Effects And Mechanism Of Macrophage In Tumor Immune Suppressive Microenvironment And Epigenetically Engineered Macrophage For Enhanced Cancer Immunotherapy

Immunotherapy emerges as a revolutionary strategy for cancer treatment and change the model of cancer therapy from directly destroying to mobilizing the immune system to attack tumor cells,which is expected to eradicate cancer.However,there still remains a number of patients with poor response to immunotherapy.The main obstacle of immunotherapy is the immunosuppressive effect of tumor microenvironment.Therefore,verifying the mechanism of immune suppressive microenvironment and exploring novel strategy to reverse immunosuppression are of great significance for immunotherapy.Tumor associated macrophages(TAMs)are the most abundant inflammatory immune cells in the tumor microenvironment,which involve in tumor initiation,invasion,metastasis,and immunosuppression.TAMs are mainly M2 type,which can generally suppress antitumor immunity.Additionally,TAMs inhibit the function of other immune cells in innate and adaptive immune response,amplify immunosuppressive effect in tumor microenvironment and ultimately accelerate tumor progression.Meanwhile,TAMs have great plasticity in response to variable microenvironmental stimuli.Hypoxia,a condition where the oxygen pressure is less than 5-10 mm Hg,represents a hallmark feature of neoplastic growth and has profound influences on the angiogenesis,invasion,metastasis,epithelial-mesenchymal transition and chemoresistance of tumor cells.However,the influence of hypoxia on TAMs still remains controversial.As the importance of hypoxia in tumor progression,we supposed that hypoxia may also be a significant factor in regulation TAMs.Since the essential role of TAMs in the immunosuppressive microenvironment,strategies targeting TAMs has emerged.Currently,targeted depletion of TAMs,inhibiting their recruitment and reprogramming of cell polarization are three commonly used strategies to enhance cancer immunity.However,all of these strategies present disadvantages of poor effect and severe side effects.Employing gene editing technology to engineer macrophage provides a promising way to enhance antitumor immune response and reprogram tumor immune environment.A large number of epigenetic marks have been identified in the genome of TAMs that are affected by tumor microenvironment.Additionally,epigenetic changes of cells are persistent,reversible and inheritable.In recent years,epigenome editing based on CRISPR/Cas9,an easily programmable genome editing technology,has renewed epigenetic editing,widen their therapeutic applications,and provided a safe and steady cell editing strategy.In this study,we develop an epigenetically modified macrophage by targeting HIF1αusing CRISPR/Cas9 system,which not only has enhanced antitumor effects itself,but releases other immune cells in tumor microenvironment.Our research brings new sight for cancer immunotherapy.ObjectiveTo find important factor that regulates immune function of tumor associated macrophage,and to epigenetically modify macrophage via CRISPR/Cas9 system and study the efficacy of the engineered macrophage.MethodsExpression and function of HIF1αin the tumor associated macrophages were analyzed by immunohistochemistry,q PCR,et al.Epigenetically silenced macrophages were engineered by recruiting the EZH2 to the promoter region with CRISPR/d Cas9-EZH2system.Effects of the engineered macrophages on tumor progression and the immune microenvironment were analyzed in subcutaneous B16-F10 melanoma syngeneic model.Results1.HIF1αacted as an important factor mediating immunosuppressive function of TAMs.C57BL/6 mice were subcutaneously inoculated with 5×10~5 B16-F10 cells and the tumor size was monitored.H&E staining shown that there were abundant inflammatory cells infiltrated in“large”tumors.Compared to“small”tumors,increased expression of HIF1αin TAMs in“large”tumors.Meanwhile,TAMs in“large”tumors were significantly polarized to M2 phenotype.To explore whether HIF1αalters the function of macrophage under hypoxic condition,we added tumor conditioned medium to RAW264.7 cells to mirror the tumor microenvironment,followed by cobalt chloride(Co Cl2)treatment.Macrophage trend to polarize to M2 phenotype as well as produce more angiogenic factors such as Vegfa,Vegfc and pdgf,under hypoxic condition.These results indicate that elevated expression of HIF1αof macrophage under hypoxic condition might contribute to tumor progression through these angiogenic factors.In other words,HIF1αcould be a candidate to reprogram macrophage for reverse immune suppressive microenvironment.2.Epigenetically silencing of HIF1αvia CRISPR/d Cas9-EZH2 system.Epigenetically silencing of HIF1αvia histone H3 methylation in the promoter region was achieved by CRISPR/d Cas9-EZH2 system,in which histone H3 methylase EZH2 was recruited to the promoter region specifically.Western blot analysis revealed that d Cas9and PCP-EZH2 were substantially expressed in the groups infected with the lentiviruses expressing the CRISPR/d Cas9-EZH2 system.Meanwhile,sg RNA was extremely overexpressed after infection as expected.Ch IP-q PCR showed methylation of H3K27.Besides,the expression of HIF1αin macrophages could be efficiently and persistently repressed by CRISPR/d Cas9-EZH2 epigenetic editing system.3.The HIF1αsilenced macrophages(HIF1αEpigenetically Repressed Macrophage,HERMs)manifested as inheritable tumor suppressing phenotype.We treated bone marrow derived macrophages(BMDMs)with tumor conditioned medium and Co Cl2,followed by lentivirus infection of the epigenetic editing system and induced polarization.HERMs inhibit the expression of growth factor and significantly have an enhanced antitumor capacity.Besides,epigenetically repressing HIF1αrobustly promoted phagocytosis of macrophage.All of these data indicated that HERMs displayed enhanced M1-like polarization and phagocytosis,with stronger anti-tumoral function in vitro.4.Intratumor adoptive transfer of HERMs remarkably inhibit tumor progression by reversing immunosuppression of tumor microenvironment.The GFP positive macrophages(GFP-M(37))were intratumorally injected in the B16-F10melanoma model to trace the cell distribution.GFP-M(37)can be detected in the tumor as long as 12 d after intratumor injection and some GFP-M(37)redistributed to the spleen and liver after intratumoral injection.Since increased HIF1a expression occurred at about day12 while macrophage polarization changed at about day 16 in the syngeneic model.H&E staining revealed that there was no obvious toxicity observed in main organs after HERMs intratumorally injection.Bioluminescence imaging and morphological observation demonstrated that intratumorally injection of HERMs had a visible decrease of tumor size in bilateral tumors.And the mice treated with HERMs had much higher survival rate.Immunofluorescence and flow cytometry showed that HERMs treatment increased the the proportion of M1-polarized macrophages,elevated the number of effector T cells,promoted T cell proliferation and activated T cells.Meanwhile,HERMs unleash the immune suppression by negative regulation of the immune checkpoints and Tregs.Additionally,tumors after treatment of HERMs had significantly reduced blood vessels.These data indicated that the HERMs remarkably activate effective T cells,reverse immune suppression and eventually inhibit tumor progression.ConclusionIn this study,we first demonstrated that elevated HIF1αin TAMs promoted tumor progression by altering the anti-tumoral effect of macrophage.Next,we have modified macrophages by epigenetically silencing HIF1αusing CRISPR/d Cas9-EZH2 system.Intratumor injection of the HERMs significantly activate innate and adaptive immune response,remarkably reduce angiogenesis,comprehensively revert the immune suppressive microenvironment,and finally repress tumor progression and prolong survival.Additionally,previous work have shown that M1 macrophages have anti-tumoral effect,while M2 macrophages have pro-tumoral effect;our study focused on the function of HIF1αand found that macrophages promoted tumor progression in a polarization incomplete dependent way.It is not that accurate to study tumor immunity simply according to macrophage polarization.Therefore,the future of cancer immunotherapy based on macrophage should pay more attention to certain molecules with key regular functions.