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.

All the steps of the protocol requiring the use of animals are in compliance with the EU Directive 63/2010 and included in an experimental protocol approved by the Institutional Animal Experimentation Committee and the Italian Ministry of Health (approval number 858/2015/PR).
1. Preparation of cancer cells
<ol>
	<li>Cancer cell culture routine (day -7; duration: 2 h)
	<ol>
		<li>Grow murine MCA205 fibrosarcoma cells in Roswell Park Memorial Institute (RPMI) 1640 growth mediu...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

<ol>
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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent+cancer-on-chip+models+to+assess+immuno-oncology+therapy.">Immunocompetent cancer-on-chip models to assess immuno-oncology therapy.</a> Adv Drug Deliv Rev. 173, 281-305 (2021).</li><li>Lorenzi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+IFNs+control+antigen+retention+and+survival+of+CD8&#945;(+)+dendritic+cells+after+uptake+of+tumor+apoptotic+cells+leading+to+cross-priming.">Type I IFNs control antigen retention and survival of CD8&#945;(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming.</a> J Immun. 186 (9), 5142-5150 (2011).</li><li>Musella, M., Manic, G., Galassi, C., Vitale, I., Sistigu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytofluorometric+assessment+of+dendritic+cell-mediated+uptake+of+cancer+cell+apoptotic+bodies.">Cytofluorometric assessment of dendritic cell-mediated uptake of cancer cell apoptotic bodies.</a> Methods Enzymol. 632, 39-54 (2020).</li><li>Lucarini, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+Type+I+Interferons+and+5-Aza-2'-Deoxycitidine+to+improve+antitumor+response+against+melanoma.">Combining Type I Interferons and 5-Aza-2'-Deoxycitidine to improve antitumor response against melanoma.</a> J Invest Dermatol. 137 (1), 159-169 (2017).</li><li>Guti&#233;rrez-Mart&#237;nez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+of+cell-associated+antigens+by+MHC+Class+I+in+dendritic+cell+subsets.">Cross-presentation of cell-associated antigens by MHC Class I in dendritic cell subsets.</a> Front Immunol. 6, 363 (2015).</li><li>Joffre, O. 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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

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.

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

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.

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.

359 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.

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle

Summary