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Effect Of Intracellular Bacteria On Breast Cells
Spencer G. Phillips, MS Biomedical Engineering, Adam Katz, MD, Nicole Levi, PhD.
Wake Forest University, Winston Salem, NC, USA.
PURPOSE – Recent research has shown that bacteria reside within cells in the tumor microenvironment, making them more proliferative than their non-infected counterparts. There is a need to understand how intracellular bacteria associated with breast cancer can be mitigated, since downstream effects might impact breast reconstruction. Here, we quantified differences in the number of intracellular colony forming units (CFUs), proliferation, cytoskeleton, and uptake of therapeutic nanoparticles (NPs) in breast cells with and without intracellular bacteria.
METHODS – Photothermal NPs were custom-designed by combining heat-generating and fluorescent polymers utilizing solvent evaporation via published methods.
Pseudomonas aeruginosa (PA) 27853 was used for infecting breast cancer cells, or normal breast epithelial cells. Bacteria were cultured with cells for 4 hours, after which cells were treated with gentamicin to eliminate extracellular bacteria. The next day, a portion of the infected cells were lysed, serially diluted, and then plated to determine CFUs/cell. The remaining infected cells were plated on a chamber slip, and after 24h, cells were incubated with 100 µg/mL of NPs for a further 24h. Following incubation, NPs were removed, and cells were stained for PA, DAPI to visualize nuclei, actin expression, and NP uptake, which was quantified via fluorescent intensity of the actin (green) and intracellular NPs (red). Infected and non-infected cells were allowed to culture for 1 week to calculate differences in doubling time.
RESULTS – Cancerous (MCF7 and MDA-MB-231) breast cells had increased proliferation rates of approximately 23% compared to their non-infected counterparts. There was no significant difference in proliferation rates between infected and non-infected non-cancerous breast epithelial cells (MCF10A). Additionally, MCF7 and MDA-MB-231 cells had a 29.1-fold and 61.7-fold increase in CFUs/cell over MCF10A cells, respectively. There was no significant difference in actin expression between infected and non-infected MCF10As; however, infected MDA-MB-231 cells had more than twice the amount of actin expression compared to their non-infected counterparts. There was no significant difference in NP uptake between infected and non-infected MCF10A cells, but there was an almost 9-fold increase in NP uptake by infected MDA-MB-231 cells compared to non-infected ones.
CONCLUSIONS – Increased actin expression in infected cancerous cells is a possible explanation for the observed morphological differences. The photothermal NPs used in this study were not functionalized with a targeting moiety, making the fact that there was increased uptake by infected MDA-MB-231 cells even more intriguing, since passive
targeting of infected cells is a potential strategy for enhanced delivery of therapeutic NPs. Additionally, increased actin expression in infected cancerous cells could potentially be leveraged for treatments of breast cancer by adding targeting moieties on the surface of NPs to increase NP-cell interactions. Finally, although infected epithelial breast cells do not contain clinically relevant amounts of intracellular bacteria, the mere presence of bacteria could be a contributing factor to increased infection rates seen in post-mastectomy breast reconstruction compared to aesthetic reconstruction, necessitating a deeper understanding of how intracellular bacteria in the breast tumor can be effectively treated.
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