Development of Salmonella strains as cancer therapy agents and testing in tumor cell lines.

Despite significant progress in the development of new drugs and radiation, deaths due to cancer remain high. Many novel therapies are in clinical trials and offer better solutions, but more innovative approaches are needed to eradicate the various subpopulations that exist in solid tumors. Since 1997, the use of bacteria for cancer therapy has gained increased attention. Salmonella Typhimurium strains have been shown to have a remarkably high affinity for tumor cells. The use of bacterial strains to target tumors is a relatively new research method that has not yet reached the point of clinical success. The first step in assessing the effectiveness of bacterial tumor therapy will require strain development and preclinical comparisons of candidate strains, which is the focus of this chapter. Several investigators have developed strains of Salmonella with reduced toxicity and capacity to deliver anti-tumor agents. Although methods for obtaining safe therapeutic strains have been relatively successful, there is still need for further genetic engineering before successful clinical use in human patients. As described by Forbes et al. in 2003, the main stumbling block is that, while bacteria preferentially embed within tumor cells, they fail to spread within the tumor and finish the eradication process. Further engineering might focus on creating Salmonella that remove motility limitations, including increased affinity toward tumor-generated chemotactic attractants and induction of matrix-degrading enzymes.

Competition among isolates of Salmonella enterica ssp. enterica serovar Typhimurium: role of prophage/phage in archived cultures.

Previously, we reported extensive diversity among survivors of Salmonella enterica ssp. enterica serovar Typhimurium that were stored for four decades in sealed agar stabs. Thus raising the question: was there selection for greater fitness among eventual survivors? To address this, we cocultured archived LT2 survivors with nonarchived (parental) LT2 strains in competition experiments. Selected archived strains outgrew a nonarchived LT2 sequenced strain. Although we initially assumed this was the result of mutations empowering greater nutritional utilization, we found phage selection was also involved. Phage fels-1 and fels-2 in supernatants were identified by primer/PCR as a putative selective force following single plaque isolations on a prophage-free strain and testing on appropriate hosts. In confirmatory experiments, instead of coculture in Luria-Bertani requiring antibiotic marker insertions, competing strains without markers were inoculated at opposite edges of motility plates. Not only did the archived LT2 population overgrow the nonarchived LT2 population, but also clear zones appeared at edges of encounters from which phage fels-1 and fels-2 (but not gifsy-1 nor gifsy-2) were recovered. However, in competitions of an archived strain with S. Typhimurium ATCC 14028, phage emerged that had a DNA base sequence segment of prophage ST64B but the sequence differed from the reported homologous segment in ST64B.

Salmonella-host cell interactions, changes in host cell architecture, and destruction of prostate tumor cells with genetically altered Salmonella.

Increasingly, genetically modified Salmonella are being explored as a novel treatment for cancer because Salmonella preferentially replicate within tumors and destroy cancer cells without causing the septic shock that is typically associated with wild-type S. typhimurium infections. However, the mechanisms by which genetically modified Salmonella strains preferentially invade cancer cells have not yet been addressed in cellular detail. Here we present data that show S. typhimurium strains VNP20009, LT2, and CRC1674 invasion of PC-3M prostate cancer cells. S. typhimurium-infected PC-3M human prostate cancer cells were analyzed with immunofluorescence microscopy and transmission electron microscopy (TEM) at various times after inoculation. We analyzed microfilaments, microtubules, and DNA with fluorescence and immunofluorescence microscopy. 3T3 Phi-Yellow-mitochondria mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria distribution in live cells. Our TEM results show gradual destruction of mitochondria within the PC-3M prostate cancer cells with complete loss of cristae at 8 h after inoculation. The fluorescence intensity in YFP-mitochondria-transfected mouse 3T3 cells decreased, which indicates loss of mitochondria structure. Interestingly, the nucleus does not appear affected by Salmonella within 8 h. Our data demonstrate that genetically modified S. typhimurium destroy PC-3M prostate cancer cells, perhaps by preferential destruction of mitochondria.