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

Summary

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.

Abstract

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.

Introduction

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 "engine" the immune system ignites to destr....

Protocol

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

1. Cancer cell culture routine (day -7; duration: 2 h)
   1. Grow murine MCA205 fibrosarcoma cells in Roswell Park Memorial Institute (RPMI) 1640 growth mediu.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microtubes	Eppendorf	30120086
100 kV e-beam litography	Vistec
100 mm Petri dishes	Greiner Bio One	664160
12-well plates	Euroclone	ET3012
15 and 50 mL tubes	Corning	352096; 352070
40 μm cell strainer	Corning	CLS431750
5 mL polystyrene tubes	Greiner Bio-One	120180
70 μm cell strainer	Corning	CLS431751
75 cm2 cell culture treated flask	Euroclone	ET7076
Adsorbent wipes
Allumin foil
anti-mouse CD107a (LAMP-1) Antibody	Miltenyi Biotec	130-111-319
anti-mouse CD25 (7D4) Antibody	Miltenyi Biotec	130-118-678
anti-mouse CD3 (17A2) Antibody	BioLegend	100206
Aptes	Sigma Aldrich	440140
BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit with BD GolgiPlug	BD Biosciences	555028
BD GolgiPlug Protein Transport Inhibitor (Containing Brefeldin A)	BD Biosciences	555029
BD GolgiStop Protein Transport Inhibitor (Containing Monensin)	BD Biosciences	554724
Bovine serum albumin (BSA)	US Biological, Salem	A1312
CD11c Monoclonal Antibody (N418)	eBioscience	12-0114-81
CD137 (4-1BB) Monoclonal Antibody (17B5)	eBioscience	17-1371-82
CD3 Monoclonal Antibody (17A2)	eBioscience	25-0032-82
CD44 Monoclonal Antibody (IM7)	eBioscience	11-0441-82
CD45 Monoclonal Antibody (30-F11)	Invitrogen	MCD4528
CD69 Monoclonal Antibody (H1.2F3)	eBioscience	48-0691-82
CD8a Monoclonal Antibody (53-6.7)	eBioscience	11-0081-82
CD8a Monoclonal Antibody (53-6.7)	eBioscience	17-0081-82
CD95 (APO-1/Fas) Monoclonal Antibody (15A7)	eBioscience	53-0951-82
Cell counting slides	Kova International	87144E
Chromium quartz masks	MB W&A, Germany
Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit	Invitrogen	C10635
CytoFLEX Flow Cytometer	Beckman Coulter
Dead Cell Removal Kit	Miltenyi Biotec	130-090-101
Dulbecco's Phosphate-Buffered Saline (D-PBS)	EuroClone	ECB4053L
EDTA	Invitrogen	AM9260G
Fetal bovine serum (FBS)	EuroClone	ECS0180L
Flowjo v10.0.7	Flowjo, LLC
Granzyme B Monoclonal Antibody (NGZB)	eBioscience	12-8898-82
H2O2	Sigma Aldrich
H2SO4	Sigma Aldrich
hotplate
Humified cell culture incubator (37°, 5% CO2)	Thermo Scientific
Ice machine	Brema Ice Makers
IFN gamma Monoclonal Antibody (XMG1.2)	eBioscience	11-7311-82
Illustrator CC 2015	Adobe Systems Inc.
ImageJ	National Institute of Health
Incucyte 2022A Software	Sartorius
Incucyte Cytotox Dye for Counting Dead Cells	Sartorius	4632
Incucyte SX5 Live-Cell Analysis System	Sartorius
JuLi Smart Fluorescent Live Cell Imaging Microscope	Bulldog Bio
Laboratory bench
Laboratory refrigerator (4°C)
Laboratory Safety Cabinet (Class II)	Angelantoni
L-glutamine 200 mM	EuroClone	ECB3004D
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit	Invitrogen	L34957
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit	Invitrogen	L10119
MACS columns	Miltenyi Biotec	130-042-201; 130-042-401
MACS separators	Miltenyi Biotec	130-042-10; 130-042-302
MCA205 mouse fibrosarcoma cell line	Sigma-Aldrich	SCC173
Microbiologically controlled animal facility equipped with Class II safety cabine
MicroCL 21R Microcentrifuge	Thermo Scientific	75002552
Microsoft Excel	Microsoft, Redmond
Mouse: C57BL/6J	The Jackson Laboratory	000664
Naive CD8a+ T Cell Isolation Kit, mouse	Miltenyi Biotec	130-096-543
Nikon ECLIPSE Ts2	Nikon Instruments Inc.
NIS-Elements BR 5.30.0064-BIT	Nikon Instruments Inc.
Optical litography	EVG
Penicillin G sodium salt and streptomycin sulfate	EuroClone	ECB3001D/1
Pipet aid	Drummond Scientific Co., Broomall, PA	4-000-201
Pipettes	Eppendorf
PKH67 Fluorescent Cell Linker Kits	Sigma-Aldrich	PKH67GL-1KT	fluorescent cell linker  kit
plastic coverslip	IBIDI	10812
Propidium Iodide	Thermo Scientific	P1304MP
Reactive Ion Etching system	Oxford plasmalab
Roswell Park Memorial Institute 1640 (RPMI 1640)	EuroClone	ECB9006L
serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)	Corning- Millipore-Sigma; St. Louis, MO	CLS4486; CLS4487; CLS4488; CLS4489
SL 16 Centrifuge Series	Thermo Scientific	75004031
Sterile scalpels, surgical forceps, scissors and pliers
Sterile tips (1–10 μL, 20–200 μL, 1000 μL)	EuroClone Spa, Milan, Italy	ECTD00010; ECTD00020; ECTD00200; ECTD01005
SU-8 3000 series	MicroChem corp, Newton, (MA)
Suite of dermal biopsy punches	Kai Medical, Tedpella
Sylgard 184	Dowsil, Dow Corning	101697
TCR beta Monoclonal Antibody (H57-597)	eBioscience	12-5961-82
Thermostatic bath
Timer
TMCS	Sigma Aldrich	92360
Trypan Blue Stain (0.4%)	Thermo Scientific	15250061
Trypsin-EDTA w/ Phenol Red	EuroClone	ECM0920
Vacuum dessicator