| ObjectiveAmong all stages of prostate cancer,bone metastasis represents an incurable stage,in which bone pain and skeletal-related events(SREs),including vertebral compressive fractures and pathologic fractures are blamed on bone destruction,and account for the worse quality of life and high rates of mortality.Of all the immune cell populations in the tumor microenvironment(TME),tumor-associated macrophages(TAMs)represent one of the most abundant cell subsets in multiple solid malignancies including prostate cancer.TAM-derived CCL5,an essential target in mediating immune escape,plays a vital role in accelerating prostate cancer stemness and bone metastasis.Remodeling the immunosuppressive TME is precisely the crucial mechanism and advantage of traditional Chinese medicine(TCM)in cancer treatment.it has been proven that FZYL formula delayed prostate cancer progression to castration-resistant prostate cancer and improved the immune function of the body.Preclinical studies have confirmed that icariin,the active component screened from FZYL formula,could target to inhibit TAMs/CCL5 in TME.Moreover,icariin has a strong ability to regulate the bone microenvironment.The purpose of this study is to determine whether icariin inhibits bone metastasis and bone destruction and to investigate whether icariin inhibits bone destruction in prostate cancer by regulating TAMs/CCL5-induced osteoclast differentiation.At present,many risk model of bone metastasis have been discussed,including PSA,Gleason score,and TNM stage.However,there is a lack of novel indicators for inclusion and even more lack of external validation sets to validate the model.In order to turn the core targets CCL5 in this experimental study into clinical application,this clinical study aims to construct a new bone metastasis diagnostic model by incorporating CCL5 and TCM syndrome type as clinical factors with other common clinical indicators,and apply external validation set to explore the model differentiation and clinical applicability.MethodsFor in vivo study,the mouse RM1-Luc bone metastasis model was built by injecting luciferase-labeled RM1 cells or RM1-Luc cells mixed with TAMs at a ratio of 1:3 into the tibia of BALB/c nude mice.The body weights and tumor diameters were measured.In vivo imaging and X-ray imaging were performed to detect the growth and bone metastasis of RM1 xenografts.the osteoblast and osteoclast activity was examined by TRAP and ALP staining.The tibia sections invaded by bone metastasis were detected by immunohistochemical method to observe whether icariin affected the expression of osteoclast-related proteins by regulating TAMs/CCL5.The homogenate of bone metastatic tumor in mice was detected by flow cytometry to observe the effect of icariin on the polarization of TAMs in vivo.The immunofluorescence assay was used to investigate the regulation of CCL5 by icariin in vivo.For bioinformatics analysis,Gene Expression Omnibus(GEO)data were used for the gene expression microarray experiment to detect the relationship between TAMs/CCL5/CCR5 axis and osteoblast.The Cancer Genome Atlas(TCGA)data were used for the clinical analysis of CD 163.We used the Log-rank test to compare the survival rates between the two groups(high CD 163 expression and low CD 163 expression).The expression of TAMs/CCL5 in the metastatic and adjacent metastatic lesion of human prostate cancer was detected by immunofluorescence.For in vitro study,the CCK8 experiment was used to investigate the icariin and CCL5 cytotoxicity in each cell line.The supernatant of RM1 was used to induce mouse mononuclear macrophage Raw264.7 into tumor-associated macrophage(TAMs).Condition medium of TAMs or Condition medium of icariin pre-treated TAMs was used to culture the pre-osteoclasts.TRAP,ALP,and F-actin ring assay were applied to investigate the osteoclast and osteoblast differentiation.Osteoclast differentiation-related proteins and genes were investigated by Western blotting and QPCR.TAMs/shCCL5,TAMs/rCCL5,TAMs/siSPIl,and TAMs/SPI1 were constructed by gene editing and applied in each group.For mechanism research,the effect of icariin on the polarization of TAMs was detected by flow cytometry.Elisa,Western blotting,QPCR,and double luciferase reporter gene assay were used to detect the effects of icariin on TAMs-derived CCL5 in the secretion,protein,transcription,and promoter levels.The pharmacological effect of icariin in the binding of SPI1 to CCL5 was verified by chromatin co-immunoprecipitation(ChIP-PCR).For clinical research,the cross-sectional study was conducted in Guangdong provincial hospitals(Ersha Hospital,Fangcun Hospital,and University City Hospital).Patients with prostate cancer diagnosed pathologically from 2011 to 2022 were collected and included bone metastasis-related information and routine clinical information.At the same time,prostate cancer tissues were collected to detect the expression of CCL5 by immunohistochemistry.In addition,the clinical data in the patient case system are sorted out and re-differentiated according to the syndrome differentiation of deficiency and excess,which is used as a modeling set.Univariate and multivariate logistic regression analysis was used to sift out the factors and build a bone metastasis classical risk model.The epidemiology and End Results(SEER)database was used as a verification set.ROC and DCA curves were applied to verify the classical risk model.Finally,two new clinical factors(CCL5 and syndrome type,)were included in the modeling set to construct a new bone metastasis diagnosis model.The discrimination evaluation analysis and clinical applicability evaluation of the new model and the classical model were compared by ROC and DCA curves.ResultsThe part of experimental research:Section 1:icariin inhibits bone metastasis and bone destruction of prostate cancer in vivo and in vitro.1.The volume of bone metastatic tumor in 20mg/kg/day icariin group and 40mg/kg/day icariin group decreased significantly,and the fluorescence signal of bone metastatic tumor decreased gradually(P<0.05),but there was no significant change in body weight between the intervention group and Saline group(P>0.05).2.Through X-ray and HE staining of the tibia in mice,it was found that the lesion of bone in icariin group was significantly less than that in Saline group.3.By TRAP/ALP double staining,it was found that compared with Saline group,20mg/kg/day icariin group and 40mg/kg/day icariin group significantly inhibited osteoclast activity(P<0.05).but had no significant effect on osteoblast activity(P>0.05).4.Raw264.7-derived pre-osteoclast cells,BMMs-derived osteoclast progenitor cells,and BMSCs-derived osteoblast progenitor cells showed no obvious cytotoxicity under the intervention of 2.5-50μM icariin by CCK8 assay.5.Concentration gradients(10,20,30μM)significantly inhibited osteoclast differentiation from Raw264.7 and BMMs(P<0.05).However,it had no significant effect on osteogenic differentiation(P>0.05).Section 2:There is a correlation between TAMs/CCL5/CCR5 axis and osteoclast in TME of prostate cancer.1.The expression of CD 163,the surface marker of TAMs,was significantly higher in prostate cancer than that in normal prostate tissue,and was related to the low progression-free survival rate.CCR5 was expressed in prostate cancer,osteoclasts and osteoblasts,and the expression level of CCL5 was positively correlated with osteoclast-related genes(P<0.05).2.Immunofluorescence and TRAP/ALP staining of pathological sections of human prostate cancer tissue and bone metastasis tissue showed that CD 163 and CCL5 were expressed in both these tissues(P<0.05),and the osteoclast activity of bone metastasis was higher than that of para-bone metastasis,but there was no significant change in osteoblast activity between the two groups(P<0.05).Section 3:Icariin inhibits RANKL-induced osteoclast differentiation in the presence of TAMs supernatant.1.The supernatant of prostate cancer cell RM1 could significantly induce the proportion of CD206+/F4/80+ cell subsets(representing TAMs subsets)(P<0.05).2.The supernatant of TAMs could promote osteoclast differentiation and formation,while the supernatant of TAMs pretreated with icariin 30μM could significantly inhibit osteoclast differentiation and formation(P<0.05).3.The supernatant of TAMs could promote the expression of osteoclast differentiation-related protein and mRNA,while the expression of osteoclast differentiation-related protein and mRNA could be significantly inhibited by icariin-pretreated TAMs supernatant(P<0.05).Section 4:Icariin inhibits M2 polarization of TAMs and inhibits CCL5 expression at the transcriptional level by inhibiting SPI1.1.By CCK8 and flow cytometry,it was found that icariin in concentration gradient did not show obvious cytotoxicity in TAMs,but significantly inhibited the M2 phenotypic polarization of TAMs(P<0.05).2.Elisa,western blotting,QPCR and double luciferase reporter gene experiments showed that icariin inhibited CCL5 expression at secretion,protein,transcription and promoter activity levels(P<0.05).3.Icariin was found to inhibit CCL5 transcription factor SPI1 protein by immunofluorescence and western blotting.ChIP-PCR further confirmed that icariin could inhibit the expression of CCL5 by inhibiting the direct binding of SPI1 to CCL5 promoter.Section 5:Icariin inhibits osteoclast differentiation by regulating TAMs/CCL5 axis.Both 20ng/ml CCL5 recombinant protein and TAMs supernatant could promote osteoclast differentiation,formation,osteoclast related protein and mRNA expression,while TAM/shCCL5 group and 30μM icariin pretreated TAMs-CM group could inhibit osteoclast differentiation,formation,osteoclast related protein and mRNA expression in TAMs supernatant.However,the inhibitory effect of icariin could be reversed by adding 20ng/ml CCL5 recombinant protein.There were significant differences in the above-mentioned comparisons(P<0.05).Section 6:CCL5 induces the differentiation and chemotaxis of pre-osteoclast cells by binding with CCR5.1.Western blotting showed that the expression of CCR5 was the lowest when it was not stimulated by RANKL,which belonged to monocyte stage.With the stimulation of RANKL,the pre-osteoclast cells gradually changed from monocytes to mature osteoclasts,and the expression of CCR5 was the highest from the second day to the fourth day during osteoclast induction.2.CCK8 showed that CCL5 intervention with concentration gradient had no obvious cytotoxic effect on osteoclast progenitor cells.3.The concentration gradient of CCL5(20-40ng/ml)could promote osteoclast differentiation and formation,while the addition of CCR5 specific inhibitor DAPTA could partially inhibit the promoting effect of CCL5(P<0.05).4.Trans well and scratch test showed that the concentration gradient of CCL5(20-40ng/ml)could promote the chemotactic migration of pre-osteoclast cells,while the addition of DAPTA could partially inhibit the chemotactic migration of CCL5(P<0.05).Section 7:Icariin inhibits PCa-induced osteolytic bone destruction in vivo by downmodulating osteoclastogenesis via inhibiting the TAMs/CCL5 pathway1.The results of tumor volume and in vivo imaging in mice showed that icariin could significantly delay the tumor growth of prostate cancer RM1-Luc bone metastatic tumor model mice by regulating TAMs/CCL5 pathway(P<0.05).2.The results of tibia X-ray,HE staining and TRAP staining in mice showed that icariin could down-regulate the osteoclast activity and inhibit bone destruction by regulating the TAMs/CCL5 signaling.It could also inhibit the expression of osteoclast-related protein(P<0.05).3.Flow cytometry and immunofluorescence assay of bone tumor slices showed that icariin could reduce the infiltration of TAMs and inhibit the expression of CCL5(P<0.05).The part of clinical research:1.There were 48 cases of deficiency syndrome and 16 cases of excess syndrome in bone metastasis group,36 cases of deficiency syndrome and 60 cases of excess syndrome in non-bone metastasis group,the difference was statistically significant(P<0.05).In bone metastasis group,there were 50 cases with high expression of CCL5 whlie only 14 cases with low expression.There were 25 cases with high expression of CCL5 in non-bone metastasis group while 65 cases with low expression.2.Univariate analysis showed that there were significant differences in T stage,N stage,PSA,Gleason score,syndrome type and CCL5 expression.The risk factors with statistical significance in the above univariate analysis were included in the multivariate logistic regression.It was found that T stage,PSA,Gleason score,and CCL5 were the risk factors for the diagnosis of bone metastasis.However,the syndrome type had no statistical significance in multivariate analysis(P>0.05).3.The classical bone metastasis diagnosis model was established based on T stage,PSA and Gleason score of the modeling set.The discrimination evaluation analysis shows that the AUC of the modeling set is 0.903 and the AUC of the verification set is 0.840.The analysis of clinical applicability evaluation showed that the DCA curve in the modeling set had a higher net benefit rate when the probability of diagnosing prostate cancer bone metastasis was between 30%and 80%.the DCA curve in the verification set showed a higher net benefit rate when the probability of diagnosing bone metastasis of prostate cancer was between 35%and 80%.4.A new bone metastasis diagnosis model was constructed based on the T stage,PSA,Gleason score of the modeling set,and CCL5 expression,which was named CCL5 model.Comparing the CCL5 model with the classical model,it is shown that the CCL5 model is superior to the classical model in terms of differentiation and clinical applicability.5.Nomogram was constructed to visualize the diagnosis model of bone metastasis of prostate cancer.ConclusionThis study systematically revealed that TAMs-derived CCL5 can induce the chemotaxis of pre-osteoclast cells and promote their differentiation into osteoclasts in prostate cancer bone metastasis.Icariin inhibits bone metastasis and bone destruction of prostate cancer by blocking osteoclast differentiation induced by TAMs/CCL5.This study not only confirmed the potential efficacy of icariin in the treatment of bone destruction in prostate cancer,but also provided a new insight for TAMs/CCL5/osteoclast axis blocking as a promising drug strategy for the treatment of bone destruction in prostate cancer.Moreover,a new bone metastasis diagnosis model was constructed based on the target CCL5.It was proved that the CCL5 bone metastasis diagnostic model was better than the classical model in terms of differentiation and clinical applicability compared with the classical model. |
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