bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1145: Biomarker-driven molecular imaging of prostate cancer using the PEG10 promoter
Authors: Shapovalova Mariya
Published: August 2019

The retrotransposon-derived paternally expressed gene 10 (PEG10) protein is ordinarily expressed at high levels in the placenta. The over-expression of PEG10 has been documented in a number of malignancies including hepatocellular carcinoma and leukemia. Recently, it was discovered that PEG10 isoforms promoted the progression of prostate adenocarcinoma to a highly lethal non-androgen receptor (AR) driven subtype called aggressive variant prostate cancer (AVPC).

In this study, we found that PEG10 was also expressed in castration-resistant PCa possessing constitutively active AR-splice variants in addition to AR-negative AVPC. We subsequently developed a molecular genetic imaging strategy for the non-invasive imaging of PCa by utilizing the cancer specificity of the PEG10 promoter to drive the expression of reporter genes. The transcriptional output of the PEG10 promoter was enhanced to enable high expression of reporter genes by multiple imaging modalities and immunoassay. By using this PEG10 promoter upstream of a reporter gene in a plasmid, we were able to detect PCa by fluorescence and positron emission tomography (PET) imaging after systemic administration of the plasmid in mice. This method also allowed for the detection of PCa by an immunoassay of mouse urine.

Our study demonstrates a pre-clinical proof-of-concept that the PEG10 promoter is a powerful and specific tool that can be utilized for non-invasive detection of highly lethal PCa subtypes. This biomarker imaging technology has the potential to improve detection and can be further modified for therapeutic applications.

Citation Format: Mariya Shapovalova. Biomarker-driven molecular imaging of prostate cancer using the PEG10 promoter [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 1145.
Abstract 1148: Recombinant cancer-stromal cells Identified in the metastatic tumor microenvironment By color-coded imaging
Authors: Nakamura Miki , Suetsugu Atsushi , Matsuura Kana , Satake Tomoyuki , Shimizu Masahito , Saji Shigetoyo , Moriwaki Hisataka , Hoffman Robert M.
Published: August 2019

Background/Aim:The relationship of cancer and stromal cells is poorly understood, including their ability to fuse or otherwise their genetic materials. Materials and Methods:Red-fluorescent protein (RFP-expressing) mouse colon 26 cancer cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors were visualized. After 14 days culture, the cells were injected into the spleen. Results:After splenic injection, colon 26 metastases were observed in the liver, ascites, and bone marrow. RFP colon cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assured to be a pre-metastatic niche. Conclusion:Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer stromal cells. The significance of which will be evaluated in future experiments.

Citation Format: Miki Nakamura, Atsushi Suetsugu, Kana Matsuura, Tomoyuki Satake, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Robert M. Hoffman. Recombinant cancer-stromal cells Identified in the metastatic tumor microenvironment By color-coded imaging [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 1148.
Abstract 1151: Real-time visualization of brain metastasis in vivo
Authors: Tsuji Takahiro , Wake Hiroaki , Ozasa Hiroaki , Haruwaka Koichiro , Ajimizu Hitomi , Yasuda Yuto , Sakamori Yuichi , Nomizo Takashi , Kim Young Hak , Hirai Toyohiro
Published: August 2019

Research into physiological phenomena has taken a leap forward by recent advanced optical bio imaging techniques. Traditionally, physiological phenomena including cell division, cell function, molecular transport and expression, and signal transduction were detected by biochemical methods. However, this information lacked high resolution spatial or temporal information. Using recent bio imaging techniques, both spatial and temporal information can be integrated across the molecular, cellular and systems levels that allow us to further investigate the hierarchical interaction of organ systems. Here, we have developed an in vivo imaging method to visualize brain metastasis of lung cancer which provides real-time information of their proliferation, metastasis and the immune reaction for cancer cells. CMT167 and LLC1 cell lines which expressed mCherry or Venus were established. The fluorescently labeled cell lines were administered into the brain via the carotid artery or direct injection to the CX3CR1-GFP mice, transgenic mice in which microglia was specifically labeled with GFP. The metastasis cancer and the immune reaction were visualized using 2-photon microscopy for 14 days. Interaction between cancer cells and microglia was observed. We identified heterogeneity of the interaction between microglia and cancer cells. Some microglia cells showed phagocytosis-like response to cancer. Other microglia touched, surrounded, and released cancer cells without phagocytotic response. These interactions between microglia and cancer cells could be observed by 3-6 hours continuous real-time in vivo imaging. Further study to address detailed mechanism for microglia reaction is in progress. This research has potential to provide the information of brain metastasis in vivo and to be a new solution to elucidate the process of immunity and niche.

Citation Format: Takahiro Tsuji, Hiroaki Wake, Hiroaki Ozasa, Koichiro Haruwaka, Hitomi Ajimizu, Yuto Yasuda, Yuichi Sakamori, Takashi Nomizo, Young Hak Kim, Toyohiro Hirai. Real-time visualization of brain metastasis in vivo [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 1151.
Abstract 1159: Association of ALDH7A1 overexpression with chemotherapy resistance and cancer stem cell in lung cancer
Authors: Yasuda Yuto , Sakamori Yuichi , Ozasa Hiroaki , Ajimizu Hitomi , Oguri Tetsuya , Maeno Ken , Tsuji Takahiro , Nomizo Takashi , Yamamoto Tomoko , Yoshida Hironori , Hirai Toyohiro , Kim Young Hak
Published: August 2019

Aldehyde dehydrogenases (ALDHs) play a major role in the oxidation of aldehydes. Certain isoforms of ALDH is related to cancer stem cells (CSCs) responsible for tumor initiating, drug resistance and metastasis. ALDH1A1 and ALDH3A1 have been known for CSC markers of lung cancer. Since these two isoforms and ALDH7A1 are involved in cellular structural and osmoregulatory function, we focused on function of ALDH7A1 in lung cancer. We established SN-38 resistant DMS53 (DMS53-SR) and SN-38 or gemcitabine resistant NCI-H23 (H23-SR or H23-GR, respectively) by intermittent and dose-escalating exposure of the agent. The expression of ALDH7A1 was upregulated in DMS53-SR and H23-SR/H23-GR compared with DMS53 and NCI-H23, respectively. ALDH7A1 expression was altered using a plasmid to overexpress ALDH7A1 in DMS53. ALDH7A1 overexpression led to a slight increase of IC50 of SN-38, upregulation of SOX2, and tumor sphere formation. Overexpression of ALDH7A1 in DMS53 have enhanced tumorigenic capacity in vivo. These data suggested that ALDH7A1 might be a marker of CSC and a therapeutic target of lung cancer.

Citation Format: Yuto Yasuda, Yuichi Sakamori, Hiroaki Ozasa, Hitomi Ajimizu, Tetsuya Oguri, Ken Maeno, Takahiro Tsuji, Takashi Nomizo, Tomoko Yamamoto, Hironori Yoshida, Toyohiro Hirai, Young Hak Kim. Association of ALDH7A1 overexpression with chemotherapy resistance and cancer stem cell in lung 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 1159.
Abstract 1171: Identifying small molecules that specifically inhibit glioma stem cells
Authors: Sloan Anthony R. , Spina Raffaella , Voss Dillon M. , Laye Sophie , Bar Eli E.
Published: August 2019

Glioblastomas (GBM) are the most common primary malignant brain tumor in adults and are recognized as one of the deadliest forms of cancer. Despite aggressive therapy consisting of maximal surgical resection followed by concomitant radiation and temozolomide (TMZ) chemotherapy, GBM remains to have a median survival time of ~ 15 months. Glioblastoma stem cells (GSCs) are suggested to play a critical role in the GBM phenotype; GSC have tumor initiation ability, propagation ability, and have been shown to be a driver in promoting GBM resistance to current therapeutic interventions. Due to these poor clinical outcomes and resistance to current treatments, more potent and preferably more specific pharmacological therapies are needed in order to target cellular-molecular pathways to eradicate GSCs and effectively treat GBM. In an effort to identify agents that specifically inhibit GSC growth, we recently conducted a drug screen of over 3,000 small molecules. HSR020913 patient derived neurospheres were treated for 72 hours with library agents at a concentration of 10 µM and cell growth was observed for a period of 5 days. We identified twelve highly potent compounds in the primary screen that inhibited the growth of HSR020913 neurospheres by over 50%. Subsequently, the potency of these compounds was tested on five additional GBM patient derived neurosphere lines (HSR040622, HSR040822, CCF3691, CCF3832, and CCF08-387) at even lower concentrations. We next sought to determine the specificity of these compounds in killing GSCs but not normal cells. To this aim we tested these compounds against immortalized human neural stem cells (v-Myc hNSCs) and normal human astrocytes (NHA). Of the twelve potent compounds identified in the primary screen, three compounds (AGSC9, AGSC11, AGSC12) showed to have negligible to minimal effect, only at higher doses, on normal neural stem cells. The respective half maximal inhibitory concentration (IC50) was determined for all the neurosphere lines utilized. Subsequent studies determined the inhibitory effect of these compounds on clonogenic capacity and enzymatic activity. Future studies are warranted to identify molecular pathways targeted by these compounds and determine their effect on tumor progression and survival in vivo.

Citation Format: Anthony R. Sloan, Raffaella Spina, Dillon M. Voss, Sophie Laye, Eli E. Bar. Identifying small molecules that specifically inhibit glioma 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 1171.
Abstract 1173: A novel high throughput platform for head & neck cancer organoids drug screening
Authors: Queiroz Karla , Driehuis Else , Bonilla Silvia , Lanz Henriëtte , Clevers Hans , Joore Jos , Vulto Paul
Published: August 2019

Head and neck (HN) cancer is a broad category of tumor types arising from various anatomic structures including the craniofacial bones, soft tissues, salivary glands, skin, and mucosal membranes. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumour recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate, a microfluidic based 3D culture plate, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. These models are expected to show better predictivity as compared to laboratory animals and conventional 2D cell culture models, without compromising throughput or ease of use. Hereby we show the establishment of HN cancer-derived organoids in 2-lane OrganoPlate, and its usefulness for phenotypic drug screenings. The aim of the study is to evaluate the 2-lane OrganoPlate as a platform for growing HN cancer organoids and drug screening. Organoid lines (T2, T3 and T4) were embedded in the 2-lane OrganoPlate as single cells in an Extracellular Matrix gel. At day 3, cultures were treated with Cisplatin or Carboplatin for 120 hours. Drug response was evaluated by assessment of morphology (phase contrast), Cell Viability (Alamar blue) and proliferation (EdU incorporation). Organoids cultures grow well under perfusion in the 2-lane OrganoPlate and different sensitivity to cisplatin is captured by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate platform offers an attractive method for growing HN cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings.

Citation Format: Karla Queiroz, Else Driehuis, Silvia Bonilla, Henriëtte Lanz, Hans Clevers, Jos Joore, Paul Vulto. A novel high throughput platform for head & neck cancer organoids 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 1173.
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

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

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

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

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

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

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

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

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

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.
Abstract 1256: Validation of Ubiquitin specific peptidases 37 as a prognostic and diagnostic marker in osteosarcoma
Authors: Singh Mayank , Batra Atul , Bakhshi Sameer , Summers Matthew
Published: August 2019

Osteosarcoma has been a challenge for the oncologist because even after surgical removal of tumors 80 percent patients go on to develop metastasis to the Lung. Systematic development of Drug has been literally on a standstill in osteosarcoma. The emergence of Adjuvant therapy after surgery for localized osteosarcoma has increased the long term survival in patients under age 40 by 60 to 70 percent however for patients who go on to develop metastasis the prognosis is poor as there are limited therapeutic options. Protein ubiquitination and deubiquitylation plays an important role in controlling protein stability. Initial studies by us points to a model in which Ubiquitin specific peptidase 37 regulates cell cycle progression. Our data indicates that USP37 expression correlates with transformation by preventing degradation of different oncoproteins. It was found that in osteosarcoma cell lines USP37 is elevated which gives survival advantage to these cells while its depletion leads to enhanced cell death in response to genotoxic stress. USP37 depletion results in reduced resolution of DNA damage foci like gamma H2AX and 53BP1 and increase in no of collapsed replication fork which indicate the reduced ability of cells to carry out constitutive DNA replication. Physically USP37 interacted with proteins involved in DNA damage repair and effected their stability. We tried to correlate our data with archived patient osteosarcoma samples by analyzing if USP37 levels corelate with patient prognosis at our center which will be further discussed. The current data adds a new dimension to this newly explored deubiquitinates and merits development of targeting strategies to explore its therapeutic potential in osteosarcoma.

Citation Format: Mayank Singh, Atul Batra, Sameer Bakhshi, Matthew Summers. Validation of Ubiquitin specific peptidases 37 as a prognostic and diagnostic marker in osteosarcoma [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 1256.
Abstract 1258: Pooled-analysis of response markers in cancer cell lines treated with tumor treating fields
Authors: Shahaf Gitit Lavi , Giladi Moshe , Schneiderman Rosa S. , Gotlieb Karnit , Zeevi Einav , Porat Yaara , Munster Mijal , Weinberg Uri , Kirson Eilon D. , Palti Yoram
Published: August 2019

Low intensity, intermediate frequency alternating electric fields (TTFields) exert an inhibitory effect on numerous cancer cell lines with some variability in the response. The goal of the present study is to compare characteristics of cell lines based on their response pattern to TTFields and to identify response markers. Forty-five different human cancerous cell lines were treated for 72 hours with TTFields at optimal cell-specific frequency at the same nominal intensity (1.7 V/cm). Cytotoxicity and clonogenic potential were determined. Functional analysis of differentially expressed genes and mutations associated with response to TTFields was performed based on the Cancer Cell Line Encyclopedia (CCLE) database. Sensitivity to TTFields was compared with pharmacological profiling (CCLE). The cytotoxic response to TTFields was found to be distributed around an average of 50% with (ranging between 14% and 86% reductions in cell counts). The clonogenic effect varied between no effect and 88% reduction in the number of colonies. Increased sensitivity to: Lapatinib, PHA-665752 and PLX-4720 was common in the group of cell lines which were more highly responsive to TTFields. Functional analysis of cell line gene expression and mutation data revealed enriched pathways related to DNA damage repair response, cell migration, hypoxia signaling and oxidative stress. This analysis of cancerous cell line response to TTFields define the optimal frequency to be applied for each cell line and demonstrates the broad effectiveness of TTFields in cells. The role of the specific mutations associated with TTFields response should be further explored in additional studies. Pharmacological profiling may offer a rational for combining specific agents with TTFields.

Citation Format: Gitit Lavi Shahaf, Moshe Giladi, Rosa S. Schneiderman, Karnit Gotlieb, Einav Zeevi, Yaara Porat, Mijal Munster, Uri Weinberg, Eilon D. Kirson, Yoram Palti. Pooled-analysis of response markers in cancer cell lines treated with tumor treating fields [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 1258.
Abstract 1260: Inhibition of DOG1, or TMEM16A, results in antitumoral effects in gastrointestinal stromal tumors (GIST) in vitro
Authors: Fröbom Robin , Berglund Erik , Bränström Robert
Published: August 2019

DOG1/TMEM16A, over-expressed in >95% of all GISTs, is a calcium-activated chloride channel that has gained attention as a promising drug target due to its involvement in several tumor-physiological processes. Two well-established human GIST cell lines (GIST-T1 and GIST882) were used to study DOG1 inhibitors effect on viability, colony formation, and cell cycle. In this study, we show that specific DOG1/TMEM16A-inhibitors (T16inh-A01 and CaCCinh-A01) decrease chloride currents, and reduces cell viability in GIST cells. However, only CaCCinh-A01 changes the distribution of the cell cycle in GIST cells, putting them into G1 cell cycle arrest, and increases sub-G1 phase population in GIST882 cells. CaCCinh-A01 strongly reduces the colony forming ability of the cells, whereas limited effect by T16inh-A01. Conclusions: DOG1/TMEM16A inhibition has antitumoral effects in GIST cells in vitro, and could potentially serve as a target for GIST therapy. CaCCinh-A01 seems to be the more potent inhibitor, presumably due to its effect not only on chloride currents but also on DOG1/TMEM16A expression.

Citation Format: Robin Fröbom, Erik Berglund, Robert Bränström. Inhibition of DOG1, or TMEM16A, results in antitumoral effects in gastrointestinal stromal tumors (GIST) in vitro [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 1260.
Abstract 1262: Relationship between RAS, BRAF, PIK3CA and location of primary tumor in urothelial carcinoma
Authors: Kim Young Saing , Lee Sang-Cheol , Hwang In Gyu , Lee Su Jin , Park Se Hoon
Published: August 2019

Purpose: In urothelial carcinoma (UC), somatic mutations in RAS, BRAF, and PIK3CA genes are suggested for prognostic markers as well as for therapeutic targets. Studies have reported that upper tract (renal pelvis and ureter) and bladder UC differ in factors including prognosis and genetic features.

Methods: We investigated the difference in clinicopathological features and oncogenic mutations (RAS, BRAF, and PIK3CA) according to the location of primary disease. In a prospective biomarker study in patients with metastatic or unresectable UC, tumor DNA was analyzed for mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CAusing Ampliseq v2 platform. We further stratified the frequency of the mutations by the location of primary tumor.

Results: Among a total of 210 patients, those with upper tract UC and bladder UC were 90 and 120 patients, respectively. Missense mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CA were present in 9 (4.3%), 1 (0.5%), 18 (8.6%), 4 (1.9%), and 32 (15.2%) patients, respectively. PIK3CA mutations were detected in 16 patients with upper tract UC (17.8%) and 16 patients with bladder UC (13.3%). Likewise, HRAS mutations were detected more frequently in upper tract UC (10/90, 11.1%) than bladder UC (8/120, 6.7%). In metastatic UC setting, PIK3CA or HRAS mutations were not associated with different overall survival.

Conclusions: PIK3CA and HRAS mutations were more frequently observed in patients with upper tract UC compared to those with bladder UC. The different genetic features need to be studied by further studies.

Note: This abstract was not presented at the meeting.

Citation Format: Young Saing Kim, Sang-Cheol Lee, In Gyu Hwang, Su Jin Lee, Se Hoon Park. Relationship between RAS, BRAF, PIK3CA and location of primary tumor in urothelial carcinoma [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 1262.
Abstract 1266: Co-expression of P-glycoprotein and ABCG2 in a cell line model reveals both independent and cooperative transporter function
Authors: Robinson Andrea , Robey Robert , Gottesman Michael
Published: August 2019

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how expression of multiple transporters might confer resistance to chemotherapy drugs. We therefore examined how P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 contribute to drug resistance in a cell line model. HEK-293 cells were transfected with vector encoding full-length ABCB1, ABCG2, or a vector containing both genes under the control of a separate promoter. Cells transfected to express both transporters (B1/G2 cells) expressed high levels of both transporters and were able to transport both the P-gp-specific substrate rhodamine 123 and the ABCG2 specific substrate pheophorbide a when examined by flow cytometry. B1/G2 cells were also cross-resistant to the P-gp substrate doxorubicin, the ABCG2 substrate topotecan as well as mitoxantrone, a substrate of both transporters. The checkpoint inhibitor prexasertib was also found to be a substrate of both P-gp and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that P-gp and ABCG2 can function independently to transport substrates. P-gp and ABCG2 were also found to function cooperatively to transport the common fluorescent substrates mitoxantrone and BODIPY®-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. This was also true in 3-day cytotoxicity assays with mitoxantrone and prexasertib. Thus, P-gp and ABCG2 can independently and cooperatively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

Citation Format: Andrea Robinson, Robert Robey, Michael Gottesman. Co-expression of P-glycoprotein and ABCG2 in a cell line model reveals both independent and cooperative transporter function [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 1266.