Antiangiogenic Therapy Enhances The Antitumor Activity Of Cytokine-induced Killer Cells By Modulating Tumor Vasculature And Tumor Hypoxic Microenvironment

Part ?Recombinant human endostatin improves the antitumor effect of CIK cells in the treatment of NSCLC[Objective]The purpose of this study was to investigate the enhancement of the antitumor effect of cytokine-induced killer(CIK)cells induced by recommibinant human endostatin(rh-endostatin)and to elucidate the underlying mechanisms by which rh-endostatin modulate tumor vasculature and hypoxic tumor environment to release the full potential of the CIK cell therapy.[Methods]1.Three different lung carcinoma models were established.A549,SPC-A1 lung adenocarcinoma xenograft and Lewis lung carcinoma were established subcutaneously in BALB/c nude mice and C57BL/6 mice respectively.For each tumor model,when tumor volume reached 100mm3,mice were randomly divided into four groups.(a)Group Control:treated with normal saline(NS);(b)Group EN,treated with rh-endostatin for consecutive 7 days;(c)Group CIK:treated with NS followed by CIK cells;(d)Group EN+CIK:treated with rh-endostatin followed by CIK cells.Tumor size was monitored as indicator of therapeutic response.2.The phonotype of CIK cells were observed by flow cytometry when CIK cells were cultured for 7,14 and 21 days.3.Immunofluorescence was performed to observe the expression of CD31 and?-SMA on tumor vasculature to evaluate tumor mean vascular density and vascular pericyte coverage.4.Evans Blue dye was injected and intravital microscope was conducted to analyze tumor vascular permeability to macromolecules.Dynamic Contrast Enhanced Magnetic Resonance Imaging(DCE-MR1)was performed to analyze tumor vascular permeability to small molecules.5.The in vivo trafficking and homing ability of CFSE-labeled CIK cells into tumor and spleen of A549 tumor bearing mice were observed by flow cytometry.6.Immunohistochemistry was performed to observe the intratumoral infiltration of CD3+ T lymphocytes in Lewis lung carcinoma.7.The infiltration of Myeloid-derived suppressor cells(MDSCs)and tumor associated macrophages(TAMs)into spleen and tumor were observed by flow cytometry.8.The hypoxyprobeTM-1 kit was used to study the effect of rh-endostatin on tumor hypoxia.A549 tumor bearing mice were treated with rh-endostatin for consecutive 7 days with normal saline as control.Ond day 3,6 and 9,mice were injected with hypoxyprobe.Immunohistochemistry was conducted to analyze tumor hypoxic area.[Results]1.In all lung tumor models,combination of rh-endostatin with adoptive CIK cells immunotherapy exert significant antitumor effect and could significantly inhibit the growth of lung carcinoma.However,CIK therapy alone or rh-endostatin therapy alone could not show obvious antitumor effect.2.After incubation for 7,14 and 21 days,phenotypes of CIK cells were detected by flow cytometry.The percentages of CD3+CD56+CIK cells were 8.93%,23.66%and 17.17%on day 7,14 and 21,respectively.3.Rh-endostatin induces structural normalization of tumor vasculature by decreasing tumor angiogenesis and improving tumor vessel maturation.4.Rh-endostatin treatment induces a decreased tumor vessel permeability to macromolecules and increased permeability to small molecules,which are hallmarks of tumor vascular normalization.5.Rh-endostatin increases the infiltration of CIK cells into tumor tissue and spleen.There was 15.95±1.64%CFSE-labeled CIK cells infiltrating into tumor from rh-endostatin treated group compared with 1.57±0.07%from control group(P=0.006).CIK cells infiltration into the spleen was also increased in rh-endostatin treated group(5.55±1.56%vs 1.63±0.22%,P=0.036).6.Rh-endostatin and CIK cells therapy alone slightly increased the level of infiltrating CD3+T lymphocytes but did not reach statistical significance.However,tumor-infiltrating CD3+T lymphocytes were significantly improved in the combinatorial group.7.Rh-endostatin significantly decreases the CD11b+ Gr-1+ MDSCs frequency in the tumor(3.21±0.19%vs 4.62±0.43%,P=0.022).However,after the treatment of rh-endostatin,MDSCs frequency in the spleen was comparable with its control(7.65±1.65%vs 7.82± 1.51%,P=0.394).No obvious difference was obtained in the lymph node(1.75±96%vs 1.77± 1.47%,P=0.984).No difference was shown in the infiltration of TAMs in tumor,spleen and lymph node between rh-endostatin treated group and control group.8.Treatment of rh-endostatin resulted in less hypoxic area,as assessed by pimonidazole staining on day 6 and day 9 in A549 lung carcinoma compared with NS controls(P=0.002 and P=0.0093,respectively).[Conclusion]Synergistic antitumor activity is achieved by combining rh-endostatin immunotherapy with adoptive CIK cells immunotherapy in the treatment of NSCLC.The underlying mechanisms mediating the antiangiogenic therapy-induced enhancement of CIK cells'efficacy may involve the normalization of tumor vasculature,reduction of tumor hypoxia and augmentation of homing ability of CIK cells to tumor and spleen tissues.Part ?Metronomic paclitaxel improves the antitumor effect of CIKcells in the treatment of NSCLC[Objectives]Metronomic chemotherapy shows promising anticancer activity by its potential antiangiogenic effect and reduced toxicity.We hypothesized that metronomic chemotherapy with PTX could improve the antitumor effect of adoptive CIK cell immunotherapy in the treatment of NSCLC.[Methods]1.Two dififerent lung carcinomas were established.A549 lung adenocarcinoma xenograft and PTX-resistant A549(A549/PTX)lung carcinoma were established subcutaneously in BALB/c nude mice,respectively.For each tumor model,when tumor volume reached 100mm3,mice were randomly divided into four groups.(a)Group Control:treated with normal saline(NS);(b)Group PTX,treated with metronomic PTX for consecutive 10 days;(c)Group CIK:treated with NS followed by CIK cells;(d)Group PTX+CIK:treated with metronomic PTX followed by transfusion of CIK cells.Tumor size was monitored as indicator of therapeutic response.2.Mice appearance,behavior,reactivity and body weight were recorded and scored daily.The status of mice was assessed by clinical appearance scoring system.3.The hypoxyprobeTM-1 kit was used to study the effect of metronomic PTX on tumor hypoxia.A549 tumor bearing mice were treated with 4mg/kg PTX for consecutive 5 days with normal saline as control.Ond day 6,mice were injected with hypoxyprobe.Immunohistochemistry was conducted to analyze tumor hypoxic area.4.Immunohistochemistry was performed to observe the intratumoral infiltration of CD3+T lymphocytes in A549 lung carcinoma.[Results]1.In both lung tumor models,combination of metronomic PTX with adoptive CIK cells immunotherapy exert significant antitumor effect and could significantly inhibit the growth of lung carcinoma.However,CIK therapy alone or metronomic PTX therapy alone eould not show obvious antitumor effect.Our results suggested that synergistic antitumor effects were obtained by combination of metronomic PTX therapy and CIK cell adoptive therapy and combined therapy could also inhibit the growth PTX-resistance NSCLC.2.Mice treated with combination of metronomic PTX and CIK cells displayed significantly better clinical scores than those in control group or monotherapy treated groups,suggesting that combination therapy could improve the health status of tumor bearing mice.3.Hypoxic tumor area was significantly reduced after the administration of PTX compared with control group(12.74±0.72%vs 38.05±3.98%,P=0.003).Reduced hypoxic tumor area may provide a better immunosupportive tumor microenvironment for transftused CIK cells.4.Tumor tissues from control group were infiltrated by only a few CIK cells.However,PTX treatment significantly increased the infiltration of CIK cells into tumor parenchyma by 2.56 times compared with NS control which may be one of the mechanisms through which combination of metronomic PTX and CIK cells exert better antitumor efficacy.[Conclusion]Synergistic antitumor effect could be achieved by combining metronomic PTX with adoptive CIK cell immunotherapy in the treatment of NSCLC.Metronomic PTX and its combination with CIK cells have been improved to be safe without causing obvious side effects and could improve the health status of tumor bearing mice.Our studies provide primary evidence that metronomic PTX improves the antitumor efficacy of adoptive CIK cell imnunotherapy by reducing hypoxic tumor area and improving the recruitment of CIK cells into tumor parenchyma.Part ?The effects of hypoxia on CIK cells and HUVECs[Objectives]The propurse of this study is to establish in vitro hypoxic culture environment and analyze the influence of hypoxia on the proliferation,migration,cytotoxicity and adhesion ability of CIK cells.[Methods]1.CIK cells were cultured in 96-well plates under normoxia or hypoxia.Forty eight hours later,CIK cells were counted by hemocytometry under light microscope.2.Immunocytochemistry was performed to observe the adhesion of CIK cells on HUVECs which are co-cultured under normoxia or hypoxia.3.Immunocytochemistiy was conducted to observe the expression of ICAM-1 and VCAM-1 on HUVECs which are cultured under normoxia or hypoxia.4.Transmigration assay was performed with TranswellTM inserts to analyze the migration ability of CIK cells across HUVECs monolayers.5.The cytotoxicities of CIK cells against target cells(A549 cells)in normoxic and hypoxic conditions were analyzed by using CytoTox 96(?)Non-Radloactive Cytotoxicity Assay-Lactate Dehydrogenase(LDH)release assay.6.Immunohistochemistry was conducted to analyze the relationship of intratumoral infiltration of CIK cells with the expression of ICAM-1 and VCAM-1.[Results]1.Under microscopy,CIK cells showed cluster-like growth.Masses of CIK cells gradually multiplied and became larger after 4 days incubation.Under light microscopy,CIK cells formed larger and more cell spheres and had a higher rate of proliferation under normoxia compared with hypoxia.2.Hypoxia could impede the adhesion of CIK cells to HUVECs.3.Hypoxia depressed the expression of ICAM-1 and VCAM-1 on HUVECs.4.Transmigration assay showed that the number of CIK cells migrating HUVECs layer under normoxia was 2.89 times more than that under hypoxia(P=0.003).5.Under normoxia,CIK cells showed moderate cytotoxicity on A549 cells,with 49.09±2.54%killing rate at 20:1 E-to-T ratio.However,hypoxia significantly decreased the cytotoxicity of CIK cells with 8.35±2.74%killing rate at 20:1 E-to-T ratio.Similarly,hypoxia notably inhibited the cytotoxicity of CIK cells against A549 cells at 40:1 E-to-T ratio(30.76±6.19%vs.77.06±12.48%,P=0.005).6.Accumulation of tumor infiltrating CIK cells was correlated with the expression of ICAM-1 and VCAM-1.More CIK cells accumulated around the ICAM-1 or VCAM-1 high expression vessels.[Conclusion]Hypoxia could inhibit the proliferation,adhesion and migration ability of CIK cells.In addition,CIK cells exert weaker cytotoxicity against tumors cells when cultured under hypoxic microenvironment.Hypoxia could also depress the expression of ICMA-1 and VCAM-1 on HUVECs,which are correlated with the intratumoral infiltration of CIK cells in vivo.Part IV VEGFR2 Phosphorylation site Y951/Y949 signaling and the role in angiogenesis[Objectives]To study the role of VEGFR2 Y951/949 signaling in angiogenesis and its influence on tumor growth and metastasis[Methods and Matherials]1.In vitro embryoic stem eells were cultured to form embryoie bodies and study the role of Y949 in angiogenesis.2.Mile assay was conducted to study the influence of Y949 on vascular permeability.3.RipTag WT mice and RipTag Y949 mutant mice were breed until they were 12-14 weeks old.Tumors were removed and measured for tumor volume.4.Immunofluorescence was conducted to analyze the expression of Fibrinogen,NG2 and podocalyxin.[Results]1.Y949 mutant inhibited the formation of EB and angiogenesis.2.Y949 mutant reduced the vascular permeability to Evans blue.3.Y949 mutant reduced the ratio of NG2/CD31 and fibrinogen/CD31 in RipTag tumor and did not have affect on fibrinogen.4.Y949 mutant inhibited the liver metastasis in RipTag tumor.[Conclusion]VEGFR2 phosphrlation site Y951/949 mutant inhibit the angiogenesis,decrease vascular permeability,inhibit tumor growth and liver metastasis of RipTag tumor.