Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

SIMPLE SUMMARY: Fibrosis, which is often caused by chronic diseases and environmental substances, is closely associated with cancer. Thus, the development of a robust method allowing for deep studies of the linkage between fibrosis and cancer is essential. Here, we tested whether our novel three-dimensional (3D) co-culture of breast epithelia and fibroblasts would be a suitable model for that purpose. We compared the phenotypic effects of L-NAME, an inhibitor of nitric oxide (NO) production, on 3D mono- and co-cultures. We previously reported that prolonged NO depletion with L-NAME caused fibrosis and tumorigenesis in mouse mammary glands. Such in vivo effects of L-NAME were well recapitulated in 3D co-cultures, but not in 3D mono-cultures of epithelia and fibroblasts. These results support not only the essential roles of the presence of the stroma in cancer development, but also the utility of this co-culture in studying the causal relationship between fibrosis and cancer. ABSTRACT: Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.