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Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer

  • Authors:
    • Luis Bugia
    • Annette Affolter
    • Johann Kern
    • Emma Sohn
    • Frederic Jungbauer
    • Jens Fleckenstein
    • Anne Lammert
    • Nicole Rotter
    • Claudia Scherl
  • View Affiliations / Copyright

    Affiliations: Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Medical Faculty Mannheim of Heidelberg University, D‑68167 Mannheim, Germany, Department of Radiation Oncology, University Hospital Mannheim, Medical Faculty Mannheim of Heidelberg University, D‑68167 Mannheim, Germany
    Copyright: © Bugia et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 151
    |
    Published online on: September 9, 2025
       https://doi.org/10.3892/or.2025.8984
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Abstract

Fractionated irradiation causes premature senescence of tumor cells. Interactions between senescence, the immune system and survival signaling are poorly understood to date. As MAP kinases are implicated in immune resistance, the present study addressed the detection of senescence‑associated modulation of postradiogenic programmed death‑ligand 1 (PD‑L1) and MAP kinase ERK1/2 expression in an in vitro and ex vivo model for head and neck squamous cell carcinoma (HNSCC). Established HNSCC cell  lines (UM-SCC-11B, UM-SCC-14C and UM-SCC-22B) were employed to study the expression levels of p21, histone  H2AX (γH2AX), PD-L1 and phosphorylated (p)ERK1/2 via immunohistochemistry following application of 4x2 Gy. Using senescence‑associated β‑galactosidase (SA‑ß‑Gal) staining, postradiogenic induction of senescence was additionally assessed. Results were validated in a 3D ex vivo HNSCC model with vital explants. Upon ionizing radiation (IR), senescence‑like subpopulations were observed in all cell lines, showing upregulation of PD‑L1 and pERK1/2 as well as of established senescence markers p21 and γH2AX. SA‑β‑Gal‑positive cells were found in all lines. These results were supported in a 3D tumor model. Fractionated IR can generate a subpopulation of HNSCC cells characterized by senescence‑typical cellular changes and marked expression of PD‑L1 and pERK1/2. Postradiogenic senescence in both 2D and 3D cancer models was possibly related to survival signaling and immune checkpoint regulation, crucial elements in tumor development and progress.
View Figures

Figure 1

Irradiation-induced senescence in
HNSCC cell lines detected by SA-β-Gal staining. (A)
Irradiation-induced senescence in HNSCC cell lines UM-SCC-11B,
UM-SCC-14C and UM-SCC-22B. Left panel: Mock-treated controls;
middle panel: irradiated cells (4×2 Gy); right panel: section
enlargement of irradiated batch. SA-β-Gal staining reveals
senescent cells (green-blue signal). Arrows (right panel) indicate
morphologic cellular features typical for senescence (enlarged and
flattened cells) (scale bar, 100 µm). (B) Quantification of
postradiogenic senescence induction in HNSCC cell lines. The y-axis
displays SA-β-Gal positive cells as a percentage of the total
number of cells (median + SEM). Experiments were performed in
triplicates. For significance calculation, Mann Whitney U test was
applied. *P=0.0286. HNSCC, head and neck squamous cell carcinoma;
SA-β-Gal, senescence-associated β-galactosidase.

Figure 2

Differential expression and
quantification of p21, pERK1/2, PD-L1 and γH2AX in HNSCC cell lines
following fractionated IR. (A) Expression of p21, pERK1/2, PD-L1
and γH2AX in HNSCC cell lines UM-SCC-11B, UM-SCC-14C and UM-SCC-22B
post IR. IHC staining of p21, γH2AX, PD-L1 and pERK1/2 after
fractionated IR (4×2 Gy) compared with mock-treated controls
displayed differential modulations of target proteins
(representative images shown). (B) Quantification of IHC results.
The IRS was obtained by visual examination. Results were quantified
using the IRS modified according to Remmele and Stegner (36). The mean ± SD value was calculated.
Statistical significance (*P<0.05) was assessed using Mann
Whitney U test. p, phosphorylated; PD-L1, programmed death-ligand
1; γH2AX, histone H2AX; HNSCC, head and neck squamous cell
carcinoma; IR, ionizing radiation; IHC, immunohistochemistry; IRS,
immunoreactive score. Scale bar=100 µm.

Figure 3

Expression and quantification of p21,
γH2AX, PD-L1, and ERK1/2 in irradiated 3D ex vivo HNSCC
tissue. (A) IHC staining of p21, γH2AX, PD-L1, pERK1/2 and tERK1/2
in irradiated 3D ex vivo HNSCC tissue cultures. Expression
patterns in an ex vivo human HNSCC tissue culture model.
Simultaneous increase of PD-L1 and p21 after fractionated IR in the
IHC, compared with mock-treated controls (representative images
shown; scale bar, 100 µm). (B) Quantification of IHC results. The
IRS was obtained by visual examination. Results were quantified
using the IRS modified according to Remmele and Stegner (36) as performed for the in vitro
data. The mean ± SD was calculated. Statistical significance
(*P<0.05) was assessed using Mann Whitney U tests. (C) pERK1/2
to tERK1/2 ratio in ex vivo samples following radiation
treatment. Bar graph showing the pERK1/2/ERK1/2 ratio in ex
vivo 1–4. IHC, immunohistochemistry; γH2AX, histone H2AX;
PD-L1, programmed death-ligand 1; p, phosphorylated; HNSCC, head
and neck squamous cell carcinoma; IR, ionizing radiation; IHC,
immunohistochemistry; IRS, immunoreactive score. Scale bar=100
µm.
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Copy and paste a formatted citation
Spandidos Publications style
Bugia L, Affolter A, Kern J, Sohn E, Jungbauer F, Fleckenstein J, Lammert A, Rotter N and Scherl C: Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer. Oncol Rep 54: 151, 2025.
APA
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J. ... Scherl, C. (2025). Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer. Oncology Reports, 54, 151. https://doi.org/10.3892/or.2025.8984
MLA
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J., Lammert, A., Rotter, N., Scherl, C."Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer". Oncology Reports 54.5 (2025): 151.
Chicago
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J., Lammert, A., Rotter, N., Scherl, C."Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer". Oncology Reports 54, no. 5 (2025): 151. https://doi.org/10.3892/or.2025.8984
Copy and paste a formatted citation
x
Spandidos Publications style
Bugia L, Affolter A, Kern J, Sohn E, Jungbauer F, Fleckenstein J, Lammert A, Rotter N and Scherl C: Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer. Oncol Rep 54: 151, 2025.
APA
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J. ... Scherl, C. (2025). Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer. Oncology Reports, 54, 151. https://doi.org/10.3892/or.2025.8984
MLA
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J., Lammert, A., Rotter, N., Scherl, C."Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer". Oncology Reports 54.5 (2025): 151.
Chicago
Bugia, L., Affolter, A., Kern, J., Sohn, E., Jungbauer, F., Fleckenstein, J., Lammert, A., Rotter, N., Scherl, C."Irradiation‑induced cellular senescence is linked to pro‑survival signaling and checkpoint regulation in a 2D and 3D model for head and neck cancer". Oncology Reports 54, no. 5 (2025): 151. https://doi.org/10.3892/or.2025.8984
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