Development Of Myc-CaP Prostate Cancer Mouse Model And Investigation Of The Relationship Among Tumor-associated Antigen NY-ESO-1, Toll Like Receptors And Prostate Cancer Progression

Part 1 Development of Myc-CaP Cell lines expressing prostate cancer tumor antigen NY-ESO-1 and its variants in syngeneic FVB mice1.1 Construction of pRV-Display based-retroviral vectors encoding prostate cancer antigen NY-ESO-1 and its variantsObjectives:To construct pRV-Display-based retroviral vectors encoding prostate cancer antigen NY-ESO-1 (abbreviated as ESO throughout the thesis) and its variants, ESOcsl, ESOcs2, ESOcs3, and LAGE-lb, as well as control genes HMGB-1 and GFP.Methods:The full-length cDNA fragment of ESO, its variants, and the control GFP and HMGB-1 were amplified using PCR on pDisplay-based vector templates. The fragments were subjected to digestion by restriction enzyme pair XhoI/BamHI or Xhol/Notl. Restriction enzyme pair XhoI/BamHI was used to digest the pRV-ESO plamid to obtain the pRV vector. The digested inserts were ligated to pRV vector. The resultant plasmids were analyzed by restriction enzyme followed by confirmation with DNA sequence analysis. Results:DNA fragments of about 1kb and vector at the size of 6kb were observed after electrophoresis of the resultant plasmids following restriction enzyme digestions. After DNA sequence analysis, the sequencing result indicated all DNA fragments inserted into the pRV vector matched the published sequences in PubMed.Conclusions:All designated plasmids were successfully constructed and named pRV-Display-ESO, pRV-Display-ESOcsl, pRV-Display-ESOcs2, pRV-Display-ESOcs3, pRV-Display-LAGE1b, pRV-Display-HMGB1, and pRV-Display-GFP. These plasmids were used in the subsequent production of retrovirus to engineer Myc-CaP prostate cancer line. 1.2 Generation of retrovirus encoding designated gene products for Myc-CaP prostate cancer engineeringObjectives:To generate retrovirus encoding designated gene products, transducer Myc-CaP cells with the retrovirus, and evaluate the engineered Myc-CaP cells.Methods:Phoenix ecotropic cells were transfected with pRV-Display plasmids expressing designated gene products using Lipofectamine 2000. The retroviral supernatant were collected at 24h,48h and 72h after transfection. Myc-CaP was transduced with each of the above retrovial supernatant followed by expansion in a CO2 incubator. The resultant Myc-CaP cells expressing the above gene products on their cell-surface were positively selected by immunomagnetic beads. The positively isolated Myc-CaP cells were verified to express the above molecules by Western blot, and the percentages of cells with cell-surface expression were quantified with flow cytometry.Results:Retrovirus produced by Phoenix ecotropic cells transfected with pRV-Display-GFP, encoding the control green fluorescent protein, was used as a reporter for evaluating retrovirus-mediated transduction efficiency. Successful transfection of Phoenix ecotropic producer line and tranduction of Myc-CaP based on reporter GFP expression was observed by inverted fluorescent microscope. GFP expression on the surface of Myc-CaP cells was stable after continued cell expansion in vitro, as evidenced by observation under inverted fluorescent microscope. NY-ESO-1 protein expression on Myc-CaP/ESO cells was confirmed by Western blot. The percentage of cells with cell-surface expression of NY-ESO-1 and its variants or control proteins in the post-sorted Myc-CaP/ESO/ESOcs1/ESOcs2/ESOcs3/LAGE1 /HMGB1/GFP cells varied from 81.8% to 86.2%.Conclusions:Retrovirus encoding cell-surface designated gene products was successfully generated, and used to transducer Myc-CaP cells. The sorted Myc-CaP cells with designated gene expression had high purity and could be used in future experiments.Part 2 Development of Myc-CaP prostate cancer mouse model and Investigation of the relationship among tumor-associated antigen NY-ESO-1, toll like receptors and and prostate cancer progression 2.1 Development of Myc-CaP prostate cancer mouse model and observation of the tumor growth of the mice with designated gene expression.Objectives:To develop the Myc-CaP prostate cancer mouse model and observe the tumor growth of the mice injected with Myc-CaP/ESO/ESOcsl/ESOcs2/ESOcs3/ LAGE1/HMGB1 cells.Methods:MycoFluor mycoplasma detection kits were used to ensure the absence of mycoplasma contamination in the Myc-CaP cells before subcutaneous inoculation. Myc-CaP cells were mixed with Matrigel at the ratio of 1:1 before being injected subcutaneously into male FVB mice at 0.5×105,2×105,8×105,16×105,32 X 105 cells respectively. Choose the optimal injection dose according to the tumor growth observation. Subcutaneously injecte FVB mice with sorted Myc-CaP/ESO /ESOcs1/ESOcs2/ESOcs3/LAGE1/HMGB1 cells, observe the tumor growth every 2-3 days and then got the tumor growth curve. Sacrifice the mice when the maximum tumor diameter reaches 1.5cm to do paraffin section and hemetoxylin and eosin staining.Results:Myc-CaP cells were not contaminated with mycoplasma. Based on observation of the tumor growth, the tumor latency period and tumor growth rate were proportional with the number of the injected tumor cells. The optimal number of cells for the Myc-CaP prostate cancer mouse modle was found to be 2×105 cell/mouse. Among the different group of mice that were injected with Myc-CaP/ESO/ESOcsl/ESOcs2/ ESOcs3/LAGE1/HMGB1 cells, we found that Myc-CaP tumor bearing NY-ESO-1 on the cell surface had the shortest latency and the fastest tumor growth rate. Its tumor latency was 15 days and tumor growth time to reach the maximal diameter of 1.5cm was 37 days on average. The tumor latency for Myc-CaP/LAGE1 Myc-CaP/ESOcs1 and Myc-CaP/ESOcs2 mice were 17 days,19 days and 19 days respectively, tumor growth time were 39 days,41 days and 43 days respectively. The tumor latency for Myc-CaP/ESOcs3,Myc-CaP/HMGB 1 and Myc-CaP mice were 21 days,21 days and 23 day respectively, the tumor growth time were 47 days,49 days and 49 days respectively. According to the paraffin section and Hemetoxylin and Eosin Staining, malignant tumor cells were more than 95% of the total.Conclusion:Myc-CaP prostate cancer model in syngeneic FVB mice was successfully established. Comparing to the wild type tumor and tumors bearing other variants, Myc-CaP expressing cell-surface NY-ESO-1 tumors had demonstrated shorter tumor latency, and enhanced tumor growth rate, Myc-CaP expressing cell-surface ESOcs3 and HMGB1 tumors had demonstrated longer tumor latency, and slower tumor growth rate, 2.2 Investigation of the relationship among tumor-associated antigen NY-ESO-1, toll like receptors and prostate cancer progressionObjectives:Investigation of the relationship among tumor-associated antigen NY-ESO-1, toll like receptors and and prostate cancer progression.Methods:(1) After 4 weeks injection, collect 30ul blood from all the 6 group of mouse's eye socket and detect IgG1 and IgG2a in the blood by ELISA. Then detect IgG1 and IgG2a of Myc-CaP/ESO,Myc-CaP/ESOcs3 and Myc-CaP/HMGB1 mice that injected with smaller dose of tumor cells in the 1st,3rd,5th,7th week after injection and draw the secretion curve. (2) Sacrifice the mice when the maximum tumor diameter reaches 1.5cm. Detect DC, MDSC and Treg by immunofluorescence frozen section and count the cell number. (3) Detect the secretion of IL-6 in Myc-CaP,Myc-CaP/ESO, Myc-CaP/ESOcs1, Myc-CaP/ESOcs2, Myc-CaP/ESOcs3, Myc-CaP/LAGE1 and Myc-CaP/HMGB1 cells by ELISA. Re-detect the secretion of IL-6 after adding different amount of LPS in the cell culture fluid.Results:IgGl and IgG2a in Myc-CaP/ESO, Myc-CaP/ESOcsi, Myc-CaP/ESOcs2, Myc-CaP/ESOcs3, Myc-CaP/LAGE1 and Myc-CaP/HMGB1 mice were significantly higher than Myc-CaP mice. IgG1 in Myc-CaP/ESO mice was the highest, IgG2a in Myc-CaP/LAGE1 mice was the lowest. IgG1 was more than IgG2a in Myc-CaP, Myc-CaP/ESO, Myc-CaP/ESOcs1, Myc-CaP/ESOcs2, Myc-CaP/ESOcs3 amd Myc-CaP/HMGB1 mice, IgG2a was more than IgG1 in Myc-CaP/HMGB 1 mice, all of which had no significant difference. After continuous detection of IgG1 and IgG2a in Myc-CaP/ESO, Myc-CaP/ESOcs3 amd Myc-CaP/HMGB1 mice injected with smaller dose of tumor cells in the 1st,3rd,5th,7th week after injection, we found both IgGl and IgG2a were growing fast in the first 3 weeks, and then keep in a stable level. According to the results, different group of mice has the similar immune reactions. (2) According to the immunofluorescent staining, mTLR4+/I-AE+DC and CD11b+/Gr1+MDSC were found in all the tumor tissue. Myc-CaP and Myc-CaP/LAGE1 mice tumor tissue has the fewest DC of 5, Myc-CaP/ESO mice tumor tissue has the most DC of 8. Myc-CaP/ESO mice tumor tissue has the fewest MDSC of 6, Myc-CaP/HMGB1 mice tmor tissue has the most MDSC of 10. The positive DC and MDSC in different group of mice have no significant difference. In addition, mTLR4 was found to be expressed in tumor cell. (3) Compared with Myc-CaP cell, the IL-6 secretion of Myc-CaP/ESO, Myc-CaP/ESOcs1, Myc-CaP/ESOcs2, Myc-CaP/ESOcs3, Myc-CaP/LAGE1 cells was significantly increased when 8 x 104 cells were detected. IL-6 secretion was highest in Myc-CaP/ESO cells and lowest in Myc-CaP/HMGB1 cells. After adding lng/ml and 100ng/ml LPS, IL-6 secretion was increased slightly without large difference.Conclusions:Different tumor growth rate in different group of mice were not correlated with IgGl and IgG2a, were not correlated with DC and MDSC as well. Tumor cells expressed mTLR4, NY-ESO-1 may be the ligand of TLRs, which could increase the IL-6 secretion and then promote the tumor growth.