(C-108) Investigating the uptake of engineered bacterial membrane vesicles by cancer cells

Introduction: Bacterial membrane vesicles (BMVs) are ~100nm particles constitutively released from bacteria, bearing membrane and intracellular components specific to the parent strain. BMVs have immunostimulatory effects which can potentially be used to break tumor immune tolerance, prompting tumor clearance and initiating anti-tumor immunomemory. Here, we have engineered Escherichia coli to produce BMVs expressing one photoactivatable fluorescent protein on the outer membrane and a different photoactivatable protein contained within the BMV. This first-of-its-kind dual-labeled BMV design not only allows for detailed investigation of BMV interactions with cancer cells, but it sets the stage for future development of engineered BMVs as a platform for cancer immunotherapy applications.


Materials and Methods: A pET-21b vector was designed to encode (1) photoactivatable red fluorescent protein PATagRFP fused to lipoprotein outer membrane protein A, which is expressed on the outside of bacteria and BMVs, and (2) photoswitchable Skylan-S fused to an outer membrane protein A signaling peptide, which causes Skylan-S to be trafficked to the periplasm where it is sorted into BMVs. The engineered vector was transformed into Escherichia coli DH5α, from which BMVs were collected via centrifugation of an overnight culture followed by ultracentrifugation of the BMV-containing supernatant. BMVs were visualized both on a transmission electron microscope as well as an Oni Nanoimager superresolution microscope to confirm production of dual-fluorescence BMVs. To investigate how these BMVs interacted with mammalian cells, non-tumorigenic cell line MCF-10a and tumorigenic cell line MCF7 cells were seeded into a 96-well plate and then incubated with the BMVs for 4 hours before imaging with a confocal microscope.


Results, Conclusions, and Discussions:

We have designed and created photoactivatable dual-fluorescence E. coli BMVs as a preliminary platform for investigating BMV-cancer cell interactions and demonstrated their interaction with both tumorigenic and non-tumorigenic cells in vitro. We have demonstrated the ability to track these BMVs as they are taken up and processed by the mammalian cells. This is the first time that bacteria have been engineered to produce dual-fluorescence BMVs, which will enable us to distinguish how the BMV membrane is processed from how the internal contents are processed. Better understanding the method of uptake and intracellular trafficking will allow us to optimize these BMVs as a platform for immunotherapy applications. Moreover, in our future work using our dual-expression plasmid we will be able to load different molecules onto the BMVs with the aim of enhancing cancer cell targeting specificity.