The last decade has seen a rapid increase in studies utilising a genetically modified probiotic, Escherichia coli Nissle 1917 (EcN), as a chassis for cancer treatment and detection. This approach relies on the ability of EcN to home to and selectively colonise tumours over normal tissue, a characteristic common to some bacteria that is thought to result from the low-oxygen, nutrient-rich and immune-privileged niche the tumour provides. Pre-clinical studies have used genetically modified EcN to deliver therapeutic payloads that show efficacy in reducing tumour burden as a result of high-tumour and low off-target colonisation. Most recently, the EcN chassis has been expanded into an effective tumour-detection tool. These advances provide strong justification for the movement of genetically modified EcN into clinical oncology trials. What is currently unknown in the field is a deep mechanistic understanding of how EcN distributes to and localises within tumours. This review summarises the existing EcN literature, with the inclusion of research undertaken with other tumour-homing and pathogenic bacteria, to provide insights into possible mechanisms of EcN tumour homing for future validation. Understanding exactly how and why EcN colonises neoplastic tissue will inform the design and testing of the next generation of EcN chassis strains to address biosafety and containment concerns and optimise the detection and treatment of cancer.

Engineered bacterial therapeutics are emerging as promising candidates for the treatment of solid cancers. However, despite effectively reducing tumour burden in different pre-clinical models, these "smart bugs" are yet to convincingly translate this efficacy to cancer treatment in humans. Here, we highlight key features of in vitro co-culture methods and how they can be used to investigate bacterial behaviour and guide the rational engineering of more effective anti-cancer bacterial therapeutics.

Engineered tumour-homing bacteria are a promising targeted treatment option for colorectal cancer (CRC). However, little is known about why they home to tumours. To investigate potential metabolic drivers of E. coli Nissle (EcN) tumour-homing, we performed targeted amino acid metabolomics using orthotopic mouse models of CRC. We identified enriched glutamate and asparagine in the tumour when compared to healthy colon tissue. To determine if the levels of glutamate and asparagine in the tumour were sufficient to support EcN survival and colonisation, we generated glutamate (ΔgdhAΔgltBD) and asparagine (ΔasnAΔasnB) auxotrophic EcN mutants, resulting in bacteria that require exogenous supplementation of these amino acids for growth. The tumour-homing ability of these strains was tested in our CRC mouse model post oral dosing. Both strains survived gut transit. EcN biodistribution analysis showed that ΔgdhAΔgltBD EcN retained tumour colonisation ability, while ΔasnAΔasnB EcN had reduced tumour colonisation. Overall, our results suggest that increased levels of glutamate, but not asparagine, provided by the tumour microenvironment supports EcN growth and tumour colonisation. Conversely, endogenous asparagine production is vital for effective EcN tumour colonisation. These results help define the metabolic mechanisms required for EcN tumour-colonisation and point to novel and targetable metabolites enriched in the CRC microenvironment that could be harnessed for further EcN engineering.