Intratumoral Delivery Of CCL25 Enhances Immunotherapy Against Triple Negative Breast Cancer By Recruiting CCR9~+T Cells

Background and Objective:The thymus-expressed chemokine(TECK,CCL25),a chemokine expressed and secreted by small intestine and thymic epithelial cells and thymus medullary dendritic cells,is chemotactic for thymocytes,lymphocytes,dendritic cells and activated macrophages.These cells recognize CCL25 uniquely through the cell surface receptor CCR9.Originally,CCR9 has been fully investigated on its function in terms of navigation and maturation of thymocytes and gut homing characteristics.Recent studies demonstrated that CCR9~+ T cells from normal small bowel and mesenteric lymph node high express CD69,furthermore,more circulating CCR9~+ T cells were activated than CCR9-cells.With other,CCR9~+ T cells isolated from PB and SB have a prominent Th1 cytokine profile which produce large amounts of IFN-?.Hepatic et al previously revealed that the aberrant high expression of CCL25 in primary sclerosing cholangitis patients outside the gut and thymus associated with infiltration of functionally activate CCR9 T cells in the liver.The CCL25/CCR9 was also powerful to license effector/memory T cells to access anatomic sites.Binding of CCR9 to CCL25 inhibits CD4 T cells differentiating to Tregs.In autoimmunity,CCR9~+ T helper cells promote expansion and survival of CD8~+ T cells through secreting IL-21 to induce diabetes.Importantly,circulating CD8~+CCR9~+ na?ve T cells were associated with a favorable prognosis in melanoma patients and decrease lung metastasis,neutralizing the CCL25 in a sarcoma with high levels CCL25 accumulation accelerate the growth of tumor35.Therefore,we hypothesized delivery CCL25 into tumor and release it into the intercellular space should be an effective therapeutic strategy for TNBC to enhance the antitumor effect of the immune modulates.CD47,a strong “don't eat me” signal,is overexpressed on a broad range of solid tumors,including TNBC,contributing to their escape from immune surveillance and destruction by phagocytes.Blockade of CD47 using antibody resulted in enhancement of macrophage phagocytosis of tumor cells,and even promote T cell-dependent antitumor immunity priming by dendritic cells38.However,as CD47 also exists on normal cells,systemic administration CD47 antibody may also affect normal cell function,leading to side effects such as anemia and the decrease of neutrophil.Intravenous delivery of si RNA targeting CD47 had been proved to effectively inhibit melanoma tumor growth and lung metastasis and reduce side effects.However,targeted delivery of RNA-based therapeutics for cancer therapy remains a challenge.Combination of two therapeutic approaches with different mechanisms has been proved to be able to cooperatively prohibit cancer development,being a promising strategy for effective treatment of cancer.Nanotechnology-based delivery system,which can carry different kinds of antitumor drugs by physical entrapment,has improved it's pharmacokinetics and biodistribution profile via enhanced permeability and retention(EPR)effect,leading to potentially enhanced anticancer effect.However,it should be attended that chemokine CCL25 and CD47 si RNA have very different physical properties,and they should be released extracellularly and intracellularly after being delivered to tumor tissue,respectively.To acquire satisfactory efficacy,an ideal nanotechnology-based drug delivery system following systemic administration should possess a number of attributes,mainly including(1)enhanced accumulation in the tumor,(2)rapidly or sufficiently extracellular chemokine release,(3)facilitated cellular internalization and release of si RNA.Therefore,it is urgently desired to design a novel drug delivery system,which should fulfill all above-mentioned attributes for promoted efficient immunotherapy against TNBC.Methods:1.We established mouse othotopic triple negative breast cancer(4T1)and detected the expression of CCL25 and CCR9 in human and mouse TNBC tissues.2.We synthesized the tumor actidity sensitive nanopaticel NP-si CD47/CCL25 entrapped si CD47 and CCL25 through electostatic interaction.3.We analyzed the celluar uptake of siCD47 and CCL25 and the silence effect of si CD47 under the neutral and mildly acidic conditions in vitro by flow cytometry,laser scanning confocal microscopy(LSCM),quantitative real time PCR and western blotting.4.We measured the tissue distribution of NPs by small animal imaging,LSCM and immunohistochemistry.5.We examined the effect of CCL25 on tumor immune microenvironment and the expression of CD47 in vivo by flow cytometry and immunohistochemistry.6.We observed the tumor growth and metastasis after i.v.injection of NPsi CD47/CCL25,and investigated the anti-tumor immune responses by flow cytometry.Finally,we examined the impact of NP-si CD47/CCL25 and NP-si NC/CCL25 on the PD-1/PD-L1 antibodies therapy effect.Results:1.Proportion of CCR9~+ T cells in Periperal blood cells is comparable and relatively low in TNBC patients and healthy women.CCL25 was undetectable in human TNBC tissues and mouse 4T1 tissues.CCR9 was undetectable in human TNBC and the ratios of CCR9~+ cells in TILs remained low.Proportion of activated cells in CCR9~+ T cells was high compared with CCR9-T cells.2.si CD47 and CCL25 were colocolized under neutral condition and seperated under in vitro.A markedly enhanced cellular uptake of si CD47 by 4T1 cells and more effective silence effect under midly acidity condtion compared to neutral condition.3.NP-si CD47/CCL25 sequentially release CCL25 to tumor stroma and recruited CCR9~+CD8~+ T cells to tumors.si CD47 was uptake by tumor cells and expression of CD47 on tumor cells was decreased.4.NP-si CD47/CCL25 delayed the formulation of 4T1 tumors and inhibited the growth of established tumors.After systematic administration,nanoparticles inhibited lung metastasis of 4T1 cells.However,NP-si CD47 or NP-si NC/CCL25 has no antitumor effect.5.Anti-tumor effect of NP-si CD47/CCL25 depend on T cells,nanoparticles decrease the propotion of MDSCs and increase CD8~+ T cells.6.NP-siCD47/CCL25 revesed the PD-1/PD-L1 antibodies resistance,PD-1/PDL1 antibodies enhanced the antitumor effect of NP-si CD47/CCL25.NPsi CD47/CCL25 was able to revese PD-1/PD-L1 antibodies resistance.Conclusion:1.CCR9~+CD8~+ T cells showed active cell phenotype,but the number of infiltration in triple negative breast cancer is limited.Human and mouse 4T1 triple negative breast cancer don't express CCL25,the ligand of CCR9.2.NP-si CD47/CCL25,a tumor acid sensitive nanoparticle,can recruit CCR9~+CD8~+ T cells to infiltrate into tumor tissue and reduce the expression of CD47 on tumor cells.3.The inhibitory effect of NP-si CD47/CCL25 on the formation,growth and distant metastasis of 4T1 tumor depends on the antitumor immune response mediated by CD8~+ T cells.4.NP-si NC/CCL25 can reverse the drug resistance of PD-L1 m Ab.The synergistic effect of NP-si CD47/CCL25 and PD-1/PD-L1 m Ab significantly enhanced the immunotherapeutic effect on 4T1 tumor.