A Study On Antitumor Effect Of Malaria Genetically Attenuated Sporozoites

Lung cancer is remained the common malignancy in world and its high morbidity and mortality means that no effective therapeutic methods are available.Non-small-cell lung cancer(NSCLC)accounts for about 85% of all lung cancer cases.Despite recent advances in surgery,chemotherapy and radiotherapy,the prognosis of patients with lung cancer is still poor.Therefore,it is necessary to develop new approaches to replace or complement the current therapies.In the current decade,some new insights in the interaction between tumors and the immune system have led to the development of immunotherapy as a fundamentally new concept for the treatment of NSCLC.However,immunotherapy for NSCLC was rather unsuccessful,because of lung cancer often present a tolerogenic microenvironment that hampers effective antitumor immunity.Malaria via deletion of preerythrocytic-stageexpressed genes(UIS3,UIS4 and Fab B/F)that play essential roles for it's growth it may result in significant growth defects in a hepatocytes and avoided malaria infection This defective malaria of preerythrocytic-stage was named genetically attenuated sporozoites.Furthermore,GAS has been reported to stimulate host immune responses such as stimulating T-cell proliferation to protection against malaria challenge.We hypothesized that whether GAS induced immune response could treat for lung cancer.To clarify this issue,we have designed the following experiment to verify,whether intravenous immunization with GAS has antitumor effect and it is the mechanism of antitumor effect.Part 1.Identification of GAS:1.1 GAS acquisition: The attenuated P.yoelii BY265 strain was provided by department of Pathogenic Biology,Third Military Medical University,China1.2 verification test of GAS: Mice were divided into two groups.Each group of mouse administration of 30 × 10~3 numbers of genetically attenuated sporozoites(GAS)and wild type(WT)sporozoites respectively.In WT group mice,at day 6 we could find parasitemia,and at day 14 the level of parasitemia up to 30%.But in GAS group,the level of parasitemia is always zero.GAS was a safely,potential vaccine.Part 2.Antitumor effect of GAS:2.1 Mice immunization and tumor inoculation: Mice were randomly divided into two groups.In intravenous immunization group,each mouse was administered with 200?l of GAS vaccines in 1.5 × 10~5/ml concentration.In non-immuniz tion group,each mouse was given 200 ul phosphate buffer saline(PBS)as background blank control.After 2 weeks administration,subcutaneous inoculated 5 × 10~5 LLC to each mouse of all groups.2.2 GAS inhibited LLC growth and prolonged the survival of tumor-bearing mice: To judge the inhibition of tumor growth,animals were examined daily until the tumors became palpable,tumor volume was determined by measuring the diameter of the tumors using calipers.The volume was calculated following formula: tumor volume(mm~3)= 1/2 × a(mm)× b~2(mm~3),where ‘a' means the long axis of the tumor and ‘b' denotes the short axis.Once,mouse died during experiment record the survival rate in mice.The result revealed GAS inhibited LLC growth(P<0.05)and prolonged the survival of tumor-bearing mice(P=0.041).Part 3.Mechanism of GAS Antitumor effect:3.1 Immunohistochemistry analysis: Mice tumor were dissected and washed once in PBS,submerged in neutral buffered 10%formalin.and transferred to 70% ethanol for paraffin embedding.Semiserial sections were cut at 4 ?m,placed on positively charged slides,and fixed in cold acetone.Immunohistochemistry was performed with antibodies for CD31,Ki-67 and TUNEL following the manufacturer's protocol.The result revealed angiogenesis was inhibited and the proportion of proliferative cells was decreased,whereas apoptotic cells were increased.3.2 Cytokine analysis by BD? cytometric bead array(CBA): Blood was collected from GAS group and PBS group of mice by eyeball bleeding on day 1,3,5 7,9 after immunization.Flow cytometry measure a panel of four murine cytokines(IL-12,IFN-?,TNF-?,and IL-6)simultaneously in a single peripheral blood sample.We found that malaria attenuated sporozoites at early stage led to rapid increases in IFN-?,TNF-? and IL-6/12 levels,peaking 3 days after immunization.3.3 Flow cytometry: Blood was collected from GAS group and PBS group of mice by eyeball bleeding on day 1,7,14,after immunization and leukocytes were purified for flow cytometry using red blood cell lysis solution following the manufacturer's protocol.Cells were labeled with antibodies for antimouse CD8-APC,CD11c-FITC,following the manufacturer's protocol.We found that GAS immunization markedly increased the percent of CD8~+ T cells in GAS group mice.