Interaction and Functional Impact of Colorectal Cancer Extracellular Vesicles on Escherichia coli

The human gut microbiota plays a vital role in regulating various physiological processes, and alterations in the composition and function of the microbial community (dysbiosis) are associated with the pathogenesis of colorectal cancer (CRC). Although the causative link between CRC and microbiota is widely investigated, the underlying microbiota-tumour interactions are not well understood, yet. It is evident that CRC-derived extracellular vesicles (EVs) have an impact on various oncogeneses processes, however, their impact on the surrounding microbiota is not clear. Therefore, this project hypothesises that EVs have an impact on the microbiota, supporting the disease-linked interactions between host and microbiota, and contributing to dysbiosis. CRC cell lines (SW480, SW620) were cultured in CELLine AD 1000 bioreactor flasks, blood was collected from CRC patients and healthy individuals, and colon tissue was collected from CRC patients. EVs were isolated from CRC-cell line culture media, blood plasma, and digested tissues by size-exclusion chromatography (SEC) and characterised by nanoparticle flow cytometry (NanoFCM), western blotting, ELISA, and transmission electron microscopy (TEM). Confocal microscopy, TEM, Flow cytometry, and EVs-enzymatic treatments were performed to assess the interactions between EVs and E. coli strains (E. coli MG1655 (Laboratory strain) and E. coli 11G5 (CRC-associated strain)). Also, the impact of the EVs on E. coli phenotypic characteristics, growth and biofilm formation, and bacterial transcriptome were assessed by turbidimetric assay, microtiter plate assay, and transcriptomic analysis, respectively. NanoFCM analysis showed a high yield of EVs with characteristic size profiles, EV markers detection confirmed the presence of EVs, and TEM revealed the double-membranous structure of EVs. TEM analysis indicated an interaction between the EVs and E. coli with clear surface binding. Flow cytometry analysis showed that E. coli-EVs interactions are disease-stage specific and bacterial-strain specific. EV treatment had an impact on bacterial phenotypic characteristics; an increase in E. coli growth and a decrease in the ability of the bacteria to form biofilm were observed. It also resulted in an upregulation of genes which are involved in bacterial motility, the flagella structure of E. coli such as fliA, and a downregulation of genes involved in the zinc-ion uptake system such as ZinT. Overall, EVs appeared to be capable of mediating CRC-microbiome interactions and altering bacterial phenotypes; bacterial growth and ability to form biofilm.