Journal of Cancer Research and Clinical Oncology

, Volume 145, Issue 2, pp 373–381 | Cite as

Spatiotemporal characteristics of fibroblasts-dependent cancer cell invasion

  • Tomoyuki Miyashita
  • Tomokazu Omori
  • Hiroshi Nakamura
  • Masato Sugano
  • Shinya Neri
  • Satoshi Fujii
  • Hiroko Hashimoto
  • Masahiro Tsuboi
  • Atsushi Ochiai
  • Genichiro IshiiEmail author
Original Article – Cancer Research



Cancer cells can invade the surrounding stroma with the aid of fibroblasts (fibroblasts-dependent invasion). The aim of this study was to explore the spatiotemporal characteristics of fibroblast-dependent invasion of cancer cells.


We performed an in vitro three-dimensional collagen invasion assay using Fluorescent Ubiquitination-based Cell Cycle indicator (Fucci)-labeled A431 carcinoma cells co-cultured with fibroblasts. We used time-lapse imaging to analyze the total cell number, frequencies of small cancer cell nests and S/G2/M phase of A431 cells in the invasion area. We compared the frequencies of small cancer cell nests and geminin (+) cancer cells within fibroblast-rich areas and fibroblast-poor areas in surgically resected human invasive squamous cell carcinoma tissue.


The total invasion number of A431 cells was significantly higher when cultured with fibroblasts than without. The formation of small cancer cell nests was observed within the invasion area only in the presence of fibroblasts. The frequency of S/G2/M phase cells was significantly higher in A431 cells when cultured with fibroblasts than without. Immunohistochemical analysis of surgically resected human invasive squamous cell carcinoma tissue revealed that the frequencies of small cancer cell nests and geminin-positive cancer cells were significantly higher in fibroblast-rich areas compared to those in fibroblast-poor areas within the same tumor region.


Our current study clearly showed that fibroblast-dependent cancer cell invasion was characterized by the progression in cell cycle and formation of small cancer cell nests.


Squamous cell carcinoma Fucci Cell cycle Invasion Fibroblasts 



Cancer-associated fibroblasts


Dulbecco’s modified Eagle’s medium


Extracellular matrix


Fluorescent ubiquitination-based cell cycle indicator


Horseradish peroxidase


Matrix metalloproteinase



This work was supported in part by the National Cancer Center Research and Development Fund (23-A-12), and JSPS KAKENHI (24659185 and 16H05311). Tomoyuki Miyashita and Genichiro Ishii designed the study; Tomoyuki Miyashita performed in vitro experiments and analyzed the data; Tomokazu Omori, Hiroshi Nakamura, Masahiro Tsuboi, Masato Sugano, Genichiro Ishii and Tomoyuki Miyashita analyzed clinical and pathological data; Shinya Neri and Hiroko Hashimoto, Satoshi Fujii and Atsushi Ochiai provided intellectual advice; Tomoyuki Miyashita, and Genichiro Ishii wrote the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Comprehensive informed consent was obtained from all individual participants included in the study.

Supplementary material

432_2018_2798_MOESM1_ESM.pptx (1.6 mb)
Supp Fig. 1: Schema of the 3-dimensional collagen invasion assay. (a) Schema of the experiment of A431 cells monoculture. A431-Fucci cells were seeded on collagen-coated dish and scratched. After that, they were embedded in collagen type I matrix and observed every 3 h. (b) Schema of the experiment of A431 cells co-cultured with fibroblasts. Same number of A431-Fucci cells and fibroblasts were seeded. Similar protocol was followed as with the A431 cells in monoculture. Blue arrow indicates the direction of invasion. Supp Fig. 2: Representative images of A431-Fucci cells. (a) Representative image of cell cycle progression in A431 cells in monoculture at 27, 39, and 45 h. White arrow indicates single invaded A431 cell with progression of cell cycle. Left panel: A431 cell in G1 phase, middle panel: A431 cell in S/G2/M phase, right panel: two daughter cells in G1 phase. (a) Representative images of cell cycle progression in A431 cells co-cultured with fibroblasts at 7.5, 15, and 27 h. White arrow indicates single invaded A431 cell with progression of cell cycle. Left panel: A431 cell in G1 phase, middle panel: A431 cell in S/G2/M phase, right panel: two daughter cells in G1 phase. Supp Fig. 3: Frequency of S/G2/M phase in A431 cells in 2D culture. The average ratio of S/G2/M phase in cancer cells at 72 h. Values are given as mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s t-test. ***p< 0.01 (PPTX 1610 KB)
432_2018_2798_MOESM2_ESM.xlsx (10 kb)
Supplementary material 2 (XLSX 10 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Tomoyuki Miyashita
    • 1
    • 2
  • Tomokazu Omori
    • 2
    • 3
  • Hiroshi Nakamura
    • 2
    • 4
  • Masato Sugano
    • 2
    • 4
  • Shinya Neri
    • 2
  • Satoshi Fujii
    • 2
  • Hiroko Hashimoto
    • 2
  • Masahiro Tsuboi
    • 3
  • Atsushi Ochiai
    • 1
    • 5
  • Genichiro Ishii
    • 1
    • 2
    Email author
  1. 1.Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier SciencesThe University of TokyoKashiwaJapan
  2. 2.Division of Pathology, Exploratory Oncology Research and Clinical Trial CenterNational Cancer CenterKashiwaJapan
  3. 3.Division of Thoracic SurgeryNational Cancer Center Hospital EastKashiwaJapan
  4. 4.Department of Pathology and Clinical LaboratoriesNational Cancer Center Hospital EastKashiwaJapan
  5. 5.Exploratory Oncology Research and Clinical Trial CenterNational Cancer CenterKashiwaJapan

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