Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy
Abstract
:1. Introduction
2. Results
2.1. Cell Death Induction by Radiotherapy in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines
2.2. Cell Death Induction by Hyperthermia in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines
No Significant Cell Death Induction by Conventional Warm-Water Heating but by Microwave Heating
2.3. Cell Death Induction by Hyperthermia and Radiotherapy in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines
Conventional Warm-Water Hyperthermia Barely Induces Further Cell Death in Combination with Normo- or Hypofractionated Irradiation, but Microwave Heating Has Additive Cell Killing Effects
2.4. Release of Danger Signal HSP70 in the Supernatant Following Radiotherapy and/or Hyperthermia
2.4.1. The Danger Signal HSP70 is Significantly Increased after Radiotherapy
2.4.2. Significantly Increased Release of Danger Signal HSP70 Directly after Hyperthermia in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines
2.4.3. Release of Danger Signal HSP70 after Hyperthermia and Radiotherapy on Day 3 and Day 5
2.5. Impact of Radiotherapy and Hyperthermia on the Expression of Immune Checkpoint Molecules
2.5.1. Modulation of Immune Checkpoint Molecules on the Tumor Cell Surface after Radiotherapy
2.5.2. Expression of Immune Checkpoint Molecules on the Tumor Cell Surface after Hyperthermia
2.5.3. Modulation of Immune Suppressive Checkpoint Molecules on the Tumor Cell Surface after Hyperthermia and Radiotherapy
2.5.4. Modulation of Immune Stimulatory Checkpoint Molecules on the Tumor Cell Surface after Hyperthermia and Radiotherapy
2.6. Numerical Simulations to Demonstrate Comparable Heating Conditions of CH and MH
3. Discussion
3.1. Augmenting Breast Cancer Cell Death by Microwave-Based Hyperthermia and Radiotherapy
3.2. Different Temperature Ranges of Hyperthermia Might be Most Beneficial for Immunomodulation
3.3. Adding Hyperthermia to Radiotherapy Dynamically and Individually Affects the Expression of Immune Checkpoint Molecules on Breast Cancer Cells
4. Materials and Methods
4.1. Closed-Loop System for Heat Treatments of Tumor Cells
4.2. Numerical Simulations and Modeling of the Heating System
4.3. Cell Lines and Cultivation
4.4. Treatments and Sampling
4.5. Cell Death Detection by AnnexinV/PI Staining
4.6. Detection of Heat Shock Protein 70 (HSP70) by ELISA
4.7. Detection of Immune Checkpoint Molecule and EGFR Expression by Multicolor Flow Cytometry
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A. Gating and Calculation Strategy for Analyses of Expression of Immune Checkpoint Molecules on The Tumor Cell Surface following Treatment with HT and/or RT
References
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Marker | Laser Color | Manufacturer | Cat. Nr. | µL Per Well |
---|---|---|---|---|
PD-L1 (CD 274) | BV 605 | BioLegend | 329724 | 0.5 1 |
PD-L2 (CD 273) | APC | BioLegend | 345508 | 0.5 1 |
EGF-Receptor | PE | BioLegend | 352904 | 0.5 1 |
ICOS-L (CD 275) | BV 421 | BD Bioscience | 564276 | 0.5 1 |
HVEM (CD 270) | APC | BioLegend | 318808 | 0.5 2 |
OX40-L (CD252) | PE | BioLegend | 326308 | 0.5 2 |
TNFRSF9 (CD137-L) | BV 421 | BioLegend | 311508 | 0.5 2 |
CD27-L (CD70) | FITC | BioLegend | 355106 | 0.5 2 |
Zombie NIR | APC-A750 | BioLegend | 423105 | 0.1 1,2,3 |
FACS-buffer | 2% FBS in DPBS (sterile) | 97.9 1,2/99.9 3 |
Parameter | 25 °C | 35 °C | 45 °C | 55 °C |
---|---|---|---|---|
76.7 | 74.0 | 70.7 | 67.5 | |
12.04 | 9.398 | 7.494 | 6.008 |
Parameter | Stainless Steel (CH) | Stainless Steel (MH) | Quartz Glass (MH) | Air (MH & CH) |
---|---|---|---|---|
Relative permeability, µr (-) | 1 | 1 | 1 | 1 |
Electric conductivity, σ (S/m) | — | 4 · 106 | — | 0.85 |
Relative permittivity, εr (-) | — | — | 3.78 - j2 · 10−4 | 1 |
Density, (kg/m³) | 7800 | 7850 | 2200 | 1.2 |
Heat conductivity, λ (W/mK) | 15 | 44.5 | 1.1 | 0.026 |
Heat capacity, cp (J/kgK) | 420 | 475 | 480 | 1006 |
Parameter | Levels | Unit |
---|---|---|
Ttarget | 37.0, 39.0, 41.0, 44.0 | °C |
m | 2.0 | mL/s |
teff * | 10 1, 20 1, 30 1, 60 | min |
normo(fractionation) 2 | 5 fractions of 2 Gy consecutively | |
hypo(fractionation) 2 | 2 fractions of 5 Gy on d0 and d3 |
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Hader, M.; Savcigil, D.P.; Rosin, A.; Ponfick, P.; Gekle, S.; Wadepohl, M.; Bekeschus, S.; Fietkau, R.; Frey, B.; Schlücker, E.; et al. Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy. Cancers 2020, 12, 1082. https://doi.org/10.3390/cancers12051082
Hader M, Savcigil DP, Rosin A, Ponfick P, Gekle S, Wadepohl M, Bekeschus S, Fietkau R, Frey B, Schlücker E, et al. Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy. Cancers. 2020; 12(5):1082. https://doi.org/10.3390/cancers12051082
Chicago/Turabian StyleHader, Michael, Deniz Pinar Savcigil, Andreas Rosin, Philipp Ponfick, Stephan Gekle, Martin Wadepohl, Sander Bekeschus, Rainer Fietkau, Benjamin Frey, Eberhard Schlücker, and et al. 2020. "Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy" Cancers 12, no. 5: 1082. https://doi.org/10.3390/cancers12051082
APA StyleHader, M., Savcigil, D. P., Rosin, A., Ponfick, P., Gekle, S., Wadepohl, M., Bekeschus, S., Fietkau, R., Frey, B., Schlücker, E., & Gaipl, U. S. (2020). Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy. Cancers, 12(5), 1082. https://doi.org/10.3390/cancers12051082