Bifidobacterium Infantis As A Delivery System For Cancer Gene Therapy: Targeting Localization And Growth In Hypoxic Region Of Tumors

Objective:Explore the targeting effect of bifidobacterium infantis on tumor tissue of nude mice model of nasopharyngeal carcinoma. Based on the foregoing, further explore the target distribution of bifidobacterium infantis in hypoxic regions of the tumor tissue. Method: Establish a nude mice model of nasopharyngeal carcinoma with a plug kit, continuously feed the mice for one to two weeks, select the tumor-bearing mice whose tumor volumes reach 100 mm3 and randomly assign them into a control group and an experiment group, each of which includes 8 mice. Perform a tail intravenous injection of bifidobacterium infantis suspension of 0.2ml(number of bacteria in the suspension totaling 3×106-6×106 cfu) to each mice in the control group, and inject aseptic PBS of 0.2ml into each mice in the experiment group. The mice are then sacrificed after seven days and 40 minutes before their deaths they are administered pimonidazole hydrochloride of 60mg/kg by intraperitoneal injection. After killing the mice by disjointing their cervical vertebrae, take their livers, lungs, kidneys, spleens, hearts and tumor tissues. Halve the tumors and the spleens. Put one half of the tumor(spleen) in formalin for fixation and another half in grinding cylinder for homogenation and mixed cultivation in plates, respectively. The rest of the organs are directly put in grinding cylinder for mixed cultivation after homogenation. Count the bacteria colonies cultivated in the plates after 72 hours of anaerobic cultivation. Compute the total number of bacteria (cfu/g) in each sample in accordance with an equation, to learn colonization of the anaerobic bacteria in the control group and the experiment group, and accumulation of the anaerobic bacteria in tumor and in other normal organs and tissues in the experiment group. Further, slice the formalin-fixed and paraffin-embedded tumor and spleen tissues, immunofluorescence stain the slices and put them under fluorescence microscope for observation and photography. Analyze the obtained images with IPP image analysis software and compute the distribution ratios of bifidobacterium infantis in hypoxic regions, necrosis regions and rich-oxygen regions of the tumor issue. Then, conduct a statistic analysis of the obtained data with SPSS 13.0 to learn the distribution characteristic of bifidobacterium infantis within the tumor. Result:Count the bacteria colonies after tissue homogenate anaerobic cultivation. For the tumor-bearing mice that have not been subject to bifidobacterium infantis injection(control group), no anaerobic bacterium is found in the homogenate anaerobic cultures of their tumor tissues and normal liver, heart, lung, spleen and kidney tissues. For the tumor-bearing mice that have been subject to bifidobacterium infantis injection(experiment group), after tissue homogenate anaerobic cultivation of their tumor tissues and normal liver, heart, lung, spleen and kidney tissues, it is found that the bifidobacterium infantis mainly accumulate in the tumor tissues and almost not accumulate in the normal liver, heart, lung, spleen and kidney tissues. Thus, a ratio of the amount of the bacterium in the tumor to the amount in normal tissue exceeding 300-500:1 is realized.1. This difference has its statistical significance (P<0.05).2. After immunofluorescence staining of the tissue slices, quantitative analysis of distribution ratios of the bifidobacterium infantis within each region of the tumor reveals that the distribution of bifidobacterium infantis in hypoxic regions of the tumor is significantly higher than its distributions in the necrotic regions and rich-oxygen regions of the tumor(P<0.01). This difference has its statistical significance. It also reveals that the distribution of bifidobacterium infantis in the necrotic regions of the tumor is significantly higher than its distribution in the rich-oxygen regions of the tumor(P<0.05).This difference has its statistical significance. Conclusion:in nude mice model of nasopharyngeal carcinoma, the tail intravenous injected bifidobacterium infantis has good targeting distribution capability for tumor tissue and is mainly distributed in hypoxic regions of the tumor. The targeting distribution capability of bifidobacterium infantis with regard to tumor tissue makes it a gene treatment carrier for delivering medicine to the tumor's hypoxic regions. Thus, this experiment may experimentally support that gene targeting treatment medicine directing to tumor hypoxic region can fully exert its anti-tumor power.