GLIIFCA 2017 Presentation Abstracts

Dissecting the Prostate Tumor Microenvironment

Joshua Lang (University of Wisconsin Carbone Cancer Center, Madison, WI)

While there are a number of pathways within cancerous cells that can lead to therapeutic resistance, researchers have also found that non-cancerous cells in these tumor lesions also play critical role in the development of resistant disease. These non-cancerous include immune cells, blood vessels, and support cells (known as stroma) that are collectively known as the tumor microenvironment (TME). Within a tumor, cancer cells “co-opt” these normal cells and use them to support cancer progression as well as therapeutic resistance. These cells promote resistance to a number of therapies, including chemotherapy and hormone therapy. We have utilized multi-color flow cytometry to optimize dissection and extraction of the multi-cellular TME. These tools are being employed in an upcoming neoadjuant clinical trial in prostate cancer to identify patients most likely to benefit from these therapies.

Optimizing DC Vaccination and Tumor Preconditioning Regimens to Improve Anti-Tumor Responses for Renal Cell Carcinoma

Jason Muhitch (Roswell Park Cancer Institute, Buffalo, NY)

Abstract: Compared to conventional cancer therapies, immune-based strategies have the potential to generate tumor-specific systemic protection long after patients have received treatment. This promise has been partly realized through advancements in immunotherapy for renal cell carcinoma (RCC) that have prolonged overall survival and improved objective response rates. However, most RCC patients still do not respond to treatment. Effective anti-tumor responses rely on dendritic cells (DC) activating tumor-specific T cells and intratumoral expression of antigens including tumor-associated antigens (TAA) for T cell-mediated tumor cell lysis. DC vaccination induces T cell-dependent immunity that can result in durable complete responses. However, preparation and efficacy of DC vaccination is hampered by interindividual, intraindividual and manufacturing variability. While DC-based therapeutic approaches have been used for more than fifteen years, the optimal starting material for DC maturation has yet to be determined. Our recent findings demonstrate that DC derived from the minor human non-classical monocyte subset have increased levels of costimulatory molecules and IL-12 production. Analysis of pretreatment samples from a completed DC clinical trial for RCC (NCT00085436) revealed circulating non-classical monocytes are increased more than two-fold in long-term survivors (> 100 months). Improvements to DC vaccination and T cell activation do not resolve tumor-intrinsic barriers to anti-tumor immunity, including limited TAA expression. Radiation, which is a long-standing approved treatment for a broad range of tumors, has the potential to overcome this impediment by releasing pro-inflammatory mediators and upregulating TAA. Our laboratory has recently determined that a single treatment of high-dose radiation delivered by stereotactic body radiation therapy (15 Gy) can increase TAA expression in RCC patient tumors in a first-of-its-kind completed pilot trial (NCT01892930). Collectively, these studies provide strategies to overcome obstacles that limit both DC activation of tumor-specific T cells and expression of TAA for the treatment of RCC patients.

Research was supported by the NCATS of the NIH under award number UL1TR001412 to University at Buffalo. Work also supported by NCI P30CA016056 involving the use of Roswell Park Cancer Institute’s Flow and Image Cytometry as well as Biostatistics Shared Resources. Research funding also provided by the Sklarow Foundation, Elsa Kreiner Memorial Fund, Fraternal Order of Eagles, and RPCI Friends of Urology.

ImmunoPET Imaging for Evaluation of Functional Tumor Immunotherapy

Heather Gibson (Wayne State University, Detroit, MI)

[]{#instrument-and-application-development-f .anchor}Recent breakthroughs in cancer immunotherapy are met with challenges to effectively gauge response. Evaluation of immunotherapy by patient outcomes requires time which may squander the opportunity to modify the therapeutic strategy. Peripheral immune analyses remain non-standardized, often require knowledge of tumor antigen(s), and may not fully reflect conditions within the tumor microenvironment. Our strategy instead focuses on the in situ detection of functional immune activity. Using positron emission tomography (PET), we are developing novel antibody-based (immunoPET) probes for non-invasive detection of key soluble biomarkers of immune activation. We have developed and evaluated an immunoPET tracer to cytotoxic effector cytokine interferon gamma (IFNγ). IFNγ is produced by activated CD8+ cytotoxic T lymphocytes (CTL), NK cells and a subset of helper CD4 T cells and is associated with anti-tumor immune activity. We show IFNγ immunoPET positively identifies immune activation in situ after administration of a HER2/neu DNA vaccine in neu-expressing TUBO xenografts and spontaneous neu transgenic (NeuT) mouse tumor models. IFNγ imaging outperforms immunoPET targeting total CD3 T cell infiltration. Detection of non-specific secondary lymphoid tissues (spleen, thymus and lymph node) is also greatly reduced when using an IFNγ probe compared to total CD3 T cell imaging. Despite the increase in IFNγ immunoPET detection, peripheral T cell response to neu is undetectable in vaccinated NeuT mice. Collectively, these results support the use of immunoPET for timely in situ evaluation of immune activity to guide clinical approach.

Tumor-Associated Exosomes Represent a Reversible T Cell Checkpoint

Gautam Shenoy (University at Buffalo, Buffalo, NY)

[]{#flow-cytometry-and-immunotherapy-histori .anchor}T cells in tumor microenvironments are rendered hyporesponsive to activation by a combination of cellular and acellular factors. The identification of these immunosuppressive factors would allow for designing strategies to block them and ultimately enhance patients’ anti-tumor immune responses by re-activating these tumor-associated T cells. We have identified membrane encapsulated extracellular vesicles with the size, biophysical and compositional properties that are characteristic of exosomes, in ascites fluids as well as solid tumors derived from ovarian cancer patients. T cells briefly pulsed with these tumor-associated exosomes during activation rapidly inhibit multiple different activation endpoints including the translocation of NFkB and NFAT into the nucleus, the upregulation of CD69 and CD107a, production of cytokines and cell proliferation. The inhibition occurs without loss of cell viability, and coincidentally with the binding and internalization of these exosomes. This exosome-mediated inhibition of T cells has been determined to be transient and reversible. We demonstrate that the tumor-associated exosome-induced T cell signaling arrest is dependent upon phosphatidylserine (PS), as a blockade of PS with anti-PS antibodies significantly reverses the inhibitory activity of the exosomes. We also report that the inhibitory activity is overcome by the addition of diacylglycerol kinase inhibitors, suggesting a possible mechanism for the T cell inhibition. We conclude that exosomes present within ovarian tumor microenvironments represent a novel T cell checkpoint, and represent a potentially viable target to block and thereby enhance anti-tumor immune responses in ovarian cancer patients.

Insights into Viruses and Viral Replication from Flow Cytometry Assays

John ‘Chip’ Tilton (Case Western Reserve University, Cleveland, OH)

[]{#biomarkers-as-guide-posts-in-patient-sel .anchor}This talk will provide an introduction to different flow cytometric assays to measure viral infection and replication of cells, with a focus on a combination reporter virus assay that allows monitoring of multiple stages of the human immunodeficiency virus (HIV) life cycle. Using this assay, we can precisely measure the susceptibility of different CD4+ T cell subsets to viral entry and productive infection, rapidly screen small molecule libraries to identify compounds inhibiting or facilitating viral entry, and sort cells based upon infection status for downstream analysis. One of the interesting findings from this assay is that viral infection of target cells is quite inefficient: only \~5-10% of resting CD4+ T cells that fuse with HIV become productively infected by the virus. I will discuss cellular and viral factors that regulate susceptibility of cells to infection and their implications for HIV treatment and prevention. Finally, I will discuss how these findings inform the rational engineering of virus-like particles for therapeutic purposes including gene therapy and RNA and protein delivery.

Rapid Enumeration and Functional Characterization of HIV-1 Particles by High-resolution Flow Cytometry

Michał Bonar (Case Western Reserve University, Cleveland, OH)

[]{#immuno-monitoring-for-clinical-trials .anchor}Recent advancements in flow cytometry allow for nanoparticle analysis with applications in virology and extracellular vesicle biology. Here, we utilized cell-free, fluorescently labeled HIV-1 particles to develop a technique for direct observation of viruses. Based on biochemical data, we further hypothesized that we could leverage the technology to evaluate whether axenic viral swarms contain noninfectious decoys as well as infection-competent virus. Fluorescently labeled – but not unlabeled – single viral particles could be identified and distinguished from exosomes on a standard cytometer. Viral particles could be sorted based on these added markers and retained the ability to infect target cells. Dedicated volumetric nanoparticle flow cytometer accurately quantified viral titers to high sensitivity rivaling that of standard PCR techniques. Moreover, viral maturation rate was successfully monitored by a novel FRET-based assay using a BD machine. We conclude that flow virometry is a ‘toolkit’ to rapidly and accurately quantify viruses, explore their lipid and protein composition diversity, and assay infectivity of individual live viral particles with unprecedented detail. It is now possible to work out the impact of viral social lives on the course of infection. We anticipate that viral heterogeneity will reveal critical insights into viral transmission and vaccine design.

2017 Carleton and Sigrid Stewart Lecture

Clinical Microbial Cytometry: A Century and a Half of Neglect

Howard Shapiro (One World Cytometry, Inc., West Newton, MA)

[]{#visualizing-obstacles-to-anti-tumor-immu .anchor}Microbes were first established as causes of infectious disease almost 150 years ago. Mycobacterium tuberculosis (MTB) was established as the cause of what remains the world’s deadliest infectious disease in 1882. Today, TB is still most commonly diagnosed by human observers using light microscopes with a stain developed in 1883. Although that reagent was suspected within a few years of failing to stain many of the MTB in a specimen, and is now known to be particularly ineffective in staining drug-resistant forms, it remains on the market. Malaria microscopy, the stain for which dates from 1905, is similarly flawed. The current trend in microbiologic diagnosis favors detecting microbial genomes and resistance genes rather than identifying pathogens at the cellular level. In recent years, however, it has been found that much resistance is phenotypic rather than genotypic, making it essential to examine the effects of drugs on live pathogens. This can now be done using simple and inexpensive cytometric equipment and reagents with a wide range of applications inside and outside the clinical laboratory; the approach, while scientifically sound, has not seemed commercially attractive.

Circulating Exosomes offer a Potential Clinical Diagnostic for Glioma Patients Receiving Survivin-targeted Immunotherapy, SurVaxM

Phillip Galbo, (Albert Einstein College of Medicine, Bronx, NY)

Introduction: Exosome transport of proteins, lipids, RNAs and DNAs mediates inter-cellular communication and can contribute to immune suppression, chemo-resistance, tumor proliferation and migration. Detailed phenotyping of exosomes can aid in the identification of their cargo, their cells of origin, and possibly their targets, thereby elucidating targetable mechanisms of action. The current study determined whether enumerating the presence of specific exosomes could be used as a marker of therapy response. In this context, Imaging Flow Cytometry was used to analyze exosomes isolated from the serum of patients diagnosed with glioblastoma, who were enrolled in a phase I clinical trial of SurVaxM; a peptide vaccine designed to target the inhibitor of apoptosis molecule survivin.

Methods: Exosomes were isolated by differential ultracentrifugation from cryo-preserved serum samples obtained at different treatment stages. Exosome preparations were morphologically verified by electron microscopy. Exosomes were phenotyped with antibodies against CD9 (a member of the tetraspanin protein family that is abundantly expressed on exosomes), glial fibrillary acidic protein (GFAP, a glial marker), and survivin (the target molecule of SurVaxM). All samples were acquired on an ImageStream-X Mkll Cytometer and analyzed with IDEAS (vs 6.2). To validate the method, staining buffer only controls, single color controls, and exosome lysis by triton X-100 were included, as well as serial sample dilutions to determine the potential contribution of event coincidence to double positive events. Samples were acquired for equal acquisition times, and exosome concentrations were determined by normalizing to sample volume acquired.

Results: Method validation demonstrated that only a background signal was obtained with staining buffer controls, that exosome- labeled signal was dissolved in the presence of triton, and that sample dilution did not affect the average staining intensity of exosomes, nor did it diminish the incidence of double positive events. CD9+/GFAP+/survivin+ exosomes were readily detectable in patient sera at all treatment stages in contrast to healthy donor serum in which they were absent. In 5 of the patients who experienced early tumor progression following SurVaxM, this was accompanied by an increase in CD9+/GFAP+/survivin+ exosomes. In contrast, 3 patients who experienced long progression-free intervals were accompanied by 63-67% reduction in CD9+/GFAP+/survivin+ exosomes. One patient with no tumor progression had a 98% reduction in serum CD9+/GFAP+/survivin+ exosomes that had been observed for 48+ months after initial vaccination.

Conclusion: This small pilot study indicates that enumeration of CD9+/GFAP+/survivin+ circulating exosomes has the potential to serve as a biomarker for treatment response of glioma patients to SurVaxM.

♫♪♫♪ What’s FLOW got to do with it? ♫♪♫♪

Lisa Green (Covance, Indianapolis, IN)

The pharmaceutical industry is challenged to translate breakthroughs in scientific discovery to novel medicines that address unmet medical needs. We must both limit development costs and deliver to patients the right drug at the right dose, for the optimal treatment course (personalized medicine). Flow cytometry is an ideal technique to make this happen at all stages of drug development and therapy. Flow methods can be developed to examine drug pharmacodynamic (PD) effects to guide dosing and patient selection. Very often these methods need to be first developed for animal species and then translated to clinical testing. Examples of flow cytometry PD assays that will be presented include cell function assays such as dye efflux, cell signaling analysis (phospho-proteins), and NK cell activity along with complex immunophenotyping analysis such as receptor occupancy, antigen specific T cells, and cell depletion studies. Successful implementation of these complex methods requires careful assay design along with consideration of a wide variety of practical factors depending on the stage of drug development (discovery, animal models, or clinical trials) and the laboratory setting (in-house or external laboratory). Challenges that will be discussed include available sample volume and stability of analyte, standardization needed for multi-site and/or longitudinal studies, and technical and scientific expertise required for sample preparation and data interpretation. Invaluable translational information can be delivered when scientific, technical, and logistical factors are appropriately addressed for each flow cytometry- based PD assay.

2017 Immudex Translational and Clinical Cytometry Lecture

Genetically-engineered DC Vaccines for Melanoma

Lisa Butterfield (University of Pittsburgh, Pittsburgh, PA)

Cancer Immunotherapy has led to a revolution in the treatment of many types of cancer. The response to checkpoint inhibition appears to depend upon an existing immune response for strong antitumor effects and tumor regressions. Cancer vaccines, based on peptides, proteins, viruses and antigen presenting cells like dendritic cells (DC) have been demonstrated to promote antitumor immunity. There are many important questions yet to address regarding how to use cancer vaccination to promote effective antitumor immunity. Recent studies on combination cancer vaccine trials, induced antitumor effects and the biology of DC vaccines will be presented.

2017 ISAC Lecture

Where Again was the Autopilot Switch?

Josef Spidlen (FlowJo, Ashland, OR)

[]{#aml-differentiation-therapy .anchor}Arguably, automated analysis methods in flow cytometry have reached the point where they can provide a solution to the challenge of analyzing large and complex data sets (Aghaeepour, et al. Nat. Methods 2013; Finak, et al. Sci. Rep. 2016; Aghaeepour et al. Cytometry 2016, Weber et al. Cytometry 2016). If chosen and used well, some of these tools show even better results with less undesirable variability than analysis performed by human experts. However, it never is that simple. The limited use of automated analysis in practice is often attributed to the burden of having to learn R. Having to learn a programming language to do biology is far from ideal, but is that the only issue? There are over 40 R libraries covering all the different aspects of flow cytometry data analysis. So why is it that computational biologists keep creating more, and also, why is it that they almost exclusively use the methods that they have created themselves? These methods are often presented as unsupervised or easy to use, but in reality, you have to spent significant time learning the implementation details and then tweaking the analysis pipeline for a specific data set to work well. This extra effort outweighs the benefits of using automated analysis unless you are doing the same experiment over and over again. A typical analysis workflow can be broken down into data pre-processing (compensation, quality control, outlier removal, normalization, transformation), gating, biomarker discovery or feature extraction (e.g., existence of certain cell populations, proportions of cells in those populations, level of expression of certain markers, various descriptive statistics on those populations), and visualization. In my talk, I will briefly review your options for these steps. With a single goal of bringing the algorithms to you, the benchtop scientists, we have been focusing on making the methods robust and easy to use. I’ll share how far we have got and where we are heading. You can also expect to get an exclusive early access to some of our new features. Your feedback will immediately drive the design aspects of what will be shared with the rest of the cytometry community.

Multidimensional Boundaries to assess Bone Marrow Populations: The Basis of Residual Disease Detection

Mike Loken (Hematologics, Inc., Seattle, WA)

Bone marrow (BM) is a complex mixture of cells from 12 different lineages, each of which may exhibit maturation. Support Vector Machines (SVM) were used to circumvent the possibility of gating bias in the assessment of quantitative immunofluorescence of reference populations identified in regenerating BM specimens obtained from patients following chemotherapy. 77 randomly selected BM specimens obtained over a period of 3.5 years from a large clinical study were divided into a training set (27) and testing set (50) to identify the quantitative expression of multiple cell surface antigens on reference cell populations. Using this approach, the variation within and between individuals for each gene product could be determined without introducing gating bias. The results of this study show that the amounts of gene products expressed on stressed BM cells are invariant from individual to individual, independent of age of the individual. Interestingly, the variance of gene product expression within an individual is greater than the variance of the means between individuals. Quantitative antigen expression on normal hematopoietic cells is a highly regulated process where the amounts of gene products are a biologic constant.

An Immune Clock of Pregnancy

Nima Aghaeepour, (Stanford University School of Medicine, Stanford, CA)

Despite the well-established impact of development during the early months of pregnancy on long-term outcomes, the biological mechanisms that govern pregnancy have not been adequately documented. Most clinical assays (e.g., those based on ultrasound) can only capture abnormalities at a late pregnancy stage and lack predictive power. The maintenance of pregnancy relies on a finely-tuned immune balance between tolerance to the fetal allograft and protective mechanisms against invading pathogens. Demonstrating the chronology of immune adaptations to a term pregnancy provides the framework for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia. In the first part of this presentation, we demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using cytometry by time-of-flight mass spectrometry (a.k.a. CyTOF), the abundance and functional responses of billions of cells from all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. An Elastic-Net model, with prior distributions extracted from literature-based knowledge of the immune system, was used to develop a predictive model of inter-related immune events that accurately captured the chronology of pregnancy. Model components highlighted existing knowledge, such as enhanced innate immune responses during pregnancy. However, the approach also revealed novel biology, including a critical role for IL-2–dependent STAT5ab signaling pathway in modulating T-cell function during pregnancy. Next, we integrated this analysis with other assays of the same cohort, including data from the transcriptome, microbiome, proteome, and metabolome to develop a holistic understanding of immunity during pregnancy. A predictive model was developed that not only significantly increased predictive power by combining all available datasets, but also revealed unique interactions between different aspects of pregnancy and immunity.

Mapping the Cellular Immunome of the Maternal-fetal Interface with Computational Flow Cytometry: Promise and Pitfalls

Aleksandar Stanic-Kostic (University of Wisconsin-Madison, Madison, WI)

Recent revolution in the understanding of innate and adaptive immunity in mucosal sites warrants re-examination of immune cells at the decidual maternal-fetal interface. As immune cells at the decidua have key regulatory roles in vascular remodeling, fetal tolerance, and protection from infection. To directly map the immunome of human and mouse maternal-fetal interface, we have developed an experimental and computational workflow allowing operator-independent identification of all major decidual immune subsets from individual specimens. Our workflow is dependent on highly-multiplexed fluorescent panels, multi-parametric flow cytometry and computational means of interpretation. To deconvolute cellular composition, we employed dimensionality reduction by Barnes Hut-modified t-distributed Stochastic Neighbor Embedding (bht-SNE) and machine-learning aided density-based clustering (DenseVM). Cell identity was assigned using CellOntology with expert review. Results revealed unexpected complexity of the decidual immunome, highlighting both the power and challenges of this approach.

Page updated on 2017-09-21 22:41:39 -0400