Anti-CD47 Blockade Synergizes With Chemo-photothermal Therapy For Enhanced Tumor Immunotherapy

Objective:During cancer treatment,metastasis is a bottleneck that is difficult to overcome.Tumor cells also utilize various mechanisms to escape the identification and clearing of immune cells.The immune checkpoint is to break the tumor escape mechanism and use its own immune system to quickly kill tumors and without side effects.CD47 is a membrane protein that highly express on the surface of tumors,which is coupled to the surface of the macrophage surface by SIRPαprotein,thereby escaping the phagocytosis of macrophages.We combined the advantages of silica nanoparticles（mSiO2）in the in vivo decay period of tumor treatment,small side effects,and combined with immunological checkpoints to design Anti-CD47-nSiO₂nanoparticles and（ICG+BEZ-235）@mSiO₂,also in the primary tumor of the tumor,phototherapy and chemotherapy,breaking the stable morphology and structure of the tumor.A two-pronged approach that synergistically accomplishes the inhibition of tumor cell growth and metastasis.Methods:In our study,we constructed two nanoparticles:Anti-CD47-mSiO₂ and（ICG+BEZ-235）@mSiO₂.in vitro,we use AM-PI staining,flow cytometry,macrophage and tumor cell co-culture to verify the effect of this combination therapy.In vivo,we implanted two tumors on both sides of C57bl/6 mice,which were set as local tumors and metastases,respectively.We use the anti-CD47-nSiO₂ nanoparticle tail vein injection to activate the body’s immune system,and also use the effect of nanoparticles on the tumor site to enhance the killing effect of macrophages on the tumor site.For（ICG+BEZ-235）@MSN,we are in local tumor injection.ICG is a photosensitizing reagent that releases a large amount of heat and inhibitor in a short time under the action of 808nm laser,thereby killing and inhibiting tumor cells growing.After treatment,we measured the size of the local tumor and metastases tumor.We also collect serum at 24h,72h,168h,measuring the levels of IL-12p40 and TNF-α.We also verified infiltration of macrophages,CD4+T cells,and CD8+T cells in metastatic tumors by flow cytometry and immunohistochemistry.Results:The immune system has an irreplaceable role in the treatment and prognosis of tumors.In order to break the immune escape mechanism of tumor cells,we used checkpoints in combination with nanotechnology.In addition,we also used photothermal therapy and chemotherapy for tumors,and verified them in vivo and in vitro.In vitro,this combination therapy effectively kills tumor cells,and flow cytometry also demonstrates that the combination of photothermal-therapy and chemotherapy can drastically kill tumors.To prepare bone marrow-derived macrophages（BMDMs）,6 weeks of BALB/c mice were sacrificed,then tibiae and femurs were collected and stored in PBS.Co-culture of macrophages and tumor cells under the effect of Anti-CD47-nSiO₂,with increase recognition and phagocytosis of tumor cells by macrophages,which also promote the amount of TNF-αin the culture medium;In vivo,we measure immune cells such as macrophages,CD8 T cells and CD4 T cells in tumors after treated mice.Compared with the control group,the left and right tumors became smaller or even disappeared.Conclusions:Macrophages,as an antigen presenting cell（APC）,play an important role in defense the cancer for people.By designing a nanoparticle,we aim to promote phagocytosis of tumor cells by macrophages,increasing the infiltration of immune cells in tumors.Through the combined strategy,the tumor was effectively inhibited in growth and metastasis.These data show that this combined tumor immunotherapy nanotechnology can inhibit tumor recurrence and metastasis and prolong the survival time of animals.Therefore,this synergistic system is a tumor treatment method with great prospects.