American Association for Cancer Research 2019
March 29-April 3, 2019
Georgia World Congress Center
Title: Quantifying cell subsets and heterogeneity in living cultures using real-time live-cell analysis
Presenter: Hinnah Campwala, Research Biologist
Session Date and Time: Monday Apr 1, 2019 1:00 PM - 5:00 PM
Location: Georgia World Congress Center, Exhibit Hall B, Poster Section 13
Poster Board Number: 7
Authors: N Bevan, T Jackson, C Szybut, L Kelsey, H Campwala, T Dale, N Dana, N Holtz, E Endsley, D Appledorn, C Schramm, L Skerlos, R Lister, D Trezise.
Essen BioScience Ltd, Welwyn Garden City, AL7 3AX UK & Essen BioScience Inc, Ann Arbor, Michigan, 48108 USA
Considerable heterogeneity exists in the properties and activity of individual cells, even in the simplest cell system. This arises from fundamental differences in the basic cell types present, genetic or epigenetic variations, the stage of cell cycle or differentiation, and the impact of each cell’s unique and dynamic local microenvironment. Such heterogeneity is mirrored by the diversity of pharmacological response at the cellular level, where even seemingly identical cells may respond differently and at different times to drug treatments and perturbagens. Accordingly, analysis at the cell-by-cell level promises valuable and additional biological insight beyond which whole population measures may deliver. Here, we describe new, enabling and industrial-scale, live-cell analysis solutions for quantifying the phenotypic biology of cell subsets in heterogeneous cultures.
Time-lapse images of cultured cancer and immune cells in 96-well microplates were automatically collected using an IncuCyte S3 live analyzer (Essen Bioscience). Using new segmentation algorithms, the boundaries of individual cells (typically 300-1000 per image) were identified in each image of the sequence. Parameters and features were extracted from single cells, such as cell area, eccentricity and fluorescence (e.g. with cell labels, cytotoxicity and apoptosis probes). Populations of cells could be identified and classified over time using custom ‘flow cytometry’ like visualisation and classification tools. Using this new approach, we demonstrate cell-by-cell analysis for a range of different primary and immortalised, adherent and non-adherent, living cells for up to 7 days in culture (e.g. Jurkat, A549, human PBMCs). This was coupled with a novel live-cell, fluorescent antibody-based labeling strategy (IncuCyte FabFluor-488) to probe for specific subsets within the cultures. Example data generated in PBMCs during T cell activation (anti-CD3/IL-2, 10 ng/mL) demonstrates the change in cell shape from small, spherical cells (average area 81 ± 0.5 μm2, eccentricity 0.57 ± 0.002) to larger, flatter cells (117 ± 4 μm2, 0.69 ± 0.004) over 120 h. With the addition of FabFluor-488-CD71, it was possible to show an associated, temporal increase in CD71 expression within the activated T cell subset (75 ± 1% of large cells were CD71 positive compared to 12 ± 1% of smaller cells at 48 h, increasing to >90% in the larger cells by 120 h). Other example data sets for subset analysis of proliferation, cell death and cell cycle measurements as well as immuno-phenotyping will be shared to illustrate the value of this approach.
Title: Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis
Presenter: Cicely Schramm, Principal Scientist
Session Date and Time: Wednesday Apr 3, 2019 8:00 AM - 12:00 PM
Location: Georgia World Congress Center, Exhibit Hall B, Poster Section 38
Poster Board Number: 9
Authors: Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn
Essen BioScience Inc, Ann Arbor, Michigan, 48108 USA
Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the Incucyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the Incucyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the Incucyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements.
Screening ex vivo conditions that increase memory T cell frequency using high throughput flow cytometry and an optimized multiplexed assay
Title: Screening ex vivo conditions that increase memory T cell frequency using high throughput flow cytometry and an optimized multiplexed assay
Presenter: Zhaoping Liu, Senior Scientist
Session Date and Time: Monday Apr 1, 2019 8:00 AM - 12:00 PM
Location: Georgia World Congress Center, Exhibit Hall B, Poster Section 10
Poster Board Number: 6
Authors: Zhaoping Liu, Andrea Gomez-Donart, & John O’Rourke
Intellicyt Corporation®, Part of the Sartorius Group. 5700 Pasadena Ave. NE, Albuquerque, NM 87113
A critical process in bio-manufacturing of adoptive cell therapies such chimeric antigen receptor (CAR) T and tumor infiltrating lymphocyte (TIL) therapies is the ex vivo expansion of T cells. Recent clinical studies show a correlation between in vivo expansion and persistence of infused T cells and patient outcomes. Additional studies show that a subset of functional memory T cells including T memory stem cells (Tscm), central memory T cells (Tcm) and other less differentiated T cell subsets are responsible for the majority of in vivo expansion and persistence leading to increased anti-tumor responses. This suggests that ex vivo protocols generating higher percentages of Tscm and Tcm in the total cell product will lead to significant clinical improvements in adoptive cell therapies.
To address the need to monitor T cell phenotype and function for improved ex vivo expansion protocols and other studies where profiling of memory subsets is crucial, we developed a robust, high content T cell memory assay. This miniaturized assay uses high throughput flow cytometry to measure cell phenotype, cell viability and effector cytokine release in the same sample well of a 96- or 384-well microtiter plate. The optimized antibody panel includes markers to identify T cells (CD3, CD4, and CD8), markers to discriminate between naive, memory, effector subsets (CD45RA, CD45RO, CD62L, and CD95), and a long-term survival marker (CD27). In addition, secreted cytokine quantitation (INFand IL-10) is performed simultaneously with the phenotypic and cell health measurements using a bead-based assay.
Proof of concepts studies were performed by stimulating peripheral blood mononuclear cells (PBMC) from multiple donors using anti-CD3/anti-CD28 coated beads. Activated T cells were expanded by culturing in serum-free media supplemented with different combinations and concentrations of IL-4, IL-7, IL-15, and IL-21 in a 96-well plate format. On Days 3 through 7 post stimulation, a small aliquot of cells and supernatant from each culture well was transferred to an assay plate and assessed for phenotype and function using the T cell memory assay. Data were acquired using the Intellicyt iQue Screener PLUS® VBR high throughput flow cytometer and analyzed using the integrated ForeCyt® software package. These data showed that the various cytokine combinations had a distinct effect on promoting T cell memory frequency, especially the Tscm cell subset, as well as effector cytokine secretion in a temporal and concentration specific manner.
These data show the T cell memory assay combined with high throughput flow cytometry is a powerful tool to rapidly screen for cytokine combinations that increase the frequency of T-memory cells during ex vivo expansion of T cells. Besides their use in optimizing cell manufacturing protocols, these tools can be easily applied to the profiling of other biologics and drugs where the monitoring of T cell memory subsets is required.