Study On Microenvironment Reconstruction And Nutrition Redistribution Based On Alkaline Arginine For Antitumor Immune Therapy

Due to its specific cell activity and metabolism,tumor microenvironment(TME)formed by tumor cells is very different from normal tissues.Tumor cells can survive,proliferate and develop in TME with poor survival conditions through their mutations,while other cells infiltrated in tumor tissues,especially immune cells,will turn into tolerant phenotypes for TME adaptation,and thus losing their anti-tumor immune function.This“self-serving”TME helps tumor cells realize immune escape.Improving this immunosuppressive TME is one of the important bases for the effective treatment of immunotherapy.Tumor cells will produce large amount of lactic acid through high intensity anaerobic glycolysis,known as“Warburg effect”.The resulting weak acidity is one of the main characteristics of immunosuppressive TME,which is closely related to tumor invasion,metastasis and tolerance to various therapies.Unfortunately,many anti-tumor immune cells cannot tolerate the weak acid environment.For example,under p H 6.0-6.5,T cells will be in a state of incapacitation,showing impaired cytolytic ability and cytokine secretion.Similarly,the same effects caused by weak acid environment also inhibit natural killer cells(NK)and macrophages.Therefore,the neutralization of acidic TME will be beneficial to save the incapacitated anti-tumor immune cells.In this study,the alkaline amino acid L-arginine(L-arg)was chosen as the neutralizer.On the one hand,L-arg is an endogenous amino acid with good biocompatibility.On the other hand,L-arg support is required for the normal function of immune cells.However,the L-arg content in TME is always deficient.The abnormal angiogenesis in tumor tissues leads to hypoperfusion leading to low L-arg supply.The rapidly proliferating tumor cells and other immunosuppressive cells will consume a large amount of L-arg,which is insufficient in TME.These two factors make the availability of L-arg to anti-tumor immune cells extremely low.In the absence of L-arg,the CD3ζchain expression and cytokine production of T cells are decreased,and the T cell cycle arrest is indused through general control non-derepressible 2/eukaryotic initiation factor-2α(GCN2/e IF2α)pathway.At the same time,L-arg is also the substrate for the synthesis of nitric oxide in M1 macrophages,and the deficiency of L-arg will also lead to the impaired function of them.Therefore,neutralizing TME with L-arg can also synchronously supply the key nutrition for immune cells and further promote the anti-tumor immune responses.To optimize acid neutralization and L-arg supplement,we prepared a kind of multivesicular liposomes(MVLs)loaded with high concentration of L-arg(L-arg@MVLs)with soy lecithin,cholesterol and vitamin E acetate.L-arg@MVLs presented stable quality,high safety,and good sustained-release ability,which could continuously neutralize acidity and supply L-arg to improve TME for a long time.However,tumor cells will also benefit from the improved TME.L-arg is one of the essential nutrients for tumor proliferation.To avoid potential tumor-promoting risks and maximize the effect of L-arg supplementation for anti-tumor immunity,the utilization of L-arg by tumor cells must be limited.The L-arg transporters are required for cells to uptake L-arg in the environment.The dominant isoforms of cationic amino acid transporters(CATs)are CAT-1 and CAT-2.In vitro experiments,we verified the dependence of different cells on these two proteins.The results showed that tumor cells were more dependent on CAT-2,while CD8~+T cells and M1 macrophages were more dependent on CAT-1.The down-regulation of CAT-2 in tumor cells inhibited the proliferation of tumor when L-arg was sufficient,and redistributed the limited L-arg to CD8~+T cells,realizing the improved activation and differentiation.We combined L-arg@MVLs with CAT-2 downregulation to enhance the anti-tumor immune responses through environmental improvement and L-arg redistribution.In the B16 melanoma model,the combined strategy promoted intratumoral infiltration,activation and differentiation of CD8~+T cells,enhanced the polarization and function of M1 macrophages,realized effective inhibition of tumor development,and showed the potential of sensitizing immunotherapy,which provided a new reference for the improvement of TME targeting environmental inhibitors and key nutritional metabolic pathways.