Endostatin Synergized With Tumor Special DC-T Cellular Therapy On Antitumor Effect And Its Possible Mechanisms

In recent years, malignant tumors have been a worldwide issue of public health. To most patients with tumors, conventional treatment strategies such as surgery, chemotherapy and radiotherapy couldn’t effectively control diseases. As an emerging antitumor therapeutic method, cellular immunotherapy has remarkable curative effect on tumors, even those refractory to chemotherapy and radiotherapy. However, its clinical benefits in cancer therapy are very modest. At present, how to improve antitumor effect of cellular immunotherapy by combination with different safty and targeted therapies is always the key point in cancer research of antitumor biotherapy.Angiogenesis is one of the basic hallmarks of cancer. Antiangiogenic therapy is a new and good prospective treatment strategy. Studies showed that monotherapy with antiangiogenic agents is failing to produce longlasting clinical responses in most patients, however, antiangiogenic molecules may contribute to persistent tumor’s remission in combination with immunotherapy. And it suggested that antiangiogenic agents synergized with cellular immunotherapy. Endostatin is regarded as one of the promising antiangiogenesis agents with less adverse side effects and seldom susceptible to the resistance to antigiogenesis. However, it’s rare to study on the role and mechanism of Endostatin playing in cellular therapeutic potency. To explore the relationship between Endostatin and cellular immunotherapy on antitumor treatment and provide a theoretical basis for raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy. The inflammatory tumor microenvironment forms a major part of tumors and plays a vital role in the antitumor effect of cellular therapy, and adoptive cytotoxic T cells would be changed into immunosuppressive regular T cells (Treg) through many immunosuppressive factors such as MDSC in the tumor microenvironment. The immune inhibitory cells (MDSC, TAM, and imDC) and proinflammatory cytokines (IL-6,Il-17, TNF-α and chemokines) promote angiogenesis and tissue remodeling and contribute to tumor development and innumosuppression in the tunor micro-environment. Recent studies demonstrated that antiangiogenic agent Sunitinib decreased immunosuppressive cells such as MDSC and Treg, immunosuppressive cytokines IL-10and TGF-β, as well as T cell inhibitory factor PD-1and reversed immunosuppression in the tumor microenvironment. However, it’s rare to study on the role and mechanism of Endostatin playing in the immune cells and related cytokines in the tumor microenvironment. In a Lewis lung cancer tumor-bearing mice model, we investigated the mechanism of Endostatin antitumor potency and provided a theoretical basis for designing the optimal strategy of Endostatin combined with cellular immunotherapy in cancer treatment.Accurate evaluation of the changes in the tumor microenvironment after antiangiogenic therapy is very important to definite the mechanism of antitumor effect and design the optimal strategy in cancer treatment. Inflammatory cells and mediators are tightly related to proangiogenesis factors in the tumor microenvironment. Both TNF-α and IL-6are the vital proinflammatory cytokines in the tumor microenvironment. TNF-α promotes CXC chemokines and their receptor CXCR2axis signaling and plays the crucial role in neuvascularization of lung tissue in pulmonary inflammation and ischemia injury. The level of TNF-α is significantly higher in the serums of patients with non-small cell lung cancer (NSCLC) than that of healthy individuals. IL-6induces VEGF transduction, proliferation of endothial cells, and tumor metastasis via STAT3signaling in many solid tumors. Studies have demonstrated that genic polymorphisms of TNF-α and IL-6are associated with increased occurring risk of some benign or malignant diseases. In the present study, we studied SNP markers in the promoter-regulating regions of TNF-α and IL-6genes in the ethnic group Han of North China, to explore the SNP polymorphisms of the TNF-α and IL-6genes relevant to occurring of lung cancer. It will further investigate the relationship between pro-angiogenic inflammatory cytokines in the tumor microenvironment and occurring of lung cancer. Moreover, we may provide the theoretical basis for the main targets and mechanism of Endostatin antitumor effect.Induction of angiogenesis requires a shift/switch towards activation/upregulation of inducers of angiogenesis over suppression of angiogenic inhibitors in the tumor microenvironment. Preclinical studies and clinical trials mainly focused on VEGF and its receptors signaling, and demonstrated that VEGF-A and its receptor VEGFR-2are key targets of antiangiogenic agents. However, it’s rare to study on role and the molecular mechanism of Endostatin antitumor effect. In the human Genome study, the data demonstrated that Endostatin down-regulated proangiogenic signaling relevant to the endothelial cells such as VEGF, HIF-lα, and TNF-a receptor (sTNFR) and so on.The data suggested that Endostatin is a multi-target antiangiogenic agent. The cells and mediators of inflammation play the crucial role in tumor angiogenesis, so they are the promising therapeutic standpoints in antiangiogenic therapy.To screen the proflammatory cytokines TNF-α, IL-6and their receptors as well as down-stream factors of their signaling pathways by Propeome Profiling microarray and definite the main targets and mechanism of Endostatin antitumor effect, so as to provide a new perspective and theoretical basis for the optimal strategy of Endostatin combined with cellular immunotherapy.Based on the previous study that we successfully cultured and achieved tumor antigen special DC-T cells of Lewis lung cancer, further demonstrated cytotoxic CD8+T cells are the main subpopulation of the DC-T cells detected by Flow cytometry (FCM). In this project, we explored the relationship between Endostatin and cellular immunotherapy on antitumor treatment, the role and molecular mechanisms of Endostatin playing its antitumor effect in the tumor microenvironment, then provided a theoretical basis for the main targets and mechanism of Endostatin antitumor effect and promoting the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy. Moreover, we invetigated the SNP polymorphisms of the TNF-a and IL-6genes relevant to occurring of lung cancer in the ethnic group Han of North China.Furthermore, we screened the main targets of Endostatin antitumor effect by Propeome Profiling microarray, we may provide a theoretical basis for the optimal strategy of raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy.Part I Endostatin Synergized with Tumor Special DC-T Cellular Therapy on Antitumor EffectOBJECTIVE To investigate the role of Endostatin playing in tumor special DC-T cellular therapy on antitumor effect.METHODS1. The transplanted Lewis lung cancer models of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into three groups, including DC-T cells combined with Endostatin group, DC-T cells group, and PBS control group.There were seven mice in each group. Since day7, the tumor-bearing mice had been administered by Endostatin or DC-T cells or PBS.The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.2. In DC-T cells combined with Endostatin group, each tumor-bearing mouse had been treated by rhEndostatin15mg/Kg/d by tail vein injection since day7after transplanted with LLCs, for14days.3. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy C57BL/6mice by Ficoll density gradient centrifugation, T lyphoytes were enriched from PBMCs by Nylon wool column method. On day5, DCs and tumor antigen from LLC by repetitive freeze thaw method were cocultured for3days, then transferred into the culture flasks containing T cells, and cocultured for24hours, tumor antigen special DC-T cells were harvested. And, the T cell subpopulation was detected by Flow cytometry (FCM).4. In DC-T cells group and DC-T cells combined with Endostatin group, each tumor-bearing mouse was treated by DC-T cells5×106by tail vein injection on day7after transplanted with LLCs.5. All the mice were sacrificed24hours later after the last time of administration. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.6. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.7. The proportions of CD8+T, mDC, TAM (M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by FCM.8. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.RESULTS:1. Compared with control group, tumor special DC-T cells inhibited the tumors growth (P=0.021); tumor special DC-T cells combined with Endostatin remarkably regressed the tumors better than PBS control (P=0.009). The data of immunohistochemical staining are shown that CD31was emplored as the marker of vascular endothelial cell and its cytoplasm was dyed yellow-brown. The Microenvironment Density in DC-T cells group was slightly lower than that in the control group (P=0.027); the MVD of tumor special DC-T cells combined with Endostatin was deeply lowest in comparison with control group (P=0.002), concurrently tumor necrosis obviously increased in comparison with control group.2. The expressions of VEGF, IL-6, IL-17in the tumors were decreased, concurrently those of IFN-γ and HIF-la were increased administered with DC-T cells compared with PBS control (P<0.05, respectively). Tumor special DC-T cells combined with Endostatin alleviated VEGF, IL-6, and IL-17, concurrently elevated IFN-y and HIF-la remarkably better than PBS control (P<0.01, respectively).3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by DC-T cells therapy, compared with PBS control(P<0.05, respectively). The changes in tumor special DC-T cells combined with Endostatin were strongly more than PBS control (P<0.01, respectively).4. Tumor special DC-T cells combined with Endostatin potently reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, enhanced that of IFN-γ, concurrently recruited CD8+T cells into the tumor tissue in comparison with control group (P<0.01, respectively); The changes in DC-T cells group were significantly more than control group(P<0.05, respectively).CONCLUSIONS:1. Tumor special DC-T cells combined with Endostatin potently reduced tumor vasculature and tumor growth, and augmented tumor necrosis, implying the better antitumor effects than monotherapy.2. Tumor special DC-T cells combined with Endostatin strongly inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors, and intensified the hypoxia in the tumors.3. Tumor special DC-T cells combined with Endostatin remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment.4. Tumor special DC-T cells combined with Endostatin effectively provided the advantage of reducing the levels of immunosuppressive cytokines in the tumor regions, further exhibited synergistic and much better antitumor effects than monotherapy strategy, making them more vulnerable to the immune reactions. Part Ⅱ Endostatin Effectively Relieved Immunosuppression of the Inflammatory Tumor Microenvironment in Lung CanerOBJECTIVE To investigate the role of Endostatin playing in immunosuppressive cells and related cytokines in the inflammatory tumor microenvironment in lung caner.METHODS1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells(LLC). The tumor-bearing mice were randomly divided into three groups, including high-dose Endostatin group (rhEndostatin15mg/Kg/d), low-dose Endostatin (rhEndostatin7.5mg/Kg/d) group, and control group(PBS0.2ml/d), all the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days.There were seven mice in each group. All the mice were sacrificed24hours later after the last time of administration. The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.2. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.3. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.4. The proportions of CD8+T, mDC, TAM(M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by Flow cytometry (FCM).5. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.RESULTS1. Compared with PBS control group, Endostatin inhibited the tumors growth (P<0.05), and there were no significant difference between high-dose and low-dose Endostatin (P>0.05); the Microenvironment Density in low-dose Endostatin group was slightly lower than that in the control group (P<0.05), that strongly reduced in high-dose Endostatin (P<0.01). There were significant difference between high-dose and low-dose Endostatin (P<0.05).2. Endostatin greatly decreased the expressions of VEGF, IL-6and IL-17, enhanced the expressions of IFN-y and HIF-la compared with control group (P<0.05, respectively) by Western blot, immunohistochemistry or ELISA. There were significant difference between high-dose and low-dose Endostatin (P<0.05).3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by Endostatin therapy, compared with PBS control(P<0.05, respectively). And there were no significant difference between high-dose and low-dose Endostatin (P>0.05).4. Endostatin significantly reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, and concurrently elevated that of IFN-γ in comparison with control group (P<0.05, respectively); There were no significant difference between high-dose and low-dose Endostatin (P>0.05).CONCLUSIONS1. Endostatin strongly reduced tumor vasculature and tumor growth, and augmented tumor necrosis.2. Endostatin inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors in dose-dependent manner, and intensified the hypoxia in the tumors.3. Endostatin remarkably inhibited immunosuppressive MDSC and TAM, remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment. 4. Endostatin effectively provided the advantage of reducing the levels of immune inhibitory cytokines in the tumor regions, further demonstrated that Endostatin efficantly reversed immunosuppression of the inflammatory tumor microenvironment in lung caner, promoted mature DC and CD8+T cells to infiltrate the tumors, so as to synergize with tumor special DC-T cellular therapy on antitumor effect. Part Ⅲ Pro-inflammatory cytokines TNF-α and IL-6gene polymorphisms were associated with occurring of lung cancerOBJECTIVE To investigate the relationship between SNP polymorphisms of pro-inflammatory cytokines TNF-α and IL-6genes and occurring of lung cancer.METHODS1.138consecutive patients with primary lung cancer were recruited in this study. The control group consisted of138healthy individuals. All of them were from the ethnic group Han of North China. The ages, sex distribution, and behaviors (such as smoking or not) between lung cancer group and healthy control group had no significant difference statistically (P>0.05). Total DNAs were isolated from Peripheral blood mononuclear cells of138lung cancer patients and138healthy individuals.2. PCR were performed and amplified-238G locus of TNF-α gene and-572C locus of IL-6gene, respectively. Further, the gene polymorphisms of two cytokines were revealed by restriction fragment length polymorphism (RFLP).3. The PCR produced fragments of-238G locus of TNF-α gene about290bp as expected. The PCR products were then subjected to BglⅡ digestion to explore if a G-to-A single base alteration occurring at-238locus.4. The PCR produced fragments of-572C locus of IL-6gene about400bp as expected. The PCR products were digested with BsrBI to explore if a C-to-G single base alteration occurring at-572C locus.5. The correlation between the polymorphisms and risks of lung cancer was indicated by odds ratio (OR) and their95%confidence interval (CI). OR values and their95%CI were calculated using a non-conditional logistic regression model, which were adjusted by ages, gender, smoking and other factors.RESULTS1Detection of SNP polymorphisms at-238G locus of the TNF-a geneThe PCR produced fragments about290bp as expected, digestion of the290bp fragments from the276individuals in both groups with BglⅡ generated three kinds of band patterns on agarose gels:(ⅰ) a band of about290bp,(ⅱ) two bands with sizes of about80and210bp, respectively, or (ⅲ) three bands with sizes of80,210, and290bp, respectively.Since BglⅡ recognizes AGATCT, generation of the80and210bp bands may be explained by a single-base G-to-A alteration occurred at-238G locus, which would generate a BglⅡ locus. Three kinds of SNP patterns were found at-238G locus of the TNF-a gene, including no alteration, a single-base G-to-A alteration at-238G locus of both alleles, a single-base G-to-A alteration at-238G locus of one of both alleles, were named as wt, G-A/G-A, and G-A, respectively.2Detection of SNP polymorphisms at-572C locus of the IL-6geneThe PCR produced fragments about400bp as expected, digestion of the400bp fragments from the276individuals in both groups with BsrBI generated3kinds of band patterns on agarose gels:(ⅰ) a band of about400bp,(ⅱ) two bands with sizes of about150and250bp, respectively, or (ⅲ) three bands with sizes of150,250, and400bp, respectively.Since BsrBI recognizes CCGCTC, generation of the150and250bp bands may be explained by a single-base C-to-G alteration at-572C locus, which would generate a BsrBI locus.These three polymorphisms, including no alteration, a single-base C-to-G alteration at-572C locus of both alleles, a single-base C-to-G alteration at-572C locus of one of both alleles, were named as wt, C-G/C-G, and C-G, respectively.3High rates of single-base G-to-A alteration at-238G locus of the TNF-a gene correlated with occurring of lung cancerIt was founded that the frequency distribution of-238G SNPs at both alleles fits the Hardy-Weinberg equilibrium (.P>0.05). Most cases possessed a wt SNP at-238G locus (113and99for healthy control group and lung cancer group, respectively). The control group and lung cancer group had similar number of G-A cases, respectively. The control group had2G-A/G-A cases, while lung cancer group had14G-A/G-A cases. It was suggested that correlations between G-A/G-A cases of-238G SNPs and occurring of lung cancer (P=0.005, OR=1.74395%CI0.232-0.682)4High rates of single-base C-to-G alteration at-572C locus of the IL-6gene correlated with tumorgenesis of lung cancer.Similar situations were also found for the SNPs on-572C locus of the IL-6gene. The healthy control group and lung cancer group had similar numbers of wt or C-G SNPs. However, the control group had three C-G/C-G cases, while lung cancer group had nine C-G/C-G cases. The increased numbers of C-G/C-G SNPs in lung cancer group suggested that the homozygous C-G/C-G SNPs on both alleles may be related to occurring of lung cancer (P=0.029, OR=1.23495%CI1.412-2.727).CONCLUSIONS1. The TNF-α and IL-6gene polymorphisms may be a critical risk for the genie susceptibility to lung cancer in the ethnic group Han of North China.2. The high rates of singlebase G-to-A alteration at-238G locus of both alleles of the TNF-α gene and high rates of single-base C-to-G alteration at-572G locus of both alleles of the IL-6gene correlated with tumorgenesis of lung cancer. Part Ⅳ Chemokines CXCL1-2/CXCR2Axis Signaling were Involved in the Antitumor Effect of EndostatinOBJECTIVE To screen the main targets of the antitumor effect of Endostatin using Proteome Profiler arrays. METHODS1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into two groups, including Endostatin group (rhEndostatin15mg/Kg/d) and control group (PBS0.2ml/d). There were seven mice in each group. All the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days. All the mice were sacrificed24hours later after the last time of administration.2. The tumor specimens were separated from tumor-bearing mice. Proteome Profiling array of tumor specimens was performed, which included19pro/anti-angigenic factors such as CCL11, CCL2, CCL3, GM-CSF, IFN-γ, IL-12p70, IL-2, IL-4, IL-6, IL-9, CXCL1, CXCL2, MCP5, SCF, sTNFRl, TIMP1and VEGF-A so on. Significance Analysis of Microarrays (SAM) soft was used for statistical analysis.3. The sample sizes of Endostatin group and control group both were expanded doubly, and the experiment was repeated for further validation. Moreover, the molecular profiling was adjusted based on the data of the first experiment. Significance Analysis of Microarrays (SAM) soft and clustering analysis were used for statistical analysis.RESULTS1. The levels of CXCL1, CXCL2, and CCL3in the tumors were remarkably enhanced (4.1fold,2.4fold, and2.3fold, respectively), however, the critical pro-angiogenic factors including VEGF-A, GM-CSF, sTNFR1/2and TIMP1had no significant change.2. Based on the above data, the Proteome profiling was adjusted to include the key pro/anti-angiogenic factors such as GM-CSF、CCL27、CXCL1、CXCL2、IL-2、 MCP5、sTNFR1、TIMP1and VEGF-A. In the repeated experiment, according to SAM soft and clustering analysis, CXCL2was still strongly elevated, concurrently VEGF-A and GM-CSF were significantly enhanced (1.964fold, 1.505fold, and2.18fold, respectively).CONCLUSIONS1. The data demonstrated that multiple proangiogenic factors had remarkable changes after receiving Endostatin therapy, and strongly supported that Endostatin have the characteristic of multi-targeted antiangiogenic efficancy.2. Besides VEGF signaling pathway, chemokines CXCL1-2/CXCR2axis signaling in the tumor microvironment participated in the antitumor effect of Endostatin.