Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. CD14+ Cell Isolation and Monocyte-Derived Macrophage Culture
2.3. Multi-Component Spheroid Culture
2.4. Brightfield Microscopy
2.5. Primary Tissue Processing and Explant Culture
2.6. Flow Cytometry
2.7. T Cell Functional Assay
2.8. LEGENDplexTM Bead-Based Immunoassay
2.9. Statistical Analyses
3. Results
3.1. NSCLC Spheroid-Conditioned Polarisation of Mφs In Vitro: Morphology and Two-Marker Analysis Based on CD64 and CD206 Expression
3.2. Ex Vivo Study of Mφ Polarisation Induced by Lung Tumour Explants, Using the Two-Marker Approach
3.3. Mφ Plasticity in the TME Model
3.4. Tumour Explant, but Not Spheroid, Co-Cultures Suppress CD8+ T Cell Responses
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AGFB | AG02603 fibroblasts |
CAFs | Cancer-associated fibroblasts |
EBV | Epstein-Barr virus |
FBS | Fetal bovine serum |
HCMV | Human cytomegalovirus |
IHC | Immunohistochemistry |
iNOS | Nitric oxide synthase |
LPS | Lipopolysaccharide |
Mφ | Monocyte-derived macrophages |
MDSCs | Myeloid-derived suppressor cells |
MFI | Mean fluorescence intensity |
MT | Transitional myeloid cells |
MU | Unpolarised myeloid cells |
NSCLC | Non-small cell lung cancer |
PBMCs | Peripheral blood mononuclear cells |
PBS | Phosphate-buffered saline |
RT | Room temperature |
TAM | Tumour-associated macrophage |
TGFβ1 | Transforming growth factor β1 |
TME | Tumour microenvironment |
TT | Tetanus toxoid |
VP | Viral peptide |
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Demographic | All Patients n = 11 |
---|---|
Age at operation, years | |
Median (Min, Max) | 72.0 (58.0, 78.0) |
Mean | 71.3 |
SD | 6.3 |
Sex | |
Male | 6 (54.5%) |
Female | 5 (45.5%) |
Histology | |
Adenocarcinoma | 3 (27.3%) |
Squamous cell carcinoma | 8 (72.7%) |
T stage | |
T2a | 4 (36.4%) |
T2b | 2 (18.1%) |
T3 | 5 (45.5%) |
N stage | |
N0 | 6 (54.5%) |
N1 | 4 (36.4%) |
N2 | 1 (9.1%) |
M stage | |
MX | 7 (63.6%) |
M0 | 3 (27.3%) |
M1b | 1 (9.1%) |
Cytokine/Chemokine Concentration (pg/mL) | ||||
---|---|---|---|---|
IFNγ | ||||
4898 | 4836 | 2780 | 5178 | |
T cells Only | <0.53 | 212.47 | 113.23 | 3.32 |
+ Explants | 0.62 | 105.40 | 150.66 | 1.55 |
+ Explants/Mφ | 1.13 | 18.65 | 209.30 | 78.56 |
TNFα | ||||
4898 | 4836 | 2780 | 5178 | |
T cells Only | <1.20 | 1.21 | 1.71 | 3.52 |
+ Explants | <1.20 | <1.20 | 1.95 | <1.20 |
+ Explants/Mφ | <1.20 | <1.20 | 1.58 | 1.58 |
IL-10 | ||||
4898 | 4836 | 2780 | 5178 | |
T cells Only | <1.09 | 1.675 | 1.18 | 1.77 |
+ Explants | 2.95 | 3.44 | 3.96 | 2.74 |
+ Explants/Mφ | 1.73 | 17.03 | 8.12 | 8.31 |
Arginase | ||||
4898 | 4836 | 2780 | 5178 | |
T cells Only | 3663.90 | 3923.90 | 2346.75 | 3975.41 |
+ Explants | 3052.86 | 3285.92 | 2346.46 | 2819.79 |
+ Explants/Mφ | 5712.62 | 4302.11 | 4212.98 | 5670.56 |
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Evans, L.; Milward, K.; Attanoos, R.; Clayton, A.; Errington, R.; Tabi, Z. Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models. Biomedicines 2021, 9, 302. https://doi.org/10.3390/biomedicines9030302
Evans L, Milward K, Attanoos R, Clayton A, Errington R, Tabi Z. Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models. Biomedicines. 2021; 9(3):302. https://doi.org/10.3390/biomedicines9030302
Chicago/Turabian StyleEvans, Lauren, Kate Milward, Richard Attanoos, Aled Clayton, Rachel Errington, and Zsuzsanna Tabi. 2021. "Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models" Biomedicines 9, no. 3: 302. https://doi.org/10.3390/biomedicines9030302