A novel radio-sensitization method for lung cancer therapy: enhanced radiosensitization induced by antigens/antibodies reaction after targeting tumor hypoxia using Bifidobacterium CURRENT POSTED

BackgroundThe hypoxic microenvironment of solid tumors reduces the susceptibility of cancer cells to radiotherapy. Current treatments are focused on the development of anti-cancer agents that selectively target tumor cells with no toxicity to healthy tissue. Bacteria colonize and destroy tumors and have emerged as biological vectors that can survive in the tumor microenvironment. MethodsIn this study, a Lewis lung carcinoma transplant mouse model was established and treated with a combination of bifidobacterium infantis (Bi), its specific monoclonal antibodies (Ab) and radiotherapy (RT). 18 F-FDG PET/CT and 18 F-FMISO PET/CT imaging were performed to monitor tumor growth and hypoxia in the tumor tissue. Phosphorylated histone (γ-H2AX), the proliferation index (Ki-67), platelet endothelial cell adhesion molecules (CD31), tumor necrosis factor-α (TNF-α), hypoxia inducible factor-1α (HIF-1α) and glucose transporter 1 (Glut-1) levels were assessed through immunohistochemistry. ResultsThe results showed that the combined treatment group (Ab+ Bi+ RT) displayed delayed tumor growth and prolonged the survival of mice. The combined treatment group also had lower levels of HIF-1α, Glut-1, and CD31 expression, and a lower uptake of FDG and FMISO. The tumors treated with the combination therapy also had lower levels of hypoxia, and increased γ-H2AX and TNF-α expression. ConclusionTaken together, these data suggest that the combination of bifidobacterium infantis and its specific monoclonal antibodies can markedly improve the efﬁcacy of radiotherapy for the treatment of lung cancer. infantis can further alter the hypoxic state of tumor tissue. In this study, we tested this hypothesis in mice using bifidobacterium infantis, an anaerobic bacteria that is non-toxic analysis were performed using SPSS 17.0 software (Chicago, Illinois, USA). Comparisons between the groups were performed using a Student’s t-test and s one-way or two-way analysis of variance (ANOVA) for more than two groups. Survival curves were plotted using the Kaplan Meier method. Data are presented as mean ± standard error (SE). For all tests, two-sided p < 0.05 and < 0.01 and considered statistically signiﬁcant. charts Prism

a small drop of each suspension was observed by fluorescence microscopy (Leica TE2000-S Microscope, Tokyo, Japan).

In vivo evaluation of the anti-tumor effects
Mice were subcutaneously injected in the right flank with 0.1 ml of LLC cell suspension at a density of 1.0×10 6 /ml. Tumor volumes were calculated as × length × width 2 . Once tumors reached an average volume of ~150 mm 3 , tumor-bearing mice were randomly divided into the following 8 groups RT. Mice were immobilized using a shielded restrainer, which allowed the tumors to be exposed whilst sparing the areas surrounding the tumor. Each tumor received irradiation at a dose of 10 Gy. Tumors were measured every two days of treatment. Behavior, diet and other states of mice in each group were also recorded. Three mice per group were sacrificed after two days of treatment and tumor tissues were harvested for γ-H2AX evaluation by immunohistochemistry. Three mice per group were sacrificed after 10 days of treatment and tumor tissues were harvested for H&E staining and immunohistochemical analysis. The remaining mice were used to observe tumor growth and survival times. All animal care and experimental procedures were performed according to our institutional Animal Care and Use guidelines.
Mice were sacrificed and the liver, heart, spleen, lungs, and kidneys of each mice were prepared for H&E staining. To evaluate the degree of damage to normal tissue, histological changes in the organs were determined.

Micro 18 F-FDG and 18 F-FMISO PET/CT imaging
To investigate the early tumor response in each group, micro 18 F-FDG PET/CT imaging (Siemens, Munich, Germany) was performed. Briefly, at least three mice per group were starved for ≥ 8 h prior to PET/CT scans, and intravenously injected with 150-250 µCi FDG (0.1-0.2 ml) for at least 30 min.
Mice were then administered inhalation anesthesia and placed in the center of the PET/CT imaging field. The parameters used were as follows: 80 kV, 500 µA; 1.5 mm slice collimation; 10 min per bed position. To evaluate the oxygen content of the tumor tissue, micro 18 F-FMISO PET/CT imaging scans (Siemens, Munich, Germany) were performed. Similarly, at least three mice in each group were intravenously injected with 100-150 µCi FMISO for ≥ 30 min prior to scanning. Irradiation methods and specific parameters were as described for PET/CT scans.

Histopathology
Tumor tissues were obtained 48 h after the initial treatment for the immunohistochemical (IHC) detection of γ-H2AX. Tumor tissues from mice in each group were harvested 10 days post-treatment for other indicators of IHC (γ-H2AX, Ki-67, TNF-α, HIF-1α and Glut-1). Specific steps included: fixation of the tumor in neutral formaldehyde for at least 24 h, followed by paraffin-embedding. Tissues were cut into 3-4 mm thick sections and H&E stained. Paraffin sections were probed with anti-mouse γ-H2AX, Ki-67, CD31, TNF-α, HIF-1α and Glut-1 primary antibodies. Sections were then labeled with secondary biotinylated goat anti-mouse antibodies as per the manufacturer's instruction (Bioworld Technology, Nanjing, China). Sections were imaged using a Leica TE2000-S microscope (Tokyo, Japan). The number of γ-H2AX, Ki-67, TNF-α, HIF-1α and Glut-1 positive cells were calculated in five randomly selected fields (400× magnification) and are shown as the number of positive/total cells. In each tumor section, microvessel density (MVD) was calculated in five randomly selected areas (at 400× magnification) as the mean of CD31-positive microvessels.

Statistical Analysis
All statistical analysis were performed using SPSS 17.0 software (Chicago, Illinois, USA). Comparisons between the groups were performed using a Student's t-test and s one-way or two-way analysis of variance (ANOVA) for more than two groups. Survival curves were plotted using the Kaplan Meier method. Data are presented as mean ± standard error (SE). For all tests, two-sided p < 0.05 and < 0.01 and < 0.001 were considered statistically significant. All charts were designed using Prism 5.0 (GraphPad, La Jolla, CA, USA).

Bacterial morphology and antibody verification
After 72 h of anaerobic culture ( Figure 1A) the plates showed colonies with a milky white, translucent appearance, generally 1-2 mm in diameter with neat edges, which conformed to the growth characteristics of Bifidobacterium infantis (rod-shaped), ( Figure 1B). Compared to the control group (bifidobacterium infantis plus non-specific antibodies), the combination of bifidobacterium infantis combination with its specific monoclonal antibody, showed red fluorescence under the fluorescence microscope ( Figure 1C), indicating that the antibody is specific.

Antitumor Effect in Lung cancer Mouse Model
We next evaluated the antitumor effects of Bifidobacterium infantis and its specific monoclonal antibody in combination with RT on the growth of LLC tumors. The maximum dose of external beam radiation that could be delivered without causing morbidity was approximately 10 Gy [17] . The mice showed no discomfort after bacteria or antibody injection. Mice in the radiotherapy group lost weight and appetite, but recovered after two days. Tumor growth curves after 16 days of treatment are shown in Figure 2A These results suggest that Ab+ Bi+ RT was effective in inhibiting tumor growth and prolonging the survival time of tumor-bearing mice. Body weight was recorded every 2 days but no significant differences were observed between groups.

Micro 18 F-FDG and 18 F-FMISO PET/CT imaging
The SUVmax values of 18 F-FDG imaging for each group were 6.12 ± 0.15 for control, 5.59 ± 0.08 for Ab, 5.28 ± 0.07 for Bi, 5.03 ± 0.04 for Ab+ Bi, 2.89 ± 0.06 for Bi + RT, 3.36 ± 0.07 for RT and 3.21 ± 0.08 for Ab + RT, 1.20 ± 0.05 for Ab + Bi+ RT. Upon comparison to other groups, mice in the Ab+ Bi+ RT group had the lowest SUVmax, suggesting lower metabolism and a more powerful antitumor effect of Ab+ Bi+ RT (P 0.001), which was consisted in general view of the tumor xenograft model ( Figure   3A). Similarly, following 18  When compared to other groups, mice in the Ab+ Bi+ RT group had the lowest SUVmax, suggestive of a low oxygen content in the tumor tissue (P 0.001).

IHC analysis
The expression of γ-H2AX was evaluated via immunohistochemistry ( Figure  The expression of CD31, an endothelial cell surface molecule that can be used to investigate MVD, was determined by immunohistochemistry ( Figure 6A).The MVD in each group were as follows: control Ab Bi Ab+ Bi RT Ab+ RT Bi+ RT Ab+ Bi + RT ( Figure 6B). The MVD of the tumor tissue derived from mice in the Ab+ Bi + RT group (0.67 ± 0.57) was significantly lower than mice in the Bi+ RT group (3.48 ± 0.87) and other groups (P 0.01 in all cases). The MVD was also significantly lower in the Ab+ Bi + RT group.
The expression of TNF-α, a marker of apoptosis, was determined by immunohistochemistry ( Figure   6A). As shown in Figure 6C, the Ab+ Bi+ RT group had significantly increased apoptotic rates (81.60% ± 3.15%) compared to the control group (3.74%±1.12%), Bi+ RT group (42.14% ± 3.13%) and other groups (P 0.001 in all cases). These data indicated that AB+ Bi dramatically enhanced radiationinduced cellular killing effects when combined with RT.
Glut-1 is an endogenous marker of hypoxia as was assessed via immunohistochemistry ( Figure 7A).
The percentage of Glut-1 positive cells differed across the different treatment groups in the order: Ab+ Bi+ RT < Bi +RT < Ab +RT <RT <Ab +Bi <Bi <Ab <Control ( Figure 7B). A reduction in the number of Glut-1 positive cells was observed in tumor tissues derived from mice in the Ab+ Bi+ RT group (13.20% ± 3.34%) compared to mice in the RT group (53.32% ± 2.35%), Ab + RT group (51.63% ± 2.97%), Bi+ RT (45.32% ± 2.76%) and other groups (p<.0001 in all cases). Taken together, these data show that the combination treatment of Ab +Bi +RT significantly improves the oxygen content of tumor tissue.
The expression of HIF-1α was evaluated via immunohistochemistry ( Figure 7A). As shown in Figure   7C, the percentage of HIF-1α positive cells in the tumor tissue derived from Ab+ Bi+ RT-treated mice

In vivo side effects
We investigated the side effects of Ab or Bi to investigate organ toxicity. After mice were euthanized, the heart, liver, spleen, lungs, and kidneys were dissected and prepared for H&E staining. As shown in Figure 8, no obvious pathologic changes were observed in any of the organs.

Discussion
Radiation therapy is one of the most broadly anti-cancer treatments acting on a wide range of tumor types [9] . The lack of oxygen in solid tumors is considered a major reason for tumor resistance to radiotherapy and chemotherapy [22] , and a driving force for malignant transformation [13,23] .
Targeting hypoxia represents an effective means to improve cancer treatment. Although the hypoxia of solid tumors has lowered treatment efficacy, the hypoxic microenvironment serves as an ideal habitat for a number of anaerobic bacteria. Anaerobic bacteria have been used experimentally as anticancer agents due to their selective growth in the hypoxic/necrotic regions of solid tumors after systemic administration. Bacteria can actively migrate from the vasculature and penetrate the necrotic region of tumors. Previous studies have shown that bacteria can enhance the therapeutic effects of radiation by killing the regions of the tumors that are resistant to radiation therapy due to their low oxygen content. Bifidobacterium is an obligate anaerobic bacterium that colonizes large tumors due to their anaerobic environment. In contrast to Clostridia, bifidobacteria are nonpathogenic, non-spore-forming, and naturally found in the digestive tract of humans and other mammals. As such, live bacterial agents can enhance the treatment of tumors [24] .
We established a lung cancer xenograft model in C57BL/6 mice to explore the radiation enhancement of Ab+Bi. The results described above confirmed our hypothesis that Ab + Bi can significantly improve radiotherapy in an experimental settings. During the study, a series of observations were used to assess the therapeutic effects of RT combined with Bi and Ab. Mice treated with Ab+Bi+RT showed the highest therapeutic effects amongst the treatment groups regarding tumor volume and survival rates. This further indicated that combination therapy was more effective in a lung cancer xenograft model. The positive effects of this treatment were also assessed using 18 F-FDG PET/CT imaging. Several studies have shown that 18 F-FDG PET/CT can recognize early decreases in glucose metabolism that are associated with progression-free survival and overall survival in many cancers [25][26][27] . Unlike normal cells, the majority of the energy used by tumor cells relies on glycolysis, mediated by glycometabolic pathways as opposed to aerobic oxidation [28] . Thus, inhibiting proliferation can weaken glycometabolism. A higher 18 F-FDG uptake is indicative of a poorer therapeutic response, whilst lower uptake suggests an improved outcome. 18 F-FDG PET/CT can therefore predict tumor malignancy in a clinical setting. Our data showed that the combination treatment group had the lowest levels of uptake compared to the Bi +RT group, although 18 F-FDG uptake in mice in the Bi+ RT group was lower compared to that of other groups. This suggested that Bi in combination with RT could inhibit tumor metabolism, but when Ab +Bi was combined with RT, the inhibition of tumor metabolism was more significant. 18 F-FMISO specifically concentrates in hypoxic tissue and is used to evaluate hypoxia in experimental [29,30] and human [31,32] tumors using positron emission tomography (PET). Our data suggest that combination therapy leads to the lowest 18 F-FMISO uptake compared to other groups, indicating that the combination of Bi with Ab can improve the oxygen content and enhance the radiation effect.
DNA is the major target of ionizing radiation that induces base damage, sugar damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), of which DSBs induce histone H2AX phosphorylation, leading to cell death. Histone γ-H2AX is an important sensor of DSBs after irradiation treatment [33,34] . In our study, the expression of γ-H2AX was investigated as a biomarker for DSBs. In this study, mice in the Ab+ Bi+ RT group had a significantly higher expression of γ-H2AX compared to mice in the other groups. Therefore, the radiation enhancement of Ab+Bi was due to enhanced X-ray-induced DSBs.
Under regular physiological conditions, angiogenesis results in a structurally well organized and highly efficient network of vasculature. However, in tumors, angiogenesis leads to a chaotic network of blood vessels, a distinctive component of the cancer microenvironment. This vasculature can be characterized by disorganization, dilation, branching, shunts, and varied diameters that result in an inconsistent blood flow and hypoxic areas and regions of high acidity [35,36] . As previously reported, a lower MVD is related to slower tumor growth and improved biologic and clinical behavior [37][38][39][40] . Mice in the Ab+ Bi+ RT group showed the lowest expression of CD-31 and Ki-67 compared to mice in other groups. These results indicate that Ab+ Bi+ RT inhibited the growth of tumor cells by inhibiting tumor angiogenesis and proliferation.
Tumor necrosis factor-α (TNF-α) participates in programmed cell death. Tumor necrosis occurs as a result of endothelial cell apoptosis caused by the deactivation of integrin α v β 3 and the disruption of the interface with the extracellular matrix (ECM), followed by T-cell activation to remove the remaining tumor cells [41,42] . Mice in the Ab+ Bi+ RT group showed the highest expression of TNF-α, suggesting that Ab+ Bi+ RT plays a significant role in tumor cell apoptosis. Glut-1 is the major glucose transporter in tumor cells that provides glucose for energy production [43] . Accordingly, Glut-1 expression closely correlates with tumor invasiveness and poor prognosis. Mice in the Ab+ Bi+ RT group showed the lowest expression of Glut-1 compared to other groups. These results indicate that the combination treatment group inhibited the growth of tumor cells by inhibiting tumor glucose uptake with a lower availability of glucose in vivo. Hypoxia not only increases tumor aggressiveness, but causes chemoradiotherapy resistance in tumor cells, which is closely related to tumor progression [44,45] . HIF-1α mediates the proliferation, migration, invasion and angiogenesis of tumor cells, and In addition, staining of the heart, liver, spleen, lung, and kidneys from each of the treatment groups was evaluated microscopically (Figure 8). Notably, no significant morphological changes in these organs were observed, indicating that the combination therapy had no obvious adverse impact on normal tissue.
Generally, when Bi was combined with RT, good radiation enhancement is observed, whilst no significant differences occurred between mice in the RT and Ab+ RT groups. Ab+ Bi + RT showed outstanding radiation enhancement in lung cancer xenograft models. Bifidobacterium infantis targets the components of tumors least sensitive to radiation, as they are poorly oxygenated. Additionally, recent experiments suggested that the damage to microvascular endothelial cells is an important component of the radiation effects [46] . Such microvascular damage would increase the niche for bifidobacterium infantis growth by creating a larger number of hypoxic areas within the tumors, thereby exacerbating bacteriolysis. When specific antibodies are combined with the bacteria implanted in the hypoxic area, radiation therapy was further Improved. Although these data are promising, some limitations should be noted. Bacteria and its specific antibodies in tumor induced apoptosis through the competition for nutrients and stimulation of the immune response. Therefore, more detailed in vivo studies on the effects of bacteria combined with its specific antibodies that target the tumor microenvironment will be further explored.