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.

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...

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

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.

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 ...

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

Research

JoVE Journal

Cancer Research

717 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.

Video: Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.