SonoBacter: Ultrasound-guided Activation and Imaging of Engineered Bacteria for Stroma Reprogramming

Bacteria have been investigated for cancer therapies, either for their abilities to produce drug molecules or enzymes that can attack tumours, or to induce immune responses that could help remove cancer cells. There are a number of issues with the use of existing bacteria-mediated cancer therapies, primarily concerning their mode of action and the danger that insufficient number of bacteria will be retain in cancer tissue and that they enter the blood system to cause infection.
Our proposal addresses these issues by focusing on reprogramming the tumour environment rather than killing cancer cells, including a means to retain the bacteria where they are injected, and providing their on-demand activation. This approach supports the innate mechanisms of cancer removal, does not lead to drug resistance and uses nature-inspired compounds that can impact the metabolism of non-cancerous cells within solid tumours rather than employing highly toxic traditional cancer drugs.
The bacteria in our study will be engineered to carry genes which can be activated by a simple chemical signal to produce plant-based compounds, known as flavonoids, that have effects on non-cancerous cells. The chemical signal molecule will be packaged within small particles encased within a material that forms a gel upon injection into the body. This gel also encapsulates the bacteria and ensures their safe delivery and retention at the injection site.
When ultrasound is applied to the capsule, the particles will burst and release the signal molecule, which in turn activates the bacteria and initiates the production of flavonoids. In this way, release of the active cargoes is controlled externally and can be initiated on-demand, thus ensuring the correct location of delivery and the desired in-tumour concentration of flavonoid compounds. In addition to bacterial activation, ultrasound can also be used for imaging, with the help of contrast agent programmed into the bacteria, allowing us to monitor non-invasively the mobility of the bacteria in the gel and their growth into and within the tumour. Furthermore, the gel material can be easily modified so that it is suitable either for injection via endoscopic needle or post-surgery insertion into the tumour, making it rapidly applicable to current medical practice.
The culmination of the study will be evaluation of bacterial anti-cancer activity in advanced 3D models of pancreatic and lung cancer, which will enable optimisation of bacterial population, active compound release and activity assessment without animal use.

The aim of proposed project is to develop engineered bacteria that can deliver flavonoids to the tumour microenvironment to initiate reprogramming of stromal cells, primarily cancer associated fibroblasts (CAFs). To ensure safe delivery, inactive bacteria will be integrated into a smart hydrogel capsules containing ultrasound-sensitive nanostructure containing initiator compound that can trigger the expression cycle of the bacteria on demand. Ultrasound will not only be utilized to regulate bacterial activation, but also to adjust the mechanical properties of encapsulating hydrogel and to image bacteria within the tumour. The latter will be achieved exploiting gas vesicle production programmed into bacteria by genetic engineering. Imaging is particularly important as it will enable assessment of migration and proliferation of bacteria when injected into tumour models. Ultrasound in combination with fluorescence microscopy, advanced microscopy and mass spectrometry will enable not only assessment of bacterial activation and growth, but also aid quantification of active compound production. All of the validation studies will be performed using 3D spheroid and organoid cultures of pancreatic cancer. Once the technology is established it will be translated to development of bacterial strains capable of production of both therapeutic payload and contrast agents, with ultimate goal of developing bacterial gels for injection and post-surgery implantation.