Anti-proliferative and pro-apoptotic effects of bacterial secreted cardiac protein
Incubation of MCF-7 human mammary carcinoma cells with growth media of bacteria bearing construct under hypoxic condition for a 48-h period, promoted both cell death and apoptosis. The results of MTT assay demonstrated that administration of the growth media of bacteria bearing construct, resulted in inhibition of growth in about 40% of MCF-7 cells which was significantly higher compared to growth media of bacteria without construct and PBS (25% and 5% inhibition respectively). (fig. 3A). The Annexin-V/7-AAD flow cytometry analysis also demonstrated that treating MCF-7 cells with the growth media of bacteria bearing construct significantly increases rate of apoptosis compare to bacteria without construct and PBS treated group (Fig. 3B).
Preferential colonization of E. coli BW25113 within tumor
Systemic administration of E. coli BW25113 in syngeneic mice bearing spontaneous mammary tumors resulted in elicit and preferential colonization of bacteria at tumor site. As depicted in figure 4A, bacteria accumulated in tumor site with an approximately 15-fold greater density compare to the liver. Furthermore, microscopic examination of tissue slides also confirmed that E. coli was present in all analyzed tumors. Accumulation of E. coli BW25113 was more significant at necrotic sites of tumor.
Confirming hypoxia-activated expression of cardiac peptides following tumor specific localization of E. coli Bw25113 usingIn vivo  fluorescence reflectance imaging
E. coli BW25113 bearing cardiac hormone expressing constructs and PBS were injected to the mice and images were collected immediately and 24 h, 3 days and 6 days post injection. As depicted in figure 4B, no significant differences in emitted fluorescent intensity was observed between two group during the first 24 h post injection. On day 3 however, emitted fluorescent from bacteria receiving group slightly grew and become a bit higher than those observed in PBS receiving group. Continuing similar trend, on day 6 post injection, emitted fluorescent from bacteria receiving group reached to its maximum and was completely restricted to the tumor site. Regarding other organelles, no significant changes in fluorescent intensity was observed during image acquisition period neither in control nor in bacteria receiving groups. These observations further confirm site specific colonization of bacteria and expression of GFP as an indicator of successful expression of construct in hypoxic condition.
Tumor growth retardation potential ofbacteria expressing cardiac protein
The delay in tumor growth was evaluated over a 24-day period after the administration of bacteria bearing construct with concentrations equal to 107 and 108 cfu/g, bacteria without construct in concentration equal to 108 cfu/g and PBS as the control group. Based on results, administration of bacteria without construct didn’t result in any significant tumor growth retardation compared to PBS receiving group. Therefore, bacteria per se were not capable of suppressing tumor growth speed. Contrarily, administration of bacteria bearing constructs, even at concentration equal to 107 cfu/g resulted in a significantly greater delay in tumor growth compare to PBS group. Furthermore, increasing the concentration of bacteria from 107 cfu/g to 108 cfu/g resulted in more enhanced tumor suppression. As depicted in figure 5A, differences in delaying tumor growth between two concentrations of bacteria bearing construct become significant on day 15 from beginning of treatment. Furthermore, the differences between delaying in tumor growth in group receiving concentrations of bacteria equal to 108 cfu/g and PBS group become significant on day 12. However, similar significancy for the group receiving bacteria bearing construct at 107 cfu/g took place on day 15. Based on these observations we concluded that first, the delay in tumor growth observed with administration of bacteria bearing construct is mainly due to the secretion of cardiac proteins. Second, increasing concentration of administered bacteria may result in enhancement of production and secretion of cardiac peptides. Administration of bacteria bearing constructs at both concentrations also significantly enhanced survival rate as observed in Kaplan-Meyer analysis (figure 5B).