Bacterial Cancer Therapy
Therapeutic bacteria have unique properties that enable them to treat metastatic disease and inoperable tumors. These organisms are motile and active penetration of tumor tissue. In contrast, chemotherapeutic molecules poorly penetrate tissue, which leaves large regions untreated and is a primary cause of patient mortality. Active motility is the only way to overcome these intrinsic transport barriers. Therapeutic bacteria are nontoxic and specifically accumulate in tumors and metastases. This targeting enables delivery directly to malignant tissue, while sparing healthy organ. Inflammation, chemotaxis and growth are the dominant mechanisms that control bacterial colonization in tumors. Inflammation increases the porosity of tumor endothelium and increases bacterial extravasation. When in tumor tissue, bacteria use chemoreceptors to sense and migrate toward small molecules produced by cancer cells. Insertion of transposons in the regulatory region of the flhDC operon of the bacterial genome prevents a sessile lifestyle and increases tissue penetration. Understanding and manipulating these mechanisms is the key to developing effective therapies that are suitable for human use and that eliminate drug-resistant tumors and metastases.
Synthetic Biology of Biomedical Tools
The microbial genome is highly plastic and malleable. Reconfiguration of biological components enables bottom-up design of novel therapeutic tools. Many anticancer compounds exist in the biological world, from mammal signaling molecules to microbial defense mechanisms. Bacteria that secrete Staphylococcus aureus alpha-hemolysin (SAH) rapidly kill cancer cells and eliminate tumors in mice. After release, SAH diffuses through tissue, which considerably increases the affected volume and overall efficacy. Bacteria that produce TNF-related apoptosis-inducing ligand (TRAIL) suppresses tumor growth and enhances survival in mice. Controlling protein expression with density-sensitive triggers promotes expression of therapeutic molecules in tumors and prevents production in healthy organs. Genetic circuits that controllably induce microbial lysis create failsafe organisms that cannot propagate in the environment and ensure patient safety. Cell-communication circuits, built from genetic components of the marine organism Vibrio fischeri, increase protein production 400 times and signal sensitivity 10,000 times. Engineered bacteria that produce and secrete fluorescent proteins can detect very small tumors. The 10 µg limit of bacterial detection is 100,000 times lower than the one-gram limit for tomographic techniques. By taking cues from the living systems, rational design of bacterial genetics will create biomedical tools that are safe and effective at treating human disease.
Epigenetic regulation has been hypothesized to control dedifferentiation of cells into cancer stems cells and enable the formation of metastatic lesions. Targeted bacteria can be used to disrupt these processes. Three major epigenetic regulators are the proteins EZH2 and NIPP1. The Forbes Lab has developed bacterial delivery vectors that specifically invade tumor cells, sense invasion, lyse, and then deliver a payload of shRNA or peptide. Silencing expression of EZH2 and NIPP1 will prevent the formation of cancer stem cells, and the subsequent propagation of metastases. Release of peptides will interrupt NIPP1 and PP1 docking and cause death in resistant tumors and established metastases. Using bacteria to interrupt intracellular protein-protein interactions will enable targeting of disease-specific signaling pathways and will play a critical role in the next generation of biological therapies.
Molecular transport defines whether a therapy will be effective or not. Often the interactions between transport mechanisms are complex and non-obvious. In some cases, either fast diffusion or strong cell binding can critically impair the performance of a therapeutic molecule. Understand these interactions requires integration of computational models, microfluidic devices, and animal models. An effective in vitro device must mimic the tissue surrounding blood vessels in tumors and enable measurement of diffusion, cell binding, and therapeutic outcome. Quantitative measurement in devices has shown that cell attachment is critical for the cancer drug, doxorubicin. This effect is masked when it is encapsulated in stealth liposomes, which release it too slowly for effective binding. Without measuring all of these mechanism simultaneously this masking effect would not have been discovered. When doxorubicin is bound to gold nanoparticles, cellular uptake, and not diffusion, controls delivery. The surface charge of the particles affects the rate of uptake: positive particles are taken up more readily than negative particles. Tools that quantify molecular transport in biological systems will be essential for designing effective therapeutics and drug-delivery systems.
Metabolic heterogeneity in tumor reduces the efficacy of cancer therapy. Limited diffusion of nutrients and oxygen from supplying blood vessels creates region of hypoxia and hypoglycemia. These poorly oxygenated regions are quiescent and are unresponsive to most cancer therapies. The combination of multiple techniques is required to accurately describe these phenomena. Fluorescence microscopy, three-dimensional in vitro models, and metabolic flux analysis enable quantification of intracellular metabolism as functions of position and time. Hypoxia-inducible-factor-1a (HIF-1a) is the primary signaling molecule that responds to available oxygen by regulating cell survival, apoptosis, and expression of glycolytic enzymes. In a diffusion limited environment, however, HIF-1a does not affect metabolism when glucose is not available. Deprivation of different nutrients affects cells differently. When glucose is limited, cells become quiescence prior to becoming apoptotic. However, without glutamine, which cells require for biosynthesis, apoptosis is quickly induced. Nutrient gradients, intracellular metabolism, and cell growth are critical tumor mechanisms should be taken into account when developing cancer therapies.
Computational Tumor Modeling
Understanding the interactions between intracellular metabolism, cell growth, and molecular transport is necessary to predict the efficacy of any therapeutic strategy. The size and complexity of this problem requires computation modeling and must integrate intracellular energy metabolism, the cell-cycle, angiogenesis, molecular diffusion, and pharmacokinetics. In average tumors, oxygen transport has a greater effect than glucose transport on the distribution of quiescent cells. Counterintuitively, tumors with less proliferating cells are more responsive to chemotherapeutics than tumors with more proliferating cells because of cell re-population. When used to interpret breast MRI images from breast cancer patients at Baystate Medical Center in Springfield, MA, computational models show that increased transvascular transport is associated with tumor aggressiveness. Based on this analysis, greater transport heterogeneity leads to poor drug responsiveness. Predicting drug responsiveness is important for patients with mid-stage tumors who are deciding between complete mastectomy and breast conservation surgery. In addition to understanding tumor behavior, computational tumor models provide critical tools for oncologists, surgeons, and cancer patients.