bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5655: Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy
Authors: Liang Ying Yu , Ramos Anderson , Laursen Henriette , Surova Olga , Lengqvist Johan , Lööf Sara , Langebäck Anette , Bacanu Smaranda , Bergh Jonas , Nordlund Pär
Published: August 2018

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development.  We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug action can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues.

 

Although CETSA was first developed for evaluating drug binding to target proteins in cells it is also useful for characterizing physiological interactions, including protein-protein, protein-metabolite and protein-nucleic acid interactions (Martinez Molina & Nordlund, Ann Rev Pharm Toxic 2016 56:141). In the proteome-wide CETSA experiment, interactions with more than 7000 proteins can be measured in parallel (Savitski, Science 2014 346;6205). This strategy therefore constitutes a novel mean for discovering key interactions determining the fate of cancer cells during therapy and resistance – interactions that can serve as biomarkers or candidates for novel drug targets in cancer therapy.

Citation Format: Ying Yu Liang, Anderson Ramos, Henriette Laursen, Olga Surova, Johan Lengqvist, Sara Lööf, Anette Langebäck, Smaranda Bacanu, Jonas Bergh, Pär Nordlund. Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy [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 5655.
Abstract 5667: High-throughput analysis of MAPK and JAK-STAT signaling in CT26 tumors using a combination of immunophenotyping and phospho-flow cytometry
Authors: Draper David , Wong Alden , Lapinski Philip , Barnes Sheri R. , Franklin Maryland Rosenfeld , Wise Scott C.
Published: August 2018

The development of robust platforms that enable the analysis of cell signaling proteins is crucial for small molecule drug development. Analysis of phospho-proteins in solid tumors is one area that remains a challenge due to the complex nature of the tissue. The most common approaches used to measure the effects of in vivo therapy are immunoblot and ELISA-based assays. However, these bulk analysis methods have drawbacks. This is primarily due to their inability to distinguish between phospho-protein levels in different cell subsets that exist in the tumor. Using the CT26 syngeneic model for colorectal cancer, we describe a novel high-throughput phospho-flow platform that can simultaneously analyze the phosphorylation status of up to 6 signaling proteins in both tumor cells and immune cells. To first validate the phospho-flow assay, splenocytes from naïve mice were treated in vitro with various stimuli that preferentially target distinct signaling proteins. We then tested the platform in vivo. To this end, CT26 tumor-bearing mice were dosed with the MEK inhibitor trametinib or vehicle. At the appropriate time point post-dosing, tumors were harvested and dissociated into single cell suspensions. Live tumor cells and CD8+ T cells were first identified by flow cytometry using antibodies against CD45, CD3, and CD8. Following fixation, MAPK and JAK-STAT signaling activity was analyzed by measuring the levels of phosphorylated MEK, ERK1/2, STAT1, STAT2, STAT5, and STAT6 proteins using phospho-flow. Our results demonstrated that trametinib triggered a reduction in both pMEK and pERK1/2 levels in tumor cells that was evident 24 hours after the final trametinib dose. We also found phosphorylated STAT1, STAT3, and STAT5 was readily detectable in tumor cells, while STAT6 phosphorylation was not obvious. In contrast, we found that the CD8+ T cell levels of pMEK and pERK1/2 were not affected by trametinib. Furthermore, when JAK-STAT signaling was analyzed, only phosphorylation of STAT3 was detected in CD8+ T cells, which was also unaffected by trametinib. These results demonstrate successful combination of immunophenotyping and phospho-protein analysis in solid tumor-derived cells using flow cytometry. This platform is a tool that can be used to provide valuable insight into the effects that small molecule inhibitors may have on distinct cell subsets within a heterogenous tumor microenvironment.

Citation Format: David Draper, Alden Wong, Philip Lapinski, Sheri R. Barnes, Maryland Rosenfeld Franklin, Scott C. Wise. High-throughput analysis of MAPK and JAK-STAT signaling in CT26 tumors using a combination of immunophenotyping and phospho-flow cytometry [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 5667.
Abstract 5670: Systemic lymphoid architecture response assessment (SYLARAS): Application to system-wide immunophenotyping of glioblastoma
Authors: Baker Gregory J. , Palaniappan Sucheendra K. , Moore Jodene K. , Davis Stephanie H. , Sorger Peter K.
Published: August 2018

Interactive flow cytometry analysis software allows users to make binary decisions on immunomarker status of single cells through arbitrary placement of one- and two- dimensional gates. However, manual gating through a point-and-click graphical user interface (GUI) is impractical for large datasets. Here we describe SYLARAS, a suite of experimental and computational tools for the systematic and chronological evaluation of lymphoid tissue architecture. SYLARAS takes multiplex single-cell data as input, bins cells in n-dimensional orthant space, and returns schematized summary statistics on immunophenotypes of interest. We programmatically curated data on 33 immunophenotypes constituting primary and secondary lymphoid tissue of immunocompetent mice bearing syngeneic, orthotopic glioblastoma (GBM) at 3 stages of tumor growth. SYLARAS permitted the discovery of tumor-induced immune signatures whose divergence from mock engrafted, age-matched controls was quantified and statistically analyzed. By integrating information on peripheral immune composition with the levels of 111 blood cytokines of the same mice, we uncovered novel cell types, cytokines, and correlation networks perturbed by the mouse GL261 glioma model. SYLARAS facilitates the discovery of immune regulatory mechanisms and their response to therapy and is broadly applicable to the fields of immuno-oncology, autoimmunity, and infectious disease.

Citation Format: Gregory J. Baker, Sucheendra K. Palaniappan, Jodene K. Moore, Stephanie H. Davis, Peter K. Sorger. Systemic lymphoid architecture response assessment (SYLARAS): Application to system-wide immunophenotyping of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5670.
Abstract 5677: Establishment and application of a panel of PBMC-humanized mouse tumor models in immune-oncology and targeted cancer immunotherapy
Authors: Zhang Lan , Wu Haochen , Chen Fei , Zhao Lianqi , An Xiaoyu , Tan Weibin , Fu Xiaoyan , Qiao Meng , Shi Qian , Yang Wenqing
Published: August 2018

Background: Monoclonal antibodies and checkpoint blocking approaches have achieved remarkable success in cancer immunotherapy in clinical practices. Besides the success of anti-PD-1 and anti-PD-L1 antibodies (such as Keytrude and Tecentriq), two bispecific antibodies, Catumaxomab and Blinatumomab have been approved to treat cancer patients, and many more bispecific antibodies are currently in pre-clinical or clinical development. To meet the increasing market needs for fast, reliable and cost effective mouse tumor model systems, we developed a panel of humanized tumor models, designated as MiXenoTM platform. MiXenoTM could be used in a broad spectrum of applications in immuno-oncology drug discovery, including targeted cancer immune-therapy.

Method: CrownBio has a collection of about 200 xenograft models, including subcutaneous, orthotropic and systemic models, which covers majority of cancer type. Gene expression and mutation status are often profiled in these models. To engage both host immune system and tumor antigens, we have developed some specific MixenoTM tumor models by inoculating tumor cells over-expressing specific anti-tumor antigens (e.g. EGFR, CD47, Braf or PD-L1) into PBMC-humanized immunocompromised mice. Reconstitution of human immune component with human PBMCs in these tumor-bearing mice provides a useful tool to evaluate the targeted immune-therapeutics including bispecific T cell engagers.

Results: To validate the MiXenoTM models for targeted cancer immunotherapy, several xenograft models have been profiled and selected based on their tumor antigens or gene expression. Models with over-expression of a variety of tumor antigens (e.g. EGFR, CD47, Braf, PD-L1, etc.) were used to develop specific MixenoTM tumor models. The immune and tumor cells were engrafted either simultaneously or sequentially. Graft versus host disease (GvHD) can be managed by optimizing the immune cell constitution and tumor cell engraftment. Some models were also validated using stand of care I/O drugs and characterized by immune-phenotyping.

Conclusions: MiXenoTM tumor models with specific tumor antigen targeting strategy are valid model systems to evaluate the human immuno-modulatory drugs including bispecific antibodies. Further studies are needed to expand model collections and to extend their applications in I/O space.

Citation Format: Lan Zhang, Haochen Wu, Fei Chen, Lianqi Zhao, Xiaoyu An, Weibin Tan, Xiaoyan Fu, Meng Qiao, Qian Shi, Wenqing Yang. Establishment and application of a panel of PBMC-humanized mouse tumor models in immune-oncology and targeted cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5677.
Abstract 5689: Characterization of immune infiltrate and checkpoint protein expression patterns in murine syngeneic tumors via multiplex immunohistochemistry
Authors: Ziello Jennifer , Klein Sarah , Crosby Katherine
Published: August 2018

Murine syngeneic tumor models are increasingly utilized for preclinical immuno-oncology studies as immunotherapeutic strategies continue to make clinical strides. However, the immunologic features of the tumor microenvironment (TME) in these models remain largely undefined. In this study, we applied a 7-color multiplex immunohistochemistry panel to visualize and quantify the immune infiltrate within formalin-fixed, paraffin-embedded RENCA, CT26.WT, and LL/2 tumor tissues derived from subcutaneous mouse models of renal cell carcinoma, colon carcinoma, and lung carcinoma, respectively. Additionally, we applied this panel to a 4T1 orthotropic primary mammary tumor and lung metastasis formed via tail vein injection. The multiplex panel included antibodies detecting CD3 and CD8 as T cell markers, F4/80 as a myeloid cell marker, the immunosuppressive receptor PD-1 as well as its ligand PD-L1, pan-keratin as a tumor mask, and DAPI as a nuclear counterstain. We characterized the tissue localization of tumor-infiltrating immune cells and analyzed the trends in co-expression and frequency patterns of immunosuppressive proteins. This study strives to better understand the underlying differences in the immunologic landscapes of these tumors, which in turn has implications for researchers studying responses to immunotherapeutic approaches and combination strategies.

Citation Format: Jennifer Ziello, Sarah Klein, Katherine Crosby. Characterization of immune infiltrate and checkpoint protein expression patterns in murine syngeneic tumors via multiplex immunohistochemistry [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 5689.
Abstract 5690: Characterization of tumor growth and immune microenvironment in humanized NOG-EXL mice implanted with A549, MDA-MB-436 and A375 cells
Authors: Ghosh Srimoyee , Fiore Anne , Eberle Christoph , Wang Sarah , Mikule Keith , McEachern Kristen , Kumar Sujatha , Jenkins David , Sharma Geeta
Published: August 2018

Syngeneic mouse models are the most extensively explored mouse models for evaluation of immune-oncology therapeutic modalities in preclinical settings. One of the major limitations with syngeneic models is that both the tumor and the immune system is murine and thus do not express any human targets. Also, this precludes the evaluation of human clinical product (antibody or protein) mandating the synthesis and use of murine surrogates. Therefore, development of models of human tumors in mice with competent human immunity became an urgent need. Tumor-bearing, humanized NOG-EXL mice are a new and valuable preclinical testing platform for immuno-oncology, to simulate trials, evaluate multiple drugs alone or in combination, and produce predictive data both in cell line derived (CDX) and patient derived (PDX) models. With the objective of understanding the tumor growth and basal immune microenvironment in Hu-NOG-EXL mice (NOG mice humanized using CD34+ cells isolated from cord blood), we implanted three different human tumor cell lines (A549: Human Lung Carcinoma; MDA-MB-436: Triple Negative Breast Cancer; A375: Human Melanoma) in Hu-NOG EXL mice procured from Taconic, NY. The tumors were collected at a tumor volume of 1000-1500mm3, and profiled for the lymphoid and myeloid compartments using flow cytometry. The corresponding splenic compartment was also profiled to monitor the correlation or lack thereof between the tumor and splenic compartments. Finally the tumors were compared and categorized based on their basal infiltrates. The results demonstrated that Hu-NOG-EXL mice could be an useful tool to dissect the immune response in human tumors. It was possible to identify and quantitate the myeloid cells which are absent in a model that has been implanted with human PBMNC's (Peripheral Blood Mono Nuclear Cells) instead of human CD34 stem cells.

Citation Format: Srimoyee Ghosh, Anne Fiore, Christoph Eberle, Sarah Wang, Keith Mikule, Kristen McEachern, Sujatha Kumar, David Jenkins, Geeta Sharma. Characterization of tumor growth and immune microenvironment in humanized NOG-EXL mice implanted with A549, MDA-MB-436 and A375 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5690.
Abstract 5696: Discovery of putative breast cancer antigens using genomics-driven immunoproteomics
Authors: Han David K.
Published: August 2018

Recent advances in cancer immunotherapeutics such as checkpoint inhibitors, chimeric antigen-receptor T cells, and tumor-infiltrating T cells (TIL) are now significantly impacting cancer patients in a positive manner. Although very promising, reports indicate no more than 25% of cases result in complete remission. One of the limitations of these treatments is the identity of cancer antigens in each patient as it is technically challenging to identify cancer antigens in a rapid fashion. Thus, identification of cancer antigens for each patient followed by targeted treatment will likely increase the efficacy of cancer immunotherapies. To achieve this goal, a combined technologies of deep genomic sequencing and personalized immune assessment platform was devised, termed Genomics-Driven Immunoproteomics (GDI). Using this technological platform, we report the discovery of 149 putative tumor antigens from human breast cancer patients. Significant numbers of these cancer antigens arise from single nucleotide variants (SNVs), as well as insertions and deletions that results into frame-shift mutations. We propose a general model of anticancer immunity and suggest that GDI platform may help identify patient-specific tumor antigens in a timely fashion for precision immunotherapies.

Citation Format: David K. Han. Discovery of putative breast cancer antigens using genomics-driven immunoproteomics [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 5696.
Abstract 5701: Verification of the methodology for evaluating tumor-infiltrating lymphocytes in colorectal cancer
Authors: Matsutani Shinji , Shibutani Masatsune , Maeda Kiyoshi , Nagahara Hisashi , Fukuoka Tatsunari , Tamura Tatsuro , Ohira Go , Yamazoe Sadaaki , Kimura Kenjiro , Toyokawa Takahiro , Amano Ryosuke , Tanaka Hiroaki , Muguruma Kazuya , Hirakawa Kosei , Ohira Masaichi
Published: August 2018

Background: The density of tumor-infiltrating lymphocytes (TILs) have been reported to reflect antitumor immune response and correlate with prognosis in malignancy. However, the methodology for evaluating the density of TILs by an immunohistochemical analysis differs among reports. The aim of this study was to verify the methodology for evaluating the density of TILs by immunohistochemical analysis and thereby identify the optimum methodology in clinical setting.

Methods: Three-hundred-thirteen patients who underwent curative operation for stage II/III colorectal cancer were enrolled. We retrospectively examined the density of TILs using immunohistochemical staining according to each method as follows: 1) subset of lymphocytes (i.e. CD4+/CD8+), 2) selected fields (i.e. at random or focusing on hot spots), 3) location in low-power field (i.e. the invasive margin [TILsIM] or the center of the tumor [TILsCT] or the surface of the tumor [TILsST]), and 4) location in high-power field (i.e. in tumor stroma [sTILs] or intra-tumor cells [iTILs] or total TILs [tTILs: sTILs+iTILs]). We then assessed the prognostic value of the density of TILsIM evaluated as described above. We also evaluated the correlation between the density of TILsIM and that of TILsCT/TILsST.

Results: In randomly selected fields, the densities of CD8+sTILsIM and CD8+tTILsIM were significantly associated with the survival. However, the densities of TILs evaluated via other methods were not associated with the survival. Furthermore, the density of CD8+TILsIM was significantly associated with that of CD8+TILsCT and CD8+TILsST.

Conclusions: We concluded that best and easiest way to evaluate the density of TILs in the clinical setting may be to assess the density of total CD8+TILs at the invasive margin in randomly selected fields.

Citation Format: Shinji Matsutani, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsunari Fukuoka, Tatsuro Tamura, Go Ohira, Sadaaki Yamazoe, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Verification of the methodology for evaluating tumor-infiltrating lymphocytes in colorectal 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 5701.
Abstract 5702: Characterization of circulating tumor cells during immune checkpoint inhibition in metastatic renal cell carcinoma and malignant melanoma
Authors: Zhang Tian , Park SinJung , Runyambo Daniella , Poellmann Michael J. , Reyes-Martinez Marco , Mahbooba Zahra , Green Rebecca , Lee Carrie , George Daniel J. , Armstrong Andrew J. , Hong Seungpyo , Wang Andrew Z.
Published: August 2018

Background: Although immune checkpoint inhibitors (ICIs) have shown efficacy in a range of solid tumors, there are no established circulating biomarkers able to identify early responders to treatment. Circulating tumor cells (CTCs) allow a non-invasive peripheral assessment of disease burden over time. We evaluated the prevalence of CTCs before and during treatment with ICIs.

Methods: Patients were enrolled prospectively at Duke Cancer Center and UNC Lineberger Cancer Center and selected for metastatic renal cell carcinoma (mRCC) and metastatic malignant melanoma (mMM) receiving standard of care immunotherapy. Using E-selectin for slowing cell rolling and a nanoparticle dendrimer based technology for cell capture with EpCAM, HER2, and EGFR, the CapioCyte platform was used for CTC capture at baseline, during treatment, and upon disease progression.

Results: We enrolled 5 subjects with mRCC and 4 subjects with mMM. Prior to treatment, subjects had a median of 293 CTCs/mL (all healthy volunteers (HVs, n=10) had <8 CTCs/mL). All subjects had more CTCs at baseline than HVs. One subject had CTCs rise within the first 4 weeks on ICI treatment, which corresponded to clinical disease progression. Several other subjects showed a rapid decrease in CTCs within 4 weeks but increased at the time of disease progression. In most cases of disease control, CTCs decreased by at least 50% at the 4-week timepoint and increased at time of disease progression.

Conclusions: CapioCyte is a sensitive platform to detect CTCs in mRCC and mMM. Based on CTC detection, many patients had early response to ICI treatment, but rapid progression corresponding to clinical progression. This pilot data suggest that CTCs can be a valuable biomarker for monitoring the clinical benefit of cancer immunotherapies.

Citation Format: Tian Zhang, SinJung Park, Daniella Runyambo, Michael J. Poellmann, Marco Reyes-Martinez, Zahra Mahbooba, Rebecca Green, Carrie Lee, Daniel J. George, Andrew J. Armstrong, Seungpyo Hong, Andrew Z. Wang. Characterization of circulating tumor cells during immune checkpoint inhibition in metastatic renal cell carcinoma and malignant 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 5702.
Abstract 5703: Immunoscore as prognostic biomarkers in patients with epithelial ovarian cancer
Authors: Lee Shin-Wha , Kim Min-Je , Kim Ju-hyun , Lee Young-Jae , Kim Yong-Man , Kim Yong-Man
Published: August 2018

Background: The analysis of single parameters alone may not provide sufficient insights about complex immune system-tumor interactions. This study is to validate the immune contexture as prognostic biomarkers in high-grade serous ovarian cancer (HGS-OC) and to find new era of immunoscore in HGS-OC.

Methods: We collected FFPE samples from 187 patients with HSOC and produced TMA samples. We accomplished the OPAL multiplex IHC assay for the quantitative analysis of immune markers, including CD4, CD8, CD20, FoxP3, PD-L1, and CK. Multiplex Biomarker Imaging and inForm® Image Analysis Software was used.

Results: FIGO stage III and IV patients were 84.5% (158/187). The optimal debulking surgery was done in 66.8% (125/187). The 3-year disease-free survival and 5-year overall survival were 35.1% and 50.0%, respectively. Any single marker was not related to the survival, including CD8, FoxP3, and PD-L1. However, high CD8:FoxP3 and CD8:PD-L1 ratios were correlated with good survival. In Cox regression model, the risk factors for HGS-OC survival were FIGO stage (HR 1.784, 95% CI: 1.295-2.457, p<0.001) and platinum resistance (HR 4.257, 95% CI: 2.753-6.582, p<0.001). Additionally, CD8:PD-L1 ratio was a favorable prognostic factor (HR 0.621, 95% CI: 0.042-0.917, p=0.017).

Conclusion: These findings indicate that although any single immune marker is not related to the survival, CD8:FoxP3 and CD8:PD-L1 ratios provide the positive correlation with the prognosis in HGS-OC; especially, CD8:PD-L1 ratio is prognostic biomarker that is comparable to clinical biomarkers. The next study for immunoscore is necessary to define immunoscore in ovarian cancer.

Citation Format: Shin-Wha Lee, Min-Je Kim, Ju-hyun Kim, Young-Jae Lee, Yong-Man Kim, Yong-Man Kim. Immunoscore as prognostic biomarkers in patients with epithelial ovarian 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 5703.
Abstract 5714: SPADE identification of novel MDSC subsets
Authors: Cole Kathryn , Britton Holly , Warkentin Phyllis , Talmadge James E.
Published: August 2018

Myeloid derived suppressor cells (MDSC's) are a heterogeneous population of myeloid cells at varying stages of differentiation that occur in increased numbers in the blood of cancer patients. The phenotypes of MDSC's, are controversial but are generally subdivided into three phenotypes: macrophage (M-), granulocytic (G-) and immature (i-) MDSC's. Our studies used a single staining tube with antibodies to linage markers (CD3, CD19 and CD56), HLA-DR, CD11b, CD14, CD15, CD16, CD33, CD34, CD45, PD-L1 and lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1). Mobilized apharesis products from lymphoma patients provided a product with a high frequency of MDSCs compared to normal donor blood samples. A total MDSC population (Lin-HLA-DR-CD11b+CD14+ and Lin-HLA-DR-CD11b+CD14-CD33+) was exported from FlowJo to Spanning tree progression analysis of density normalized events (Spade) in Matlab. This analysis identified nine MDSC sub-populations as nodes. Impressively, cells from the non-MDSC populations, including monocytes and granulocytes did not occur within these nodes. The SPADE analysis identified four M-MDSC nodes, four G-MDSC nodes and one iMDSC node. LOX-1 expression was limited to two G-MDSC and one M-MDSC subset. The nodes were differentiated based on CD14, CD15, CD33, CD16, LOX-1, and CD34 expression. The total populations were SPADE analyzed by overlaying non-MDSCs populations onto MDSCs and monocytes and granulocytes subset to nodes independent of MDSC nodes. The assessment of MDSC function used intracellular arginase-1 and nitric oxide synthetase-2 expression with the expected differential expression consistent with the M and G-MDSC subsets. The proportions of the different MDSC nodes varied between cancer patients and were generally at least a log higher than in the blood of normal donors. In summary, a SPADE analysis can cleanly identify MDSCs from monocytes and granulocytes and provide insight into novel MDSC subsets.

Citation Format: Kathryn Cole, Holly Britton, Phyllis Warkentin, James E. Talmadge. SPADE identification of novel MDSC subsets [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 5714.
Abstract 5715: Monitoring immune cells-mediated cytolysis through impedance technology allows kinetic analysis of reagent efficacy
Authors: Cerignoli Fabio , Xi Biao , Guenther Garret , Muir Lincoln , Lamarche Brandon , Shields Jason , Abassi Yama
Published: August 2018

In vitro characterization of immunotherapy reagents efficacy and combination synergies is necessary before moving to expensive animal models and clinical studies. Current standard assays like Chromium-51 release, ATP-based luminescence cell monitoring or flow cytometry are difficult to implement in a high throughput environment and are based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we validate an impedance-based platform for monitoring cytotoxic activity of immune cells in the context of cancer immunotherapy assays. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates, without the use of labeling or cell modification. We present a large set of validation including CD19 Car T models, EpCAM/CD3 BiTE antibodies and checkpoint combination therapies with anti PD-1/PD-L1 and CTLA-4 antibodies. Data comparison with Annexin V staining/Flow Cytometry shows perfect correlation between the drop in impedance signal and % of apoptotic cells. Long time monitoring (> 7 days) in combination with low effector:target ratios reveal the occurrence of resistance mechanisms in target cells and exhaustion in effector cells. Such dynamic response has been observed in animal models and patient clinical trials, but it is difficult to monitor in short time assays. Overall, our results demonstrate the value of such label-free approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays but that is highly relevant when testing combinations. Thanks to the availability of 384-wells format and minimal sample handling, the technology is ideally suited for applications in large reagent validation screening or therapeutic protocol validation directly on patient samples.

Citation Format: Fabio Cerignoli, Biao Xi, Garret Guenther, Lincoln Muir, Brandon Lamarche, Jason Shields, Yama Abassi. Monitoring immune cells-mediated cytolysis through impedance technology allows kinetic analysis of reagent efficacy [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 5715.
Abstract 5739: Targeting EZH2 histone methyltransferase activity alleviates inflammatory bowel disease
Authors: Long Haixia , Zhou Jie , Wang Zhongyu , Huang Shuo , Zhu Bo
Published: August 2018

Objective: Inflammation plays crucial roles in the pathogenesis of inflammatory bowel disease (IBD). Enhancer of zeste homolog 2 (EZH2)-mediated trimethylation of histone 3 lysine 27 (H3K27m3) is critical to immune regulation. However, evidence is lacking to address the effect of EZH2 enzyme's activity on intestinal immune responses during IBD. This study aimed to explore the effects of targeting EZH2 methyltransferase activity on IBD progression in mice.

Design: EZH2's enzymatic activity inhibitor GSK343 or GSK126 were delivered by intravenous injection to C57BL/6 mice at the onset, or during the ongoing of IBD, induced by dextran sodium sulfate (DSS). Azoxymethane (AOM)/DSS protocol was applied to trigger colitis-associated colorectal cancer (CAC). Immune cells in the colon of colitis mice were analyzed by flow cytometry, immunofluorescence staining and myeloid-derived suppressor cells (MDSCs) deletion experiments. We differentiated mouse hematopoietic progenitor cells (HPCs) into MDSCs in vitro to investigate the effect of GSK343 on MDSCs generation.

Results: Inhibition of EZH2 activity significantly attenuated IBD-related symptoms and inflammation, substantially delaying the onset of CAC. Injection of GSK343 increased the number of functional MDSCs in the colons during IBD. Deletion of MDSCs abrogated the protective effects of GSK343 on IBD. Moreover, inhibition of EZH2 activity promoted the generation of MDSCs from HPCs.

Conclusion: Inhibition of EZH2 activity alleviates DSS-induced colitis by promoting MDSC development, and might serve as a new therapeutic strategy to treat IBD. Additionally, EZH2 inhibition enhances MDSC generation, therefore, EZH2 enzymatic inhibitors now in clinical trials for cancer treatment may hinder beneficial anticancer immunity responses.

Citation Format: Haixia Long, Jie Zhou, Zhongyu Wang, Shuo Huang, Bo Zhu. Targeting EZH2 histone methyltransferase activity alleviates inflammatory bowel disease [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 5739.
Abstract 5740: PD-L1 overexpression induced by oncogenic KRAS and BRAF mutations through MAPK pathway activation in non-small cell lung cancer
Authors: Miura Yosuke , Sunaga Noriaki , Bao Pinjie , Girard Luc , Tsukagoshi Yusuke , Masuda Tomomi , Kasahara Norimitsu , Sakurai Reiko , Kaira Kyoichi , Hisada Takeshi , Minna John , Yamada Masanobu
Published: August 2018

Mutant KRAS and BRAF are important oncogenic drivers in non-small cell lung cancer (NSCLC), however, effective treatment strategies for most of these mutants have not been established. Immuno-oncology clinical trials have shown promising results of anti-programmed death 1 (PD-1) antibodies for a subset of NSCLC patients and tumor expression of programmed-death ligand 1 (PD-L1) is regarded as a predictive marker for anti-PD-1 therapy. We found that CD274 mRNA and PD-L1 protein expression were reduced in NSCLC lines by small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, and also by pharmacologic inhibitors of MEK and ERK. In addition, there was a positive correlation between CD274 expression and KRAS expression in NSCLC lines, and analyses of The Cancer Genome Atlas (TCGA) database revealed that NSCLCs with high KRAS expression exhibited higher levels of CD274 compared to the tumors with low KRAS expression among KRAS-mutant NSCLCs. Similarly, siRNA-mediated BRAF knockdown and inhibitors of BRAF and MEK reduced PD-L1 expression in BRAF-mutant NSCLC cells, suggesting that PD-L1 expression was also upregulated by oncogenic BRAF. Mechanistically, these results suggest that oncogenic KRAS and BRAF induce PD-L1 overexpression in NSCLCs by activation of the MEK-ERK pathway. For clinical translation, our results suggest that: 1. in NSCLCs the combination of KRAS and PD-L1 overexpression could be a surrogate predictive marker for anti-PD-1 treatment; and 2. that since the goal of checkpoint inhibitor immuno-oncology therapy is to inhibit the PD-L1 axis, that a combination of anti-PD-1 and targeted therapy to inhibit the MEK-ERK pathway could be beneficial.

Citation Format: Yosuke Miura, Noriaki Sunaga, Pinjie Bao, Luc Girard, Yusuke Tsukagoshi, Tomomi Masuda, Norimitsu Kasahara, Reiko Sakurai, Kyoichi Kaira, Takeshi Hisada, John Minna, Masanobu Yamada. PD-L1 overexpression induced by oncogenic KRAS and BRAF mutations through MAPK pathway activation in non-small cell 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 5740.
Abstract 5758: Generation and characterization of novel monoclonal antibodies against overexpressed CD109 on pancreatic cancer cells for use in diagnosis and therapy
Authors: Arias-Pinilla Gustavo A. , Dalgleish Angus , Mudan Satvinder , Bagwan Izhar , Walker Tony , Modjtahedi Helmout
Published: August 2018

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, and several have been approved for the treatment of human cancers; however, none have approval for pancreatic cancer. Our aim was to discover novel overexpressed cell surface antigens in human pancreatic cancer cells using mAb technology. We have generated two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and mass spectrometry, we discover that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumor cell growth in vitro or cell migration. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumor cells by Western blot and immunohistochemistry. Immunohistochemistry of human pancreatic carcinoma arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumor specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research should unravel the therapeutic potential of the humanized forms or the conjugated versions of such antibodies in patients whose tumors overexpress CD109 antigen.

Citation Format: Gustavo A. Arias-Pinilla, Angus Dalgleish, Satvinder Mudan, Izhar Bagwan, Tony Walker, Helmout Modjtahedi. Generation and characterization of novel monoclonal antibodies against overexpressed CD109 on pancreatic cancer cells for use in diagnosis and therapy [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 5758.
Abstract 5767: Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy
Authors: Mitsunaga Makoto , Nishimura Takashi , Ito Kimihiro
Published: August 2018

Background

Photoimmunotherapy (PIT) is a new class of molecular targeted cancer theranostics, which employs monoclonal antibody (mAb) conjugated to a photosensitizer, IR700, that is activated by focal near-infrared (NIR) light irradiation leading to necrotic cell death by cell membrane rapture where mAb-IR700 conjugates binds to target membrane proteins specifically. We have previously demonstrated HER2-targeted PIT employing trastuzumab-IR700 conjugates for HER2 expressing cancers. However, some cancer cells were survived partly because of tumor heterogeneity and inhomogeneous micro-distribution of mAb-IR700 conjugates. In this study, we developed a new type of PIT agents targeting VEGFR-2 expressed on vascular endothelium in a tumor and evaluated the feasibility by comparing conventional PIT in vitro and in vivo.

Methods

HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. HER2-targeting trastuzumab and VEGFR-2-targeting ramucirumab were conjugated to IR700. Cells were treated with mAb-IR700 conjugates followed by NIR light irradiation after washing the cells. LIVE/DEAD assay was performed to assess short-term phototoxicity and trypan blue dye exclusion assay to assess long-term phototoxicity. Next, mouse tumor xenograft models were created for in vivo PIT. Tumor-bearing mice were randomized and treated with mAb-IR700 conjugates followed by NIR light irradiation under anesthesia. Antitumor effects were monitored by measuring tumor diameters with a caliper.

Results

PIT utilizing ramucirumab-IR700 conjugates did not induce phototoxic effect in vitro because of the absence of membranous expression of VEGFR-2 in NCI-N87 cells, while PIT utilizing trastuzumab-IR700 conjugates induced rapid phototoxic effect because of the strong membranous expression of HER2 in NCI-N87 cells. By contrast, antitumor effects were observed in NCI-N87 xenograft tumors in vivo utilizing both ramucirumab- and trastuzumab-IR700 conjugates followed by NIR light irradiation. Microscopic analysis of the harvested tumors showed decreasing tumor micro-vessel densities only when tumors were treated with PIT utilizing ramucirumab-IR700 conjugates but not trastuzumab-IR700, which represents a different mechanism than that of conventional PIT targeting antigens expressed on the tumor cell membrane.

Conclusion

We demonstrated a new mechanism of PIT utilizing ramucirumab-IR700 conjugates. As VEGFR-2 is upregulated in many types of solid cancers, this method may be considered as being applicable to various types of cancers in future clinical settings.

Citation Format: Makoto Mitsunaga, Takashi Nishimura, Kimihiro Ito. Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy [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 5767.
Abstract 5786: Grabbing GRB2: The use of liposome-incorporated Grb2 antisense oligonucleotides as a novel therapy in gynecologic malignancies
Authors: Lara Olivia D. , Rodriquez-Aguayo Cristian , Bayrakter Emine , Amero Paola , Mangala Selanere , LaFargue Chris , Tari-Ashizawa Ana , Lopez-Berestein Gabriel , Sood Anil
Published: August 2018

Prexigebersen (liposome-incorporated nuclease resistant antisense oligonucleotides specific for GRB2, L-GRB2) has proven highly effective in the treatment of leukemia, however its application in solid malignancies including gynecologic cancer has yet to be investigated. Given the biological significance of GRB2, we aim to assess the effect of prexigebersen in pre-clinical models. We tested the clinical significance of GRB2 using publicly available high-throughput data. We investigated the expression of GRB2 in a series of in vitro (Western Blotting) experiments in high-grade serous (HGSC) and uterine (UC) carcinoma models. We also tested in vivo (orthotopic mouse model) the biological effects of prexigebersen in HGSC models (OVCAR 5), first in a dose-defining experiment then in combination with standard dose paclitaxel. We first examined GRB2 using TCGA and GISTIC data and found alterations (amplifications, deletions and mutations) in 6% (33 of 594) of HGSC cases and 5% (25 of 547) of UC cases. Amplifications of GRB2 are associated with a decreased patient survival (p<0.01) in UC, suggesting it to be an attractive therapeutic target. GRB2 was expressed in most ovarian (HEYA8, OVCAR5, SKOV3, and OVCAR3) and uterine (MFE 319, Ishikawa and Hec1a) cancer cell lines tested. We performed a dose-defining in vivo experiment on an orthotopic mouse model of ovarian cancer (OVCAR 5). We found a maximum decrease in GRB2 expression at a dose of 15 mg/kg prexigebersen and a dose dependent decrease in tumor burden (p<0.05). We then performed an additional in vivo model in which prexigebersen was combined with standard dose paclitaxel. There was an eighty-six percent decrease in tumor burden (p<0.05), and multinodular burden (p<0.01) in the combination prexigebersen/paclitaxel group compared to control. Prexigebersen shows promising preclinical efficacy and may be a novel therapeutic strategy in gynecologic malignancies.

Citation Format: Olivia D. Lara, Cristian Rodriquez-Aguayo, Emine Bayrakter, Paola Amero, Selanere Mangala, Chris LaFargue, Ana Tari-Ashizawa, Gabriel Lopez-Berestein, Anil Sood. Grabbing GRB2: The use of liposome-incorporated Grb2 antisense oligonucleotides as a novel therapy in gynecologic malignancies [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 5786.
Abstract 5796: Modulation of transcription factor landscape through BET family protein inhibition is dependent on specific driver mutations in pancreatic cancer
Authors: Weldon Mikele G. , Wang Albert R. , Beyer Hope A. , Stiles Shannon G. , Iyer Gopal
Published: August 2018

Targeting of bromodomain and extra-terminal domain (BET) family protein activity has the potential to reduce proliferation in many cancers. However, changes in transcription factor (TF) landscape can be influenced by different mutational profiles of specific cancers. Within this mutational background, BET proteins can target these TFs and create an oncogenic phenotype. To exploit these altered TF readouts, we tested the hypothesis that inhibition of BET activity in different genetic mutations of pancreatic cancer will lead to antiproliferative effects. Towards this, we treated pancreatic cancer cell lines that had mutations in KRAS, CDKN1A, SMAD4 and TP53 with a bromodomain inhibitor. We showed that cell lines with CDKN1A deletion and wild-type SMAD4 were highly resistant to BET inhibition and had a high nanomolar IC50 concentration. Through targeted transcription factor gene expression profiling of over 400 TFs, we discovered that LMO2, GTF2B, ID1, and CREB1 were upregulated, while SALL4, TCF4, and TP63 were downregulated in these cell lines. This suggests that these TFs may be partly responsible for resistance to BET inhibition. Furthermore, we showed dramatic changes in TF landscapes in pancreatic cancer cells sensitive to BET treatment compared to the resistant cells. This indicates that for antiproliferative effects in pancreatic cancer cells, modulating BET activity may be necessary to switch on or off specific TFs. Together, these results suggest that BET inhibitors may be useful in pancreatic cancer treatment and that mutation-specific differences in TF targets of BETs may dictate the progression of pancreatic cancer.

Citation Format: Mikele G. Weldon, Albert R. Wang, Hope A. Beyer, Shannon G. Stiles, Gopal Iyer. Modulation of transcription factor landscape through BET family protein inhibition is dependent on specific driver mutations in pancreatic 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 5796.
Abstract 5797: BET inhibitor synthetic lethality in malignant peripheral nerve sheath tumors
Authors: Cooper Jonathan M. , Patel Amish J. , Chen Zhiguo , Liao Chung-Ping , Chen Kun , Wang Yong , Le Lu Q.
Published: August 2018

BET bromodomain inhibitors have emerged as a promising therapy for numerous cancer types in pre-clinical studies, but molecular indicators of tumor response are lacking. Through modeling tumor evolution by studying genetic lesions underlying the development of Neurofibromatosis Type 1 (NF1)-associated Malignant Peripheral Nerve Sheath Tumors (MPNST), a lethal sarcoma with no effective medical treatment, we identified a BRD4 dependency that serves as a controlled model system to delineate mechanisms of sensitivity or resistance to BET bromodomain inhibitors in this disease. In this study, we show that loss of NF1 and p53 is associated with increased BRD4 protein levels and partial sensitivity to BET inhibition in MPNST. Strikingly, genetic depletion of BRD4 profoundly sensitizes MPNST cells to diverse BET inhibitors in culture and in vivo, while wild-type cells are spared. Collectively, genetic and pharmacological inhibition of BRD4 reveals that BET inhibitor resistance in MPNST requires wild-type BRD4 to support growth in a bromodomain-independent manner. Our findings provide a framework for understanding BET inhibitor sensitivity and resistance within distinct molecular subsets of MPNST. In addition, discovery that a synthetic lethality exists between BET inhibition and reduced BRD4 levels nominates a BRD4-low patient subpopulation as that which might best respond to a BET inhibitor therapeutic strategy for MPNST.

Citation Format: Jonathan M. Cooper, Amish J. Patel, Zhiguo Chen, Chung-Ping Liao, Kun Chen, Yong Wang, Lu Q. Le. BET inhibitor synthetic lethality in malignant peripheral nerve sheath 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 5797.