Spatiotemporal characteristics of fibroblasts-dependent cancer cell invasion
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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.
KeywordsSquamous cell carcinoma Fucci Cell cycle Invasion Fibroblasts
Dulbecco’s modified Eagle’s medium
Fluorescent ubiquitination-based cell cycle indicator
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.
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.
Comprehensive informed consent was obtained from all individual participants included in the study.
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