bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3669: Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis
Authors: Cuya Selma M. , Pilgrim Adeiye , Rathi Komal , Chen DongDong , Schnepp Robert
Published: August 2019

Background: Patients with high-risk neuroblastoma endure an extremely intense, multidrug treatment regimen and yet, approximately half of them die, thus mandating a better approach to this cancer. Our laboratory is investigating the overarching hypothesis that RNA binding proteins (RBPs), which play key roles in balancing self-renewal, differentiation, and cell proliferation, are deregulated in neuroblastoma, and drive oncogenic signaling networks. Therefore, RBPs may constitute novel targets for therapeutic development. In this work, we focus on Musashi 2 (MSI2), an RBP that has been shown to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumors, notably in acute myeloid leukemia and in colon cancer. The role of MSI2 in neuroblastoma, however, has not been assessed and is the focus of this investigation.

Methods: To test this hypothesis, we used in silico analysis of neuroblastoma gene expression datasets annotated with clinical parameters. Additionally, we used shRNAs to manipulate transcripts of interest in neuroblastoma cells and measured effects on cell proliferation, cell cycle status, and apoptosis.

Results: Gene expression profiling revealed that MSI2 is robustly expressed in neuroblastoma cell lines, tumors, and patient derived xenografts, within both the MYCN and non-MYCN amplified context. Immunoblotting in a representative panel of lines confirmed the expression of MSI2 at the level of protein. High MSI2 expression was associated with more advanced stages of neuroblastoma across multiple independent datasets and was correlated with worse survival. Depletion of MSI2 using four independent shRNAs led to 2 to 3-fold decreased proliferation across four cell lines that was due in part to increased apoptosis. Another RBP, ELAVL1, is also expressed within neural lineages, and has been shown to regulate MSI1 expression. We hypothesized that ELAVL1 might similarly promote MSI2 expression and we demonstrated that, ELAVL1 depletion led to decreased MSI2 expression.

Conclusions: MSI2 is robustly expressed in multiple models of neuroblastoma and appears to drive increased proliferation and survival. Currently, we are investigating the functional interplay between ELAVL1 and MSI2 providing a foundation for designing strategies to target the ELAVL1-MSI2 interaction for therapeutic effect.

Citation Format: Selma M. Cuya, Adeiye Pilgrim, Komal Rathi, DongDong Chen, Robert Schnepp. Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3669.
Abstract 367: The connection of vitamin D3 induced cytotoxicity with the vitamin D3 receptor
Authors: Hoshi Takaya , Abe Yuta , Sugiyama Ikumi , Sadzuka Yasuyuki
Published: August 2019

[Purpose] This study aims to evaluate the possible antitumor effect of vitamin D. Vitamin D is a group of lipophilic compounds comprising of vitamin D2 and vitamin D3. Vitamin D3 has been shown to play an important role in humans. It has been previously reported that the onset risk of malignant melanoma and leukemia can be decreased by maintaining high vitamin D3 levels in the body. In addition, the cytotoxic effect of vitamin D on breast cancer cells has also been shown. In this study, the relationship between the cytotoxicity of vitamin D3 and vitamin D3 receptor (VDR) expression on melanoma and leukemia cells was examined.

[Method] B16F10 melanoma (B16F10) and P388 leukemia (P388) cells were used in this study. Western blot analysis was performed to determine VDR expression in each cell type. To evaluate cytotoxicity, B16F10 or P388 cells were seeded at 1×105 cells/mL, and calcitriol as vitamin D3 (range 9 ng/mL-90 μg/mL) was added at once. After two days, cytotoxicity was evaluated by colorimetric analysis using Cell Counting Kit-8. Additionally, the cytotoxicity of P388 cells was determined following the combined treatment of vitamin D3 and retinoic acid.

[Results and Discussion] Western blot analysis revealed that both B16F10 and P388 cell types, expressed VDR with a higher expression in P388 cells. In P388 cells, there was a significant reduction in cell viability following vitamin D3 treatment, which decreased to 10% at 90 μg/mL. In contrast, the viability of B16F10 cells following vitamin D3 treatment only slightly decreased. Interestingly, these results suggest that the cytotoxicity of vitamin D3 correlates with VDR expression. VDR reportedly forms a heterodimer with the retinoid X receptor. However, the presence of retinoic acid did not alter vitamin D3 cytotoxicity. Therefore, it was considered that cytotoxicity induced by vitamin D3 was not connected with the formation of the VDR-retinoid X receptor heterodimer. In conclusion, there was a higher expression of VDR in P388 cells compared with B16F10 cells. This higher expression correlated with a higher level of vitamin D3 cytotoxicity, suggests an antitumor effect of vitamin D3. This study proposes the possibility of using vitamin D3 as a novel treatment for leukemia and other cancers.

Citation Format: Takaya Hoshi, Yuta Abe, Ikumi Sugiyama, Yasuyuki Sadzuka. The connection of vitamin D3 induced cytotoxicity with the vitamin D3 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 367.
Abstract 3677: Downregulation of FOXO3a by DNMT1 promotes breast cancer stem cell properties and tumorigenesis
Authors: He Zhimin , Liu Hao , Song Ying , Zheng Guopei
Published: August 2019

Breast cancer stem cells (BCSCs) are tumor initiating cells that can self-renew and are highly tumorigenic and chemoresistant. Recent evidence indicated that FOXO3a plays an important role in regulating CSCs properties. However, the biologic function and detailed molecular mechanism of FOXO3a in BCSCs remains largely elusive. Here, we demonstrated that FOXO3a was hypermethylated and downregulated in breast cancer cells and tissues. Overexpression of FOXO3a potently attenuated the BCSCs properties in vitro and inhibited tumorigenesis in vivo, whereas knockdown of FOXO3a increased these properties. Further mechanistic investigation revealed that FOXO3a was functionally linked to the inhibition of the FOXM1/SOX2 pathway. Moreover, we found that SOX2 directly transactivated DNMT1 gene expression and then feedback inhibited FOXO3a expression. Clinically, we observed a significant inverse correlation between FOXO3a and FOXM1 or SOX2 expression levels. Loss of FOXO3a expression also predicted poor prognosis in breast cancer. In addition, we demonstrated that inhibition of DNMT activity suppressed tumor growth via regulating of FOXO3a/FOXM1/SOX2 signaling in breast cancer. Our findings suggest an important role of DNMT1/FOXO3a/FOXM1/SOX2 pathway in regulating BCSCs properties, identifying potential therapeutic targets for breast cancer.

Keywords: breast cancer, cancer stem cell, FOXO3a, DNMT

Note: This abstract was not presented at the meeting.

Citation Format: Zhimin He, Hao Liu, Ying Song, Guopei Zheng. Downregulation of FOXO3a by DNMT1 promotes breast cancer stem cell properties and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3677.
Abstract 368: Next generation DNA aptamers with prolonged stability and antitumor activity
Authors: AMERO Paola , Rodriguez-Aguayo Cristian , Chaudhari Rajan R. , Zhang Shuxing , Sood Anil K. , Lopez-Berestein Gabriel
Published: August 2019

Aberrations in Gas6/AXL signaling is associated with many human diseases including ovarian cancer, where in patients expressing high levels of AXL have shorter overall survival than patients expressing low levels. Aptamers are short synthetic oligonucleotides (DNA or RNA) that can bind with highest specificity to various other macromolecules such as peptides, proteins and carbohydrates, based on their tertiary structures. Aptamers are similar in their functionality to antibodies in their specificity;

easier to manufacture than antibodies; have minimal side effects; and thus hold a high promise as therapeutics. The typical issues with the use of aptamers as therapeutic agents are their selectivity, stability, and bioavailability. We present the development of the next generation DNA aptamers, targeting p-AXL specifically to as a therapeutic in ovarian cancer (OC) by chemical modifications to their primary sequence. Improved bioavailability and stability, based on the comparative analysis the library of 17 DNA aptamers that differed in the position and number of fluoro- and thio- modifications on their nucleotide sequence, were used as the criteria for the selection and characterization of the best aptamer candidates to target p-AXL. Two of the best candidates, GLB-G25 and GLB-A04 thus chosen with the highest bioavailability and stability, when tested in ovarian cancer cell lines, decreased the migration and invasion of cells in vitro. Treatment of ovarian cancer orthotopic murine model animals with modified DNA aptamer candidates chosen above significantly reduced tumor growth and the number of metastases. The candidates, GLB-G25 and GLB-A04 show highest potential to be developed into therapeutics to target p-AXL that is elevated in several cancers including ovarian cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Paola AMERO, Cristian Rodriguez-Aguayo, Rajan R. Chaudhari, Shuxing Zhang, Anil K. Sood, Gabriel Lopez-Berestein. Next generation DNA aptamers with prolonged stability and antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 368.
Abstract 3685: HOTAIR regulation and functionality in ovarian cancer stem cells
Authors: Wang Weini , Fang Fang , Ozes Ali , Nephew Kenneth P.
Published: August 2019

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. We have previously shown that epigenetic changes in OCSCs play a role in post-therapy OCSC persistence and demonstrated that is it possible to target OCSC using epigenetic therapies. HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and to be overexpressed in many types of cancers, including high-grade serous OC (HGSOC). HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and due to its histone methyltransferases activity plays a key role in chromatin remodeling. Because HOTAIR is a known epigenetic regulator of differentiation and developmental genes in OC and other cancers, we hypothesize that HOTAIR is a key epigenetic regulator in OCSCs and therapeutically targeting HOTAIR in OCSCs will prevent tumor relapse. The goal of this study is to understand the role of HOTAIR in regulating OCSCs and further dissect the mechanism of action of HOTAIR in OCSCs. Aldehyde dehydrogenase (ALDH) activity and FACS was used to separate OCSCs from non-OCSCs in a panel of HGSOC cell lines (OVCAR3, CAOV3, OVCAR5, Kuramochi, COV362). Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSC cells compared to non-OCSC cells. Knockout of HOTAIR using CRISPR-Cas9 system significantly decreased OCSC population and stemness-related phenotypes, including spheroid formation and colony formation ability. Overexpression of HOTAIR in HGSOC cells significantly increased these stem-like characteristics. Furthermore, targeting HOTAIR using peptide nucleic acid (PNA), which blocks interaction with Enhancer of Zeste Homologue 2 (EZH2), significantly decreased the OCSC population, indicating HOTAIR functions through EZH2 in regulating OCSCs. Combining the HOTAIR-targeting PNA with other epigenetic inhibitors, including the hypomethylating agent guadecitabine and the EZH2 inhibitor GSK503, significantly decreased proliferation and colony formation ability of HGSOC cells. We suggest that a better understanding of HOTAIR will facilitate identifying epigenomic alterations and chromatin landscape that contributes to OCSC phenotypes. Targeting HOTAIR in combination with epigenetic therapies may represent a therapeutic strategy to prevent tumor relapse.

Citation Format: Weini Wang, Fang Fang, Ali Ozes, Kenneth P. Nephew. HOTAIR regulation and functionality in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3685.
Abstract 3687: Colorectal cancer stem cell expressing POU5F1 promotes liver metastasis
Authors: Miyoshi Norikatsu , Fujino Shiki , Sasaki Masaru , Saso Kazuhiro , Takahashi Hidekazu , Haraguchi Naotsugu , Hata Taishi , Matsuda Chu , Mizushima Tsunekazu , Mori Masaki , Doki Yuichiro
Published: August 2019

Background: The gene POU5F1, encoding the POU domain, class 5, is a transcription factor and the expression is found in embryonic stem cells. POU5F1 gene controls self-renewal and differentiation, and regulates pluripotency and proliferation in embryonic stem cells. However, the role of POU5F1 in metastatic CRC (mCRC) is not well known. We focused on the relationship between POU5F1 gene expression and liver metastasis of CRC and clarify the role and abilities of POU5F1-positive CRC cells.

Methods: The study included 158 patients with CRC who underwent surgery from 2009 to 2011. The correlations between the POU5F1 gene expression and the clinical parameters were assessed, and liver-metastasis-free survival (LMFS) was evaluated in these patients. POU5F1-EGFP-positive cells were established by the construct with lentiviral vector, and we examine their sub-population and ability. The capacity to form liver metastasis in vivo was examined using CRC cell lines and primary cultured CRC cells.

Results: In the survival analysis, LMFS was significantly poor in the high POU5F1 expression group compared to the low-expression group (P=0.008). Multivariate analyses showed that POU5F1 expression (P=0.015) and TNM stage (p<0.001) significantly correlated with LMFS. POU5F1-EGFP-positive cells highly expressed stem-cell-associated markers and they had self-renewal and differentiation abilities. POU5F1-high cells actively formed liver metastasis.

Conclusion: The POU5F1expression correlated with liver metastasis in CRC patients. POU5F1-positive cells have the self-renewal and differentiation abilities as cancer stem cells. POU5F1 contributes to the ability forming liver metastasis in CRC.

Citation Format: Norikatsu Miyoshi, Shiki Fujino, Masaru Sasaki, Kazuhiro Saso, Hidekazu Takahashi, Naotsugu Haraguchi, Taishi Hata, Chu Matsuda, Tsunekazu Mizushima, Masaki Mori, Yuichiro Doki. Colorectal cancer stem cell expressing POU5F1 promotes liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3687.
Abstract 3692: The effect of endothelin 1 on cancer stem cell development
Authors: Seno Akimasa , Seno Masaharu
Published: August 2019

Although we have been demonstrating the induction of cancer stem cells from ESCs or human/mouse iPSCs reprogrammed from normal fibroblasts using cancer cell conditioned media, the exact inducing factor is still unknown. Previously, we compared the gene expression profiles among hiPS/ESC, cancer stem like cells induced from hiPSC with cancer cell conditioned media, cancer stem cells obtained from cancer tissue or cell culture, and cancer cells. In this study, the expression of endothelin 1 was found commonly upregulated in different cancer stem cell groups. To investigate the roles of endothelin 1 in the development of cancer stem cells, the significance of endothelin 1 signaling including endothelin 1 in the cancer cell conditioned media and the expression of endothelin 1 receptors in iPSCs as well as in the developed cancer stem cells were quantitatively and qualitatively evaluated.

Citation Format: Akimasa Seno, Masaharu Seno. The effect of endothelin 1 on cancer stem cell development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3692.
Abstract 3697: Optimization of human cancer cell xenografts into zebrafish larvae for high-throughput drug screening
Authors: Dockins Stephen R. , Blackburn Jessica S. , Haney Meghan G. , Wilson Bradley
Published: August 2019

The use of zebrafish in cancer xenograft models has grown rapidly in recent years. This is primarily due to the fact that this model takes advantage of the ease of in vivo imaging and the high-throughput screening capabilities that zebrafish have to offer. However, researchers have yet to come to a consensus on standardized procedure to utilize zebrafish for xenograft of human cells. This study aims to optimize a zebrafish xenografting protocol for various human cancers with the intention of performing high-throughput drug screening.

We tested the survival of 3 different zebrafish strains when incubated at 28°C, 34°C and 37°C. There was no difference in survival between the AB, SAT, and Casper strains of zebrafish at any temperature. Human leukemia, breast, lung, colon, and brain cancer cell lines were fluorescently labelled with Vybrant DiI cell staining dye and injected at a concentration of 100, 250, 500, or 1000 cells per 2nL droplet volume into dechorionated 2-day-old zebrafish embryos. The injection sites used were the brain, caudal vein, duct of Cuvier (DC), eye, pericardium, perivitelline space (PVS), and yolk sac. As expected, injections became more difficult and time consuming when using higher cell counts as the injector needle clogged more often. Similarly, injection sites that required more precision like the caudal vein or eye took the longest to inject successfully and had lower engraftment rates than sites like the yolk or DC. We determined that using 500 cells per injection was optimal as engraftment level was comparable to 1,000 cell injections but had improved survival and reduced injection time. Optimal injection site varied between cell lines, but overall, injections into the duct of Cuvier resulted in consistently high survival and engraftment rates with one of the lowest injection times.

We are in the process of determining the extent to which the injection site affects chemotherapy response on human cells implanted into zebrafish. We are also preforming a high-throughput drug screen on human leukemia cells implanted into zebrafish to provide proof-of-principle that these methods are useful in identifying novel anti-cancer compounds. In total, this work will establish standard operating procedures for use of xenograft in zebrafish, providing new opportunities in personalized medicine and drug discovery.

Citation Format: Stephen R. Dockins, Jessica S. Blackburn, Meghan G. Haney, Bradley Wilson. Optimization of human cancer cell xenografts into zebrafish larvae for high-throughput drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3697.
Abstract 3703: Development of oncopig urothelial carcinoma cell lines
Authors: Qazi Aisha , Thomas Faith M. , Chaki Sulalita , Robertson Noah , Patel Shovik , Schachtschneider Kyle M. , Schook Lawrence B.
Published: August 2019

Urothelial carcinoma (UC), more commonly known as bladder cancer, affects approximately 81,000 people a year in the United States and is the fourth most common type of cancer among men. Age also plays a tremendous factor, as 90% of people affected by bladder cancer are older than 55, and if the cancer has metastasized the 5-year survival rate is 35%. This requires the advancement of early detection and treatment methods for bladder cancer. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgene. In this study, we standardized a protocol for the development and growth of Oncopig UC cell lines that faithfully recapitulates in vitro and in vivo features of human bladder cancer.

The focus of this preliminary research was to successfully create OCM UC cell lines that mimic features of human UC. OCM urothelial cell lines (n=7) were isolated from whole bladders collected from male Oncopigs at euthanasia. OCM urothelial cell lines were exposed to Cre recombinase 48 hours post isolation to induce expression of KRASG12D and TP53R167H transgenes, resulting in development of OCM UC cell lines. Differences in migration rates were observed between OCM urothelial and UC cell lines, confirming cellular invasiveness. RT-PCR was performed after several passages to confirm KRASG12D and TP53R167H gene expression, which was observed in OCM UC but not urothelial cell lines. Uroplakin II and pan-Cytokeratin (PCK-26) are markers for UC and urothelial cells, respectively, and are used to diagnose UC clinically. OCM UC cells stained positively for Uroplakin II and PCK-26, confirming their identity as malignant UC cells. Future work is needed to confirm OCM and human UC display similar histological and molecular features, and to develop strategies for in vivo OCM UC tumor development.

Citation Format: Aisha Qazi, Faith M. Thomas, Sulalita Chaki, Noah Robertson, Shovik Patel, Kyle M. Schachtschneider, Lawrence B. Schook. Development of oncopig urothelial carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3703.
Abstract 3704: CRISPR screening in a xenograft model for in vivo drug moa analysis
Authors: Melton Michele , Bast Athena , Leeson-Payne Alastair , Blanck Maximilian , Lawo Steffen , Stacy Deeds , Cross Benedict C.S.
Published: August 2019

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification.Until now, these exquisitely powerful screening technologies have been deployed predominantly in simple in vitro analyses. Whilst these in vitro approaches allow for high quality hit discovery and at whole-genome level scale, they are not able to adequately capture the impact of tumour microenvironment and heterogeneity on the genetic perturbations under study. To address this, we present here the development and validation of a platform for pooled CRISPR knock-out screening in xenograft models of tumour growth.

A critical step in appropriate study design is the evaluation of the cell uptake following implantation. Since this directly contributes to the tumour growth and cell number, understanding cell survival dynamics post-implantation is necessary to determine the appropriate experimental scope. We used a barcoding approach coupled to next generation sequencing to determine the response and contribution of individual A375 melanoma cells to final tumour composition and used this to calibrate the experimental design for robust pooled-based CRISPR screening analysis.

Following the determination of experimental conditions, a proof-of-concept drug mechanism of action analysis was conducted using a custom designed cancer genome CRISPR knock-out library. A375 cells were first infected with the CRISPR library and selected ex vivo ahead of implantation and treatment of established tumour-bearing nude mice with a defined therapeutic regime of the BRAFV600E selective inhibitor, Vemurafenib. Tumours were harvested and the identification of genes which selectively modulate the tumour response to drug were identified by next generation sequencing using our optimised screening analysis platform.

Our results provide a robust experimental validation and define a novel platform which will allow the rapid and definitive in vivo evaluation of both drug and gene interactions using powerful CRISPR-based screening tools.

Citation Format: Michele Melton, Athena Bast, Alastair Leeson-Payne, Maximilian Blanck, Steffen Lawo, Deeds Stacy, Benedict C.S. Cross. CRISPR screening in a xenograft model for in vivo drug moa analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3704.
Abstract 3708: Multiomic validation of canine simple carcinoma as a model for human basal-like breast cancer
Authors: Watson Joshua L. , Feng Yuan , Zhao Shaying
Published: August 2019

Roughly 20% of all breast cancer (BC) cases in humans are classified as basal-like breast cancer (BLBC), the deadliest subtype with a five-year survival prognosis lower than any other subtype. BLBC usually lacks the clinically targetable receptors estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). One of the main challenges in BLBC research is the lack of a spontaneous cancer model. To address this deficiency, we performed a comprehensive dog-human multiomic comparison. Briefly, we characterized the genomic and transcriptomic alterations of >100 spontaneous canine mammary cancer cases, including simple carcinoma (SC), a common subtype in pet dogs, from our own data and published studies by others. We then evaluated the dog-human molecular homology by comparing our canine findings to the studies of BLBCs from The Cancer Genome Atlas (TCGA).

Our study shows a strong dog-human molecular homology. First, cross-species PAM50 classification indicates significant grouping of canine SC with human BLBC, but not other BC subtypes. This is supported by unsupervised clustering of a panel of recently published ~1800 cancer genes that characterize human BLBC. Second, gene set enrichment analysis with 44 BC molecular signatures, 20 of which are BLBC-specific, indicates that canine SCs have the same enrichment patterns as human BLBCs. Third, canine SCs show the same overall copy number alteration (CNA) patterns as human BLBCs. Specifically, canine SCs have significantly more amplified/deleted genes and a much greater percentage of the genome with CNA than other mammary cancer subtypes of the dog, which is also true for human BLBC. Furthermore, canine SC shares significantly more amplified/deleted genes with BLBC than any other BC subtypes. Importantly, these shared genes are strongly enriched in known BLBC signatures, including the 8q23-24 amplicon (containing the oncogene MYC) and chromosomal instability regulation. Finally, as with the PAM50 analysis, unsupervised cross-species clustering with amplified/deleted genomic regions and genes indicates a strong homology between canine SCs and human BLBCs.

In conclusion, canine SC faithfully recapitulates the molecular features of human BLBC, and can effectively serve as a much-needed spontaneous cancer model for BLBC research. Canine SC will accelerate basic research, e.g., providing a novel dog-human comparison strategy for cancer driver-passenger discrimination. Canine SC will also accelerate the bench-to-bedside translation by bridging the gap between preclinical studies in mice and human clinical trials.

Citation Format: Joshua L. Watson, Yuan Feng, Shaying Zhao. Multiomic validation of canine simple carcinoma as a model for human basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3708.
Abstract 3714: Efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic, ovarian and renal tumors
Authors: Hardt Olaf , Agorku David , Langhammer Anne , Zickgraf Franziska , Geist Felix , Noll Elisa M. , Eisen Christian , Bosio Andreas , Sprick Martin R. , Trumpp Andreas
Published: August 2019

Cancer cell lines are widely used as in vitro models to study tumor biology and for efficacy testing of novel anti-cancer therapeutics. In the past, most of this work has been done using established cell lines that have been cultured for decades. Extensive in vitro propagation has been shown to lead to the acquisition of multiple genetic and epigenetic alterations in cell lines, leading to decreased heterogeneity and the lack of tumor initiating as well as multi-lineage differentiation capacity. Consequently, established cell lines only insufficiently resemble the characteristics of tumors and thus have limited applicability as in vitro models for example. To overcome this hurdles, cell lines can be derived from primary cancer biopsies. However, this process is very inefficient for most tumor entities. In addition, most of the media used include largely undefined serum, such as FBS, which has been shown to drive primary tumor cell cultures to a more differentiated state when used over multiple passages. We have developed advanced, serum-free media for derivation and expansion of tumor cell lines from pancreatic, ovarian or renal tumors. Our media have been optimized concerning formulation, stability, and usability and allow for efficient generation of primary cell lines from both, patient and xenotransplanted tumors. Primary cell lines derived with our media retained their tumorigenic potential and could therefore be xenotransplanted and propagated in immunodeficient mice. Notably, the resulting tumors closely resembled the initial patient tumor as shown on the histomorphological as well as functional level. Moreover, the primary cell lines closely resembled essential characteristics of the parental tumor in vitro, including expression of subtype-specific markers, cellular heterogeneity, as well as genetic and epigenetic signatures. Taken together, we have developed serum-free medium for efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic, ovarian and renal tumors, allowing for the establishment of easily accessible in vitro as well as corresponding xenografting vivo models. This facilitates the translation of in vitro findings directly into in vivo settings, allowing for more reliable pre-clinical modelling.

Citation Format: Olaf Hardt, David Agorku, Anne Langhammer, Franziska Zickgraf, Felix Geist, Elisa M. Noll, Christian Eisen, Andreas Bosio, Martin R. Sprick, Andreas Trumpp. Efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic, ovarian and renal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3714.
Abstract 3716: Conditional reprogramming technology for Next Generation Living Biobanks (NGLB)
Authors: Liu Xuefeng
Published: August 2019

As one of the next generation biobanks, living biobanks (iPSC, organoids, CRC, PDX, etc.) and related functional analyses are rapidly growing in basic research, clinics, and industry (especially immune-oncology and targeting therapies) as well. Most recently NCI launched PDMR (patient-derived model repository) using PDX, CRC and organoid cultures, NCI/ATCC/Broad/Sanger launched HCMI (Human Cancer Models Initiative) using Organoids and CRC cultures. In this study, we established a conditional reprogramming (CR) protocol which allows to generate cell cultures from cryopreserve fresh tissue specimens from surgery, core biopsies, needle biopsies, and brushed cells.It has been extremely difficult to generate primary cultures from tumor specimens, it has not been possible to expand and indefinitely propagate cells derived from adult tissues while retaining lineage-commitment, normal growth control and differentiation potential. The CR method rapidly expands both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of cell cultures from both normal and tumor tissue (many types) is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 x 106cells in 5 days from a core biopsy of breast tumor. Normal cultures retain a normal karyotype and differentiation potential and cell lines derived from tumors retain their tumorigenic phenotype. We were also able to generate their corresponding fibroblast cultures from the same specimens. These fibroblasts can be immortalized by exogenously expressed hTERT. This approach allows to establish in vitro "epithelial-stroma interactions" to study normal cell differentiation and tumor microenvironment. The ability to produce inexhaustible cell populations from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (termed as NGLB - next generation living biobanks) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool (or a living biomarker) for precision cancer medicine. CR cells can be also an inexhaustible resource to generate CR cells derived iPSC, organoids, and xenografts (CDXs).

Citation Format: Xuefeng Liu. Conditional reprogramming technology for Next Generation Living Biobanks (NGLB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3716.
Abstract 3720: Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine cancer
Authors: Wen Yun-Fei , Wang Ying , Ma Shaolin , Raicu Anca Chelari , Baggerly Keith A. , Sood Anil K.
Published: August 2019

Purpose: Extra-pituitary prolactin (PRL) is involved in development and progression of uterine carcinoma. However, the underlying mechanisms of PRL/PRLR axis in uterine cancer are not well understood, and current means of targeting this axis are limited. Here, we used a potent PRL antagonist (G129R) to interfere with oncogenic activities of uterine cancer cells and further inhibit tumor growth.

Experimental Design: We performed integrated analyses by comparing expression of PRLR isoforms in a broad panel of tumors and normal countparts using TCGA and GTex databases. Therapeutic effects of G129R treatment in uterine cancers were assessed in both orthotopic PTENwt and PTENmut models. Additionally, we carried out reverse phase protein assay (RPPA) at in vivo condition from the resulting tumors and at in vitro conditions to interrogate downstream targets and mechanism of action for G129R. Further functional analyses including cell viability, cell cycle and nascent protein synthesis were accessed in two types of uterine cancer cells under serum-depleted condition.

Results: We found that PRLR_S1a, a short form of PRLR that is generated from alternative splicing during transcription, is expressed in human uterine cancers to a greater extent than other isoforms. Blocking activities of the PRL/PRLR_S1a axis with the antagonist G129R significantly reduced growth and progression of both orthotopic PTENwt and PTENmut uterine tumors. Data from reverse-phase protein arrays (RPPA) by in the resulting tumors showed that pathways involved in epithelial-mesenchymal transition such as PI3K/mTOR activities were significantly downregulated by G129R treatment. In addition, based on the RPPA data from the in vitro uterine cancer cells treated with G129R as monotherapy or in combination with PRL, we discovered a novel function of G129R in initiating cell death mediated by nuclear FOXO3a and inducing the G0/G1 arrest in both PTENwt and PTENmut uterine cancer cells. Furthermore, G129R decreased the nascent protein synthesis in these uterine cancer cells mediated by transcriptional suppressor EIF-4EBP1. Collectively, our findings reveal a new role of the PRLR_S1a isoform in uterine cancer and identify G129R as a potential therapeutic in uterine cancer.

Citation Format: Yun-Fei Wen, Ying Wang, Shaolin Ma, Anca Chelari Raicu, Keith A. Baggerly, Anil K. Sood. Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3720.
Abstract 3722: Novel Optical Coherence Tomography (OCT) technology for no-invasive 3D ex vivo drug activity profiling
Authors: Dhar Sumeer , Ueda Akihiro , Muira Takemitsu , Kuromi Yasushi , Mori Yuki , Fujimoto Hiroki
Published: August 2019

Drug discovery and early to late drug development process have relied on 2D monolayer based in vitro assay platforms and small animal models. The limitations posed by 2D in vitro assays for evaluation of targeted and immunomodulatory agents have prompted researchers to develop novel 3D in vitro and ex vivo platforms utilizing the tumor microenvironment like conditions for better understanding of mechanisms of novel targeted and immunomodulatory drug/compound entities. Therefore, rapid development of robust 3D ex vivo cell culture as drug efficacy testing system for drug discovery, development and profiling of standard of care and Immunotherapeutic molecules have led to understanding the relevance of these platforms within the scientific and clinical community. 3D ex vivo platforms are being diligently evaluated as better predictive drug efficacy testing tools in preclinical as well as clinical space in the quest for profiling of novel anticancer entities (as single or two drug combinations). Within this context, there has been growing need in improved imaging and analysis of complex 3D structures (Multicellular tumor spheroids/Organoids) containing multiple cellular components such as, Tumor cells, stromal cells, various immune cells to monitor signaling pathways and assess immune events including T cell infiltration as drug efficacy measurement. At SCREEN LifeScience, we have developed a novel OCT based technology for imaging and analysis of complex 3D structures, such as, spheroid /organoid for growth & morphological profiling, quantification of internal cavities, drug sensitivity testing to capture the events leading to tumor cell death and assessing mechanism of drug action. Additionally, this technology allows us perform large tissue imaging, non-invasive monitoring of macro and sprouted neo-vasculature without the need for fluorescent staining for providing quantitative information about the vascular morphological changes, thereby allowing for the evaluation of anti-angiogenic drugs in real time. In summary, the OCT technology shown to be robust and versatile technology useful for studying multiple disease models, including cancer. Its utility in regeneration medicine is being further evaluated as a versatile non-invasive platform.

Citation Format: Sumeer Dhar, Akihiro Ueda, Takemitsu Muira, Yasushi Kuromi, Yuki Mori, Hiroki Fujimoto. Novel Optical Coherence Tomography (OCT) technology for no-invasive 3D ex vivo drug activity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3722.
Abstract 3728: Inverse effect of 28Si and 56Fe radiation on intestinal tumorigenesis vs. carcinogenesis in APC1638N/+ mice
Authors: Kumar Santosh , Suman Shubhankar , Kallakury Bhaskar V.s. , Moon Bo-Hyun , Fornace Albert J. , Datta Kamal
Published: August 2019

Space radiation is a major risk factor for gastrointestinal (GI) cancer in astronauts and is a matter of concern. Earlier we have reported higher relative effects for intestinal tumorigenesis in APC1638N/+ mice after exposure to different heavy ion radiation spanning a range of LET values. The purpose of the current study was to histologically grade intestinal tumors into adenoma vs. adenocarcinoma and assess relative effects of different heavy ion radiation on tumorigenesis vs carcinogenesis. Tumors from male APC1638N/+ mice 150 d after exposure to whole-body sham, γ (0.3 keV/μm), 12C (13 keV/μm), 28Si (70 keV/μm), or 56Fe (148 keV/μm) radiation were harvested, fixed, paraffin embedded, and sectioned. Radiation doses were 0.1 Gy, 0.5 Gy and a equitoxic dose of 2.0 Gy γ-rays. H&E stained tumor sections from at least 50 tumors per study group were used for histological grading into adenoma and adenocarcinoma. Hotspot mutations for p53 and Kras genes were assessed using targeted PCR followed by amplicon sequencing in 20 tumors per study group. Our previous published data showed that among the three LETs tested relative intestinal tumorigenic effects (relative to γ-rays) peaked at a LET of 70 keV/μm (28Si) with 13 keV/μm (12C) showing the least effect and 148 keV/μm (56Fe) showing an intermediate effect. In contrast, the current study demonstrates that the frequency of adenocarcinoma peaked after 56Fe with the least effect after 12C and an intermediate effect after 28Si. Relative to γ-rays, the carcinoma frequencies were 42.4+0.9, 13.2+0.61 and 8.0+1.14 higher after 56Fe at doses of 0.1, 0.5 and 2.0 Gy respectively. Whereas the relative carcinoma frequencies of 34.2+1.35, 14.0+0.95 and 9.0+2.25 were observed after 28Si at doses of 0.1, 0.5 and 2.0 Gy respectively. While Kras mutation analysis of tumors did not show any difference among radiation types, p53 mutation analysis demonstrate a higher number of mutations in exon 5 in 56Fe relative to 28Si samples. Collectively, our data show that while LET-dependent tumorigenic effects followed a parabolic shape, carcinogenic effects followed a more linear-like pattern with highest adenocarcinoma percentage and frequency at 148 keV/μm (56Fe). Although inverse effects of LET on tumorigenesis vs. carcinogenesis could in part be attributed to the beams’ differential physical properties, our mutation data on p53 supports differential effects of the beams on tumor suppressor mutation.

Citation Format: Santosh Kumar, Shubhankar Suman, Bhaskar V.s. Kallakury, Bo-Hyun Moon, Albert J. Fornace, Kamal Datta. Inverse effect of 28Si and 56Fe radiation on intestinal tumorigenesis vs. carcinogenesis in APC1638N/+ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3728.
Abstract 3733: Development of photoimmunotherapy for colon cancer in nude mice models
Authors: Hollandsworth Hannah , Amirfakhri Siamak , Filemoni Filemoni , Razemjooie Sahar , Yazaki Paul , Hoffman Robert , Bouvet Michael
Published: August 2019

Introduction: Photoimmunotherapy (PIT) uses tumor specific monoclonal antibodies conjugated to photosensitizer phthalocyanine dye IR700 to produce cytotoxicity. To date, there is no published data on the use of PIT in colorectal cancers, which could be useful as an adjunct to surgery to prevent local recurrence, or as a primary therapy.

Methods: Humanized anti-CEA antibody (m5A) was conjugated to IR700CW dye (LI-COR). In vitro PIT was performed using a human colon cancer cell line LS174T. Cancer cells (2x103) were seeded on a 96-well plate and incubated for 24 hours. m5A-IR700CW containing media was added to the cells. Cells received either 2 minutes of PIT or no treatment. PIT was administered at 4 J/cm2, 8 J/cm2 or 16 J/cm2. Cell viability was measured on a microplate reader with CellTiter 96 Aqueous One Solution (Promega, Madison, WI). Subcutaneous implantation of LS174T was performed on the bilateral flanks of four male nude mice. Tail vein injection of 25 micrograms of m5A-IR700CW reconstituted in 100 microliters PBS was performed for each mouse. Animal imaging was performed with a LI-COR Pearl Trilogy imaging system. Right flank tumors of three mice were treated with a 690 nm laser at 150 mW/cm2 for 30 minutes, delivering a total of 270 J/cm2. A non-treated mouse served as the control. Images were obtained prior to PIT, immediately after PIT and 10 minutes, 20 minutes, 30 minutes and 24 hours after treatment. Maximum fluorescence intensity was measured at each time point. Tumor size was measured prior to treatment, 24 hours, 48 hours, 5 days and 10 days after treatment.

Results: In vitro PIT was effective to kill the cancer cells in a dose response manner. In vivo PIT decreased tumor fluorescence intensity in a time-dependent manner. PIT arrested tumor growth in all mice, compared to the control, which demonstrated continuous tumor growth.

Conclusion: PIT affects cancer cell death in vitro and arrests tumor growth in colon cancer models.

Citation Format: Hannah Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Sahar Razemjooie, Paul Yazaki, Robert Hoffman, Michael Bouvet. Development of photoimmunotherapy for colon cancer in nude mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3733.
Abstract 3740: Ionizing radiation-induced transcriptomic changes in triple-negative breast cancer cells reveal putative mechanisms of treatment resistance
Authors: Lin Victor T. , Lama-Sherpa Tshering D. , Samant Rajeev S. , Shevde Lalita A.
Published: August 2019

Breast cancer is the most common malignancy and the second leading cause of cancer-associated deaths in women. Of these, triple-negative breast cancers (TNBCs) remain the most difficult to treat. Unlike hormone-driven breast cancers, TNBCs that remain in remission past the five-year mark rarely relapse and are considered functionally cured. Thus, investigating mechanisms of treatment resistance in order to improve remission rates is of paramount importance. In this study, we use RNA-Seq to analyze global transcriptomic changes in TNBC cells in response to DNA damage induced by ionizing radiation (IR). Applying gene set enrichment analysis (GSEA), we have identified a number of expression signatures that represent putative mechanisms of resistance to treatment. In prior work, we have demonstrated the role of the transcription factor Gli1, the terminal effector of the Hedgehog (Hh) signaling pathway, in regulating the expression of DNA repair proteins in response to genotoxic damage with cisplatin. We have previously shown that Hh inhibition blunts the expected upregulation of specific proteins involved in single-strand DNA repair in ovarian cancer cells. Here, we expand on our earlier studies by examining TNBC cells with high levels of Hh activity and querying how global transcriptomic changes in response to IR are altered by Hh inhibition. Our data suggest that rational manipulation of these pathways may sensitize TNBCs to treatment and improve the functional cure rate of these tumors.

Citation Format: Victor T. Lin, Tshering D. Lama-Sherpa, Rajeev S. Samant, Lalita A. Shevde. Ionizing radiation-induced transcriptomic changes in triple-negative breast cancer cells reveal putative mechanisms of treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3740.