| Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially the stromal cells, in the progression of cancer. However, the role of normal epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. Here, I used in vitro and in vivo co-cultivation approaches, whereby I mixed GFP-tagged MCF-10A cells (G2B-10A), as a model of benign mammary epithelial cells (MECs), and RFP-tagged MDA-MB-231-T1AS cells (R2-T1AS), as a model of breast cancer cells, to address whether benign MECs alter the transformed phenotype of human breast cancer cells. My initial approach, matrigel co-cultures, suggested that the benign and the malignant cells mutually influence their morphogenesis on matrigel. Further in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, I used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells +/- PFA-fixation, revealed that G2B-10A cells promoted a ∼3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, I found that G2B-10A cells +/- PFA induced R2-T1A cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells. Taken together, these data shift our understanding of adjacent normal cells in the cancer process, from passive, noncontributory cells to an active and tumor-promoting cell population that may have significant effects early, when the benign cells outnumber malignant cells. |
