Experimental Study On The Inhibition Of Triple Negative Breast Cancer By Lactic Acid Bacteria

Background and Propose:Triple-negative breast cancer(TNBC)is a very aggressive cancer.accounting for about 15-20%of breast cancers.It is characterized by high recurrence and metastasis rate,poor efficacy and poor prognosis.Surgery and radiotherapy are the conventional treatments,but the efficacy is poor.Based on the tumor heterogeneity of TNBC,more detailed molecular typing may provide new drug references,and new targeted therapies and immunotherapy are under continuous development.with some new drugs such as PARP inhibitors Olaparib and Talazoparib.and ADC drug Sacituzumab govitecan being approved for specific patients.Overall,however,the available therapies are still relatively scarce.Lactic acid bacteria(LAB),one of the common intestinal commensal bacteria,has the ability to ferment lactose or glucose to produce lactic acid and has been widely used in the fermented food industry.It is closely associated with inflammation,immune regulation and has been shown anti-tumour effects in colorectal cancer,pancreatic cancer and melanoma.LAB can be classified into several genera,and those that have received more attention include Lactobacillus.Lactococcus.Enterococcus and Pediococcus.Some of these strains have shown the ability to affect breast cancer.They can affect breast cancer cell proliferation and apoptosis,and inhibit breast tumour growth in vivo in studies with potential therapeutic value.But there are relatively few studies on the inhibition of TNBC by LAB.We attempted to investigate the therapeutic potential of LAB in TNBC.We selected three strains of LAB.PB-01.PB-02 and PB-03.from the genera Pediococcus.Enterococcus and Lactobacillus.In vitro experiments,we cultured 4T1 cells with cellfree supernatant of the strains to assess their effects on cell function and apoptosis.In vivo experiments wetreated mice with fresh bacterial solution by gavage to assess their effects on tumours and immune environment.Methods:1.In vitro experiments:Cell-free supernatants of the selected bacteria were extracted and adjusted to pH 7.3.The supernatants were diluted with RPMI 1640 medium containing 10%fetal bovine serum and 1%penicillin,and concentration gradients of 30μL/mL,60μL/mL,90 μL/mL,120 μL/mL and 150 μL/mL were set.Finding the appropriate concentration to inhibit the proliferation of 4T1 cells by CCK-8.then used it in scratch test.Transwell migration and invasion test,and apoptosis assay to evaluate the effects on cell function and apoptosis.Investigating the effects on apoptosis-related genes Bcl-2,Bad.Bax,Fas-R,Cas-3,Cas-8,and Cas-9 by real-time quantitative PCR.2.In vivo experiments:(1)Testing the tolerance of the selected bacteria in the GI environment by artificial gastric juice and bile salt.Fresh bacterial solution was made at a concentration of 5×109 CFU/ml and each mouse was treated with 200 μL once a day to ensure the administration of 1 ×109 CFU of live bacteria.(2)Establishment of tumor-bearing mouse model:6-8 weeks old BALB/c female mice were used and the second left breast pad was injected with 1×106 4T1 cells to construct a TNBC model.(3)Treatment of 4T1 tumors with the selected bacteria and their effects on the immune environment:Mice were randomly divided into four groups of eight mice each,and treatment with fresh bacterial solution by gavage was started on the 5th day of tumour injection and continued for 10 days.Measured tumor volumes and weighed the mice three times a week.The changes of immune cells in the tumor and spleen were detected by flow cytometry.(4)Treatment of in vivo tumors by PB-03 combined with chemotherapy:Mice were randomly divided into 4 groups,named control group(G1),nab-paclitaxel group(G2),PB-03 group(G3)and combined drug group(G4),10 mice in each group.G3 and G4 were treated by gavage with fresh bacterial solution for 7 days before tumour injection,and all mice were injected on the 8th day.G3 and G4 were treated with fresh bacterial solution until the end of the experiment,and on the 12th day G2 and G4 were given continuous tail vein injections of nab-paclitaxel at a dose of 10 mg/kg for 5 days.Measured tumor volumes and weighed the mice three times a week.The experiment ended on the 21st day,and the measurements were analysed to draw conclusions about the effectiveness of the treatment.Results:1.All three supernatants showed significant inhibition of 4T1 cell proliferation after treatment with each concentration gradient after 6h(P<0.05).After 24h.the inhibitory effect of PB-01 and PB-02 supernatants remained significant at each concentration gradient(P<0.05).but PB-03 supernatant had no significant inhibitory effect at 30μL/mL and 60 μL/mL(P>0.05).only significant at 90 μL/mL,120 μL/mL.and 150μL/mL concentrations(P<0.001).After 24h,all three supernatants achieved more than 30%inhibition at 150 μL/mL.which met the experimental requirements and was therefore selected for follow-up in vitro experiments.2.In the scratch test,the PB-03 supernatant significantly inhibited the migratory ability of 4T1 cells after 6h of treatment(P<0.01),while the PB-01 and PB-02 groups were not significant(P>0.05).After 24h of treatment,all three supernatants showed significant inhibitory effects(P<0.05).In the Transwell migration and invasion test,the migratory and invasion ability of 4T1 cells was significantly inhibited after 24h of treatment by all three supernatants(P<0.0001).3.In the apoptosis assay,apoptosis of cells was significantly enhanced after 24h treatment by all three supernatants(P<0.0001).RT-PCR results suggested that PB-01 supernatant up-regulated Cas-3 gene expression in 4T1 cells(P<0.001).and PB-02 and PB-03 supernatants up-regulated Bax,Bad,Cas-3 and Cas-9 gene expression(P<0.05).4.All the selected bacteria well tolerated in the human gastrointestinal environment.PB-03 had significant tumor suppressive effect and reduced tumor weight(P<0.05),while the other two had no effect(P>0.05).Flow cytometry suggested that PB-01 significantly reduced the proportion of Tregs(P<0.01)and macrophages(P<0.05)in the tumor,PB-02 significantly reduced the proportion of macrophages(P<0.05)in the tumor,and PB-03 did not result in significant immune cell changes(P>0.05).PB-01 significantly increased CD103+CD4+T cells(P<0.05),but PB-02,PB-03 did not significantly affect splenic immune cells(P>0.05).5.Treatment of 4T1 tumors by PB-03 combined with chemotherapy showed significant tumor suppression in all three experimental groups(P<0.05).but there was no synergistic therapeutic effect between PB-03 and nab-paclitaxel.The combination of the two did not enhance the efficacy or change the weight loss of mice caused by nabpaclitaxel.Conclusions:PB-01,PB-02,and PB-03 inhibited 4T1 cell proliferation,migration,and invasion ability in vitro,promoted 4T1 cell apoptosis,and up-regulated apoptosis-related genes Bax,Bad,Cas-3,and Cas-9.PB-03 significantly inhibited 4T1 tumor growth in mice,but no synergistic effect with nab-paclitaxel was confirmed.PB-01 and PB-02 in vivo treatment did not show significant tumor suppressive effects,but PB-01 decreased Tregs and macrophages in tumors and increased CD103+CD4+T cells in spleens,and PB-02 downregulated macrophages in tumors.