Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in ductal carcinoma in situ of the breast.

Abstract

Ductal carcinoma in situ (DCIS) is a precursor form of breast cancer. 13%-50% of these lesions will progress to invasive breast cancer, but the individual progression risk cannot be estimated. Therefore, all patients receive the same therapy, resulting in potential overtreatment of a large proportion of patients. The role of the tumor microenvironment (TME) and especially of fibroblasts appears to be critical in DCIS development and a better understanding of their role may aid individualized treatment. Primary fibroblasts isolated from benign or malignant punch biopsies of the breast and MCF10DCIS.com cells were seeded in a 3D cell culture system. The fibroblasts were cultured in a type I collagen layer beneath a Matrigel layer with MCF10DCIS.com cells. Dye-quenched (DQ) fluorescent collagen I and IV were used in collagen and Matrigel layer respectively to demonstrate proteolysis. Confocal microscopy was performed on day 2, 7, and 14 to reveal morphological changes, which could indicate the transition to an invasive phenotype. MCF10DCIS.com cells form smooth, round spheroids in co-culture with non-cancer associated fibroblasts (NAFs). Spheroids in co-culture with tumor-associated fibroblasts (TAFs) appear irregularly shaped and with an uneven surface; similar to spheroids formed from invasive cells. Therefore, these morphological changes represent the progression of an in situ to an invasive phenotype. In addition, TAFs show a higher proteolytic activity compared to NAFs. The distance between DCIS cells and fibroblasts decreases over time. The TAFs seem to play an important role in the progression of DCIS to invasive breast cancer. The better characterization of the TME could lead to the identification of DCIS lesions with high or low risk of progression. This could enable personalized oncological therapy, prevention of overtreatment and individualized hormone replacement therapy after DCIS.