Eradication of Metastatic Renal Cell Carcinoma after Adenovirus-Encoded TNF-Related Apoptosis-Inducing Ligand (TRAIL)/CpG Immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC

Superoxide Enhances the Antitumor Combination of AdMnSOD Plus BCNU in Breast Cancer

Overexpression of manganese superoxide dismutase (MnSOD) can sensitize a variety of cancer cell lines to many anticancer drugs. Recent work has shown that cancer cells can be sensitized to cell killing by raising peroxide levels through increased manganese superoxide dismutase (MnSOD) when combined with inhibition of peroxide removal. Here we utilize the mechanistic property of one such anticancer drug, BCNU, which inhibits glutathione reductase (GR), compromising the glutathione peroxidase system thereby inhibiting peroxide removal. The purpose of this study was to determine if anticancer modalities known to produce superoxide radicals can increase the antitumor effect of MnSOD overexpression when combined with BCNU. To enhance MnSOD, an adenoviral construct containing the cDNA for MnSOD (AdMnSOD) was introduced into human breast cancer cell line, ZR-75-1. AdMnSOD infection alone did not alter cell killing, however when GR was inhibited with either BCNU or siRNA, cytotoxicity increased. Futhermore, when the AdMnSOD + BCNU treatment was combined with agents that enhance steady-state levels of superoxide (TNF-α, antimycin, adriamycin, photosensitizers, and ionizing radiation), both cell cytotoxicity and intracellular peroxide levels increased. These results suggest that the anticancer effect of AdMnSOD combined with BCNU can be enhanced by agents that increase generation of superoxide

Ad5mTRAIL+CpG induces humoral immunity without autoimmunity.

<p>(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 <i>p</i>, 0.05; ** indicates <i>p</i><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.</p

Local combinatorial Ad5mTRAIL+CpG therapy leads to regression of primary renal tumors.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031085#s2" target="_blank">Methods</a>. Mean values from 4–10 mice per group, combined from 3 individual experiments, are shown. Asterisks indicate <i>p</i><0.05 for that treatment group vs normal, tumor-free kidney weights. Statistical <i>p</i> 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 <i>p</i> = 0.11, for Ad5mTRAIL+CpG –CD8 vs Ad5mTRAIL+CpG –CD4 <i>p</i> = .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.</p

IR administration of Ad5mTRAIL+CpG stimulates CD8-dependent eradication of metastatic Renca tumors.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031085#pone-0031085-g002" target="_blank">Figure 2</a> (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 (<i>p</i> = 0.182), suggesting tumor eradication was nearly complete. Ad5mTR+CpG vs PBS at d 21, <i>p</i> = 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 <i>p</i> = 0.186; Ad5mTR+CpG vs Ad5mTR+CpG with CD8 depletion <i>p</i> = 0.015; Ad5mTRAIL+CpG – CD8 vs Ad5mTRAIL+CpG – CD4 <i>p</i> = .037; PBS vs Ad5mTR+CpG with CD8 depletion <i>p</i> = 0.573; PBS vs CpG <i>p</i> = 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.</p

Renca s.c. tumor challenge produces only local tumor growth.

<p>(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.</p