(557c) Genetically Modified Bacteria Actively Secrete Recombinant Protein Biomarker for Early Detection of Solid Tumors

An attenuated strain of Salmonella typhimurium has been engineered to actively secrete Zoanthus sp. green fluorescent protein (ZsGreen) in a controlled manner for use in in vitro and in vivo tumor models for early detection of cancer. Current methods of cancer detection involve a primary screen for abnormal levels of native biomarkers, which might indicate cancer and other diseases. These preliminary screens often yield false results, requiring an invasive biopsy that is often painful and discomforting, to confirm diagnosis. Secretion of an exogenous recombinant protein from within tumor tissue allows cancer detection through noninvasive blood sampling without uncertainty in the results. It has been shown previously that systemically administered Salmonella typhimurium localize in solid tumor tissue 2000 fold more than in healthy tissue (Low et al. 1999). Once colonization of the bacteria in the tumor is achieved, triggered production and secretion of a protein biomarker is transported back to the blood stream for detection. Fluid transport in tumor interstitial space has been shown to have outward flowing pressure gradients hindering systemic administration of small molecule chemotherapeutics. These transport limitations act in favor of export of protein from within tumor tissue to the blood stream. A secreted biomarker, produced from the bacteria, within tumor tissue, would indicate the presence of cancer without invasive procedures and require a simple blood draw for detection. In this study a gram negative type III secretion system(T3SS) signal was fused to the n-terminus of ZsGreen protein (MvZsGreen) yielding a 3-fold increase in secretion in wild type strain (SL1344) and 5-Fold increase in an attenuated strain (VNP20009) of S. typhimurium. Over half of the protein produced is secreted from the attenuated strain (pBAD_MvZsG_VNP) under control of the L-arabinose inducible promoter (pBAD).  Time-lapse fluorescent microscopy with viability staining revealed that a majority of cells (85%) are viable after 36hrs of culture and release protein at twice the rate than dead cells. Immunoblotting measurements and fluorescence measurements of secreted recombinant MvZsGreen diluted in blood was shown to have detection limits of 1:10 and 1:1000 respectively.  A previously developed micro-fluidic 4T1 tumor model (Toley et al. 2011) was used to determine transport rates of protein through tissue and detection limit on the secreted protein from actual tumor tissue under triggered induction using the L-arabinose inducible system.  Increased secretion from VNP20009 over the wild type S. typhimurium is extremely beneficial in reducing virulence while improving protein production and transport for detection. Viability studies revealed two mechanisms of protein release in culture with an increased rate in active secretion through T3SS compared to cell death related diffusion. This allows production and secretion of biomarker, MvZsGreen, as the bacteria proliferate within the tumor tissue, which is key for monitoring cancer growth for prolonged periods of time. MvZsGreen has higher sensitivity for detection via fluorescence than immunoblotting  allowing for more accurate determination of the presence of a tumor mass. Micro-fluidic studies revealed favorable MvZsGreen transport through solid tumor tissue and subsequent detection. This system demonstrates the ability of bacteria to produce recombinant protein in a controlled manner deep within tissue, secrete that protein, and have the protein transport through tumor tissue to the blood stream for detection.  These studies demonstrate the feasibility of a engineered bacterial tool for early detection and diagnosis of cancer.