Over the last decade the cancer research field has seen a number of advances aimed at increasing the efficacy of treatments, reducing toxic side effects, and decreasing attrition rates of drug candidates. The first is the rise of immunotherapies which incorporate a patient’s own immune system to combat the cancer. In particular, adoptive immunotherapy techniques activate a patient’s T cells ex vivo against specific tumor antigens before infusing the activated T cells back into the patient to target and destroy tumor cells selectively. A second focus has been the incorporation of three dimensional (3D) in vitro testing models. Traditional use of 2D models many times led to attrition rates of drug candidates for cancer reaching 95%, stemming from in vitro drug efficacy values that did not translate to the clinic, as well as unforeseen toxicity issues. 3D models, by comparison, better mimic in vivo conditions found within the target patient, including oxygen and nutrient gradients, increased cell-to-cell and cell-to-ECM interactions, and non-uniform exposure of cells within a 3D structure to the test molecule. In this webinar, the attendee will learn how commonly performed in vitro cancer research procedures can be performed using advanced digital widefield microscopy.

Three areas of focus will be covered:

1. Monitoring directed T cell activation and cell mediated cytotoxicity using co-cultured T cells and 3D target cancer cell models

2. 3D tumoroid invasion within a hydrogel

3. Use of stem cell derived small intestinal organoids for toxicity assessment

Proper label-free and fluorescence image capture techniques will be demonstrated, including the use of z-stacking and montaging to accurately visualize the 3D structures. Image and cellular analysis methods will also be shown to quantify metrics such as stem cell organoid budding, extent of tumoroid invasion, and T cell activation and induced target cell cytotoxicity.

The cytolytic activity of Immune cells toward cancer cells is finely regulated through several pathways and receptors. Although end point assays have been routinely used to measure cell killing, they have intrinsic limitations which led to new in vitro potency assays that are more sensitive and better predictive of in vivo outcomes. The introduction of kinetic approaches has tremendously expanded the dynamic range and resolution of potency determination, allowing the development of safer drugs. In this webinar, Dr. Cerignoli will provide an overview of the Agilent xCELLigence Real Time Cell Analysis technology and its utility in cell mediated cytolysis.  He will describe the advantages and limitations of impedance and imaging alone approaches and highlight the importance of comprehensive multiparametric analysis.

For Research Use Only. Not for diagnostic procedures.

Learning Objectives:

1. Comprehend advantages of combining real time impedance and live cell imaging in your immuno-oncology workflow

2. Identify Important factors to consider when designing cell-mediated cytolysis xCELLigence experiments 3. Understand specific cellular phenomena from kinetic data analyzed with RTCA Software

With the recent successes of immunotherapies, a fresh breeze of optimism in the fight against cancer has reinvigorated the industry. Perhaps the greatest legacy will be the realization of the need to understand the interplay within the tumor microenvironment between tumor malignancy and protective immunity. Targeting the liabilities of tumors by ‘tuning’ cellular networks back into homeostatic harmony may just be the key to effective, durable therapies.

Targeting and eliminating pathogenic cells is not good enough on its own to consistently achieve long-term survival. For example, immune cells that can eliminate tumors must persist in hostile, immunosuppressive microenvironments throughout tumor elimination and be capable of renewing protective surveillance. With the emergence of quantitative, live-cell analysis platforms researchers can make the necessary sensitive, time-resolved measurements to monitor the temporal components of an ever evolving cellular network.

This talk will showcase new concepts and strategies to target cancer through an understanding of the immune response. We will discuss purpose-built applications that enable real-time measures of immune cell function, fate and fitness. This tool bench provides the capability to measure, engineer and control immune cell function, thus enabling translational researchers and developers to achieve the necessary level of therapeutic potency and safety

Learning Objectives:

1. Define new concepts and understanding of the interplay between tumors and immunity

2. Identify new solutions to model and study tumor immunity over time

3. Key strategies being tested to target tumor liabilities through immune-based drug design

B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. Here we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α vs CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T-cell populations in vitro. However, CD8α/CD28-CAR T cells consistently outperformed other CAR T-cell populations in three animal models, resulting in a significant survival advantage. We next explored if adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T-cell expansion in vivo and improved antitumor activity in 1 of 4 evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

Learning Objectives:

1. Understand B7-H3 as a promising immunotherapy target found at high levels on many tumor types

2. Identify Optimal CAR design as a determined empirically for each new construct

The identification of novel drug targets and the development of next generation therapeutic strategies remain elusive goals for cancer researchers. We believe that the aberrant molecular events driving cancer development and progression can be exploited to devise novel therapeutic strategies that are highly selective towards cancer cells. Chromosome instability (CIN) is a form of genome instability that induces ongoing changes in chromosome complements and thus, is a driver of cell-to-cell heterogeneity. CIN is prevalent in many cancer types and is associated with cellular transformation, intratumoral heterogeneity, metastasis, the acquisition of drug resistance and poor patient prognosis. Despite all these associations, the aberrant genes, proteins and cellular processes (i.e. molecular determinants) giving rise to CIN remain largely unknown. To expand our basic understanding of the molecular determinants of CIN and cancer mandated the use of single-cell approaches capable of quantifying the cell-to-cell heterogeneity induced by CIN. Accordingly, we created several quantitative imaging microscopy approaches and employed them to evaluate hundreds of candidate CIN genes. Here, we present these approaches and their application in the discovery and characterization of a subset of CIN genes with pathogenic implications for cancer. We subsequently discuss the use of additional quantitative approaches to identify novel drug targets that selectively target and kill cancer cells harboring defects in CIN genes. Collectively, our work provides novel insight into the pathogenic origins of cancer that is crucial to develop the next generation of drug targets aimed at better combating the disease.

Learning Objectives:

1. Define chromosome instability (CIN) and describe its relationship with cancer.

2. Detail three quantitative imaging microscopy approaches used to identify CIN genes.

3. Describe how quantitative imaging microscopy approaches can be used to identify novel drug targets.

The major function of mitochondria in cellular homeostasis has been the generation of ATP through oxidative phosphorylation. However, we have previously demonstrated that mitochondria can serve as signaling organelles by releasing low levels of reactive oxygen species (ROS) that are essential for hypoxic activation of HIF, antigen activation of T cells, cellular differentiation and proliferation of cancer cells. Our recent findings indicate that mitochondria also release TCA cycle metabolites that are necessary for chromatin and DNA modifications. We will present our current findings on how mitochondria affect cellular function beyond ATP production through ROS and TCA cycle metabolites in controlling cancer and brain metabolism.

Learning Objectives:

Discover:

1. Mechanisms for mitochondria to act as signaling organelles.

2. The role of mitochondria in the TCA cycle and chromatin and DNA modifications.

3. How mitochondria affect cellular function beyond ATP production through ROS and TCA cycle metabolites in controlling cancer and brain metabolism.

Background: Acute liver injury (ALI) is characterised by hepatocyte death and liver inflammation. Macrophages play a pivotal role in ALI by phagocytosing dead cells and producing pro-regenerative signals. We have previously demonstrated that phagocytic cargo-dependent activation of phosphoSTAT3 is required to maintain phagocytosis efficiency in macrophages. In this study, we aim to understand the molecular mechanisms underlying the pro-phagocytic function of phosphoSTAT3.

Methods: Phagocytosing bone marrow derived macrophages (BMDMs) treated with and without a phosphoSTAT3 inhibitor, or from mice deficient for the cargo digestion step of phagocytosis (Gpnmb-), were analysed using flow cytometry, transmission electron microscopy (TEM), and qPCR. We used paracetamol overdose (POD) as mouse model for ALI. We FAC-Sorted and analysed infiltrating macrophages from livers at early stages of POD (10h post-overdose) with the nCounter® technology (Nanostring) and using proteomics.

Results: Gpnmb- mice are unable to regenerate their liver efficiently after POD: infiltrating phagocytosing macrophages FAC-Sorted from these mice showed increased expression of senescence-related genes, such as Tnfrsf1b and Cdkn1a, but not of apoptosis-related genes, such as Card9. This suggests that macrophages unable to process the internalised cargo enter immuno-senescence, a condition characterised by elevated production of inflammatory cytokines and reduced phagocytosis. TEM and qPCR analyses showed that both Gpnmb- and phosphoSTAT3 inhibitor-treated BMDMs develop a senescent phenotype during phagocytosis, suggesting that cargo digestion-dependent phosphorylation of STAT3 prevents immuno-senescence in phagocytosing macrophages. Autophagy is a mechanism widely used by cells to cope with stress: interestingly, TEM and qPCR analyses unveiled a lower activation of autophagy in Gpnmb- and phosphoSTAT3 inhibitor-treated BMDMs.

Conclusions: Phagocytosing macrophages require phosphoSTAT3 activation.

Learning Objectives:

1. Understand the use of flow cytometry-based medium throughput screening assays as an essential tool to investigate macrophage phagocytosis

2. Describe the role of pSTAT3 as a pro-phagocytic pathway in macrophages

3. Explain that other pathways are controlled downstream of pSTAT3, including autophagy and senescence

Coordination of immune cell metabolic programs with cell fate and state is a fundamental determinant of immune responses. Emerging evidence highlights that the interplay between immune signaling and metabolic programming orchestrates the activation and differentiation of T cells that further impact the outcome of immune responses. In particular, immunometabolism has key roles in shaping the functional fitness of both regulatory and effector T cells in the tumor microenvironment. Here I will discuss our recent findings regarding how mTOR and other signaling pathways bridge nutrient signals and cell metabolic programs to direct T cell priming and function, and the implications in immune tolerance and antitumor immunity. I will also discuss the use of systems immunology and functional genomics approaches including pooled CRISPR screening to investigate metabolic signaling and identify new therapeutic targets in cancer therapy.

Learning Objectives:

Discover how:

1. Immunometabolism has key roles in shaping the functional fitness of both regulatory and effector T cells in the tumor microenvironment.

2. mTOR and other signaling pathways bridge nutrient signals and cell metabolic programs to direct T cell priming and function, and the implications in immune tolerance and antitumor immunity.

3. The use of systems immunology and functional genomics approaches including pooled CRISPR screening is powerful to explore metabolic signaling and discover new therapeutic targets in cancer therapy.

Advances in genetic engineering have improved specificity, safety and potency of T-cell receptor (TCR) and Chimeric Antigen Receptor (CAR-T) based therapies for solid and non-solid tumours. These developments have allowed the manufacturing and commercialisation of these immunotherapy ATPMs (advanced therapy medicinal products). However, there is a current need to develop more robust product release assays to shorten product release time, including methods to ensure the identity, safety or potency of the product. Current methodologies to evaluate product potency require cell labelling, are labour intensive and only provide information about product’s specific cytotoxic effect at one selected time-point. The xCELLigence platform overcomes these limitations by measuring electrical impedance over time. In this presentation we will discuss how to develop and qualify the use of a high-throughput impedance-based system to measure T-cell cytotoxicity for both TCR or CAR-T approaches using either suspension or adherent target cell lines.

Current methods to treat malignant brain tumors are highly intrusive, leading to poor quality of life with abysmal patient survival rates. Combined with radiation therapy, first/second-line DNA damaging chemotherapeutics marginally improve patient survival. However, high rates of drug-resistance and recurrence are due to development of intra/intertumoral heterogeneity due in part from the emergence of chemoradioresistant clones. The targeted use of DNA repair inhibitors to sensitize and augment tumour cell killing is an emerging tool in cancer therapy. However, the biochemical and functional readout of combining chemotherapeutic drugs with one or more DNA damaging inhibitors is limited by use of traditionally low-throughput analytical techniques that preclude their adequate interrogation and translational potential.

We have developed novel high-throughput DNA repair assay methods, which allow us to combine multiple DNA repair inhibitors and genotoxic agents in order to identify synergistic interactions that sensitize CNS tumours to chemoradiotherapeutics. This combinatorial analysis approach combines DNA damage, cell cycle and cell viability readouts for determining drug selection and dosing effectiveness.

Part 1 of my talk will detail our development of a high-throughput comet assay (HTCOMET) that enables simultaneous, unbiased multi-well/sample DNA damage analysis. Using this strategy, we have compared drug/repair inhibitor responses amongst patentderived glioblastoma multiforme (GBM) and medulloblastoma (MB) cell lines. Using this approach we have found differential sensitivities amongst tumor types and amongst tumor lines. Validation studies have identified key molecular differences that prescribe these differential sensitivities.

Part 2 of my talk will outline our development of the high-throughput γH2AX analysis (HT-γH2AX) whereby γH2AX imaging and foci quantification has enabled a detailed examination of poly(ADP-ribose) polymerase inhibitor (PARPi) use in GBM lines in conjunction with the first-line DNA alkylating agent, Temozolomide (TMZ), and secondline DNA Toposiomase-1 inhibitor, Topotecan (TPT). This analysis has enabled a new level of molecular insight into the properties of these drugs whereby γH2AX foci analysis combined with predictive markers of the DDR pathway and cell cycle analysis (in both dose-escalating and time-dependent manners) identify specific properties in select PARPis that can synergize with chemotherapeutics to better enable tumor cell killing.

Our novel high-throughput DNA damage assay methodology enables molecular dissection and understanding of the interplay of DNA repair pathways with broad implications to fundamental science and clinical treatment. Fast, efficient and reproducible analyses, drug-screening and validation can be achieved using patientderived tumor specimens and live cells to identify targeted and/or personalized therapies for improved patient outcomes.

Learning Objectives:

1. To understand the cellular DNA damage response and types of DNA repair pathways

2. To identify DNA damage-specific repair pathways that brain tumor cells use to resolve damage induced by chemotherapy which act to evade cell death.

3. To describe how conventional low-throughput DNA repair methodologies can be streamlined using unbiased semi-automated high-throughput techniques to enable drug combination synergy analysis and drug discovery through the lens of DNA damage repair – a fundamental outcome of most anti-cancer chemotherapeutics.

Metabolic reprogramming is critical for governing proper T cell immune responses, including activation, production of effector molecule, migration and differentiation. The metabolic stress T cells could encounter in tumors has emerged as a microenvironmental challenge to tumor-infiltrating T cells. Moreover, the metabolic insufficiency has been observed in tumor-infiltrating T cells associated with declined effector functions. However, it remains unclear why tumor-infiltrating T cells lose metabolic fitness and the detailed impacts of metabolic insufficiency on modulating T cell differentiation. In my talk, I will describe how the tumor microenvironment could influence metabolic fitness and differentiation program in tumor-infiltrating T cells and further provide ideas on boosting T cell anti-tumor responses via metabolic interventions.

Learning Objectives:

Discover:

1. Theory and techniques for systems immunology and metabolic control of T cell tolerance and antitumor immunity

2. How nutrient sensing and signaling in Tregs and the tumor microenvironment could influence metabolic fitness and differentiation program in tumor-infiltrating T cells

3. Potential approaches for boosting T cell anti-tumor responses via metabolic interventions.

Three dimensional (3D) spheroidal cell models have become a mainstay in life science research to provide the most in vivo-like environment. Performing media exchanges and washes with unattached spheroids can be problematic due to the risk of accidental spheroid removal. In this webinar we will introduce the MultiFlo™ FX's new AMX™ Automated Media Exchange Module, a novel automated, peristaltic pump-based tool that allows these procedures to be carried out in a controlled and consistent manner that eliminates spheroid disruption and removal, simplifying long-term 3D experimental procedures.

In this webinar, Dr. Luke O'Neill will discuss an overview of immunology and immunometabolism in infectious disease. The past 10 years or so has seen a major resurgence of interest in immunometabolism. Metabolic changes in immune cell activation has become a focus and the role of glycolysis and Krebs Cycle in the altered phenotype of macrophages and T cells has been explored in detailed. Metabolite such as succinate and itaconate have been probed and the potential to target metabolic change for is gaining momentum.

Learning Objectives:

1. PKM2 as key regulator of inflammatory macrophage activation and Th17 cell function

2. Itaconate as anti-inflammatory metabolite that targets multiple inflammatory processes, notably NLRP3

3. Broader context of how viruses subvert immunometabolism and how this might be useful in current studies

Mouse models have been essential for establishing the role of the T cells in anti-tumor responses, but they have limited utility in translational studies for the testing of candidate therapeutics due to lack of cross-reactivity of many human antibodies to mouse receptors. Most of the human cell-based models for studying anti-tumor T cell responses, however, have significant biological or practical limitations that constrain their usefulness. This talk will discuss the development of a model system to study antigen-specific T cell-mediated cytotoxicity of HLA-matched human tumor cells based on memory anti-viral T cells, and how optimizing this model using real-time instrumentation has led to new understanding of the interplay among the dynamic variables that impact the interactions between tumor cells and T cells.

Learning Objectives:

1. Approaches to modeling and studying HLA-restricted anti-tumor T cell responses without access to primary tumor samples

2. Variables that can dramatically impact these types of studies

3. How using both real time impedance measurement and live cell imaging has impacted our understanding of the dynamics of these assays

Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases (Hanahan et al., 2011). The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges, including the choice of an appropriate cell model. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types; therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format. Other hurdles include accurate mechanism of action determination, and proper capture and analysis of kinetic reader-based data and microscopic images during the tumor invasion process.

In this webinar we will demonstrate a method to perform 3D tumor invasion assays and mechanism of action determinations. Tumor invasion tracking was performed via digital microscopy and cellular analysis using a novel cell imaging multi-mode reader. The combination presents an accurate, yet easy-to-use method to assess target-based and phenotypic effects of new, potential anti-metastatic drugs.

In this webinar, Dr. Yuting Sun discusses her research supporting a Phase 1 clinical trial advancing cancer therapies, targeted at metabolic vulnerabilities. Metabolic rewiring is a hallmark of cancer. However, the plasticity of preexisting regulatory circuit compromises the effectiveness of targeted therapies. We postulate that leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. In this presentation, we discuss strategies in targeting two distinct metabolic vulnerabilities- FHmut cancer and glycolysis-deficient cancer.

Learning Objectives:

1. Targeting redox balance in FHmut cancer

2. Targeting OXPHOS in glycolysis deficient tumor

3. Utilization of seahorse assay supporting Phase 1 clinical trial (for research use)

In this webinar we provide a detailed review of available methods for measuring cell proliferation, with a particular focus on recent assay innovations, including both 2D and 3D formats. This information will assist researchers in selecting the most appropriate and effective system for measuring cell proliferation based on a broad range of factors. Additionally, we cover important considerations for setting up and running cell proliferation assays, including techniques for improving the accuracy and reproducibility of results.