bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4017: Inhibition of mutagenicity of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine)by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa
Authors: Alva Rayford , Wong Brian Yuen Yau , Tanner John , Wan Yanlingxue , Uppala Padma Tadi , Hayes Ryan
Published: August 2018

Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. Recent research revealed that these two herbs were included in most of the herbal cancer treatment formulas in Taiwan hospitals. We previously showed that SB and OD inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 (AFB1) and benzo(a)pyrene (BaP) bioactivated by Aroclor 1254-induced rat S9. PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is produced from high cooking temperature of creatine, sugars, and amino acids. It was demonstrated positive result in the Ames mutagenicity test. PhIP is one of the most abundant heterocyclic amines (HCAs) in cooked meat and has been announced as "reasonably anticipated to be a human carcinogen" by the U.S. Department of Health and Human Services National Toxicology Program. Cancer of the prostate, breast, lung, esophagus, stomach, and colorectum may be related to high intake and high exposure to PhIP. In this study, the effects of aqueous extracts of SB and OD on PhIP-induced mutagenesis using Salmonella typhimurium TA98 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. Our results showed that SB and OD significantly inhibited the mutagenicity of PhIP (both 0.1 and 1µg/plate) bioactivated by S9 (800 mg/plate) in a dose-response fashion (p< 0.05). The percent inhibition of revertants formation of these two herbs against 0.1µg/plate of PhIP were 45%, 77%, and 87% for SB (1.5, 3, and 6 mg/plate) and 21%, 80%, and 95% for OD (1.5, 3, and 6 mg/plate), while the percent inhibition against 1 µg/plate of PhIP were similar to that of the 0.1 µg/plate (45%, 80%, 90% for SB and 44%, 81%, 93% for OD, respectively). There was a slight additive inhibition effect of the herbs in combination (SB + OD) at higher concentrations (60%, 85%, and 90%) against both concentrations of PhIP. These results suggested that OD and SB contain antimutagenic phytochemicals towards PhIP-induced mutagenicity. Further study of their modulation effects on metabolism and DNA binding of PhIP is warranted to reveal the potential anti-mutagenicity mechanism and their chemopreventive property against PhIP and potentially against other HCAs.

Citation Format: Rayford Alva, Brian Yuen Yau Wong, John Tanner, Yanlingxue Wan, Padma Tadi Uppala, Ryan Hayes. Inhibition of mutagenicity of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine)by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4017.
Abstract 4021: The evaluation of carcinogenicity evidence of dibenzanthracenes
Authors: Tsai Feng , Hsieh Jennifer C. , Osborne Gwendolyn , Sandy Martha
Published: August 2018

Dibenzanthracenes (DBAs) are identified as carcinogens and are on the California Proposition 65 list. DBAs are five-ring polycyclic aromatic hydrocarbons (PAHs). This chemical group consists of three isomers: DB[a,c]A, DB[a,h]A, and DB[a,j]A. DBAs are products of the incomplete combustion or pyrolysis of organic matter and can be formed during high temperature cooking. They are air pollutants present in engine exhaust, tobacco and marijuana smoke, and may also contaminate soil and water. DB[a,h]A is a well-studied International Agency for Research on Cancer (IARC) Group 2A carcinogen, whereas fewer studies relevant to carcinogenicity have been conducted with DB[a,c]A and DB[a,j]A. Carcinogenicity evidence from multiple data streams was integrated in the hazard identification of this chemical group. These data streams include animal cancer bioassays, mechanistic studies and other relevant data, including high-throughput studies (e.g., ToxCast), toxicogenomic studies, and information from quantitative structure-activity relationship (QSAR) models. Structure activity comparisons across the three DBAs and related carcinogenic PAHs served to bridge the data gaps for the two less studied isomers. The results of our analyses showed that each of the DBAs share similar biological activities in animals: all induce tumors in mice, are mouse skin tumor initiators, form DNA adducts, are genotoxic, and form metabolites that are also genotoxic and initiators of mouse skin tumors. All three DBAs activate the aryl hydrocarbon receptor (AhR) in reporter gene assays, and DB[a,h]A and DB[a,c]A have been shown to bind to the AhR. In addition to strong evidence of genotoxicity, other carcinogenic mechanisms are likely also operative, including receptor activation (e.g., AhR), immune suppression, altered cell proliferation, apoptosis, and cell cycle regulation. There are strong structure-activity similarities among the DBAs and several carcinogenic 4-, 5- and 6-ring PAHs, including benzo[a]pyrene and dibenzo[a,h]pyrene. In addition, DB[a,c]A and DB[a,j]A were predicted to be mutagenic and carcinogenic in several QSAR models, including VEGA, Lazar and OECD QSAR Toolbox. Integration of the evidence from these multiple data streams resulted in the identification of the DBAs as a chemical group as carcinogens.

Citation Format: Feng Tsai, Jennifer C. Hsieh, Gwendolyn Osborne, Martha Sandy. The evaluation of carcinogenicity evidence of dibenzanthracenes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4021.
Abstract 4027: Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells
Authors: Miura Kentaro , Ito Ken-ichi , Oba Takaaki , Iesato Asumi
Published: August 2018

Pemetrexed (MTA) is a folate antimetabolite used for treatment of non-small cell lung cancer and plural mesothelioma. Although several molecules such as thymidylate synthase (TS) or ATP-binding cassette transporter 11 (ABCC11) have been reported to be involved in the mechanisms of resistance to MTA, precise mechanisms underlying resistance to MTA remains unknown. To elucidate the mechanisms of resistance to MTA in lung cancer, we established MTA resistant clones in PC9 and H1993 lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Then, gene expression profiles were compared between PC9 and PC9-MTA cells by using microarray analyses, and several genes including fibroblast growth factor 2 (FGF2) were identified. Elevated expression of FGF2 in PC9-MTA cells and H1993-MTA cells were confirmed by western blot analyses. Knockdown of FGF2 by siRNA partially restored the sensitivity to MTA both in PC9-MTA and H1993-MTA cells. In addition, anti-FGFR1 inhibitor (PD173074) restored MTA sensitivity particularly in PC9-MTA cells. On the other hand, the expression of TS was increased in PC9-MTA and H1993-MTA cells, and the inhibition of TS with the TS-targeting siRNA restored the sensitivity to MTA. However, with regard to the expression of FGF2 and TS, the inhibition of FGF2 did not modify the expression of TS, and vise versa. Furthermore, a simultaneous inhibition of FGF2 and TS by siRNAs in the MTA-resistant cells restored MTA sensitivity to approximately the level observed for the parental cell lines. With regard to the factors related to the signal transduction pathway, the phosphorylation of Erk and Akt in H1993-MTA cells was decreased compared with those in the parental cells. When H1993-MTA cells were treated with FGF2-targeting siRNA or anti-FGFR1 inhibitor, the increase of phosphorylation of ERK and AKT were induced. These data indicated the possibility that both FGF2 and TS could confer MTA resistance in the lung cancer cell lines in an independent manner, and that modification of signal transduction through FGF2-FGFR1 pathway might be involved in the development of MTA resistance in lung cancer cells.

Citation Format: Kentaro Miura, Ken-ichi Ito, Takaaki Oba, Asumi Iesato. Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4027.
Abstract 4048: Comprehensive analysis of the suppressed tumor immune microenvironment of CIN gastro-esophageal adenocarcinomas
Authors: Derks Sarah , Xu Xinsen , Klerk Leonie de , Fleitas Tania , Liu Kevin , Liu Yang , Margolis Claire A. , Dietlein Felix , Chiaravalli Anna Maria , Silva Annacarolina da , Freeman Gordon , Rodig Scott J. , Allen Eliezer M. Van , Li Bo , Fu Jingxin , Wang Jin , Liu Shirley X. , Bass Adam J.
Published: August 2018

Background and aims: Gastro-esophageal adenocarcinoma (GEA) is a common and lethal disease where immune checkpoint inhibitors have demonstrated efficacy within a minority of patients. Prior studies have identified distinct molecular classes of GEA, characterized by specific features that would be predicted to influence responses to checkpoint therapy. We here perform a comprehensive analysis of tumor immune associations in GEAs to further define the tumor immune microenvironment in distinct classes of GEA and to identify additional candidate mediators of immune evasion that may impact response to immunotherapy efficacy. Methods: Tumor immune interactions in GEAs were studied first using computational analyses to infer immune cell subsets from RNAseq data generated from The Cancer Genome Atlas (TCGA). These data were complemented by additional focused studies spanning immunohistochemistry, gene expression profiling and flow cytometry on archival tissue samples and from freshly resected GEA tumor samples. Results: Our results confirmed substantial heterogeneity in the composition of the tumor immune microenvironment between distinct GEA subtypes. As shown earlier, tumors with microsatellite instability (MSI) or Epstein-Barr virus (EBV) harbored larger T cell infiltrates in contrast to those with chromosomal instability (CIN), the most common class of GEA. We then focused greater attention on the CIN cohort, finding a spectrum of phenotypes including tumors with ‘immune hot' and ‘immune cold' states. The ‘hot' cases were more often from Western patients and had greater degrees of DNA methylation. Notably, these tumors often showed co-expression of secondary immune checkpoint proteins, as confirmed by flow cytometry. There was no association between number of infiltrating T cells and mutational or neoantigen load within the CIN GEA subgroup. Immunological cold CIN GEAs were characterized by a higher level of chromosomal instability, enrichment of cell cycle pathways, specifically amplification of cyclin E1, CCNE1, and enrichment of an unfolded protein response.

Citation Format: Sarah Derks, Xinsen Xu, Leonie de Klerk, Tania Fleitas, Kevin Liu, Yang Liu, Claire A. Margolis, Felix Dietlein, Anna Maria Chiaravalli, Annacarolina da Silva, Gordon Freeman, Scott J. Rodig, Eliezer M. Van Allen, Bo Li, Jingxin Fu, Jin Wang, Shirley X. Liu, Adam J. Bass. Comprehensive analysis of the suppressed tumor immune microenvironment of CIN gastro-esophageal adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4048.
Abstract 4063: Target immunosuppressive myeloid cells to enhance cancer immunotherapy
Authors: Lu Xin
Published: August 2018

A significant fraction of advanced prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint blockade (ICB) using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/ programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types. However, mCRPC showed overwhelming de novo resistance to ICB, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumor immune evasion. Circulating MDSC abundance correlates with PSA levels and metastasis in PCa patients. Mouse models of PCa show that MDSCs (CD11b+ Gr1+) promote tumor initiation and progression. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of ICB agents together with targeted agents that neutralize MDSCs yet preserve T cell function. Here we developed a novel chimeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and dactolisib, also showed minimal anti-tumor activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when ICB was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of IL-1ra and suppression of MDSC-promoting cytokines secreted by PCa cells. These observations illuminate a clinical path hypothesis for combining ICB with MDSC-targeted therapies in the treatment of mCRPC.

More recently, through mass spectrometry we uncovered a new mechanism by which MDSCs employ potent reactive nitrogen species (RNS) to inactivate key molecules in the T cell activation signaling pathway. Agents that neutralize RNS generate significant anti-tumor effect when combined with ICB in prostate cancer.

Citation Format: Xin Lu. Target immunosuppressive myeloid cells to enhance cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4063.
Abstract 4065: Suppression of myeloid cell arginase activity leads to therapeutic response in Kras mutant lung cancer by activating anti-tumor immunity
Authors: Miret Juan J. , Kirschmeier Paul , Wu Min , Huang Wei , Walker William , Palakurti Sangeetha , Saccomano Nick , Hammerman Peter S. , Wong Kwok-kin , Akbay Esra
Published: August 2018

Metabolic changes in the tumor microenvironment impair the generation of an efficient anti-tumor immune response. Reduction of specific amino acids like arginine and tryptophan limit anti-tumor immune responses, contributing to a tumor generated immune suppressive environment. Arginine levels are critical for lymphocyte proliferation and function, and they are regulated by arginase 1 & 2 and nitric oxide synthase. Arginase 1 activity has been associated with most of the immunosuppressive effects resulting from arginine depletion and the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant established tumor models. RNA sequencing of sorted cell populations from immunocompetent genetically engineered KRAS G12D mutant mouse lung tumors, showed that arginase 1 expression is elevated in the myeloid cell population in the tumor microenvironment. Ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSCs) from mouse tumors hindered T cell function by depleting arginine. These MDSCs expressed elevated levels of arginase 1, and inhibition of its activity by an Arg1/2 inhibitor, compound 9, restored effector T cell function. Treatment of a genetically engineered KRAS mutant mouse model with compound 9 increased arginine levels in blood and tumor and led to an increased number of tumor T cells and a significant reduction in tumor volume after 1 week of treatment. Compound 9 had no growth inhibitory activity across several murine and human KRAS mutant lung cancer cell lines in vitro. We also determined by immunohistochemistry that elevated levels of arginase 1 were expressed in granulocytic myeloid cells from KRAS mutant lung cancer patient samples. Given the data reported here and by others, an arginase inhibitor with excellent potency and in vivo target engagement could represent an important new immunotherapeutic agent. We have recently discovered and are fully characterizing molecules that demonstrate excellent potency and selectivity along with oral bioavailability in rodents that provide good arginase target coverage at modest doses.

Citation Format: Juan J. Miret, Paul Kirschmeier, Min Wu, Wei Huang, William Walker, Sangeetha Palakurti, Nick Saccomano, Peter S. Hammerman, Kwok-kin Wong, Esra Akbay. Suppression of myeloid cell arginase activity leads to therapeutic response in Kras mutant lung cancer by activating anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4065.
Abstract 4067: Toll-like receptor 2 deficiency enhances KRAS-driven lung cancer
Authors: Liu Chia-Hsin
Published: August 2018

Chronic infection and inflammation have been suggested to have a strong association with lung cancer in clinical and epidemiologic studies. Toll-like receptor 2 (TLR2) mediates inflammatory response and is essential in regulating innate and adaptive immunity. TLR2 has also been reported to play either pro-tumorigenesis or tumor-suppressive role depending on the oncogenic driver mutations and cell types. However, the role of TLR2 in lung cancer, especially in the lung adenocarcinoma harboring KRAS mutations, remains unknown. In an analysis using human lung adenocarcinoma cohort (TCGA RNAseq dataset), our results revealed that KRAS-mutant lung tumors showed lower TLR2 mRNA expression than the paired adjacent normal lung tissue. Furthermore, lung adenocarcinoma patients with lower TLR2 expression displayed worse survival outcome. To functionally test the role of TLR2 during KRAS-driven lung tumorigenesis, we generated a mouse model of lung adenocarcinoma mutated for K-ras, with and without TLR2 inactivation (K-rasG12D/+ TLR2-/- and K-rasG12D/+TLR2+/+, respectively). Interestingly, the tumor number and tumor area significantly increased in K-rasG12D/+ TLR2-/- mice compared to K-rasG12D/+TLR2+/+ mice. In addition, the inflammatory cells in bronchoalveolar lavage (BAL), especially the alveolar monocytes, significantly increased in K-rasG12D/+ TLR2-/- mice. Microdissected lung tumors from K-rasG12D/+ TLR2-/- mice showed significantly higher inflammatory chemokines and cytokines gene expression (Ccl1, Ccl2, Cxcl1, Cxcl2, Cxcl5, Csf1, IL-6, Tnf) than those from K-rasG12D/+TLR2+/+mice. Additionally, Cxcl1 was significantly higher in K-rasG12D/+ TLR2-/- when BAL fluid was analyzed for cytokines/chemokines by Luminex assays. Our results suggest that TLR2 deficiency may promote KRAS-mutation driven lung tumorigenesis through increased cancer-related inflammation. Furthermore, the expression may serve as a prognostic biomarker for which the lower TLR2 expression is associated with worse survival outcome in lung adenocarcinoma patients with KRAS mutation.

Citation Format: Chia-Hsin Liu. Toll-like receptor 2 deficiency enhances KRAS-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4067.
Abstract 4077: A new mouse model of gastric adenocarcinoma: A valuable tool for preclinical studies
Authors: Till Jacob , Addai Prince , Bhagwat Neha , Yee Stephanie , Carpenter Erica , Yoon Sam , Ryeom Sandra
Published: August 2018

We recently generated the first model of metastatic mixed-type gastric adenocarcinoma that closely recapitulates the human disease. Our model combines conditional loss of E-cadherin (Cdh1fl/fl) and p53 (Trp53fl/fl) with oncogenic Kras (KrasLSL-G12D/+) activation and yellow fluorescence protein (Rosa26LSL-YFP/LSL-YFP) expression in cells of the gastric parietal cell lineage (Atp4b-Cre). Median survival is 76.5 days (range 64-91 days, SD 7.2) with all mice developing local lymph node metastases and lung metastases, half developing paratracheal lymph node metastases, and 20% developing liver metastases. We previously provided proof of concept for the preclinical utility of our model by demonstrating extended survival upon treatment with a MEK inhibitor (PD0325901). Here we demonstrate the utility of this model to investigate the role of the microbiome in gastric carcinogenesis and to determine the use of circulating tumor cells (CTCs) as biomarkers of metastatic disease. The specific pathogen H. pylori is known to be causal in gastric adenocarcinoma. Further, many studies have implicated the microbiome in colon carcinogenesis. However, no studies have investigated the role of the normal microbiome in gastric carcinogenesis in the absence of H. pylori infection. We treated our model of mixed-type gastric adenocarcinoma with a commonly utilized antibiotic cocktail to deplete the gut microbiota and confirmed depletion of the microbiota by qPCR in stomach and fecal contents at necropsy. Microbiome depletion resulted in a 7.5-day increase in median survival (n=6) over vehicle treated mice (n=5, p=0.04). These data suggest a role for the normal microbiota in gastric cancer progression regardless of H. pylori infection. Given the broadly metastatic phenotype of our model, we examined whether the number of CTCs correlated with metastatic burden. Because our gastric cancer model incorporated a yellow fluorescence protein (YFP) as a marker of tumor cells, we used flow cytometry to measure CTCs in cardiac blood samples from terminal mice. We detected CTCs (defined as DAPI-;CD45-;YFP+) in all terminal mice and correlated CTC counts with lung metastatic burden. Mice with macrometastatic disease in the lung had >90 CTCs per 100 μl blood (n=3) as compared to mice with micrometastatic disease of the lung which showed <15 CTCs per 100 μl blood (n=4, p=0.008). These data suggest that our new mouse model of metastatic gastric adenocarcinoma offers a valuable tool for a variety of preclinical studies.

Citation Format: Jacob Till, Prince Addai, Neha Bhagwat, Stephanie Yee, Erica Carpenter, Sam Yoon, Sandra Ryeom. A new mouse model of gastric adenocarcinoma: A valuable tool for preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4077.
Abstract 4091: Knockout of genes in the DNA mismatch repair pathway to model colorectal cancer hypermutation
Authors: Jin Abbey Y. , Westcott Peter , Smith Olivia , Cruz Amanda , Beytagh Mary Clare , Jacks Tyler
Published: August 2018

Human colorectal cancer (CRC) is characterized by a high burden of somatic mutations, particularly in tumors harboring defects in the DNA mismatch repair (MMR) pathway. However, genetically-engineered mouse models (GEMMs) of cancer to date harbor a very low burden of mutations. Importantly, recent clinical trials have shown that MMR deficient tumors respond favorably to anti-PD-1 immune checkpoint blockade, and it is thought that PD-1 suppression reinvigorates existing T cell clones against neo-antigens associated with hypermutation in these tumors. In order to develop a more clinically relevant GEMM of CRC in which to study the immune response, MMR genes will be knocked out via CRISPR-Cas9 to induce hypermutation. To identify optimal Cas9 single guide RNAs (sgRNAs) against the MMR genes Msh2, Msh3, Msh6, and Mlh1, sgRNAs were designed using the Broad Institute algorithm and knockout efficiency assessed in a reporter cell line by Western blot. In order to induce colon tumors in vivo, colonoscopy-guided submucosal injection was employed to deliver lentivirus encoding sgRNAs against a MMR gene and Apc into mice carrying a constitutive Cas9 allele. Apc is inactivated in over 80% of human CRC, and is an initiating event in CRC in humans and mice. Using a sgMsh2-sgApc dual targeting lentivirus, we were able to efficiently generate colon tumors harboring complete knockout of Apc and near complete knockout of Msh2. Deep sequencing of the targeted loci revealed predominantly loss-of-function frameshift mutations at Apc and Msh2, but also a high degree of polyclonality. Lower titer injections of virus will be explored to induce more clonal tumors. Mlh1, Msh3, and Msh6 will also be targeted in vivo, and the mutational effects of MMR gene loss will be assessed by whole-exome sequencing of tumors. This rapid and flexible GEMM of hypermutant CRC may facilitate a more clinically relevant assessment of the role of the immune system in CRC development, as well as provide improved platforms for testing immunotherapies.

Citation Format: Abbey Y. Jin, Peter Westcott, Olivia Smith, Amanda Cruz, Mary Clare Beytagh, Tyler Jacks. Knockout of genes in the DNA mismatch repair pathway to model colorectal cancer hypermutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4091.
Abstract 4097: Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system
Authors: Manfredonia Celeste , Muraro Manuele Giuseppe , Hirt Christian , Mele Valentina , Governa Valeria , Papadimitropoulos Adam , Daester Silvio , Soysal Savas D. , Droeser Raoul A. , Mechera Robert , Oertli Daniel , Rosso Raffaele , Bolli Martin , Terracciano Luigi M. , Spagnoli Giulio C. , Martin Ivan , Iezzi Giandomenica
Published: August 2018

Colorectal cancer (CRC) is a leading cause of cancer-related death, often diagnosed at advanced stages. Conventional chemotherapeutic regimens have limited success rates, and a major challenge is represented by the lack of adequate in vitro models predicting patient responsiveness to defined treatments, possibly guiding therapeutic decisions. Non-malignant cells, including mesenchymal and immune cells, critically affect development, progression and drug responsiveness of human CRC. However, tumor drug responses are still evaluated on culture systems, such as 2D tumor cell monocultures or on tumor xenografts, which do not preserve all cellular components of in vivo tumor microenvironment.In this work, we have investigated the suitability of a perfusion-based bioreactor for 3D culture of primary CRC samples. Freshly excised CRC specimens were cut into fragments, inserted between two collagen type I sponges in a “sandwich-like” format and cultured for three days in a perfused-based system or under static conditions.We show that cultures under perfusion result in significantly higher maintenance of tissue integrity as compared to static cultures, with preservation of whole tumor microenvironment components, including cancer cells, mesenchymal stromal cells and a fraction of immune cells. Tumor tissues cultured under perfusion displayed an almost intact architecture with viable and proliferating tumor cells. Stromal cells were also maintained in proportions similar to those of original tumors and were fully viable, as indicated by their responsiveness to microenvironmental stimuli, such as IL-17. In addition, immune cells were also partially preserved, and were capable to release effector cytokines upon activation. Importantly, perfusion-based cultures proved suitable for testing sensitivity of primary tumor cells to chemotherapies currently in use for CRC and revealed heterogeneous responsiveness across different samples.

[C.M. and M.G.M. contributed equally to this work.]

Citation Format: Celeste Manfredonia, Manuele Giuseppe Muraro, Christian Hirt, Valentina Mele, Valeria Governa, Adam Papadimitropoulos, Silvio Daester, Savas D. Soysal, Raoul A. Droeser, Robert Mechera, Daniel Oertli, Raffaele Rosso, Martin Bolli, Luigi M. Terracciano, Giulio C. Spagnoli, Ivan Martin, Giandomenica Iezzi. Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4097.
Abstract 4103: Utility of multispectral optoacoustic tomography in imaging pancreatic tumors using a uPA-probe
Authors: Chiba Akiko , Samykutty Abhilash , Smith Mary , McNally Lacey
Published: August 2018

Background: Pancreatic cancer has the lowest 5-year survival rate of all cancer types. Current methods of pancreatic cancer screening, diagnosis and treatment have failed to improve outcome in the last few decades. Potential explanation for this failure may be explained by the hypovascularized nature of pancreatic cancer, making conventional chemotherapy and imaging modality suboptimal. There is a potential for improved imaging using a new imaging modality, multispectral optoacoustic tomography (MSOT). We hypothesize that a using urokinase plasminogen activator (uPA) contrast agent while using MSOT imaging would improve identification of orthotopic pancreatic cancer in xenografts. Methods: Expression of uPA receptor (uPAR) were evaluated in pancreatic tumor cell lines, Panc1, S2CP9, MiaPaca-2 and S2VP10 using western blot. A uPA targeted contrast agent was created using standard bioconjugation methods with the Hilite 750 near infrared (NIR) dye. Five SCID mice were orthotopically implanted with S2CP9 (1.5 x 105) and 5 additional SCID mice were implanted with MiaPaCa2 (2.0 x 106) pancreatic tumor cells. When tumor size reached 3mm, 200µL of 100 nM uPA-750 probe or 750 dye alone was injected into mice. Biodistribution and accumulation of the uPA-750 probe was visualized using MSOT imaged at 2-hour intervals for 8 hours and at 24 hours. Accumulation of uPA-750 probe was evaluated in the tumor, liver, and kidney using NIR fluorescent imaging. Results: All pancreatic cancer cell line expressed uPAR with the highest in S2CP9 cells (3.0x), S2VP10 (2.5x), Panc1 (1.77x) and MiaPaca-2 (1.3x). In vivo, peak intensity of uPA-750 probe was successfully detected using MSOT at 4 hours, within the pancreas tumor in slices from 37mm-41mm. uPA-750 probe was undetectable within the tumor after 8 hours. Using ex vivo NIR fluorescence imaging, uPA-probe signal was detected only within the pancreatic tumor, however, no signal was detected in the liver or kidney. Conclusion: This study demonstrated MSOT imaging using uPA-probe as a contract agent may lead to improved pancreatic cancer detection which may lead to improvement in overall outcome.

Citation Format: Akiko Chiba, Abhilash Samykutty, Mary Smith, Lacey McNally. Utility of multispectral optoacoustic tomography in imaging pancreatic tumors using a uPA-probe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4103.
Abstract 4117: Imaging hypoxia: Development of a PET-optical smart probe
Authors: Klockow Jessica L. , Hettie Kenneth S. , Chin Frederick T.
Published: August 2018

Hypoxia is a characteristic trait of many aggressive tumors. Identification of hypoxic tissues is important for establishing patient treatment plans and prognoses. We show progress towards the development of a hypoxia-activated molecular probe that combines near-infrared (NIR) fluorescence and positron emission tomography (PET) imaging. The fluorescence emission enables facile scrutiny of probe function in vitro and positron emission facilitates translation for clinical use. The probe is a small molecule that possesses a nitro-aromatic group which is sensitive to hypoxic environments. The caged smart probe is non-fluorescent due to photo-induced electron transfer from the nitro-aromatic group. Nitroreductase, an enzyme which is upregulated in hypoxic environments, activates the fluorescence by reducing the nitro group, thereby disabling the quenching effect. Wet chemistry techniques were used to develop the probe which was analyzed by UV/Vis and fluorescence spectroscopies. In vitro and in vivo imaging experiments were also performed. The probe demonstrated membrane permeability and lysosomal accumulation in glioblastoma cells. Additional favorable characteristics include NIR emission and low toxicity to cells. The fluorescence was detected in subcutaneous murine cancer models providing evidence of potential clinical translation toward applications like image-guided surgery. In short, the probe was synthesized in good yield from commercially-available materials and showed promising optical characteristics in cells and animal models. Future work involves F-18 radiolabeling of the probe followed by microPET imaging.

Citation Format: Jessica L. Klockow, Kenneth S. Hettie, Frederick T. Chin. Imaging hypoxia: Development of a PET-optical smart probe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4117.
Abstract 4123: Pancreatic cancer imaging and interventional PTT with a human bispecific scfv antibody via click chemistry
Authors: wang qian , li dan , wang kun , liu chunyan , gao mingyuan , zhao xinming , tian jie
Published: August 2018

Early diagnosis and therapy intervention remains a meaningful task for prolonging pancreatic cancer lifetime and improving prognosis. Successful development of so many highly specific targets emerge, for accurate diagnosis and precise treatment. Herein, we generated bispecific antibody fragment by connecting anti-EGFR scfv and anti-VEGF scfv, via click ligation of TCO and tetrazine. Firstly, Cellvizo imaging was acquired by AF488/647 labeled anti-EGFR scfv and anti-VEGF scfv, respectively, displayed overexpression and metabolic process in real time. IVIS and 3D MSOT imaging of mice bearing anthropogenic cell line of bxpc3(EGFR+/VEGF+) subcutaneous and orthotopic and tumors with Irdye800 labeled Bs-(scfv)2 revealed a more significantly enhancement and long circulation of tumor uptake and tumor-to-background ratio compared with each special scfv group(T/B ratio of 12.1±3.4 at 4h post-injection, n=5 in IVIS image). Thus, we validated that dual targeting of VEGF and EGFR provides a high affinity and specificity by Irdye800-labeled-Bs-(scfv)2. Finally, we successfully replaced irdye800 with PLA-PEG-NHS@ICG (combined or uncombined GEM) and conjugated Bs-(scfv)2 for interventional PTT, compared with GEM treatment, a 18% higher median survival rate of PTT combined GEM has been achieved. Our finding offer a preclinical proof concept for Bs-(scfv)2 PTT in clinical trial in future, due to effective bio-distribution and high affinity.

Citation Format: qian wang, dan li, kun wang, chunyan liu, mingyuan gao, xinming zhao, jie tian. Pancreatic cancer imaging and interventional PTT with a human bispecific scfv antibody via click chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4123.
Abstract 413: Emerging principles in synthetic lethality in glioblastoma
Authors: Hoellerbauer Pia , Arora Sonali , Kufeld Megan , Carter Lucas , Girard Emily J. , Feldman Heather , Corrin Philip , Olson James M. , Paddison Patrick J.
Published: August 2018

Synthetic lethality occurs when mutations in two otherwise nonessential genes are combined to cause lethality. Because cancer is a disease of genetic alteration, synthetic lethality has been heralded as a method to identity candidate therapeutic targets, e.g., where a target gene could be "synthetic lethal" with a cancer driver mutation. To define synthetic lethal relationships in glioblastoma (GBM), we have performed multiple focused-set and genome-wide CRISPR-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs) and nontransformed human neural progenitor cells. Because GSCs isolates likely represent a sub-clone of the original tumor and we can determine GSCs' genetic and epigenetic makeup, it is possible to address the concept of synthetic lethality for GBM. To this end, we recently performed comprehensive CRISPR-Cas9 retests of all scoring GBM lethal genes (>900) from screens in three patient isolates with different and overlapping genetic drivers. We then performed secondary retests of high-priority gene targets in 13 GSC harboring various alterations commonly found in GBMs, e.g., EGFRamp, NF1mut, PIK3CAmut, PTENloss/mut, TP53mut, etc. The results were surprising, first in what we did not find. We failed to find synthetic lethal targets for TP53loss/mut, RB1mut, or TERT expression, suggesting that synthetic lethal relationships for these alterations may not exist for GBM. Second, NF1mut interactors defined a broader class of synthetic lethal targets with general overactivity of the RTK/Ras pathway, which can arise from various activating lesions. Third, candidate synthetic lethal relationships can be observed, but, so far, only with EGFRamp, MYC/MYCNamp, and PTEN/PIK3CA alterations. Thus, in general our results suggest that the majority of synthetic lethal relationships in GBM arise from oncogenic activation of the RTK/Ras and PI-3 kinase pathway or amplification of MYC/MYCN. (Synthetic lethal targets will be revealed and discussed at the meeting.)

Citation Format: Pia Hoellerbauer, Sonali Arora, Megan Kufeld, Lucas Carter, Emily J. Girard, Heather Feldman, Philip Corrin, James M. Olson, Patrick J. Paddison. Emerging principles in synthetic lethality in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 413.
Abstract 4131: Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors
Authors: Nes Johan V. , Groningen Tim V. , Koster Jan , Valentijn Linda , Zwijnenburg Danny , Westerhout Ellen M. , Hamdi Mohamed , Versteeg Rogier
Published: August 2018

Background: Core Regulatory Circuitries (CRCs) of lineage transcription factors associate with super-enhancers and are drivers of cellular identity. We recently showed that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal (MES) cells and committed adrenergic (ADRN) tumor cells resemble cells from developmental stages of the adrenergic lineage.

Results: We identified the unique super-enhancer (SE) landscapes of MES and ADRN-type cells. SEs associated with lineage transcription factors that identified the Core Regulatory Circuitries (CRC) for each cell type. MES cells were resistant to chemotherapy. Accordingly, MES cells were strongly enriched in post-treatment samples, suggesting that MES cells could play a role in resistance and relapse development. MES and ADRN cells of isogenic origin spontaneously interconverted, showing the transcriptional plasticity of CRCs. We studied the mechanism of MES and ADRN transdifferentiation. The MES TFs PRRX1 and NOTCH were expressed as single genes in ADRN cells. Each of the genes induced a step-wise reprogramming of the ADRN transcriptome towards a MES state. The transcriptional switch was accompanied by genome-wide remodeling of the epigenome to a MES enhancer state. MES TFs repressed super-enhancers of ADRN core TFs, leading to transcriptional downregulation of the ADRN CRC. Deregulated enhancers associated with Polycomb repression. The transdifferentiation was initially reversible, but became gradually stabilized. The transgenes activated an endogenous feed-forward loop including ligands, receptors and co-factors from the NOTCH signaling route. Ultimately, this endogenous NOTCH-cascade maintained a transgene-independent MES state. Super-enhancers in stable MES cell lines associated with NOTCH receptors and co-factors, consistent with NOTCH driving MES lineage identity. Both NOTCH-induced MES cells and ADRN cells were tumorigenic in vivo.

Conclusions: Our results demonstrate that single TFs from the MES CRC impose transdifferentiation via remodeling of the epigenetic and transcriptional landscape of ADRN cells, mimicking spontaneous interconversion. Plasticity of CRCs and lineage identity may have profound implications for treatment strategies in neuroblastoma.

Citation Format: Johan V. Nes, Tim V. Groningen, Jan Koster, Linda Valentijn, Danny Zwijnenburg, Ellen M. Westerhout, Mohamed Hamdi, Rogier Versteeg. Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4131.
Abstract 4139: SNAI2 inhibition promotes myogenic differentiation and prevents tumorigenic features of embryonal rhabdomyosarcoma
Authors: Pomella Silvia , Carcarino Elena , Cossetti Cristina , Locatelli Franco , Rota Rossella
Published: August 2018

Pediatric Rhabdomyosarcoma (RMS) is a soft tissue sarcoma of childhood that in its high risk form has a dismal prognosis. RMS includes two histological subtypes, embryonal and alveolar, which differ clinically and molecularly. Both are characterized by the expression of Myogenic Regulatory Factors (MRFs) such as MYOD and MYOG but tumor cells are unable to differentiate in skeletal muscle cells and proliferate indefinitely. For this reason, differentiation therapies have anticancer potential in this tumor. Interestingly, one of the cause of the inability of MRFs in inducing myogenic-like differentiation is related to the loss of transcriptional activity of these factors partly due to the lack of availability of DNA binding sites and/or of capability to form transcriptional complexes. We demonstrate here that the repressor SNAI2 is upregulated in embryonal RMS (ERMS) patients and cells lines compared to their normal counterparts. Silencing of SNAI2 in 2 ERMS patient-derived cell lines using a puromycin pLKO lentiviral vector expressing a SNAI2 shRNA results in a slowdown of cell proliferation associated to p21Cip1 and Myogenin upregulation starting 3 days post-selection, as compared to control vector. These molecular changes are followed by the induction of Myosin-Heavy Chain and the formation of myogenic-like multinucleated structures reminiscent of myogenic fibers. Moreover, colony formation and anchorage independent growth are both strongly impaired. Finally, SNAI2 depleted cells form smaller spheres when cultured in cancer stem cells conditions. Therefore, SNAI2 down-regulation could have therapeutic value in ERMS. Studies are ongoing to verify the restoring of MYOD transcriptional activity and to identify genes regulated by SNAI2 in our system.

The work is supported by Associazione Italiana Ricerca sul Cancro project IG15312 to RR.

Citation Format: Silvia Pomella, Elena Carcarino, Cristina Cossetti, Franco Locatelli, Rossella Rota. SNAI2 inhibition promotes myogenic differentiation and prevents tumorigenic features of embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4139.
Abstract 414: Broad range chromosomal abnormality detection through Bionano genome mapping
Authors: Hastie Alex R.
Published: August 2018

Current methods for detection of balanced structural variation can be broken down into two categories: traditional cytogenetics and molecular methods. Cytogenetics may include chromosomal karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray and adaption of them. Molecular methods primarily include NGS sequencing based methods. Bionano genome mapping, an optical mapping approach in a NanoChannel array system, is a method that combines the advantages of different categories while solving many of the limitations. Compared to cytogenetics, Bionano mapping is high throughput and removes manual interpretation, it also has much higher resolution, detecting balanced events as small as about 30 kbp compared to multi-megabases needed for cytogenetic approaches, and unbalanced events starting at 1 kbp. NGS based methods often are limited by read lengths that cannot provide unambiguous information across repeat elements longer than individual reads. This limitation results in significantly reduced sensitivity for balanced variation and abnormalities as well as for insertions and even some categories of larger deletions. This is particularly true in highly medically relevant locations where segmental duplications mediate chromosomal abnormalities. Genome mapping using Bionano Genomics' Saphyr System offers high-throughput, genome-wide visualization of extremely long DNA molecules in their native form. It allows researchers to interrogate genomic structural variations (SVs) in the range of one kilobase pair and above. We present several in silico and biological validation experiments that demonstrate the sensitivity and specificity of Bionano mapping for the detection of insertions, deletions, inversions and translocations compared to benchmark studies using short read and long read sequencing. We also show a new direct labeling method that dramatically improves assembly contiguity such that chromosome arms can be assembled into single maps. Bionano mapping is a fast and cost effective method for the detection of a broad range of traditionally refractory SVs across the human genome.

Citation Format: Alex R. Hastie. Broad range chromosomal abnormality detection through Bionano genome mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 414.