Fig 1.
The adherence and growth inhibition by P. mirabilis in vitro.
(A) P. mirabilis adhered to 4T1, MDA-MB-231, MCF7, and CHO cells in vitro. (B) The cells were counted by stained with Trypan Blue. Bacteria treatment showed dose- and time-dependent antiproliferative effects in 4T1 cell lines (C) In the colony formation assay, P. mirabilis inhibited colony formation of breast tumor cells and CHO cells by crystal violet staining. Data were expressed as the mean ± SD.
Fig 2.
Body distribution of P. mirabilis in tumor-bearing mice.
(A) The bacteria were detected in tumors and other organs, including heart, liver, spleen, lung and kidney at 0, 6, 24, 48, 72, and 96 hours after P. mirabilis injection. The number of bacteria in the liver, spleen and other organs gradually decreased with time; however, the number of bacteria in the tumor tissues increased. The means ± SEM of five mice per group were shown. (B) Surface ulceration of an allograft tumor was observed 24 hours after P. mirabilis treatment. (C) Morphological analysis of the tumor sections demonstrated tumor cell death and inflammatory cells infiltrated after bacteria intravenous injection.
Fig 3.
The therapeutic effect of P. mirabilis in 4T1 solid tumor model.
(A) Bacterial treatment suppressed tumor growth at the end of the experiment. Quantitative analysis demonstrated that the volume (B) and weight (C) of tumors in the P. mirabilis and CTX treatment groups were significantly less than those in the control group (p < 0.05). Corresponding to the images in (D) and (E) quantitative analysis of Ki-67 staining further confirmed that tumor proliferation was significantly inhibited in the treatment group (p < 0.05). (F) The body weights of mice significantly decreased at the beginning of treatment, but at the end of the experiment, they were not significantly different among these groups. (J) Morphological analysis of mice kidney tissues: there were no obvious differences in morphology between the P. mirabilis treatment group and the PBS control group. Data were expressed as mean ± SD.
Table 1.
Liver function detection of mice after P. mirabilis treatment.
Fig 4.
P. mirabilis suppressed spontaneous pulmonary metastases in vivo.
Bacterial treatment inhibited tumor spontaneous pulmonary metastases by observing fresh lung tissues (A) at the end of the experiment. (B) Quantitative statistics of metastatic foci showed significant differences between P. mirabilis treatment group and PBS control group (n = 6) (p < 0.05). (C) Quantitative statistics showed lung weight of mice in the control group was significantly higher than those in the P. mirabilis treatment group (p < 0.05). (D) Those results were further confirmed by analyzing H&E stained sections. Data were expressed as the mean ± SD.
Fig 5.
P. mirabilis treatment regulated the immune system in vivo.
Results of IHC indicated that the expression of NKp46 (A) and CD11c (B) in spleen sections was significantly increased after 24 hours bacteria treatment (p < 0.05). There were no differences of the expression of CD11b (C) and Ly-6G (D) between the control groups and the groups after 24 hours bacteria administration. (E) Quantitative analysis of immunohistochemistry staining for the expression of NKp46, CD11c, CD11b, and Ly-6G in the mice spleen tissues, respectively.
Fig 6.
P. mirabilis treatment alters markers of hypoxia in the tumor microenvironment.
(A) Western blotting demonstrated reduced expressions of CA IX and HIF-1a in the bacteria treatment group after 21 days of treatment; (B) The result was confirmed by immunohistochemistry for CA IX and HIF-1a in tumor sections of treated and control mice. CA IX positivity was localized in the cytoplasm and HIF-1a in the nucleus. (C and D) Quantitative analysis of immunohistochemistry staining indicated that CA IX and HIF-1a expression were lower in the bacteria treatment group than those in the control group (p < 0.05).