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  • 11
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    Online Resource
    Abstract 1656: Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft model
    American Association for Cancer Research (AACR) ; 2017
    In:  Cancer Research Vol. 77, No. 13_Supplement ( 2017-07-01), p. 1656-1656
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 77, No. 13_Supplement ( 2017-07-01), p. 1656-1656
    Abstract: The recent successes of immunotherapeutic approaches to the treatment of melanoma and the promise of similar treatments in a variety of other cancers underscore the importance of the immune system in cancer. Indeed, effective therapeutic design and evaluation require a comprehensive understanding of the interplay between the immune compartment and the proliferating tumor cells that comprise the tumor microenvironment. A humanized mouse strain engrafted with cancerous tissue from a patient derived xenograft (PDX) tumor provides researchers with a highly sophisticated tool, ideally suited to facilitate the design of treatment strategies that prevent tumor evasion of immune cells and that improve cytotoxic responses. Severely combined immunodeficient mice such as NOD scid gamma (NSGTM) and triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSGTM-SGM3) are proven hosts for the engraftment of human tumors and establishment of human immune system components following hematopoietic stem cell (CD34+) transplantation. The endogenous expression of cytokines that support the development of myeloid lineages and regulatory T cells potentially represents a substantial improvement over standard NSG mice. Here we employ four 14-color flow cytometry panels to perform a comprehensive and detailed analysis of the entire immune system. The four panels are designed to fully characterize specific branches of the immune system: 1) T cells 2) NK cells/dendritic cells/B cells 3) myeloid lineages, and 4) immune checkpoint markers. Blood, spleen, and bone marrow tissue from both NSG and NSG-SGM3 mice were evaluated at 9, 16, 21, and 31 weeks of age using each of the four phenotyping panels. Our results indicate that the triple transgenic NSG-SGM3 mice exhibit a more completely humanized immune system compared to NSG mice, with specific improvements in the distribution of T-cell subsets and overall representation of the myeloid lineage. NSG mice engrafted with allogeneic human tumors represent a valuable preclinical testing platform for immuno-oncology. Citation Format: Aaron J. Middlebrook, Eileen Snowden, Warren Porter, Friedrich Hahn, Mitchell Ferguson, Brian Soper, James Keck, Joan Malcolm, Shannon Dillmore, Smita Ghanekar, Rainer Blaesius. Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2017-1656
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2017-1656
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2017
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  • 12
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    Abstract 3949: Deep immunophenotyping using flow cytometry of dissociated cells from Barrett's esophagus and matched adjacent squamous epithelium defines distinct phenotypic clusters
    American Association for Cancer Research (AACR) ; 2017
    In:  Cancer Research Vol. 77, No. 13_Supplement ( 2017-07-01), p. 3949-3949
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 77, No. 13_Supplement ( 2017-07-01), p. 3949-3949
    Abstract: Barrett's esophagus (BE) is defined as metaplasia of the squamous epithelium to a specialized columnar epithelium with risk factors of gastroesophageal reflux and obesity and a predilection for middle-age and older white males. BE progresses through stages of dysplasia (low-grade and high-grade) before developing into esophageal adenocarcinoma. Challenges remain in early detection and predicting which patients may progress to dysplasia. Here we describe a method by which we compare human clinical IRB-approved BE biopsies and adjacent normal squamous epithelium using tissue dissociation and deep immunophenotyping by flow cytometric collection and analysis. A cassette of canonical epithelial or tumor stem cell-associated targets (EpCAM, CD24, CD44, CD49f, Her2/neu, CD133, CD90, CD166, and CD29), immune cell markers (CD3, CD45, CD127, HLA-DR, CD16, CD56, CD4, CD8, CD25, and CD19), as well as targets associated with myeloid derived suppressor cells (CD14, CD15, CD33, CD11b, HLA-DR, CD31 and CD86) were used to discern differences across subjects and between cellular compartments in normal and BE tissue. The Barrett’s samples show a majority population with a characteristic phenotype (EpCAM+CD133lowCD49fhigh) when compared with normal squamous tissue samples (EpCAM-CD133-CD49flow). The samples separate into two discrete groups using hierarchical clustering based on differential surface marker expression of combined epithelial and immune cell markers, but also reveal unexpected, shared phenotypes for some normal and BE samples. Principal component analysis supports this grouping and was used to identify more compelling targets for categorization, such as CD133 and CD49f. The resulting expression and distribution of targets offer a phenotypic fingerprint characterizing both the epithelial cell and immune cell compartment. Besides providing the potential for revealing clinically relevant differences between BE and normal tissue, as well as across subjects, the discovered surface immunophenotypes can be used to target specific subpopulations from dysplastic tissue for further molecular investigation. A deeper understanding of the role of such specific subpopulations should increase the prospects for more complete understanding of BE and its progression. Citation Format: Friedrich G. Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, William S. Dillmore, Stephanie S. Yee, Taylor Black, Maureen DeMarshall, Aaron Middlebrook, Smita Ghanekar, Anil Rustgi, Erica L. Carpenter, Rainer Blaesius. Deep immunophenotyping using flow cytometry of dissociated cells from Barrett's esophagus and matched adjacent squamous epithelium defines distinct phenotypic clusters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3949. doi:10.1158/1538-7445.AM2017-3949
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2017-3949
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2017
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  • 13
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    Abstract B11: Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry
    American Association for Cancer Research (AACR) ; 2015
    In:  Molecular Cancer Therapeutics Vol. 14, No. 12_Supplement_2 ( 2015-12-01), p. B11-B11
    Details
    In: Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 14, No. 12_Supplement_2 ( 2015-12-01), p. B11-B11
    Abstract: The model of solid tumors as monolithic entities under the control of a handful of driver genes which seemed to dominate the general perception of cancer for many years is increasingly being replaced by that of highly diverse ecosystems of cells. Multiple tumor subpopulations, as well as attending non-cancerous cells such as fibroblasts, endothelial cells, and cells from the immune compartment populate this ecosystem, communicate with each other and all influence clinically relevant decision points throughout tumorigenesis. Progress in analyzing and characterizing this highly heterogeneous, complex “society of cells” has been remarkably enhanced using the strengths of flow cytometry. A rather comprehensive characterization of individual cells within a population, known as deep phenotyping, is becoming possible with recent advances in multi-parametric staining, collection, and analysis of solid tissue derived cells. This technology is enabling researchers to identify multiple targets within a single sample more efficiently than with more traditional methods. We have developed a process whereby single cells are liberated from solid tumors through a combination of mechanical and enzymatic treatments and then interrogated by flow cytometry using deep phenotyping. Our chosen marker panels include targets (CD24, CD44, CD49f, EpCAM, CD166, CD133, CD184, HER2/Neu) which have been used to investigate properties relevant for cancer stem cells (CSC), Endothelial Mesenchymal Transition (EMT) and Tumor Microenvironmental (TME) processes but options are built into the design to accommodate less well characterized targets such as GD2, CD73, Notch receptors, EphB2, and c-Met. We have identified discrete subpopulations in breast cancer patient-derived xenograft (PDX) tumors in mice, demonstrating consistent and reproducible results which constitute a distinct immunophenotypic fingerprint for every model. Building on the experience with PDX derived biopsies and adding markers targeting the immune system we applied our work flow to clinical breast cancer tissue. Our results demonstrate that the multi-dimensional analyses enabled by a surface marker panel reveals differences in highly characterized subpopulations, some at less than 1% of the total population, that remain hidden by more conventional assessment methods. Citation Format: Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Shannon Dillmore, Aaron Middlebrook, Shahryar Niknam, Peter Llontop, Smita Ghanekar, Rainer Blaesius. Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B11.
    Type of Medium: Online Resource
    ISSN: 1535-7163 , 1538-8514
    URL: Article
    DOI: 10.1158/1535-7163.TARG-15-B11
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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    SSG: 12
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  • 14
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    Abstract 2113: The effects of enzymatic digestion on epitope detection by flow cytometry
    American Association for Cancer Research (AACR) ; 2018
    In:  Cancer Research Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2113-2113
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2113-2113
    Abstract: The heterogeneous nature of solid tumors, coupled with the relatively small sample size of available biopsies, has led to an emerging need to glean as much information as possible from these valuable specimens. Current approaches to solid tumor analysis fail to completely reveal the diverse range of cellular compartments that comprise the tumor microenvironment. A comprehensive approach to tumor interrogation requires efficient tissue dissociation to facilitate analysis at the single-cell level. In contrast to current methods, single-cell analysis of tumor derived cell suspensions by flow cytometry has the potential to provide a more complete understanding of the many subpopulations within the tumor microenvironment and the cell to cell interactions that govern this space. In order to prepare a cell suspension from solid tumor tissue, most investigators subject the tumor sample to a combination of mechanical and enzymatic dissociation modalities. Regardless of the combination of enzymes used for dissociation, there exists a degree of non-specific proteolytic cleavage. In the work presented here, we set out to quantify the non-specific cleavage associated with a proprietary enzyme mixture developed for tumor dissociation. Using a mixed sample that included peripheral blood mononuclear cells and several cell lines, we were able to generate a sample that expressed approximately 90% of our current antibody catalog. By staining our sample with a rudimentary 3 color panel, we were able to identify 10 individual cell populations based on marker expression and/or scatter. This composite sample was left untreated or exposed to our propriety mixture of dissociation enzymes and then subsequently screened for all 251 antigens in our catalog. The impact of non-specific proteolytic cleavage on surface marker detection was gauged in terms of median fluorescence intensity of positive population and stain index. Our results indicate that of the 251 surface markers tested in our experimental series, less than 10 percent were significantly affected by enzymatic digestion. Of those markers that were affected only a fraction of them were affected to such an extent that it was impossible to detect positive expression. These results underscore the importance of understanding the factors that can influence surface marker detection and bolster our confidence that this particular mixture of enzymes is imparting minimal impact on surface marker detection. Citation Format: Aaron J. Middlebrook, Caitlin Austin, Daniel Santos, Eileen Snowden, Warren Porter, Friedrich Hahn, Mitchell Ferguson, Rainer Blaesius, Smita Ghanekar. The effects of enzymatic digestion on epitope detection by 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 2113.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2018-2113
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2018
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    detail.hit.zdb_id: 410466-3
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  • 15
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    Abstract 2197: Surface marker and gene expression profiling of tumors from PDX models of small cell lung cancer with varying sensitivity to growth inhibition by the p70S6K/AKT1/3 inhibitor M2698
    American Association for Cancer Research (AACR) ; 2018
    In:  Cancer Research Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2197-2197
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 2197-2197
    Abstract: Small Cell Lung Cancer (SCLC) is characterized by rapid tumor growth and currently, there are few therapeutic options. As part of the development of M2698, a potent, selective inhibitor of p70S6K and AKT 1/3, the compound was tested in a preclinical screen of 45 patient derived xenograft (PDX) models of SCLC. In this screen, two mice were implanted subcutaneously with tumors for each model; one mouse was treated with vehicle while the other was treated with M2698 25 mg/kg QD po and tumors were measured 2x/week until the tumor in the vehicle-treated mouse reached ~1200 mm3. Tumor control (tumor stasis or regression) was seen in 12 (27%) of the models. From these data, a subset of models that were the most and the least sensitive to M2698 (n=7) were selected for implantation into a new set of mice. Tumors were profiled once they reached ~800 mm3 for tumor cell surface markers potentially correlating with sensitivity to M2698 seen in the previous study. PDX model systems are capable of recapitulating the intra-tumor heterogeneity (ITH) observed in the original patient tumor. The relationship between ITH and drug response/acquired drug resistance has been clearly demonstrated. Thus, a more detailed examination of this complexity in PDX tissue enables one to elucidate the underlying mechanisms in a reproducible, biologically relevant system. We have previously shown that cell surface marker profiles of PDX derived tumor tissue demonstrate high intra-model reproducibility whereas each model has a unique profile. In addition, some markers have a distinct heterogeneity. Gene expression profiling of tumor cell subpopulations identified by some of these specific markers suggest different biological roles for these subpopulations with relevance to their drug responses. Here we show surface marker and gene expression profiles of multiple PDX models for SCLC. We have evaluated 50+ markers commonly used to identify tumor initiating cells (e.g. CD44, CD90, CD133, CD166, CD184), EMT or aggressiveness (e.g. CD166, EphB2, CD324, CD325) or drug targets (e.g. CD184, EGFR, Her2) to establish extensive marker profiles. Our data reveal that surface marker profiles in SCLC PDX models are consistent among tumors from mice bearing the same model, show major differences from other PDX (e.g. breast) cancer types and heterogeneity exists for several markers of interest. By correlating gene expression and surface marker profiles in the current preclinical study, we aim to illuminate the interactions of major cell subpopulations within SCLC PDX tumor tissue and also investigate potential markers of sensitivity to growth inhibition by treatment with M2698. Overall, our workflow of deep phenotyping may provide tools for sample characterization, quality control and possibly study of cellular response to varying selective pressures such as drug challenges. Citation Format: Friedrich Hahn, Warren Porter, Eileen Snowden, Mitchell Ferguson, Frances Tong, William S. Dillmore, Anderson Clark, Hong Zhang, Rainer Blaesius. Surface marker and gene expression profiling of tumors from PDX models of small cell lung cancer with varying sensitivity to growth inhibition by the p70S6K/AKT1/3 inhibitor M2698 [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 2197.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2018-2197
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2018
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  • 16
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    Growth, differentiation capacity, and function of mesenchymal stem cells expanded in serum-free medium developed via combinatorial screening
    Elsevier BV ; 2013
    In:  Experimental Cell Research Vol. 319, No. 10 ( 2013-06), p. 1409-1418
    Details
    In: Experimental Cell Research, Elsevier BV, Vol. 319, No. 10 ( 2013-06), p. 1409-1418
    Type of Medium: Online Resource
    ISSN: 0014-4827
    URL: Article
    DOI: 10.1016/j.yexcr.2013.04.004
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2013
    detail.hit.zdb_id: 1466780-0
    SSG: 12
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  • 17
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    Structure of Gαi1 Bound to a GDP-Selective Peptide Provides Insight into Guanine Nucleotide Exchange
    Elsevier BV ; 2005
    In:  Structure Vol. 13, No. 7 ( 2005-07), p. 1069-1080
    Details
    In: Structure, Elsevier BV, Vol. 13, No. 7 ( 2005-07), p. 1069-1080
    Type of Medium: Online Resource
    ISSN: 0969-2126
    URL: Article
    DOI: 10.1016/j.str.2005.04.007
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2005
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    SSG: 12
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  • 18
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    Correction: Flow based single cell analysis of the immune landscape distinguishes Barrett’s esophagus from adjacent normal tissue
    Impact Journals, LLC ; 2019
    In:  Oncotarget Vol. 10, No. 49 ( 2019-08-20), p. 5119-5119
    Details
    In: Oncotarget, Impact Journals, LLC, Vol. 10, No. 49 ( 2019-08-20), p. 5119-5119
    Type of Medium: Online Resource
    ISSN: 1949-2553
    URL: Issue
    URL: Article
    DOI: 10.18632/oncotarget.v10i49
    DOI: 10.18632/oncotarget.27161
    Language: English
    Publisher: Impact Journals, LLC
    Publication Date: 2019
    detail.hit.zdb_id: 2560162-3
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  • 19
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    Flow based single cell analysis of the immune landscape distinguishes Barrett’s esophagus from adjacent normal tissue
    Impact Journals, LLC ; 2019
    In:  Oncotarget Vol. 10, No. 38 ( 2019-06-04), p. 3592-3604
    Details
    In: Oncotarget, Impact Journals, LLC, Vol. 10, No. 38 ( 2019-06-04), p. 3592-3604
    Type of Medium: Online Resource
    ISSN: 1949-2553
    URL: Issue
    URL: Article
    DOI: 10.18632/oncotarget.v10i38
    DOI: 10.18632/oncotarget.26911
    Language: English
    Publisher: Impact Journals, LLC
    Publication Date: 2019
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  • 20
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    Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins
    Springer Science and Business Media LLC ; 2004
    In:  Purinergic Signalling Vol. 1, No. 1 ( 2004-12), p. 43-49
    Details
    In: Purinergic Signalling, Springer Science and Business Media LLC, Vol. 1, No. 1 ( 2004-12), p. 43-49
    Type of Medium: Online Resource
    ISSN: 1573-9538 , 1573-9546
    URL: Article
    DOI: 10.1007/s11302-004-4748-1
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2004
    detail.hit.zdb_id: 2172143-9
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