CyTOF Mass Cytometry Day

October 15, 2019
9:00 am - 5:00 pm
Eric P. Newman Education Center (EPNEC) Auditorium (Medical Campus)

A day of Mass Cytometry and Imaging Mass Cytometry talks, resources, and networking for researchers both familiar and unfamiliar.

Register for free:  bit.ly/CyTOFDay2019

Program

(see registration link for abstracts and any updates to program)

8:30 am–9:00 am

  • Registration: Name badge & lanyard table
  • BREAKFAST: American Continental
  • Step-right-up Q&A Table: Ask an application scientist
  • Resource Tables: Indica Labs and Visiopharm

9:00 am

Welcome and Opening Remarks

“Powering Health Insights with Mass Cytometry”

Fluidigm Mass Cytometry Team

“Overview of Immumonitoring Lab”

Stephen Oh, MD/PhD, Assistant Professor, Department of Med – Hematology

9:10 am–9:35 am

“Adoptively transferred Memory-like NK cells expand and persist MHC-compatible environment”

Melissa Berrien-Elliott, PhD, Instructor of Medicine, Washington University School of Medicine

Abstract: Allogeneic memory-like NK cell adoptive therapy has been shown to be effective at inducing remissions in patients with relapsed or refractory (rel/ref) acute myeloid leukemia (AML). However, these transferred cells are eliminated by the recipients recovering immune system after a few weeks, thereby restricting the window of opportunity ML NK cells have to perform their anti-tumor functions. Here we examine HCT donor-matched ML NK cell persistence in patients treated with reduced-intensity HCT transplant. Using mass cytometry, we can distinguish between graft-derived and transferred ML NK cells, which persist for months, in this MHC-compatible setting.

9:40 am–10:15 am

“High risk glioblastoma cells revealed by machine learning and single cell signaling profiles”

Jonathan Irish, PhD, Assistant Professor of Cell and Developmental Biology and Pathology, Microbiology and Immunology; Scientific Director of the Cancer & Immunology Core and the Mass Cytometry Center of Excellence, Vanderbilt University School of Medicine

Abstract: Glioblastoma is well described genomically, but expression of lineage and signaling proteins has not been extensively characterized in single cells. Here, 34 different phospho-proteins, transcription factors, and lineage proteins were evaluated in millions of cells from resected glioblastomas using mass cytometry and a new, unsupervised, machine learning algorithm that identifies single-cell phenotypes and assesses clinical risk stratification as a continuous variable. This Risk Assessment Population IDentification (RAPID) approach identified tumor cell subsets that independently and continuously stratified patient outcomes, were distinguished by abnormal co-expression of lineage proteins, and harbored opposing signaling axes, suggesting the opportunity to match precision medicine approaches to these prognostic signatures. Once revealed by high-dimensional cytometry, the glioblastoma signaling profiles were apparent in tissue microarray cores using traditional pathology approaches. Pre-print: https://www.biorxiv.org/content/10.1101/632208v3

10:20 am–10:45 am

“Integrating spatial and signaling information to identify actionable phenotypes in brain tumors”

Rebecca Ihrie, PhD, Assistant Professor, Cell & Developmental Biology and Neurological Surgery, Vanderbilt University School of Medicine

Abstract: Gliomas develop and present in multiple anatomically distinct locations within the brain, and the precise location of these tumors affects both patient prognosis and treatment options. However, the effects of spatially segregated cells of origin and the sites of tumor growth on signaling biology are underexplored relative to the genomic features of gliomas. To address this need, we have begun studies of key signaling features and lineage proteins using suspension and imaging mass cytometry of three brain tumor types with different spatial distributions and prognoses. In addition to inter-tumoral differences in lineage marker expression and signaling biology, different per-cell signaling phenotypes are found in distinct patterns within individual tumors, suggesting potential targeted approaches to therapy. This presentation will discuss key considerations for custom panel design, reagent validation, and subsequent analyses of high-dimensional cytometry in the study of progenitor cell biology and tumors within the brain.

10:45 am–11:00 am – BREAK

11:00 am–11:25 pm

“Remodeling of Intratumoral Lymphoid and Myeloid Compartments by Effective Cancer Immunotherapies”

Jeffrey Ward, MD/PhD, Assistant Professor of Medicine, Washington University School of Medicine

Abstract: Immune checkpoint therapies (ICT) effectively control tumor progression and improve patient survival in diverse tumor types, however, many patients either do not respond, or have responses of limited duration. To gain insights into the molecular and cellular mechanisms of action of anti-PD-1 and anti-CTLA-4 antibody therapies, we employed the complementary high dimensional techniques of mass cytometry and single cell RNA sequencing (scRNASeq) to define differences across populations of tumor infiltrating immune cells from syngeneic murine tumors during unrestrained tumor growth or effective ICT. Using panels of metal-labeled antibodies to detect surface and intracellular markers as well as MHC Class I tetramers to enable detection of cancer neoantigen-specific CD8+ T-cells, we uncovered significant remodeling of both lymphoid and myeloid compartments during the process of tumor rejection in a manner at least partially dependent on interferon gamma. Characterization of these defined populations using scRNASeq analysis allowed a comparison of murine macrophage populations to their human counterparts, providing information on additional pathways for potential therapeutic intervention.

11:30 am–12:00 pm

“Panel Discussion: Mass Cytometry 101: Getting started with MC/IMC”

Panelists

  • Akil Merchant, MD/PhD
  • Denis Schapiro, PhD
  • Jonathan Irish, PhD
  • Stephen Oh, MD/PhD

12:00 pm–12:45 pm – LUNCH: Light-Plate Sandwiches & Salads

Lunchtime presentations (optional):

  • Main Auditorium: Human Immune Monitoring Panel, Michelle Poulin, PhD, Fluidigm
  • Breakout Room 1: Mass Cytometry 101
  • Resource Tables: Registration for three-station October 16th workshop
  • Q&A Table: Ask an application scientist

12:50 pm–1:15 pm

“Interrogating Dysregulated Cytokine Signaling Networks in Myeloproliferative Neoplasms”

Stephen Oh, MD/PhD, Assistant Professor of Medicine, Co-Head, Immunomonitoring Laboratory, Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine

Abstract: Pending

1:20 pm–1:55 pm

“Highly multiplexed single cell spatial analysis of the tumor microenvironment in lymphoma”

Akil Merchant, MD/PhD, Associate Professor and Director of Imaging Mass Cytometry Shared Resource, Cedars-Sinai Medical Center

Abstract: Recent clinical successes with checkpoint inhibitors and immuno-oncology agents have demonstrated the importance of the immune system in controlling and treating cancers. However, responses to checkpoint inhibitors in lymphoma are highly variable. For example, two recent clinical trials with the PD-1 targeting antibody nivolumab demonstrated response rates in Hodgkin lymphoma of 87%, while responses in diffuse large B cell lymphoma (DLBCL) were less than 10%. We hypothesize that a better characterization of the spatial architecture of the tumor microenvironment (TME) in lymphoma will help explain differences in responses to PD1/PDL-1 inhibitors and guide future targeted immunotherapies for these patients. In the present study, we characterized TME components, including immunophenotypes, frequency and spatial interaction, in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution. Using this approach, we have identified subtypes of CD8 T cells that strongly predict overall survival and suggest novel targets for combination clinical trials. To describe the spatial architecture of the tumor microenvironment, we developed a novel clustering approach to group CD8 cells by their distance to tumor and other components of the microenvironment. Using this approach, we were able to demonstrate phenotypic differences in activation (ki-67, granzyme B) or exhaustion (PD-1, TIM3, LAG3) in these different spatial clusters and associate these with response to chemotherapy. Finally, we show that sub-setting our analysis of CD8 phenotypes based on their spatial location to other cells improved our ability to predict overall survival in the cohort. Together, these results show that deep profiling of the immune architecture of the tumor microenvironment can predict clinical outcomes in DLBCL, and that spatial analysis of immune cells should be explored as biomarkers for lymphoma patients treated with immunotherapies.

1:55 pm–2:15 pm – BREAK

2:15 pm–2:40 pm

“Revealing Site-specific Tumor Microenvironment Features of Metastatic Pancreatic Cancer with Mass Cytometry”

Won Jin Ho, MD, Hematology-Oncology Fellow, Elizabeth Jaffee Lab, Johns Hopkins School of Medicine

Abstract: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies are limited with dismal five-year survival of 5-10%. PDAC most commonly metastasizes to liver and lung, and interestingly, the site of involvement has been differentially associated with prognoses and responses to systemic therapies. By leveraging mass cytometry and RNAseq, we identify key differences between the liver and lung tumor microenvironments (TME) in a preclinical mouse model of metastatic PDAC. We then recapitulate these findings in matched human lung and liver samples using immunohistochemistry and RNAseq analysis. Deeper understanding of the distinctive features within the metastatic TMEs may lead to novel targets for therapy and/or inform improved therapeutic strategies against metastatic pancreatic cancer.

2:45 pm–3:10 pm

“Investigating phenotypic heterogeneity of chemoresistant triple negative breast cancer using imaging mass cytometry”

Amanda Rinkenbaugh, PhD, Postdoctoral Fellow, Helen Piwnica-Worms Lab, MD Anderson Cancer Center

Abstract: Shifts in tumor phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful cancer therapies. Intrinsic and adaptive mechanisms of drug resistance suggest that treatment strategies need to account for the presence of phenotypically diverse subpopulations and interactions between them that alter tumor biology. Recent work from our lab characterized a reversible, drug-tolerant state in a subset of patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), following chemotherapy treatment. While the residual tumors exhibited a unique transcriptome and altered microenvironment, the pre-treatment and regrown tumors were largely indistinguishable in bulk transcriptome and proteome analyses. We hypothesize that localized neighborhoods of tumor cells possess specialized phenotypes which represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the tissue. We have constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. Analysis of tumors throughout chemotherapy treatment via IMC recapitulate previously described changes in residual tumors such as the altered microenvironment, while also identifying novel phenotypic shifts in regrown tumors, such as increased MAPK signaling localized to discrete neighborhoods. Our PDX collection also includes sequential pairs derived from fine needle aspirates taken before and after chemotherapy treatment. Comparison of multiple pre-/post-chemotherapy pairs also indicates spatially constrained MAPK activation emerging after treatment, suggesting this phenomenon may be a feature of chemoresistant TNBC. Taken together, our findings suggest that unique signaling niches arise following treatment, and may promote tumor fitness during or after exposure to treatment to contribute to tumor evolution.

3:15 pm–3:50 pm

“‘Google Maps’ for tissue biology—How to find topographic biomarkers”

Denis Schapiro, PhD, Independent Fellow, Harvard Medical School and Broad Institute

3:50 pm–4:30 pm

Mingle: Post-Seminar Networking

Q&A Table: Ask an application scientist

Resource Tables: Indica Labs and Visiopharm

Sponsored by: Fluidigm & Washington University in St. Louis
Co-sponsored by: The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs