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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster H19 - Investigating Neurite Outgrowth Influenced by Breast Cancer Progression Using In Vitro Models of Collagen I Hydrogel

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  In the United States, 1 in 8 women will be diagnosed with breast cancer sometime in their life [1]. One of the causes of poor prognosis in breast cancer patients is tumor innervation, where adjacent nerve axons infiltrate tumors, resulting in increased tumor aggression [2]. One factor influencing tumor innervation is the neurotrophin neural growth factor (NGF), secreted by breast cancer cells that stimulate the growth of parasympathetic, sympathetic and sensory nerves [3]. 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.

Materials and

Methods:  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. 8mm wells were made with polydimethylsiloxane (PDMS) and placed on a glass slide which was then placed on a petri dish. 1% polyethyleneimine (PEI) was then added to the wells for 10 minutes with each well receiving 50 ul followed by 0.01% glutaraldehyde was added to the wells for 30 minutes. Finally the wells were washed twice with deionized water. Collagen pregel solution was diluted from 10 mg/ml to 5mg/ml using 10% (v/v) M199, 10% (v/v) media, neutralized to a pH of 7.4 using 1M NaOH, and the remainder with 1x PBS. The gels were incubated for 30 minutes to cure. PC-12 cells were seeded on top of all gels for four hours before adding media. Culture conditions included: PC-12 monoculture and media supplementation from MCF cultures. Samples supplement of NGF was added on day 0 then all cultures changed media after 72 hours, where NGF supplementation stopped, with media supplementation cultures receiving the media from the co cultures. Samples were fixed on day 8 of the cultures. Immunofluorescence staining was used with neurofilament and beta III tubulin being used to identify perineural invasion. DAPI was used as a nuclear counterstain.

Results, Conclusions, and Discussions: 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. Here we demonstrated that supplementation of MCF cultures influence the PC-12s cells in a similar way to NGF. 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. This knowledge allows for more insight on the relationship between neurite outgrowth influenced by breast cancer progression.

Acknowledgements (Optional):  This work was supported by the National Science Foundation through award number EWFD 2243953 and the National Institutes of Health through the award number R37CA279722.

References: 1. Breast cancer facts & stats 2024 - incidence, age, survival, & more [Internet]. 2024 [cited 2024 Jul 19]. 2. Gregory E, Dugan R, David G, Song YH. The biology and engineered modeling strategies of cancer-nerve crosstalk. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 2020 Dec;1874(2):188406. 3. Pundavela J, Roselli S, Faulkner S, Attia J, Scott RJ, Thorne RF, et al. Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer. Molecular Oncology. 2015 May 14;9(8):1626–35.