Tumor Microenvironment Remodeling-Antitumor Efficiency And Antitumor Mechanism Of Anaerobic Selective Salmonella And Its Variant Mediated Delivery Of HPRG And ShSTAT6

Tumor microenvironment which composed of the tumor cells,stromal cells and extracellular matrix is a relatively stable local microenvironment and provide material foundation to tumor growth,progression and metastasis.Tumor tissue hypoxia and and immunosuppression are the major biological characteristics of the tumor microenvironment.Therefore,anti-tumor effect can be achieved by improving tumor tissue hypoxia and and immunosuppression.Tumor tissue hypoxia is one of the basic characteristics of the tumor microenvironment.The main cause of tumor tissue hypoxia is tumor vascular abnormalities.Tumor vascular abnormalities not only exhibit increasing of vessel density but aslo show its structure and function abnormalities.HPRG as a cysteine protease inhibitors,which can significantly inhibited tumor angiogenesis and promote tumor vessel normalization.However,systemic administration of HPRG have poor targeting and low bioavailability.Salmonella VNP20009 is a facultative anaerobic gram-negative strains,which may accumulate in solid tumor,carry exogenous plasmid and expressed exogenous protein.Our previous studies show that VNP20009 can significantly inhibit tumor angiogenesis.Therefore,we choose VNP20009 to mediate HPRG protein expression and exert anti-tumor effect.In the first part of this study,we engineered VNP20009 to express HPRG under the control of a hypoxia-induced NirB promoter in tumor tissues.We evaluated the efficacy and mechanism of the VNP20009-mediated targeted expression of HPRG(VNP-pNHPRG)on tumor growth in primary and metastatic tumor models.We found that VNP-pNHPRG can preferentially accumulate in tumor tissues and effectively control HRPG expression in tumor tissues.Anti-tumor results showed that VNP-pNHPRG can significantly inhibit tumor growth,delay tumor double time and prolong survival time in 4T1 breast cancer mice than that of PBS and VNP-pN.Futhermore,VNP-pNHPRG can significantly inhibit accumulation of B16F10 tumor cell in lung tissue and prolong survival in B16F10 metastasis model than that of PBS and VNP-pN.H&E staining showed F1OB 16 tumor cell invasion and necrosis in lung tissues of PBS,VNP,and VNP-pN mice,but slight tumor cell invasion and inflammation in VNP-pNHPRG mice after 30 d.The study futher analyzed the anti-tumor mechanism of VNP-pNHPRG according to biological function of HPRG protein.Immunofluorescence assay showed that tumor tissue vascular density/areas in mice inoculated with VNP-pNHPRG significantly decreased compared with that in mice inoculated with PBS,VNP and VNP-pN.The ressults were futher tested by three-dimensional reconstruction of two-photon microscopy.Except to inhibiting tumor vascular density/areas,VNP-pNHPRG can significantly promote tumor vessel normalization.Two-photon microscopy assay showed that the branched structure of tumor vessel in mice inoculated with VNP-pNHPRG exhibited two branches,but three or several branches in mice inoculated with PBS and VNP-pN.Double staining for CD31 and desmin in tumor tissue sections facilitated the analysis of tumor vessel normalization.The results show that tumor vessels in VNP-pNHPRG mice were covered by numerous pericytes.By contrast,pericytes of tumor vessels in PBS,VNP,and VNP-pN mice were few or rare.Tumor vessel morphology were analyzed by SEM.Unlike tumor vessels in PBS,VNP,and VNP-pN mice,the walls of tumor vessels in VNP-pNHPRG mice showed smooth and regular ultrastructures.In summary,VNP-pNHPRG can enhance anti-angiogenic effects of VNP and promote the transformation of tumor vessels from abnormality to normality.Normalization of tumor vessel can improve tumor oxygenation.Immunofluorescence assay showed the HIF-la protein level in VNP-pNHPRG mice was significantly down-regulated compared with that in PBS,VNP,and VNP-pN mice.HIF-1Α accumulates and binds to HIF-1β,thus forming the active HIF complex that can initiate modulated expression of numerous angiogenesis genes such as VEGF and Ang-2 in hypoxic conditions.The real time PCR assay showed that VEGF and Ang-2 mRNA level in VNP-pNHPRG mice were also significantly down-regulated compared with those in PBS,VNP,and VNP-pN mice.In summary,VNP-pNHPRG can inhibit tumor growth and tumor metastasis mainly through the inhibition of tumor angiogenesis,normalization of tumor blood vessels and improvement of tumor hypoxic conditions.Immunosuppressive is another feature of tumor microenvironment.Reversing tumor immune suppression environment can effectively enhance the anti-tumor immune effect of immune cells to kill tumor cells.In the second part,we are tying to improve immunosuppressive microenvironment to achieve the anti-tumor effect.The studies show that STAT6 not only mediated immune response but also plays an important role in cell proliferation,apoptosis and tumorigenesis.In this study,it has been found that the level of STAT6 expression in breast tumor tissues is 7 times compared with the adjacent normal breast tissues.In order to verify that stat6 maybe involved in the proliferation and migration of 4T1 breast cancer cells,RNA interference technology was employed to silence the expression of STAT6 in 4T1 cells.Our results show that compared with the control group silencing stat6 led to the decreased proliferation and migration of 4T1 cells,indicating STAT6 may play an important role in the occurrence,development and metastasis of breast cancer cells.So targeted silencing STAT6 may inhibit tumor growth and metastasis of 4T1 breast cancer cells.Although salmonella can both carry plasmid encoding shRNA and preferentially accumulate and invade the tumor,VNP20009 remains within the salmonella-containing vacuole following invasion,within which they are unable release the interference plasmid and hence fail to regulate targeted protein.Therefore in order to silence the STAT6 expression by salmonella-based STAT6 shRNA,we construct a mutant namely VNP(PhoP/Q-)by inactivating the PhoQ/PhoP two-component regulatory system in VNP2009,which is necessary for salmonella survival within the cells,enablingit to silence the expression of STAT6 by effectively infecting cells and releasing interference plasmid.Firstly we analyzed the biological function of VNP(PhoP/Q-)strains by High Content Screening System,colony counting and immunofluorescence.All of them showed that compared with VNP20009 which hardly released the interference plasmid and rarely EGFP were observed,the ability of VNP(PhoP/Q-)strains to survive within the cells were considerably decreased and the expression of EGFP in the cytoplasm were noticeable after VNP(PhoP/Q-)strains invading the tumor cells and releasing interference plasmid.This showed that VNP(PhoP/Q-)can be used as an interference delivery vector.We next examined the therapeutic effect of VpSi-STAT6 in mouse on 4T1 breast cancer.The results showed that VpSi-STAT6 significantly reduced tumor volume,prolong the tumor doubling time,increase the survival time of mice and inhibit tumor lung metastasis which suggests that VpSi-STAT6 has a good therapeutic effect on 4T1 breast cancer.In addition,The studies in vitro and in vivo showed that VpSi-STAT6 can induce the 4T1 tumor cells and tumor tissue to release large amounts of CCL5.CCL5 is chemotactic factor of mononuclear macrophage which can induce mononuclear macrophage infiltration into tumor tissue.This study showed that CD4+,CD8+T lymphocytes,NK cells,DC cells and macrophages signifanctly enhanced in mice treated with VpSi-STAT6 than that of PBS and VpSi-Vector.The results showed that these cells may be involved in the antitumor effect of VpSi-STAT6.We then respectively co-cultured 4T1 STAT6 infected by VNP(PhoP/Q-)for 4 hours and 4T1 STAT6 with T lymphocytes and analyzed the expression of Thl/Th2 specific cytokines and transcription factor in 4T1 breast cancer cell and T lymphocytes by the real-time quantitative PCR.The results showed that compared with 4T1STAT6+cells,Thl specific cytokines including IL-12 and IFN-r were significantly upregulated while the Th2 specific cytokines including IL13 and IL10 were dramatically reduced,whereas IL4 was not detected,chemokine CCL5 was significantly upregulated,and CCL2 levels didn’t change.Compared with T lymphocytes co-cultured with 4T1STAT6+cells,TH1 cytokines(IL-12 and IFN-r)were significantly upregulated,whereas TH2 cytokines(IL-13,IL-4 and IL-10)did not change,while expression level of the transcription factor JAK1 and T-bet associated with activating TH1 cells was significantly increased.Thl/Th2 specific cytokines of the tumor tissue homogenates in vivo was consistent with in vitroexperiments.Thl cytokines of the group treated by VpSi-STAT6 was greatly increased than that of VpSi-Vector group,while th2 cytokines except IL-4 were significantly reduced.These suggested that the Thl/Th2 cytokine expression profile may be altered by VpSi-STAT6 which drive T helper 1(Thl)-cell polarization and activate the transcription of TH1 cytokines.Thl cytokine-enriched microenvironment enhance tumor cell killing which may partially explain the anti-tumor effect of VpSi-STAT6.Macrophages have heterogeneity which cell phenotype was transformed during different cytokine stimulation.This study shows M2 type macrophages in breast tumor tissue were reversed to M1 type macrophages when 4T1 tumor mice were treated with VpSi-STAT6.The results were futher conformed by VNP(PhoP/Q-)stimulated M2 STAT6 silent RAW 264.7 cell in vitro.M2 type phenotype transformation of macrophages in breast tumor tissue was one of the reasons anti-tumor effect of VpSi-STAT6.In addition,VpSi-STAT6 promoted the activation of DC cells in tumor tissue and may enhanced antigen presenting ability to mediated cell cytotoxic effect of CD8+T lymphocytes cells.A large number of infiltrating NK cells as congenital killer cells,can directly kill the tumor.In summary,VpSi-STAT6 inhibits the growth and metastasis of 4T1 breast cancer cancer.Except to STAT6 silencing in 4T1 cells inhibited cell proliferation and migration,VpSi-STAT6 improved is tumor immune microenvironment and increases anti-tumor immune response in tumor tissue.The study provided new ideas and reference for the treatment of breast cancer.