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Bystander effects induced by the interaction between urothelial cancer cells and irradiated adipose tissue-derived stromal cells in urothelial carcinoma

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Abstract

Cell–cell interactions between cancer cells and neighboring adipose tissue-derived stromal cells (ATSCs) are known to regulate the aggressiveness of cancer cells. In addition, the radiation-induced bystander effect is an important modulator of cancer cell kinetics. Radiation therapy is often given for urinary cancer, but the biological effects of the irradiated cancer stroma, including adipose tissue, on urothelial carcinoma (UC) remain unclear. We investigated the bystander effect of irradiated ATSCs on UC using a collagen gel culture method to replicate irradiated ATSC–cancer cell interactions after a single 12-Gy dose of irradiation. Proliferative activity, invasive capacity, protein expression and nuclear translocation of p53 binding protein-1 (53BP1) were analyzed. Irradiated ATSCs significantly inhibited the growth and promoted the apoptosis of UC cells in comparison to non-irradiated controls. The invasiveness of UC cells was increased by irradiated ATSCs, but not irradiated fibroblasts. Nuclear translocation of 53BP1 protein due to the bystander effect was confirmed in the irradiated group. Irradiated ATSCs regulated the expressions of the insulin receptor, insulin-like growth factor-1 and extracellular signal-regulated kinase-1/2 in UC. In conclusion, the bystander effect of irradiated ATSCs is a critical regulator of UC, and the actions differed depending on the type of mesenchymal cell involved. Our alternative culture model is a promising tool for further investigations into radiation therapy for many types of cancer.

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Acknowledgements

We thank M. Itoh, T. Sakumoto, S. Morito, M. Nishida, F. Mutoh and S. Nakahara for their excellent technical assistance.

Funding

This work was funded by the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research (Grant no. 18K09138).

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Authors and Affiliations

  1. Department of Urology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga, 849-8501, Japan

    Maki Kawasaki, Kei Nagase, Kazuma Udo, Shohei Tobu & Mitsuru Noguchi

  2. Division of Pathology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga, 849-8501, Japan

    Shigehisa Aoki & Mihoko Rikitake-Yamamoto

  3. Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjo, Saga, 840-8502, Japan

    Masaya Kubota & Takayuki Narita

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  1. Maki Kawasaki
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Correspondence to Maki Kawasaki.

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There are no conflicts of interest. This research was supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research (No. 18K09138).

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13577_2022_668_MOESM1_ESM.eps

Supplementary file1 Figure S1. Extracellular matrix structure of collagen gels cultured for 7 days or 14 days. (a) Transmission electron microscopy image of a non-irradiated collagen gel cultured for 7 days. (b) Electron micrograph of an irradiated collagen gel cultured for 7 days. Non-irradiated collagen fibers exhibited helical structures, whereas the irradiated collagen gel consisted mainly of finely fragmented fibers. (c) The elastic moduli of non-irradiated and irradiated (12 Gy) collagen gels with or without NIH3T3 cells on day 7 and day 14. When compared with gels not containing NIH3T3 cells, gels embedded with NIH3T3 cells had a significantly lower elastic modulus on day 7 and a significantly higher elastic modulus on day 14 (both non-irradiated and irradiated conditions). (EPS 728 KB)

13577_2022_668_MOESM2_ESM.eps

Supplementary file2 Figure S2. Irradiation and mesenchymal cells regulate the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and the expressions of the insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) in RT4 cells. Total IR expression was significantly greater in RT4 cells co-cultured with non-irradiated ATSCs than in RT4 cells monocultured on a non-irradiated gel. Irradiated ATSCs decreased the phosphorylated/total IR ratio in RT4 cells compared to the monoculture group. Total IGF-1R expression was significantly greater in RT4 cells co-cultured with non-irradiated ATSCs than in RT4 cells monocultured on a non-irradiated gel or RT4 cells co-cultured with irradiated ATSCs. Irradiation of ATSCs significantly decreased the phosphorylated/total IR ratio in RT4 cells. Co-culture with ASTCs increased total JNK expression in RT4 cells under both non-irradiated and irradiated conditions, and irradiated ATSCs decreased total JNK expression in RT4 cells compared to non-irradiated ATSCs. The phosphorylated/total JNK ratio was increased by irradiation in the presence and absence of ATSCs and by co-culture with ATSCs under both non-irradiated and irradiated conditions. Total Akt expression in RT4 cells was increased by co-culture with ATSCs under both non-irradiated and irradiated conditions. In the absence of ATSCs, irradiation decreased the phosphorylated/total Akt ratio in RT4 cells. Both non-irradiated and irradiated ATSCs significantly increased the phosphorylated/total Akt ratio in RT4 cells compared to the respective monoculture group. Non-irradiated and irradiated ATSCs significantly upregulated total mTOR expression in RT4 cells compared to the respective monoculture group. Irradiation significantly decreased total mTOR expression in RT4 cells both in the presence and absence of ATSCs. There were no significant change in the phosphorylated/total mTOR ratio in RT4 cells under any experimental conditions. Relative expression is depicted as the ratio of the target protein expression to α/β-tubulin expression. Data represent the mean ± SD of 5 determinations. *P < 0.05, **P < 0.01, ***P < 0.001. (EPS 895 KB)

13577_2022_668_MOESM3_ESM.eps

Supplementary file3 Figure S3. Irradiation and mesenchymal cells regulate the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and the expressions of the insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) in T24 cells. Total IR and IGF-1R expressions in T24 cells were significantly increased after co-culture with ATSCs under both non-irradiated and irradiated conditions. The phosphorylated/total IR ratio and phosphorylated/total IGF-1R ratio were significantly greater in T24 cells co-cultured with non-irradiated ATSCs than in T24 cells monocultured on a non-irradiated gel or T24 cells co-cultured with irradiated ATSCs. Total JNK expression in T24 cells was significantly increased after co-culture with ATSCs under both non-irradiated and irradiated conditions. The phosphorylated/total JNK ratio was significantly greater in T24 cells monocultured on a non-irradiated collagen gel than in T24 cells monocultured on an irradiated gel or T24 cells co-cultured with non-irradiated ATSCs. Total Akt expression in T24 cells was significantly reduced by ATSCs under both non-irradiated and irradiated conditions, and by irradiation both in the presence and absence of ATSC co-culture. Non-irradiated and irradiated ATSCs significantly increased the phosphorylated/total Akt ratio in T24 cells compared to the respective monoculture group. Non-irradiated and irradiated ATSCs significantly upregulated total mTOR expression in T24 cells in comparison to the respective monoculture group. The phosphorylated/total mTOR ratio in T24 cells was not affected by irradiation or ATSCs. Relative expression is depicted as the ratio of target protein expression to α/β-tubulin expression. Data represent the mean ± SD of 5 determinations. *P < 0.05, **P < 0.01, ***P < 0.001. (EPS 892 KB)

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Kawasaki, M., Nagase, K., Aoki, S. et al. Bystander effects induced by the interaction between urothelial cancer cells and irradiated adipose tissue-derived stromal cells in urothelial carcinoma. Human Cell 35, 613–627 (2022). https://doi.org/10.1007/s13577-022-00668-7

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