inducing cancer cell death and evaluating cd8 t cells activation through co-culture and flow cytometry analysis

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Over the past few decades, immunotherapy has been at the forefront of cutting-edge options for cancer treatment. Harnessing the immune system with antitumor purposes has provided powerful proof-of-concept for patient benefit across diverse hematologic and solid malignancies with historically poor prognoses. As immunotherapy is offering an opportunity for otherwise hard-to-treat cancers, it is experiencing an astoundingly rapid pace of progress. Such progress can be, at least in part, attributed to the refined understanding of the interplay between cancer cells and immune cells. This interplay resembles a feed-forward &#34;engine&#34; the immune system ignites to destroy cancer cells, the so-called cancer-immunity cycle. This anticancer immune response progresses across three main levels: recognition, processing, and reaction. Firstly, in the phase of recognition, tumor antigens (Ags) produced during tumor formation are released by dying cancer cells in the tumor microenvironment (TME, step 1) and engulfed by tumor-infiltrating dendritic cells (DCs, step 2). Next, in the phase of processing, DCs present the epitopes of the captured tumor Ags through the major histocompatibility complex (MHC) molecules, express on their surface higher levels of costimulatory molecules (step 3), and move to tumor-draining lymph nodes (dLNs) to cross-present their cargo to na&#239;ve CD8 T cells (CD8N, step 4). All these steps converge in the final process of reaction during which tumor Ag-specific cross-primed CD8 T cells (CD8C-P) are activated, mature into effector CD8 T (CD8E) cells, and undergo a clonal expansion (step 5). CD8E cells then leave the dLNs and home through the blood to the TME (step 6) where they specifically recognize and bind to cancer cells through the interaction between their T cell receptor (TCR) and their cognate tumor Ags, release cytotoxic molecules [i.e., interferon (IFN)-&#947;, perforins and granzymes (Grzs)] and kill cancer cells (step 7)1,2. Cancer cell killing leads to the release of further tumor Ags to fuel the cancer-immunity cycle. As a matter of fact, through all these steps, the immune system destroys and rejects cancer cells far more often than supposed. However, in cancer patients, at least one of these steps does not work properly. We and others showed that cancer cells seek to stall the immune response by either evolving into more aggressive and immune-privileged variants3,4,5 or hampering T-cell effectiveness6,7.
Cancer research and cancer drug development both rely on experimental models that allow the study of the relationship between cancer and immune cells, the so-called onco-immunology. Here are described fast, reliable, reproducible, and low-cost in vitro models that comprehensively reproduce each step of the onco-immunology cycle and is offered a rapid and clear view of the phenotypic and functional feature sets of immunosurveillance and eventually immunoediting.
Multiparametric flow cytometry (mFC) is one of the most successful single-cell analytical tools in fundamental cancer research, diagnosis, and translational research in cancer clinical trials. As it allows to simultaneously capture more features in each cell, mFC has earned its place as a gold-standard analysis platform in onco-immunology. It couples high sensitivity and specificity with the possibility to measure multiple protein expression patterns and functional properties quickly and reproducibly at a single cell level from heterogeneous and even heterotypic cell suspensions, as those from the TME8,9,10. As both phenotypic and functional expression patterns are time-sensitive, careful attention to experiment design, the selection of suitable panels, controls, and titred antibodies, and to appropriate sample processing and instrumentation use are critical for the reliability, comparability, and reproducibility of results and to confidently interpret experiment outcome11.
Tumor-on-a-chip microfluidic devices (ToCs) model the TME by allowing in vitro microscale biomimetics of cancer and immune cell dynamics and interplays12,13,14,15. Specifically, ToCs are multichannel microfluidic cell-culture devices able to host diverse cell types organized in either two-dimensional (2D) or three-dimensional (3D) culture settings and able to model with high fidelity and to control with high precision, key structural and functional units such as heterotypic cellular interactions and flows of chemical gradients that physiologically occur in the TME12,13,14,15. In particular, immune chemoattraction and trajectories as well as immune cell interaction with cancer cells, can be monitored in real-time and quantified by time-lapse microscopy and automated tracking analysis5,12,13,14,15,16. Furthermore, ToCs offer the possibility to both analyze and manipulate crucial processes regulating cancer onset and progression and response to therapy17.
In this article, mFC with ToCs are combined to study all the levels of the anticancer immune response going through DC-mediated phagocytosis of cancer Ags (steps 1-3), T cell cross-priming (step 4), activation and clonal expansion (the last by means of 5-ethynyl-2&#39;-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition [click] technology, a highly sensitive and accurate methodology, step 5), CD8E cell homing to the TME (step 6) and, finally CD8E-cell-mediated cancer cell killing (step 7, Figure 1).
This work contributes to the effort toward establishing simple, fast, and reliable standard protocols to study the cancer-immunity cycle. The improvement and integration of mFC and ToC models into cancer research, TME dynamics, and response to therapy hold great potential as these models provide biological fidelity along with experimental control. Hence, this protocol helps recreate, in a stepwise manner, the cancer-immunity cycle by making it possible to characterize, monitor, and timely maneuver the roles of individual cell players and their reciprocal interactions upon natural and acquired immunosurveillance. This ultimately will help refine, reduce, and replace animal studies while providing critical insights to outsmart tumors and guide clinical care. Finally, mFC and ToC advantages and limitations are critically discussed and compared with state-of-the-art technologies (e.g., high plex spatial analyses at single cell and even sub-cellular resolution) to push onco-immunology research and therapy forward.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

This protocol allows simple, faster, and reliable monitoring and characterization of each step of the cancer immunity cycle, and thus led to the identification of the mechanisms responsible for keeping the balance between cancer-immune surveillance and immune evasion. The interaction and evolution of cancer and immune cells in the tumor microenvironment have emerged as crucial factors detecting therapeutic outcomes. Currently, several technologies are advancing research in our field, including flow cytometry, single-cell transcriptomics, special transcriptomics, proteomics, tumor-on-a-chip models, and in-vivo models.Our protocol offers significant advantages over other techniques as it provides a simple, fast, reliable, and low cost method for monitoring and characterizing each step of the cancer immunity cycle. In the future, we want to study the dynamics of the tumor microenvironment and immunogenic and immune therapies.

Inducing Cancer Cell Death and Evaluating CD8 T Cells Activation Through Co-Culture and Flow Cytometry Analysis

To begin, plate 3 times 10 to the power of 6MCA205 fibrosarcoma cells in 10 milliliters of complete growth medium in a 100 millimeters Petri dish. Irradiate cells with ultraviolet light and incubate at 37 degrees Celsius with 5%carbon dioxide for at least six hours to let them die. Wash in vitro differentiated dendritic cells, or DCs, twice, at 1100G for four minutes in a complete growth medium.Count the bone marrow-derived empty DCs using the Trypan blue dye exclusion test. Centrifuge the irradiated cancer cells at 1100G for five minutes. Set the co-culture of empty DCs with irradiated apoptotic cells at a two to one ratio in a 12-well plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide.The next day, wash the cells twice at 1100G for five minutes in 10 milliliters of complete growth medium in 15 milliliter tubes. For cell surface staining, re-suspend the bone marrow-derived DCs in 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees Celsius in the dark. After washing the cells with FACS buffer, add 100 microliters of PBS containing vitality fixable near-infrared dye for 30 minutes.Wash the cells once with PBS before re-suspending them in the FACS buffer. Identify the cells of interest based on their FSCA and SSCA properties. Then plot FSCA and FSCH to exclude cell doublets and clumps from the analysis.Plot 780 to 60 nanometer emission band pass filter area and SSCA to remove dead cells. Using 575 to 26 and 530 to 30 nanometer emission band pass filters, detect cell positivity for CD11C marker and PKH67 fluorescent cell linker in two parameter density plots. After counting, culture the CD8 T cells with bone marrow-derived DCs that had taken up apoptotic MCA205 cells at a two to five to one ratio at 37 degrees Celsius and 5%carbon dioxide for 72 hours.Add EdU to the co-culture medium at 10 micromolar final concentration and incubate for 16 to 20 hours. Centrifuge the CD8 cross prime twice at 1100G for five minutes and stain for surface markers in cold FACS buffer for 20 minutes at four degrees Celsius in the dark. After washing the cells twice in FACS buffer, re-suspend them in 100 microliters of PBS containing the vitality fixable aqua dye for 30 minutes.Wash the cell suspensions once with three milliliters of 1%BSA in PBS in flow tubes. Add 100 microliters of 4%paraformaldehyde for 15 minutes for cell fixation. Incubate the cells in 100 microliters of saponin-based permeabilization and wash reagent for 15 minutes.Add 500 microliters of the reaction cocktail and incubate for 30 minutes in the dark. After washing, re-suspend the cells in 100 microliters of saponin-based permeabilization and wash reagent. Identify the cells of interest based on their FSEA and SSEA properties.Then plot FSEA and FSEH to exclude cell doublets and clumps from the analysis. Plot 525 to 50 nanometer emission band pass filter area and SSCA to remove dead cells. Using 530 to 30 and 575 to 26 nanometer emission band pass filter density plots, detect cells'positivity for CD8a and CD3.Analyze cells for Alexa Fluor 647 EdU incorporation in a single parameter histogram using a 660 to 620 nanometer band pass emission filter. CD11c positive dendritic cells effectively engulfed apoptotic MCA205 cancer cells at 37 degrees Celsius, demonstrating temperature-dependent phagocytosis in early cancer immunity cycle stages. After phagocytosis, dendritic cells showed increased levels of CD86 and MHC-II along with reduced levels of PDL1.The clonal expansion of CD8 positive T cells once activated by mature dendritic cells was evident with approximately 20%proliferation rate. Using tumor-on-chip models, the chemotactic response of these T cells towards cancer cell released alarmins was observed.

inducing-cancer-cell-death-evaluating-cd8-t-cells-activation-through

Research

JoVE Journal

Cancer Research

33 ビュー. まず、100ミリメートルのシャーレに10ミリリットルの完全増殖培地に6MCA205線維肉腫細胞の3倍10をプレートします。細胞に紫外線を照射し、摂氏37度で5%二酸化炭素と少なくとも6時間インキュベートして細胞を死滅させます。in vitroで分化した樹状細胞(DC)を1100Gで2回、完全増殖培地中で4分間洗浄します。トリパンブルー色素排除試験を使用し...