Harnessing colorectal tumour enriched glutamate to improve E. coli Nissle tumour specificity

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 (Δ gdhA Δ gltBD ) and asparagine (Δ asnA Δ asnB ) 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 Δ gdhA Δ gltBD EcN retained tumour colonisation ability, while Δ asnA Δ asnB 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.