Targeting CAMKⅡ To Reprogram Tumor-associated Macrophages And Inhibit Tumor Cells With An Injectable Hybrid Peptide Hydrogel

Objective:Tumor-associated macrophages（TAMs）are an important part of the immunosuppressive tumor microenvironment,and play an important role in the occurrence and development of tumors.The ideal way to target TAMs is not to clear them,but to reshape TAMs from tumor-promoting M2 type to anti-tumor M1 type.We found for the first time that KN93,a specific inhibitor of CAMKII,has both direct tumor suppressive activity and the ability to reshape TAMs to polarize to anti-tumor M1macrophages.However,the application of these small molecule inhibitors in vivo often encounters the problems of low cell uptake efficiency,short cycle time,and low level of induced immunogenic cell death,making it difficult to effectively activate the antitumor immune response in vivo.Therefore,we screened out a melittin hydrogel scaffold,MR₅₂,with good gelling properties and antitumor activity,loaded with KN93(MR₅₂-KN93,MRK),and explored the antitumor effect of MRK on melanoma and malignant ascites and the impact on the remodeling of TAMs,and explored the relevant mechanism of MRK exerting dual effects.Methods:First,the effect of KN93 on the proliferation of B16F10 cells was verified by CCK8 experiments;the effect of KN93 on the polarization of bone marrow-derived macrophages（BMDMs）was detected by flow cytometry;RNA-seq detected transcriptome m RNA levels of BMDMs treated with KN93.At the same time,MR₅₂ with the best characteristics was selected from a series of melittin-（RADA）n hydrogel scaffolds.Next,CCK8,clone formation experiments,cell cycle and apoptosis experiments verified MRK’s antitumor activity;flow cytometry was used to detect the role of MRKs in remodeling the polarization of BMDMs to M1-type macrophages.In addition,by measuring the levels of reactive oxygen species,adenosine triphosphate,and calreticulin in B16F10 cells after MRK treatment,it was determined whether MRK caused immunogenic cell death;flow cytometry and confocal experiments were conducted to investigate whether MR₅₂promoted Cy5 uptake by cells.Subsequently,animal experiments were used to explore the antitumor and immunoregulatory effects of MRK in vivo.Intratumoral injection of MRK in a melanoma mouse subcutaneous tumor model to study its inhibitory effect on melanoma;flow cytometry to detect the proportion of dendritic cells in inguinal drainage lymph nodes,macrophages and T cells in tumor and PD-L1 Expression of tumor-associated macrophages effected by MRK.A mouse liver cancer ascites model was used to study the effect of MRK alone or MRK combined with PD-1 monoclonal antibody on malignant ascites.Results:KN93 can effectively inhibit the proliferation of melanoma cells and reshape the polarization of BMDMs to M1.MR₅₂ hydrogel has a network of nanofiber structures,which can directly kill tumor cells and control drug release.Compared with free KN93,MRK can significantly enhance the killing effect on tumor cells in vitro and induce immunogenic death of tumor cells.The mechanism may be that MR₅₂ promotes the uptake of MRK-loaded KN93 by B16F10 cells.In vivo injection of MRK significantly inhibited the growth of subcutaneous melanoma,promoted the maturation of DC in the draining lymph node area,significantly reduced the number of M2 macrophages in the tumor microenvironment,and significantly increased the infiltration of cytotoxic T cells.At the same time,PD-L1 expression of macrophages was up-regulated after MRK treatment.MRK combined with PD-1 monoclonal antibody therapy has a better inhibitory effect on mouse melanoma.In a mouse model of malignant ascites with poor conventional treatment effect,the MRK administration group significantly prolonged the survival time of the mice compared with the control group,and MRK combined with PD-1 monoclonal antibody therapy further extended the mouse survival time and produced a higher cure rate.Conclusion:In this study,we have created a KN93-encapsulated melittin hydrogel MRK with good biodegradability and biocompatibility,which can continuously deliver KN93and promote the uptake of KN93 by tumor cells.MRK kills tumor cells while reprogramming TAMs,improving the immunosuppressive tumor microenvironment,and combined with PD-1 monoclonal antibody therapy can significantly prolong the survival of mice with malignant melanoma and liver cancer malignant ascites,which is expected to transform into clinical applications.