Salmonella typhimurium engineered to produce CCL21 inhibit tumor growth
Markus LoeZer · Gaelle Le’Negrate · Maryla Krajewska · John C. Reed
Received: 3 April 2008 / Accepted: 24 June 2008 / Published online: 17 July 2008
© Springer-Verlag 2008
Summary:
Intravenously-applied bacteria tend to accumulate in tumors and can lead to tumor regression. However, most bacteria were found to be toxic except attenuated Salmonella typhimuriu.. Clinical trials showed that S. typhimurium had no significant adverse effects in humans, but it did not have significant anti-tumor activity. Researchers hypothesized that engineering S. typhimurium to express a chemokine, CCL21, would have anti-tumor activity. CCL21 is a small secreted molecule that attracts lymphocytes, dendritic cells, natural killer cells, therefore the tumor might be eliminated with these phagocytes. To investigate this hypothesis, researchers engineered an attenuated strain of S. typhimurium to produce the chemokine CCL21.
methods:
Western blotting was used to test CCL21 protein expression. In vivo experiment, by injecting S. typhimurium with or without the vector for CCL21 into the tumor of mice that were developed with CT-26 colon carcinoma cells or D2F2 breast carcinoma cells, was used to test tumor size/amount and metastases. Immunohistoligy and ELISA were used to examine phagocytes at the sitesd and other cytokine proteins induced by CCL21, respectively.
Results:
Results showed that only the engineered S. typhimurium expressed CCL21 both in the pellet and supernatants. They found that, attenuated S. typhimurium expressing CCL21 significantly inhibited the growth of primary tumors and pulmonary metastases in preclinical models of multi-drug-resistant murine carcinomas, while control bacteria did not. Histological analysis of tumors showed marked inflammatory cell infiltrates in mice treated with CCL21-expressing but not control bacteria. Levels of cytokines and chemokines known to be induced by CCL21 [e.g., interferon-γ (INF γ), CXCL9, and CXCL10] were significantly elevated in tumors of mice treated with CCL21- expressing but not control S. typhimurium.
The anti-tumor activity was found to be dependent on CD4- and CD8-expressing cells, based on antibody-mediated in vivo immuno-depletion experiments. Anti-tumor activity was achieved without evidence of toxicity. In summary, chemokine-expressing bacteria may provide a novel approach to cancer immunotherapy for effective and well tolerated in vivo delivery of immunomodulatory proteins.
problem: Researchers did not do an experiment to test toxicity quantitatively. The evidence of toxicity was merely based on that fact that mice didn't show obvious abnormalities with the injection with bacteria.Therefore, there might be toxicity in the bacteria but be able to eliminate with the liver detoxification function.
Significance:
Engineered bacteria used as a chemokine CCL21secreting factory with anti-tumor functions. Due to S. typhimurium’s safety profile, the bacteria do not harm the human body, so it’s a feasible way for drug delivery. Since it’s a living organism and they can accumulate up to 10^9 colony forming unit per gram tumor tissue, there is no need to concern about diffusion and concentration problems in effectively target the tumor. After eliminating the tumor, these bacteria might be eliminated by the immune system of host. However, the bacteria relies heavily on the immune system in reducing tumors, there is one concern that the immune system might target the bacteria colony instead of the tumor, leading to ineffectiveness in treatment.
3 comments:
Do they mention how the bacteria were attenuated? Is it possible that through recombination with other bacteria that are in our body, the introduced Salmonella typhimurium could become harmful? I think this is a concern with all bacteria-based therapeutics. Also, do they somehow make the bacteria "invisible" to the immune system? And if not, would it be a possibility that these therapeutic bacteria could be eliminated before any chemokine is produced.
It would be interesting to follow this approach to anti-cancer therapies. I know that currently it is extremely hard to get drugs and materials approved by the FDA, but what the paper suggests is using an artificially modified bacteria. I can't imagine how long it would take to get the FDA to approve such an approach; everything from genetic recombination to problems with sterile technique would generate a nightmare situation.
to answer simina's question,
no, the paper does not mention how the bacteria were attenuated. but attenuation is used in vaccine a lot, i think this one is processed in the same manner. basically what it does is to reduce the pathogenicity of the bacteria/virus, but keep it vital. in this case, probably make the bacteria less vital by passaging several times in tissue culture, Embryonated eggs or Live animals. you can wikipedia about how the whole idea. you raise a good point about avoiding the immune response. actually, s. typhimurium accumulates in the tumor site after injecting into the mice veins, i think the bacteria itself likes the tumor site, maybe like high temperature or the specific PH or, something unique about the specific bacteria. however, i think natural bacteria does not trigger immune response at the tumor site, b/c immune cells might not notice they are there. but engineered bacteria will secret CCL21. immune cells will come and may attack bacteria. this is one of the challenges of this groups of scientists is facing. they did not address it. they focus on testing if secretion of CCL21 to induce immune response will eliminate the tumor.
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