Application Of Sodium Alginate Microspheres In Fecal Microbiota Transplantation And Tumor Antigen Immunotherapy

Purpose:Following the study of treating mouse ulcerative colitis(UC)with fecal microbiota transplantation(FMT),sodium alginate microspheres(SAM)were made to study its application in FMT and immune activation with mouse melanoma cell B16 antigen.We hope to development a novel method to treat melanoma.Methods:1.BALB/C mice were randomly assigned into groups.Dextran sulfate sodium(DSS)was used to make UC model.The method of Orifice Bath Solidification was used to make microspheres containing the fecal microbiota(FM).These microspheres were used to treat UC mice through gavage.Another group of mice were treated with FMT.The body weight and disease activity index(DAI)were measured.Mouse colons were taken for histochemistry study.Changes in microbiota were studied using 16 S rRNA gene sequencing.2.C57BL/6J mouse model of melanoma was established.Dendritic cells(DC)activated by B16 cell antigens inhibit melanoma.Tumor growth was followed and compared between groups.Mouse spleen tissues were studied by histopathology.SAM containing B16 cell antigens were made using the Micro Emulsion method.Microspheres were used to immunize mice and then serum levels of IFN-γ,IL-12,IL-6 and TNF-α were studied by enzyme-linked immunosorbent assay(ELISA).Results:1.Microspheres containing fecal microbiota were made with an average diameter of about 500 μm and this is suitable for gavage.Acid resistance and enteric solubility test showed that SAM is resistant to the strong acid environment of stomach.It disintegrated in gut fluid and colon fluid to release its content.After mouse UC model was established using DSS,mice showed a significant drop in weight,significant increase in DAI.Treatments were started seven days after DSS treatment.Mouse body weight began to increase the second day following treatment with FMT and SAM containing FM and DAI began to decrease.The treatment was continued for 7 days.In control mice and mice treated with control microspheres,the body weight kept decreasing and DAI kept increasing.These control mice died 11 days after treatment or were euthanized because of severe diarrhea(3 days after DSS stopped).Histochemistry results showed ulceration,bumps and decreased number of crypts in the colon mucosal surface in UC mice.After treated with FMT or SAM containing FM for 7 days,the colon mucosa appeared to be similar to that of the normal mice with a smooth surface.16 S rRNA gene sequencing results showed that there are more Spiral bacillus,Escherichia coli and Bacteroides,but less lactobacillus in UC mice as compared with normal mice.After treated with SAM containing FM,the numbers of Spiral bacillus,Escherichia coli and Bacteroides were reduced while that of lactobacillus was increased in UC mice.2.Results of mouse melanoma experiments: mice inoculated with B16 cell antigens-activated dendritic cells(DC)showed no tumor growth;mice inoculated with B16 cells for 1 week followed by another inoculation with B16 cell antigens-activated DC showed significantly reduced tumor size as compared with the mice inoculated with B16 cells only.C57BL/6J mice inoculated with B16 melanoma for 14 days showed significantly changed spleen structure with increased spleen follicles;mice inoculated with B16 cell antigens-activated DC for 14 days had significantly decreased spleen follicles.Mice inoculated with SAM containing B16 cell antigens showed significantly increased serum IFN-γ and IL-12 levels.Conclusion:Treating UC mice with SAM containing FM showed certain beneficial effects and can be considered as an alternative of FMT;melanoma cell antigens-activated DC can inhibit the tumor growth;immunizing mice with SAM containing B16 cell antigens can increase serum levels of IFN-γ and IL-12 and has potential anti-tumor effects.