Secretions From Hypochlorous Acidtreated Tumor Cells Delivered In A Melittin Hydrogel Potentiate Cancer Immunotherapy

【Objective】To screen out the most immunostimulatory tumor cell-derived secretions（CDS）,that is,HOCl-CDS,obtained from tumor cells treated with hypochlorite（HOCl）.Loading HOCl-CDS into the melittin-capsulated hydrogel to construct the HOCl-CDS hydrogel.The characterization,anti-tumor effect and immune activation effect of HOCl-CDS hydrogel are studied in vivo and in vitro.【Methods】Ⅰ.Fabrication and characterization of HOCl-CDS hydrogel.1.To obtain HOCl-CDS:Melanoma cell B16F10 was treated with hypochlorite（HOCl）,cisplatin,radiotherapy,photodynamic therapy and hypoxia to obtain five kinds of tumor cell-derived secretion（CDS）.Flow cytometry was used to detect the degree of bone marrow-derived dendritic cells（BMDCs）maturation and polarization of bone marrow-derived macrophages（BMDMs）stimulated by these five kinds of CDS in vitro.As a result,the CDS with the strongest immunostimulatory effect was chosen.2.Fabrication of HOCl-CDS hydrogel:HOCl-CDS was loaded into melittin capsulated hydrogel（melittin-RADA24,MELR）to obtain HOCl-CDS hydrogel.3.Characterization of HOCl-CDS hydrogel.The nanostructure,mechanical properties and sustained release effect of HOCl-CDS hydrogel were characterized by transmission electron microscope,rheological detection and in vivo bioluminescent imaging.Ⅱ.In vitro antitumor activity and immune activation effect of HOCl-CDS hydrogel.1.The effect of HOCl-CDS hydrogel on the proliferation of B16F10 cells was detected by CCK8 assay.2.The effect of HOCl-CDS hydrogel on BMDCs maturation was detected by flow cytometry.3.The effect of HOCl-CDS hydrogel on the polarization of BMDMs was detected by flow cytometry.3.Immunostimulatory cytokines in HOCl-CDS were detected by ELISA.4.The expression of PD-L1 on BMDCs and BMDMs stimulated by HOCl-CDS hydrogel was detected by flow cytometry.Ⅲ.In vivo The anti-tumor and immune activation effect of HOCl-CDS hydrogel.1.Anti-tumor effect:Subcutaneous melanoma model on mice was established.After intratumoral injection of HOCl-CDS hydrogel,the changes of tumor growth were dynamically observed,and the tumor growth curve and survival curve of tumor-bearing mice were drawn.Fluorescence intensity was detected by in vivo bioluminescent imaging to evaluate tumor growth.2.Immune activation effect:The maturation of DC in inguinal lymph nodes and the polarization of tumor-associated macrophages and T cell infiltration in tumor microenvironment were detected by flow cytometry.3.Biosafety:HOCl-CDS hydrogel was injected subcutaneously,and the changes of body weight were recorded.Blood samples were collected for blood routine test and blood biochemical test,and heart,liver,spleen,lung and kidney were taken for tissue section and H&E staining.4.The anti-tumor effect and immune activation effect of HOCl-CDS hydrogel combined with PD-1 antibody.（1）Anti-tumor effect:Subcutaneous melanoma model on mice was established,and treated with HOCl-CDS hydrogel combined with PD-1 antibody.The tumor growth was observed to evaluate the therapeutic effect.（2）Immune activation effect:The infiltration of Th1 and CTL in tumor microenvironment was detected by flow cytometry.【Results】Ⅰ.Fabrication and characterization of HOCl-CDS hydrogel.1.To obtain HOCl-CDS:The percentages of BMDCs maturation stimulated by CDS treated with HOCl,photodynamic therapy,radiotherapy,cisplatin and hypoxia were 26.1%,19.7%,22.4%,16.9%and 11.9%,respectively.When these five kinds of CDS stimulated BMDMs,the mean fluorescence intensity of M1-related surface marker CD86 decreased in turn.2.Fabrication of HOCl-CDS hydrogel:Dissolve MELR polypeptide and HOCl-CDS solution in Na Cl solution with a final concentration of 0.9%,mix well and stand at 4℃for 4-6 hours to form a gel;3.Characterization of HOCl-CDS hydrogel.（1）HOCl-CDS hydrogel showed a fibrous reticular structure with a diameter of11.8±1.2 nm under transmission electron microscopy.（2）Rheological detection showed that the viscosity and hardness of HOCl-CDS hydrogel remained stable at a fixed low strain rate of 0.1%,and gradually recovered when the strain rate was restored to 0.1%after high strain rate breaking.（3）In vivo bioluminescent imaging showed that the fluorescence signal intensity of MELR-Cy7 hydrogel coated with fluorescein Cy7 was 7.9 times（p<0.01）,3.3 times（p<0.05）,3.4 times（p<0.01）of that of free Cy7 group at 6h,24h,3d,5d and 7d,respectively.Ⅱ.In vitro antitumor activity and immune activation effect of HOCl-CDS hydrogel.1.CCK8 assay showed that the cytoxic rates of HOCl-CDS hydrogel on B16F10cells at the 1^st,3^rd,5^th and 7^th day were 73.9%,83.5%,86.5%and 90.1%,respectively.2.Flow cytometry showed that the percentage of BMDCs maturation stimulated by HOCl-CDS hydrogel was 69.5%,which was 2.0 times of that of PBS group（p<0.01）,1.5 times of that of MELR hydrogel group（p<0.01）and 1.2 times of that of HOCl-CDS solution group（p<0.05）.When BMDMs was stimulated by HOCl-CDS hydrogel,HOCl-CDS solution and MELR hydrogel,the average fluorescence intensity of M1-related surface marker CD86 decreased successively.3.ELISA detection and ATP detection showed that the HOCl-CDS contained more immunostimulatory cytokines than the Untreated-CDS:TNF-α,IL-1α,GM-CSF,M-CSF,MIP-3α,MIP-1α,ENA78,CD40L,MCP1,HMGB1 and ATP.4.After co-incubation of HOCl-CDS hydrogel with BMDCs/BMDMs,the expression of PD-L1 on BMDCs/BMDMs surface increased.Ⅲ.In vivo anti-tumor and immune activation effect of HOCl-CDS hydrogel.1.Anti-tumor effect:In the animal experiment,the subcutaneous tumor volume of melanoma in the HOCl-CDS hydrogel group was smaller than that in the PBS group（p<0.001）.2.The proportion of mature DC in inguinal lymph nodes in HOCl-CDS hydrogel group was 6.2 times of that in PBS group（p<0.001）,the proportion of M1/M2 in tumor microenvironment was 3.5 times of that in PBS group（p<0.001）,the infiltration rate of Th1 in tumor microenvironment was 2.5 times of that in PBS group（p<0.001）,and that in CTL group was 16.3 times of that in PBS group（p<0.05）.3.After injection of HOCl-CDS hydrogel,there was no significant difference in red blood cell,white blood cell and platelet count between HOCl-CDS hydrogel group and PBS group（p>0.05）;as well as AST,ALT,BUN;and no pathological injury was found in heart,liver,spleen,lung and kidney tissue sections by HE staining.4.The anti-tumor effect and immune activation of HOCl-CDS hydrogel combined with PD-1 antibody.（1）When melanoma was treated with HOCl-CDS hydrogel combined with PD-1antibody,the tumor volume in combined treatment group was smaller than that in HOCl-CDS hydrogel group（p<0.05）and PD-1 antibody group（p<0.05）.（2）The infiltration rate of Th1 in combined group was 10.4 times of that in the PBS group（p<0.0001）and CTL was 17.8 times of that in the PBS group（p<0.001）.【Conclusion】In this study,the most immunostimulatory tumor cell-derived secretions（CDS）,was screened from hypochlorite,photodynamic therapy,radiotherapy,cisplatin and hypoxia treatment,that is,HOCl-CDS obtained by hypochlorite treatment.The multifunctional HOCl-CDS hydrogel constructed by loading HOCl-CDS into melittin-capsulated hydrogel has good sustained release effect,biosafety,anti-tumor activity and immune activation effect,and can also enhance the efficacy of PD-1 antibody,indicating the potential value of HOCl-CDS hydrogel in tumor immunotherapy.