| Background:The spontaneous canine tumor is an ideal animal model for human cancer research,therefore it is very crucial to further investigate the molecular character,gene expression,and treatment of canine mammary tumor(CMT).A variety of bacteria have been used as agents and vectors as cancer therapy.The component of bacterial antitumor effect may be native bacterial toxicity,sensitization of the immune system and competition for nutrients.Objectives:The purpose is to research the effects of Proteus species on cancer growth and lung metastasis in CMT,and further to learn if the bacteria can regulate immune system and tumor hypoxia as a role on their antitumor effect.Methods:1.The CMT cells were established and purified from primary tumor by primary cultures and characterization by the ultrastructure analysis,growth studies and doubling time,karyotype analysis,soft agar assay,immunohistochemistry(IHC),immunocytochemistry(ICC),transplantation into mice,and mycoplasma detection.2.The cell adherence,growth inhibition,and colony formation assay were used to evaluate the effect of bacteria on breast tumor cell and normal cell in vitro.The ability to proliferate and accumulate in tumor,the efficacy and safety of the bacterial treatment in murine tumors were figured out after bacteria intravenous injection.3.The lipopolysaccharide(LPS)was extracted from P.mirabilis and injected to mice bearing tumor to figure out the effect of LPS on the tumor growth inhibition and lung metastasis by count the tumor volume and lung metastasis foci.Then ELISA,IHC and western analysis were then performed on excised tumors to study the mechanism of bacteria therapy.Results:1.In the present study,a novel triple negative canine mammary cancer cell line was successfully established,named CMT-7364.CMT-7364 cells showed stable growth for more than 100 passages.The primary tumor was diagnosed as the intraductal papillary carcinoma and a triple negative CMT by IHC.The cell line was characterized by epithelial origin.The karyotype analysis confirmed the canine origin of this cell line.The cell line was triple negative which showed negative immunoreactivity to Estrogen Receptor(ER),Progesterone Receptor(PR)and Human Epidermal Growth Factor Receptor-2(HER-2),and Epithelial-Mesenchymal Transitions(EMT)of the tumor cells.In vivo experiment,tumor mass and spontaneous metastases in the lung were found after injecting CMT-7364 cells to nude mice and immune-complete mice.2.In vitro,P.mirabilis had a non-specific effect of the growth inhibition on the tumor cell.In vivo,P.mirabilis localized preferentially to tumor tissues.And the number of bacteria in the tumor was increased with time and last a few days.P.mirabilis could remarkably suppress the growth of primary breast cancer and spontaneous pulmonary metastasis of mouse mammary tumor models in 21 days treatment,and this bacterial therapy was certainly safe.3.Furthermore,the result showed LPS had no effect on primary tumor and spontaneous pulmonary metastasis.Furthermore,the immune system was slightly sensitized by this bacteria therapy.The expression of carbonic anhydrase IX(CA IX)and hypoxia inducible factor-la(HIF-la)in the tumor tissue,surrogates for hypoxia,was significantly lower in the treated group than the control group mice by IHC and western analysis.Conclusion:In summary,the CMT-7364 cell line is a novel triple negative cell line that has been established,and has the tumorigenesis and metastasis potential on the immune-competent mice.Those findings demonstrated that Proteus mirabilis may a promising bacterial strain for used against primary tumor growth and pulmonary metastasis,and the immune system and reduction of tumor hypoxia may contribute to the antineoplastic and anti-metastatic effects observed. |
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