bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2799: Development of a mouse model of KRAS mutated colorectal cancer tumorigenesis
Authors: Maitra Radhashree , Venkatesh Madhu Kumar , Augustine Titto Alby , Chandy Carol , Liu Qiang , Goel Sanjay
Published: July 2017

Introduction Development of a preclinical animal model that mimics KRAS mutated colorectal cancer disease etiology is essential for validation of new therapeutic interventions. Colorectal cancer patients with tumors harboring KRAS mutation are excluded from receiving anti EGFR monoclonal antibodies, and currently have no alternate FDA approved treatment options available. Ongoing research requires validations of different modalities on a robust preclinical study platform that will resemble the disease phenotype and genetic profile. In this context we have developed viable animal mouse model of colorectal cancer bearing KRAS mutation that authentically serves as a model for the disease.

Methodology: The C57 BL/6 mice with truncated APC floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate APC f/f KRAS +/f mouse colony. In another set of breeding APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after second round of inbreeding. The final model of the disease was generated by the cross of the two aforementioned colonies when viable APC f/f KRAS +/f CDX2-Cre-ERT2 were genotyped and characterized. The model animals were tamoxifen induced to generate tumors. Micro-PET scan was used to detect and measure tumor volume. H & E staining was done to establish neoplasm and immunohistochemistry was performed to determine histological similarities with human FFPE biopsies. The MSI/MSS status was also determined.

Results Ø The newly developed animal model develops colonic tumors upon induction with Tamoxifen. Ø The tumors were malignant as confirmed by H and E staining. Ø The tumors showed a higher radioactive FDG uptake (SUV) in micro-PET scan Ø The tumors resembled human colorectal cancer tissue Ø Once tumor is generated the animals died of cachexia and rectal bleeding

Conclusion: Heterozygous mutation of KRAS oncogene along with deletion mutation of Adenomatous polyposis coli (APC) gene is embryonically lethal in animal models. We have successfully developed a viable animal model of KRAS mutated colorectal cancer with tamoxifen inducible tumors harboring KRAS and APC mutations. The tumors mimic the human disease type both genetically and immunohistochemically. This model can serve as a robust preclinical platform for understanding the disease mechanism at the molecular level as well for evaluation of various therapeutic interventions.

Citation Format: Radhashree Maitra, Madhu Kumar Venkatesh, Titto Alby Augustine, Carol Chandy, Qiang Liu, Sanjay Goel. Development of a mouse model of KRAS mutated colorectal cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2799. doi:10.1158/1538-7445.AM2017-2799
Abstract 2810: Using CRISPR/Cas9 to generate primary soft tissue sarcoma in genetically engineered and wild-type mice
Authors: Huang Jianguo , Chen Mark , Whitley Melodi J. , Kuo Hsuan-Cheng , Walens Andrea , Mowery Yvonne M. , Mater David V. , Eward William , Cardona Diana M. , Luo Lixia , Ma Yan , Nelson Christopher E. , Robinson-Hamm Jacqueline N. , Gersbach Charles A. , Dodd Rebecca D. , Kirsch David G.
Published: July 2017

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for pre-clinical studies, but this approach is costly and time-consuming. Here, we show that the CRISPR/Cas9 system can be used to efficiently complement existing GEMMs of sarcoma and generate primary sarcomas in wild type mice. Mice with the genotype KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft tissue sarcomas. These tumors arose with kinetics similar to those generated using the Cre-loxP system to activate oncogenic Kras and delete Trp53 alleles. Additionally, we injected an adenovirus containing Cas9 and sgRNAs targeting Nf1 and Trp53 into the sciatic nerve of wild type mice. These mice formed malignant peripheral nerve sheath tumors (MPNSTs) in the same timeframe as MPNSTs generated using the Cre-loxP system to delete Nf1 and Ink4a/Arf alleles in GEMMs. These data demonstrate that CRISPR/Cas9 can be used to generate soft tissue sarcomas in wild type mice. Moreover, these results suggest that this technology can complement existing GEMMs for rapid assessment of tumor-modifying genes. These tools should decrease the time and expense associated with employing autochthonous mouse models of sarcoma for preclinical research.

Citation Format: Jianguo Huang, Mark Chen, Melodi J. Whitley, Hsuan-Cheng Kuo, Andrea Walens, Yvonne M. Mowery, David V. Mater, William Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Using CRISPR/Cas9 to generate primary soft tissue sarcoma in genetically engineered and wild-type mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2017-2810
Abstract 2812: Development and characterization of the ultra immunodeficient B6;129-Rag2
tm1Fwa
IL2rg
tm1Rsky/DwlHsd (R2G2) mouse model
Authors: Wildt Sheryl J. , McClellan Jamie , Horn Mandy
Published: July 2017

The “R2G2” (B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd) knockout mouse is the latest advancement to provide an alternative option in the highly immunodeficient mouse model category for the oncology, immunology and other biomedical research communities. This model was generated by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. The R2G2 model is not only ultra immunodeficient, but provides a model that is less leaky and more tolerant to gamma radiation than traditional SCID models. This model is particularly useful for both tumor transplantation and humanization studies. Herein we describe the development and characterization of the R2G2 model, which includes breeding history, growth curve, CBC/Chemistry, flow cytometry and both huHSC and huPMBC humanization studies.

Citation Format: Sheryl J. Wildt, Jamie McClellan, Mandy Horn. Development and characterization of the ultra immunodeficient B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2812. doi:10.1158/1538-7445.AM2017-2812
Abstract 2825: Gene expression profile in genetically engineered mouse model of lung cancer correlates with that in human lung adenocarcinoma
Authors: Lakshmanan Imayavaramban , Rachagani Satyanarayana , Ponnusamy Moorthy , Chugh Seema , Dutta Samikshan , Muniyan Sakthivel , Batra Surinder , Kishore Apar
Published: July 2017

Introduction: Despite recent advances in the understanding of the biology of lung cancer, there are no agents that can target Kras, the most common mutation seen in lung adenocarcinoma. Development of genetically engineered mouse (GEM) models with krasG12Dand p53R172H+ will help us to understand the biology of kras mutated lung cancer and develop specific targeted therapeutic agents.

Material and Methods: We have developed two GEM models of lung adenocarcinoma harboring the KrasG12D mutation [KrasG12D;AdCre (KA) and KrasG12D; p53R172H;AdCre (KPA)] by conditional activation in the lung. We performed RNA-seq analysis from KA and KPA tumor tissues and compared them to normal lungs from littermate controls. The results were compared with publicly available human data (TCGA, cBioportal). In addition, we have developed two novel syngeneic cell lines from KA and KPA tumors, which will be used to test the efficacy of candidate drugs.

Results: Hyaluronan binding protein (Habp2) was top most upregulated gene [LogFC 12.7 (KA) 10.4 (KPA)] in both KA and KPA tumor tissues compared to normal. The TCGA and cBioportal data suggest Habp2 is overexpressed in lung adenocarcinoma as opposed to other histologic subtypes. We then analyzed Habp2 associated gene signature and observed that fibrinogen alpha chain (FGA), fibrinogen gamma chain (FGG), hyaluronan synthase 1 (HES1), serpin peptidase inhibitor, clade E1(Serpine1) were also upregulated in both models. On comparison of KA and KPA tumors, we observed that St6galnac1(ST6 N Acetylgalactosaminide Alpha-2,6 Sialyltransferase 1) was upregulated only in the KPA model, suggesting that p53R175H mutation leads to St6galnac1 overexpression.

Conclusion: Hyaluronan binding protein (Habp2) was top most upregulated gene in the KA and KPA models, while St6galnac1expression appeared to be associated with p53R175H mutation. Gene expression data from our GEM models of lung cancer agree with the TCGA and cBioportal analyses. Thus these GEM models seem to replicate human lung cancer and can be used to test drugs for in vivo therapeutic efficacy, prior to use in humans.

Citation Format: Imayavaramban Lakshmanan, Satyanarayana Rachagani, Moorthy Ponnusamy, Seema Chugh, Samikshan Dutta, Sakthivel Muniyan, Surinder Batra, Apar Kishore. Gene expression profile in genetically engineered mouse model of lung cancer correlates with that in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2825. doi:10.1158/1538-7445.AM2017-2825
Abstract 2837: Differential expression of Protocadherin-7 (PCDH7) in prostate cancer (PCa) cells: a direct association between PCDH7 and castrate resistant PCa
Authors: Shishodia Gauri , Koul Sweaty , Dong Qin , Koul Hari K.
Published: July 2017

Introduction: PCa is the most commonly diagnosed disease in American men and the second leading cause of death among them. Most of PCa deaths are a result of distant metastasis and due to emergence of castrate resistant PCa. Protocadherin-7 (PCDH7), a Cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer (NSCLC). PCDH7 has been shown to mediate brain metastasis of breast cancer. Overexpression of PCDH7 was reported to be associated with poor clinical outcome of NSCLC. However, there is no report on involvement of PCDH7 in PCa tumorigenesis and metastasis. In the present study we evaluated expression of PCDH7 in androgen dependent and androgen independent PCa cells and in TCGA and Trento/Cornell/Broad 2016 clinical data sets.

Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. Protein expression was checked by Western Blotting. mRNA levels were checked by quantitative real time PCR.

Results: Our data reveal high PCDH7 protein expression in PC3 cells and low levels of PCDH7 in LNCaP and RWPE1 cells. qRT-PCR confirmed an increased transcript level of PCDH7 in PC3 and low levels in LNCaP cells. We also observed elevated transcript levels for PCDH7 in castrate resistant and Enzalutamide refractory PCa (22Rv1) cells. These data suggest a direct relationship between castrate resistance and PCDH7 expression. Our results also show an inverse relationship between PCDH7 and E-Cadherin and a direct relationship between PCDH7 and EMT markers, indicating expression of PCDH7 is associated with tumor progression. Interestingly, there was no correlation between PCDH7 with tumor grade or metastasis from TCGA data, but analysis of NEPC (Trento/Cornell/Broad 2016) data set revealed amplification of PCDH7 in 9% of NEPC tumors. Collectively, these data suggest PCDH7 expression may be associated with advanced PCa in general and NEPC phenotype PCa in particular.

Conclusion: Our results suggest direct association of PCDH7 expression and castrate resistance in PCa cell lines. Role of PCDH7 in castrate resistant PCa needs further evaluation.

Citation Format: Gauri Shishodia, Sweaty Koul, Qin Dong, Hari K. Koul. Differential expression of Protocadherin-7 (PCDH7) in prostate cancer (PCa) cells: a direct association between PCDH7 and castrate resistant PCa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2837. doi:10.1158/1538-7445.AM2017-2837
Abstract 284: The trending of lymph node dissection for esophageal squamous cell carcinoma over 40 years in China
Authors: Chen Pei Nan , Zhou Fu You , Chen Hong Mei , Feng Xiao Shan , Sun Lei , Yuan Ling , He Ping , Pan Ying , Jiang Tao , Wu Min Jie , Gao Wen Jun , Wei Jin Chang , Wang Li Dong
Published: July 2017

The accumulated evidences have indicated the importance of lymph node dissection (LND) in improving survival for esophageal squamous cell carcinoma (ESCC). However, it is largely unclear in terms of standard lymph node dissection, e.g., how many is suitable and which node station is more important, etc. The present study was undertaken to summarize the trending of lymph node dissection for ESCC over 40 years at a single institute in northern China. A total of 27,260 ESCC patients were enrolled in this study from 1974 to 2016, including 16,565 males with a mean age of 59.72±7.82 and 10695 females with a mean age of 60.3±8.51. All the patients were from the ESCC database in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University (1973-2016). All the patients had pathological LND records, including the number dissected. The patients were classified into four period groups based on the diagnosed time, group I (1974 to 1986, 367/1.3%), group II (1987 to 1996, 3182/11.7%), group III (1997 to 2006, 10, 494/38.5%) and group IV (2007 to 2016, 13,217/48.5%). The SPSS21.0 software was used to evaluate the trending changes of LND at different periods. The results showed that the total number of LND/the mean number by each patient from group I to group IV was 3,000/8.43, 22,001/7.07, 87,740/8.36, and 159,340/12.06, respectively (p<0.0001). Further analysis indicated that the total number of LND/the mean number by each patient from group I to group IV in males was 1,634/7.93, 13,428/6.99, 53,143/8.53 and 100,073/12.19, respectively and 1,366/8.48, 8,573/6.80, 34,597/8.11, 59,267/11.83, respectively in females. The present results indicate an apparent increasing in number of LND from 1987 to 2016 for esophageal cancer surgical treatment in China. Further analysis is needed to correlate the LND and metastasis and survival. [National High-Tech Research and Development Program of China (SQ2015AA0202183), Major Science and Technology Projects of Henan Province (161100311300).]

Note: This abstract was not presented at the meeting.

Citation Format: Pei Nan Chen, Fu You Zhou, Hong Mei Chen, Xiao Shan Feng, Lei Sun, Ling Yuan, Ping He, Ying Pan, Tao Jiang, Min Jie Wu, Wen Jun Gao, Jin Chang Wei, Li Dong Wang. The trending of lymph node dissection for esophageal squamous cell carcinoma over 40 years in China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 284. doi:10.1158/1538-7445.AM2017-284
Abstract 2840: Integrative approach to biomarker discovery by performing comparative analysis of two cancers, hepatocellular carcinoma and endometrioid endometrial carcinoma, using genetics and transcriptomics from RNA sequencing data
Authors: Billaud Jean-Noel , Tugendreich Stuart , Toburen Debra
Published: July 2017

Hepatocellular Carcinoma (HCC) and Endometrioid Endometrial Carcinoma (EEC) are two lethal diseases of public health importance worldwide. Using QIAGEN’s RNA sequencing solution, we were able to highlight key molecular and biological processes that indicate similarities in the tumor progression toward metastasis between one EEC patient and the three HCC patients. We have identified ITGB1-001 isoform as a potential common biomarker between these two complex cancers. We have also shown these two cancers share activated pathways such as actin cytoskeleton signaling, and RAF1 as an upstream regulator indicating similar transcriptional program between these patients. Furthermore, we have identified as a potential therapeutic target the master regulator CTGF that connects a network of 286 downstream targets to invasion of tumor cells. We have filtered a set of unique variants in the EEC patient that affect a network of upstream regulators common with HCC. This approach may be useful in the context of precision or personalized medicine. Examining the gene expression in tumors from groups of patients with each disease revealed that at a molecular level, early stages of EEC resemble established HBV-positive, HCV-negative, liver cirrhosis positive HCC.

Citation Format: Jean-Noel Billaud, Stuart Tugendreich, Debra Toburen. Integrative approach to biomarker discovery by performing comparative analysis of two cancers, hepatocellular carcinoma and endometrioid endometrial carcinoma, using genetics and transcriptomics from RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2017-2840
Abstract 2846: Identify urinary biomarkers for colorectal carcinoma liver metastasis
Authors: Cai Meng , Sun Yulin , Gao Jiajia , Zhao Lina , Liu Fang , Sun Wei , Zhou Zhixiang , Zhao Xiaohang
Published: July 2017

Colorectal cancer (CRC) is the third leading prevalent cause of death from cancer in adults, and more than one third of CRC patients are accompanied by liver metastasis. The disease begins as a benign adenomatous polyp, and it subsequently develops into an advanced adenoma and gradually progresses to an invasive cancer. The driving factors behind CRC comprise a series of successive accumulated gene mutations that follow the order “APC-KRAS-TP53-DCC”. Moreover, urine is an important display window for tumor-derived exosomes. Here, to explore the roles of TP53 in CRC and identify novel urinary biomarkers for colorectal cancer metastatic to the liver, a comprehensive proteomic analysis of exosomal proteins from HCT116 TP53-wild type (WT), TP53-knockout (KO) and constructed TP53 (R273H)-mutant (MT) cells, as well as urine samples from CRC patients with and without liver metastasis was performed using the isobaric tag for relative and absolute quantitation (iTRAQ)-2D-LC-MS/MS strategy. The exosomes from the MT and KO cells exhibited significantly reduced sizes compared with the WT cells. A total of 3437 protein groups with ≥2 matched peptides were identified. Specifically, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) was consistently down-regulated in the exosomes from the MT and KO cells. Functional studies demonstrated that low HGS levels were responsible for the decreased exosome size. TP53 regulated HGS expression and thus HGS-dependent exosome formation. Furthermore, the HGS expression was gradually increased concomitant with CRC carcinogenesis and was an independent poor prognostic factor. In addition, HGS was one of the differentially expressed urine proteins between CRC liver metastasis and healthy individuals. In conclusion, HGS mediates TP53-regulated exosome formation. HGS may serve as a novel histological and urinary prognostic biomarker and a candidate target for therapeutic interventions in CRC.

Citation Format: Meng Cai, Yulin Sun, Jiajia Gao, Lina Zhao, Fang Liu, Wei Sun, Zhixiang Zhou, Xiaohang Zhao. Identify urinary biomarkers for colorectal carcinoma liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2017-2846
Abstract 2856: Measuring tumor metabolism in cancer spheroids and biopsies
Authors: Tee Sui Seng
Published: July 2017

Introductory Sentence

Real-time metabolic flux can be measured non-invasively using hyperpolarized magnetic resonance spectroscopy in cancer spheroids as well as tumor biopsies.

Experimental Procedures

LnCAP cells were resuspended in a 1:1 mixture of Matrigel:alginate (1x105 cells/ml) and grown in a porous hollow fiber (A/G Technology Corporation, NY). [1-13C] pyruvate was polarized in a SpinLab (General Electric, NY) before dissolving with appropriate buffers. NMR studies were performed on a 1 Tesla Magritek Spectrometer (Magritek, CA). HP spectra were acquired (10o degree flip angle with repetition time of 4s for 25 scans in total). Cell tracking used the manual tracking function in ImageJ. Xenograft biopsies were obtained from LnCAP tumors using a 2mm punch.

Data Summary

LnCAP cells seeded sparsely enable cancer spheroid formation remain viable as assessed by Live/DEAD fluorescent staining. Histological sectioning revealed spheroids of approximately 200 µm in diameter. Direct deposition of these threads into a 5mm NMR tube allows measurement of metabolic flux using hyperpolarized [1-13C] pyruvate resulting in the formation of [1-13C] lactate. To modulate metabolism, we used an allosteric AKT inhibitor, MK2206. This drug resulted in relocalization of AKT from the plasma membrane as well as a reduction in cell motility from 0.72 ± 0.04 to 0.46 ± 0.03 μm/s (n=5, p<0.05). Administration of hyperpolarized pyruvate resulted in approximately 30% of lactate production 24 hr after treatment. Histological staining of spheroids after treatment revealed sustained decrease of AKT phosphorylation after treatment. These measurements are in good accordance with measurements of lactate secretion in the same cells grown under standard conditions, with MK2206 resulting in loss of AKT Ser473 phosphorylation as early as 2 hr post-treatment, followed by a decrease in lactate secretion in the media from 5.16 ± 0.2 to 1.35 ± 0.05 mM/106 cells (n=3, p>0.05) 24 hr after treatment.

To ensure that these methods are applicable clinical settings, LnCAP xenograft biopsies (~60mg) were deposited directly into an NMR tube to measure hyperpolarized [1-13C] pyruvate metabolism. Line-width of the pyruvate peak was measured to be 1.72 ± 0.19 Hz (n=3) in the first spectra, demonstrating good homogeneity of the magnetic field and conversion to lactate was observed. Future experiments will focus on measuring the metabolism of patient-derived xenografts and effects of treatment response.

Conclusions

Metabolic measurements using hyperpolarized pyruvate can be performed on cancer spheroids as well as biopsies, allowing quantification of metabolism to complement on-going clinical trials utilizing hyperpolarized MR imaging.

Citation Format: Sui Seng Tee. Measuring tumor metabolism in cancer spheroids and biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2856. doi:10.1158/1538-7445.AM2017-2856
Abstract 2863: Magnetic resonance imaging based analysis of tumor growth and vascular parameters in animal model of GBM following IV formulated of HET0016 treatments
Authors: Arbab Ali S. , Jain Meenu , Achyut Bhagelu , Angara Kartik , Rashid Mohammad H. , Iskander Asm , Borin Thaiz F. , Zhi Wenbo , Ara Roxan , Lebedyeva Irina , Bagher-Ebadian Hassan
Published: July 2017

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 a selective CYP450 inhibitor has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Tumor growth and vascular parameters (tumor blood volume, permeability and, extravascular and extracellular space volume) were determined by in vivo dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI). The pharmacokinetics of HPßCD-HET0016 were evaluated in plasma and tumor tissues using IV and IP routes of administration. IV treatment with HPßCD-HET0016 decreased tumor growth, tumor blood volume, permeability and, extravascular and extracellular space volume, when compared with the vehicle group (p<0.05). Similar growth inhibition was also observed in syngeneic GL261 GBM (p<0.05). Survival studies using patient derived xenografts of GBM (PDX), showed prolonged survival to 25-27 weeks in animals treated combinedly with focal radiation, IV HET0016 and TMZ (p<0.05). Administration of a single dose resulted in 7-fold higher levels of HET0016 in the IV group in plasma and 3.6-fold higher levels in tumor tissue at 60 min compared to that of IP route. We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA) and migration (MHC-1) in the treated group (p<0.05). Decreased tumor growth was associated with reduced expression of pro-angiogenic markers (IL-8, MCP-1, VEGF, HIF-1α, and VE-Cadherin), inflammation (p-NFκB), intermediates of MAPK pathway (p-AKT, p-ERK and p-STAT1, EGFR), arachidonic acid metabolism (COX-1, CYP4A11) and increased expression of anti-angiogenic markers (Ang2, Angiostatin and Tie-2). Our results indicate that the improved IV formulation of HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, migration, and neovascularization. Furthermore, HET0016 significantly enhanced sensitivity of TMZ and prolonged survival in PDX GBM.

Citation Format: Ali S. Arbab, Meenu Jain, Bhagelu Achyut, Kartik Angara, Mohammad H. Rashid, Asm Iskander, Thaiz F. Borin, Wenbo Zhi, Roxan Ara, Irina Lebedyeva, Hassan Bagher-Ebadian. Magnetic resonance imaging based analysis of tumor growth and vascular parameters in animal model of GBM following IV formulated of HET0016 treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2017-2863
Abstract 2871: Non-invasive monitoring of the efficacy of anticancer therapeutic agent in lung orthotopic xenograft models of NSCLC using Bio-Luminescence Imaging (BLI)
Authors: Dey Sanchareeka , Kalainayakan Sarada Preeta , Ghosh Poorva , Zhang Li
Published: July 2017

Non-Small Cell Lung Cancer (NSCLC) patients have a poor rate of survival owing to diagnosis at a later stage. This necessitates development of novel therapeutic strategies that limit rapid progression of NSCLC and imaging techniques that monitor such changes in vivo. There has been a significant growth of bioluminescence imaging applications in small laboratory animal models in recent years, owing to its high sensitivity, efficiency, and non-invasive nature.

Our lab has previously demonstrated that a therapeutic agent that disrupts mitochondrial respiration effectively hampers proliferation of NSCLC cell lines in vitro. With an effort to investigate the potential of this therapeutic agent in clinical settings, we used a lung orthotopic xenograft mouse model along with bioluminescence imaging (BLI) to continuously monitor the efficacy of the drug in vivo. Briefly, we implanted NSCLC cell line transduced with lentiviral particles carrying luciferase sequence, in the left lung of 6-8 weeks old female NOD/SCID mice to generate lung orthotopic xenografts. The therapeutic agent was administered intravenously over several weeks. Tumor growth was then compared between the untreated versus the treated mice, non-invasively through bioluminescence imaging (Perkin Elmer’s IVIS Lumina III Imager). On sacrificing the mice, the lungs were harvested, formalin fixed and paraffin embedded for Immunohistochemistry.

Our BLI data show that in the mice treated with the therapeutic agent, tumors are localized to the left lung (site of implantation) as opposed to the control mice where the tumors metastasized to the right lung. The BLI data indicates significant decrease in radiance (total flux in photons per second) in mice subjected to treatment when compared to control mice.

We show that the therapeutic agent significantly inhibits progression and metastasis of human NSCLC cell line orthotopically injected in the lungs of NOD/SCID mice. The results suggest that it might be potent anti-cancer drug as it is effective in a model that recapitulates the natural environment of lung tumorigenesis. Immunohistochemical studies are underway to discern the mechanistic details.

Citation Format: Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh, Li Zhang. Non-invasive monitoring of the efficacy of anticancer therapeutic agent in lung orthotopic xenograft models of NSCLC using Bio-Luminescence Imaging (BLI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2871. doi:10.1158/1538-7445.AM2017-2871
Abstract 2874: Inhibition of tumor angiogenesis by Salmonella typhimurium A1-R in the in vivo Gelfoam color-coded imaging assay
Authors: Kiyuna Tasuku , Tome Yasunori , Murakami Takashi , Zhao Ming , Kanaya Fuminori , Hoffman Robert M.
Published: July 2017

We previously demonstrated a color-coded angiogenesis imaging model using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice in which nascent blood vessels express GFP. In this assay, we demonstrated osteosarcoma promotes angiogenesis. We report here that Salmonella typhimurium A1-R (S. typhimurium A1-R) inhibits angiogenesis of osteosarcoma cells in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation, skin flaps were made and 143B human osteosarcoma cells, expressing red fluorescent protein (RFP), were injected in the transplanted Gelfoam®. After establishment of tumor in the Gelfoam®, the control group mice were treated with PBS by tail vein injection. The treated group was treated by S. typhimurium A1-R, also by tail vein injection. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® in order to allow imaging of vascularization. The OV100 variable magnification small animal imaging system and FV1000 confocal fluorescence microscope were used to visualize vascularization. Nascent blood vessels grew in the Gelfoam® in a time-dependent manner. A random 3 fields were quantified in each group. The mean length of ND-GFP-expressing blood vessels in the Gelfoam® in mice were treated with S. typhimurium A1-R was 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean total length of ND-GFP-expressing blood vessels in the Gelfoam® in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with S. typhimurium A1-R had shorter vessels than the control group (on day-28, P<0.05). There was not a significant difference in tumor size between the 2 groups, thereby indicating a specific inhibition of angiogenesis by S. typhimurium A1-R. In the present study, we reported that S. typhimurium A1-R inhibited angiogenesis of human osteosarcoma cells in the in vivo Gelfoam® color-coded imaging assay.

Citation Format: Tasuku Kiyuna, Yasunori Tome, Takashi Murakami, Ming Zhao, Fuminori Kanaya, Robert M. Hoffman. Inhibition of tumor angiogenesis by Salmonella typhimurium A1-R in the in vivo Gelfoam color-coded imaging assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2017-2874
Abstract 2877: IKK-mediated signaling controls prostate tumor initiating cells
Authors: Conard Sara E. , Ebbs Aaron , Baldwin Albert S.
Published: July 2017

The heterogeneous nature of prostate cancer tumors is thought to play an important role in the decreased effectiveness of existing therapies. Tumor initiating cells (TICs) are capable of self-renewal and comprise a subset of the tumor mass. These cells are proposed to drive the growth and metastasis of tumors, and are considered to be resistant to traditional cytotoxic and targeted therapies. Several studies have shown that NF-κB signaling is increased in recurrent prostate cancer and enriched in prostate TICs. We sought to determine the potential of an IKK/NF-κB-driven mechanism for inherent or acquired resistance by IKK-mediated control of a subset of prostate tumor initiating cells. These studies were performed by using established prostate cancer cell lines, murine prostate organoids, and a murine prostate cancer animal model. We have found that inhibition of IKKα and IKKβ, upstream regulators of noncanonical and canonical NF-κB signaling, block self-renewal of several PTEN-deficient prostate cancer cell lines. Furthermore, we have also found that IKK is important for tumorigenicity and stemness seen in prostate cancer cells as measured by colony formation and extreme limiting dilution assays. Loss of canonical NF-κB (p65/RelA) decreased stemness while loss of noncanonical did not alter tumorsphere formation. Interestingly, Pten-/- tumors with loss of Ikkα or Ikkβ displayed decreased levels of self-renewing cells as measured by CD49fhigh expression, a known prostate basal cell marker. Isolated murine Pten-/- cells were 2x more efficient than Pten-/-Ikkα-/- or Pten-/-Ikkβ-/- cells in forming prostate organoids suggesting that loss of Ikk decreased the number of self-renewing cells needed for formation. Taken together, we conclude that IKK-mediated signaling is important for maintenance of prostate tumor initiating cells and further studies will address whether IKK-mediated signaling provides a mechanism for evading current therapies.

Note: This abstract was not presented at the meeting.

Citation Format: Sara E. Conard, Aaron Ebbs, Albert S. Baldwin. IKK-mediated signaling controls prostate tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2877. doi:10.1158/1538-7445.AM2017-2877
Abstract 2878: Anti-cancer effects of retinoic acid in CRC occurs via decreased growth of ALDH+ cancer stem cells and increased differentiation of stem cells to neuroendocrine cells
Authors: Modarai Shirin R. , Opdenaker Lynn M. , Gupta Anindita , Fields Jeremy Z. , Boman Bruce M.
Published: July 2017

Our studies on tissues from colon cancer patients show that in the development of colorectal cancer (CRC), stem cell (SC) overpopulation underlies tumor initiation and progression. Because aldehyde dehydrogenase (ALDH) is a marker for SCs in several tissues and is a key enzyme in retinoid acid (RA) signaling, we investigated the role of RA signaling in normal and malignant colonic SCs. Our overall hypothesis is that RA signaling regulates growth and differentiation of ALDH+ colonic SCs and dysregulation of RA signaling contributes to SC overpopulation in CRC development. To begin to investigate underlying mechanisms, we analyzed CRC cell lines/tissues to see if retinoid receptors RXR and RAR are exclusively expressed in ALDH+ SCs and if the RA pathway components change during CRC development. We determined whether RA signaling influences SC proliferation, differentiation, self-renewal, and SC population size. RXR & RAR were selectively expressed in ALDH+ colonic SCs, but not in MCM2+ proliferative cells, suggesting that RA signaling mainly occurs in SCs. Western blotting/immunostaining of CRCs revealed that RA signaling components, including RAR and RXR, become overexpressed in parallel with ALDH overexpression, which coincides with the known overpopulation of ALDH+ SCs that occurs during CRC development. Treatment of cells with RA ligands (ATRA, 9-cis RA) inhibited proliferation, reduced sphere formation, decreased ALDH+ SC population size, and induced differentiation along the neuroendocrine cell (NEC) lineage. Taken together, our findings indicate that: (1) retinoid signaling, by regulating ALDH+ colonic SCs, reduces SC proliferation, self-renewal and SC population size, and promotes SC differentiation to NECs, and (2) dysregulation of RA signaling in colonic SCs likely contributes to overpopulation of ALDH+ SCs and CRC growth. Thus, RA signaling, by inducing increased differentiation of the SC population, suggests a novel therapeutic target.

Citation Format: Shirin R. Modarai, Lynn M. Opdenaker, Anindita Gupta, Jeremy Z. Fields, Bruce M. Boman. Anti-cancer effects of retinoic acid in CRC occurs via decreased growth of ALDH+ cancer stem cells and increased differentiation of stem cells to neuroendocrine cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2017-2878
Abstract 2880: GANT61-mediated constraint of Fibulin-5/prorenin receptor/BMI1 signaling impacts nasopharyngeal carcinoma stemness and metastasis
Authors: Tasi Hsin-Ting , Chen Chang-Han
Published: July 2017

Background: Cancer stem cells (CSCs) of NPC exhibit higher tumorigenesis and metastatic potential. Novel therapies based on molecular targets of CSCs have become the focus to cure NPC. GANT61, a GLI1 inhibitor, has been reported to exhibit potent anticancer effect on many cancers; here, we evaluated whether GANT61 has inhibitory effects on NPC-CSCs and further explored the possible mechanism.

Methods: Condition medium was used to enrich NPC stem-like cells. The NPC stem-like cells characteristics were examined by Q-RT-PCR, Western blotting, MTT, BudU, colony formation, sphere formation, and subcutaneous and metastatic xenograft models. Genetic, pharmacological and biochemical approaches were applied in NPC stem-like cells to investigate molecular signals, to assess the efficacies under the inhibitors treatment, and to examine the correlation among the molecules in vitro, in vivo and NPC specimens.

Results: In the present study, we had demonstrated that NPC tumorsphere cells (NPC-TCs) possess CSC properties. The proliferation, colony formation, spheroid formation, migration, and invasion of NPC-TCs were dramatic prevented in the presence of GANT61 in vitro and in vivo. ChIP reveals that Gli1 preferentially binds to the Fibulin-5 promoter, and Fibulin-5 transcriptional/post-transcriptional levels in NPC-TCs were regulated upon GANT61 treatment. However, ectopic expression of Fibulin-5 in NPC-TCs not only enhanced self-renewal and ALDH1 expression, but also reduced the effects of GANT61-eilicted anti-tumor in vitro and in vivo. We further identified that a novel signaling, prorenin receptor/BMI1 pathway was modulated by Fibulin-5 and involved in Fibulin-5-raised self-renewal capacity of NPC-TCs in vitro and in vivo. Notably, GANT61 inhibited NPC-TCs phenotypes was associated with suppression of Fibulin-5/prorenin receptor/BMI1 signaling. Finally, a significant correlation was observed among GLI1, Fibulin-5, AT1R and BMI1 in the specimens of NPC.

Conclusions: Our data highlight that Fibulin-5/prorenin receptor/BMI1 signaling has critical role in maintaining stem-like properties of NPC-CSCs and can be efficiently targeted by GANT61 representing a preclinical therapeutic strategy to repress NPC-CSCs.

Note: This abstract was not presented at the meeting.

Citation Format: Hsin-Ting Tasi, Chang-Han Chen. GANT61-mediated constraint of Fibulin-5/prorenin receptor/BMI1 signaling impacts nasopharyngeal carcinoma stemness and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2017-2880
Abstract 2881: CXCL1 and CCL5, induced by ionizing radiation, reprogram nontumorigenic cancer cells into cancer stem cells in breast cancer
Authors: Bailleul-Dubois Justine , Bidan Nadège , Mouttet-Audouard Raphaëlle , Arcicasa Mélanie , Hannebicque Karine , Takayama Yuki , Meignan Samuel , Bourhis Xuefen Le , Lagadec Chann
Published: July 2017

Identification of cancer stem cells (CSC) in solid tumors - with self-renewal, multipotency, tumorigenesis, and therapy resistance capacities - has opened path to new targeting therapeutic approaches. However, CSC targeting alone might not be sufficient to eradicate a tumor. Indeed, recent studies showed that cancer cells are plastic, and conventional therapies, such as radiotherapy, can lead to cancer cells (non-CSC) reprogramming into iCSC (induced-CSC). The goal of our work is to identify the molecular mechanisms responsible for treatment-induced CSC emergence. First, we have shown that conditioned media from irradiated non-CSC is sufficient to induce iCSC reprogramming. These results suggest that cell plasticity might be actively regulated by diffusible factors secreted by irradiated cells. By using proteins arrays and ELISA, we demonstrated that the secretion of a specific cocktail of chemokines is induced by ionizing radiation, such as CXCL1 and CCL5. Interestingly, recombinant CXCL1 and CCL5 treatments increase the sphere forming capacity (SFC) of isolated non-CSC treated population. Concomitantly, treatment with neutralizing antibodies targeting CXCL1 and CCL5 leads to a decreased CSC number (ALDH+ cells). We also studied the expression of the corresponding chemokines receptors, by flow cytometry. First, we saw that reprogrammable ALDH- cells are enriched for CXCL1 and CCL5 receptors expressing cells compare to unsorted population or ALDH+ population (CSC). We analysed the reprogramming potential of isolated ALDH-/receptor-positive cells versus ALDH-/receptor-negative cells. The ALDH-/receptor-positive-derived cell population is more able to form spheres and overcomes the receptor-negative-derived population when the two populations are mixed and tested for their sphere forming capacity. The use of pharmacological inhibitors against the receptors induce a slight decrease of CSC. Taken together, our results indicate the involvement of chemokines, in particular CXCL1 and CCL5, in the reprogramming mechanism. To validate the implication of CXCL1 and CCL5 and in a pre-clinical perspective, we are currently beginning an in vivo experiment to study the effects of CXCL1 and CCL5 inhibition on tumor development and on CSC enrichment in vivo.

Citation Format: Justine Bailleul-Dubois, Nadège Bidan, Raphaëlle Mouttet-Audouard, Mélanie Arcicasa, Karine Hannebicque, Yuki Takayama, Samuel Meignan, Xuefen Le Bourhis, Chann Lagadec. CXCL1 and CCL5, induced by ionizing radiation, reprogram nontumorigenic cancer cells into cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2881. doi:10.1158/1538-7445.AM2017-2881
Abstract 2887: Macrophage conditioned medium promotes colon cancer stem cell phenotype via the hedgehog signaling pathway
Authors: Fan Fan , Wang Rui , Boulbes Delphine R. , Zhang Huiyuan , Watowich Stephanie S. , Bhattacharya Rajat , Xia Ling , Ye Xiang-Cang , Ellis Lee M.
Published: July 2017

Background: Contradicting studies have shown that increased macrophages infiltration in colorectal cancer (CRC) could either result in anti-tumoral or in pro-tumoral effects. Because of these inconsistencies, we sought to determine the role of conditioned medium from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance.

Methods: Murine (CT26) and human (HCP-1) CRC cell lines were treated with conditioned media (CM) from lipopolysaccharide (LPS)-activated murine macrophages. Viability studies were performed using the MTT assay, annexin V-FITC assay, and Western blot analysis. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay.

Results: The results showed that 1) murine LPS-activated macrophage CM induced apoptosis in murine and human CRC cells, 2) murine and human LPS-activated macrophage CM secreted paracrine factors that promoted the CSC phenotype in murine and human CRC cells via the Sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal and 3) these effects were blocked by depletion of SHH in macrophage CM.

Conclusions: Our data show that LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway.

Citation Format: Fan Fan, Rui Wang, Delphine R. Boulbes, Huiyuan Zhang, Stephanie S. Watowich, Rajat Bhattacharya, Ling Xia, Xiang-Cang Ye, Lee M. Ellis. Macrophage conditioned medium promotes colon cancer stem cell phenotype via the hedgehog signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2887. doi:10.1158/1538-7445.AM2017-2887
Abstract 2891: Retinoic acid directs breast cancer cell state changes through regulation of TET2-aPKC pathway
Authors: Wu Meng-Ju , Chang Chun-Ju
Published: July 2017

The key molecular mechanism governing the cancer cell state (stem cell-like state vs. differentiation state) to control the cancer stem cell (CSC) pool remains elusive. This study provides the first evidence showing that all-trans retinoic acid (ATRA) induces the interaction and chromatin recruitment of a novel RARB-TET2 complex to epigenetically activate a specific cohort of gene targets, including MiR-200c. TET2-activated miR-200c further targets and suppresses aPKC, a cell polarity protein that plays a pivotal role in directing asymmetric division of mammalian stem cells to sustain the stem cell pool. Our data reveals that pharmacological concentration of ATRA effectively down-regulates aPKC through activation of miR-200c, leading to a decrease of the stem cell-like populations from non-tumorigenic mammary epithelial cells and non-aggressive breast cancer cells. However, aggressive breast cancer cells that manifest TET2-miR200c dysregulation sustain a CSC pool highly resistant to ATRA, where inhibition of aPKC directs the resistant CSCs to the luminal cell-like state and sensitization to Tamoxifen, resulting in abrogation of mammary tumor growth and progression. Together, these findings elucidate a novel RARB-TET2-miR200c-aPKC signaling pathway that directs cancer cell state changes and also provide previously unidentified therapeutic implications for aPKC inhibitors in diminishment of breast CSCs to eradicate breast cancer.

Citation Format: Meng-Ju Wu, Chun-Ju Chang. Retinoic acid directs breast cancer cell state changes through regulation of TET2-aPKC pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2017-2891