Investigating Neurite Outgrowth Influenced by Breast Cancer Progression Using In Vitro Models of Collagen I Hydrogels

Through the use of vitro modeling of tumor progression, using collagen I hydrogels and progressing cell lines, a demonstration of the interactions between breast cancer cells and nerve cells can be investigated. With these models, our goal is to compare the measurement of outgrowth of nerve cells through the use of supplemented NGF and co cultures of breast cancer cells. Human MCF-CA1a.cl1, -AneoT, and -10A, adipose-derived stem cells (ASCs), and rat PC-12 cells, were all passaged from 2D cultures and cultured in a collagen I hydrogen at a cell density of 250,000 cells/ml of media. Collagen hydrogels were then made from a warm mammalian cell procedure. PC-12 cells were seeded on top for four hours after gels were made then media was added. NGF for NGF supplemented cultures was added on day 0 and after 72 hours was removed through a media change. Gels were fixed after 8 days of culture. Immunofluorescence staining was used with neurofilament and beta III tubulin being used to identify perineural invasion. DAPI was used as a nuclear counterstain. Immunofluorescence imaging showed that there was zero outgrowth when no NGF was added. However, outgrowth did appear when NGF was added as well as in all MCF media cultures. Nonetheless, there was no significant difference in the percentage of PC-12s with outgrowth per field of view. The AneoT media had the highest average percentage of PC-12s with outgrowth (51.5%), as well as the highest average length of outgrowth (37.04 µm). Measuring the neurite length, all PC-12 cultures treated with MCF media only were significantly higher than no NGF treatment. By comparing the length of outgrowth and the percentage of outgrowth, we found that AneoT cells had a significant influence on the length of outgrowth.