Abstract
Purpose
The clinical outcome of head and neck squamous cell carcinoma (HNSCC) remains poor, partly due to the presence of resistant cancer stem cells (CSCs) which are responsible of recurrences. CSCs have low EGFR expression and, conversely, overexpress the anti-apoptotic Bcl-2 protein, which is involved in resistance to apoptosis and the invasion/migration capacities of tumour cells.
Methods
The combination therapy of ABT-199, a Bcl-2 inhibitor, cetuximab an EGFR inhibitor, and radiation using an HNSCC model (SQ20B cell line) and its corresponding CSC subpopulation were evaluated in vitro (2D/3D cell proliferation; invasion/migration and apoptosis using videomicroscopy) and in vivo.
Results
Cetuximab strongly inhibited 2D and 3D cell proliferation, as well as invasion/migration, only in non-CSC-SQ20B cells, whereas ABT-199 selectively inhibited these mechanisms in SQ20B/CSCs. The combination of irradiation + cetuximab + ABT-199 increased the inhibition of the 2D and 3D cell proliferation, invasion/migration, and resistance to apoptosis in both cell sub-populations. In addition, in a nude mouse model with heterotopic tumour xenograft, a treatment combining cetuximab + ABT-199 with fractional irradiation strongly delayed the tumour growth and increased in vivo lifespan without side effects.
Conclusion
Based on the present results, this triple combination therapy may represent a new opportunity for testing in clinical trials, particularly in locally advanced HNSCC.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank all participants in the experiments and acknowledge the contribution of the flow cytometry platform of the SFR BioSciencesGerland-Lyon Sud (UMS3444/US8).
Funding
This work was supported by the LABEX PRIMES (ANR-11-LABX-0063) of Université de Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). It was also financed by La Ligue Contre le Cancer Comité de la Loire.
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JBG, SE, NM, and CRL designed the study. JBG, SE, NV, and CM did the 2D and 3D experiments. JBG, SE, SL, GA, and CM performed the in vivo experiments. JBG, SE, MAG, AG, ASW, CM, and GA did the collection and assembly of the data. Resources and funding were obtained by CRL, NN, and JBG. The original writing draft was prepared by JB and SE, and corrected by DA, GA, NM, and CRL.
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Supplementary Information
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432_2021_3593_MOESM1_ESM.pdf
Supplementary file1 (PDF 153 KB) Supplementary Data 1. Statistical analysis of cell proliferation measured at 120h in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. *P < 0.05; **P < 0.01; ***P < 0.001.
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Supplementary file2 (PDF 178 KB) Supplementary Data 2. Microscopic observation (10×) of the spheroid growth of SQ20B cells and SQ20B/CSCs. (A) SQ20B 3D-spheroid growth observed as red fluorescence by phase contrast video microscopy at 2, 5, 7, 10 and 14 days. (B) SQ20B 3D-spheroid growth observed as red fluorescence by video microscopy at 2, 5, 7, 10 and 14 days.(C)SQ20B/CSC 3D-spheroid growth observed as red fluorescence and phase contrast video microscopy at 2, 5, 7, 10 and 14 days. (D)SQ20B/CSC 3D-spheroid growth observed in 10× red fluorescence video microscopy at 2, 5, 7, 10 and 14 days.
432_2021_3593_MOESM3_ESM.pdf
Supplementary file3 (PDF 154 KB) Supplementary Data 3. Statistical analysis of 3D-spheroid growth in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments.Cell 3D-spheroid growth was measured as the mean fluorescence intensity (RCU) at 10 days. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM4_ESM.pdf
Supplementary file4 (PDF 107 KB) Supplementary Data 4. Statistical analysis of caspase 3/7 activation in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. Cellular apoptosis was measured at 120h and is expressed as a percentage of activated caspase 3/7. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM5_ESM.pdf
Supplementary file5 (PDF 151 KB) Supplementary Data 5. Statistical analysis of migration assays of SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. Cell migration was measured by relative wound density at 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM6_ESM.pdf
Supplementary file6 (PDF 151 KB) Supplementary Data 6. Statistical analysis of invasion assays of SQ20B cells (A) and SQ20B/CSC (B) exposed to the various treatments. Cell invasion was measured by relative wound density at 48 h. *P < 0.05; **P < 0.01; ***P < 0.001.
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Guy, JB., Espenel, S., Louati, S. et al. Combining radiation to EGFR and Bcl-2 blockade: a new approach to target cancer stem cells in head and neck squamous cell carcinoma. J Cancer Res Clin Oncol 147, 1905–1916 (2021). https://doi.org/10.1007/s00432-021-03593-8
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DOI: https://doi.org/10.1007/s00432-021-03593-8
Keywords
- HNSCC
- Cancer stem cells
- Epidermal growth factor receptor
- Bcl-2
- Radiation therapy
- Cetuximab
- ABT-199