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Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis

  • Authors:
    • Taisei Ichihara
    • Yoshihiro Hayashi
    • Takuya Sato
    • Mitsuko Iguchi
    • Makoto Toi
    • Hironaga Satake
    • Ichiro Murakami
  • View Affiliations / Copyright

    Affiliations: Department of Medical Oncology, Kochi Medical School Hospital, Nankoku‑shi, Kochi 783‑8505, Japan, Science Research Center, Kochi University, Nankoku‑shi, Kochi 783‑8505, Japan, Diagnostic Pathology Department, Kochi Medical School Hospital, Nankoku‑shi, Kochi 783‑8505, Japan, Department of Pathology, Kochi Medical School, Nankoku‑shi, Kochi 783‑8505, Japan
    Copyright: © Ichihara et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].
  • Article Number: 35
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    Published online on: December 19, 2025
       https://doi.org/10.3892/or.2025.9040
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Abstract

Krebs von den Lungen‑6 (KL‑6), a high‑molecular‑weight glycoprotein, is frequently elevated in patients with cancer; however, its precise role in the clinical progression of breast cancer (BC) remains unclear. Tumor hypoxia has been recognized as a critical driver of cancer progression. The present study aimed to investigate the effects of hypoxia on KL‑6 expression and its contribution to the invasive behavior of BC cells. Immunohistochemical analysis of KL‑6 and hypoxia‑inducible factor‑1α (HIF‑1α) was performed in 30 clinical BC tissue samples, and their expression levels were correlated with patient outcomes. BC cell lines (MCF‑7 and MDA‑MB‑231) were used for in vitro analyses, including immunofluorescence, western blotting, wound healing assays, and three‑dimensional spheroid cultures under normoxic and hypoxic conditions, as well as chemically induced hypoxia using cobalt chloride (CoCl2). The subcellular localization of KL‑6 was further examined through immunoelectron microscopy. In clinical specimens, high KL‑6 expression was significantly associated with increased recurrence or metastasis, as was elevated HIF‑1α expression. Although MCF‑7 cells exhibited higher basal KL‑6 expression, marked upregulation was observed in MDA‑MB‑231 cells under hypoxic spheroid conditions, where KL‑6 co‑localized with HIF‑1α, particularly within invasive cellular protrusions. Functional inhibition of KL‑6 suppressed the migration of MCF‑7 cells. Treatment with CoCl2 significantly induced KL‑6 and HIF‑1α expression in MCF‑7 cells. Ultrastructural analysis confirmed the localization of KL‑6 on cell membranes and within protrusive structures. Collectively, these findings demonstrated that hypoxia enhances KL‑6 expression in BC cells, partly mediated by HIF‑1α, and that KL‑6 contributes to tumor cell invasion.
View Figures

Figure 1

IHC expression of KL-6 and HIF-1α in
breast cancer tissues. (A-F) Representative IHC images showing KL-6
(A-C) and HIF-1α (D-F) expression in tissue samples from patients
5, 12 and 14. Scale bar, 50 µm. IHC, immunohistochemical; KL-6,
Krebs von de Lungen; HIF-1α, hypoxia-inducible factor-1α.

Figure 2

KL-6 expression in breast cancer cell
lines. (A) Immunofluorescence analysis shows that most MCF-7 cells
express KL-6 (a). Only a few MDA-MB-231 cells express KL-6 (b).
KL-6 localization is observed as dot-like signals on the plasma
membrane (c). KL-6 is localized at the membrane edges and cell
protrusions (d). Scale bar, 200 µm (a and b); 50 µm (c and d). (B)
Western blot analysis demonstrates lower KL-6 protein expression in
MDA-MB-231 cells compared with MCF-7 cells. (C) Quantification of
KL-6 positive cells in each cell line. KL-6, Krebs von de
Lungen.

Figure 3

Effect of anti-KL-6 treatment on
MCF-7 cell migration (wound healing assay). (A) Representative
images of wound healing assays showing the impact of anti-KL-6
antibody treatment on MCF-7 cell migration. Scale bar, 500 µm. (B)
Quantitative comparison of relative wound closure areas between
control and anti-KL-6-treated groups. KL-6, Krebs von de
Lungen.

Figure 4

KL-6 expression in breast cancer
spheroids. (A) Immunohistochemical analysis of KL-6 expression in
three-dimensional spheroids. In MCF-7 spheroids, KL-6 is expressed
at the spheroid periphery (a and c), with strong expression
observed peripherally and internally in some spheroids (b and d).
KL-6-positive regions are indicated by arrows. Scale bar, 50 µm.
(B) Western blot analysis revealing higher KL-6 expression in
MDA-MB-231 spheroids compared with MCF-7 spheroids. KL-6, Krebs von
de Lungen.

Figure 5

Time-dependent KL-6 expression and
hypoxia in MDA-MB-231 spheroids. (A and B) IHC analysis of KL-6
expression in MDA-MB-231 spheroids at 2 days (A) and 6 days (B)
after seeding, showing a mosaic-like expression pattern at both
time points. (C and D) IHC staining for hypoxia-inducible factor-1α
(C), a hypoxia marker, demonstrates nuclear localization at day 6
(D). Fluorescent hypoxia probe imaging confirms pronounced hypoxia
within the spheroid core at day 6. Scale bar, 50 µm. KL-6, Krebs
von de Lungen; IHC, immunohistochemical.

Figure 6

KL-6 expression in MDA-MB-231 cells
and spheroids embedded in the matrix. (A and B) Immunofluorescent
staining shows nuclear hypoxia-inducible factor-1α localization in
most MDA-MB-231 cells within the matrix. Nuclei were counterstained
with 4′,6-diamidino-2-phenylindole. (C and D) KL-6
immunohistochemical staining of MDA-MB-231 cells and spheroids
embedded in the matrix. KL-6 expression is observed on cell
protrusions extending from the spheroids into the surrounding
matrix. Scale bar, 50 µm. KL-6, Krebs von de Lungen.

Figure 7

Hypoxia-induced upregulation of KL-6
expression. (A) Changes in hypoxia markers and KL-6 expression in
CoCl2-free (a, c and e) and CoCl2-added (b, d
and f) groups; after 12 h of CoCl2 addition, the
fluorescence intensities of LOX-1 (a and b) and HIF-1α (c and d)
increased, indicating hypoxia. Similarly, KL-6-positive cells were
markedly increased in treated samples (e and f). Scale bar, 50 µm.
(B) Quantification of KL-6-positive cells shows a significant
increase in the CoCl2-treated group compared with the
untreated control group. KL-6, Krebs von de Lungen;
CoCl2, cobalt chloride.

Figure 8

Immunoelectron microscopy of KL-6 in
MDA-MB-231 cells and spheroids. (A-D) KL-6 immunogold labeling of
cells (A and C) and spheroid surfaces (B and D). Positive gold
particles were observed on the cell membrane, in cell protrusions,
and around the cells (C). Positive staining of spheroid and cell
surfaces with cell protrusions (D). Scale bar, 20 µm (A and B) and
10 µm (C and D). KL-6, Krebs von de Lungen.
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Copy and paste a formatted citation
Spandidos Publications style
Ichihara T, Hayashi Y, Sato T, Iguchi M, Toi M, Satake H and Murakami I: Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis. Oncol Rep 55: 35, 2026.
APA
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., & Murakami, I. (2026). Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis. Oncology Reports, 55, 35. https://doi.org/10.3892/or.2025.9040
MLA
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., Murakami, I."Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis". Oncology Reports 55.2 (2026): 35.
Chicago
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., Murakami, I."Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis". Oncology Reports 55, no. 2 (2026): 35. https://doi.org/10.3892/or.2025.9040
Copy and paste a formatted citation
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Spandidos Publications style
Ichihara T, Hayashi Y, Sato T, Iguchi M, Toi M, Satake H and Murakami I: Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis. Oncol Rep 55: 35, 2026.
APA
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., & Murakami, I. (2026). Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis. Oncology Reports, 55, 35. https://doi.org/10.3892/or.2025.9040
MLA
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., Murakami, I."Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis". Oncology Reports 55.2 (2026): 35.
Chicago
Ichihara, T., Hayashi, Y., Sato, T., Iguchi, M., Toi, M., Satake, H., Murakami, I."Hypoxia‑mediated Krebs von den Lungen‑6 expression in breast cancer: Implications for tumor invasion and metastasis". Oncology Reports 55, no. 2 (2026): 35. https://doi.org/10.3892/or.2025.9040
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