enhancing cd8 t cell responses through re-stimulation with cancer cells and evaluation of tumor reactivity

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.

Enhancing CD8 T Cell Responses Through Re-stimulation with Cancer Cells and Evaluation of Tumor Reactivity

After recovering CD8 cross-primed from coculture, centrifuge them at 1100 g for 10 minutes in 10 milliliters of complete growth medium. Resuspend 1 x 10 to the power of 7 total cells in 100 microliters of dead cell removal microbeads, and incubate for 15 minutes at room temperature. Place separation columns in the magnetic separator, and rinse them with binding buffer.Transfer cell suspensions into separation columns, and collect the flow-through. After washing the column, collect the unlabeled cells that pass through, and combine them with previously collected flow-through. Centrifuge enriched live CD8 cross-primed at 1100 g for five minutes in a complete growth medium.After counting, culture CD8 cross-primed cells with fresh MCA205 cells at a two-to-one ratio in 12 well plates, and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Before recovering CD8 effector, add monensin and brefeldin to the cancer immune cell cocultures for six hours. Then recover CD8 effector, and centrifuge twice at 1100 g for five minutes.Resuspend cells in three different primary antibody mixes prepared in cold FACS buffer, and incubate for 20 minutes at four degrees Celsius, protecting from light. Add 100 microliters of fixation solution to the cell palate from Mix C and incubate for 20 minutes at four degrees Celsius in the dark. Resuspend the cells in a cold wash buffer, and incubate for 30 minutes at four degrees Celsius.After washing cells twice in FACS buffer, add 100 microliters of PBS containing the Vitality Fixable Aqua Dye to cell pellets for 30 minutes. Identify 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 525/50 nanometer emission band-pass filter and SSCA to remove dead cells, Using 660/20 and 780/60 nanometer emission band-pass filters, detect cell positivity for CDAA and CD3 in two parameter density plots. Further, analyze cells for CD44, CD25, and CD69 markers for CD137 marker, and for interferon gamma positive and granzyme B for Mix A, B, and C, respectively. For tumor killing assay, supplement the coculture medium with a real-time cell death quantification dye.After warming the plate to 37 degrees Celsius in the live cell analysis system, perform data scanning every two hours for up to 72 hours. Centrifuge the cells twice at 1000 g for five minutes. Stain cells for surface markers with 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees celsius in the dark.Then add the vitality dye propidium iodide to stain the cells for a gating strategy. 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.Use a 450/50 nanometer emission band pass filter to detect cancer cells negative for CD45. In a single parameter histogram, using a 610/620 nanometer band-pass emission filter, analyze cells for propidium iodide incorporation as mean fluorescent intensity. Through multi-parameter flow cytometry, the surface expression levels of the early activation marker CD69, late activation markers CD44 and CD25, membrane expression of CD137, and CD107, tumor reactivity markers were enhanced.The intracellular levels of cytotoxic molecules, granzyme B, and interferon gamma positive were progressively and significantly increased, reaching a peak of expression at 72 hours of coculture. Cognate cocultured MCA205 cells underwent significant levels of CD8 effector mediated death.

enhancing-cd8-t-cell-responses-through-re-stimulation-with-cancer

Research

JoVE Journal

Cancer Research

21 Görüntüleme. Kokültürden çapraz astarlanmış CD8'i geri kazandıktan sonra, 10 mililitre tam büyüme ortamında 10 dakika boyunca 1100 g'da santrifüjleyin. 100 mikrolitre ölü hücre çıkarma mikro boncuklarında 1 x 10'u toplam 7 hücrenin gücüne yeniden süspanse edin ve oda sıcaklığında 15 dakika inkübe edin. Ayırma kolonlarını manyetik ayırıcıya yerleştirin ve bunları bağlama tamponu ile durulayın.Hücre süspansiyonlarını ayırma kol...