(A) Renca-Luc cells were injected s.c. and whole-body bioluminescent images were taken on d 37. Shown are 3 tumor-free controls and 3 tumor-bearing mice. (B) Total light flux values acquired via BLI on d 37 for individual tumor-bearing mice (n = 6). The mean background light flux for 8 tumor-free mice is also shown. Data are cumulative from 2 independent experiments. (C) Total light flux values for individual excised lungs taken from tumor-free or tumor-bearing mice, as indicated.

(A) Parental Renca cells were injected IR, and weights of excised kidneys in grams (gm) were recorded on d 23. Representative data from >5 independent experiments are shown (n = 5 mice per group). (B) H&E-stained sections from one normal tumor-free kidney, or tumor-bearing kidneys harvested at the times indicated. Areas of dense purple staining indicate renal tumors. Scale bar = 200 µm. (C, D) Quantitation of visible lung tumors from 13 individual mice. Lungs were inflated with India Ink en bloc to allow visualization of white lung tumor nodules. (E, F) Total light flux values for individual excised kidneys (E) and lungs (F) taken from mice on d 7 after IR Renca-Luc challenge or tumor-free (ctrl) mice.

(A) Parental Renca cells were injected IR, followed by PBS or Ad5mTRAIL (Ad5mTR) +CpG on d 7. Kidneys were excised on d 12 and analyzed by flow cytometry to assess T cell infiltration. Dot plots show the percentages of live CD8⁺ and CD4⁺ T cells per kidney, as well as expression of CD44 and CD62L on the gated CD8⁺ and CD4⁺ T cells. (B, C) Mice were challenged as in (A) and PBS, Ad5mTR and/or CpG was given on d 7. Tumor-challenged kidneys were excised and weighed on d 23. CD4 and CD8 depletions were performed as described in Methods. Mean values from 4–10 mice per group, combined from 3 individual experiments, are shown. Asterisks indicate p<0.05 for that treatment group vs normal, tumor-free kidney weights. Statistical p values are shown for PBS vs CpG, PBS vs Ad5mTR+CpG, Ad5mTR+CpG vs Ad5mTR+CpG with CD4 depletion, and Ad5mTR+CpG vs Ad5mTR+CpG with CD8 depletion. For PBS vs CpG alone or Ad5mTR alone p = 0.11, for Ad5mTRAIL+CpG –CD8 vs Ad5mTRAIL+CpG –CD4 p = .14. (C) In addition, the excised kidneys were processed for H&E staining. Areas of dense purple staining indicate renal tumors. Scale bars in upper panels = 2.0 mm.

(A) Parental Renca cells were injected IR, followed by administration of the indicated therapies IR on d 7. Spleens were excised and live cells counted on d 12. Mean live splenocyte counts from 1 experiment, representative of 4, are shown (n = 4 mice per group). For PBS vs Ad5mTRAIL p = 0.17; for PBS vs CpG p = <0.001; for Ad5mTRAIL+CpG vs Ad5mTRAIL p = 0.06; for Ad5mTRAIL+CpG vs CpG p = 0.16. (B–D) Flow cytometric analyses of spleens are shown; numbers indicate the percentages of live splenic B cells (B), CD4 and CD8 T cells (C) or CD11c^high DC (D) (* indicates p<0.05 versus PBS controls).

(A) Total serum IgG concentrations obtained on d 12 from the indicated treatment groups. Only Ad5mTR+CpC resulted in a significant increase in serum IgG versus tumor-free control mice. (B) Anti-dsDNA serum concentrations obtained on d 12, 20, and 48 from Ad5mTR+CpG-treated mice. (For both A and B, * indicates p, 0.05; ** indicates p<0.01). (C, D) Representative photomicrographs of H&E stained sections of the contralateral kidney (C) and liver (D) taken from one Ad5mTR+CpG-treated mouse (of a total of 7 analyzed) at d 102 after IR tumor challenge. Scale bar indicates 200 µm.

(A) Parental Renca cells were given IR, followed by administration of PBS or Ad5mTR+CpG IR on d 7. Flow cytometric analyses examining T cell infiltration were performed on excised lungs on d 12. Numbers indicate the frequencies of live cells within the gated regions. (B) Mice were challenged as in (A), followed by PBS or Ad5mTR+CpG on d 7. Lungs were excised on d 21, and surface lung nodules were enumerated as in Figure 2 (n = 15 mice per group, combined from 3 independent experiments). (C, D) Renca-LUC cells were given IR, followed by administration of PBS or Ad5mTR+CpG IR on d 7. (C) Mean total light flux values from 5 mice per indicated treatment group are shown from d 6–21. The mean light flux value for 5 tumor-free mice is shown at d 23 to denote background level of detection. Light flux values for Ad5mTR+CpG-treated mice at d 21 are statistically insignificant to those from tumor-free mice (p = 0.182), suggesting tumor eradication was nearly complete. Ad5mTR+CpG vs PBS at d 21, p = 0.032. (D) Mean total light flux values measured on d 21 for 3–10 mice per group, combined from 2 individual experiments, are shown. Ad5mTR+CpG vs Ad5mTR+CpG with CD4 depletion p = 0.186; Ad5mTR+CpG vs Ad5mTR+CpG with CD8 depletion p = 0.015; Ad5mTRAIL+CpG – CD8 vs Ad5mTRAIL+CpG – CD4 p = .037; PBS vs Ad5mTR+CpG with CD8 depletion p = 0.573; PBS vs CpG p = 0.440. (E) Mice were challenged as in (A), followed by PBS or Ad5mTR+CpG on d 7. Survival data from 10 mice per group are shown through d 72. (F) Mice were challenged on d 0 with Renca-Luc, treated on d 7 with Ad5mTRAIL+CpG, then re-examined at d 102 by BLI for signs of tumor recurrence. Of 15 tumor-challenged mice, 11 survived to d 102, and 8 of these showed no evidence of tumor re-growth.