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Page 12 of 25 Torres et al. J Cancer Metastasis Treat 2018;4:4 I http://dx.doi.org/10.20517/2394-4722.2017.49

Clostridium spores have low immunogenicity and can colonize multiple organs after systemic
administration [187] . However, once they germinate, they induce an inflammatory response with infiltration
[99]
of immune cells with oncolytic effects . These strains have been employed in genetic engineering as
vectors for cytokines secretion such as TNF-α [188] , IL-12 [124] and IL-2 [189] , achieving high concentrations
inside tumor tissue without systemic toxicity. Also C. novyi-NT and C. sporogenes increase secretion of
specific antibodies against hypoxia inducing factor-1 (HIF-1), main component observed in hypoxia
response regulation inside tumors [125] .

Salmonella: multi-use bacterium
Salmonella enterica serivar tyhimurium (S. typhimurium) is one of the most studied bacterium for its
adaptative qualities leading to new strains with bacterial engineering showing antitumor activity [119] . In
the beginnings of the 21st century, phase I clinical trials were conducted to show their efficacy with gene
modification via deletion in msbB and purI genes. The msbB gene is required for lipid A synthesis and its
deletion was made to reduce TNF-α related toxicity, preventing septic shock [190] . On the other hand, by
deletion of purI gene, the bacterium became able to colonize tumors more selectively. All of this made the
strains depend on purine external sources for survival restricting their growth to areas with substantial
cell renewal [191] . Tumor tissues with their purine rich activities would be perfect regions for their selective
colonization [191] . Salmonella typhimurium VNP20009 is one of the main strains in experimental studies
originated from this theory.

This study results showed the maximum tolerated dose of this bacteria, its toxicity limit dose, and adverse
effects by increasing production of proinflammatory cytokines. The observed adverse effects included
thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, nausea, vomit, elevated alkaline
phosphatase and hypophosphatemia. However, tumor colonization was detected only in 3 patients, and no
tumor regression was observed [110] . Despite the fact that the study did not show promising results, it was the
start line for prospect studies to find doses that could be adjusted for efficiency and tumor localization and
for other therapeutic features.

In order to increase this bacterium therapeutic effect, a study was initiated to use them as vectors in tumor
gene therapy [192] . A pilot study was performed with an attenuated and gene modified Salmonella strain
with expression of E. coli CD, called suicide prodrug-activating enzyme [193] . These genes were integrated in
VNP20009 chromosome through Donnenberg and Karper method resulting in TAPET-CD strain [175] . The
mechanism of action of this enzyme consists in conversion of 5-fluorocytosine (5-FC, antifungal agent with
limited cytotoxicity) to 5-fluorouracil (5-FU, cytotoxic antimetabolite capable of producing cellular apoptosis)
[193] . No promising results were obtained 2 out of 3 treated patients did not present any tumor regression but an
[175]
improvement of their disease was observed .
Another example from S. typhimurium is strains would be A1-R, which currently is on preclinical studies
[14]
against different cancerous tissues such as prostate , pancreatic [194,195] , glioma [196] , colorectal [197] , and
ovary [198] . S. typhimurium A1-R colonization seems to be more selective and effective than other strains and
less toxic than VNP20009 strain. It also has safer systemic administration than C. novyNT [199] . Therefore,
clinical trials for this strain are coming. A1-R is a gene modified strain, auxotrophic for leucine and
arginine by nitroguanidine mutagenesis (NTG)-preventing healthy tissue invasion. It was utilized in animal
models with prostatic cancerous cells PC3 and also in humans showing tumor regression, inhibition and
[14]
prevention of cancer .
Bacteria therapeutic use has been confirmed in cancer models with stem cell characteristics. This represents
the only method capable to reduce in vivo tumor sizes in relation to chemotherapy (5-FU in monotherapy,
cisplatin and gemcitabine). The efficacy increased when combined with 5-FU [195] . S. typhimurium A1-R