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

Abstract

Advances in genetic engineering of non-pathogenic Escherichia coli (E. coli) have made this organism an attractive candidate for gene delivery carrier. 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 cell spreading in the 3D interstitial space of tumors. These findings are useful for researchers to develop novel strategies for improvement of oncolysis or bacteria-mediated gene delivery in cancer treatment.

Microfluidic 3D Cell Culture: Potential Application For Tissue-Based Bioassays

“…Other polymeric materials, such as poly (methyl methacrylate) (PMMA), polycarbonate (PC) and polystyrene (PS) are also biocompatible materials for microdevice substrates [52,53]. Over the recent decades, numerous polymer-based devices have been designed and fabricated for microfluidic cell cultures, with different structural features, such as microchannels [5,13,19,47,54-58], micro-wells [3,11,59], micropillars [10,14,17,18,20,60-63] and cell retention chambers. Most of them were designed to optimize medium flow and oxygen perfusion throughout the cell culture and diminish the effect of medium-starved necrotic regions.…”

A microfluidic system for investigation of extravascular transport and cellular uptake of drugs in tumors

“…Using the image of cells immediately after they were loaded into the central channel, the diameter of cells was measured. Its mean ±SD were 14.3 ± 1.5 µm ( n = 41), suggesting that the average volume of a cell was 1,526 µm 3 if the shape of cells was assumed to be spherical (Elliott et al, 2011). Therefore, the volume fraction of cells in the central channel was approximately 51% upon loading or the volume fraction of extracellular space was 49%, which was consistent with the in vivo data of solid tumors reported in the literature (Krol et al, 1999).…”

“…As a result, both dead and resistant cells are observed simultaneously within the same system. In addition to the applications discussed above, our model can be used to investigate mechanisms of spreading of replication-competent viruses or bacterial cells in studies of oncolysis or gene delivery in solid tumors (Elliott et al, 2011). The viruses and bacteria, carrying genes for fluorescent proteins, can be visualized in 3D using confocal or two photon microscopy.…”

“…The culture was maintained at 37°C, and 95% air and 5% CO 2 (Elliott et al, 2011). Unless otherwise stated, all cell culture reagents were obtained from Gibco, Invitrogen (Grand Island, NY).…”

Microfluidic Device to Quantify the Behavior of Therapeutic Bacteria in Three-Dimensional Tumor Tissue

“…Other groups have used similar devices to show that Salmonella have a preference for cancer hepatocytes compared to normal cells [11], and expression of invasin increases proliferation of E coli. in 3D tumor tissue [12]. …”