Exploration Of New Anti-tumor Mechanism Of Chloroquine: Mobilizing Macrophage-dependent Anti-tumor Immunity

Objective:The 2018 Nobel Prize in Physiology or Medicine is awarded to the discovery of cancer therapy by inhibition of negative immune regulation,which push tumor immunotherapy forward to a new era.Currently,the tumor immunotherapy mainly includes PD-1 or CTLA-4 antibodies for immune checkpoints and CAR-T cells,all of these were based on activation of T cells to obtain an adaptive immune response.However,a large number of innate immune cells infiltrated in the tumor immune microenvironment and they can identify and attack tumor cells in the first place.So,study these innate immune cells will help us to in-depth understand tumor immunosuppression and develop new immunotherapy strategies.Especially,macrophage,as the most infiltrated innate immune cells in tumor immune microenvironment,did not play its original role in phagocytosis of tumor cells,presenting tumor antigens,and killing tumor cells.On the contrary,they were educated to M2-like tumor-associated macrophages(TAMs)by tumor cells to promote tumor growth,metastasis,and immune escape.Therefore,targeting M2?TAMs into anti-tumor M1-TAMs,remodeling the tumor immune microenvironment,and promoting anti-tumor immunity of T cells,will open a new avenue for tumor immunotherapy.Methods:We cultured bone marrow-derived macrophages in vitro,LPS/IFN-? was used to induce M1 macrophages,and M2 macrophages were polarized by IL-4.The lysosomal pH value and the lysosomal quantity of the two types of macrophages was analyzed.Next,we chose chloroquine(CQ.a lysosomal specific inhibitor)to target the lysosomes of M2 macrophages for altering macrophage phenotype.Furthermore,the mechanism of macrophage phenotype with CQ was unveiled by specific small molecule inhibitors and CRISPR/Cas9 technology.Meanwhile,the metabolic mode of M2 macrophages induced by CQ was detected by seahorse cellular energy metabolism analyzer,and CHIP-QPCR was used to clarify the mechanism that the metabolic mode reprogramed in M2 macrophages by CQ.Moreover,we used mouse subcutaneous melanoma model and liver cancer ascites model to verify the anti-tumor effect of CQ,explore the relationship between the anti-tumor effect of CQ and macrophage by macrophage depletion experiment in vivo.Moreover,we studied the relationship between CQ and tumor antigen-specific killing in OVA-B16 and OT-I CD8 T cell models.Finally,the tumor immune microenvironment after CQ treatment was analyzed by flow cytometry,and Treg or MDSCs depletion were combined with CQ to further enhance the anti-tumor immunity.Results:(1)CQ-mediated antitumor effect is T-cell dependent.Firstly,we demonstrated the antitumor effect of CQ in B16 melanoma-bearing mouse model and H22 liver malignant ascites mouse model.However,the tumor-inhibiting effect of CQ against B16 melanoma and H22 hepatocarcinoma was lost in T-cell-deficient nude mice or the wide type mice with T-cell depletion by CDS antibodies.These data suggest that the antitumor effect of CQ appears to be ascribed to its mobilizing T-cell immunity.(2)CQ-induced antitumor T-cell immunity is macrophage dependent.Further,we found that CQ had no effect on T-cell proliferation or activation,and did not be able to promote the maturation and activation of dendritic cells.More intriguingly,we found that macrophage depletion disrupted the inhibitory effect of CQ on B16 melanoma,as well as H22 hepatocarcinoma growth.In addition,CQ can induce tumor-infiltrated CD8+T cells to secrete more IFN-?,while enhancing the tumor killing ability of tumor-specific T cells.These findings suggest that CQ initiates a macrophage-dependent pathway that enhances anti-tumor T-cell immunity.(3)CQ resets tumor-associated M2 macrophages to M1 phenotype.Since the anti-tumor effect of CQ is dependent on macrophages,we have also confirmed that CQ can reverse the M2 macrophage phenotype to Ml type in vitro.More importantly,CQ reversed TAMs in vivo and the reset-M2 cells promote T cell proliferation in vitro.Additionally,CQ re-educated human monocyte-derived M2 macrophages to M1 type.(4)M1 macrophages are induced by CQ activated p38 and NF-?B.By detecting the classical MAPK signaling of macrophage activation,combined with specific small molecule inhibitors and CRISPR/Cas9 gene knockout technology,we found that CQ induced the M1 macrophage phenotype using p38/NF-?B signaling.(5)Lysosomal Ca2+release activates p38 and NF-?B.Furthermore,studies on lysosomes,the CQ-specific target,have found that CQ increased the lysosomal pH value of M2 macrophage and triggered the release of calcium into the cytosol via the Mcolnl channel,thereby activating p38/NF-?B signaling pathway.(6)Ca2+-activated TFEB reprograms macrophage metabolic mode.We found that CQ is capable of promoting anaerobic glycolysis in macrophages and this is due to the fact that cytosolic calcium promotes the entry of TFEB into the nucleus and the TFEB transcription factor initiates these key enzymes in the glycolytic metabolic pathway.(7)CQ-reset macrophages ameliorate tumor immunosuppression.The macrophage-dependent antitumor effect of CQ induced the reduction of two major types of immunosuppressive cells(MDSCs and Treg)in the tumor immune microenvironment.Meanwhile,CQ combined with low-dose cyclophosphamide or CD25 neutralizing antibody completely eliminates Treg and further amplified the antitumor effect,and combined with Gr-1 neutralizing antibodies to deplete MDSCs can also augment the antitumor effect of CQ.Conclusion:Chloroquine,an old anti-malarial drug,increases M2-like TAMs lysosomal pH,causing Ca2+release via the lysosomal Ca2+channel Mcolnl,which induces the activation of p38 and NF-?B,thus polarizing TAMs to produce inflammatory cytokines.Meanwhile,released Ca2+activates TFEB that reprograms macrophage metabolism towards glycolysis.At this time,the M2-like TAMs reversed by CQ not only have the M1 phenotype,but also possess glycolysis metabolic model to provide energy to sustain its antitumor function.These macrophages promote T cells to exert antitumor effects in tumor microenvironment,especially the tumor-specific T cells,and also reprogram the tumor immune microenvironment,relieve immunosuppression,and further amplify antitumor immunity.