| In various malignant tumors,metastasis is responsible for over 90%of tumor-induced death.Though emerging technologies provided early detection of tumor metastasis or even warning of high metastatic risk before the actual occurrence of metastasis,clinical treatment on metastasis prevention lags far behind.Pieces of evidence have illustrated that primary tumor-induced the formation of pre-metastatic niche(PMN)in distal organs by producing pro-metastasis factors.Given the fundamental role of PMN in the development of metastases,this study focused on the process of PMN establishment and metastasis development and proposed a metastasis-preventive treatment to intervene PMN,that is,to take effective measures to curb the formation of PMN before the actual occurrence of tumor metastasis.The pathological process of PMN formation including the alterations in cellular and molecular characteristics was investigated over time.Dynamic changes in the pathological process of the matrix microenvironment and the immune microenvironment were monitored.The increased gathering of?-smooth muscle actin~+(?SMA~+)activated fibroblasts was observed,with the up-regulation of extracellular matrix components fibronectin(FN),matrix metalloproteinases(MMPs)as well as the pro-angiogenic factor(VEGF),angiogenesis and increased vascular leakage induced by matrix remodeling and the incomplete structure of neovasculature.The constant increase of MDSCs recruitment was demonstrated to exacerbate the immunosuppressive microenvironment.Based on the high expression of MMPs in PMN,this study developed a MMP-responsive post-processed self-assembling peptide FR17,aiming to prevent tumor metastasis by regulating and intervening in PMN formation through targeting-delivery of immunomodulatory drug and in-situ assembled peptide.Peptide FR17 is a branched-chain polypeptide consisting of three parts:(1)the main chain ffky with self-assembly property;(2)the immunomodulatory peptide thymopentin(TP5);(3)MMP2 or MMP9-degradable peptide PLGLAG,which is applied to link TP5 to the side-chain of ffky.FR17could be specifically triggered to assemble in-situ in PMN,i.e.,PMN-activated post-assembling,and release TP5 at the same time.After enzymatic degradation,FR17produces the self-assembling fragment sequence FG8(ffk(GPLG)y),which would fold to construct nano-sized films,here termed as"peptide nano-blanket",through intermolecular hydrogen bonding,?-?stacking and other interatomic forces or intermolecular interactions.To evaluate whether FR17 is able to intervene in the PMN formation and whether it has the potential to realize the preventive treatment of tumor metastasis,the melanoma lung metastasis models with induced PMN were established.The anti-metastatic efficacy of FR17 was investigated in the PMN model and the post-surgery lung metastasis model.Consequently,FR17 administration effectively inhibited pulmonary PMN formation and postoperative metastasis of melanoma,prolonged the survival time of model mice.The above experimental results support the prevention and treatment strategy proposed in this study that tumor metastasis can be impeded by inhibiting the formation of the pre-metastatic microenvironment.Meanwhile,in order to reveal the respective roles of the two sequence components(i.e.,the self-assembling peptide segment FG8 and the immunomodulator TP5)released after the enzyme-responsive degradation of FR17 in PMN,a scrambled PMN-activated post-processed self-assembling peptide s FD17without the immunomodulatory bioactivity was designed and employed as the peptide assembly control.s FD17 exhibited almost the same inhibitory effect as FR17 on metastasis,which indicated that the inhibitory effect of FR17 on melanoma lung metastasis was mainly contributed by the in-situ assembled"peptide nano-blanket",while the free drug TP5 exhibited almost no substantial inhibitory effect on the occurrence and development of lung metastases.Therefore,the author next explored the mechanisms behind the above-mentioned significant inhibitory effect on metastasis achieved by"drug-free carrier",i.e."peptide nano-blanket".Based on the previous research foundation of PMN,the activation of tissue-resident fibroblasts can be regarded as the tipping point of PMN initialization.FR17 degraded by the overexpressed MMPs would assemble to form"peptide nano-blanket"in situ,which was found to inhibit the activation of PMN-associated fibroblasts induced by TDSF in PMN.FR17 and s FD17 exhibited a similar inhibitory effect on fibroblast activation,which was mainly reflected by the limit on the overactivated cellular functions,including cell proliferation,the expression of FN,MMPs,VEGF and other matrix remodeling proteins,migration and collagen contractility.What's more,the prophylactic administration of FR17 or s FD17 significantly reduced the activation ratio of fibroblasts in the lung tissues of PMN model mice after FR17 or s FD17 administration,with the down-regulation of extracellular matrix components,decrease on collagen deposition and cross-linking.Referring to the vital role of tumor-associated fibroblasts in the regulation of tumor vascular and matrix remodeling,the author further explored whether the"peptide nano-blanket"can affect vascular endothelial cells via mediation of PMN-associated fibroblasts,thereby regulating angiogenesis and vascular integrity in PMN.On the one hand,the study verified the critical role of PMN-associated fibroblasts in regulating the proliferation,angiogenesis,and intercellular junctions of vascular endothelial cells in PMN by secreting proteins,controlling the modeling of the pre-metastatic matrix microenvironment and vascular integrity.On the other hand,experimental results also proved that"peptide nano-blanket"has a protective effect on fibroblasts,that is,the"peptide nano-blanket"restricts the activation process of fibroblasts induced by TDSF.Both FR17 and s FD17 effectively inhibited the proliferation activity of vascular endothelial cells and restricted their angiogenesis ability in vitro,maintaining the integrity of vascular endothelial cell adhesion junctions and reducing vascular leakage.Next,the study further explored the effect of FR17 on MDSC recruitment in PMN.FR17 effectively inhibited the recruitment of MDSCs in the PMN lung.Through different cellular pathways,the two major components of FR17,TP5 and the in-situ assembled"peptide nano-blanket"exerted inhibitory effects respectively on the immunosuppressive microenvironment.The co-localization of extracellular matrix components and MDSCs confirmed that the"peptide nano-blanket"restrained MDSC recruitment by down-regulation of extracellular matrix components through the inhibition of fibroblast activation.In addition,the transcriptome analysis of PMN-MDSCs recruited to the lung in different treatment groups demonstrated that the regulation effect of FR17 was mainly related to the activation of immune response pathway,cytokine production involved in immune response,regulation of leukocyte and lymphocyte activation,leukocyte chemotaxis and migration.Among them,the effect on the chemotaxis and migration of immune cells is mainly related to the in-situ assembled"peptide nano-blanket",while the effect on immune cell differentiation and T cell activation is mainly related to TP5.Based on the observation of the pathological process and molecular biological characteristics of PMN lung,a PMN-activated post-assembling peptide FR17 was developed.The peptide was able to assemble to construct the"peptide nano-blanket"in response to PMN in situ,exhibiting significant prevention and treatment on tumor metastasis,revealing certain potential in the application of inhibiting postoperative recurrence and metastasis of malignant tumors.Alongside the cellular pathway of TDSF---pre-metastasis associated fibroblast---vascular endothelial cells---MDSC,the study revealed that the in-situ assembled"peptide nano-blanket"inhibited the activation of fibroblasts in the early stage of PMN,inhibited the formation and development of the pro-metastasis matrix microenvironment,suppressing matrix remodeling,angiogenesis and vascular leakage,reduced MDSC recruitment to improve the immunosuppressive microenvironment,so as to prevent and inhibit the occurrence of metastasis by intervening in the formation of PMN.This study revealed the critical regulatory role of PMN-associated fibroblasts in PMN,offering a promising target for futural development on PMN intervention,and providing a PMN post-processed self-assembled functional bio-platform with microenvironment regulating capacity for the delivery of anti-tumoral or anti-metastatic drugs. |
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