A.S. is supported by AIRC (IG #28807) and by PRIN (#P2022YE2MX). M.M. is supported by the AIRC-FIRC fellowship (#25558). A.D.N. is supported by the Innovation Ecosystem Rome Technopole ECS00000024 funded by the EU - Next Generation EU, PNRR Mission 4 Component 2 Investment 1.5.

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

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The authors have no conflicts of interest to disclose.

Monitoring anticancer immune response is of utmost importance to elucidate and understand the intricate molecular and cellular interplays acting in the TME and supporting a constant battle for supremacy23. Here, we detail a simple mFC- and ToC-assisted protocol for the monitoring and characterization of the steps constituting the cancer-immunity cycle. With minor variations, this protocol, based on murine cell lines and mouse-derived immune cells, can be adapted to both human immortalized and even...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtubes&#160;</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>100 kV e-beam litography</td>
			<td>Vistec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dishes&#160;</td>
			<td>Greiner Bio One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Euroclone</td>
			<td>&#160;ET3012</td>
			<td></td>
		</tr>
		<tr>
			<td>15 and 50 mL tubes</td>
			<td>Corning&#160;</td>
			<td>352096; 352070</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS431750</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene tubes&#160;</td>
			<td>Greiner Bio-One</td>
			<td>120180</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flask</td>
			<td>Euroclone</td>
			<td>&#160;ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Adsorbent wipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allumin foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD107a (LAMP-1) Antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-111-319</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD25 (7D4) Antibody&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-118-678</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD3 (17A2) Antibody</td>
			<td>BioLegend</td>
			<td>100206</td>
			<td></td>
		</tr>
		<tr>
			<td>Aptes</td>
			<td>Sigma Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit with BD GolgiPlug</td>
			<td>BD Biosciences</td>
			<td>555028</td>
			<td></td>
		</tr>
		<tr>
			<td>BD GolgiPlug Protein Transport Inhibitor (Containing Brefeldin A)</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BD GolgiStop Protein Transport Inhibitor (Containing Monensin)</td>
			<td>BD Biosciences</td>
			<td>554724</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological, Salem</td>
			<td>A1312</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c Monoclonal Antibody (N418)</td>
			<td>eBioscience</td>
			<td>12-0114-81</td>
			<td></td>
		</tr>
		<tr>
			<td>CD137 (4-1BB) Monoclonal Antibody (17B5)</td>
			<td>eBioscience</td>
			<td>17-1371-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 Monoclonal Antibody (17A2)</td>
			<td>eBioscience</td>
			<td>25-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 Monoclonal Antibody (IM7)</td>
			<td>eBioscience</td>
			<td>11-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11)</td>
			<td>Invitrogen</td>
			<td>MCD4528</td>
			<td></td>
		</tr>
		<tr>
			<td>CD69 Monoclonal Antibody (H1.2F3)</td>
			<td>eBioscience</td>
			<td>48-0691-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a Monoclonal Antibody (53-6.7)</td>
			<td>eBioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a Monoclonal Antibody (53-6.7)</td>
			<td>eBioscience</td>
			<td>17-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD95 (APO-1/Fas) Monoclonal Antibody (15A7)</td>
			<td>eBioscience</td>
			<td>53-0951-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting slides</td>
			<td>Kova International</td>
			<td>87144E</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium quartz masks</td>
			<td>MB W&#38;A, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit</td>
			<td>Invitrogen</td>
			<td>C10635</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS)</td>
			<td>EuroClone</td>
			<td>ECB4053L</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>EuroClone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo v10.0.7</td>
			<td>Flowjo, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Granzyme B Monoclonal Antibody (NGZB)</td>
			<td>eBioscience</td>
			<td>12-8898-82</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hotplate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humified cell culture incubator (37&#176;, 5% CO2)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice machine</td>
			<td>Brema Ice Makers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IFN gamma Monoclonal Antibody (XMG1.2)</td>
			<td>eBioscience</td>
			<td>11-7311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator CC 2015</td>
			<td>Adobe Systems Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte 2022A Software</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Cytotox Dye for Counting Dead Cells</td>
			<td>Sartorius</td>
			<td>4632</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte SX5 Live-Cell Analysis System&#160;</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JuLi Smart Fluorescent Live Cell Imaging Microscope</td>
			<td>Bulldog Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory bench</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory refrigerator (4&#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Safety Cabinet (Class II)</td>
			<td>Angelantoni</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM</td>
			<td>EuroClone</td>
			<td>ECB3004D</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201; 130-042-401&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separators</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-10; 130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MCA205 mouse fibrosarcoma cell line</td>
			<td>Sigma-Aldrich</td>
			<td>SCC173</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiologically controlled animal facility equipped with Class II safety cabine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroCL 21R Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002552</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel&#160;</td>
			<td>Microsoft, Redmond&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Naive CD8a+ T Cell Isolation Kit, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-543</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon ECLIPSE Ts2</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements BR 5.30.0064-BIT</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical litography</td>
			<td>EVG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt and streptomycin sulfate</td>
			<td>EuroClone</td>
			<td>ECB3001D/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td>Drummond Scientific Co., Broomall, PA</td>
			<td>&#160;4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PKH67 Fluorescent Cell Linker Kits</td>
			<td>Sigma-Aldrich</td>
			<td>PKH67GL-1KT</td>
			<td>fluorescent cell linker&#160; kit</td>
		</tr>
		<tr>
			<td>plastic coverslip</td>
			<td>IBIDI</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Thermo Scientific</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive Ion Etching system</td>
			<td>Oxford plasmalab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td>EuroClone</td>
			<td>ECB9006L</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td>
			<td>Corning- Millipore-Sigma; St. Louis, 
			MO</td>
			<td>CLS4486; CLS4487; CLS4488; CLS4489</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16 Centrifuge Series</td>
			<td>Thermo Scientific</td>
			<td>75004031</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels, surgical forceps, scissors and pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tips (1&#8211;10 &#956;L, 20&#8211;200 &#956;L, 1000 &#956;L)</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECTD00010; ECTD00020; ECTD00200; ECTD01005</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3000 series</td>
			<td>MicroChem corp, Newton, (MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suite of dermal biopsy punches</td>
			<td>Kai Medical, Tedpella</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dowsil, Dow Corning</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR beta Monoclonal Antibody (H57-597)</td>
			<td>eBioscience</td>
			<td>12-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMCS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>92360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA w/ Phenol Red</td>
			<td>EuroClone</td>
			<td>ECM0920</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

co-culture in vitro systems to reproduce the cancer-immunity cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

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

The ability of CD11c+ DCs, the widely known phagocyte subset specialized for cross-presentation21,22 and gated as shown in Figure 2A, to engulf apoptotic bodies from UV-irradiated MCA205 cancer cells that were previously labeled with the PKH67 fluorescent cell linker was evaluated by mFC. As expected, CD11c+ DCs efficiently captured apoptotic MCA205 cells in vitro at 37 &#176;C but not at 4 &#176;C (<st...

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

co-culture-in-vitro-systems-to-reproduce-the-cancer-immunity-cycle

Research

JoVE Journal

Cancer Research

553 Views.  Università Cattolica del Sacro Cuore. Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.