I was recently at dinner with a family friend, who has survived stomach cancer, but lost his wife to liver cancer two and a half years ago. He is an interesting man, who worked for a large oil company for many years, travelling Saudi Arabia and other areas of the Middle East before retiring about 20 years ago. He has an incredible wealth of knowledge in almost all matters, and there are very few conversations to which he cannot contribute. So, with this in mind and his own past suffering with cancer, it is safe to say that when he starts talking about a novel cancer therapeutic, he probably has done his research and knows a little about the subject matter. I was told that the Telegraph had printed an article on Salmonella and its ability to flag tumours to the immune system. Stories about novel cancer therapeutics often appear in newspapers and social media, and I rarely give them much thought, but this left me somewhat intrigued so I thought it would be interesting to look into it further.
The use of bacterial preparations to stimulate the immune system in cancer patients has been a contested subject for over a hundred years, since William Coley began routinely injecting streptococcal organisms into bone cancer and sarcoma patients. Prior to this, Coley lost one of his first patients due to widespread metastases, despite a forearm amputation in response to a malignant tumour. This deeply moved him and, having trawled through the literature, he found a correlation between concurrent bacterial infection and tumour regression. Results from his bacterial treatment of cancer patients suggested that treatment with “Coley’s toxin” lead to the regression of tumours. However since the advent of chemotherapy and radiotherapy, the use of Coley’s toxin gradually disappeared (McCarthy, E.F., 2006).
Immunology has progressed quite a lot since Coley carried out this work; the mechanisms involved are now better understood and it is for this reason that interest in this area of research has been reignited.
One of the main drawbacks of chemotherapy is its inability to target tumours specifically; this leads to high off-target toxicity in non-cancerous cells and low tumour penetration with the chemotherapeutic. However, the use of bacteria provides unique mechanisms by which site-specific treatment of tumours may be possible.
The natural ability of bacteria to sense their environment through chemoattractants, and then actively follow chemical gradients whilst crossing biological barriers means they are able to penetrate tumour tissue. Metabolically-active, genetically-modified bacteria are also able to perform specific tasks once at the tumour site, such as the production of immunomodulatory molecules (cytokines) or enzymatic conversion of a pro-drug into an active therapeutic (St Jean, A.T., 2008). Bacterial vectors are also inherently immunostimualtory, as Toll-like receptors (TLRs) expressed by innate immune cells recognise bacterial-expressed virulence factors such as peptidoglycan and LPS. This leads to downstream activation of DCs, which travel from the local tumour environment to draining lymph nodes and activate adaptive immune responses through presenting tumour antigens to T-cells (Chorobik, P., 2013).
So, why is Salmonella a favourable candidate for potential bacterial therapy? Salmonella Typhimurium have been shown to have a high affinity for tumour cells and their facultative anaerobic nature means they can happily infiltrate the hypoxic areas of tumours, but they have also been shown to target non-hypoxic regions and metastases. Salmonella spp. are highly motile and can therefore penetrate into therapeutically-resistant regions of tumours, and have been shown to be preferentially attracted to such areas. Salmonella also displays direct tumour-killing activity, as they compete for nutrients and also stimulate primary and secondary immune responses. Toxins produced by the bacteria may have apoptotic effects on tumour cells, and intracellular infection with Salmonella can lead to cell death through autophagy. The combination of all these attributes can lead to reduction in tumour size (Chang, W.W., 2014).
In order to develop new, Salmonella-based vector strains for the administration of therapies, they must be attenuated/altered to stimulate an appropriate immune response. Both S. Typhimurium and S. Typhi are responsive to attenuation, and roughly 50 genes can be inactivated to produce a specific profile of virulence factors, which lends them to being used as appropriate vectors for therapeutics.
The successful use of Salmonella in reducing tumour size in murine models of cancer has been well documented in the literature. Attenuated Salmonella has been shown to work in combination with cisplatin to demonstrate an additive effect on the reduction of tumour size in mice (Lee, C.H., 2005). These results show the impact of untransformed attenuated bacteria as a result of its inherent ability to augment immune responses. Multiple studies using S. Typhimurium, genetically engineered to express pro-inflammatory mediators (e.g. TNF, IL-18) or chemokines (CCL21) also demonstrate similar success in treating tumours in murine models of cancer (Chorobik, P., 2013).
However, the story is not quite so successful when it comes to looking at similar studies in humans. An attenuated strain of S. Typhimurium (VNP20009) has been tested in a phase I study in which metastatic cancer patients were dosed intravenously with the bacteria. None of the 25 patients experienced cancer regression, significant levels of circulating TNF were measured in the peripheral blood and tumour colonisation with Salmonella was only observed in biopsies from three of the 25 patients (Toso et al., 2002). These results are in contrast with all of the animal models and could be a result of limited tumour-specific targeting by the bacteria.
The more recent developments in this field of research, which prompted the news media to publish articles suggesting that Salmonella can cure cancer, uses a much less virulent strain of bacteria, with a much higher lethal dose. This means that larger concentrations of the bacteria can be used without the side effects observed in the original phase I study by Toso et al. The bacteria are also engineered to overexpress and inducibly secrete Vibrio vulinficus flagellin B (flaB), which stimulates innate immune responses through the TLR 5 pathway and in this way acts as an excellent adjuvant for immunotherapy. Three days post infection, levels of intratumoural bacteria were 10000 fold higher than other organs, and it was at this time point that the FlaB payload was delivered through induction with L-arabinose. As a result of this, the off-target toxic effects are massively reduced and targeted therapy is achieved (Zheng, J.H., et al., 2017). So far, this research utilising Salmonella’s innate ability to target tumours, as well as inducibly secrete the therapeutic looks promising in mice, but we will have to wait and see if this is developed for human trials.
In summary, bacteria have been used as immunomodulators for cancer therapy for a long time, but the more recent advances in immunology and molecular biology mean that we are now able to further tailor microbes to create potentially viable therapeutics. The more recent studies look promising in mice, and perhaps the use of genetically engineered bacteria to deliver therapeutics to tumour sites will be used routinely in the future. However, the only recent study in humans shows that the mouse models are not always indicative of how these therapies will fare in man. The unique ability of bacteria to specifically colonise tumour sites and then deliver their payload means they are ideal candidates for tumour-specific therapy, so advances in this area of research will hopefully lead to novel and viable therapies for cancer in the near future.
Blog written by Will Pearce
Chang, W.W. and Lee, H.C., (2014), Salmonella as an Innovative Therapeutic Antitumor Agent, nt. J. Mol. Sci. 15(8), 14546-14554
Chorobik, P. et al., (2013); Salmonella and cancer: from pathogens to therapeutics, Acta Biochim Pol. 60 (3):285-97
Lee, C.H.; Wu, C.L.; Tai, Y.S.; Shiau, A.L (2005) Systemic administration of attenuated Salmonella choleraesuis in combination with cisplatin for tumor therapy. Mol. Ther. 11, 707–716
McCarthy, E.F., (2006); The Toxins of William B. Coley and the Treatment of Bone and Soft-Tissue Sarcomas, Iowa Orthop J, 26: 154-158
St Jean, A.T., (2008); Bacterial therapies: completing the cancer treatment toolbox, Curr Opin Biotechnol, 19: 511-517
Toso JF1, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, Sherry RM, Topalian SL, Yang JC, Stock F, Freezer LJ, Morton KE, Seipp C, Haworth L, Mavroukakis S, White D, MacDonald S, Mao J, Sznol M, Rosenberg SA (2002), Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol. 2002 Jan 1;20(1):142-52
Zheung, J.H., Nguyen, V.H, Jian., S.N., Park, S.H., Tan, W., Hong, S.H., Shin, M.G., Chung, I.J., Hong, Y., Bom, H.S., Choy, H.E., Lee, S.E., Rhee, J.H., Min, J.J., (2017), Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin, Science Translational Medicine Vol. 9, Issue 376,