Target gene therapy is one of the promising treatments of cancer due to its high effectiveness and safety [17, 29]. However, a crucial difficulty in performing this therapy is the lack of specific delivery system [29, 30]. Hypoxic regions are characteristics of solid tumors in rodents and occur with high frequency in many types of human tumors. Tissue oxygen electrode measurements taken in cancer patients show a median range of oxygen partial pressure of 10 mmHg to 30 mmHg in tumors, with a significant proportion of readings below 2.5 mmHg, whereas those in normal tissues range from 24 mmHg to 66 mmHg [31, 32]. The hypoxic environment advocates a basis for using anaerobe as specific delivery carrier.
Bifidobacterium infantis is a kind of Bifidobacteria that is strictly anaerobic . Many studies have found that they could specifically target to the anaerobic environment of the tumor center. Kimura et al.  injected Bifidobacterium bifidum intravenously into tumor-bearing mice to study its specific targeting property. They sacrificed the mice 48 and 96 hours after the injection and cultured the tumor tissue and other tissues. Kimura et al.  found that there were massive Bifidobacterium bifidum growing in the tumor tissue and no bacteria growing in other tissues, such as liver, spleen, kidney, lung, blood, bone marrow, muscle, and so on. Yazawa et al.  used Bifidobacterium longum as delivery system for gene therapy on tumor-bearing mice. They sacrificed 6 to 8 mice on 1, 2, 3, 4, and 7 days after the treatment,and cultured the lung, liver, spleen, kidney, heart, and tumor tissues. They found that Bifidobacterium longum could only specifically target the tumor tissue. In this study, the tumor and other tissues were also cultured after the mice were sacrificed. Many white colonies were observed in the medium culturing tumor tissue, whereas no colony was found growing in the medium culturing other tissues, such as heart, liver, lung, kidney, and spleen. Thus, Bificobacterium infantis has a very good targeting property to the anaerobic environment of tumor tissues.
Bifidobacterium infantis is a Gram-positive, domestic, and non-pathogenic bacteria found in the lower small intestine and large intestine of humans and some other mammalian animals. These intestinal organisms are believed to have health-promoting properties for their host, including increase of the immune response , inhibition of tumor growth, inhibition of carcinogenesis, and protection of the host against viral infection . In addition, Bifidobacterium can be killed easily by antibiotics or in oxygen environment in vitro or in vivo. Therefore, it is very safe to use Bifidobacterium infantis as delivery carrier. In this study, there were no obvious side effects on tumor-bearing mice after the intravenous delivery of Bifidobacterium infantis during the whole experiment process, which further proves its safety.
Antiangiogenesis therapy is one of the most important strategies for treating cancer [5, 13]. Tumor angiogenesis is a complex of coordinated interactions between numerous proteins involved in different signaling pathways. Each step provides an opportunity for therapeutic intervention. Angiogenesis mediated by VEGF constitutes a new target for anticancer therapy, which has been explored through different forms of intervention aimed at blocking tumor neovascularization. Strategies to inhibit tumor angiogenesis include inhibition of angiogenic factor production and their receptors, inhibition of the VEGF signaling pathway, inhibition of the binding between VEGF and its receptors, and inhibition of intracellular transduction of the VEGF signal [10, 13, 31]. The KDR is VEGFR-2, which is one of the most important receptors for binding with VEGF. The structure of VEGFR-2 contains an intracellular part, a transmembrane part, and an extracellular part which are all essential for signal transporting . The sKDR is the soluble form of the extramembrane part of VEGFR-2, which has same high affinity for VEGF but does not conduct signal. Thus, sKDR can bind with VEGF and compete with normal VEGFR-2 and can function as dominant negative by forming inactive heterodimers with membrane-spanning VEGF receptors [12, 38]. To date, many studies have proven that the specific binding of sKDR with VEGF could significantly inhibit the blood vessel formation in tumor tissue, thereby inhibiting the growth, proliferation and migration of tumor [15, 23, 31, 38].
In this study, in vitro experiment showed that the Bifidobacterium infantis-mediated sKDR prokaryotic expression system was successfully constructed and could express sKDR at both gene and protein levels. The products of this system could significantly inhibit the growth of HUVECs induced by VEGF. In addition, the in vivo experiment showed that Bifidobacterium infantis could specifically target to tumor tissue and express sKDR after intravenously injected. The blood flow signals and MVD of the tumor in group c were both lower than those of the other two groups, showing that the blood vessel formation in group c was significantly inhibited. At the same time, the tumors of mice in group c showed more serious necrosis rate and less growth rate. The mice in group c also exhibited longer survival time. All these results showed that the Bifidobacterium infantis-mediated sKDR prokaryotic expression system possessed anti-angiogenetic effects that further caused tumor necrosis and anti-tumor effects. Although this system could significantly reduce the MVD, increase the tumor necrosis and show greater tumor inhibitive rate, the effects on tumor volume and tumor weight seems to be week (Figures 4, 5A). We think the combined treatment of Bifidobacterium infantis-mediated sKDR prokaryotic expression system and chemotherapy or radiotherapy may be more effective and need further study in our future experiment.
In this study, Bifidobacterium infantis itself showed anti-tumor effects in some way. The results showed that the tumor volume in pTRKH2-PsT group was smaller than that in the control group, and the MVD and signals of blood flow in the tumor were less in pTRKH2-PsT group compared with the control group. The mechanism remains uncertain. Bifidobacterium infantis may be able to stimulate inflammatory response, induce the accumulation of immune cells, and promote the secretion of antitumor factors by macrophage. The structure of Bifidobacterium infantis cell wall had antitumor effects . The local proliferation of Bifidobacterium infantis competing with the nutrition of tumor cells may contribute to its antitumor effects as well. However, the expression of sKDR may play a more significant role, based on a comparison of group b and group c.