1. Background and Significance Currently there are no efficacious therapies for renal cell carcinoma (RCC). Despite recent

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1 1. Background and Significance Currently there are no efficacious therapies for renal cell carcinoma (RCC). Despite recent advances in development of protective immune-based therapies for RCC, the prognosis for individuals with metastatic disease remains poor [1, 2]. For the last two decades immunotherapies for RCC focused on approaches using interferon alpha (IFN- ) or interleukin-2 (IL-2) [3]. Unfortunately, use of IFN- has not resulted in any complete responses in patients, and IL-2 has only led to a complete response in ~5% of patients [3, 4]. Therapeutic advances have been made in recent years that target pathways important for tumor progression, such as the tyrosine kinase inhibitors sorafenib and sunitinib, which increase survival [5]. Though these therapies have shown promise, most patients maintain progressive tumors. Currently, localized treatment for RCC is surgical resection; however, most patients present with advanced RCC that includes tumor metastases beyond the primary tumor. With the primary metastatic site of RCC being the lung [1], it is imperative that new therapies combat both primary and metastatic tumor sites. The induction of cancer cell death using chemotherapy or radiation has been a common therapeutic option for most cancers for many years, but these agents have limited tumor specificity and are frequently associated with extensive toxicity. The death-inducing receptors of the tumor necrosis factor (TNF) receptor superfamily and their cognate ligands of the TNF superfamily have garnered much interest in recent years for their therapeutic potential in numerous diseases, including cancer. Within the TNF family, TNF-related apoptosis-inducing ligand (TRAIL) is being heavily investigated as a cancer immunotherapeutic molecule because it has the unique ability to induce cell death in a broad range of cancer cell lines, while having little to no toxicity against normal cells [6-8]. Preclinical studies have demonstrated the safety of large systemic doses of TRAIL, as well as the evaluation of TRAIL s antitumor activity in vivo [9]. As 1

2 an alternative to systemic administration of TRAIL protein, our lab developed a nonreplicative recombinant adenovirus encoding the TRAIL cdna (Ad5TRAIL) that also selectively induces tumor cell death [10-12]. Ad5TRAIL has proven efficacious in vitro against several cancer types [13-15], and in vivo[16-18] resulting in regression of the primary tumor after local administration. It is important to remember that advanced RCC is metastatic; therefore, an efficacious immunotherapy administered at the primary tumor site must also be able to induce systemic antitumor immunity to eliminate any known and/or undetected metastases that exist. For a robust antitumor immune response to develop, tumor antigen (Ag) processing and presentation must be increased [19]. Dendritic cells ingest (phagocytose) dead tumor cells, and process and present exogenous tumor-derived Ag to CD8 T cells resulting in T cell activation and acquisition of various effector functions [19]. Based on the ability of Ad5TRAIL to robustly induce tumor cell death, we examined the hypothesis that increasing the amount of tumor Ag available (through Ad5TRAIL-induced tumor cell death) would increase CD8 T cell activation, leading to systemic tumor-specific immunity and enhanced tumor clearance [20]. Results published by VanOosten et al showed systemic antitumor immunity can indeed be induced following local, intratumoral treatment with Ad5TRAIL in a mouse model of RCC [21]. Despite the induction of a systemic antitumor immune response and primary tumor regression, complete tumor elimination only occurred in ~40% of the treated animals [21]. The current research in the lab is focused on the inhibition of protective antitumor immunity by regulatory cell populations, and the investigating methods to overcome these obstacles. Both cell-mediated innate and adaptive immunity are essential for preventing primary tumor outgrowth and rejecting transplanted experimental tumors [22, 23]. Despite the fact that activated immune cells exist in cancer patients, the immune system often fails to prevent tumors and limit 2

3 their spread. Regulatory immune cells such as regulatory T cells (Treg) [24, 25], tumorinfiltrating dendritic cells (TIDC) [26], and myeloid-derived suppressor cells (MDSC) [27] prevent or dampen the immune system from mounting sufficient antitumor immune responses. Treg are specifically recruited into the tumor microenvironment where they are proposed to inhibit effector T cell function, leading to enhanced tumor outgrowth [28]. TIDC are a recently characterized mature, myeloid dendritic cell with the potent ability to suppress the CD8 T cellmediated antitumor response [26]. Distinct from the myeloid DC phenotype of TIDC, are MDSC. MDSC are a mixed population comprised of immature precursors of macrophage, granulocytes, and myeloid DC. MDSC accumulate systemically and intratumorally during outgrowth of many human and mouse tumors, and diminish protective antitumor responses mediated by CD8 T cells [27, 29, 30]. A recent study of 306 RCC patients found tumor infiltration of MDSC type cells that was associated with poor prognosis and death [31]. Another study analyzed peripheral blood samples from RCC patients, finding decreased L-arginine concentrations and impaired T cell function - both linked to the presence of MDSC [32]. An in depth assessment in an experimental model of advanced RCC to determine the extent to which MDSC and TIDC suppress local and systemic effector function and whether or not immunotherapy (Ad5TRAIL) can augment these functions has not been demonstrated. Based on the background and gap of knowledge in this scientific field, our long-term goal is to develop novel and effective immunotherapeutic strategies to treat advanced RCC. Our objectives are twofold: 1) to develop an efficacious immunotherapeutic treatment regimen for metastatic RCC by stimulating effector T cells function via Ad5TRAIL into an established primary tumor; and 2) to understand the impact of tumor-infiltrating myeloid cells (MDSC, TIDC) on tumor progression and metastasis. Our central hypothesis is that localized 3

4 intratumoral Ad5TRAIL administration and the ensuing tumor cell death, in combination with therapies designed to overcome tumor-derived immunosuppression, will lead to systemic protective antitumor immunity of mice bearing primary and metastatic RCC tumors. This hypothesis is based not only on the aforementioned data, but also our preliminary data showing subcutaneous intratumoral treatment with Ad5TRAIL plus an immunostimulatory CpG oligonucleotide (CpG) that generates systemic CD8 Tcell-mediated antitumor immunity, which leads to increased survival. CpG ODN is used to activate the dendritic cells to allow for activation of the innate and adaptive immune responses [33]. Our rational for these studies is that the identification of novel immunotherapeutic protocols for using Ad5TRAIL in experimental preclinical models of RCC will establish a solid foundation for eventual clinical trials. Therefore, my research will examine the in vivo effects of intratumoral Ad5TRAIL/CpG administration on tumor progression/regression, metastasis, myeloid cell infiltrating populations and CD8 T cell-mediated antitumor responses. 4

5 2. Procedure/Technique To test the effects of intratumoral Ad5TRAIL/CpG administration on tumor progression/regression and metastasis, we will examine the hypothesis that intratumoral administration of Ad5TRAIL/CpG at the primary tumor site will lead to tumor regression and enhanced metastatic tumor clearance in the lungs. To evaluate this we will use the following tumor challenge schedule with female Balb/c mice: Day 0: administration of RCC tumor cells 2x10 5 Renca cells (murine spontaneously arising RCC cell line) or Renca-Luc (Renca cells expressing luciferase) given intrarenally (directly into kidney) Day 7: intrarenal (I.R.) immunotherapy administration group 1: AdTRAIL + CpG, group 2: AdTRAIL, group 3: CpG, group 4: control adenoviral vector encoding irrelevant gene AdLacZ and group 5: PBS control o Day 14/21: Harvest organs for cell infiltration tumor bearing kidney, contra lateral kidney (internal control), spleen, and lungs (site of metastases) o Longitudinal assessment of tumor progression/regression: Renca-Luc cells allow us to longitudinally monitor tumor progression/regression without having to sacrifice animals. At various times following tumor implantation and immunotherapy an intra peritoneal (i.p.) injection of Luciferine, the substrate for luciferase, was given to each mouse to observe luciferase expressing cells (cancer cells) within the live mouse. Xenogen IVIS full body imaging instrument located in the microscopy core, was used to determine the tumor burden via luciferase activity. 5

6 A. We first will determine the effects of Ad5TRAIL/CpG therapy on Renca MDSC and TIDC populations, by harvesting organs on days 14 and 21 post tumor implantation and 7 and 14 days post immunotherapy. Single cell suspensions will be prepared by homogenization followed by enzyme-mediated digestion. To dissociate cell aggregates cells will be incubated in PBS with 10% serum and 5mM EDTA for 15 minutes. The cells from each sample were processed and analyzed as follows: 1. The number and percentage of Renca MDSC, TIDC will be determined by flow cytometry according to the following surface markers: CD11b + CD11C + Ly6G - Ly6C - for TIDC and CD11b + CD11C - Ly6G - Ly6C + (monocytic) or CD11b + CD11C - Ly6G + Ly6C + (granulocytic) for MDSC. Viable cell counts for each preparation were determined by trypan blue exclusion and manual counting prior to staining. 2. The maturation state (phenotypic analysis) of these cells will be examined by staining for expression of mature activated phenotypic markers, I-A d, CD40, CD80, CD86, and CD54, by flow cytometry. 3. The stimulatory and inhibitory capacity of TIDC and MDSC will be determined in vitro through the use of standard radioactive [ 3 H] thymidine incorporation proliferation assays. As T cells divide they incorporate the radioactive thymidine is assessed using a cell harvester, and beta counter. TIDC and MDSC will be sort purified utilizing the phenotypic markers listed (1) from tumor-bearing mouse spleens and kidneys. Control splenic dendritic cells (spdc) from naïve BALB/c mice and control splenic T cells from DUC18 mice will be purified via magnetic bead selection. The T cells from DUC18 mice have a T cell receptor specific for the terk peptide, therefore when DC are allowed to phagocytose the terk peptide they 6

7 present it to the T cells to prime them for proliferate. Co-culture conditions consisted of DUC18 T cells + spdc alone as a control, DUC18 T cells + spdc +TIDC, and DUC18 T cells + spdc + MDSC. Cultures will be treated with [ 3 H] thymidine after 72 hours, and harvested 18 hours later to measure the proliferation of DUC18 T cells. 4. Determine the inhibitory mechanism used by RCC MDSC and TIDC by utilizing the same co-culture conditions listed above with various inhibitors. MDSC and TIDC utilize two metabolic pathways to suppress T cell proliferation: arginase and inos. We will supplement the media with L-arginine and utilize inos inhibitors to test this. We will then look for restoration of DUC T cell proliferation when in the presence of TIDC or MDSC. In addition, arginase and inos activity can be examined via radioactive L-arginine uptake and nitrite/nitrate release into culture supernatants via fluorometric assay kit. B. To determine tumor progression/regression luciferase activity will be monitored via IVIS imaging at various time points before and after immunotherapy. Live Image Software will be used to determine the tumor burden based on total light flux emitted from the tumor site of each mouse. At end time points (>21 days) mice were sacrificed after an i.p. injection of luciferine and organs harvested. The organs were examined for tumor burden via IVIS imaging. C. Next, we will determined the effects of immunotherapy on RCC metastasis to the lungs. Lungs will be inflated at an end time point (>21 days) with India ink and left in Fekete s solution over night. This technique allows for the manual quantitative evaluation of lung metastases, as the metastases will not take up India ink and appear white on the black lung. 7

8 D. We will also determine the extent to which depletion of TIDC and MDSC enhances antitumor immunity after intratumoral Ad5TRAIL/CpG administration at the primary tumor site. TIDC and MDSC depletion can performed administration of monoclonal antibodies (mab): anti-cd11c mab for TIDC, and anti-gr-1 for MDSC. Mice are given 250µg mab i.p. 1 and 2 days prior to tumor implantation. Tumor progression/regression and metastases outgrowth are determined as previously mentioned. 3. Results/Discussion Myeloid cell populations increase at the primary tumor site, spleen and lung during RCC I.R. tumor challenge (Figure 1). Intratumoral administration of Ad5TRAIL+CpG therapy 7 days post tumor challenge significantly decreased tumor progression and burden in mice (Figure 2). Intratumoral administration of Ad5TRAIL+CpG inhibited lung metastasis in RCC tumor bearing mice (data not shown). Mice treated with either CpG +/- Ad5TRAIL had a decreasing trend of MDSC at the primary tumor site and a significant decrease in the spleen (Figure 3A). MDSC and TIDC sorted from the primary tumor site of RCC challenged mice significantly suppressed T cell proliferation (Figure 3B). Interestingly, we also found a significant decrease in the percentage of MDSC in the spleen following CpG immunotherapy alone (Figure 3A). This led us to look at the phenotype and function of CpG-stimulated MDSC that had been sort purified from primary tumors. In vitro, CpG-stimulated MDSC exhibited a matured phenotype: upregulation of I-A d, CD40, CD80 and CD86 (Figure 4A). CpG-stimulated MDSC also exhibited less inhibition of in vitro T cell proliferation (Figure 4B). We have been able to demonstrate the efficaciousness of Ad5TRAIL/CpG therapy in an experimental model of advanced RCC. Not only have we demonstrated tumor regression, but also an inhibition of metastasis. Ad5TRAIL/CpG therapy decreased the amount of MDSC and 8

9 TIDC at the primary tumor site, spleen, and metastatic lung, suggesting less suppression of the antitumor immune response which was supported by tumor regression. To further understand effects on the antitumor immune response following Ad5TRAIL/CpG therapy in an RCC model, tumor-ag-specific CD8 T cell responses will need to be examined. Further investigation will elucidate the mechanisms by which CpG therapy alone augments MDSC phenotype and function, and the extent to which depletion of MDSC and TIDC in combination with Ad5TRAIL monotherapy will result in tumor regression. We will also further examine the heterogeneity of the MDSC population, and determine if distinct subsets of MDSC are necessary for tumor outgrowth, metastasis, and suppression of the antitumor immune response. This research has advanced the understanding of immunotherapy effects in vivo to not only inhibit tumor growth, but also to prevent metastasis. We are also beginning to elucidate the mechanisms by which immunosuppressive immune myeloid cells suppress the antitumor immune response. By understanding these mechanisms we can pinpoint targets for immunotherapies for cancer patients. This research has directly advanced the scientific field towards potential efficacious cancer therapies for RCC patients. 9

10 A. B. Figure 1: Myeloid Cell populations increase with tumor burden. A) Representative gating for CD11c + CD11b + TIDC, CD11c - CD11b + MDSC, and conventional dendritic cells CD11c + CD11b -, in the spleen and kidney of a naive and tumor bearing mouse. B) Cell frequency of MDSC population day 7, 14 and 18 post tumor challenge. Figure 2: Tumor Burden following tumor challenge (day 0) and immunotherapy (day 7). A) IVIS images for Luciferase activity post tumor challenge (p.c.). B) Tumor burden calculated from total light flux utilizing Image Software 10

11 A. B. A B. Figure 3: MDSC population is increased and functional. A) MDSC and conventional DC population frequencies in the kidney and spleen of tumor bearing mice 23 days p.c. B) DUC18 T cell proliferation assay with control spdc +/- MDSC and TIDC from primary tumors at the indicated ratios. *p<.05 between control DUC18 Tcell + spdc cultures (10:1) and DUC18 T cell + spdc + MDSC cultures Figure 4: CpG Stimulation alters MDSC phenotype and function. A) Phenotype profile after 24 hr incubation of sort purified whole MDSC (top) or granulocytic and monocytic (bottom) populations with CpG ODN. (Blue-unstimulated, purple- stimulated). B) Proliferation assay with DUC18 T cells and spdc +/- MDSC sort purified from either mice treated with CpG or PBS 7 days p.c.. * p<.05 between control DUC18 Tcell + spdc cultures (10:1) and DUC18 T cell + spdc + MDSC cultures 11

12 References 1. Ritchie, A.W. and G.D. Chisholm, (4): p Jemal, A., et al CA Cancer J Clin, (2): p Yang, J.C. and R. Childs, J Clin Oncol, (35): p Atzpodien, J., et al, J Clin Oncol, (7): p Motzer, R.J., et al., N Engl J Med, (2): p Marsters, S.A., et al., Recent Prog Horm Res, : p Pitti, R.M., et al., J Biol Chem, (22): p Walczak, H., et al., Nat Med, (2): p Ashkenazi, A., et al., J Clin Invest, (2): p Griffith, T.S., Methods Mol Biol, : p Griffith, T.S., et al., (5): p Griffith, T.S. and E.L. Broghammer, Mol Ther, (3): p Armeanu, S., et al., Cancer Res, (10): p Yang, F., et al., Med Oncol, (2): p Holoch, P.A. and T.S. Griffith, (1-3): p Griffith, T.S., et al., Curr Gene Ther, (1): p Lin, T., et al., (52): p Ren, X.W., et al., Cancer Gene Ther, (2): p Robinson, B.W., et al., Immunol Cell Biol, (6): p Nowak, A.K., et al., J Immunol, (10): p VanOosten, R.L. and T.S. Griffith, Cancer Res, (24): p van der Most, R.G., et al., Cancer Res, (2): p Trinchieri, G., D.P. Aden, and B.B. Knowles, Nature, (5558): p Gavin, M.A., et al., Nat Immunol, (1): p Weiner, H.L., Immunol Rev, : p Norian, L.A., et al., Cancer Res, (7): p Ochoa, A.C., et al., Clin Cancer Res, (2 Pt 2): p. 721s-726s. 28. Curiel, T.J., et al., Nat Med, (9): p Corzo, C.A., et al., J Immunol, (9): p Ko, J.S., et al., Cancer Res, (9): p Webster, W.S., et al., Cancer, (1): p Zea, A.H., et al., Cancer Res, (8): p Kemp, T.J., B.D. Elzey, and T.S. Griffith, J Immunol, (1): p