IHC and histological examination
At the end of the 24-day treatment period, mice receiving PBS and E. coli BW25133 strain bearing construct with concentration of 108 cfu/g were sacrificed and tumors equivalent in size from both groups were selected for performing immunohistological analyses. Collected tumors were cut in to 7 µ m thick section on a microtome and stained using anti-Ki67 monoclonal antibody (ab15580, Abcam, USA), a marker for tumor proliferation; anti-MMP9 monoclonal antibody (ab38898, Abcam, USA), an enzyme involved in inducing angiogenesis and metastasis; anti-VEGF receptor 2 monoclonal antibody (ab2349, Abcam, USA), a proangiogenic growth factor; anti-CD31/PECAM-1 monoclonal antibody (ab24590, Abcam, USA), recognizing platelet–endothelial cell adhesion molecule-1 (PECAM-1) expressed on surface of endothelial cells; anti-CD8 monoclonal antibody (ab209775, Abcam, USA), an specific marker for cytotoxic T-cells; anti-CD4 monoclonal antibody (ab221775, Abcam, USA), an specific marker for helper T-cells; and their specific horseradish peroxidase-conjugated secondary antibody. Antigen recovery was performed in citrate buffer pH 6.0 and then blockade of endogenous peroxidase, as well as non-specific proteins was performed by 40 min incubation with 3% of H2O2 and another 40 min with 3% FBS. At the end, tumor sections were incubated with mentioned antibodies and extend of antigen expression was evaluated using horseradish peroxidase (HRP)-conjugated streptavidin and 2-Solution DAB kit (Life Technologies) according to the companies’ manual instructions. Finally, each antigen’s expression level was quantified using ImageJ software (NIH, Bethesda, MD, USA). Results were expressed as the mean of at least 5 tumor sections for each antigen.
H&E staining was also performed for evaluating tumor infiltrating lymphocytes (TILs) according to the previously established protocols. In Brief, initially, percentage of stromal lymphocytes were determined by two separate observers and then stromal TILs were measured as the percentage of immune cells with mononuclear immunological infiltrate characteristics. Findings were classified based on three cut-off points for TIL proportions including 10%, 30% and 50%.