Date Published: June 29, 2017
Publisher: Public Library of Science
Author(s): Carola Murphy, Elizabeth Rettedal, Panos Lehouritis, Ciarán Devoy, Mark Tangney, Paulo Lee Ho.
Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNFα) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic E. coli strain MG1655 was investigated as a tumour targeting system in order to produce TNFα specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNFα producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNFα production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models (CT26 colon, RENCA renal, and TRAMP prostate) as evidenced by tumour volume and survival analyses. A pattern of pro-inflammatory cytokine induction was observed in tumours of treated mice vs. controls. Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNFα expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours.
The efficacy of current anti-cancer small drug chemotherapeutics is limited because of the narrow therapeutic index inherent in most of the drugs employed to treat cancer which leads to systemic damage of healthy tissue and side effects upon treatment. For this reason, alternative therapies for the treatment of cancer that aim to localize the therapeutic agent to the site of the tumour are been investigated. TNFα was identified in 1975 when it was discovered that a substance from the sera of animals that were challenged with BCG and endotoxin could kill mouse cells in vitro and induce haemorrhagic necrosis of transplantable mouse tumours in vivo . Subsequently, TNFα was investigated as a therapeutic agent for cancer treatment. However, due to severe systemic toxicity it was soon abandoned for systemic use, only to be revisited later in the settings of isolated limb perfusion to treat inoperable cancer . The inherent high level of toxicity of TNFα poses health risks, and therefore it is essential that if it is to be used for treating cancer it must be confined to the tumour site in a highly controlled manner. Biological vehicles have been examined for this purpose in the context of cancer gene therapy, and TNFα delivery by viruses such as adeno-associated virus  or adenoviruses have shown promise. TNFerade is a serotype 5 adenovirus that expresses TNFα under the control of the early growth response gene (egr-1) promoter that responds to radiation, which has been examined in Phase 3 clinical trials for advanced prostate cancer [4–7]. In this approach, while the biological delivery vehicle is not confined to tumours, TNFα production is restricted via physically targeted radiation induction of the egr-1 promoter to express the TNFα transgene.
To our knowledge, this is the first in vivo study that demonstrates TNFα delivery by bacteria to experimental murine tumours. E. coli MG1655 was employed to deliver and produce the therapeutic biomolecule TNFα inside murine tumours. Initially, both i.t. and i.v. routes of administration were tested to compare tumour targeting, and MG1655 performed well in both. For more clinical potential (for inaccessible and metastatic cancers), the i.v. route was chosen and MG-TNFα was administered to mice bearing CT26 tumours for further qualitative and quantitative analysis. MG-TNFα was capable of targeting tumours and proliferating as evidenced by BLI, IF and cfu counts. TNFα production within CT26 tumours was confirmed by ELISA and therapeutic studies indicated that MG-TNFα can impede tumour growth without inducing significant systemic toxicity.