Abstract

Senescent fibroblasts have been found to promote the growth of both premalignant and malignant cells in vitro and in vivo, but the mechanism has not been well studied yet. The aim of this dissertation is to identify the factor(s) and mechanism(s) responsible for the promotion effect of senescent fibroblasts on tumor formation.

Fibroblasts with bleomycin-induced senescence promote tumorigenesis by stimulating initial tumor growth via matrix metalloproteinases. Human fibroblast HCA2 cells with senescence induced by bleomycin were used in the study. Bleomycin-induced senescent HCA2 cells overexpress proteases and cytokines known to be upregulated by replicatively senescent fibroblasts. Bleomycin-induced senescent HCA2 cells stimulated the growth of cocultured tumor cells. They also promoted tumor growth, especially the proliferation of early-stage tumors, when subcutaneously cotransplanted with human breast cancer MDA231 cells in immunodeficient mice. Significant extracellular fluid accumulation occurred in 5-day tumors, especially in those cotransplanted with senescent HCA2 cells. The pro-tumorigenesis effect of bleomycin-induced senescent HCA2 cells was related to their action on extracellular fluid accumulation in early-stage tumors. Inhibition of matrix metalloproteinase function by a synthetic matrix metalloproteinase inhibitor did not affect the pro-proliferation effect of bleomycin-induced senescent HCA2 cells in vitro, yet did reduce tumor size, proliferation and extracellular fluid accumulation in early-stage tumors in vivo, suggesting that matrix metalloproteinases may mediate the pro-tumorigenesis effect of senescent fibroblasts.

Fibroblasts with bleomycin-induced senescence promote tumor proliferation, rather than angiogenesis and invasion, via hepatocyte growth factor. Hepatocyte growth factor expression and secretion increased in bleomycin-induced senescent fibroblast lines. Exogenous hepatocyte growth factor dose-dependently stimulated the proliferation of both human breast cancer MDA231 and human prostate cancer PC-3 cells in 3-dimensional collagen gel culture; inhibition of hepatocyte growth factor by a neutralizing antibody compromised the promotion effect of bleomycin-induced senescent HCA2 cells on the growth of cocultured tumor cells in vitro, suggesting that hepatocyte growth factor is involved in the pro-proliferation effect of senescent fibroblasts. Bleomycin-induced senescent HCA2 cells stimulated tubule formation by cocultured endothelial cells. However, tubule networks were not able to be maintained by senescent fibroblasts. Intratumoral microvessel density did not show a significant difference in tumors subcutaneously transplanted with MDA231 cells alone or cotransplanted with either nonsenescent or senescent HCA2 cells. In addition, bleomycin-induced senescent HCA2 cells showed less promotion effect on MDA231 cell invasion through Matrigel than nonsenescent HCA2 cells. These findings suggest that the effect of senescent fibroblasts on tumor angiogenesis and invasion may not contribute much to their pro-tumorigenesis actions.

The promotion effect of fibroblasts on tumor progression is independent of senescence in a subrenal capsule transplantation model. The kidney has unique fluid dynamics and a rich vasculature compared with other organs. When MDA231 cells were transplanted alone or with either nonsenescent or bleomycin-induced senescent HCA2 cells beneath the kidney capsule of immunodeficient mice, tumors showed rapid progression with occurrence of kidney invasion by day 9 and distant metastasis by day 25. Tumors without HCA2 cells demonstrated long-term, dramatic extracellular fluid accumulation and a poor vasculature. Both nonsenescent and senescent HCA2 cells promoted tumor growth and invasion, and their promotion effects did not show significant differences. The present study indicates that the pro-tumorigenesis effect of senescent fibroblasts is site-specific in experimental models. The promotion effect of fibroblasts on tumor progression is likely to be independent of their senescent state in the subrenal capsule transplantation model.