Studies have shown enhanced survival of ovarian cancer patients in which the tumors are infiltrated with tumor infiltrating lymphocytes and natural killer cells showing the importance of immune surveillance and recognition in ovarian cancer. Therefore, in this study, we tested cellular immunotherapy and varying combinations of cytokines (IL-2 and/or pegylated-IFNα-2b) in a xenograft mouse model of ovarian cancer. SKOV3-AF2 ovarian cancer cells were injected intra-peritoneally (IP) into athymic nude mice. On day 7 post-tumor cell injection, mice were injected IP with peripheral blood mononuclear cells (PBMC; 5 × 10(6) PBMC) and cytokine combinations [IL-2 ± pegylated-IFNα-2b (IFN)]. Cytokine injections were continued weekly for IFN (12,000 U/injection) and thrice weekly for IL-2 (4000 U/injection). Mice were euthanized when they became moribund due to tumor burden at which time tumor and ascitic fluid were measured and collected. Treatment efficacy was measured by improved survival at 8 weeks and overall survival by Kaplan-Meier analysis. We observed that the mice tolerated all treatment combinations without significant weight loss or other apparent illness. Mice receiving PBMC plus IL-2 showed improved median survival (7.3 weeks) compared to mice with no treatment (4.2 weeks), IL-2 (3.5 weeks), PBMC (4.0 weeks), or PBMC plus IL-2 and IFN (4.3 weeks), although PBMC plus IL-2 was not statistically different than PBMC plus IFN (5.5 weeks, p > 0.05). We demonstrate that cytokine-stimulated cellular immune therapy with PBMC and IL-2 was well tolerated and resulted in survival advantage compared to untreated controls and other cytokine combinations in the nude-mouse model.

Introduction: Expression of specificity protein 1 (Sp1) transcription factor is negatively associated with survival in some cancer patients. Survivin, a member of Inhibitor of Apoptosis Protein family, causes resistance to chemo- and radiation therapy. Tolfenamic acid (TA), a NSAID, targets Sp1 and inhibits survivin expression in some cancer models. Our aims were to examine the expression of Sp1 and survivin in ovarian cancer (OC) specimens and compare the in vitro anticancer response of cisplatin and TA alone and in combination. Methods: Tissues from clinical specimens (16 advanced stage tumors, 3 normal) were used to isolate RNA and prepare protein extracts. Sp1 and survivin expression was measured using qPCR and Western blots. OC cell lines (ES-2, SKOV3-AF2) were used for in vitro assays. Anti-proliferative response of cisplatin and TA was measured at 24, 48 and 72 h, post-treatment. Cells were treated with DMSO (vehicle), TA (25/50/100 µM) or cisplatin (5/10/20/50 nM), and cell viability was assayed using CellTiter Glo kit. Subsequently, cells were treated with optimized doses of TA (50 µM) or cisplatin (20 nM) for 48 h. Caspase-3/7 activity was measured using Caspase-3/7 Glo kit and Sp1, survivin, and c-PARP expression was determined by Western blots. Cell viability, Sp1 expression, and caspase-3/7 activity were assessed following individual and combination treatment of TA and cisplatin. Results: qPCR revealed that all tumor samples have increased survivin expression (>5 fold) and the majority showed increased Sp1 expression (2-3 fold). Consistent with the qPCR data, Western blot confirmed that all (survivin) or majority (Sp1) of specimens have high expression levels. TA and cisplatin showed a dose- and time-dependent inhibition of cell viability. TA caused 75% (ES-2) and 25% (SKOV3-AF2) growth inhibition while cisplatin caused ∼20% inhibition in both cell lines. Combination TA and cisplatin treatment resulted in maximum inhibition (ES2: >90%; SKOV3-AF2: 75%), showing a synergistic effect. These data confirmed that both agents up-regulated c-PARP and caspase-3/7 indicating activation of apoptotic pathways. However, only TA caused a significant decrease in Sp1, survivin, and c-Met expression. Increased inhibition of cell growth following combination treatment is consistent with increased caspase-3/7 activity and decreased Sp1 expression. Conclusions: Sp1 and survivin expression in OC specimens confirmed that targeting these molecules/pathways would be therapeutically relevant. TA enhanced the response of cisplatin when used in combination. Sp1 regulates survivin expression and targeting survivin with TA might render the OC cells radiation-sensitive. TA is less toxic than cisplatin but causes greater anticancer activity over cisplatin. Further studies to evaluate the mechanism(s) using proteomic and molecular profiling approach are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4656. doi:1538-7445.AM2012-4656

Introduction: Epithelial ovarian cancer is the leading cause of death among gynecologic malignancies, and two-thirds of patients present with stage III and IV disease. Unfortunately, the vast majority of advanced-stage ovarian cancer (OC) patients will relapse, require ongoing treatment, and eventually succumb to chemotherapy-resistant disease. The aim of this study was to test cellular therapy in combination with cytokines (IL-2 and/or pegylated-IFNα-2b) in a xenograft mouse model of OC. Methods: SKOV3-AF2 OC cells (1x106) were injected intraperitoneally (IP) into athymic nude mice (n=40). Day-7 post OC cell injection, mice were IP injected with mononuclear cells (MC; 5x106) and cytokine combinations [IL-2 (4,000 U) ± pegylated-IFNα-2b (IFN; 12,000 U); n=5 per group]. Cytokine injections were continued weekly for IFN and thrice weekly for IL-2. Mice were sacrificed when they became moribund due to tumor burden at which time solid tumor and ascitic fluid was measured and collected. Total RNA was isolated from tumor samples and reverse transcription (RT) reactions were performed. Gene expression of Survivin, DNA mismatch repair homolog (MLH1), cysteine-rich 61 (CCN1), and suppressor of cytokine signaling 1 (SOCS1), which have been shown to play roles in the pathobiology of OC, was analyzed by quantitative (q)RT-PCR using Taqman probes. Results: The in vivo model demonstrated that mice tolerated all the treatment combinations well (PBS, IL-2, IFN, IL-2+IFN, MC, MC+IL-2, MC+IFN, MC+IL-2+IFN) without significant weight loss or other cytotoxicities. Mice receiving MC+IL-2 treatment showed improved survival at 9-weeks post-tumor cell injection (60%, p<0.05) vs. mice without treatment (20%), IL-2 (0%), or MC (20%). There was no significant survival advantage in mice receiving MC+IFN (40%) or MC+IL-2+IFN (20%). Harvested tumors consisted of poorly differentiated surface epithelial carcinoma, growing in solid nests and sheets of large cells with a moderate amount of amphophilic or clear cytoplasm. Tumor cells focally were pleomorphic and multinucleated. Mitoses were frequent with abnormal forms present indicating a high rate of proliferation. We found that all tumors analyzed expressed Survivin, MLH1, CCN1, and SOCS1; however, there was no significant correlation of gene expression level (> 3-fold change) with time to sacrifice, tumor volume, or ascitic fluid production. SOCS1 gene expression, which is known to down-regulate proangiogenic molecules, was up-regulated in IFN treated animals (3-fold increase) but not control animals or animals receiving IL-2. Conclusions: IFNα-2b up-regulates SOCS1 expression in our in vivo mouse model. Cytokine-stimulated cellular immune therapy produces an anti-tumor effect in vivo. This data strongly supports development of cellular therapy (autologous or allogeneic) and is a potentially useful therapeutic strategy for the treatment of OC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3518. doi:1538-7445.AM2012-3518