bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2004: Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer
Authors: Lin Chang-Ching , Lo Miao-Chia , Moody Rebecca , Stevers Nicholas , Tinsley Samantha , Gasparyan Mari , Wicha Max , Sun Duxin
Published: August 2018

Cancer stem cells (CSCs) are a small population of cancer cells that are capable of self-renewal and tumor initiation. As a result, targeting CSCs is a potential therapeutic strategy for preventing cancer relapse and metastasis. Mixed lineage kinase 4 (MLK4), a serine/threonine kinase, is known to regulate mesenchymal glioma stem cells and to drive tumorigenesis in colorectal cancer. We found that MLK4 was highly expressed in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes according to the database from The Cancer Genome Atlas. Furthermore, multiple datasets indicated that higher expression of MLK4 was associated with poor prognosis in breast cancer. Despite the correlation of MLK4 and clinical outcome, the function of MLK4 in breast cancer is still not known. In this study, we found that knockdown of MLK4 significantly decreased secondary mammosphere formation, the CD44+/CD24- CSC population, invasion and migration in TNBC cell lines. In addition to suppressing CSC phenotypes in vitro, knockdown of MLK4 also inhibited tumor growth in NOD/SCID mice. Secondary transplantation of tumor cells demonstrated that silencing of MLK4 significantly decreased tumor-initiating cell frequency. As accessed by qPCR, knockdown of MLK4 led to decreased expression of mesenchymal genes (CD44 and VIM) and concomitant increased expression of epithelial genes (CDH1 and claudin genes). Immunohistochemical staining revealed that knockdown of MLK4 in xenografts resulted in increased expression of CK19, a luminal-epithelial marker. These studies indicate that MLK4 plays an important role in the function of mesenchymal-like CSCs in TNBC. Silencing of MLK4 in TNBC cell lines can transform CSC in these tumors to a more differentiated epithelial cell state. Together, these studies identify that MLK4 is a potential therapeutic target for CSCs in TNBC.

Citation Format: Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Mari Gasparyan, Max Wicha, Duxin Sun. Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast 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 2004.
Abstract 2005: CSCs induced from iPSCs indicate the trace of cancer cell culture conditioned media
Authors: Seno Akimasa , Kasai Tomonari , Tokutaka Heizo , Seno Masaharu
Published: August 2018

Cancer stem cells (CSCs) show drug resistance and could be a source of cancer cells. Although these cells are recently considered more important than ever in cancer studies, the extremely small number of CSCs in actual cancer tissues makes the analysis difficult. Previously, we demonstrated that CSCs could be differentiated from induced pluripotent stem cells (iPSCs) in the presence of cancer cell conditioned media. We have confirmed the conditioned media from various types of cancer cell lines were successful to convert iPSCs into CSCs showing self-renewal property, differentiation potential, and tumorigenicity. To identify their differences between the generated CSCs, gene expression was compared by spherical self-organizing map (sSOM). Comparing with more than 1000 cells including stem cells, induced CSCs, CSCs obtained from cancer tissue or cancer cell line, and cancer cells or cancer tissues, known cancer stem cell markers were found not enough to classify CSCs. In the sSOM analysis, “ideal probe” (IP), which was supposed to characteristically be expressed in each group, was set to nomiate the marker gene. The distance close to the IP depicted some candidate marker genes in each group. The gene expression commonly shared among different types of CSCs was also searched. This IP revealed known cancer related gene Endthelin 1 as a commonly expressed gene. Moreover, when the CSCs were compared with the parental hiPSC and cancer cells, of which conditioned media was used for the conversion, sSOM showed that CSCs were not always differentiated towards their origin, but which genes should be responsible for generating CSCs. These results indicate that the induced CSCs should be the initial state of cancer cells and imply some important role of Endothelin 1 in CSC development.

Citation Format: Akimasa Seno, Tomonari Kasai, Heizo Tokutaka, Masaharu Seno. CSCs induced from iPSCs indicate the trace of cancer cell culture conditioned media [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 2005.
Abstract 2006: Tobacco smoke-induced Marcks phosphorylation promotes inflammatory and stemness properties in lung cancer
Authors: Yang David C. , Wu Yichin , Huang Mu-Jen , Pinkerton Kent E. , Wu Reen , Chen Ching-Hsien
Published: August 2018

Exposure to tobacco smoke is a major risk factor associated with lung cancer development and progression; however, the mechanisms of how tobacco smoke activates pathways promoting cancer malignancy remain to be established. Previously, phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) was shown to promote malignancy of neoplastic disease, but it is still unclear how phospho-MARCKS is upregulated in lung cancer. Herein, we investigated if phospho-MARCKS is elevated in response to smoke exposure and plays a key role in smoke-related lung cancer. In a screen of 200 patients with lung cancer, we confirmed that strong phospho-MARCKS staining was observed in lung cancer specimens from smokers and was positively correlated, as compared to non-smokers. In addition, we demonstrated that increased smoking pack-year was noted in lung cancer patients with high phospho-MARCKS. Surprisingly, there is an apparent trend that patients with both tobacco use and high phospho-MARCKS levels had the worst overall survival. MARCKS was found to become activated in airway epithelium and lung cancer in response to smoke both in vitro and in vivo. MARCKS inhibition significantly attenuated smoke-induced oncosphere formation and migratory capability. Mechanistically, upregulation of phospho-MARCKS by smoke occurred in parallel with increased inflammatory cytokine expression (e.g. TNF-α, IL-8) and stemness marker expression (e.g. Oct4, Nanog, CD133, Sox2). Conversely, targeting of MARCKS phosphorylation with the MPS peptide resulted in down-regulation of smoke-induced stemness and cytokine genes. Our data suggests that phospho-MARCKS plays a role in modulating smoke-mediated lung cancer progression and is a potential prognostic biomarker in smoke-related lung cancer.

Citation Format: David C. Yang, Yichin Wu, Mu-Jen Huang, Kent E. Pinkerton, Reen Wu, Ching-Hsien Chen. Tobacco smoke-induced Marcks phosphorylation promotes inflammatory and stemness properties in 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 2006.
Abstract 2018: Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT)
Authors: Srivastava Kirtiman , Smith Victoria , Fernandez Eric A. , Breen Conor , McCloskey Karen D.
Published: August 2018

Background: Loss of the transcription factor, p63 is associated with poor prognosis in muscle-invasive bladder cancers. The current study aimed to investigate the tumor-suppressive role of p63 in urothelial cancers.

Methods: A 3-dimensional (3D) organotypic raft model of urothelial cancer was established by culturing human urothelial cancer cell lines (HT1376, T24) on human bladder fibroblast-embedded collagen-I. Control rafts contained normal primary human urothelial cells (HUC). Cells were also cultured as monolayers on collagen-I coated dishes before harvesting total RNA and protein for qPCR and Western blot analyses respectively.

Results: Non-invasive HUC and HT1376 had an epithelial phenotype, characterized by compact and cuboidal morphology whereas invasive T24 cells were elongated with a spindle morphology, typical of mesenchymal cells and indicative of epithelial-to-mesenchymal transition (EMT), which is necessary for invasion. The number of invasive incidents in 3D rafts established with T24 cells was higher compared to those with HUC or HT1376 cells (N=3, p<0.05). Consistent with this, the invasive cell line had reduced mRNA and protein expression of total p63 (N=3), a differentiation marker, and the epithelial marker E-cadherin (N=3), in addition to upregulated expression of the mesenchymal markers N-cadherin (N=3) and fibronectin (N=3) and pERK-Y204 (N=2) compared to the non-invasive cells.

Treatment of invasive T24 cells with histone deacetylase (HDAC) inhibitors (vorinostat (pan) or entinostat (HDAC1,3)) attenuated the number of invasive incidents (N=3, p<0.05), and restored protein expression of total p63 (N=3) and E-cadherin (N=2) while normalizing pERK-Y204 (N=2 for T24). The tumor-suppressive properties of the ΔNp63α isoform were studied by adenoviral-mediated overexpression of ΔNp63α in T24 cells, which mimicked the effects of HDAC inhibitors and diminished EMT by rescuing E-cadherin expression while depleting fibronectin (N=2). Overexpression of ΔNp63α also reduced the number of invasive incidents in 3D models established with T24 cells (N=2).

Conclusion: Invasive T24 cells exhibited an EMT phenotype, which coincided with depleted p63 expression. Rescue of ΔNp63α expression by treatment with HDAC inhibitors or through experimental overexpression attenuated the EMT phenotype and cell invasion.

Citation Format: Kirtiman Srivastava, Victoria Smith, Eric A. Fernandez, Conor Breen, Karen D. McCloskey. Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT) [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 2018.
Abstract 2020: SAHA and EGCG affect metastatic potential to varying degrees in three triple-negative breast cancer cell lines
Authors: Lewis Kayla A.
Published: August 2018

Although triple-negative breast cancers (TNBC) have distinct molecular aberrations, tumors of the same histological grade may have dramatically different responses to the same therapy. TNBC tumors are basal-like, aggressive, and harbor a proclivity to metastasize to the brain and lungs. By modifying the cancer epigenome we have shown it is possible to reduce the metastatic potential of triple-negative breast cancer. Suberoylanilide hydroxamic acid (SAHA, clinically Vorinostat) is a histone deacetylase (HDAC) inhibitor, and epigallocatechin-3-gallate (EGCG) is a green tea polyphenol that acts as a DNA methyltransferase (DNMT) inhibitor. In combination, we found that 3 μM SAHA and 5 μM EGCG alter mammosphere formation, and the expression of E-cadherin and N-cadherin, which are genes involved in the epithelial-to-mesenchymal transition (EMT). Epigenetic enzyme activity was also assessed to determine if SAHA and EGCG were capable of modulating the functionality of DNMTs and HDACs. Because our previous work has identified a decrease in cellular viability with the combination of SAHA and EGCG, we aimed to investigate the effect on cell cycle arrest and genes involved in cell cycle progression. Our prior findings have indicated that the combination of SAHA and EGCG decreases the expression level of miR-221/222, which is known to directly inhibit translation of p27 and PTEN. p27 is involved in regulating the transition from G1 to S phase, and with the combination of SAHA and EGCG the expression of p27 was significantly increased, suggesting potential cell cycle arrest between G1 and S phase. Because the PTEN/Akt pathway plays a role in progression from G1 to S phase through p-Akt, we also investigated changes in the expression of PTEN with SAHA and EGCG. Together our findings are reflective of tumor heterogeneity; despite the same breast cancer subtype they respond differently to chemotherapeutics. Further studies will include research into specific epigenetic modifications associated with the epithelial-like reversion seen in this project.

Citation Format: Kayla A. Lewis. SAHA and EGCG affect metastatic potential to varying degrees in three triple-negative breast cancer cell lines [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 2020.
Abstract 2037: E-cadherin enhances immune control of metastatic melanoma
Authors: Shields Bradley D. , Koss Brian , Mahmoud Fade , Byrum Stephanie D. , Tackett Alan J.
Published: August 2018

The advent of checkpoint blockade therapies has transformed the clinical management of metastatic melanoma. These monoclonal antibodies, targeted against CTLA-4 and PD-1, have shown durable and long-lasting responses in a subset of melanoma patients. However, response rates remain incomplete, even when using combination therapy (CTLA-4 plus PD-1). Identifying predictive or controlling features of responsiveness has been urgent goal in oncology research. Our group and others have previously shown responding tumors display high levels of E-cadherin, a cell-cell adhesion molecule which defines an epithelial-like phenotype. The varying clinical responses observed to checkpoint blockade in metastatic melanoma, paired with the knowledge that E-cadherin expression correlates with such responses, provides impetus to study the significance of E-cadherin in the context of checkpoint blockade. Here, we present a novel B16F10 melanoma mouse model in which E-cadherin is exogenously expressed (B16.E-cad). The parental B16F10 line does not express E-cadherin and is poorly immunogenic and notoriously resistant to treatment with immune checkpoint inhibitors. Thus, B16.E-cad and the vector control model epithelial-like (responsive) and mesenchymal-like (non-responsive) phenotypes respectively. We find, compared to vector control, B16.E-cad exhibits delayed tumor growth, reduced metastatic potential, and increased overall survival in vivo, despite similar proliferation rates in vitro. Analysis of immune cell infiltration (FACS) performed on tumors grown in C57/BL6 mice revealed no differences between vector, B16.Ecad and B16.SIY. Conversely, transplantation of B16.E-cad into Rag1-/-mice abrogated tumor growth delay indicating an immune-mediated tumor response. Additionally, B16.Ead tumors displayed greater responses to combination checkpoint inhibitor therapy. Further, imunohistochemistry for E-cadherin performed on patient pretreatment tumor biopsies revealed negative staining was predictive of a non-responsive status. These data indicate an epithelial-like phenotype in melanoma tumors facilitates tumor immunosurveillance and responsiveness to immune checkpoint blockade.

Citation Format: Bradley D. Shields, Brian Koss, Fade Mahmoud, Stephanie D. Byrum, Alan J. Tackett. E-cadherin enhances immune control of metastatic melanoma [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 2037.
Abstract 2044: Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) modulates VEGF-C and VEGF-D secretion in the triple negative breast cancer cell line MDA-MB-231
Authors: Caceres Sara , Little Andrew C. , Yates Joel A. , Gilani Rabia A. , Merajver Sofia D.
Published: August 2018

Breast cancer commonly gives rise to lymph node metastases, and it has been suggested that tumor cell migration is facilitated by the generation of new lymphatic vessels, or lymphangiogenesis. Lymphatic endothelial cell hyaluronic acid receptor 1 (LYVE-1) is a hyaluronic acid (HA) receptor commonly used as a lymphatic vessel endothelial marker. LYVE-1 has been observed in a variety of breast cancer epithelial cells, and its lymphangiogenic function remains unclear. LYVE-1 has been casually linked to the vascular endothelial growth factors VEGF-C and VEGF-D, which are well appreciated for their roles in lymphangiogenic responses. Therefore, we hypothesize that LYVE-1 may impact lymphangiogensis via a VEGF-C/VEGF-D mediated pathway. We utilized LYVE-1-targeted RNAi in MDA-231 cells followed by HA stimulation and measured production and secretion of both VEGF-C and VEGF-D by western blot and ELISA, respectively. Surprisingly, LYVE-1 knockdown resulted in elevated levels of basal VEGF-C secretion, while LYVE-1 knockdown followed by HA stimulation decreased secreted levels of VEGF-C, with no observed changes in intracellular levels when compared to their controls. Conversely, LYVE-1 knockdown displayed no significant changes in basal VEGF-D levels, although, secreted VEGF-D levels were elevated following HA stimulation. Collectively, our preliminary findings suggest that LYVE-1 modulates VEGF-C/D secretion, potentially defining a novel function for LYVE-1 in MDA-231 triple negative breast cancer epithelial cells.

Citation Format: Sara Caceres, Andrew C. Little, Joel A. Yates, Rabia A. Gilani, Sofia D. Merajver. Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) modulates VEGF-C and VEGF-D secretion in the triple negative breast cancer cell line MDA-MB-231 [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 2044.
Abstract 2045: Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer
Authors: Dhandapani Muthu , Goldman Aaron , Kumar Naveen , Prasad Peeyush , Jash Eshna , Singh Itender , Gopinath Kodaganur S. , S Krishnamurthy , P Prabhusankar , Sehrawat Seema
Published: August 2018

Background: Breast cancer is the leading cause of cancer-related mortality in women. Triple negative breast cancer (TNBC) is one of most aggressive subtypes, which is often accompanied by metastasis, a feature that requires development of new blood vessels (neovasculature) to disseminate tumor cells throughout the body. We recently reported that down-regulation of exchange factor directly activated by Cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in proliferation and inhibition of cell migration.

Methods: Here, using gene knockdown by siRNA and a 3-dimensional in-vitro TNBC co-culture model with Human Vascular Endothelial Cells (HUVECs), we studied microvascular density and the role of EPAC1. Angiogenesis proteome profiling was employed to examine protein networks that were regulated by EPAC1 in the TNBC cell line, MDA-MB-231. Immunofluorescence along with Electric cell-substrate impedance sensing (ECIS) was used to study vascular permeability. To confirm these findings in a translational context, we employed a human tumor explant assay that predicts clinical response to therapy (CANscriptTM) and studied CD34+ nodes to determine how neovasculature associates to drug response.

Results: We determined that downregulation of EPAC1 in the TNBC cell line, MDA-MB-231, leads to reduction of proteins involved in cell migration, metastasis, angiogenesis and adhesion. Interestingly, we determined that loss of EPAC1 leads to increased expression of negative regulators in these same pathways. Immunofluorescence imaging showed that EPAC1 downregulation in MDA-MB-231 cells leads to diminished expression of proteins such as Paxillin, MENA, MMP-9 and tubulin. indicated that EPAC1 role in vascular permeability. Finally, CANscriptTM demonstrated a link between response to therapy and microvascular density, which was reduced under drug pressure in patients that were predicted to respond to treatment.

Conclusion: Our results suggest that EPAC1 is a driver of microvascular density in the tumor microenvironment, a feature that may play a key role in distant metastasis and therapy failure. Ex-vivo modeling of neovasculature may be a novel strategy to predict clinical response and distant metastasis.

Citation Format: Muthu Dhandapani, Aaron Goldman, Naveen Kumar, Peeyush Prasad, Eshna Jash, Itender Singh, Kodaganur S. Gopinath, Krishnamurthy S, Prabhusankar P, Seema Sehrawat. Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast 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 2045.
Abstract 205: Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies
Authors: Russo Mariangela , Lamba Simona , Lorenzato Annalisa , Sogari Alberto , Corti Giorgio , Rospo Giuseppe , Montone Monica , Lazzari Luca , Arena Sabrina , Oddo Daniele , Linnebacher Michael , Sartore-Bianchi Andrea , Pietrantonio Filippo , Siena Salvatore , Nicolantonio Federica Di , Bardelli Alberto
Published: August 2018

Clinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.To test our assumption, we first generated a panel of CRC cells resistant to commonly used targeted agents, including EGFR and BRAF inhibitors. Treatment with targeted therapies, although initially effective, fuels clonal evolution and further amplifies molecular diversity. Exome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, associated with distinct genomic landscapes.Functional and pharmacological modulation of WNT signaling induces cell death in CRC preclinical models from patients that relapsed during treatment, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT ligands secretion resulted in translocation of β-catenin from the cytoplasm and nucleus to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones.In summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumors face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

Citation Format: Mariangela Russo, Simona Lamba, Annalisa Lorenzato, Alberto Sogari, Giorgio Corti, Giuseppe Rospo, Monica Montone, Luca Lazzari, Sabrina Arena, Daniele Oddo, Michael Linnebacher, Andrea Sartore-Bianchi, Filippo Pietrantonio, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli. Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies [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 205.
Abstract 2051: Perfusable 3D angiogenesis in a high-throughput microfluidic culture platform
Authors: Bircsak K M. , Duinen V van , Trietsch S J. , Zonneveld A J. van , Hankemeier T , Saleh A , Vulto P
Published: August 2018

The transition from 2D to 3D cell culture is a first step towards more physiologically relevant in vitro cancer models. To adequately capture the complex tissue architectures observed in vivo, 3D microfluidic techniques incorporate and achieve long-term gradient stability, continuous perfusion and patterning of cancer cell layers as stratified co-cultures. We used a standardized high-throughput (n=40) microfluidic 3D tissue culture platform called the OrganoPlate® to generate precisely controlled gradients, without pumps, ideal for growing blood vessels and inducing controlled 3D angiogenic sprouting. The blood vessel is grown against an extracellular matrix (ECM) gel with cancer cells and is subsequently exposed to pro- and antiangiogenic compounds to direct sprouting towards 3D cancer cell clusters. Utilizing high-content confocal time-lapse imaging and analysis, angiogenic potential was measured in various cancer models. The exposed vasculature shows many of the important hallmarks of cancer angiogenesis found in vivo, including tip cells induction and migration and stalk cells formation. Importantly, the stalk cells develop a perfusable lumen that is connected to the parental vessel as demonstrated with perfusion of high-molecular-weight (150KD) fitc-dextran through microvascular structures. This model will be used as an in vitro cancer screening platform to unravel the important drivers in angiogenesis and vasculogenesis and the mechanism of action of antiangiogenic compounds. By combining this culture platform with mural cells, cell-cell interactions can be studied. In parallel, we will combine this 3D cancer angiogenesis platform with our current Tumor-on-a-Chip models to create tissue models with integrated vasculature.

Citation Format: K M. Bircsak, V van Duinen, S J. Trietsch, A J. van Zonneveld, T Hankemeier, A Saleh, P Vulto. Perfusable 3D angiogenesis in a high-throughput microfluidic culture platform [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 2051.
Abstract 2069: Therapeutic targeting of ependymoma as informed by oncogenic enhancers
Authors: Mack Stephen C. , Excellence Global Ependymoma Network of
Published: August 2018

Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas commonly resist chemotherapy and lack available targeted agents for clinical trial development. In the case of genomically balanced ependymomas, and those driven by fusion oncoproteins, we hypothesized that the chromatin landscapes could uncover oncogenes that would inform actionable targets for therapy and reveal specific transcriptional circuitries to identify molecular origins of the disease. We therefore interrogated the oncogenic drivers of ependymoma by characterizing the active regulatory landscapes of 42 primary tumors through an integrative analysis of H3K27ac ChIP-seq, RNA-seq, whole-exome sequencing (WES), whole genome sequencing (WGS), copy number profiling, and DNA methylation profiling. Across the landscape of ependymal tumors, enhancer mapping revealed putative oncogenes, including NFIX, MIRLET7BHG, FGFR1, WEE1, and MTSS1L, and delineated subgroup specific enhancer lesions. Reconstruction of enhancer networks permitted the identification of core transcription factors (TFs) that establish ependymoma cell state, and lineage-specifying TFs that dictate molecular subgroup identity. Lineage-associated TFs point to distinct spatio-temporal origins of ependymoma subgroups such as FOXJ1 TF activity and expression observed preferentially in subsets of hindbrain ependymomas. To translate our results into a potential clinical paradigm, we demonstrate cancer dependencies on super enhancer associated genes and lineage TFs, and establish the utility of chromatin landscape analysis to predict novel targets for cancer therapy.

Citation Format: Stephen C. Mack, Global Ependymoma Network of Excellence. Therapeutic targeting of ependymoma as informed by oncogenic enhancers [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 2069.
Abstract 2078: Analysis of DNA structural variations in context of the gene regulatory landscape of pediatric ALL
Authors: Oksa Laura , Vouttilainen Miikka , Vuorenmaa Tapio , Laukkanen Saara , Lahnalampi Mari , Lohi Olli , Heinäniemi Merja
Published: August 2018

Acute lymphoblastic leukemia (ALL) is the most common pediatric leukemia. It comprises of multiple subtypes with combinations of chromosomal rearrangements, indels and copy number variations. Furthermore, there is a significant number of somatic mutations located outside of protein coding regions of the genome. These DNA alterations can be classified based on whether they 1) introduce novel transcription factor binding sites, 2) create aberrant (super-)enhancers, or 3) lead to the loss of regulatory boundaries at oncogene loci upon disruption of CTCF binding sites.

We performed whole genome sequencing (WGS) on two T-ALL and 16 pre-B ALL patients from diagnostic and remission samples, and measured transcriptional activity using Global Nuclear Run-on sequencing (GRO-seq). These results were integrated with chromatin topology information and transcript annotations to yield comprehensive characterization of the gene regulatory landscape. The effect on gene expression was further analyzed based on monoallelic expression.

Altogether, we found 49 translocations, 153 deletions, 9 duplications and 22 inversions across the pre-B ALL samples. Majority of the changes were located within the annotated gene regions. At 25 of these 233 sites, we found evidence that active enhancers resulted in transcription-coupled genetic instability at DNA breakpoints. In a pre-B ALL case, our analysis successfully captured complex translocation event involving ETV6 (ETV6-MUTYH, ETV6-chr7, ETV6-chr21, ETV6-TXNRD2), and deletion at chr22 that hit to active promoters of PATZ1 and DRG1 genes leading to monoallelic expression of these genes.

In T-ALLs, we found 13 translocations (8 intergenic) and 9 deletions. In a T-ALL case, we found that translocation between TCR-beta locus and chr9 brought an active promoter to the proximity of TMEM38B resulting in increased gene expression. We also found a deletion in active promoter at chr9 leading to decreased expression of MTAP and CDKN2A genes. Moreover, a large deletion in chr11 brought oncogenic transcription factor LMO2 and RAG1 close to each other.

In summary, we present a genome-wide approach to link structural variation events with gene regulatory features in ALL cells. This extends previous approaches that have mainly focused on genetic changes at coding regions.

Citation Format: Laura Oksa, Miikka Vouttilainen, Tapio Vuorenmaa, Saara Laukkanen, Mari Lahnalampi, Olli Lohi, Merja Heinäniemi. Analysis of DNA structural variations in context of the gene regulatory landscape of pediatric ALL [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 2078.
Abstract 208: Multi-omics analysis in primary cell cultures reveals the genomic basis of phenotypic diversity within tumors
Authors: Liu Sixue , Yang Zuyu , Li Guanghao , Luo Yanting , Gong Qiang , Wu Xin , Li Tao , Zhang Zhiqian , Xing Baocai , Xu Xiaolan , Lu Xuemei
Published: August 2018

To uncover the functionally essential variations related to tumorigenesis and tumor progression from large amount of cancer genomics data is still challenging due to the genetic diversity between patients, and extensive inter- and intra-tumor heterogeneity at the genomic, epigenomic, and transcription levels. Multiple primary cultures derived from phenotypically distinct subpopulations of a single patient may not only represent the diversity and heterogeneity of tumor cell types and states (such as metastasis and drug-resistant), but also narrow the analysis in genomic basis of phenotypic diversity down to the relatively small

number of variations between subpopulations with the same genetic background. Performing multi-omics sequencing, we identified variations in single nucleotide, copy number, DNA methylation and gene expression among four primary cell cultures from primary and recurrent tumors in a hepatocellular carcinoma patient. We observed the discrepancy between the phylogenetic relationships revealed by single nucleotide variations (SNVs) and transcriptional profiles in the four cell cultures. The differences in gene expression, which indicated the distinct cellular characteristics and tumorigenic capability in one of the two recurrent cell clones, were correlated to the copy number alterations (CNAs) and DNA methylation variation in gene body and confirmed by the characterization of cellular phenotype. Our multi-omics analysis in heterogeneous clones derived from an individual revealed that CNAs and epigenomic changes, rather than SNVs, dominantly contribute to the phenotypic diversity among subpopulations in tumors, which could shed light on developing new cancer diagnosis and treatment strategies based on gene dosage and epigenetic modification

Citation Format: Sixue Liu, Zuyu Yang, Guanghao Li, Yanting Luo, Qiang Gong, Xin Wu, Tao Li, Zhiqian Zhang, Baocai Xing, Xiaolan Xu, Xuemei Lu. Multi-omics analysis in primary cell cultures reveals the genomic basis of phenotypic diversity within tumors [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 208.
Abstract 2080: Role of differentially expressed micro RNAs in non-WNT, non-SHH medulloblastoma
Authors: Paul Raikamal , Jalali Rakesh , Sridhar Epari , Moiyadi Aliasagar , Shirsat Neelam
Published: August 2018

Medulloblastoma is a common malignant brain tumor in children. It has four distinct molecular subgroups namely WNT, SHH, Group 3 and Group 4. The molecular subgroups differ in their clinical characteristics including age, incidence of metastasis and survival. Group 3 and Group 4 medulloblastomas have overlapping gene expression profile but are distinct in their clinical behavior. Group 3 medulloblastomas have the worst overall survival rates whereas Group 4 medulloblastomas have intermediate survival. Group 3 and Group 4 tumors are not associated with any known signaling pathway and due to their overlapping expression profiles it is often difficult to accurately identify the subgroup, which is very crucial for the appropriate treatment design. MiR-592, located in the intronic region of GRM8 gene, is over expressed in Group 4 medulloblastoma along with its host gene, thus acting as a good marker for Group 4 tumors. MiR-204, on the other hand is expressed from the 6th intron of TRPM3 gene and expressed in some Group 3 and majority of Group 4 medulloblastomas. In order to delineate the role of miR-592 and miR-204 in medulloblastoma biology, effect of expression of these microRNAs on medulloblastoma cell growth and behavior were investigated. The micro RNAs were expressed in a lentiviral mediated inducible manner in Group 3 medulloblastoma cell lines D283, D425 and D341 and their effects on cell proliferation, anchorage-independent growth and invasion potential were studied. MiR-592 expression had marginal effects on the anchorage independent growth potential and the tumorigenic potential of the Group 3 cell line where as miR-204 expression led to significant decrease in the anchorage independent growth, tumorigenic potential, as well as invasion potential of the medulloblastoma cell lines. Further in order to decipher the molecular mechanisms underlying these micro RNA mediated effects, RNA-sequencing analysis of miR-592 and miR-204 expressing medulloblastoma cells were performed to identify genes that are differentially expressed upon micro RNA expression. The results helped us to identify the molecular pathways that are being affected by the micro RNA expression and how they affect the malignant behavior of medulloblastoma cells.

Citation Format: Raikamal Paul, Rakesh Jalali, Epari Sridhar, Aliasagar Moiyadi, Neelam Shirsat. Role of differentially expressed micro RNAs in non-WNT, non-SHH medulloblastoma [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 2080.
Abstract 2082: High-throughput droplet digital PCR for MYCN amplification detection in FFPE neuroblastoma samples: Cost-effective, rapid, feasible, and reliable alternative to FISH
Authors: Somasundaram Dinesh Babu , Aravindan Sheeja , Yu Zhongxin , Jayaraman Muralidharan , Tran Ngoc TB , Li Shibo , Herman Terence , Aravindan Natarajan
Published: August 2018

MYCN amplification directly correlates with the clinical course of neuroblastoma (NB) and poor patient survival, and serves as the most critical negative prognostic marker. Although FISH remains the gold standard for clinical diagnosis of MYCN status in NB, its limitations warrant the identification of rapid, reliable, less technically challenging, and inexpensive alternate approaches. In the present study, we examined the concordance of droplet digital PCR (ddPCR, in combination with IHC) with FISH for MYCN detection in FFPE NB samples. In 112 NB cases, ddPCR analysis demonstrated a 96-100% concordance with FISH. Consistently, IHC grading revealed 92-100% concordance with FISH. Comparing ddPCR with IHC, we observed a concordance of 95-98%. The results demonstrate that MYCN amplification status in NB cases can be assessed with ddPCR, and suggest that ddPCR could be a technically less challenging method of detecting MYCN status in FFPE specimens. More importantly, these findings illustrate the concordance between FISH and ddPCR in the detection of MYCN status. Together, the results suggest that rapid, less technically demanding, and inexpensive ddPCR in conjunction with IHC could serve as an alternate approach to detect MYCN status in NB cases, with near-identical sensitivity to that of FISH.

Funding: NIH 1P20GM103639-01 from the COBRE Program of NIH; OUHSC Department of Radiation Oncology Research Development funds

Citation Format: Dinesh Babu Somasundaram, Sheeja Aravindan, Zhongxin Yu, Muralidharan Jayaraman, Ngoc TB Tran, Shibo Li, Terence Herman, Natarajan Aravindan. High-throughput droplet digital PCR for MYCN amplification detection in FFPE neuroblastoma samples: Cost-effective, rapid, feasible, and reliable alternative to FISH [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 2082.
Abstract 2106: Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq
Authors: Imler Elliot , Babic Milos , Adams Deanna , Shepard Peter , Yeakley Joanne , Nagle Raymond B. , Seligmann Bruce
Published: August 2018

We previously reported on use of the whole transcriptome TempO-Seq® assay to profile gene expression from 1 mm2 focal areas of 5 µm thick FFPE sections of normal and cancerous tissue to identify disease biomarkers and mechanistic pathways. We are now presenting data demonstrating that the TempO-Seq assay can be performed as an “in situ” assay on slides by an automated slide stainer, followed by counterstaining. Then, using an automated digital imaging platform, areas as small as 30 μm in diameter within the FFPE section can be profiled, permitting the gene expression data to be correlated directly to the specific morphology of that focal area. The pathologist uses a computer interface to select areas for profiling during the course of the histologic examination of the section, and then the TempO-Seq probes are automatically recovered from those region(s) of interest. After depositing them into PCR tubes, the assay is completed with PCR amplification and sample barcoding before pooling the samples into a library for sequencing. Analysis of the sequencing data is carried out automatically to report results. There is no use of laser capture or destruction of the tissue. The TempO-Seq processed slides can be archived and additional areas may be sampled at a later date. In this study, we sample replicate areas of matched normal vs. cancerous tissue, measuring gene biomarkers of clinical utility, gene fusions, and SNPs. We present gene expression profiles for replicate areas of pure stroma, single normal glands, single high-grade PIN glands, and single cancerous glands from prostate FFPE, demonstrating the statistical power of the TempO-Seq in situ assay as well as the correlation to focal histology and the use of focal differential expression to elucidate molecular pathways involved in the transition from normal tissue to cancer. We report measurements of clinically relevant biomarkers and signatures from clinical FFPE samples, plus therapeutic targets and drug resistance genes. This new approach demonstrates that complex molecular tests can be carried out by any pathologist in their own lab, and makes moot the issues of “% cancer” and amount of tissue required for testing. The approach we present brings extraction-free complex molecular testing of FFPE into the pathology lab and takes molecular pathology to a new level of simplicity, focal precision and correlation to morphology.

Citation Format: Elliot Imler, Milos Babic, Deanna Adams, Peter Shepard, Joanne Yeakley, Raymond B. Nagle, Bruce Seligmann. Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq [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 2106.
Abstract 2108: Detection and molecular profiling of leptomeningeal disease in melanoma
Authors: Smalley Inna , Evernden Brittany , Law Vincent , Kenchappa Rajappa , Puskas John , Ryzhova Elena , Tran Nam , Etame Arnold , Sahebjam Solmaz , Magliocco Anthony , Forsyth Peter , Smalley Keiran S.
Published: August 2018

Approximately 5% of melanoma patients develop leptomeningeal disease (LMDz), a highly symptomatic complication with a dismal survival of 8-10 weeks. The aim of this study was to determine whether diagnosis and personalized treatment for melanoma-LMDz could be improved by assessing patient-derived cerebrospinal fluid (CSF) specimens. Circulating tumor cells in CSF (CSF-CTCs) were detected by Veridex CellSearch® System and the circulating melanoma cell kit, based on anti-CD146 and anti-high molecular weight melanoma associated antigen (HMW-MAA-PE (MEL-PE)). Of the 12 patients with definitive LMDz diagnosis, all but 1 patient (92%) had CSF-CTCs (range 23-3055 CTCs/ml). In contrast, only 3/8 (37%) melanoma patients without LMDz diagnosis had CSF-CTCs detected, with significantly lower CTC counts per ml CSF (range 0.13-0.6 CTCs/ml). Ex vivo studies of CSF uncovered that although patient-derived CSF does not appear to act as a chemo-attractant or a stimulant of invasion to initiate melanoma migration to the leptomeninges, it significantly reduced the ability of BRAF inhibitors to induce apoptosis in established melanoma cell lines (p<0.005). Patient-derived CSF composition is complex but includes many growth factors and mediators of adhesion and TGFβ signaling. In melanoma cells, the CSF stimulated signaling through mTOR/AKT and STAT. BRAF inhibition amplified these signals further. We show that ex vivo expansion of isolated CSF-CTCs is possible for ~25% of samples, in the presence of FBS, FGF and EGF. At this time, there is a very limited understanding of the mechanisms underlying melanoma metastasis to the leptomeninges, a truly devastating and rapidly terminal complication of melanoma. This is the first report of patient-derived CSF eliciting a protective effect in melanoma cells, suggesting melanoma cells find a protective niche in the central nervous system. Furthermore, we demonstrate that patient-derived CSF biopsies serve as a critical tool for improving diagnosis and personalized treatment for melanoma-LMDz patients.

Citation Format: Inna Smalley, Brittany Evernden, Vincent Law, Rajappa Kenchappa, John Puskas, Elena Ryzhova, Nam Tran, Arnold Etame, Solmaz Sahebjam, Anthony Magliocco, Peter Forsyth, Keiran S. Smalley. Detection and molecular profiling of leptomeningeal disease in melanoma [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 2108.
Abstract 2111: The tumor microenvironment of preclinical tumor models may have an impact on the therapeutic index of nanotherapeutics
Authors: Ferretti Stephane , Beckmann Nicolau , Holzer laura , Obrecht Michael , Berger Marjorie , Jensen Michael Rugaard
Published: August 2018

Rapid development in nanotechnology allows the incorporation of multiple therapeutic agents (e.g. liposomes) into nanoparticle with a size range from 1 to 1000 nm. These nanocarrier systems provide new approaches to treat aggressive malignancy. Nano-based delivery systems hold an advantage over traditional small molecule therapy in that they can deliver drugs preferentially to tumors due to the enhanced permeability and retention (EPR) effect. On one hand, high permeability of the tumor vessels and a lack of functional lymphatic vessels results in the EPR effect, driving nanoparticle extravasation. On the other hand, these phenomena lead to high interstitial fluid pressure (IFP), limiting nanoparticle extravasation. In this study, we have used dynamic-contrast enhanced (DCE) MRI with small (Dotarem, 5 nm) and large (Vistarem, 25 nm) contrast agents (CAs) to examine tumor vessel permeability in SJSA-1 osteosarcoma tumor-bearing rats. An inversion-recovery true fast imaging with steady state precession (TrueFISP) was employed to determine the transfer constant Ktrans in subcutaneous and intra-tibia tumor models. When the tumor cells were injected in both niches within the same animal, Ktrans was significantly lower in intra-tibia tumors with both CAs, and blood vessels significantly less and larger compared to subcutaneous tumors. Moreover, Ktrans negatively correlated with tumor bioluminescence for the intra-tibia model, suggesting that EPR decreased inversely with tumor size, while the opposite was noticed for the subcutaneous tumors. These data demonstrate for the first time that the EPR of tumors with a similar size within the same animal can be influenced by the microenvironment. Moreover, they show that the therapeutic index of a nanotherapeutics may be different if it's determined with either an orthotopic or ectopic model.

Citation Format: Stephane Ferretti, Nicolau Beckmann, laura Holzer, Michael Obrecht, Marjorie Berger, Michael Rugaard Jensen. The tumor microenvironment of preclinical tumor models may have an impact on the therapeutic index of nanotherapeutics [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 2111.