The effectiveness of most chemotherapeutics is limited by their inability to penetrate deep into tumor tissue and their ineffectiveness against quiescent cells. Motile Salmonella typhimurium, which are specifically attracted to compounds produced by quiescent cancer cells, could overcome this therapeutic barrier. We hypothesized that individual chemoreceptors target S. typhimurium to specific tumor microenvironments. To test this hypothesis, we used time-lapse fluorescent microscopy and tumor cylindroids to quantify the accumulation of chemotaxis machinery knockouts, including strains lacking individual cell surface chemoreceptors, chemotaxis signal transduction pathway enzymes, and the flagella and motor assemblies. To measure the extent of apoptosis induced by individual bacterial strains, caspase-3 activity was measured as a function of time. Our results showed how chemoreceptors directed bacterial chemotaxis within cylindroids: the aspartate receptor initiated chemotaxis toward cylindroids, the serine receptor initiated penetration, and the ribose/galactose receptor directed S. typhimurium toward necrosis. In addition, strains lacking proper flagella constructs, signal transduction proteins, or active motor function did not chemotax toward tumor cylindroids, indicating that directed chemotaxis is necessary to promote accumulation in tumors. By deleting the ribose/galactose receptor, bacterial accumulation localized to tumor quiescence and had a greater individual effect on inducing apoptosis than wild-type S. typhimurium. This new understanding of the mechanisms of Salmonella migration in tumors will allow for the development of bacterial therapies with improved targeting to therapeutically inaccessible regions of tumors. [Cancer Res 2007;67(7):3201-9]

Multi-drug resistance greatly limits the efficacy of conventional blood-born chemotherapeutics, which have limited ability to penetrate tumor tissue and are ineffective at killing quiescent cells far from tumor vasculature. Nonpathogenic, motile bacteria can overcome both of theses limitations. We hypothesize that the accumulation of S. typhimurium in tumors is controlled by two mechanisms: (1) chemotaxis towards compounds produced by quiescent cancer cells and (2) preferential growth within tumor tissue. We tested this hypothesis by quantifying the relative contributions of these mechanisms using the tumor cylindroid model, which mimics the microenvironments of in vivo tumors. Time-lapse fluorescence microscopy was used to measure the accumulation of GFP-labeled S. typhimurium into cylindroids of different size. Cylindroids larger than 500 microm in diameter contain quiescent cells, whereas cylindroids smaller than 500 microm do not. Spatio-temporal profiles of bacterial concentration were fit to a mathematical model to calculate two parameters that describe bacterial interaction with tumors: intratumoral bacterial growth, M, and intratumoral bacterial chemoattraction, K. It was observed that S. typhimurium is attracted to cylindroids and accumulate at long time points in the central region of large cylindroids. Both intratumoral bacterial growth and chemotaxis were significantly greater in large cylindroids, suggesting that quiescent cells secrete bacterial chemoattractants and the presence of necrotic and quiescent cells enable S. typhimurium to replicate in tumor tissue. In this study, several mechanisms of S. typhimurium accumulation in solid tumors have been quantified, which we believe is an important step in the development of bacterial-based therapeutics to target tumor quiescence.

The heterogeneity of cellular microenvironments in tumors severely limits the efficacy of most cancer therapies. We have designed a microfluidic device that mimics the microenvironment gradients present in tumors that will enable the development of more effective cancer therapies. Tumor cell masses were formed within micron-scale chambers exposed to medium perfusion on one side to create linear nutrient gradients. The optical accessibility of the PDMS and glass device enables quantitative transmitted and fluorescence microscopy of all regions of the cell masses. Time-lapse microscopy was used to measure the growth rate and show that the device can be used for long-term efficacy studies. Fluorescence microscopy was used to demonstrate that the cell mass contained viable, apoptotic, and acidic regions similar to in vivo tumors. The diffusion coefficient of doxorubicin was accurately measured, and the accumulation of therapeutic bacteria was quantified. The device is simple to construct, and it can easily be reproduced to create an array of in vitro tumors. Because microenvironment gradients and penetration play critical roles controlling drug efficacy, we believe that this microfluidic device will be vital for understanding the behavior of common cancer drugs in solid tumors and designing novel intratumorally targeted therapeutics.

The spatial arrangement of cellular metabolism in tumor tissue critically affects the treatment of cancer. However, little is known about how diffusion and cellular uptake relate to intracellular metabolism and cell death in three-dimensions. To quantify these mechanisms, fluorescent microscopy and multicellular tumor cylindroids were used to measure pH and oxygen profiles, and quantify the distribution of viable, apoptotic and necrotic cells. Spheroid dissociation, enzymatic analysis, and mass spectrometry were used to measure concentration profiles of glucose, lactate and glutamine. A mathematical model was used to integrate these measurements and calculate metabolic rate parameters. It was found that large cylindroids, >500μm in diameter, contained apoptotic and necrotic cells, whereas small cylindroids contained apoptotic but not necrotic cells. The center of cylindroids was found to be acidic but not hypoxic. From the edge to the center, concentrations of glucose, lactate and glutamine decreased rapidly. Throughout the cell masses lactate was consumed and not produced. These measurements indicate that apoptosis was the primary mechanism of cell death; acidity was not caused by lactic acid; and cell death was caused by depletion of carbon sources and not hypoxia. The mathematical model showed that the transporter enzymes for glucose and lactate were not saturated; oxygen uptake was limited by intracellular metabolism; and oxygen uptake was not limited by membrane-transport or diffusion. Unsaturated transmembrane uptake may be the cause of both proliferative and apoptotic regimes in cancer. These results suggest that transporter enzymes are excellent targets for treating well oxygenated tumors.