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Torres et al. J Cancer Metastasis Treat 2018;4:4 I http://dx.doi.org/10.20517/2394-4722.2017.49 Page 7 of 25
[96]
characterized by genomic instability, angiogenesis and metastatic qualities ; leading to new approaches
against this feature [97-100] . Strict anaerobic bacteria with spore germination qualities, such as Clostridium,
cannot proliferate in highly oxygenated environments, restricting their colonization to hypoxic and
necrotic regions of the tumor [101] . This is affected by tumor morphology, with central necrotic areas and
well perfused cells in the periphery, allowing anaerobic bacteria to proliferate in the center. Following this,
the immune system gets activated and makes a peripheral ring of immune cells prepared to eradicate the
tumor completely [102] .

Facultative anaerobic bacteria such as Salmonella act differently. They are capable to identify and penetrate into
tumors by detecting chemotactic factors including molecular gradients of serine, aspartate, and ribose [86,103] .
Necrotic cancer cells release these compounds after being exposed to hypoxia for prolonged periods of
[103]
time . A strain of obligate anaerobic Salmonella has been associated with antitumor features by replacing the
[104]
asd gene with recombinant technology. Making the gene expressed only with hypoxia-inducible promoters
to maximize selective colonization. Selective colonization consists of the bacteria’s ability to be confined to the
[105]
tumor chaotic vasculature . An increase in cytokines production such as TNF-α is observed in response to
primary colonization of bacteria that leads to a secondary colonization [106] . In addition to this, auxotrophic
microorganisms-capable to grow in environments with nutrients produced only in tumor nests- have been
synthesized with mutations generated from null alleles lacking biological capability [107,108] .

Recently, bacterial motility has shown to be critical in tumor colonization. Many bacteria have flagella
that work with consumption of energy [109] . Bacteria use this feature to migrate and stay for longer periods
[83]
of time on places distal to tumor vasculature, in contrast to passive transport with chemotherapy . In
addition to this, differences between diffusion and pressure gradients limit movement of molecules by
passive transport and most of this happens on poorly perfused tumor areas [105] .

Immunostimulation in tumor microenvironment
There is no bacterium capable of completely inhibiting tumor growth just through colonization [110] .
However, it represents an important prospect for cancer treatment as an immunostimulator or as a vector
for therapeutic components that can be released inside a tumor [111,112] [Table 1].

The main theory for this approach comes from the bacterial intrinsic ability to immunostimulate after
colonizing tumor tissue. They can proliferate inside the tumour where an increased activity of the immune
+
+
system has been observed. Neutrophils, T CD8 and CD4 cells recruitment, cytokine and chemokine
release, potentiate immune response with no effect on the surrounding healthy tissue [130] . This approach has
shown better results than conventional therapy since it can affect healthy and cancerous tissue altogether.

Bacteria have one of the largest genomes that exist. They can express multiple therapeutic transgenes
and increase immune activity with cytokines and tumor antigens presentation [131] . They can transfer
those genes to eukaryotic cells and get expressed or repressed [132,133] . Systemic administration of cytokines
such as IL-2, IL-8, and CCL21Al may show certain limitations related to their short half-life and adverse
effects [134] . Their manufacture is highly expensive and they lack tumor orientation, which may cause severe
systemic inflammatory reactions restricting their clinical use [134] . In contrast, gene modified bacteria are
manufactured with low expenses, can be directed to specific tumor tissue, and may be easily eradicated with
antibiotics [114,135] . Bacteria in situ cytokine production may benefit those with difficult DNA recombination
methods and/or protein instability in production and purification. To achieve oncolytic activity genes are
introduced to increase cytokine expression and promote tumor regression [113] .

Antitumor activity can be achieved without significant toxicity and related to inflammatory cell
infiltration such as granulocytes, T lymphocytes and NK cells. Induction of intratumor production of