New insights on the pathogenesis of COVID-19

7:00–7:30 pm PDT

Presented By: Olaf Rötzschke, PhD

The SIgN Mass Cytometry Platform (MCP) was originally established by Evan Newell, PhD, one of the pioneers of this technology. It is now part of the SIgN Immunomonitoring Platform and has recently established a Centre of Excellence (CoE) with Fluidigm. As a dedicated technology platform for mass cytometry, it is supported by highly trained personnel and has been supporting research within SIgN, across the A*STAR research entities and in public and private R&D communities. While it provides service to academic and commercial customers, it also played a crucial role in the immune monitoring of the recent COVID-19 outbreak. SARS-CoV-2 affected Singapore at an early stage of the pandemic. Blood samples of hundreds of COVID-19 patients had been processed and analyzed by the various technology platforms at SIgN, including the MCP. In this presentation a brief overview on the SIgN Immunomonitoring Platform and an update on the key findings of profiling of PBMC and plasma samples of COVID-19 patients in Singapore will be shared.

Imaging Mass Cytometry Tumor Microenvironment Applications

7:30–8:00 pm PDT

Presented By: Akhila Balachander, PhD

Understanding spatial localization of immune cells in the context of complex tumor microenvironment is key to characterizing disease progress and monitoring response to immunotherapy. Distribution of host immune cells in solid tumors determines their interaction in situ with tumor cells and with other components of the microenvironment to elicit appropriate immune responses. Analysis of the multicomponent tumor ecosystem therefore requires deep spatial phenotyping capabilities made possible by IMC technology. As recent adopters of spatial-omics, the talk will touch upon our preliminary efforts towards setting up the IMC technology at SIgN in the context of our broader approach to analyzing the tumor microenvironment.

Live-cell barcoding with Cd-CD45 antibodies

4:00–4:30 pm PDT

Michelle Poulin, PhD

Creating CyTOF panels just got even easier: Maxpar® Panel Designer v2.0

4:30–5:00 pm PDT

Kevin Brown, PhD

Drop-in expansion panels for the Maxpar® Direct assay

5:00–5:30 pm PDT

Noah Saederup, PhD

Insights into the tumor microenvironment: high-dimensional single-cell spatial analysis using the Hyperion™ Imaging System

5:30–6:00 pm PDT

Andrew Quong, PhD

Interrogating spatially-resolved biomarkers in the tissue microenvironment with quantitative image analysis on Imaging Mass Cytometry datasets

2:30–3:00 pm PDT

Presented By: Trevor McKee, PhD

Research efforts focused on understanding tumor heterogeneity and interrogating the tumor microenvironment have increased substantially in recent history. Studying these in the tissue context requires the ability to stain for multiple markers simultaneously. The technique of Imaging Mass Cytometry provides the ability to multiplex more than 30 markers simultaneously on tissue sections. However, a major challenge for the effective quantitative analysis of multiplex images arising from Imaging Mass Cytometry data is the availability of robust image analysis algorithms, using image segmentation to break the whole image into subregions representing distinct tissues and single cells. Working closely with biological scientists, we have developed an analytical pipeline to isolate individual cells according to biological domain knowledge, incorporating distinct cellular morphology and biomarker information into analytical segmentation strategies. The goal of this work is to advance the accuracy and robustness of image analysis strategies to permit greater depths of interrogation of rich, highly multiplexed Imaging Mass Cytometry datasets.

Developing an Analysis Pipeline for Mass Cytometry Studies

3:00–3:30 pm PDT

Presented By: El-ad David Amir, PhD

Analysis is the biggest bottleneck in transforming CyTOF® data into meaningful biological insights. The analysis pipeline is a conceptual tool from statistics and data science: Input goes through a series of predetermined and logical processing steps, producing a relevant output product. In this talk I will present the concept of an analysis pipeline and its application to mass cytometry data. A common pipeline involves data cleaning, feature generation and hypothesis testing, with various visualizations layered over all three steps. I will then review suggested methods for some of the components. For example, I will present a taxonomy for clustering algorithms that can help compare the different methods and frame them in the context of your project. Furthermore, I will address common analysis dilemmas, such as: Which clustering algorithm should I pick? How many clusters do I need? How do I deal with potential batch effects? Finally, I will draw attention to some of the pitfalls researchers often miss when analyzing their data. The target audience for this presentation is researchers in industry and academia of all levels (from undergraduate students to directors and PIs) who are working with mass cytometry.

FAUST: A new interpretable machine learning approach for automated gating

3:30–4:00 pm PDT

Presented By: Raphael Gottardo, PhD

From data to insight: Explore the full potential of high dimensional data by leveraging machine learning algorithms

1:30–2:00 pm PDT

Presented By: Qianjun Zhang, MS

The increasingly complicated high-dimensional datasets generated by advanced cytometry instruments has imposed great challenges in terms of data analysis. Data management, visualization and reproducibility are among the challenges every researcher is facing. In this session, we will present the Cytobank platform, a cloud-based cytometry data analysis solution that enables biologists to perform machine learning algorithms like viSNE, SPADE, FlowSOM and Citrus at their fingertips without programming. We will explore the power of those algorithms to make discoveries in the next generation of biomarkers and rare cell populations. Cytobank also promotes easy collaboration and data sharing among colleagues with efficient data management features. You will also get a peek at the soon-to-be-released new illustration feature.

Utilizing FCS Express™ for high-dimensional data reduction with CyTOF®: working with t-SNE and new tools on the horizon

2:00–2:30 pm PDT

Presented By: Sean Burke, MS

FCS Express is optimized for the visualization, display and analysis of high-dimensional data from Fluidigm systems. In this primer we will discuss how to access and use the tools in FCS Express to easily create publication-ready t-SNE charts and other visualizations to better understand highly multiparametric datasets. We will also discuss new tools and algorithms for high-dimensional analysis that will be released to researchers in the next update of FCS Express.

Maxpar Pathsetter: a comprehensive, automated, and flexible analysis for your Maxpar Assay and beyond

2:30–3:00 pm PDT

Presented By: Thiru Selvanantham, PhD and Beth Hill, PhD

Live fireside chat on IMC analysis

3:00–3:30 pm PDT

Presented By: Jared K. Burks, PhD and Trevor McKee, PhD

An engaging, open, vendor-neutral discussion with two IMC analysis leaders, sponsored by Visiopharm.

Introduction to Indica Labs and HALO IMC Analysis

3:30–4:00 pm PDT

Presented By: Donald Allen, BS

Understanding the immunosuppressive functions of cancer-associated fibroblasts in lung cancer

12:00–12:30 pm PDT

Presented By: Handan Xiang, PhD

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the stromal component in the tumor microenvironment. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, their roles and potential mechanisms by which they may contribute to immune suppression in lung squamous cell carcinoma remain largely unexplored. By leveraging patient genomic data and Imaging Mass Cytometry™, we identified a positive correlation between CAF and monocytic myeloid cell abundances as well as a significant spatial interaction between these two cell populations. In-depth analyses highlight a pivotal role of CAFs in regulating monocyte recruitment and differentiation into myeloid-derived suppressor cells.

Media smooth muscle cells reprogramming into mesenchymal stem cells drives aortic aneurysm formation

12:30–1:00 pm PDT

Presented By: Pei-Yu Chen, PhD

Using Imaging Mass Cytometry™ in combination with advanced single-cell RNA sequencing/AI data analysis, we demonstrated the existence of a disease-prone population of smooth muscle cells in the normal aorta. Under pathological conditions, this population of cells undergoes transformation into mesenchymal stem-like and macrophage-like cells and then differentiates in bone, cartilage and fat-forming cells. These cells replace normal cellular constituents of the aortic wall leading to the loss of the wall integrity and aneurysm development. The identification of disease-prone cell populations completely transforms our understanding of disease biology and opens the possibility of development of disease- and patient-specific therapies.

Unraveling the spatial distancing between immune compartments of COVID-19 tissues using IMC™

1:00–1:30 pm PDT

Hiranmayi Ravichandran, MS

Deciphering the immune landscape in the COVID-19 infected lung tissue is necessary to understand the host response to the pathogen. Using a panel of 34 antibodies, Imaging Mass Cytometry was applied on infected and noninfected lung specimens to present the preliminary evidence of tissue dynamics in its spatial resolution. By observing the lung at unprecedented detail, we gain better understanding of both SARS-CoV-2 infected lesions and the process of lung healing, which will enable and inform better management of the disease.

B cells and tertiary lymphoid structures (TLS) promote immunotherapy response

1:30–2:00 pm PDT

Presented By: Rafet Basar, MD

HLA-E and NKG2A as a novel immune checkpoint axis in bladder cancer

2:00–2:30 pm PDT

Presented By: Amir Horowitz, PhD

For more than 40 years, Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the only approved first-line therapy for treatment of high-risk non-muscle invasive bladder cancer (NMIBC), which represents ~70% of all diagnosed bladder cancer. BCG can induce durable responses, but a significant subset of patients will have recurrence or progression of disease. Immune checkpoint blockade (ICB) with anti-PD-1/PD-L1 antibodies has changed the treatment landscape for muscle-invasive bladder cancer (MIBC) and in BCG-resistant NMIBC and can induce durable responses unprecedented in the context of historical treatment approaches. In this study, we provide evidence of adaptive immune resistance unique to NMIBC, whereby IFN-g produced by activated, tumor-infiltrating NKG2A+ PD-1+ CD8 T cells enhances HLA-E and PD-L1 expression on tumors, resulting in an inhibitory interaction that may distinguish response to BCG therapy from recurrence of tumor. Using mass cytometry and Imaging Mass Cytometry (IMC™), we demonstrate that activated NK cells and CD8 T cells drive primary resistance through early IFN-g production that potently triggers elevated HLA-E and PD-L1 expression on tumors and progressive exhaustion due to chronic stimulation over the lengthy course of BCG therapy. Our preliminary findings provide a mechanism of BCG resistance and identify a novel approach to combination ICB targeting NKG2A and PD-L1 blockade.

Suspension by day, imaging by night: how to efficiently manage your system and optimize utilization

Presented By: Jared K. Burks, PhD

Mass cytometry, be it suspension or imaging, opens the doors to a large user base. How can one efficiently utilize the system for the greatest research impact and support for your facility? How hard is it to switch between suspension and imaging modes? How does one support the reagent needs of both options?

Comparing fluorescent flow and mass cytometry optimization and implementation workflows

Presented By: Matthew Cochran, MS

The URMC FCR sought to compare both mass and fluorescent flow cytometry in practical terms such as time (preparation of sample and acquisition), cell recovery and cost of experiments to help guide our user base in choosing which technique was best for their needs. As a case study we partnered with the LungMAP project to help them achieve their goal of maximizing the information learned from precious lung samples. In this presentation we will quickly review this case study and the outcomes and future directions of this work.

High-throughput mass cytometry in a clinical research setting

Presented By: Emily Thrash, PhD

Biomarker discovery in clinical cancer research is limited by the inherent complexity of the disease and the immune response, as well as a lack of validated immune-phenotyping methodology. With the capability to measure up to 50 parameters from a single cell, mass cytometry using CyTOF® is well-poised to overcome these constraints and provide greater breadth and depth to cellular phenotyping. In a clinical setting, with large sample numbers collected at different timepoints, reduction of technical variability and in-depth analysis of cellular immune response is critical. By combining reference sample spike-in and palladium-based mass tag cell barcoding sample, our group has optimized a high-throughput workflow for generating reproducible and consistent CyTOF data. In our lab, we routinely use this workflow to comprehensively analyze activation, exhaustion, memory and trafficking cellular phenotypes in the peripheral immune system of cancer patients receiving therapy, and to detect biomarker candidates.

Engaging with pathologists on IMC projects: keys to success

Presented By: Akil Merchant, MD

Characterizing distinctions in DARC-related Tumor immune MicroEnvironment (DARC TiME)

10:30 am–11:00 am PDT

Presented By: Melissa Davis, PhD

Davis will present findings emerging from her breast cancer research, where she has identified that an African ancestry allele and the recently discovered tumor expression of a gene named DARC (ACKR1) are linked to the tumor-specific immune/inflammatory response. She has utilized Imaging Mass Cytometry to characterize spatial distinctions among ethnically diverse patient groups.

Exploring the immune tumor microenvironment of hepatocellular carcinoma with Imaging Mass Cytometry

11:00–11:30 am PDT

Presented By: Won Jin Ho, MD

While there has been a remarkable progress in the number of FDA-approved systemic therapy agents for hepatocellular carcinoma, their precise effects on the tumor microenvironment are yet to be elucidated. Using Imaging Mass Cytometry™, we conduct in-depth profiling of the tumor microenvironment of hepatocellular carcinomas that have been neoadjuvantly treated with a combination of cabozantinib and nivolumab at Johns Hopkins. We first segment and cluster the resulting single-cell dataset based on 31 parameters. Then we analyze the cellular and spatial features of the tumor microenvironment to understand the differences between pathologic responders and nonresponders. Our work provides insights on what constitutes an immunologically active immune microenvironment in hepatocellular carcinomas.

On measuring photons and ions: Impact on panel design, signal detection and data quality

“Alas poor photon, ion him well.”  

10:30–11:00 am PDT

Presented By: Tim Bushnell, PhD, MBA

Flow cytometry is one of the most powerful single-cell analytical techniques. With the current generation of instruments, 30 or more parameters can be measured simultaneously. In traditional fluorescence flow cytometry, the reporter used is a fluorescent molecule that is measured using a photomultiplier tube or more recently with avalanche photodiodes. With the release of the CyTOF mass cytometer, the reporter used is a metal, and the detection system is a time-of-Flight mass cytometer. When it comes to designing a multidimensional panel, the steps involved for both technologies are similar. Due to specific issues to related measuring photons, there are additional considerations when using fluorescent reporters, adding to the complexity of panel design compared to using metal tags and mass detection. This presentation will discuss the similarities and differences in the panel design process for these two techniques, with an emphasis on how the method of detection of the reporter marker impacts these decisions.

Monitoring immunotherapy with a mass cytometry receptor occupancy assay

11:00–11:30 am PDT

Presented By: Gerd Haga Bringeland, MD, PhD

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Immunotherapy with the monoclonal antibody natalizumab prevents MS disease activity by binding to integrins on circulating leukocytes, thereby blocking leukocytes from entering the CNS. Receptor occupancy is a promising biomarker for monitoring and individualizing natalizumab therapy. I will present a method for accurate measurement of receptor occupancy with high-dimensional mass cytometry and show how I employed the method to study the individual efficacy of immunotherapy with natalizumab in MS patients.

Process development and clinical assessment of CAR T cell products

11:30 am–12:00 pm PDT

Presented By: Greg Hopkins, BS

CAR T cell immunotherapy is a therapeutic approach that involves genetically modifying the patient’s own immune cells ex vivo in order to direct these cells to a tumor target and eliminate tumor cells. bb21217 is a BCMA-directed investigational CAR T cell therapy being developed for the treatment of relapsed or refractory multiple myeloma. bb21217 is manufactured using a PI3K inhibitor (bb007), which is intended to increase in vivo persistence of CAR+ T cells. The safety and efficacy of bb21217 is being explored in the phase 1 CRB-402 study. Here we report the methodology for mass cytometry analyses used to identify key phenotypic features associated with bb2117 CAR+ T cells. CyTOF® uncovered key memory- and effector-associated markers and identified memory-like T cell populations in BCMA-directed CAR+ T cells manufactured in the presence of bb007. CyTOF and unsupervised clustering analysis are powerful tools to understand the biology of cellular products and potentially identify biomarkers associated with clinical outcomes in CAR T-treated patients.

Single-cell mass cytometry identifies mechanisms of resistance to immunotherapy in AML

6:30–7:00 am PDT

Presented By: Shelley Herbrich, PhD

The mechanisms of resistance to immunotherapies in patients with acute myeloid leukemia (AML) are not well-characterized, and biomarkers for improved immunotherapeutic strategies are critical. In this presentation, we characterize the baseline immune landscape and potential mechanisms of resistance using single-cell CyTOF mass cytometry profiling of serially collected samples from refractory/relapsed AML patients undergoing therapy with hypomethylating agents combined with PD-L1 inhibition. Single-cell characterization of these primary bone marrow samples reveals novel insights into the immune checkpoint distribution on AML cells while simultaneously allowing for the evaluation of T cell composition and fitness.

Understanding CD19 negative relapse following CAR T therapies in acute lymphoblastic leukemia

7:00–7:30 am PDT

Presented By: Kara Davis, DO

Chimeric antigen T (CAR T) cells have demonstrated impressive efficacy in refractory and relapsed B cell malignancies, including B cell lymphoblastic leukemia. However, loss of the target antigen, CD19, has been shown to be a mediator of relapse for some patients. We have used mass cytometry and single-cell RNA sequencing to understand whether rare CD19-negative cells exist for patients before they receive CAR T cells and to identify cell types and their features associated with relapse following CAR T therapy for relapsed acute lymphoblastic leukemia.

Mass cytometry reveals distinct immune signatures marking progression from mild to severe COVID-19

7:30–8:00 am PDT

Bernd Bodenmiller, PhD

Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. We used 40-plex mass cytometry and targeted serum proteomics to profile innate and adaptive immune cell populations from peripheral blood of patients with mild or severe COVID-19 and healthy controls. Our data provide new insights into the pathological immune responses that are likely key mechanism in severe COVID-19, further supporting investigation of targeted anti-inflammatory interventions in severe COVID-19.

Live-cell barcoding with Cd-CD45 antibodies

6:05–6:30 am PDT

Presented By: Michelle Poulin, PhD

Creating CyTOF® panels just got even easier: Maxpar® Panel Designer v2.0

6:30–7:00 am PDT

Presented By: Kevin Brown, PhD

Drop-in expansion panels for the Maxpar® Direct™ assay

7:00–7:30 am PDT

Presented By: Noah Saederup, PhD

Insights into the tumor microenvironment: high-dimensional single-cell spatial analysis using the Hyperion™ Imaging System

7:30–8:00 am PDT

Presented By: Andrew Quong, PhD