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Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma

  • Authors:
    • Yuto Kitano
    • Tomoharu Miyashita
    • Takeo Shimasaki
    • Isamu Makino
    • Yoshio Endo
    • Yasuhiko Yamamoto
    • Noriyuki Inaki
    • Shintaro Yagi
  • View Affiliations / Copyright

    Affiliations: Department of Hepato‑Biliary‑Pancreatic Surgery and Transplantation/Pediatric Surgery, Kanazawa University, Kanazawa, Ishikawa 920‑8641, Japan, Medical Research Institute, Kanazawa Medical University, Uchinada, Ishikawa 920‑0293, Japan, Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920‑1192, Japan, Department of Biochemistry and Molecular Vascular Biology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Ishikawa 920‑8641, Japan, Department of Gastrointestinal Surgery/Breast Surgery, Kanazawa University, Kanazawa, Ishikawa 920‑8641, Japan
    Copyright: © Kitano et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 26
    |
    Published online on: December 3, 2025
       https://doi.org/10.3892/or.2025.9031
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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrous stroma, within which a subset of fibroblasts function as cancer‑associated fibroblasts (CAFs) and contribute to tumor progression. Some of these fibroblasts undergo senescence and promote malignancy through the senescence‑associated secretory phenotype (SASP). The present study investigated SASP factor expression in senescent fibroblasts within the PDAC microenvironment and evaluated their impact on tumor progression. The expression levels of the senescence marker p16 and the SASP factor interleukin‑6 (IL‑6) were assessed using fluorescence immunostaining in resected specimens from 90 patients with PDAC who underwent pancreaticoduodenectomy. Senescence was induced in primary human pancreatic fibroblasts via X‑ray irradiation in vitro, followed by evaluation of SASP factor expression. These senescent fibroblasts were then co‑cultured with human pancreatic cancer Panc‑1 cells to assess their effects on cancer cell invasion, migration and proliferation. Immunostaining demonstrated the presence of p16‑ and IL‑6‑expressing fibroblasts in the PDAC stroma of patient samples. A positive correlation was observed between p16 and IL‑6 expression levels in fibroblasts. Notably, increased expression levels of IL‑6‑positive fibroblasts were associated with reduced postoperative survival. Multivariate analysis identified high IL‑6 expression and lymph node metastasis as independent prognostic indicators of poor outcome. In co‑culture experiments, senescent fibroblasts enhanced Panc‑1 cell invasion, migration and proliferation. These findings suggested that senescent fibroblasts within the PDAC stroma, with high SASP factor expression, contribute to tumor aggressiveness and are associated with poor prognosis. The present study demonstrated that IL‑6‑expressing senescent fibroblasts are potential prognostic markers and therapeutic targets in PDAC, therefore the targeted elimination of senescent cells may represent a promising therapeutic strategy.
View Figures

Figure 1

Molecular mechanisms of cellular
senescence. DNA damage induces cellular senescence, characterized
by either apoptosis or irreversible cell cycle arrest. Senescent
cells release SASP factors, which promote chronic inflammation and
are involved in carcinogenesis and the development of age-related
diseases. SASP, senescence-associated secretory phenotype.

Figure 2

Evaluation of p16- and IL-6-positive
cells in the stroma of pancreatic ductal adenocarcinoma. (A)
Representative immunofluorescence staining of a pancreatic cancer
resection specimen (αSMA, green; p16, red; DAPI, blue; scale bar,
50 and 25 µm). In the pancreatic cancer stroma, pancreatic
fibroblasts (αSMA-positive) expressing the senescence marker p16
were identified (yellow arrows). (B) Representative
immunofluorescence staining of a pancreatic cancer resection
specimen (p16, red; IL-6, green; DAPI, blue; scale bar, 100 and 25
µm). Fibroblasts positive for p16 and the senescence-associated
secretory phenotype factor IL-6 were observed in the pancreatic
cancer stroma (yellow arrows). (C) A significant positive
correlation between the expression intensity of p16- and
IL-6-positive fibroblasts in the pancreatic cancer stroma
(Pearson's correlation coefficient, r=0.585; P<0.0001).

Figure 3

Relationship between IL-6 expression
intensity in the pancreatic ductal adenocarcinoma stroma and
prognosis after pancreaticoduodenectomy. (A) A significant negative
correlation between the expression intensity of IL-6-positive
fibroblasts in the pancreatic cancer stroma and overall survival
(Pearson's correlation coefficient, r=−0.336; P=0.0012). (B) A
receiver operating characteristic curve based on the expression
intensity of IL-6-positive fibroblasts in the pancreatic cancer
stroma and overall survival (AUC=0.754, sensitivity=0.736,
specificity=0.734). The IL-6 H-score cut-off value was 179;
patients with H-scores <179 were defined as the IL-6 low
expression group, and those with scores ≥179 as the IL-6 high
expression group. (C) Survival curves for patients with pancreatic
cancer with low (solid line) and high (dotted line) -IL-6
expression (log-rank test, P=0.00002). Representative pancreatic
cancer specimen with (D) weak, (E) moderate and (F) strong IL-6
positivity (scale bar, 100 µm). (G) Non-tumorous pancreatic
specimen (scale bar, 100 µm).

Figure 4

Establishment of human pancreatic
fibroblast cell lines and induction of cellular senescence and the
senescence-associated secretory phenotype. (A) Pancreatic
fibroblasts established from resected pancreatic specimens. Cells
showed a spindle-shaped morphology with cytoplasmic protrusions
(AE1/3-negative, αSMA- and vimentin-positive; scale bar, 10 µm).
(B) SA-β-gal staining of IR pancreatic fibroblasts (10 Gy)
increased over time (scale bar, 50 µm). (C) SA-β-gal staining of
pancreatic fibroblasts 9 days increased with gradient irradiation
(3, 6 and 10 Gy; scale bar, 50 µm). (D) Western blot analysis of
p16 protein expression in IR pancreatic fibroblasts (10 Gy X-rays
on days 0, 3, 6 and 9) showed increased p16 protein expression from
day 3 onward compared with the non-IR group. (E) Western blot
analysis showed a gradual increase of IL-6 protein expression from
day 3 onwards to day 9 post-irradiation. (F) Reverse
transcription-quantitative PCR confirmed increased IL-6 mRNA
expression after irradiation over 9 days of culture. Data are
presented as mean ± SD (n=6). (G) Immunofluorescence of pancreatic
fibroblasts. IL-6 expression was increased on day 9 (IL-6, red;
DAPI, blue; scale bar, 50 µm). *P<0.01 and **P<0.001. ns, not
significant; H&E, hemoxylin and eosin; SA-β-gal,
senescence-associated β-galactosidase; aSMA, a smooth muscle actin;
ACTB, β-actin; IR, irradiated.

Figure 5

Co-culture experiments of Panc-1
cells with IR human pancreatic fibroblasts or their culture
supernatants. (A) Representative images of pancreatic fibroblasts
(10 Gy IR or non-IR) that were co-cultured with Panc-1 cells (scale
bar, 100 µm), and (B) the number of cells that invaded through the
gel and membrane was counted (n=6). (C) A confluent monolayer of
Panc-1 cells was scratched with a pipette, and the culture
supernatant of IR or non-IR pancreatic fibroblasts was added (scale
bar, 100 µm). (D) After 12 h, the area of cell migration was
measured (n=6). *P<0.05 and **P<0.01. (E) Panc-1 cells were
cultured with culture supernatants of IR or non-IR pancreatic
fibroblasts, and proliferation was assessed after 24 and 48 h using
the MTT assay (n=8). Data are presented as mean ± SD. *P<0.05,
**P<0.01 and ***P<0.001. aSMA, a smooth muscle actin; Ctrl,
control; IR, irradiated.
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Copy and paste a formatted citation
Spandidos Publications style
Kitano Y, Miyashita T, Shimasaki T, Makino I, Endo Y, Yamamoto Y, Inaki N and Yagi S: Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma. Oncol Rep 55: 26, 2026.
APA
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y. ... Yagi, S. (2026). Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma. Oncology Reports, 55, 26. https://doi.org/10.3892/or.2025.9031
MLA
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y., Inaki, N., Yagi, S."Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma". Oncology Reports 55.2 (2026): 26.
Chicago
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y., Inaki, N., Yagi, S."Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma". Oncology Reports 55, no. 2 (2026): 26. https://doi.org/10.3892/or.2025.9031
Copy and paste a formatted citation
x
Spandidos Publications style
Kitano Y, Miyashita T, Shimasaki T, Makino I, Endo Y, Yamamoto Y, Inaki N and Yagi S: Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma. Oncol Rep 55: 26, 2026.
APA
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y. ... Yagi, S. (2026). Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma. Oncology Reports, 55, 26. https://doi.org/10.3892/or.2025.9031
MLA
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y., Inaki, N., Yagi, S."Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma". Oncology Reports 55.2 (2026): 26.
Chicago
Kitano, Y., Miyashita, T., Shimasaki, T., Makino, I., Endo, Y., Yamamoto, Y., Inaki, N., Yagi, S."Prognostic significance of fibroblast senescence and senescence-associated secretory phenotype factor expression in the tumor microenvironment of pancreatic ductal adenocarcinoma". Oncology Reports 55, no. 2 (2026): 26. https://doi.org/10.3892/or.2025.9031
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