Results: 5

1.
Figure 5

Figure 5. Increased expression of immunosuppressive molecules in prostate cancer. From: Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Spontaneously arising prostate tumors harvested from groups of young (≤ 8 weeks old, n = 3) and old (≥ 24 weeks old, n = 3) mice, fixed in RNAlater and were homogenized at 4°C in 10 μl/mg buffer RLT with β-mercaptoethanol. Total RNA was isolated using a QIAGEN RNeasy kit according to the manufacturer’s instructions. Complementary DNA was generated using this RNA as a template, then quantitative real-time PCR performed.

Andrew Gray, et al. Vaccine. ;27(Suppl 6):G52-G59.
2.
Figure 4

Figure 4. The cytokine/chemokine expression profile of spontaneous TRAMP prostate tumors changes over time. From: Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Spontaneously arising prostate tumors harvested from groups of young (≤ 8 weeks old, n = 5) and old (≥ 24 weeks old, n = 4) were homogenized at 4°C in 10 μl/mg sterile PBS containing 1x protease inhibitors. Cytokine levels were quantified with a custom 32-plex Milliplex MAP mouse cytokine immunoassay (Millipore, Billerica MA) using the Bio-Plex multiplex system (Bio-Rad, Hercules, CA). The * symbol indicates p < 0.05.

Andrew Gray, et al. Vaccine. ;27(Suppl 6):G52-G59.
3.

Figure 2. From: Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Figure 2a. Relatively increased numbers of Tregs in periphery of TRAMP mice with increasing age. Splenocytes isolated from groups of young (≤ 8 weeks old, n = 3), middle-aged (16–20 weeks old, n = 4) and old (≥ 24 weeks old, n = 3) TRAMP mice were washed and stained with anti-mouse CD3-FITC and anti-mouse CD4-PE/Cy7. Cells were fixed and permeablised overnight, then stained with anti-mouse FOXP3-PE and analysed by flow cytometry. Events were collected gated on live, CD3+ cells and then further gated on the CD4+FOXP3 fraction and the CD4+FOXP3+ fraction. The * symbol indicates p < 0.05.
Figure 2b. Increased infiltration of CD4+ T cells and Treg into the prostate tumor with increasing age. Tumor infiltrating lymphocytes isolated from groups of young ( 8 weeks old, n = 3), middle-aged (16–20 weeks old, n = 4) and old ( 24 weeks old, n = 3) TRAMP mice were washed and stained with anti-mouse CD3-FITC, anti-mouse CD8-PE/Cy5 and anti-mouse CD4-PE/Cy7. Cells were fixed and permeablised overnight, then stained with anti-mouse FOXP3-PE and analysed by flow cytometry. Events were collected gated on live, CD3+CD8 cells and then further gated on the total CD4+FOXP3 fraction and the CD4+FOXP3+ fraction. The‡ symbol indicates p < 0.001.

Andrew Gray, et al. Vaccine. ;27(Suppl 6):G52-G59.
4.
Figure 3

Figure 3. CD4+CD25+ T cells isolated from prostate tumor draining lymph nodes are functionally suppressive. From: Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

T cells from tumor-draining lymph nodes isolated from young (≤ 8 weeks old, n = 3) TRAMP mice were purified into CD4+CD25 (responder) and CD4+CD25+ populations by magnetic separation. 5×104 CD4+CD25+ T cells were cocultured for 72 hours with 5×104 allogenic T cell-depleted splenocytes from C3H mice in complete RPMI supplemented with 1 ug/ml activating anti-mouse CD3 antibody, either alone or with autologous CD4+CD25 responder T cells in 1:2, 1:4, 1:8 and 1:16 ratios. As positive controls for maximal proliferation, 5×104 CD4+CD25 responder T cells were cocultured with 5×104 allogenic T cell-depleted splenocytes from C3H mice in complete RPMI supplemented with 1 ug/ml activating anti-mouse CD3 antibody. 3H-thymidine (1 μg/well) was added in the last 8 hours of culture. Responder cell proliferation was measured by 3H-thymidine incorporation. The relative proliferation index of responder cells for each mouse at each Treg:Tresponder ratio was calculated by dividing the mean Tresponder proliferation at each ratio by the maximal proliferation (Tresponders cultured in the absence of Treg) of Tresponders in that animal.

Andrew Gray, et al. Vaccine. ;27(Suppl 6):G52-G59.
5.

Figure 1. From: Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Figure 1a. Therapeutic vaccination against prostate tumor-associated antigens results in long term survival in TRAMP mice. Groups of 20 male 8 week old TRAMP mice were vaccinated by helium-driven gene gun at day 0 with either 2 ug mPSCA-pcDNA or 2 ug empty vector and boosted at days 15 and 60 with 106 IU mPSCA-VRP and 106 IU GFP-VRP, respectively. Groups of 20 male 16 week old mice were vaccinated by helium-driven gene gun with either 2 ug mSTEAP-pcDNA or 2 ug empty vector and boosted at days 15 and 60 with 106 IU mSTEAP-VRP and 106 IU GFP-VRP, respectively.
Figure 1b. Therapeutic vaccination against mPSCA results in reduced prostate tumor burden in TRAMP mice. All surviving mPSCA-vaccinated TRAMP mice and their age-matched controls were euthanized at day 340 and necropsy performed. Representative images of a prostate tumor isolated from a negative control animal (left, “Control”) and that of an mPSCA-vaccinated mouse (right, “mPSCA”) are shown.
Figure 1c. Therapeutic vaccination against prostate tumor-associated antigens results in superior survival when administered to TRAMP mice with PIN lesions. The difference between the cumulative survival of each group of vaccinated mice and the cumulative survival of their age-matched controls was calculated at each time point at which one or more mice died. This value was termed “delta survival” and was plotted against survival time. Linear regression analysis was performed on each data set, and the slopes of the regression lines compared.

Andrew Gray, et al. Vaccine. ;27(Suppl 6):G52-G59.

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