Rainer Blaesius

Abstract 1656: Comprehensive evaluation of human immune system reconstitution in NSGTMand NSGTM-SGM3 toward the development of a novel Onco-HuTMxenograft model

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

Abstract B11: Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry

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.

Abstract 227: Characterization of single cells from dissociated solid tumors

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. Here we demonstrate an efficient workflow that enables comprehensive single-cell analysis of solid tumors from breast cancers. Using tumors from clinical samples and mouse models, we evaluated different dissociation and processing techniques for their effects on cellular viability and surface marker expression. Solid tumors were dissociated into single-cell suspensions using a combination of mechanical dissociation and enzymatic digestion. Phenotypic distribution and morphology of cells within the tumor microenvironment was evaluated using flow cytometry. As this approach evolves, and a knowledge base of relevant surface markers is established, this technology has the potential to significantly impact how tumor biopsies are processed to get multiparametric information at a single cell level. Citation Format: Aaron J. Middlebrook, Shahryar Niknam, Joyce Ruitenberg, Albert J. Mach, Maria Suni, Warren Porter, Friedrich Hahn, Eileen Snowden, Rainer Blaesius, Smita Ghanekar. Characterization of single cells from dissociated solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 227. doi:10.1158/1538-7445.AM2015-227