Proliferation behavior of E. coli in a three-dimensional in vitro tumor model

Advances in genetic engineering of non-pathogenic Escherichia coli (E. coli) have made this organism an attractive candidate for gene delivery vehicle. However, proliferation and transport behaviors of E. coli in three-dimensional (3D) tumor environment are still unclear. To this end, we developed a novel microfluidics-based tumor model that permitted direct in situ visualization of E. coli in a 3D environment with densely packed tumor cells (B16.F10 or EMT6). The E. coli was engineered to co-express two proteins invasin and mCherry (inv(+)) so that they had the ability to enter mammalian cells and could be visualized via fluorescence microscopy. E. coli expressing mCherry alone (inv(-)) was used as the control counterpart. The inv(-) bacteria proliferated to a higher extent than inv(+) bacteria in both the 3D tumor model and a 2D monolayer culture model. Meanwhile, the proliferation appeared to be tumor cell type dependent since bacteria did not proliferate as well in the EMT6 model compared to the B16.F10 model. These differences in bacterial proliferation were likely to be caused by inhibitors secreted by tumor cells, as suggested by our data from the bacterial-tumor cell monolayer co-culture experiment. The bacterial proliferation provided a driving force for E. coli spreading in the 3D interstitial space of tumors. These findings are useful for researchers to develop novel strategies for improvement of bacteria-mediated oncolysis or gene delivery in cancer treatment.