| Backgrounds:With the development of cancer research,immunotherapy,especially immune checkpoint block(ICB)therapy,has been proved to be effective in the treatment of many solid tumors,including colorectal cancer(CRC).However,the effectiveness of ICB therapy is limited by the variability of the immune response of different patients and the sensitivity to tumor types.Therefore,it can be said that only a small number of patients can really benefit from the treatment of immunotherapy.However,subsequent studies have found that the limitation of the ICB therapy is closely related to the number and composition of intestinal bacteria in the human body to a certain extent.It is expected to improve this limitation by regulating the composition and number of intestinal bacteria in the human body.Therefore,the combined application of intestinal bacteria and ICB therapy has attracted the attention of scholars at home and abroad.The traditional methods of delivering bacteria into the body can be roughly divided into:intravenous administration,perfusion administration(anal/urethral),intraperitoneal injection administration,and oral administration.Compared with oral administration,injection method is likely to cause bacteria into the bloodstream,and then cause a series of serious infectious diseases such as bacteremia;and the infusion method will reduce the patient's compliance.Therefore,oral administration can be said to be the best method of bacterial delivery for patients with the highest compliance,convenience,and safety.However,due to the highly acidic environment of the stomach and the presence of bile salts in the intestinal tract,direct delivery of bacteria through the oral cavity will result in a very serious loss of bacterial activity.Therefore,selecting biomaterials with acid resistance and pH-responsive release characteristics to encapsulate these bacteria into microcapsules is an effective method to reduce the death of bacteria during the passage of the gastrointestinal tract and control the release of these bacteria in the intestinal tract.Aims:In this study,Bifidobacterium was selected as the research object.In order to understand the characteristics and functions of this bacterial family in more detail,Bifidobacterium longum,Bifidobacterium breve and Bifidobacterim adolescentis were selected from the subspecies level.And prepared chitosan/alginate microgel as a bacterial carrier to deliver bifidobacteria,thereby protecting bacteria from gastric acid in the stomach,and releasing the entrapped Bifidobacterium after reaching the colonic intestine.Furthermore,Bifidobacterium-microgel and small molecule programmed cell death ligand 1(Programmed cell death ligand 1,PD-L1)inhibitors were further combined to study the anti-tumor effect of secretory substance and immune level in the Bal/bc mouse orthotopic rectal cancer model.We explored the above system in vitro and in vivo.Methods:In vitro,via the 24h MTT experiment of the bacterial liquid,the pH detection of the bacterial liquid,and the short-chain fatty acid(SCFA)component detection experiment of the bacterial liquid,we studied the inhibition effect of three Bifidobacteria on tumor cells and the properties of bacteria fluid and SCFA.After microgels were prepared,via using confocal laser scanning microscope(CLSM),scanning Electron Microscope(SEM),and bacteria count experiment in the initial microgels in vitro,the appearance and encapsulation efficiency of the microgels were characterized by microscopic observation,live and dead staining experiment.Then,microgels were placed in simulated gastric fluid(SGF)(37?,2h)and simulated intestinal fluid(SIF)(37?,until Rupture)to simulate the digestion process in vitro.Moreover,we detected the number of surviving bacteria and the number of bacteria released during this process.And we also calculated the swelling rate and rupture rate of the microgels.In vivo,in order to simulate the tumor growth environment more realistically,we chose CT-26 mouse colon cancer cells to construct an orthotopic rectal cancer animal model under the rectal mucosa of Bal/bc mice.The treatment was started 14 days after the inoculation of tumor,denoted as D0.In D0,D5,D10,D15 and D20,small animal in vivo imaging equipment was used to observe the tumor growth in mice.At the same time,Bifidobacterium-microgels were delivered by gavage on D0,D7 and D14.For the combination therapy study,PD-L1 small molecule was injected intraperitoneally on the basis of this experiment,and then administered at D0,D4,D8,D12 and D16.Through the tumor anatomy photography,tumor weighing and in vivo imaging experiments,the tumor suppression effect of each group was compared.Immediately,the biological safety of the treatment plan was verified by measuring the weight of the mouse and testing the function of liver and kidney.Moreover,flow cytometry,tumor-related cytokines,and SCFA respectively showed the anti-tumor effects of the treatment from the level of immunity and secretion and metabolism.In addition,HE staining,Caspase-3 and Ki67 immunofluorescence staining were used to explore the proliferation and apoptosis of tumor tissues.Through immunofluorescence staining of tumor tissue PD-L1 protein and RNA Scope technology of Bifidobacterium in the distal and proximal rectal mucosa of the tumor,we showed the content of PD-L1 in the tumor of each experimental group to study the inhibition of Bifidobacterium and PD-L1.At the same time,results of RNA Scope test can reveal the distribution of Bifidobacteria in the intestines,so as to study the aggregation and distribution characteristics of Bifidobacteria.Results:In vitro,we have preliminarily confirmed that the secretion function of three species of Bifidobacteria can inhibit the growth of CT-26 mouse colon cancer tumor cells,and confirmed the acetic acid secretion function of three kinds of Bifidobacteria.The related characterization of the prepared microgels in vitro confirmed that the encapsulation keeps the activity of bacteria and the Bifidobacteria can be entrapped efficiently.In addition,chitosan/alginate microgels can protect Bifidobacteria from gastric acid,have good encapsulating performance and efficient delivery performance.Compared with free bacteria almost all dying in SGF,the microgels can completely rupture 18 hours after protecting the Bifidobacteria in vitro simulation process to ensure that the bacteria can safely reach the colon,and has a good bacterial delivery performance.In animal models(in vivo),relevant experiments have confirmed that the three kinds of Bifidobacterium can inhibit the growth of tumor,and the effect is further strengthened in the combination with PD-L1 inhibitors.In addition,we have confirmed that neither the Bifidobacterium-microgels nor the PD-L1 inhibitor would produce toxicity in the body and had good biological safety.Moreover,flow cytometry,tumor-related cytokines,and SCFA tests respectively confirmed from the level of immunity and secretion and metabolism that the treatment plan can promote the level of anti-tumor immunity and cause the increase of butyrate(anti-tumor substance)in the body.HE staining,Caspase-3 and Ki67 immunofluorescence staining confirmed that the treatment plan inhibited tumor proliferation and promoted tumor apoptosis.In addition,the PD-L1 protein immunofluorescence staining of tumor tissues and the RNA Scope technology of Bifidobacterium in the distal and proximal rectal mucosa of the tumor confirmed that Bifidobacterium can promote the high expression of PD-L1 and improve the efficacy of PD-L1 inhibitors.Bifidobacteria in the colon have a certain degree of aggregation around the tumor and have certain tumor targeting properties.Conclusions:1.The chitosan/alginate microgels prepared by surface coating method can be used as a microcarrier for Bifidobacterium.In the preparation process,it can maintain the activity of most bacteria,and can play an obvious protective role against bacteria in the environment of SGF and SIF respectively.And it can be maintained for up to 18h before it ruptures completely in this study.The encapsulation scheme ensures that the bacteria can reach the colon safely and efficiently,and has good bacterial delivery performance.2.In vivo,we constructed a mouse orthotopic rectal cancer model to study the anti-tumor effects of Bifidobacterium-microgels alone or in combination with PD-L1 small molecule inhibitors.The experimental results confirmed that each kind of Bifidobacterium-microgels has a certain degree of anti-tumor effect,especially the Bifidobacterium longum group,and the combination with PD-L1 inhibitor can further improve the anti-tumor effect.The anti-tumor efficacy is manifested in changes in the level of immune cells,an increase of anti-tumor-related cytokines,and bacterial anti-tumor metabolites.At the same time,the combined treatment is internally related and possesses good biological safety.3.In vivo experiments have confirmed the antitumor effect of small molecule PD-L1 inhibitors,which is similar to the monoclonal antibody immune checkpoint inhibitors(ICIs)currently used in experiments or clinic.Because of the advantages of low price and good absorption,small molecular ICIs can be better used.The results of this study confirmed the efficacy of small molecular ICIs.In summary,these three kinds of Bifidobacteria-microgels alone or in combination with PD-L1 small molecule inhibitors for the treatment of orthotopic rectal cancer animal models can effectively inhibit tumor growth and induce tumor killing effect from the secretion level and immune regulatory level.In addition,this treatment can significantly inhibit tumor proliferation and promote tumor apoptosis,which has good biological safety and promotion value.At the same time,Bifidobacterium-microgels and PD-L1 inhibitors still have intrinsic correlation,which has the theoretical basis of combined application,and can provide new ideas and better ways for tumor treatment. |
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