Joyce J. Ruitenberg

VACUTAINER ® CPT™ and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples

BackgroundFor immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNγ expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.ResultsThe results indicate that cryopreserved PBMC samples tested for CMV- and HIV- specific interferon-gamma (IFNγ) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNγ response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.ConclusionThese data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.

Rapid assessment of in vitro expanded human regulatory T cell function.

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

A rapid flow cytometric method for the detection of intracellular cyclooxygenases in human whole blood monocytes and a COX-2 inducible human cell line.

We have developed a flow cytometric method for the detection of intracellular cyclooxygenases (COX) in human whole blood monocytes and a COX-2 inducible human cell line. COX-2 is induced by endotoxin activation of whole blood monocytes or by the addition of fetal bovine serum (FBS) to a serum-deprived human fibroblastoid cell line, CCD-1070Sk. Cells are permeabilized with FACS Lysing Solution (FLS) containing saponin (Sap), stained intracellularly with COX-2 and COX-1 monoclonal antibodies (mAbs) and analyzed flow cytometrically. Intracellular COX-2 is specifically detected in endotoxin-stimulated CD14(+) monocytes in whole blood and in the inducible cell line. The specificity of COX-2 and COX-1 binding is demonstrated by competitive inhibition studies in cells and binding studies on protein-conjugated beads. In addition, a two-color reagent combination is described which simultaneously detects COX-2 and COX-1. We conclude that specific, intracellular COX-1 and COX-2 expression can be readily identified by flow cytometry in whole blood monocytes and cultured cells. The relative rapidity, ease of use and small sample volume required by this assay makes it a suitable methodology for studying COX expression in both preclinical and clinical research settings.

The gamma T-cell antigen receptor

The gamma-TCR is encoded by genes composed of V, J, and C elements that demonstrate a limited potential for recombinational diversity. These genes are rearranged, transcribed, and translated into proteins early during thymic ontogeny. Lymphocytes express gamma-TCR proteins on the plasma membrane only in association with the CD3 complex. gamma-TCR glycoproteins usually associate with another non-gamma glycoprotein, designated delta-TCR, to form a heterodimer receptor. Both non-disulfide-bonded and disulfide-bonded gamma/delta-TCR heterodimers have been identified on the plasma membrane of human T lymphocytes. On certain gamma-TCR-bearing T cell lines, a delta-TCR protein cannot be visualized by autoradiography. It is possible that delta-TCR proteins are associated with gamma-TCR glycoproteins on these cell lines but are not efficiently radiolabeled. Alternatively, it has been suggested that homodimers of gamma-TCR proteins can assemble with CD3 and be expressed on the plasma membrane of these cells. In adult lymphoid tissues, the majority of T lymphocytes expresses a CD3, alpha/beta antigen receptor, whereas only a minor subset (less than 5% of peripheral blood lymphocytes, lymph node, spleen, and thymocytes) express a CD3, gamma/delta antigen receptor. IL-2-dependent cell lines of both murine and human CD3, gamma/delta T cells have been established. Most CD3, gamma/delta T cell lines mediate cytotoxicity against a broad spectrum of tumor-cell targets, although the functional significance of this observation remains unclear. Cytotoxicity is apparently not restricted by or directed against MHC antigens. Antibodies against CD3 or gamma-TCR can induce proliferation and IL-2 secretion and can either augment or inhibit cytotoxicity, demonstrating that the gamma/delta-TCR is a functional receptor. The ligand recognized by this receptor has not been identified. The physiological role of T lymphocytes expressing gamma/delta-TCR, the molecular and structural properties of delta-TCR, and the relationship between CD3, alpha/beta T lymphocytes and CD3, gamma/delta T lymphocytes are the major unresolved questions that will be the primary focus of further experimentation.

Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors

BackgroundMonocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.MethodsUsing flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFα+IL-1β+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.ResultsCryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.ConclusionOur data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

Flow cytometric method for analysis of cytokine expression in clinical samples

Abstract Evaluation of cytokine expression is becoming an increasingly useful approach for defining cell-mediated immunity. The interest in characterizing the role of these important molecules in a wide range of clinical conditions has provided impetus to develop new methods to assess cytokine expression in clinical samples. The whole blood flow cytometric procedure described in this report is compatible with both qualitative and quantitative analysis of cytokine expression in clinical samples and is potentially a useful method for evaluating the role of cytokines in disease pathogenesis.

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

Abstract 2009: Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models

Functional and genetic heterogeneity in tumor tissue has been a well described phenomenon for many decades but only recently emerged as a potentially crucial contributor to cancer development and progression. The correlation between cellular heterogeneity and aggressiveness, metastatic potential and drug susceptibility of a cancerous lesion have led to models in which the existence of multiple clonal cell lineages is a central feature enabling a neoplastic lesion to overcome selective pressures caused by the surrounding tissues’ defensive capabilities as well as therapeutic interventions. In addition, the role of the tumor microenvironment as an integral part of tumorigenesis was recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed an integrated workflow for dissociation and flow cytometric analysis and sorting for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast cancer biopsies we have demonstrated the ability to identify distinct immunophenotypes for each model and use this analysis to isolate distinct subpopulations. Our successful optimization of a variety of well characterized surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) provides a basis for effective fingerprinting of cancer cells from a variety of sources. In an effort to demonstrate the potential of FACS sorting of solid tumor derived cell populations we have interrogated sorted fractions by NGS as well as RT-PCR array analysis and show distinct genotypic as well as gene expression signatures for each subgroup. The evidence provided by our data suggests that the single cell focused approach flow cytometry has traditionally enabled in hematological cancers is accessible for solid tumors as well and may unlock valuable biological insights. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Frances Tong, Stewart Jurgensen, Chang Chen, Daphne Clancy, Jamal Sirriyah, John Alianti, Perry Haaland, Shannon Dillmore, Jeff Baker, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models. [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 2009. doi:10.1158/1538-7445.AM2015-2009

Abstract B37: Phenotypic analysis of single cells dissociated from solid tumors

Current methods for solid tumor analysis may be inadequate for addressing both the heterogeneity of tumor cells and the components of the tumor microenvironment. Comprehensive analysis requires the ability to analyze tumor biopsy specimens at the single-cell level. A better understanding of how the tumors are behaving at a cellular level, in relation to the other cells within the tumor microenvironment, could lead to more accurate and specific treatment, and better patient prognosis. Evaluation of tumor derived single cells by flow cytometry could provide unique information that is not readily obtained from current methods. Here we demonstrate the single-cell analysis of solid tumors from breast and colorectal cancers. Since single-cell analysis of solid tumors may not occur at the collection site, it is important that tumors be preserved in order to retain their characteristics during transport. Using tumors from PDX mouse models, and human samples, different preservatives were evaluated for their effects on cellular viability and surface marker expression. Following shipment of the tumor samples in preservation solutions, the solid tumors were dissociated into single-cell suspensions using enzyme cocktails containing collagenase. Phenotypic evaluation was performed using flow cytometry after staining the single cells with monoclonal antibody panels specific for either tumor or immune cells. The results indicate that the dissociation method did not seem to adversely impact the expression of surface proteins. We demonstrate that it is feasible to analyze dissociated tumor cell populations with relevant surface markers. Analysis of the data revealed distinct phenotype patterns for single cells dissociated from breast and colorectal cancers. Further extensive evaluation of heterogeneity in these tumor types could reveal phenotypic signatures that may be clinically relevant. Citation Format: Joyce J. Ruitenberg, Aaron Middlebrook, Maria Suni, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Rainer Blaesius, Smita A. Ghanekar. Phenotypic analysis of single cells dissociated from solid tumors. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B37. doi:10.1158/1538-7445.CHTME14-B37

Abstract A198: Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models.

Functional and genetic heterogeneity in tumor tissue was first observed over 50 years ago. Today, tumor heterogeneity is frequently evoked in describing the pathway from pre-cancerous lesions to aggressive, metastatic cancer. During this progression, multiple clonal lineages are thought to arise, leading to subpopulations of the tumor showing different metastatic profiles and susceptibility to anti-cancer therapy. In addition, the role of the tumor microenvironment became recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is increasingly compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed dissociation methods for solid tumor tissue which allows flow cytometric analysis as well as sorting to provide cells for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast, colorectal and lung cancer biopsies we have demonstrated efficient dissociation, surface marker analysis and nucleic acid purification from sorted populations. Conditions have been optimized for a range of relevant surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) which are suitable to identify cells predicted to have stem cell, endothelial, epithelial or immune cell functions, respectively. Through sequencing of subpopulations identified by their phenotype we have demonstrated the compatibility of our workflow with downstream analysis methods such as Next Generation Sequencing (NGS). Our RNA stability measurements suggest that gene expression analysis is equally feasible. Our data provide a standardized basis for in depth investigation of subpopulations of cells from solid tumors with various molecular techniques. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A198. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Tina Marmura, Frances Tong, Shannon Dillmore, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A198.