the rarity and complexity of GISTs. In an effort to overcome this challenge, we have established PDX models to recapitulate the original patient tumor. In this work, we selected four primary GIST samples based on mutational and clinical profile. PDX models were developed in immunocompromised mice and were further characterized by immunohistochemistry, sequencing and pharmacological efficacy. We then evaluated the efficacy of chemotherapeutic agents in these models. Our data demonstrates that these preclinical PDX models could be used in evaluation and development of novel drug candidates for GIST.

Citation Format: Jill Ricono, Chelsea Mullins, Praveen Nair, Cyrus Mirsaidi, Thomas Broudy. Development of patient-derived xenograft (PDX) models for gastrointestinal stromal tumors (GIST) as a pre-clinical platform for drug development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3221. doi:10.1158/1538-7445.AM2015-3221
Abstract 3222: Biomarker discovery through bioinformatic analysis of genomic profiles of PDX models with different responses to cancer therapies
Authors: Jiang Jingjing , Yu Tengfei , Yan Ying , Du Wei , Tan Tingting , Yang Xuqin , Gu Jiali , Hua Liang , Ye Katherine Xin , Gu Zhenyu
Published: August 2015

Patient derived xenograft (PDX) tumor models have been proved to recapitulate the complexity and heterogeneity of their corresponding human tumors by phenotypic and genomic characterization, and thus become to be widely used in recent years in preclinical setting to facilitate drug discovery, translational studies and clinical trials support. Results from increasing number of preclinical studies, especially mouse trials, with PDX models in the past several years, have also demonstrated close correlation between drug response profiles with PDX models and clinical outcomes.

GenenDesign has established over 800 PDX tumor models and derived around 100 resistance models to drugs of interest. Genomic profiling data of PDX models are acquired at hot-spot mutation, gene expression, gene copy number and RNA/Exome sequence levels. Through our in-house efforts, PDX models of different tumor types were tested with related SOCs and clinical candidates in biomarker-driven multi-drug multi-arm clinical trial settings. So far, more than 1200 data sets have been generated, including responses to targeted inhibitors against HER2, EGFR, FGFRs, c-Met/ALK, cell cycle regulators, Ras/Raf pathway, PI3K/Akt pathway, epigenetic targets, as well as chemotherapy drugs.

In this study, we use bioinformatic tools to compare the genomic profiles of NSCLC PDX models based on their response profiles to multiple chemotherapy drugs. Biomarker signatures associated with SOC treatment sensitivity or resistance are revealed from bioinformatic analysis and being tested with both new NSCLC PDX models and clinical cohorts.

The combination of genomic profiles and drug response information to multiple chemo/targeted therapies of over 800 PDX models at GenenDesign would help biomarker discovery for companion diagnosis to meet the increasing needs for precision medicine.

Citation Format: Jingjing Jiang, Tengfei Yu, Ying Yan, Wei Du, Tingting Tan, Xuqin Yang, Jiali Gu, Liang Hua, Katherine Xin Ye, Zhenyu Gu. Biomarker discovery through bioinformatic analysis of genomic profiles of PDX models with different responses to cancer therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2015-3222
Abstract 3228: Establishment and characterization of orthotopic mouse models for human uveal melanoma hepatic metastases
Authors: Ozaki Shinji , Terai Mizue , Kageyama Ken , Cheng Hanyin , Ohara Masahiro , Aplin Andrew E. , Mastranjelo Michael J. , Sato Takami
Published: August 2015

Uveal melanoma (UM) is a rare type of melanoma although it is the most common primary ocular malignant tumor in adults. Nearly half of patients with primary UM subsequently develop systemic metastasis, preferentially to the liver. Currently, there is no effective treatment for UM hepatic metastasis and the prognosis is universally poor. The major challenge in designing a treatment strategy for UM hepatic metastasis is the lack of suitable animal models. We have developed two orthotopic mouse models for human UM hepatic metastases: (1) Direct hepatic implantation model (intra-hepatic dissemination model), and (2) Splenic-implantation model (hematogenous dissemination model) and investigated the tumor genesis in the liver. A human UM cell line, established from a hepatic metastasis and NSG mice were used for development of in vivo tumor models.

In direct hepatic implantation model, a localized tumor developed in the liver in all cases and intrahepatic dissemination was subsequently seen in about half of cases. On the other hand, in splenic implantation model, multiple hepatic metastases were observed after splenic implantation. Hepatic tumors subsequently developed intra-abdominal metastasis; however, lung metastases were not seen. These findings are consistent with those observed in human UM hepatic metastases. In addition, there are broad extracellular matrix connecting the blood vessels lined with endothelium in the hepatic tumors. These structures are assumed to be so called “vasculogenic mimicry (VM)” formed by the the tumor cells themselves. Notably, we found tumor cells in the liver begin to form VM, despite a small number of them and eventually develop a broad network of VM in the tumor. Thus, VM might contribute to the microcirculation in the tumor preceding development of vessels lined with endothelium. Moreover, we have generated orange-red fluorescence protein (td-Tomato fluorescent protein) expressing cell line using the same UM cell line and developed the same mouse models. This allowed us to monitor the growth of hepatic metastasis over time by in vivo live-imaging system.

These orthotopic mouse models offer useful tools to investigate the biological behavior of human UM cells in the liver.

Citation Format: Shinji Ozaki, Mizue Terai, Ken Kageyama, Hanyin Cheng, Masahiro Ohara, Andrew E. Aplin, Michael J. Mastranjelo, Takami Sato. Establishment and characterization of orthotopic mouse models for human uveal melanoma hepatic metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2015-3228
Abstract 3229: Distinct types of cetuximab resistant KRAS mutant of colorectal cancer revealed by an integrated sensitivity assay
Authors: Tashiro Takahiro , Okuyama Hiroaki , Endo Hiroko , Kawada Kenji , Ohue Masayuki , Sakai Yoshiharu , Inoue Masahiro
Published: August 2015

Purpose: Cetuximab, an anti-EGFR antibody, is clinically used in colorectal cancer (CRC) patients. Several clinical trials revealed that KRAS mutant is resistant to cetuximab. As KRAS is one of the downstream molecules in EGFR pathway, KRAS mutants are thought to be resistant to cetuximab due to the constitutive activation of downstream signaling, although mechanism of resistance in KRAS-mutant CRC remains to be elusive. We developed a primary culture system, CTOS method, by which cancer cells are prepared and cultured from patient tumors, maintaining histological and molecular characteristics of original tumors (Kondo 2011 PNAS). CTOS method can be applied to patient tumors as well as mouse xenografts. In this study, we performed integrated sensitivity assays by in vivo/in vitro CTOS sensitivity assay, and CTOS signaling assay in order to investigate characteristics of KRAS mutant CRC cells.

Methods: Nine CTOS lines (3 KRAS-wild type, and 6 KRAS-mutant) were analyzed. For in vivo sensitivity assay, growth suppression by cetuximab was evaluated in mouse xenografts derived from CTOSs. CTOSs were classified into three groups according to the response (Regression: tumors became smaller than initial size; Intermediate; tumors became smaller than untreated control but bigger than initial size; Progression: no effect). For in vitro sensitivity assay, growth suppression by cetuximab was evaluated in CTOSs. For signaling analysis, CTOSs were treated with EGF and/or cetuximab, and phosphorylation of EGFR, AKT, and ERK were evaluated.

Results: Regression in vivo was found in the tumors originated only from KRAS-wt (2/3). KRAS-mutant CTOSs were able to be clearly divided into two groups. Type I (2 CTOSs) showed Progression in vivo, no response in vitro, and no response in signaling assay. Even the phosphorylation of EGFR was not inhibited by cetuximab treatment. On the other hand, Type II (4 COTSs) showed Intermediate in vivo, moderate response in vitro, and variable responses in signaling assay. ERK phosphorylation was inhibited by cetuximab treatment in all 4 CTOSs.

Conclusions: Using integrated in vivo and in vitro assays, KRAS mutant was able to be divided into two distinct groups according to the response to cetuximab. Resistant mechanism of KRAS mutant rather than MAPK pathway activation can be studied in this integrated assay system.

Citation Format: Takahiro Tashiro, Hiroaki Okuyama, Hiroko Endo, Kenji Kawada, Masayuki Ohue, Yoshiharu Sakai, Masahiro Inoue. Distinct types of cetuximab resistant KRAS mutant of colorectal cancer revealed by an integrated sensitivity assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3229. doi:10.1158/1538-7445.AM2015-3229
Abstract 3231: An orthotopic xenograft model of sarcoma metastasis demonstrates essential role of tumor microenvironment for metastasis
Authors: Goldstein Seth D. , Hayashi Masanori , Albert Catherine M. , Jackson Kyle W. , Loeb David M.
Published: August 2015

Sarcoma metastasis is a multi-step process that includes migration, intravasation into the circulation, extravasation and invasion to distant tissue, and distant tumor growth, and because commonly used xenograft models do not account for every step in the process, we sought to develop a novel metastasis model system. Our technique involves implanting small fragments of patient-derived xenografts into either the tibia or gastrocnemius muscle of NOD/SCID/IL-2Rγ knock-out mice and amputating the affected limb when the tumor reaches at least 1 cm in diameter. Mice are then followed for the development of distant metastases. We found that Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma xenografts all undergo distant metastasis when implanted in an orthotopic location, but do not metastasize after subcutaneous implantation, even when the subcutaneous tumor is excised and mice are observed long-term post-excision. We also investigated whether the difference in metastatic potential is related to cell intrinsic or cell extrinsic factors. Analysis by immunofluorescence revealed that tumors are infiltrated by macrophages regardless of site of implantation, but that the polarization of these macrophages differs. We also found differences in expression of arginase and NOS2 based on site of xenograft implantation. Thus, we have demonstrated that sarcoma patient-derived xenografts undergo spontaneous distant metastasis when implanted in an orthotopic location, but not when implanted in a heterotopic location, highlighting the essential role of the tumor microenvironment in determining metastatic potential. This model has significant benefits compared with the more commonly used tail vein injection model of metastasis, which bypasses the requirements for local invasion and vascular intravasation, processes that are amenable to study using our model. We have begun to use this model to investigate the mechanisms by which tumor microenvironment influences metastasis, and also are using this model for preclinical drug evaluation to determine a therapy's effect not only on growth of the primary tumor, but also on development of metastasis.

Citation Format: Seth D. Goldstein, Masanori Hayashi, Catherine M. Albert, Kyle W. Jackson, David M. Loeb. An orthotopic xenograft model of sarcoma metastasis demonstrates essential role of tumor microenvironment for metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3231. doi:10.1158/1538-7445.AM2015-3231
Abstract 324: High throughput triculture: A breast cancer spheroid model for drug screening
Authors: Benton Gabriel J. , DeGray Gerald , Arnaoutova Irina , Kleinman Hynda K. , George Jay
Published: August 2015

Many drugs that show promise in preclinical evaluation fail miserably during clinical trials, and the inability of current preclinical models to properly predict drug response may be attributed to oversimplified or non-existent tumor microenvironments. To provide a more physiologically predictive model for drug screening, we have developed an in vitro, 384 well, triculture system that presents key chemical (i.e. low pH, low glucose, low oxygen), extracellular matrix (ECM), and cellular (tumor cells, stromal cells and endothelial cells) attributes of tumor tissues. By using ECM proteins, we can promote the proper physiology for each of these cell types in triculture. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Breast tumor microtissues spontaneously assemble when breast cancer cells are co-cultured with hMSCs under low adhesion conditions. Once formed, these microtissues are deposited onto preformed tubule networks comprised of HUVECs and hMSCs and are embedded within a hydrogel composed of ECM proteins. Cellular interactions and dissemination are monitored via fluorescence microscopy, and cell proliferation of the breast cancer cells is quantified using a fluorescence plate reader. The breast cancer cells exhibit cell-cell interactions with endothelial tubules and stromal cells, forming microtissues. Furthermore, the response to anti-cancer drugs, paclitaxel and fluorouracil, in an in vitro triculture system is similar to the response for the same drugs in xenografts model, unlike 2D or 3D monoculture. This high throughput triculture cancer model will provide more insight into pathological mechanisms and can better predict response to anti-cancer drug treatment.

Citation Format: Gabriel J. Benton, Gerald DeGray, Irina Arnaoutova, Hynda K. Kleinman, Jay George. High throughput triculture: A breast cancer spheroid model for drug screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 324. doi:10.1158/1538-7445.AM2015-324
Abstract 3240: Understanding clonal complexity of a tumor xenograft model via cellular barcoding technology
Authors: Caushi Justina X. , Bhang Hyo-eun C. , Li Jie , Kao Iris , Krishnamurthy Radhakrishna Viveksagar , Cooke Vesselina G. , Korn Joshua M. , Ruddy David A. , Kasibhatla Shailaja , Stegmeier Frank
Published: August 2015

Recent advances in next-generation sequencing have revealed the presence of genetic heterogeneity and clonal evolution within tumors. Intratumoral heterogeneity has been implicated in the disease progression, metastasis, therapeutic responses and development of drug resistance. Although cancer cell line xenograft models have been extensively used in cancer research to test drug efficacy, it is unknown how much clonal heterogeneity is maintained in the process of cell line xenograft establishment. In order to quantitatively assess the clonal complexity in xenograft models, here we applied a cellular barcoding technology using the HCC827 cell line. HCC827 is a non-small cell lung cancer cell line containing an exon 19 deletion which has been shown to be clinically relevant due to its initial response to EGFR inhibitors followed by resistance. This barcoding tool allowed us to label each individual cell with one unique DNA barcode via lentiviral infection and monitor the clonal heterogeneity within the implanted cell population by quantifying the number of unique barcodes. We were able to estimate the percentage of implanted clones that actually contributed to the formation of cell line xenografts. This study provides valuable insight on the clonal diversity present in xenograft models, which will further elucidate the heterogeneous nature of tumors.

Citation Format: Justina X. Caushi, Hyo-eun C. Bhang, Jie Li, Iris Kao, Viveksagar Krishnamurthy Radhakrishna, Vesselina G. Cooke, Joshua M. Korn, David A. Ruddy, Shailaja Kasibhatla, Frank Stegmeier. Understanding clonal complexity of a tumor xenograft model via cellular barcoding technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2015-3240
Abstract 3241: Tumor-targeting Salmonella typhimurium A1-R inhibits peritoneal dissemination of ovarian cancer and prolongs survival of the tumor-bearing nude mice
Authors: Matsumoto Yasunori , Miwa Shinji , Zhao Ming , Zhang Yong , Yano Shuya , Uehara Fuminari , Yamamoto Mako , Hiroshima Yukihiko , Toneri Makoto , Bouvet Michael , Matsubara Hisahiro , Hoffmann Robert
Published: August 2015

Peritoneal dissemination of cancer arising from an intra-abdominal organ has poor prognosis and is treatment resistant. A peritoneal dissemination nude mouse model of ovarian cancer was made with the human ovarian cancer cell line SKOV-3-GFP. Tumor-targeting Salmonella typhimurium A1-R (A1-R) was administrated i.v. or i.p. to the tumor-bearing nude mice. Survival of the tumor-bearing nude mice was prolonged by i.v. administration of A1-R (p = 0.025) as well as by i.p. administration of A1-R (p = 0.00003), compared with the untreated control. There was no significant difference in survival between i.v. and i.p. administration (p = 0.22). However, i.p. administration was less toxic. The present results suggest A1-R has clinical potential for what is currently a highly-treatment-resistant disease.

Citation Format: Yasunori Matsumoto, Shinji Miwa, Ming Zhao, Yong Zhang, Shuya Yano, Fuminari Uehara, Mako Yamamoto, Yukihiko Hiroshima, Makoto Toneri, Michael Bouvet, Hisahiro Matsubara, Robert Hoffmann. Tumor-targeting Salmonella typhimurium A1-R inhibits peritoneal dissemination of ovarian cancer and prolongs survival of the tumor-bearing nude mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3241. doi:10.1158/1538-7445.AM2015-3241
Abstract 3243: Notch inhibitors and chemoradiation in an orthotopic glioblastoma model
Authors: Yahyanejad Sanaz , Granton Patrick , van Hoof Stefan , Barbeau Lydie , Theys Jan , Verhaegen Frank , Vooijs Marc
Published: August 2015

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. Aggressive multimodal treatment using surgery followed by radiotherapy and chemotherapy extends the median survival of GBM patients to approximately one year after diagnosis. Treatment is not curative because of the intrinsic and acquired radiation resistance of a subpopulation of tumor cells. Notch inhibition has been shown to impair the tumorigenic capacity of these cells as well as enhance their sensitivity towards radiation. Therefore, we investigated if a highly potent and clinically approved Notch pathway inhibitor (g-secretase inhibitor) improves tumor control when combined with radiotherapy and chemotherapy in an orthotopic GBM mouse model.

To investigate the treatment efficacy of combinations treatments between standard of care treatment (radiotherapy and temozolomide) and Notch inhibitors, we used a three dimensional spheroid growth assay using both established and primary human glioma cell lines in which spheroid volume growth delay was quantitatively monitored. In addition, we assessed the expression of the putative glioma stem cell marker CD133 using flow cytometry. Furthermore, therapeutic efficacy of these combination treatments was als assessed in vivo in an orthotopic glioma tumor model (U87-luc) wherein tumor progression was evaluated using bioluminescence (BLI) and contrast-enhanced micro-computed tomography (CT) imaging. A small animal precision irradiation platform (PXI, XRAD 225Cx, CT, USA) was used for micro-CT imaging and irradiation delivery of conformal doses between 2- 10 Gy to intracranial tumors in mice receiving single or combination treatments with Notch inhibitors and chemotherapy.

Results: Notch blockade alone did not affect the sphere volume compared to control, whereas combination treatment with radiation and / or temozolomide resulted in a substantial spheroid growth delay (p = 0.004). Irradiation enhanced the expression of the stem cell marker CD133, while Notch blockade reduced CD133 expression. In intracranial tumours we found a strong correlation between CT and BLI imaging of tumor growth (Pearson coefficient (r) = 0.85, p = 0.001). The potential of Notch inhibition combined with precision radiotherapy and chemotherapy in our orthotopic GBM model is currently being determined and the outcome of this study will be presented.

Citation Format: Sanaz Yahyanejad, Patrick Granton, Stefan van Hoof, Lydie Barbeau, Jan Theys, Frank Verhaegen, Marc Vooijs. Notch inhibitors and chemoradiation in an orthotopic glioblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2015-3243
Abstract 3246: Novel luciferase labelling technique to improve imaging of orthotopic prostate and pancreatic cancer models
Authors: Bernoulli Jenni , Tuomela Johanna , Bozza Matthias , Fagerlund Katja M. , Suominen Mari I. , Morris George , Halleen Jussi M. , Harbottle Richard
Published: August 2015

Bioluminescent-labelling allows sensitive non-invasive sequential imaging of tumor development and early metastasis. However, current methods for the genetic modification of cells typically use integrating genotoxic viruses that can potentially disrupt the molecular behavior of cancer cell lines due to their random nature of integration. Here, we utilized a non-viral DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably modify cells to be further used in xenograft studies to allow long term expression without affecting cell behavior or silencing over cell divisions. Human PC-3 prostate cancer cells and BxPC-3 pancreatic cancer cells were stably transfected with a pCAG-LUC-S/MAR and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and molecular integrity of the DNA vector. For in vivo studies, PC-3 cells were inoculated orthotopically into the prostate, and BxPC-3 cells into the pancreas using athymic and BALB/c nude mice. For comparison, similar experiments with the corresponding study designs were performed with the non-labelled parental cell lines. The luciferase transfected cells maintained their original properties with stable expression. Luciferase-labelled PC-3 and BxPC-3 cells inoculated orthotopically into the prostate and pancreas, respectively, were successfully followed for 5 weeks with non-invasive bioluminescence imaging by IVIS. The results demonstrated high-quality follow-up of tumor growth compared with tumor models using non-labelled cells. In conclusion, S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and sustained transgene expression at high level. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essential non-invasive imaging platform that improves substantially efficacy testing of anticancer drug candidates.

Citation Format: Jenni Bernoulli, Johanna Tuomela, Matthias Bozza, Katja M. Fagerlund, Mari I. Suominen, George Morris, Jussi M. Halleen, Richard Harbottle. Novel luciferase labelling technique to improve imaging of orthotopic prostate and pancreatic cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2015-3246
Abstract 3251: Regulation of TNBC kinome by the metastasis suppressor Raf kinase inhibitory protein (RKIP)
Authors: Yesilkanal Ali E. , Frankenberger Casey , Rabe Daniel , Johnson Gary L. , Rosner Marsha R.
Published: August 2015

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with very low survival rates. Metastatic progression of tumors is the major cause of death in TNBC patients. Metastatic spread of tumor cells is generally associated with resistance to therapy and poor prognosis. Therefore, there is a clinical need for understanding the molecular and cellular basis of metastasis and developing effective therapies against metastatic progression. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor in TNBC, which acts as a homeostatic factor that regulates kinase activity. RKIP blocks invasion in vitro and experimental metastasis in vivo. Therefore, RKIP constitutes a powerful tool for studying metastatic signaling pathways in cancer cells. RKIP is known to regulate the activity of certain kinases such as Raf-1, GRK2, NIK, and GSK3β, affecting major pathways such as MAPK, NFκB, and WNT. However, our preliminary data suggests that RKIP affects a much larger part of the TNBC kinome. Using proteomics approaches, we have identified putative novel kinase targets of RKIP, and used bioinformatics analysis to build RKIP-regulated signaling networks. Elucidating RKIP's function at a systems level will reveal the interplay between key metastatic signaling cascades and potentially identify novel anti-metastatic target combinations in TNBCs.

Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel Rabe, Gary L. Johnson, Marsha R. Rosner. Regulation of TNBC kinome by the metastasis suppressor Raf kinase inhibitory protein (RKIP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2015-3251
Abstract 3261: Cytochrome p450 3a5 manipulates the progression of hepatocellular carcinoma via selectively targeting ros/mtorc2/p-akt (s473) signaling
Authors: chen lei , Wang hongyang
Published: August 2015

CYP3A5, one of the cytochrome P450 superfamily members, is involved in the metabolism of drugs, carcinogens, and endogenous molecules. Previous studies mainly focused on the role of CYP3A5 polymorphism or altered enzyme activity during carcinogenesis. However, the potential physiological function of CYP3A5 in tumor progression remains largely unknown. In our present study, the expression of CYP3A5 was firstly reported frequently down-regulated in not only 159 clinical HCC samples but also 4 external HCC cohort(n = 695). Tumors with reduced level of CYP3A5 were more likely to have aggressive characteristics (vascular invasion and poor differentiation), together with the worse overall survival time and time to disease recurrence. The exogenous expression of CYP3A5 was associated with the induction of TIMP1/2 and dysfunction of MMP2/9, which suppressed HCC migration and invasion in vitro and in vivo mechanically through inhibiting AKT signaling. Strikingly, the selective inhibition of AKT phosphorylation at Ser473(instead of Thr308) residue was found in CYP3A5-stable cells with an mTORC2-dependent manner. Co-immunoprecipitation and kinase assay further confirmed that mTORC2 kinase activity rather than Rictor/mTOR complex integrity was necessary for the inhibition of p-AKT(S473). Importantly, CYP3A5-induced ROS accumulation was identified as the key factor for the regulation of mTORC2 activity, in line with the decreased GSH/GSSG contents in most clinical HCC samples with relatively lower metastatic capacity. Together, these findings highlighted the novel function of CYP3A5 in the regulation of tumor metastasis via enhancing ROS level and in turn arresting mTORC2/p-AKT(S473) signaling, which may serve as a potential prognostic biomarker and therapeutic target for HCC.

Citation Format: lei chen, hongyang Wang. Cytochrome p450 3a5 manipulates the progression of hepatocellular carcinoma via selectively targeting ros/mtorc2/p-akt (s473) signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3261. doi:10.1158/1538-7445.AM2015-3261
Abstract 3275: A novel approach to treat medulloblastoma: The omega-3 fatty acids DHA and EPA reduce medulloblastoma tumor growth in vitro and in vivo
Authors: Ljungblad Linda M. , Johnsen John-Inge , Wickström Malin , Kogner Per , Gleissman Helena
Published: August 2015

Background: The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exert antitumoral properties shown in several cancer types. DHA is found in abundance in the brain and is of importance in normal brain development and maintenance of function including cognitive functions.

Medulloblastoma is the most common malignant CNS tumor of childhood. Intensive multimodal treatment is necessary including surgery, radiotherapy and chemotherapy. However, treatment often results in significant neurological sequelae and the risk of resistant relapses is still significant. The neuroprotective and antitumoral properties of omega-3 could therefore be of great benefit. Our aim was to investigate the effect of DHA and EPA on medulloblastoma in vitro and in vivo.

Methods: In a panel of medulloblastoma cell lines the cytotoxic activity of DHA and EPA was studied using cell viability assays. The molecular mechanisms were characterized using cell- and molecular biology techniques. Mice with human medulloblastoma xenografts were treated with; DHA alone or with DHA and EPA for 30 days from time of tumor take while tumor growth was monitored. Control mice were fed with normal chow. The incorporation of fatty acids was analyzed in tumors, erythrocytes and brain tissue by LS-MS/MS.

Results: DHA and EPA induced medulloblastoma cell toxicity with IC50 values ranging from 1.9 to 68 μM in six medulloblastoma cell lines. DHA reduced the prostaglandin E2 production, indicating a possible mechanism of action. In vivo, omega-3 supplementation resulted in significant reduction of tumor growth when established tumors were treated. DHA levels were increased in both tumor tissue and erythrocyte membrane compared to control. The in vivo treatment was non-toxic.

Conclusions: DHA and EPA exert toxic effect on medulloblastoma cells in vitro and reduce tumor growth in vivo. These omega-3 fatty acids could therefore be good candidates for improving current therapy and at the same time protect healthy neurons from therapy-induced toxicity promoting cognitive function in survivors.

Thus, omega-3 fatty acid supplementation has the potential of reducing tumor growth as well as reducing sequelae.

Citation Format: Linda M. Ljungblad, John-Inge Johnsen, Malin Wickström, Per Kogner, Helena Gleissman. A novel approach to treat medulloblastoma: The omega-3 fatty acids DHA and EPA reduce medulloblastoma tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2015-3275