Many cancer therapies exist today. They can be divided into systemic and local treatments. Systemic approaches are based on administering a therapeutic to the whole organism. The used substances have different levels of specificity for tumor cells and typically significant off-target effects. Local treatments involve either manual removal of malignant tissue by surgical interventions or irradiation. While systemically administered toxins can damage off-target sites in the body, local treatments oftentimes lack precision in terms of excision of the whole tumor tissue or are themselves carcinogenic, such as radiotherapy.

We develop a system that combines the advantages of both strategies, while minimizing their disadvantages. A non-pathogenic strain of Salmonella is used due to its inherent ability to populate solid tumors upon intravenous (systemic) administration. The strain will be equipped with a thermoinducible toxin secretory pathway, so that a spatially controlled exogenous signal can activate toxin release. To further enhance the treatment specificity, the production of an imaging contrast agent will be triggered by a quorum sensing module. Thereby the medical doctor can confirm bacteria have colonized the correct location and be assured that at least a minimally required dose of the toxin will be produced, thus avoiding potential sub- dosing. This way, a highly potent anti-cancer therapeutic is delivered, but activated only in the malignant tissue, thus minimizing damage to healthy cells.

Introduction

In 1971, former US-president Richard Nixon declared the “war on cancer”. A vast number of insights and new treatments have since been created, but despite these astounding advancements, classical strategies such as chemotherapy, surgery and radiation therapy remain the backbone of cancer therapy. These approaches share a number of drawbacks: first, they are prone to incomplete killing of cancer cells and thus often result in the recurrence of the disease. One reason for this is the possible development of resistance to the therapeutic molecules. Second, they exert considerable stress on the patient by damaging off-target tissue. This is common in chemo- and radiotherapy, where the whole body is exposed to toxic agents with a certain preference for cancer cells, or the healthy tissue between tumor and skin gets exposed to carcinogenic radiation. The toxicity or dose of those two common procedures are limited by the maximally tolerated negative effects on healthy tissue. Therefore, a subpopulation of tumor cells might survive and develop resistance to the therapy. Surgical procedures fundamentally prevent development of evolutionarily acquired resistance. But especially in difficult areas, they are not always feasible, and pose the risk of bleeding, pain, infections or anesthesia related reactions. Furthermore, it is challenging for the surgeon to differentiate tumor tissue from healthy tissue, resulting in incomplete excision of the tumor. The residing cancer cells after surgery can easily lead to regrowth of the tumor.

We aim to overcome some of the most crucial problems by engineering a non-pathogenic strain of Salmonella. These bacteria have been shown to reach tumors through the blood stream and grow preferentially in hypoxic environments,4 which results in a ~900-fold accumulation of Salmonella in cancer microenvironments compared to healthy tissues.5 The presence of bacterial cells close to tumor sites further enhances the recruitment of additional immune cells, thereby directing the immune response to immunoevasive cancer cells.

We envision to harvest the therapeutic potential and develop the bacteria to act as anti-cancer agents in a tissue-specific, safe and controlled manner. The engineered bacteria will have a series of functions, which ensure safety and control. First, they locate in the tumor and grow to a certain population density. This function is inherent in the therapeutic strain and will not be engineered. Second, they produce and sense a cell-to-cell communication molecule. Only when a quorum population size is reached, the Salmonella will then express an enzyme that produces a contrast agent, visible with the standard medical imaging method PET. This will ensure that only sufficiently large populations of the therapeutic strain produce a signal and therefore reduces the risk of false positive signals. Furthermore it assures presence of enough therapeutic bacteria, which is required to avoid sub-dosing with the potential to cause resistant cancer cells. Upon examination of the clinical image, a doctor can locally increase the temperature by focusing ultrasound to the bacterial population. This will activate an engineered temperature sensitive pathway of the therapeutic bacteria, leading to the release of a cytotoxic peptide in a precisely defined area.

With this approach, tissue damage is reduced, while the anti-cancer effect is maximized. We will develop and test the mentioned functional pathways in E. coli. In a next step, the abilities could be adapted to a Salmonella strain with the inherent ability to populate tumor tissue.

Detailed Project Description

Bacterial treatments were among the first tools in the medical inventory used in the battle against cancer. In the late 1890s, William B. Coley of New York Cancer Hospital, treated cancer patients with Streptococcus pyogenes, leading to the tumor regression. The basis of the bacterial infection’s therapeutic effect was completely vague, but recent studies have proven the preference of certain bacteria to populate the hypoxic and amino acid rich microenvironment of tumors. In that context, Salmonella tiphimurium AR-1 auxotrophic for leucine-arginine strain has been developed, offering increased tumor cell targeting. We will develop the tools and pathways that a cancer-targeting Salmonella would need in E. coli. The constructed parts could be easily adapted in further experiments to work in an appropriate therapeutic strain.

Cancer Toxin

We propose to use the engineered microorganisms to deliver an anticancer payload. Azurin (p18) effector domain has proven potent as an anticancer peptide, as it is currently tested in Phase I clinical trials. To ensure secretion of the protein, we will fuse the toxic protein p18 with a SopE protein, involved in the Type III secretory pathway of Salmonella.

Quorum sensing and contrast agent

Cancer treatment will always be supervised by a medical doctor. This requires control of the state of the treatment. Two mechanisms are implemented in our strain to control the treatment. The first checkpoint is a quorum sensing (QS) module. A positron emission tomography (PET) scan contrast agent is produced upon sufficient colonization of the tumor. To achieve this, the QS module produces a small but constant amount of N-acyl homoserine lactones (AHL). AHL can diffuse easily through tissues and be sensed by an AHL sensor also present in the QS module. If the population density of the engineered bacteria exceeds a threshold, the QS module will trigger the expression of thymidine kinase (TK), an enzyme which phosphorylates a diffusible contrast agent ([124I]FIAU). The phosphorylated molecules will be trapped in the bacterial cell, leading to an increased contrast in the medical image.6 The physician can then assess the localization of the bacteria in 3D, which in principal correlates well with the tumor. In the next step of the treatment, the physician can trigger the production of Azurin (p18) fusion protein by the engineered strain.

Thermosensor and focused ultrasound

Focused ultrasound is a novel treatment approach, recently introduced to clinics. It non-invasively rises temperature in a small tissue volume with high precision. However, when used alone, high temperatures are required to ablate a tumor, thus inevitably leading to damage in the surrounding tissue. In our approach only a slight temperature increase is needed to site-specifically activate a thermosensitive pathway which leads to secretion of cytotoxic agents by the engineered bacteria, thus minimize side effects related to high temperatures.4 We propose to construct a RNA-based temperature sensor (TS) with an activation temperature of around 41 oC, which can be reached by the focused ultrasound, but is below cytotoxic levels. This is the second checkpoint of the treatment. The expression of the therapeutic Azurin (p18) fusion protein is engineered to depend on activation of the QS module and the activation of the TS module. This way, the cytotoxic Azurin gets only delivered by those bacteria residing in the target tumor tissue, minimizing risk of systemic damage.

We build new BioBricks for the temperature sensor and we tune the quorum sensing to work in a defined manner. Furthermore, we will engineer a toxin secretion pathway by coupling the known anti-cancer peptide Azurin to a secretion pathway. Electronics hardware developments will accompany these building blocks to demonstrate that their practical use is possible. A home-made ultrasound transducer will enable us to stimulate the bacteria in conditions as close as possible to in-vivo ones. To emulate the thermal activation process of our strain, we plan to focus ultrasound on a petri-dish inoculated with our thermosensible bacteria and locally trigger the expression of a reporter gene.

Implications and Applications

Depending on the kind of tumor and how advanced it is, different treatment protocols are used. Cancer treatments can mainly be divided into local and systemic strategies. Local treatment modalities include surgery and radiation therapy, usually applied to the primary tumor site and possibly to large, solitary metastatic nodules. Systemic treatment typically involves intravenous application of a pharmaceutical agent, either in form of classical chemotherapy or, more recently, monoclonal antibodies aimed at activating patients’ own immune system to act against the tumor. Typically, different treatment modalities will be combined to achieve the highest possible tumor cell killing rate while avoiding side effects. Although most cancers have seen a significant increase in survival over the years with these strategies, statistics clearly suggest novel treatment modalities are still sought after.

Surgery, as a mainstay of local treatment, is unlikely to become obsolete anytime soon, as this remains the only way to remove large masses of neoplastic tissue. However, micro-metastases in the vicinity of tumors, not visible to the surgeon’s eye, carry a large risk of relapse. In addition, not all sites are accessible for surgery, and surgical manipulation of certain organs, such as the pancreas, carries a great risk of potentially lethal side effects. Additionally, surgical procedures are risky in general, especially for ill patients such as those suffering from cancer. Multiple surgical procedures that would be needed to remove both the primary tumor and metastatic nodules are therefore not feasible. As for radiation therapy, recent technological advances have made this treatment modality more attractive than ever. However, physical characteristics of beams used to deliver the dose make it impossible to avoid exposure of normal tissue located between the skin and tumor site. Normal tissue irradiation is related to both acute dose-limiting toxicities and long-term increase of risk for secondary malignancies.

The aim of our project is to design a tumor-targeting, cytotoxic agent-delivering microorganism to overcome limitations of current local treatment modalities. Inherent tumor targeting capabilities (primarily due to ability to survive only in neoplastic microenvironment) and safety due to knock-out of specific pathogenic genes make the specific strain of Salmonella an ideal candidate.

Upon intravenous administration, Salmonella will localize in the primary tumor site and metastatic nodules. Once a desired population density is reached, a signal will become visible to the doctor in an imaging modality available in the clinics (PET). Securing a minimal population density is needed in order to deliver a minimal amount of cytotoxic agent to avoid mutations related to sub-dosing of the tumor and leading to resistance to therapy. Once a signal is visible in both the tumor and metastases, the doctor will cause local (non-damaging to normal tissue) increase in temperature via focused ultrasound. This, in turn, will activate secretion of the cytotoxic agent by our bacteria in targeted tissue only, while avoiding any secretion in normal tissues. Depending on the specific situation, treatment can simply be repeated as necessary. Once the treatment cycle is finished (or upon clinical signs of infection) the treatment can easily be abrogated by antibiotic administration.

In conclusion, we propose a novel treatment modality for local killing of both solid tumors and solitary metastatic nodules, previously never achievable in one simple, non-invasive step. In our opinion, this way of safe local drug delivery to multiple neoplastic sites while avoiding normal tissue toxicities is a potentially valuable tool in the fight against cancer and offers significant advantages over existing local treatment modalities. Additionally, achieving a good local control allows for a decrease in doses of systemic therapy, therefore offering advantages not only for confined disease, but also for late-stage, disseminated cancer.

Team:ETH Zurich/Description

ETH_Zurich