bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 1178: Image-based quantification of tumor-immune cell interactions in 3D cultures
Authors: Goverse Gera , Beztsinna Nataliia , Yan Kuan , Price Leo , Daszkiewicz Lidia 
Published: August 2019

Abstract
INTRODUCTION Increasing numbers of cancer patients that benefit from immunotherapies and maintain durable response requires understanding of cellular mechanisms that govern anti-tumor immune responses. However, the mechanisms of action of these treatment modalities are not fully understood and the progress in this direction is hampered by a lack of appropriate pre-clinical testing models that are both clinically relevant and suitable for routine screening of drug candidates. Therefore, we developed a robust in vitro assay that allows image based analysis of 3D cultures in a high-throughput set-up. Here, immune cells are co-cultured with cancer cells in a 3D environment which recapitulates the tumor micro-environment and its complex cellular interactions. Functional read-outs, such as active migration of immune cells towards tumoroids, infiltration of immune cells into the tumoroids and their killing lead to a better understanding of the immune-modulatory profile of different immunotherapies.
MATERIAL and METHOD Tumoroids generated from cancer cell lines (e.g. breast, prostate) and colorectal cancer organoids (from HUB Organoid Technology) were cultured in protein hydrogel. Different immune cells, such as PBMCs, T cells, dendritic cells or macrophages, with and without activation were added to the 3D culture and their infiltration into tumoroids and subsequent killing was visualized using high-content microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software.
RESULTS and DISCUSSION Image-based analysis enabled the discrimination of immune-tumor cell interactions in 3D cultures. These results demonstrated the effect of immune cell targeting on the tumor progression. Different levels of immune cells infiltration and killing of tumoroids and organoids were measured depending on the activation status of immune cells. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays.
CONCLUSION Our image-based platform described here allows for analysis of immunotherapy effects on different cell types that engage in a more physiologically relevant spatial setting than when culturing them in traditional 2D cultures. Visualization and quantification of these tumor-immune cells interactions offer a highly powerful tool for cancer immunotherapy drug developers to understand the mechanism of action of their treatments and ultimately translating to a better clinical performance.
Citation Format: Gera Goverse, Nataliia Beztsinna, Kuan Yan, Leo Price, Lidia Daszkiewicz. Image-based quantification of tumor-immune cell interactions in 3D cultures [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 1178.
Abstract 1188: Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies
Authors: Ratnam Nivedita M. , Sonnemann Heather M. , Gilbert Mark R. , Giles Amber J. 
Published: August 2019

Abstract
Glioblastoma remains a lethal brain cancer, and current treatment provides only modest survival benefit. Immunotherapy is currently being investigated as a potential treatment for this disease, but immune cell trafficking to tumor is hampered. Glioblastoma are immunologically “cold”, lacking infiltrating lymphocytes. Tumors epigenetically silence the expression of chemokines that attract lymphocytes to evade immune attack. Previous studies in ovarian cancer demonstrated that the histone methyltransferase inhibitor, GSK126, increased T cell infiltration into the tumor, promoting immune-mediated tumor suppression. Lymphocyte infiltration was driven by elevated expression of interferon gamma (IFNγ)-induced chemokines CXCL9 and CXCL10. In the current study, we evaluate the ability of GSK126 to similarly reverse the silencing of CXCL9 and CXCL10 and thereby improve immunotherapy outcome for glioblastoma. We first determined that treatment of murine and human glioblastoma cell lines with GSK126 increased IFNγ-mediated gene expression of CXCL9 and CXCL10 by qPCR and protein by ELISA. Subsequently, transwell migration assays performed using human and murine T cells demonstrated increased T cell migration when exposed to conditioned medium from tumor cells treated with GSK126 and IFNγ. In addition, T cells exposed to GSK126 and IFNγ up regulated CXCR3, the receptor for CXCL9 and CXCL10 and further increased their expression of IFNγ. The in vivo efficacy of GSK126 was tested in combination with anti-PD-1 antibody in C57Bl/6 mice subcutaneously implanted with the syngeneic glioma cell line CT2A. Mice treated with a combination of GSK126 and anti-PD-1 antibody showed a significant decrease in tumor volume with treatment, providing an overall survival benefit compared the anti-PD-1 or GSK126 alone. Taken together, our data suggests a therapeutic role for GSK126 in glioblastoma that could enhance current immunotherapy regimens by reversing the epigenetic changes that enable immune cell evasion and encourage immune cell trafficking to the tumor.
Citation Format: Nivedita M. Ratnam, Heather M. Sonnemann, Mark R. Gilbert, Amber J. Giles. Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies [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 1188.
Abstract 1190: Tissue slice culture platform: A slice of reality in drug development
Authors: Burke Shannon , Garcon Fabien , Ryan Kelli , Harper James , Wilkinson Robert W. 
Published: August 2019

Abstract
Immuno-oncology (IO) therapies for cancer have recently delivered step-change improvements in patient outcomes, but the major proportion of patients remain unresponsive or become refractory to IO treatment. Because IO-based approaches necessarily rely on the interplay of multiple cell types, including immune cells, and other elements that constitute the tumor microenvironment (TME), innovative assays are critical to generate further translational insight. Indeed, results from in vivo mouse models and in vitro assays with human cell lines often do not correlate with clinical efficacy and as such, do not always represent relevant avatars. We have leveraged recent advances in tumor slice culture (TSC) techniques to enable in vitro mechanistic and novel biomarker testing/identification studies. TSC involves the in vitro culture of thick slices (200-300 μm) of fresh tissue prepared using a vibratome and is currently the only in vitro assay system which retains the 3-dimensional tissue architecture, complete with cell-cell and cell-matrix interactions. We are now regularly using the TSC together with tumor samples derived from patients and human cancer cell line derived xenograft or syngeneic mouse cancer models, to generate exciting pre-clinical data for several Oncology programs, including oncolytic viruses, modified RNA, bispecific antibodies and antibody-drug conjugates. For instance, the TSC platform has allowed us to analyse the strong type I IFN-driven inflammatory response initiated by infection of tumor nests within patient samples and demonstrate the consequent infiltration of immune cells from the surrounding stroma. Thus, using a tissue phenomics approach incorporating a range of endpoint assays, including multiplex whole-mount imaging, transcriptomics and cytokine expression, the data generated using the TSC platform will inform the underlying biology and translational strategies of multiple therapeutic candidates.
Citation Format: Shannon Burke, Fabien Garcon, Kelli Ryan, James Harper, Robert W. Wilkinson. Tissue slice culture platform: A slice of reality in drug 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 1190.
Abstract 1196: Induction of NKG2D ligand expression by CD8+ T cells through a nuclear factor-kappa B and P300/CBP-associated factor-dependent mechanism
Authors: Hu Jiemiao , Xia Xueqing , Li Shulin 
Published: August 2019

Abstract
The ligands for the natural-killer group 2 (NKG2D) protein are frequently expressed on tumor cell surfaces, rendering tumor cells susceptible to NKG2D-dependent immune cell attack. However, cancer cells escape from immune surveillance by downregulating NKG2D ligands. We previously discovered that the engagement of activated CD8+ T cells and tumor cells induces NKG2D ligands on tumor cells, but the underlying mechanism remains to be defined. TCGA database analyses suggested that the expression of CD40 and CD137L (their cognate receptors are CD40L and CD137, respectively, expressed on CD8+T cells) is associated with pan-NKG2D ligand expression in sarcoma and colorectal adenocarcinoma patients (Pearson’s correlation coefficient r=0.2225 and 0.1947, respectively; and P=.0003 and .0002, respectively). In agreement with these human data analysis results, both in vivo mouse tumor models and in vitro cell assays validated that upon the engagement with the cognate receptors, the tumor necrosis factor receptor superfamily (TNFRSF), particularly CD137 ligand and CD40, initiates the activation of nuclear factor-kappa B (NF-κB) signaling in tumor cells even in the absence of CD8+ T cells. The same as tumor and CD8+ T contact-dependent NKG2D ligand induction, this CD137L/CD40-mediated signaling activation was associated with elevated levels of acetyltransferase P300/CBP-associated factor (PCAF), whereas inhibition of phosphorylated NF-κB abrogated PCAF induction. Although the stimulation of CD137L/CD40-mediated signaling by cognate receptors is vital for NKG2D ligand upregulation, the inflammatory cytokines, including interferon γ (IFNγ) and tumor necrosis factor α (TNFα) released from CD8+ T cells also facilitate NKG2D ligand-induced immune surveillance. These inflammatory cytokines not only trigger NF-κB signaling and PCAF activation, and they also boost T cell chemotaxis, recruit large numbers of CD8+T cells to tumor sites, and result in high yield expression of NKG2D ligands. Collectively, our results unveil a novel mechanism of NKG2D ligand upregulation involving reverse signaling of CD40 and CD137L on in tumor cells, which along with inflammatory cytokines IFNγ and TNFα, stimulate downstream NF-κB and PCAF activation. Understanding this mechanism may help to develop induced NKG2D ligand-dependent T-cell therapy against cancers.
Citation Format: Jiemiao Hu, Xueqing Xia, Shulin Li. Induction of NKG2D ligand expression by CD8+ T cells through a nuclear factor-kappa B and P300/CBP-associated factor-dependent mechanism [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 1196.
Abstract 1208: Comparative analysis between DNA vs RNA-based T cell repertoire profiling for cancer immunotherapy
Authors: Kalyana-Sundaram Shanker , Traini Christopher , Halsey Wendy , Jackson Heather , Bhattacharya Sabyasachi , Yanamandra Niranjan , Sathe Ganesh , Livi George 
Published: August 2019

Abstract
The T cell receptor (TCR), a defining structure of T cells is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an immune response. The extreme diversity of the TCR repertoire represents a major analytical challenge. Massively parallel high throughput sequencing is commonly deployed to characterize various key features of T cell receptors including their clonal expansion and repertoire diversity. Genomic DNA is the most widely employed starting material to characterize the TCR repertoire. DNA is highly stable and contains a fixed copy number per cell, which allows for better quantification of TCR clones. However, the inclusiveness is a major limitation of using genomic DNA along with the PCR amplification challenges and sequencing errors due to recombined gene segments within the context of ‘unused’ segments and introns. Here we deploy RNA-based TCR profiling as an alternate strategy to overcome the major limitations associated with the DNA-based approach. Employing RNA with a simplified PCR amplification strategy allows a more comprehensive identification of unique TCR variants with greater sensitivity. It also provides information about expression levels of TCR sequences, and more importantly, excludes the non-productive sequences that are functionally irrelevant. In addition, Unique Molecular Indexing (UMIs) enables the determination of the absolute count of RNA transcripts processed in a sample, allowing straightforward error corrections.
Citation Format: Shanker Kalyana-Sundaram, Christopher Traini, Wendy Halsey, Heather Jackson, Sabyasachi Bhattacharya, Niranjan Yanamandra, Ganesh Sathe, George Livi. Comparative analysis between DNA vs RNA-based T cell repertoire profiling for cancer immunotherapy [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 1208.
Abstract 1215: Improved T cell activation bioassays for development of bispecific antibodies and engineered T cell immunotherapies
Authors: Stecha Pete , Garvin Denise , Hartnett Jim , Swanson Brad , Fan Frank , Cong Mei , Cheng Zhi-jie Jey 
Published: August 2019

Abstract
T Cell-mediated immunity is driven by activation of T cell which results in long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.
Citation Format: Pete Stecha, Denise Garvin, Jim Hartnett, Brad Swanson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Improved T cell activation bioassays for development of bispecific antibodies and engineered T cell immunotherapies [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 1215.
Abstract 1237: Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model
Authors: Prasad Sahdeo , Srivastava Sanjay 
Published: August 2019

Abstract
More than 90% of patients die of pancreatic cancer due to metastasis. Among the cytokines, CXCL12 and its CXC chemokine receptor 4 (CXCR4) are known to play a role in invasion and metastasis in many types of cancer. Thus, agents that can interrupt the CXCR4/CXCL12 signaling cascade have the potential to suppress cancer metastasis. In the present study, we demonstrate that gambogic acid (GA), a xanthone from Gamboge hanburyi, down-regulated CXCR4 expression in pancreatic cancer cells in a dose- and time-dependent manner. We found that the decrease in CXCR4 by GA was not specific to cell type since its expression was abrogated in multiple myeloma cell lines and in colorectal, head and neck, prostate, and breast cancer cell lines. Furthermore, down-regulation of CXCR4 was not due to proteolytic degradation since proteasome inhibitors had no effect. Instead, the down-regulation of CXCR4 was due to transcriptional regulation, as indicated by the corresponding down-regulation of mRNA expression. This xanthone also inhibited the invasion of pancreatic tumor cells in vitro. Down-regulation of CXCR4 and prevention of cell invasion by the xanthone were both found to be mediated through the reactive oxygen species (ROS) as N-acetylcysteine, a ROS quencher, abolished these effects. When examined in an orthotopic nude mouse model of human pancreatic cancer, intraperitoneal administration of GA suppressed both tumor growth and metastasis. Immunohistochemical analysis of tumor tissue showed that besides decreasing the level of CXCR4, GA also reduced the expression of VEGF and MMP-9, which are also linked to invasion and metastasis. Thus, our results demonstrate gambogic acid’s potential in suppressing tumor invasion and metastasis through down-modulation of CXCR4/CXCL12 signaling pathways.
Citation Format: Sahdeo Prasad, Sanjay Srivastava. Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model [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 1237.
Abstract 1239: Structure activity relationship of alexidine analogs for the inhibition of collective invasion
Authors: Arnst Jamie , Commander Rachel , Marcus Adam 
Published: August 2019

Abstract
Lung cancer is the leading cause of cancer-related deaths in the US with an overall 5-year survival of only 15-20%. Metastatic disease is a major contributor to poor survival and novel therapeutics that can selectively target invasive tumor cell populations are lacking. Metabolic reprogramming contributes to tumor progression with effective therapies targeting cancer metabolism available. However, it remains unclear whether targeting cancer metabolism will prove valuable in inhibiting invasive cancer populations. Recent studies have demonstrated anticancer activity by the mitochondria-targeting bioactive small molecule alexidine dihydrochloride. Our group has previously found that alexidine specifically inhibits the a highly invasive subpopulation of H1299 previous identified in our lab by decoupling and decreasing collective invasion. Here we report the structure activity relationship (SAR) between analogs of alexidine on the inhibition of collective invasion in NSCLC cell lines. For these studies, we utilized an in vitro 3D invasion model where NSCLC cell lines were allowed to form spheroids, and then were embedded into Matrigel and allowed to invade over 48h in the presence or absence of the analogs. Using this technique, we identified two novel alexidine analogs with improved efficacy at inhibiting the invasion of H1299 NSCLC cells. Additionally, we examined the phosphorylation of pyruvate dehydrogenase (PDH), the gatekeeper for the entrance of pyruvate into the TCA cycle, following treatment with the alexidine analogs. We observed a significant correlation between PDH phosphorylation and inhibition of invasion following treatment with the analogs. In conclusion, we have identified two novel alexidine analogs with improved inhibition of invasion in NSCLC cell lines in an in vitro model.
Citation Format: Jamie Arnst, Rachel Commander, Adam Marcus. Structure activity relationship of alexidine analogs for the inhibition of collective invasion [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 1239.
Abstract 1247: Screening the membrane proteome to determine antibody specificity and discover new immunomodulatory targets
Authors: Huston-Paterson Duncan 
Published: August 2019

Abstract
The Membrane Proteome Array (MPA) is a cell-based expression array of more than 5,300 membrane proteins that enables the screening of preclinical antibodies for off-target binding and the deorphaning of phenotypic antibodies to identify their associated targets. Each membrane protein is expressed in live cells in its native conformation with appropriate post-translational modifications. Binding interactions are tested and validated using high-throughput flow cytometry, providing a comprehensive assessment of antibody binding across the membrane proteome. Membrane proteins in the MPA are fully functional and can be used for phenotypic screening to identify new therapeutic targets. Using a T-cell co-culture approach, we identified dozens of novel co-stimulatory proteins, as well as many well-validated proteins known to be involved in T-cell activation. The proteins identified in this screen represent novel therapeutic targets across multiple membrane protein classes and likely function on diverse axes of T-cell regulation.
Citation Format: Duncan Huston-Paterson. Screening the membrane proteome to determine antibody specificity and discover new immunomodulatory targets [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 1247.