bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2334: Anti-angiogenic activity of anthrax lethal toxin using the mouse retina as a model system
Authors: Bromberg-White Jennifer L. , Boguslawski Elissa , Duesbery Nicholas S.
Published: August 2012

Our laboratory has previously shown that inhibition of MKK pathways by anthrax lethal toxin (LeTx) inhibits tumor growth and angiogenesis, reduces VEGF secretion, and results in rapid cessation of blood flow to tumors in melanoma and sarcoma xenograft model systems. To gain insight into the anti-angiogenic nature of LeTx, we utilized both developmental and pathological models of vascularization in the murine retina. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay in retinal vascular development followed by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. Using a model of oxygen-induced retinopathy, western analysis revealed that MAPK activation preceded the onset of hypoxia and development of neovascularization in this model system. Intravitreal injection of LeTx during hypoxia resulted in a persistent lack of development and progression of all pathologic aspects of neovascularization, while having minimal effect on macrophage recruitment. VEGF levels remained at an elevated level even with the persistent lack of neovascular growth and continued hypoxic environment, implying a VEGF-independent mechanism for the effects of MKK inhibition. Additionally, immature retinal vasculature during normal development was persistently destroyed following MAPK inhibition, while mature vessels from adult animals were unaffected. Our studies not only reveal that MKK signaling plays a key role in retinal angiogenesis but also that perturbation of MKK signaling by LeTx can profoundly alter vascular morphogenesis. The persistent destruction of immature vessels during normal vascular development in the retina and in the model of oxygen-induced retinopathy suggests an extreme sensitivity of immature vessels to MKK signaling inhibition. Since MKK inhibition prevents oxygen-induced retinopathy by a mechanism independent of the VEGF-targeting strategies, this approach may be used in conjunction with existing strategies utilizing VEGF inhibitors to prevent pathologic angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2334. doi:1538-7445.AM2012-2334
Abstract 234: Snapshot of MAP kinase and related signal transduction pathways from biopsied cells using a novel non-optical assay technology - a proof of concept study
Authors: Dickerson W. Matthew , Alderman Edward M. , Beausang Lee Ann , Leong Kristen , Saab Ashley
Published: August 2012

Aberrant activity of the MAP and PI3 kinases is implicated in many forms of cancer. The ability to gather quantifiable information on the activation-state of these kinases from limited in-vitro and in-vivo tumor samples would accelerate drug development and ultimately, the treatment of cancer. Here non-optical, AMMP (acoustic membrane microparticle) technology is used to quantitate the activity state of multiple kinases including EGFR, c-RAF, MEK, ERK, AKT, p38 and JNK in a single assay plate. Lysates from multiple unstimulated tumor cell lines were compared with those from the same cell lines specifically stimulated with ligands to several well-known surface receptors for expressed changes in their phosphorylation states. The data show that the AMMP assay technology can be used to monitor the MAP Kinase pathway activation from EGFR stimulation through ERK phosphorylation following EGF stimulation. Additionally, the activation states of PI3 kinase, p38 and JNK were determined from the same lysate samples. Subsequently, assays were developed to monitor kinase dimerization (e.g., MEK-ERK) by rearranging the pairing of antibodies from the kinase activity assays described above. Using fewer than 2000 cells per assessment, multiple kinases were measured, in their native states, in a single assay using AMMP technology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 234. doi:1538-7445.AM2012-234
Abstract 2343: Establishment of patients’ primary non-small cell lung cancer xenograft models for test of anticancer drugs
Authors: Liu Changnian , Sun Daqiang , Li Wenwei , Zhou Wen , He Fang , Zhou Rui , Zhang Xun
Published: August 2012

BACKGROUND Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But tumor cell lines have a relatively low transplantability and resulted in a limited number of tumor models available for selection of right models for preclinical testing novel agents based on their antitumor mechanism. Recently, we have developed a large number of patient primary non-small cell lung cancer (NSCLC) xenograft models by transplanting patients’ fresh tumor tissues into nude mice, which have been employed for preclinical test of anticancer agents. METHODS The fresh NSCLC tissues were collected from local hospitals. The tumor fragments of 1-2 mm were subcutaneously implanted in the flanks of the nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). All therapeutic efficacy experiments used female mice bearing passage 5 xenografts. The test drugs included cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, tarceva, and iressa. RESULTS A total of 142 patient primary NSCLC samples were implanted into nude mice, and 71 primary tumor models were established with a tumor taking rate of 50% for the first passage. The tumor taking rates were higher (80-100) in the later passages. The therapeutic efficacy of the test drugs in these models is consistent with their clinical findings. The histopathology and gene sequence of the established primary tumor xenografts were analyzed; their architecture, histopathological morphology, and genomic mutation status from five generations of xenografts retained the patients’ original tumor characteristics. CONCLUSIONS The patient primary NSCLC models have been employed for preclinical test of standard clinically used and novel anticancer agents. The primary tumor models retain a similarity in histology and genomic mutation status to their patients’ original tumors. They may predict more relevant clinical response rate and higher correlation with clinical findings than use of traditional xenograft models established from long-term cultured cancer cell lines. Especially, they have advantages for test of target-oriented therapeutics in new drugs development programs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2343. doi:1538-7445.AM2012-2343
Abstract 2346: Lung cancer progression by efficient cre-mediated deletion of Pten and Smad4
Authors: Akkanti Bindu , Cho Sung-Nam , Jin Nili , Matzuk Martin , Wistuba Ignacio , DeMayo Francesco
Published: August 2012

Introduction: Lung cancer is the leading cause of cancer death in the world and the incidence of pulmonary adenocarcinoma, the most common type of lung cancer in the United States has been increasing in recent years. Mouse models have played a crucial role in understanding the tumor biology and progression of disease. The need for more clinically relevant preclinical mouse models for lung cancer is clear. Most mouse models for lung cancer use adenoviral delivery or cre recombinase models that result in development of cancers of the distal air ways. The tumors resulting from these mouse models are usually a mixed phenotype with mice developing adenocarcinoma, squamous cell carcinoma and benign adenomas depending on the combination of genetic alterations. We wanted to study lung cancer in the setting of more efficient cre that is present in the proximal as well as the distal lung epithelium. Utilizing this cre-system, we created a double knockout murine model with conditional ablation of pten and smad4 genes and wanted to compare tumor development and progression in these mice when compared to wildtype or smad 4 d/d or pten d/d mice. Methods: All mice were maintained at our core facility under approved protocols. The efficient cre system was accomplished by inserting the iCre recombinase into the Clara Cell Secretory protein locus. Tissues were collected at the time of sacrifice and compared to wild-type mice. Methods include Microarray analysis, RT-PCR, Immunohistochemistry and CHIP-Seq. Results: When compared with wildtype mice, smad 4 d/d or pten d/d alone mice, we have recognized that this particular mouse model has reproducibly developed proximal lung cancers that stain negative for SPC, a marker for distal airways. Additionally, this mouse model has developed metastatic disease, with metastasis to stomach. The immunohistochemistry results confirmed that the metastasis site was positive for TTF-1. Immunohistochemistry for EMT makers show that there is gradual loss of E-Cadherin. Conclusions: We have successfully created a mouse model with CCSPicre/Pten/Smad4 that results in proximal lung tumor development that advances to metastatic disease. Previous studies showed that tissue specific knockout of Smad 4 results in malignancy including gastric polyposis, pancreatic cancer, mammary gland epithelial hyperproliferation and SCC of the skin. Our results add to the previous findings in that loss of Smad4 in conjunction Pten may provide a novel platform to study lung cancer especially as it relates to proximal lung cancers that metastasize.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2346. doi:1538-7445.AM2012-2346
Abstract 2348: Suppression of metastasis of intratesticular inoculation of B16FO melanoma cells by a novel nutrient mixture in male athymic nude mice
Authors: Roomi M. Waheed , Roomi Nusrath W. , Rath Matthias , Niedzwiecki Aleksandra
Published: August 2012

Testicular cancer (TC) is rare, but still represents one of the most common diseases in young men between the ages of 20-45. However, men of any age can develop this disease. The incidence in Caucasians is greater than in African Americans. Risk factors include undescended testis, Klinefelter syndrome, and HIV positive patients. If left untreated, it is almost certainly fatal. Metastasis is the major cause of cancer death. The most common place for TC to spread in the body is to the lung. In this investigation, we studied the effect of a novel nutrient mixture (NM) containing ascorbic acid, amino acids and green tea extract that has been shown to exhibit anti-cancer activity, on inhibition of B16FO melanoma cells inoculated intratesticularly. Male athymic mice (n=12), 10-12 weeks of age, were inoculated with half a million B16FO melanoma cells in 100 µL of PBS into the right testis; the left testis was left untreated. After inoculation, the mice were randomly divided into two groups. Group A (n=6) was fed a regular mouse chow diet, while the mice in Group B (n=6) were fed the same diet but supplemented with 0.5% NM. Four weeks later the mice were sacrificed and the abdominal cavity was opened. Mice in the control group (Group A) exhibited extensive metastasis in the peritoneal cavity, which was totally masked by B16FO melanoma cells. The testis was severely enlarged and replaced by invading malignant melanoma cells. The remaining testicular tissue was represented by necrotic seminiferous tubules. The capsular region of the testis was severely infiltrated with a population of mixed cells. In contrast, in the NM fed group (Group B), there was no evidence of peritoneal metastasis, but the testes were enlarged. Seminiferous tubules in the area of invasion showed evidence of degeneration. In all groups, there was no metastasis to liver, kidney and spleen. However, severe lung metastasis was observed in the control group (2 out of 6) and mild in the test group (2 out of 6). In conclusion, these results suggest that NM has potential to suppress tumor metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2348. doi:1538-7445.AM2012-2348
Abstract 2352: Molecular profiles of head and neck cancer tumorgrafts
Authors: Kimple Randall J. , Blitzer Grace C. , Torres Alexandra D. , Yang Robert Z. , Lorenz Laurel D. , Armstrong Eric A. , Soriano Benjamin J. , Lambert Paul F. , Harari Paul M.
Published: August 2012

Purpose: We have developed a primary tumorgraft system of squamous cell carcinoma of the head and neck with relevance for investigating mechanisms of therapeutic resistance, understanding differential responses to standard therapy in HPV-positive and HPV-negative cancers, and testing novel therapeutics. To better understand the power, limitations and relevance of our tumorgraft system, we have initiated molecular characterization of these tumors. Methods: Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor at the time of surgery. Tumor tissue was obtained, washed in antibiotic rich media, manually disaggregated, and subcutaneously implanted into mice (NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ) for initial tumorgraft establishment. Subsequent mouse-to-mouse passages were performed and formalin-fixed/paraffin embedded as well as flash frozen tissue was harvested for subsequent molecular analyses. Expression of p16(Ink4a), p53, pRB, and EGFR was assessed by immunohistochemistry(IHC). HPV status and subtype was assessed using MY09/MY11 and GP5/GP6 degenerate nested polymerase chain reaction and Sanger sequencing. Transcriptionally active HPV infection was assessed by qRT-PCR for the HPV oncogenes E6 and E7. Mutation status of p53 was assessed by PCR-based exon amplification and Sanger sequencing. Results: To date, thirty primary tumorgrafts have been established with robust growth in eighteen (60%). p16(INK4A) expression was identified in approximately one-third. The majority of HPV-positive tumorgrafts contained HPV-16 and were transcriptionally active by mRNA expression profiling. HPV-positive tumors showed low p53 and RB expression by IHC. As anticipated, mutations in p53 were seen more frequently in HPV-negative tumors. Conclusions: These primary human tumorgrafts represent a diverse range of head and neck cancers. Both HPV-positive tumors and HPV-negative tumors have been successfully propagated with a diverse range of molecular characteristics. These primary tumorgrafts will provide a valuable platform for testing novel therapeutics as well as radiation and chemotherapy response profiles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2352. doi:1538-7445.AM2012-2352
Abstract 2357: Novel transgenic animal model of salivary gland tumors
Authors: Cruz-Monserrate Zobeida , Ji Baoan , El-Naggar Adel K. , Logsdon Craig D.
Published: August 2012

Background: Salivary gland malignancies represent 1-2% of human head and neck cancers with approximately 3,300 new cases per year in the United States. Surgery is the best treatment, however must tumors are too advanced at discovery and are unresectable. Furthermore, many surgeries are not margin negative and thus they recur. In addition, advanced tumors metastasize, which makes them fatal. The lack of research tools and the limited number of cases per year makes it difficult for investigators to study the development and progression of these tumors. The aim of this study was to characterize a novel inducible mouse model of salivary gland tumors, which develops by the activation of oncogenic K-RasG12V. Methods: We developed transgenic mice with Cre regulated mutant K-RasG12D and a specific tamoxifen regulated Cre (Ela-CreER). Double transgenic animals and littermate controls were treated with tamoxifen over 3 consecutive days and after 3, 8, and 15 days they were sacrificed and analyzed histologically. Results: Tamoxifen fed animals with mutant K-RasG12D expression in the salivary gland develop tumors with intense fibrosis and inflammatory cell infiltration. Histologically the tumors resembled human high grade salivary sarcomas. Conclusions: Our results show the development of a new animal model to study the role of K-Ras in the progression of salivary gland tumors and could be a useful tool for future salivary gland tumor studies. In particular it should serve as a useful preclinical model to test for drugs against inflammatory response pathways induced by activated K-RasG12V in salivary gland tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2357. doi:1538-7445.AM2012-2357
Abstract 2358: ERK1/2 regulation of CD44 modulates aggressiveness in a new mouse model of oral cancer
Authors: Uppaluri Ravindra , Judd Nancy , Winkler Ashley , Law Jonathan , Murillo-Sauca Oihana , Sunwoo John
Published: August 2012

Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) causes significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, six unique C57BL/6 mouse OSCC cell lines were generated from 7,12-dimethylbenz(≥)anthracene (DMBA) induced murine primary OSCCs. The cell lines were designated as mouse oral cancer (MOC1, 2, 7, 10, 22 and 23) and 5/6 (excluding MOC23) formed tumors when transplanted into immunocompetent wild-type (WT) mice. Analyses of tumor growth in mice showed that MOC2, MOC7 and MOC10 grew more rapidly and metastasized to regional lymph nodes, while MOC1 and MOC22 grew much slower and did not metastasize. This aggressive growth was correlated with ERK1/2 activation, which in addition, was found to induce CD44 expression- a molecule whose expression is associated with EMT and putative cancer stem cells. Inhibiting MEK, the upstream activator of ERK1/2, led to decreased CD44 expression and promoter activity and also decreased cellular migration and invasion. Conversely, enforced activation of MEK1 led to enhanced CD44 expression and promoter activity. CD44 was required for the aggressive growth since reduction of CD44 levels significantly attenuated in vitro migration and in vivo tumor formation of MOC2 and MOC10 cell lines. These results in the mouse model were extended to freshly resected human OSCC where a strict relationship between ERK1/2 phosphorylation and CD44 expression was also identified. Thus, using a novel immunocompetent mouse model, we identify CD44 as an essential mediator of the aggressive tumor-promoting effects of ERK1/2, which highlight the potential efficacy of targeting this molecular pathway in head and neck cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2358. doi:1538-7445.AM2012-2358
Abstract 2361: Establishing a patient-derived heterotransplant mouse model of head and neck squamous carcinoma (HNSCC)
Authors: Peng Shaohua , Sen Banibrata , Mazumdar Tuhina , Myers Jeffrey , Bell Diana , Williams Michelle , El-Naggar Adel , Johnson Faye M.
Published: August 2012

There are approximately 47,000 new cases of head and neck cancer diagnosed in the United States each year. The survival rate has not improved significantly in the past 30 years. The development of new therapies for HNSCC and the understanding of its biology both depend upon clinically relevant animal models. There are several existing animal models for HNSCC that include chemically-induced cancer models, syngeneic models using animal cancer cells, transgenic mice, and xenograft models using cultured human HNSCC cell lines injected into immunocompromised mice, either subcutaneously or orthotopically. An increasingly promising xenograft model, the heterotransplant model, is developed by surgically implanting tumor tissue directly from a patient into an immunocompromised mouse. The resulting tumor is serially passaged in vivo and never cultured on plastic. We have transplanted 20 HNSCC primary tumors from untreated patients directly into mice that included 17 tongue tumors, one maxillary gingival tumor, and two from the floor of the mouth. Although several tumors grew initially in mice, only 2 of 20 (10%) established tumors that could be serially passaged and used for therapeutic and mechanistic studies. One cell line has been established from a tongue heterotransplant (HOSC1). The two established heterotransplants maintained the histological appearance of the original tumors. To test the biological and molecular signaling response of HNSCC to targeted therapy, we utilized one heterotransplant that was passaged into 40 mice. Both the Src inhibitor dasatinib and the JAK inhibitor INCB16562 modestly inhibited tumor growth; the combination was significantly more effective than the single agents. Likewise, the tumors treated with the combination had significantly more apoptosis and less proliferation than control tumors. Consistent with our published in vitro results, Src inhibition did not result in STAT3 inhibition, but JAK inhibition abrogated STAT3 activation. Src was inhibited in vivo by dasatinib. Our results demonstrate that the heterotransplant model of HNSCC has promise as an in vivo model to test the efficacy of anti-cancer drugs. The low “take rate” is problematic but will be addressed in the future by intra-operative biopsies and larger tumor specimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2361. doi:1538-7445.AM2012-2361
Abstract 2363: Comparison and characterization of two orthotopic glioblastoma in vivo mouse models with human U87 and LN-229 cells using in vivo bioluminescence imaging and magnetic resonance imaging (MRI)
Authors: Lingnau Andreas , Hoffmann Steffen , Weidensteiner Claudia , Reichardt Wilfried , Kording Fabian , Obodozie-Schaefer Cynthia , Schaechtele Christoph
Published: August 2012

Glioblastoma is the most common and most aggressive type of malignant brain tumors. The prognosis is poor, with a median survival time of several months only. Despite steady progress in treatment of glioblastomas this tumor entity is still not curable. Thus, there is an urgent need for the development of novel effective therapies, and for appropriate in vivo models to characterize them. Most often novel antiglioblastoma drugs are tested in subcutaneous xenograft mouse models using various human or murine glioblastoma cell lines. To generate more relevant, orthotopic brain cancer in vivo models, we stably transduced the human glioblastoma cell lines U-87 and LN-229 with fire fly luciferase suitable for in vivo bioluminescence imaging. To initiate orthotopic growth, nude mice were anesthetized and placed in a stereotactic fixation device. Through a burr hole cells were implanted intracranially using a Hamilton syringe. Once to twice a week we used bioluminescence imaging to continually monitor tumor growth in vivo. However, as magnetic resonance imaging (MRI) is the most relevant tool to monitor oncological therapy in glioblastomas clinically, we adapted this translational aspect and analysed the mice in parallel using a dedicated animal MRI System. MRI offers detailed information about growth characteristics, size, blood supply and interaction with the surrounding tissue of the tumor. To achieve this, we performed T2-weighted imaging, and diffusion weighted imaging (DWI) weekly to visualise the tumors as well as necrosis and edemas. The use of both methods allowed independent and complementary characterization of the tumors. When exponential growth of the orthotopic tumors was observed, and the tumors reached critical sizes necropsy was done. In vivo bioluminescence imaging revealed only a very dominant signal in the region of the brain where the primary tumor was located. To address potential metastases in more detail we analysed a set of different resected organs and tissues for in vitro luciferase activity followed by extensive histological examination. Currently, we test different anti-glioblastoma agents in these orthotopic models to evaluate an antitumoral effect as well as a potential reduction of the edemas comparing the results in both models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2363. doi:1538-7445.AM2012-2363
Abstract 2367: Neural progenitor cells deficient in BAX and BAK manifest progressive hyperplasia and tumorigenesis
Authors: Katz Samuel G. , Fisher Jill K. , Bronson Roderick T. , Ligon Keith L. , Walensky Loren D.
Published: August 2012

The pro-apoptotic BCL-2 family proteins BAX and BAK serve as essential gatekeepers of the intrinsic apoptotic pathway and, when activated, transform into pore-forming homo-oligomers that permeabilize the mitochondrial outer membrane. Deletion of Bax and Bak causes marked resistance to many death stimuli in a variety of cell types. Bax−/−Bak−/− mice are predominantly non-viable and survivors exhibit multiple developmental abnormalities characterized by cellular excess, including accumulation of neural progenitor cells in the periventricular, hippocampal, cerebellar, and olfactory bulb regions of the brain. To explore the long-term pathophysiologic consequences of BAX/BAK deficiency in the central nervous system, we generated Bak−/− mice with conditional deletion of Bax in Nestin-positive cells. Aged mice manifest progressive brain enlargement with a profound accumulation of NeuN- and Sox2-positive neural progenitor cells within the subventricular zone niche. One-third of the mice develop frank masses comprised of the progenitor neurons, and in 20% of these cases, more aggressive, hypercellular tumors emerged. Interestingly, 60% of NestinCreBaxfl/flBak−/− mice harbor high-grade tumors within the testis interstitium, a peripheral site of Nestin expression. This mouse model of apoptotic blockade highlights the constitutive role of BAX/BAK in long-term regulation of Nestin-positive progenitor cell pools, with loss of function predisposing to adult-onset tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2367. doi:1538-7445.AM2012-2367
Abstract 2367A: Understanding the role of stem-like tumor cells in mouse models of high-grade gliomas
Authors: Barrett Lindy E. , Benezra Robert
Published: August 2012

High-grade gliomas (WHO grade III-IV) are typified by poor therapeutic response and rapid lethality. Since the discovery of glioma cells with properties reminiscent of neural stem cells, there has been a great deal of interest in understanding their contributes to tumor growth and therapeutic resistance. Self-renewal is the defining characteristic of both normal and cancer stem cells, and it has been hypothesized that glioma cells with self-renewal capacity contribute disproportionately to tumor growth compared to other, more differentiated lineages. However, analyses are complicated by the lack of reliable markers to definitively identify and functionally characterize each distinct cell lineage. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes regulate self-renewal and multipotency of adult neural stem cells. Here, using mouse models of high-grade glioma driven by PDGF- or mutKRAS, we show that high Id1 expression (Id1high) identifies self-renewing, multipotent tumor cells. By contrast, low Id1 expression (Id1low) identifies progenitor-like cells with proliferative potential but limited self-renewal capacity. While Id1high cells generate tumors upon orthotopic transplantation, Id1low cells do so with shorter latency and higher penetrance. Further, eliminating tumor cell self-renewal through targeted deletion of ID genes has modest effects on overall survival, while knockdown of the bHLH transcription factor, Olig2, within the Id1low population, results in a significant survival benefit. Together, these data highlight an important distinction between self-renewal capacity and tumor growth potential in two mouse models of gliomagenesis, underscoring the importance of non-stem cell lineages in disease progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2367A. doi:1538-7445.AM2012-2367A
Abstract 2371: Circulating cancer associated cells co-express epithelial and hematopoietic markers in metastatic breast cancer (MBC) patients (pts)
Authors: Lustberg Maryam B. , Balasubramanian Priya , Miller Brandon , Villa Alejandra Garcia , Xue Wei , Wu Yongqi , Chalmers Jeffery J. , Shapiro Charles L.
Published: August 2012

Background: Circulatingtumor cells (CTCs) are most commonly isolatedby using positive selection enrichment targeting the epithelial cell adhesion activating molecule (EpCAM). Using positive selection, a CTC is defined as EpCAM and cytokeratins (CK) positive (+) and CD45 negative (-). However, other rare cancer associated cells (RCACs) including EpCAM - and/or CD45 + cells have been reported (van de Stolpe, 2011). In order to investigate these RCACs, a prospective study in MBC using multi-parameter flow cytometry and immunocytochemistry (ICC) was undertaken. Methods:Forty blood samples from MBC pts were collected prior to initiation of a new line of chemotherapy, after one cycle, or at time of progression. Twenty one (52%) samples were from pts with estrogen receptor (ER) -, progesterone receptor (PR) -, and HER2neu non-overexpressing, or triple negative breast cancer (TNBC); nineteen (47%) were from pts with ER/PR +, HER2neu nonoverexpressing disease. Blood was processed using immunomagnetic negative depletion (Yang, 2009). Multiparameter, flow cytometry analysis was conducted prior to, and after depletion of CD45 + cells. Multistep, sequential labeling and fixation for surface markers was performed, followed by permeablizition for CK. ICC staining for EpCAM, CD45, CK 8/ 18/19 and DAPI was performed. Results: Either very low or no CK + events were present in blood samples from healthy volunteers (n=5). Various post-enrichment events were identified. These populations were also present by ICC with confocal microscopy. Conclusions: In addition to CTCs, RCACS that are CD45 + and/or EpCAM - are detectable in MBC pts and more frequently present in TNBC pts. These populations, only detectable by negative depletion, may be clinically significant as ongoing studies evaluate the predictive capabilities of blood based biomarkers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2371. doi:1538-7445.AM2012-2371
Abstract 2375: Rapid unbiased enrichment (negative selection) of circulating non-hematopoietic tumor cells directly from whole blood
Authors: Peters Carrie , Fadum Jodie , Woodside Steve , McQueen Karina , Thomas Terry
Published: August 2012

There is increasing interest in analyzing circulating non-hematopoietic tumor cells (CTC) in peripheral blood to evaluate disease progression or response to treatment; however, CTC enrichment is required prior to most analytic procedures. The ideal enrichment method would be rapid, permit a high recovery of viable CTC, and would be independent of the expression of specific epithelial cell surface markers, since CTCs in the peripheral blood may be undergoing EMT (epithelial mesenchymal transition) and may not express epithelial markers. RosetteSep™ CD45 depletion of hematopoietic cells directly from whole blood meets these criteria. However, RosetteSep™ enrichment of CTC involves density gradient centrifugation, which entails careful layering of the sample over the density gradient medium and careful pipetting to remove the enriched cells after centrifugation. Centrifugation must be performed with the brake off to avoid disturbing the enriched cell layer, further lengthening the process. SepMate™, a centrifugation tube with a specialized insert, was developed to minimize mixing of the sample with the density gradient medium, thus allowing rapid layering of the sample on the density gradient medium and easy pouring off of the enriched cells after centrifugation. We compared CTC enrichment using RosetteSep™ and the standard tubes and protocol with RosetteSep™ using SepMate™ tubes and reduced cocktail incubation and spin times on 5 donor whole blood samples seeded with ∼1% CAMA cells. Purity of viable (PI negative) CTC obtained with SepMate™ with RosetteSep™ was 85 ± 7%; purity of CTC obtained with RosetteSep™ alone was 91 ± 5% (no significant difference, paired t test, p=0.050). Purity of CTC obtained with density gradient centrifugation only (no RosetteSep™), either with or without SepMate™, was 4 ± 2%. There was no significant difference in the recovery of enriched CTC under any of the conditions tested (RosetteSep™ ± SepMate™, SepMate™ alone, density gradient separation alone, Tukey-Kramer test, p>0.05). CTC enrichment was accomplished in <40 min using SepMate™ with RosetteSep™. Simplifying the layering and layer removal step makes the entire process easily scalable to processing multiple samples simultaneously. Utilizing this method, CTCs can be enriched directly from whole blood without bias regarding their surface antigen expression. Large samples can be rapidly volume-reduced in preparation for detailed examination in a microfluidic device. Finally, enriched CTC are not labeled with antibodies or beads; there is nothing to interfere with subsequent further enrichment, culture, or evaluation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2375. doi:1538-7445.AM2012-2375
Abstract 2382: SOX17 promoter methylation in CTC and cell free DNA isolated from plasma of breast cancer patients
Authors: Chimonidou Maria , Strati Areti , Malamos Nikos , Georgoulias Vasilis , Lianidou Evi S.
Published: August 2012

Introduction: Molecular characterization of Circulating Tumor Cells (CTC) is a hot topic nowadays. SOX17 plays a very critical role in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Silencing of SOX17 due to its promoter methylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. We have recently shown that the promoter of this gene is methylated in CTC (Chimonidou et al, Clin Chem 2011). The aim of the present study was to evaluate whether SOX17 promoter methylation in CTC, is associated with the methylation pattern of this gene in matched cell free DNA isolated from plasma of breast cancer patients. Patients and methods: We firstly developed a highly sensitive and specific Methylation Specific Real-time PCR method for SOX17. The methylation status of SOX17 was examined in paired specimens of DNA isolated from CTC and cell free DNA (cfDNA) circulating in plasma of the same individuals. 58 patients with operable breast cancer, 21 patients with verified metastasis, and 12 healthy individuals used as controls were enrolled in our study. For CTC analysis, DNA was extracted from EpCAM positive immunomagnetically isolated CTC fraction, while cfDNA was isolated from 200μL of plasma from the same individuals. DNA extracted from isolated CTC and cfDNA was modified by a Sodium Bisulfite (SB) conversion reaction and subjected to methylation specific real time PCR. Results: In CTC, promoter methylation of SOX17 was observed in 22/58 (37.9%) of patients with operable breast cancer, 12/21 (57.1%) of patients with verified metastasis and 1/12 (8.3%) of healthy individuals. In the matched plasma samples, promoter methylation of SOX17 was observed in 23/58 (39.6%) of patients with operable breast cancer, 11/21 (52.4%) of patients with verified metastasis and 1/12 (8.3%) in healthy individuals. By comparing these results we found that promoter methylation of SOX17 in CTC was significantly correlated with promoter methylation of SOX17 in cell free DNA isolated from plasma of the same patients (P<0.005). Conclusion: We show for the first time that promoter methylation of SOX17 in CTC, is associated with the methylation pattern of this gene in matched cell free DNA isolated from plasma of breast cancer patients. Our findings suggest a new dimension to the origin of cell free DNA and indicate a correlation between cell free DNA and CTC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2382. doi:1538-7445.AM2012-2382
Abstract 2383: A new approach to isolate label-free and viable circulating tumor cells for monitoring cancer patient treatment response
Authors: Lin Sanger Hung-Chih , Wu Min-Hsien , Hsu Hung-Chih , Hsieh Chia-Hsun , Cheng Ju-Chien , Tseng Ching-Ping
Published: August 2012

The number of circulating tumor cells (CTCs) has been demonstrated as a good indicator for cancer progression and for monitoring treatment response of cancer patients. Most of the current methods for detecting CTCs were based on positive selection using the antibody specific to epithelial cell surface marker. In this study, we established a novel PowerMag system for negative selection and enrichment of CTCs by depletion of CD45+ cells. The efficacy of PowerMag system in CTCs isolation was first analyzed by spiking prostatic PC3 cancer cells into the peripheral blood from healthy volunteers. Our data revealed that, when compared with the methods that have been reported, the PowerMag system had high sensitivity (6-7 log10 enrichment), comparable recovery rate (50-60%), broad detection range (more than 3 log10), and stable and reproducible experimental results (r2=0.999). Notably, the cancer cells obtained from PowerMag system were label-free and viable with the capability for proliferation. To validate the feasibility of PowerMag system in clinical application, the numbers and characteristics of CTCs from 38 metastatic cancer patients with a total of 175 blood samples were determined. At least two populations of CTCs with the surface markers of EpCAM+CD45−CD146− and EpCAM−CD45−CD146− were identified. The detection rate reached 100% for the patients in the baseline level before treatment. The change in CTCs numbers for both cell populations appears to correlate with patient clinical outcomes after chemotherapy. As a whole, the PowerMag system is proven to be capable of isolating CTCs from whole blood effectively and economically, by which time- consuming sample pretreatment and costly equipments are not required. PowerMag has paved an alternation rout to monitor the progression and treatment response of cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2383. doi:1538-7445.AM2012-2383
Abstract 2384: Assessment of PITX2, BMP4, FGF4, ERalpha, ERbeta promoter methylation in the circulating tumor cells (CTCs) can predict metastasis and survival in primary invasive duct carcinoma (PIDC) patients
Authors: Bahnassy Abeer A. , zekri Abdel-Rahman N. , Salem Salem E. , Aziz Hala , Moustafa Abeer E. , Gaafar Rabab M.
Published: August 2012

Recent studies have focused on hematogenous tumor cell dissemination as predictive factors of breast cancer metastasis and several methods for detecting circulating tumor cells (CTCs) in peripheral blood were described. However, in early-stage breast cancer, molecular characterization of CTCs and their prognostic value is not well established. We previously found a significant correlation between DNA promotor methylation (PM) of PITX2, BMP4, FGF4, ERalpha, ERbeta and breast cancer recurrence and metastasis (unpublished data). Therefore, we sought to compare the frequency of PM of these genes in the CTC and the primary breast tumors (PBT) in relation to recurrence, progression free and overall survival rates (DFS& OS) CTCs were collected from the 10 cm peripheral blood of 80 primary invasive duct carcinoma (PIDC) patients by immunomagnetic beads separation. The number of CTCs was determined by flow cytometry (FCM) and RT-PCR (using CK-19, mammaglobin and CD44) for confirmation and comparison. Patients were first tested for CTC counts immediately before surgery, prior to therapy, at first follow-up (4-6 weeks later) and after 6 months. PM of PITX2, BMP4, FGF4, ERalpha, ERbeta was assessed by methylation specific PCR (MSP) in PBTs and CTCs and correlated with metastasis and survival. The number of CTCs ranged from 0-500/10 ml blood. The concordance between FCM and RT-PCR was 98% (p=0.01). At first assessment, 56 patients have β10 CTCs/10 ml blood and exhibited a significantly shorter progression-free survival (PFS) (12.7 months versus 20.6 months) and overall survival (OS) (28.1 months versus 49.5 months) than women with 10 CTCs was 16.1 months, compared to greater than 36 months in the cohort with <10 CTCs. PM of PITX2, BMP4, FGF4, ERalpha and ERbeta, was reported in 42.90%, 50.00%, 51.20%, 63.10% and 46.40% compared to 63.15%, 73.75%, 62.5%, 81.2% and 78.75% in CTCs. PM of BMP4, and ERbeta was associated with reduced OS and PFS. Conclusions: 1) The presence of CTCs in the blood of PIDC patients can impact standard treatment and future research, 2) FCM provides a simple and accurate technique for enumerating CTCs in peripheral blood, 3) the significant increase in PM frequency of all studied genes except FGF4, suggests that aberrant PM may contribute to increased aggression and more liability to metastasis. More studies are still needed to assess the impact of CTCs in PIDC patients and how they might contribute to improving individualized cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2384. doi:1538-7445.AM2012-2384
Abstract 2385: A highly sensitive detection system of genetic alterations in circulating tumor cells using a telomerase-specific replication-competent adenovirus
Authors: Shigeyasu Kunitoshi , Hashimoto Yuuri , Morikawa Tatsuya , Mori Yoshiko , Sun Dong-Sheng , Kagawa Shunsuke , Uno Futoshi , Tazawa Hiroshi , Nagasaka Takeshi , Kyo Satoru , Mizuguchi Hiroyuki , Urata Yasuo , Fujiwara Toshiyoshi
Published: August 2012

Background: Malignant tumor cells often develop metastatic nodules at distant organs through circulation in blood flow and result in poor prognosis. Detection of circulating tumor cells (CTCs) is clinically an important issue to evaluate the metastatic potential of primary tumors in cancer patients. Furthermore, confirmation of genetic alterations in CTCs provides us the critical information to clarify the molecular mechanism in the acquisition of metastatic capability of tumor cells and to treat tumor metastasis by molecular targeted therapy. We recently developed a telomerase-specific replication-selective adenovirus (OBP-401; TelomeScan) expressing the green fluorescence protein (GFP). OBP-401-mediated GFP induction method was a simple and highly sensitive imaging system to detect a small population of viable CTCs in the peripheral blood. In this study, we assessed the potential of OBP-401-mediated CTC detection system to analyze the genetic alterations in CTCs. Methods: We used two types of human colon cancer cell lines SW480 and HCT116 that have the mutations in codon 12 and 13, respectively, of the K-ras gene. The detection method involved a 4-step procedure, including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, the isolation of GFP-positive tumor cells using a fluorescence-activated cell sorting (FACS), and a confirmation of genetic mutations by direct sequencing. DNA was extracted from the captured cells using FACS and we investigated the mutation status in the K-ras gene by direct sequencing. Results: When only 10 cells of SW480 or HCT116 cells were added in a 5 ml of blood obtained from healthy volunteer, a total of 2-12 GFP-positive cells were captured from OBP-401-infected blood samples using FACS Aria. We confirmed the mutations in codon 12 and 13 of the K-ras gene in the FACS-captured cells from blood samples containing SW480 and HCT116 cells, respectively, by direct sequencing. Conclusion: These results suggest that OBP-401-mediated GFP induction and FACS-based capture system are useful methods to isolate small population of CTCs from peripheral blood samples and to evaluate the genetic alterations in CTCs. The combination method using OBP-401 and FACS would be a reliable strategy for the surveillance of genetic alterations in CTCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2385. doi:1538-7445.AM2012-2385