Role of Hypoxia and the Adenosine System in Immune Evasion and Prognosis of Patients with Brain Metastases of Melanoma: A Multiplex Whole Slide Immunofluorescence Study
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Separating Tumor, Stroma, and CTL in Preparation for the Analysis
2.2. The Occurrence of Hypoxic Areas in the Examined Tumor Sections
2.3. Expression of the ADO-Generating Enzymes CD73 and CD39 and Associated Findings
2.4. Density and Distribution of CTL
2.5. Hypoxia-Induced Immune Evasion in Spatial and Statistical Analysis
2.6. Prognostic Significance of the Investigated Markers
3. Discussion
4. Patients and Methods
4.1. Patient Cohort
4.2. Immunofluorescence Staining
4.3. Quantitative Analyses
4.4. Spatial Analyses
4.5. Statistical Testing
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Ethics Approval and Consent to Participate
Consent for Publications
Abbreviations
A2AR | A2A-receptor |
ADO | Extracellular adenosine |
CA-IX | Carbonic anhydrase IX |
CCR | CC chemokine receptors |
CNS | Central nervous system |
CTL | Cytotoxic T-lymphocyte |
CTLA-4 | Cytotoxic-T-lymphocyte protein 4 |
DAPI | 4′,6-diamidino-2-phenylindole |
GLUT | Glucose transporter |
H&E | Hematoxylin and eosin |
HIF | Hypoxia-inducible factor |
HRP | Horseradish peroxidase |
ICD | Immunogenic cell death |
ICI | Immune checkpoint inhibitor |
IF | Immunofluorescence |
IHC | Immunohistochemistry |
IL | Interleukin |
IPI | Ipilimumab |
LAG3 | Lymphocyte-activation gene 3 |
LN | Lymph node |
mAb | Monoclonal antibody |
MBM | Melanoma brain metastases |
OS | Overall survival |
PD-1 | Programmed cell death protein 1 |
ROI | Region of interest |
RT | Radiotherapy |
STX | Stereotactic brain radiotherapy |
TIL | Tumor-infiltrating lymphocytes |
TME | Tumor microenvironment |
WBRT | Whole-brain radiotherapy |
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Clinicopathological Features | N (%) |
---|---|
Median age at initiation of RT (range) | 59 (29–83) |
Gender | |
Female | 10/33 (30.4%) |
Male | 23/33 (69.6%) |
Primary tumor | |
Melanoma type | |
Cutaneous | 28/33 (84.8%) |
Ocular | 1/33 (3.0%) |
Mucosal | 2/33 (6.1%) |
Unknown primary site | 2/33 (6.1%) |
Breslow depth 1 (median) | 2.2 mm |
Clark-level 1 (median) | IV |
BRAF mutated | 14/33 (42.4%) |
Metastatic lesion | |
Biopsy sites (N = 69) | |
Lymph node (LN) | 24/69 (34.8%) |
Skin and subcutaneous tissue | 29/69 (42.0%) |
Lung | 3/69 (4.3%) |
Central nervous system (CNS) | 6/69 (8.7%) |
Liver | 1 (1.4%) |
Gastrointestinal tract | 3 (4.3%) |
Other (Bone, Palate mucosae, myocard) | 3 (4.3%) |
Time between histological resection and IPI initiation (median) | 8 months |
RPA class (median) | 2 |
Quantitative data from immunofluorescence analysis | |
% CD8+ CTL median (range) | 4 (1–37) |
% GLUT-1high tumor cells median (range) | 42 (17–82) |
% GLUT-1low tumor cells median (range) | 20 (2–58) |
% CD73+ tumor cells median (range) | 7 (0–72) |
Treatments | |
Previous treatments | |
Treatment-naïve | 3/33 (9.1%) |
Previously treated | 30/33 (90.9%) |
Treatment received | |
Chemotherapy | 10/33 (30.3%) |
Radiotherapy | |
| 14/33 (42.4%) 12/33 (36.3%) 7/33 (21.2%) |
Ipilimumab | 33/33 (100%) |
Anti-PD1/anti-PD-L1 inhibitors | 5/33 (15.1%) |
BRAF ± MEK inhibitors | 8/33 (24.2%) |
IPI prior to RT | 18/33 (54.5%) |
Time between IPI initiation and RT (median) | 1 month |
Follow-up | |
Median survival upon RT (range) | 6 months (0–73 months) |
Deceased | 29/33 (87.8%) |
Antigen | Primary AB (Cat.-No., Clone, Species, Dilution, Incubation) | Primary AB Supplier | Fluorochrome (Cat.-No.) | Fluorochrome Supplier | Staining Pattern |
---|---|---|---|---|---|
CD73/NT5E | 13160, D7F9A, rabbit (mono), 1:50, for 1 h at 28−30 °C | Cell Signaling Technology, Danvers, MA (USA) | TSA Plus Cyanine 5 (NEL745001KT) | PerkinElmer, Waltham, MA, USA | See main text |
GLUT-1 | GTX62480, EPR3915, rabbit (mono), 1:500, overnight at 4 °C | GeneTex, Irvine, CA (USA) | Alexa Fluor 488 Tyramide (B40953) | Thermo Fisher Scientific, Waltham, MA, USA | Membranous |
CD8 | M7103, CD8/144B, mouse (mono), 1:50, for 1 h at 28−30°C | DAKO, Glostrup, Denmark A/S (DK) | TSA Plus Cyanine 3 (NEL744001KT) | PerkinElmer, Waltham, MA, USA | Membranous |
Na+-K+- ATPase | ab76020, EP1845Y, rabbit (mono), 1:100, for 1 h at 28–30 °C | Abcam, Cambridge (UK) | Alexa Fluor 647 Tyramide (T20951) | Thermo Fisher Scientific, Waltham, MA, USA | Membranous |
CD34 | ab81289, EPR373Y, rabbit (mono), 1:100, overnight at 4 °C | Abcam, Cambridge (UK) | Alexa Fluor 488 Tyramide (B40953) | Thermo Fisher Scientific, Waltham, MA, USA | Membranous (endothelial cells) |
CD45 | M0701, 2B11+PD7/26, mouse (mono), 1:50, for 1 h at 28−30 °C | DAKO, Glostrup, Denmark A/S (DK) | TSA Plus Cyanine 3 (NEL744001KT) | PerkinElmer, Waltham, MA, USA | Membranous |
CD39/ENTP1 | NBP2-45447, OTI2B10, mouse (mono), 1:100, for 1 h at 28–30 °C | Novus Biologicals, Littleton, CO (USA) | TSA Plus Cyanine 5 (NEL745001KT) | PerkinElmer, Waltham, MA, USA | See main text |
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Mayer, A.; Haist, M.; Loquai, C.; Grabbe, S.; Rapp, M.; Roth, W.; Vaupel, P.; Schmidberger, H. Role of Hypoxia and the Adenosine System in Immune Evasion and Prognosis of Patients with Brain Metastases of Melanoma: A Multiplex Whole Slide Immunofluorescence Study. Cancers 2020, 12, 3753. https://doi.org/10.3390/cancers12123753
Mayer A, Haist M, Loquai C, Grabbe S, Rapp M, Roth W, Vaupel P, Schmidberger H. Role of Hypoxia and the Adenosine System in Immune Evasion and Prognosis of Patients with Brain Metastases of Melanoma: A Multiplex Whole Slide Immunofluorescence Study. Cancers. 2020; 12(12):3753. https://doi.org/10.3390/cancers12123753
Chicago/Turabian StyleMayer, Arnulf, Maximilian Haist, Carmen Loquai, Stephan Grabbe, Matthias Rapp, Wilfried Roth, Peter Vaupel, and Heinz Schmidberger. 2020. "Role of Hypoxia and the Adenosine System in Immune Evasion and Prognosis of Patients with Brain Metastases of Melanoma: A Multiplex Whole Slide Immunofluorescence Study" Cancers 12, no. 12: 3753. https://doi.org/10.3390/cancers12123753