A Novel Tumor Vaccine Based On Tumor-Derived Microparticles Crosslinked With Lipopolysaccharide For Lung Adenocarcinoma Treatment

BackgroundTumor antigens can stimulate anti-tumor response through the recognization,uptake and presentation of immune cells,which is an essential process in the occurrence of antitumor response.As the understanding of cancer and its immune has been deepened,with the help of proper adjuvants,effective delivery carriers and administration methods,tumor antigens can be recognized and activate anti-tumor immune response in the body.Inorganic vectors such as liposomes have been applied in the delivery of antigens,showing good targeting ability and anti-tumor efficacy with adjuvant modification and drug loading.However,low histocompatibility of these inorganic materials could limit their application in clinical practice.Therefore,a more efficient and safer system is needed.Tumor-derived microparticles(TMPs)are secreted from tumor cells,containing tumorassociated antigens.TMPs have natural targeting,immunogenicity and high histocompatibility.It has been proven that TMPs could be uptaken by immune cells,such as macrophages and dendritic cells in the tumor microenvironment,influencing their functions.Based on the features of TMPs,theoretically,they can be further improved and applied as a promising platform for tumor antigen delivery.Therefore,we decided to create a TMPs-based nanomaterial as platform for delivery of tumor antigens.To promote stronger immune repose,adjuvants also play essential roles.Adjuvants are auxiliary parts of vaccines that can be together with the vaccine or pre-injected into the body and enhance the body’s immune response to antigens or alter the type of immune response.Lipopolysaccharide(LPS)is an active component of the bacterial endosome and exists in bacterial outer membrane vesicles.It has been revealed that LPS is included in the family of the efficient catalytic agents of immune cells,involving CD8+T cells through interaction with TLR4.Several studies have shown that it could perform as a potent adjuvant in different vaccines.Previous studies have confirmed the anti-tumor effect of bacterial outer membrane vesicles and proven that they could be easily uptaken by immune cells.All in all,LPS can function as an effective adjuvant and camouflage TMPs to increase uptake and recognition.Therefore,we modify TMPs with LPS using cationic crosslinker PEI to construct novel tumor vaccine TMPs-PEI-LPS to enhance tumor therapeutic effects.Methods1.Preparation and characterization of TMPs-PEI-LPS1.1 Synthesis of TMPs-PEI-LPS via supercentrifugation and co-incubation;1.2 Fluorescence microscopy and flow cytometry methods were applied to identify the success of TMPs-PEI-LPS synthesis;1.3 The morphology and size of TMPs-PEI-LPS were evaluated by transmission electron microscopy(TEM)and Dynamic light scattering(DLS);1.4 The markers of TMPs-PEI-LPS were identified by western blot(WB)and flow cytometry.2.Exploration of anti-tumor mechanism of TMPs-PEI-LPS in vitro2.1 DC2.4 cells were incubated with pre-fluorescently labeled TMPs-PEI-LPS,and then the cellular uptake was detected by fluorescence microscopy and flow cytometry;2.2 DC2.4 cells were treated with TMPs-PEI-LPS,before the maturation of these cells was assessed by flow cytometry and ELISA;2.3 Original BMDCs were extracted from the limb bone marrow of C57BL/6 mice.After 8 days of stimulation with GM-CSF,BMDCs were collected and treated with TMPsPEI-LPS.Subsequently,the maturation of BMDCs was confirmed by the experiment involving flow cytometry and ELISA;2.4 The apoptosis of RAW264.7 cells was detected by flow cytometry,after they were stimulated with TMPs-PEI-LPS;2.5 The polarization of RAW264.7 cells was estimated by flow cytometry,after they were stimulated with TMPs-PEI-LPS.3.Anti-tumor effect and mechanism of TMPs-PEI-LPS in vivo3.1 Firstly,LLC subcutaneous tumor models were constructed.DIO-labeled TMPs-PEILPS were injected into C57BL/6 mice via subcutaneous injection or tail intravenous injection,to detect the distribution of the nanomaterials through in vivo imaging;3.2 DIO-labeled TMPs-PEI-LPS were injected into LLC subcutaneous tumor models subcutaneously and intravenously respectively.Lymph nodes and spleen were obtained to prepare single-cell suspension.Afterwards,flow cytometry was used to evaluate the uptake rate and immunostimulating activity;3.3 After mice bearing subcutaneous tumors were treated with TMPs-PEI-LPS,vital organs and serum were obtained to evaluate biosafety of the tumor vaccine via performing H&E staining of ograns and estimating serum biochemical indexes;3.4 While mice were intervened by TMPs-PEI-LPS,volume of subcutaneous tumors and body weight of each mouse were kept track of to evaluate the efficacy;3.5 In the end of intervention,in situ immunofluorescence staining was carried out to assess the infiltration of immune cells in the tumor tissue.Results1.Preparation and characterization of TMPs-PEI-LPS1.1 TMPs-PEI-LPS was synthesized successfully;1.2 Under TEM,TMPs-PEI-LPS was elliptical and had intact vesicle membrane.The average particle size was 480.6nm;1.3 TMPs-PEI-LPS expressed protein markers of tumor-associated micropaticles,including CD63 and TSG101.2.Anti-tumor mechanism of TMPs-PEI-LPS in vitro2.1 The uptake of TMPs-PEI-LPS by DC2.4 cells were highly enhanced after 24 h stimulation;2.2 TMPs-PEI-LPS promoted the maturation of DC2.4 cells;2.3 BMDCs maturation could be induced by TMPs-PEI-LPS;2.4 RAW264.7 cells secreted TNF-α and were polarized to M1 phenotype after stimulation of TMPs-PEI-LPS,and inducing apoptosis of LLC.3.Anti-tumor efficacy and mechanism of TMPs-PEI-LPS in vivo3.1 Aggregation of TMPs-PEI-LPS was shown in the subcutaneous tumor area,indicating targeting ability;3.2 TMPs-PEI-LPS accumulated in lymph nodes and spleen,and enhanced the immune activity of these tissues;3.3 TMPs-PEI-LPS was proved to possess biological safety;3.4 TMPs-PEI-LPS inhibited the growth of subcutaneous tumor;3.5 TMPs-PEI-LPS promoted infiltration of immune cells related to anti-tumor immunity in tumor microenvironment.ConclusionAn electronic crosslinking agent PEI was used to modify TMPs with LPS to design a new tumor antigen presentation system named as TMPs-PEI-LPS.This novel platform makes full use of antigenicity of TMPs and immune-stimulating factor LPS,promoting the recognition and uptake of immune cells for tumor antigens.It is simple to synthesize and is demonstrated to be safe.Moreover,it possesses the ability to summon anti-tumor immunity.Notably,the plasticity of TMPs has ample space for optimization.Therefore,this novel tumor vaccine is prospective and potential in tumor therapy.