Effects Of STLR9~+ Neutrophils In Subcutaneous Tumor Nodules On Tumor Microenvironment And Tumor Development In Mice

Over the past few decades,tumor immunotherapy has greatly improved the therapeutic prognosis of various malignancies,but its benefits remain limited to a subset of patients and tumor types.The main reason is the paucity of lymphocytes in the tumor microenvironment(TME),also known as“cold tumor".While in TME,neutrophils often serve as initiating cells that can trigger DCs and T cells to infiltrate and activate,playing an important role in changing the tumor microenvironment from "cold",to "hot".Neutrophils are traditionally viewed as the first line of defense against the invasion of pathogenic microorganisms,and participating in the clearance of pathogenic microorganisms.In fact,neutrophils are also potent antitumor immune cells in neoplastic diseases.The function of neutrophils has plasticity,and whether it presents an antitumor or a tumor promoting phenotype is related to the tumor microenvironment and the stage of the tumor.Neutrophils at early stages of tumorigenesis are anti-tumor,whereas those at late stages of tumorigenesis have predominantly pro-tumor effects.At the early stage of tumor microenvironment formation,the utilization of immune agonists to suppress the pro tumor activity of neutrophils,enhance their anti-tumor phenotype,and thereby guide lymphocyte enriched and activated tumor microenvironment formation to inhibit tumor growth,seems to be a promising approach for tumor therapy.There are many subtypes of neutrophils,and the functions of neutrophils are not the same in different subtypes.In recent years,it has been shown that human and mouse neutrophils express surface TLR9(sTLR9),and such neutrophils are referred to as sTLR9+neutrophils.In infectious diseases,sTLR9+neutrophils are a subpopulation that plays an anti-inflammatory role by expressing IL-10.However,the role of sTLR9+neutrophils during tumor progression remains unclear.In a study exploring the optimal therapeutic strategy for tumor vaccine inhibition of subcutaneous tumor in mice,it was unexpectedly found the formation of sTLR9+neutrophil-enriched tumor nodules induced by tumor cells at the inoculation site of tumor cells.In this study,we intended to use the sTLR9 expressed on neutrophils as an entry point,and analyzed the level of sTLR9+ neutrophils and their expression of immunosuppressive or immune promoting molecules during tumor progression by using tumor nodules rapidly induced by subcutaneous injection of tumor cells in mice as an experimental mode.Furthermore,we applied CpG ODN to modulate the expression of sTLR9 on neutrophil in tumor nodules,and then observed the effects on tumor growth and TME formation in mice.The results were as follows:1.The formation of subcutaneous neutrophil-infiltrated nodules induced at the site of tumor cell inoculationIn the process of exploring the optimal antitumor strategy of tumor tissue lysates combined with CpG ODN,it was found that subcutaneous inoculation of tumor cells in mice seemed to induce some kind of antitumor immune response locally upon injection.Therefore,we subcutaneously(s.c.)inoculated 2×106 CT26 colon carcinoma cells(CT26 cells)into the groin area of BALB/c mice.Unexpectedly,at 24 h post inoculation,a nodule was noticed at the inoculation site.The nodules,with a size about 4 mm × 2.5 mm(length × width),were coated with an intact and transparent membrane.For gaining an insight into the nodule,cells in the nodule were harvested and analyzed by immunofluorescence and flow cytometry.The inoculated CT26 cells,which could be recognized by anti-Hsp70 antibodies,were dispersed among the nodule cells.More precisely,CD45+leukocytes constituted 64.5%of the nodule cells.Among the CD45+leukocytes,98%-99%are Ly6G+neutrophils,and 1%-2%are F4/80+macrophages.The results revealed that inoculating tumor cells could rapidly induce a subcutaneous nodule at inoculation site.The nodules were massively infiltrated with neutrophils,and therefore designated as neutrophil-infiltrated tumor nodules(referred to as tumor modules).To discovery correlations of the number of inoculated tumor cells and tumor nodule appearance,we s.c.inoculated the mice with 2 × 104,2 ×105 and 2 ×106 CT26 cells respectively,and checked the tumor nodule formation up to 13 days.At 24 h post inoculation,tumor nodules developed in all of the mice inoculated with 2 ×106 CT26 cells.At day 3 or day 7 post inoculation,the tumor nodules occurred in two out of five mice received 2 × 104 CT26 cells,and in four out of five mice received 2 × 105 CT26 cells,respectively.Therefore,we used 2×106 CT26 cells per mouse inoculation to carry out the following experiments.To explore the universality of various tumor cells in inducing the tumor nodules,we inoculated B16 melanoma cells(B16 cells)or H22 hepatocellular carcinoma cells(H22 cells)into the groin areas of mice respectively,and observed the tumor nodule occurrence.Consistently,the B16 and H22 cells also induced tumor nodules,indicating that short-term induction of tumor nodule formation by the inoculation of tumor cells was a common phenomenon.2.sTLR9+neutrophils in tumor nodules and their characterization in tumor developmentBecause of the curiosity about the sTLR9 molecules expressed on neutrophils,we detected whether the neutrophils in tumor nodules could express sTLR9 or not.It was revealed that approximate 22%of neutrophils in tumor nodules at 24h post-inoculation with CT26 cells were sTLR9+neutrophils.Noticeably,when the tumor nodules developed into solid tumors with the average size of 100-150 mm3 or 1000mm3 at day 7 or day 13 post-inoculation,respectively,sTLR9+neutrophils constituted of 47.5%or 90.8%of Ly6G+neutrophils in the tumor tissues at day 7 or day 13 post inoculation.In parallel,sTLR9+F4/80+macrophages in the tumor nodules/tumor tissues increased consistently.Similar changes in sTLR9+ neutrophils and sTLR9+ macrophages were also detected in the tumor nodules or tumor tissues induced by B16 cells or H22 cells.The observations imply the sTLR9+ neutrophils are involved in the progression of tumors.To further probe the role of sTLR9+neutrophils in tumor progression,we kinetically detected the expression of PD-L1,IL-10 and TNFα in sTLR9+neutrophils and sTLR9-neutrophils in the tumor nodules or tumor tissues.Comparatively,the expression of PD-L1/IL-10 in sTLR9+neutrophils was 1.7-fold or 4-fold higher than that in sTLR9-neutrophils at day 7 post inoculation.At day 13 post inoculation,PDL1/IL-10 expressing sTLR9-neutrophils were barely detected.Differently,At day 13 post inoculation,the number of TNFα+sTLR9-neutrophils was three times higher than the number of TNFα+sTLR9+neutrophils.To sum up,the results support the presumption that sTLR9+neutrophils may favor tumor progression.3.Downregulation of sTLR9 on neutrophils affects tumor growth in tumor bearing miceTo research whether the downregulation of sTLR9 on neutrophils in tumor nodules was beneficial to the induction of neutrophil activation,we used a self-designed CpG ODN,CpG 5805,as a tool to regulate sTLR9 on neutrophils in tumor nodules.We found that the percentage of sTLR9+neutrophils was obviously reduced from 25%to 15%at 6 h while the percentage of ICAM1+neutrophils markedly increased from 35%to 60%at 6 h,respectively,after CpG 5805 injection.To explore whether the downregulation of sTLR9 on neutrophils in tumor nodules at the early stage of tumor development contributed to the inhibition of tumor growth,two strategies were adopted to administer CpG 5805 treatment locally at the site of CT26 cell inoculation(Strategy 1 and Strategy 2:the former started treatment when tumor nodules had formed,and the latter when tumor tissues had formed),and then the tumor growth and survival of tumor-bearing mice were dynamically monitored.We found that the treatment with CpG 5805 in both strategies all significantly inhibited the tumor growth compared with PBS treatment.Comparatively,CpG 5805 injection at strategy 1 was more efficient,and prolonged the survival of tumor-bearing mice to a greater extent(p=0.0027).To further confirm the finding above,we chose an inhibitory ODN with CCT repeats(CCT ODN)as a control to inhibit TLR9 migration,in order to observe whether the sTLR9 and ICAM1 on neutrophils would be affected.The result revealed that the sTLR9+neutrophils in tumor nodules did not decrease,nor did ICAM1+neutrophils increase,and could not inhibit tumor growth,with a trend of tumor growth curves similar to that of the PBS group.By the same way in B16-bearing model mice,CCT ODN also had no effect on inhibiting the tumor growth.Together,the results suggested that the reduction of sTLR9 on neutrophils in tumor nodules at the early stage of tumor development contributes to the formation of an anti-tumor microenvironment.In addition,we investigated the effect of downregulating neutrophil sTLR9 on tumor killing ability of neutrophils in vitro.We harvested peripheral blood from BALB/c mice and purified the neutrophils peripheral blood,and then established a coculture system of neutrophils and CT26 cells.When CpG 5805 was added to the well plates for 24h,the expression of sTLR9 on neutrophils was downregulated,and the killing ability of neutrophils against CT26 cells was enhanced.To sum up,the downregulation of sTLR9 on neutrophil could contribute to the inhibition of tumor growth.sTLR9 appears to be an immune checkpoint molecule on neutrophils and serves as a novel target for tumor therapy.4.Downregulating the sTLR9 on neutrophils affects tumor immune microenvironment and systemic immune response in tumor bearing miceTo investigate whether downregulating the sTLR9 on neutrophils in tumor nodules contributed to the formation of an anti-tumor immune microenvironment,we detected the percentage and activation of neutrophils,dendritic cells(DCs)and CD8+T cells in tumor nodules of CT26 cell inoculated mice post 24 h of three injections of CpG 5805 under strategy 1.We found that Ly6G+neutrophils and CD8+T cells were markedly increased in tumor nodules of mice treated with CpG 5805.Among the increased Ly6G+neutrophils,sTLR9 expression was obviously reduced and the ICAM1 expression were markedly increased.Compared with PBS group,the percentage of CD80+/CD86+DCs was doubled in the tumor nodules after CpG 5805 treatment.Meanwhile,in the tumor nodule cells of mice treated with CpG 5805,the percentage of 4-1BB+CD8+T cells and IFNy+cells upregulated by about 1/3,respectively.The result reveals that the reduction of sTLR9 on neutrophils in tumor nodules can indeed induce the formation of an antitumor immune microenvironment.Based on our understanding of the immune activation effect of TLR9 agonist,we speculate that CpG 5805,in addition to the above anti-tumor effect,should also have the effect on the immune cells in tumor draining lymph nodes(TDLNs)and spleens.Therefore,we tested the lymphocyte and its activation index of TDLNs and spleens,and found that after CpG 5805 treatment,the volumes of TDLNs and spleens of mice treated with CpG 5805 were significantly larger.In addition,in TDLNs and spleens,we found that CpG 5805 promoted the activation of DC cells,NK cells and CD8+T cells.These results are consistent with previous explanations of the antitumor effect induced by CpG ODN.In this study,we unexpectedly found a subcutaneous nodule in the groin areas of mice inoculated with tumor cells.The nodule was developed 24 h after the inoculation,filled with tumor cells and massively recruited neutrophils,being designated as neutrophil-enriched tumor nodules(termed as tumor nodules).22%of the neutrophils in tumor nodules are sTLR9+neutrophils.With tumor progression,sTLR9+ neutrophils were sustainably increased in tumor nodules/tumor tissues,and approximately 90%of neutrophils at day 13 after tumor cell inoculation expressed sTLR9,and sTLR9+neutrophils highly expressed PD-L1 and IL-10.The downregulation of the expression of sTLR9 on neutrophils in tumor nodules of mice contributed to awaken the viability of neutrophils,inducing antitumor microenvironment formation.This study provides insights into understanding the role of sTLR9+ neutrophils in tumor development,especially in the early stages of tumor development,and provides a new idea on tumor immunotherapy.