CD44 is a cell membrane protein involved in normal cell function (cell-cell and cell-matrix adhesion). However, one notable isoform, CD44v6, seems to play a major role in cancer progression, facilitating cell migration and invasion and is commonly upregulated on the surface of cancer cells. The RIOT system uses CD44v6 as a spatial marker, and recognition of this protein via a CD44v6 specific antibody allows anchoring of the engineered bacteria on the surface, triggering the expression of invasin and LLO for subsequent invasion.
Many complex biological processes lead to all forms of cancer, and their significant hallmarks include an ability to resist cell death, prolonged signalling, the origination of angiogenesis and metastasis. Another observable trait of cancer is described by the Warburg effect where a higher rate of glycolysis increases lactic acid production.
In proliferative cell types such as cancer, the bulk of the pyruvate from glycolysis is moved from the mitochondria to create lactate via lactate dehydrogenase - a process usually initiated only when oxygen supply is decreased. This production of lactate in the presence of oxygen is a direct consequence of the Warburg effect, resulting in a microenvironment with an increased lactate concentration.
Invasin is derived from the bacteria Yersinia pestis which allows selective invasion of cells that express β1-integrins. Listeriolysin O (LLO) on the other hand, is expressed in Listeria monocytogenes and allows the bacteria to break out of the endosome after entry into mammalian cells. Combining the traits of these two genes, our engineered bacteria is capable of entering the cytosol of cancer cells to potentially release a cytotoxic drug.
Initially, small amounts of N-Acyl homoserine lactones (AHLs) are produced by our engineered bacteria and they freely diffuse in and out of the cell. In the tumor region where the cell density is expected to be high, the concentration of AHL also increases, and past a threshold level, LuxR binds to AHL. When this happens, the LuxR-AHL complex goes on to further activate expression of LuxI, which then produces more AHL. This results in a positive feedback loop that increases both the concentration of LuxI as well as our desired proteins, invasin and LLO.
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Biomolecule delivery systems are often plagued by problems such as non-specific targeting and low bioavailability. We sought to design a novel system that can sense and respond to specific stimuli present in the microenvironment of pathogenic cells. We engineered a dual-sensor bacterium that can sense increased metabolite levels in its microenvironment and then respond by delivering biomolecules into target cells. As a proof-of-concept, we engineered Escherichia coli to detect increased level of lactate in biological fluid, then respond by attaching itself to a cancer cell marker, and subsequently release biomolecules into the cell. A biosafety kill switch will be activated when there is insufficient lactate present, thus minimizing non-specific targeting.
Our proposed system has the flexibility to be engineered to detect other metabolites by changing the gene promoter and also detect different cell types by targeting other cell receptors. In addition, the modular nature of our system also allows part of the lactate sensing mechanism to be used as a diagnostic kit, especially for detecting for elevated levels of lactate in biological fluids such as blood or serum from patients with suspected cases of sepsis or lactic acidosis. This method of detection can be carried out without any specialized equipment or impoverished areas.