PI3K? Inhibitor Enhance The Therapeutic Efficacy Of CA4 Nanomedicine In Breast Cancer By Regulating Tumor-associated Macrophages

Background: Vascular disrupting agents(VDAs)are a class of effective vascular targeting drugs,which target the established tumor vasculature and cause shutdown of tumor vessels,ultimately leading to tumor cell necrosis.CA4(Combretastatin A4),the leading VDA,is a microtubule-depolymerizing agent leading to cytoskeletal destabilization,followed by morphological changes and blood flow reduction.In order to improve the therapeutic efficacy of CA4,our research group utilized the nanocarrier-based carriers to develop a poly(L-glutamic acid)-combretastatin A4(PLG-CA4),which could significantly inhibit tumor growth in many tumor-bearing animal models.However,studies have reported that vascular targeting drugs increase the level of hypoxia in solid tumors,limiting the therapeutic efficacy of drugs.Hypoxia is a vital stimulus of the tumor microenvironment.In hypoxic microenvironment,tumor cells remodel the infiltrating immune cells to induce immunosuppressive effect,so that tumor cells escape immune recognition and accounting for immune surveillance.Tumor-associated macrophages(TAMs)constitute the dominant myeloid-derived immune cells of the tumor microenvironment,derived from the circulating monocytes.This versatile cell type displays high plasticity and heterogeneity,and show two phenotypes under different microenvironment.Studies have indicated that TAMs display immunostimulatory M1-like phenotype in early stages of some tumors,while polarize towards the immunosuppressive M2-like phenotype in most advanced tumors,promoting the tumor survival,proliferation,and metastasis.Hypoxia is a potential stimulus for macrophage recruitment;more importantly,macrophages in hypoxic microenvironment are more likely to show M2-like phenotype.TAMs in the hypoxic milieu mediate the resistance of anti-tumor drugs and tumor relapse.Targeting TAMs may enhance the therapeutic efficacy of vascular targeting drugs.Many studies have demonstrated that gamma isoform of phosphoinositide 3-kinase(PI3K?)plays an important role in macrophage recruitment and function.PI3K? originates from the PI3 K family and is restricted to leukocytes.PI3K? inhibitor could reduce macrophage recruitment to decrease the number of M2 phenotype,attenuating immunosuppressive effect and promoting anti-tumor response.Pharmacological blockade of PI3K? in cancer mouse xenograft models significantly decrease macrophages trafficking into tumors,while restore the number and activity of cytotoxicity T lymphocytes,inhibiting tumor growth and prolonging survival time.Objective: In this study,we choose the well characterized and documented 4T1 murine breast cancer model,and investigated the impact of PLG-CA4 in TAMs.We also discussed the effect of PLG-CA4 combined PI3K? inhibitor therapy in tumor microenvironment by regulating macrophages.Methods 1.The effect of PLG-CA4 on macrophages in breast cancer microenvironment and its mechanism: After treatment with PBS or PLG-CA4 in 4T1 murine breast cancer model,immunohistochemical staining was performed in the tumor tissues with CD31 and HIF1-alpha;Hematoxylin and eosin staining was used to observe the morphological changes of tumor tissues;flow cytometry was used to detect the infiltrating leukocytes,macrophages and M2 phenotype.2.The effect of PI3K? inhibitor on PLG-CA4-induced macrophages in breast cancer microenvironment: After treatment in 4T1 murine breast cancer model,flow cytometry was used to detect the infiltrating leukocytes,macrophages and M2 phenotype,T lymphocytes and cytotoxic T lymphocytes in the microenvironment;immunohistochemical staining was performed in the tumor tissues with Vegfa,CD31 and MMP9;real time PCR was used to examine the transcription levels of Vegfa,Granzyme B,Fas L and MMP9.3.The effect of PI3K? inhibitor on the therapeutic effect of PLG-CA4 in breast cancer: After treatment in 4T1 murine breast cancer model according to the therapy regime,tumor size,mouse weight and survival time were observed;immunohistochemical staining was performed in the tumor tissues with Ki67;Hematoxylin and eosin staining was used to observe the morphological changes of tumor tissues,lung and liver.Results: 1.The effect of PLG-CA4 on macrophages in breast cancer microenvironment and its mechanism.Compared with the control group,PLG-CA4 decreased the of CD31-positive microvessel density(P < 0.001),and induced tumor cells necrosis in the central part of tumor tissue;while increased the percentage of HIF1-alpha-positive cells(P < 0.05).There was no difference in the infiltrating leukocytes and macrophages,but PLG-CA4 increased the percentage of M2 macrophages(P < 0.05).2.The effect of PI3K? inhibitor on PLG-CA4-induced macrophages in breast cancer microenvironment.Compared with the control group,PI3K? inhibitor decreased the percentage of tumor-associated macrophages(P < 0.001);reduced the Vegfa-positive areas in microenvironment(P < 0.001),and downregulated the transcription levels of Vegfa(P < 0.01);also decreased the microvessel density(P < 0.05).Compared with PLG-CA4,PLG-CA4 combined with PI3K? inhibitor decreased the number of M2-like macrophages and increased the number of cytotoxicity T lymphocyte(P < 0.05).Compared with the control group and PLG-CA4 group,the transcription levels of Granzyme B and Fas L in the tumor microenvironment of PLG-CA4 combined with PI3 K gamma inhibitor group were up-regulated(P < 0.01;P < 0.05).Compared with PLG-CA4,PLG-CA4 combined with PI3K? inhibitor increased the MMP9 positive staining area and the transcription level of MMP9 in extracellular matrix(P < 0.01;P < 0.05).3.The effect of PI3K? inhibitor on the therapeutic effect of PLG-CA4 in breast cancer.PLG-CA4 combined PI3K? inhibitor had the most significant effect on inhibiting tumor growth(PLG-CA4: P < 0.05;PI3K? inhibitor: P < 0.01;the control group,P < 0.001),and the weight changes of mice in each group had no significant difference.In H&E staining,cell necrosis appeared to different extent in therapeutic group,and the necrosis area was most obvious in the combined group.In Ki67 staining,positive cells were decreased in therapeutic group,and the least was in combined group.On day 30 of therapy regimen,the therapeutic agent-treated groups presented a reduction in the pulmonary metastatic sites,and combination of PLG-CA4 and PI3K? inhibitor obviously weakened the lung metastasis.To observe metastasis in liver,there were multiple metastatic nodules and invasion of hepatic vessels in the control group;the treatment group attenuated the liver metastasis,while the combined treatment group had only a small number of hepatic vessels and liver metastasis.PLG-CA4 combined PI3K? inhibitor had the most significant effect on prolonging survival time in tumor-bearing mice(PLG-CA4: P < 0.05;PI3K? inhibitor: P < 0.05;control group: P < 0.001).Conclusions: 1.PLG-CA4 effectively inhibits tumor vasculature and cause necrosis of breast cancer tissues.However,PLG-CA4 increases the level of hypoxia in tumor microenvironment and induces M2 polarization of tumor-associated macrophages.2.PI3K? inhibitor significantly reduces the number of M2-like macrophages induced by PLG-CA4,while increases the number of cytotoxic T lymphocytes in breast cancer microenvironment.3.The combination of PLG-CA4 and PI3K? inhibitor suppress breast tumor growth,induce cell necrosis in 4T1 breast cancer,reduce lung and liver metastasis of breast cancer,and prolong the survival time of tumor-bearing mice.