Abstract B51: Gemcitabine-induced interleukin-8 expression is a surrogate marker for effects on pancreatic ductal adenocarcinoma cells and is associated with tumor microenvironment remodeling.

Background: The clinical standard of care for pancreatic ductal adenocarcinoma (PDAC) is treatment with gemcitabine (Gem), but this treatment provides meager benefits. Recent studies suggested that the stromal environment may hamper drug delivery. To date no surrogate marker has been identified that indicates whether Gem has directly affected PDAC tumor cells. In order to identify such a marker, we investigated the effects of Gem on PDAC cell gene expression in vitro and then analyzed human PDAC tissues from Gem-treated and untreated patients. Methods: To mimic clinical treatments, 12 PDAC cell lines were treated with 5uM Gem for three hours and then allowed to grow in the absence of Gem for 24hrs before mRNA expression analysis (Illumina gene expression array). IHC (immunohistochemistry), WB (Western blotting) and ELISA (enzyme-linked immunosorbent assay) were used to quantify protein expression. Apoptosis and cell cycle were analyzed by FACS. Cell growth was analyzed with MTS reagent. SiRNA and ShRNA mediated silencing were utilized to study the therapeutic value of various Gem-induced genes. Results: Gem treatment altered gene expression in both Gem-sensitive and Gem-resistant cells in vitro. One of the highly induced genes was interleukin-8 (IL-8), QPCR and WB confirmed high levels of IL-8 mRNA and protein induction. ELISA studies indicated that the IL-8 was secreted into conditioned media of Gem-treated cells in vitro. Gem-induced IL-8 expression was transcriptionally controlled by NFkB and C/EBP (CCAAT/enhancer-binding protein). IHC revealed strong expression of IL-8 in human PDAC tissues obtained from neoadjuvant Gem-chemotherapy (strong expression 8 cases; 42.1%, week expression 11 cases; 57.9%, negative 0 case) in comparison to those patients that received only surgery (strong expression 0 case, week expression 14 cases; 77.8%, negative 4 cases; 22.2%). This indicates that Gem is able to directly modulate gene expression in human tumors. PDAC cells and HPSC (human pancreatic stellate cells) expressed the CXCR1 receptor of IL-8 but not CXCR2. In contrast, HUVEC (human umbilical vein endothelial cells) and MLECs (mouse lung endothelial cells) expressed both CXCR1 and CXCR2. Growth of each cell line was stimulated by exogenous recombinant human IL-8. Conditioned media from Gem-treated cells and recombinant IL-8 stimulated in vitro angiogenesis (polygon formation) in HUVEC and MLECs and alpha-smooth muscle actin expression in HPSC. Neutralizing antibody against IL-8 reduced PDAC, HPSC cell growth and PDAC cells migration. Stable silencing of IL-8 in Capan2 cells, which develop an abundant stroma, led to a significant reduction of tumor growth in orthotopic tumor models evaluated by non-invasive bioluminescence imaging. Histologically, IL-8 silenced tumors showed reduced micro-vessel density, as well as highly reduced desmoplasia associated with necrosis. Conclusions: 1) Gem treatment induces IL-8 synthesis and secretion from pancreatic cancer cells. 2) IL-8 can serve as a surrogate marker indicating Gem effects. 3) IL-8 functions as a growth factor not only for cancer cells but also for cells in the tumor microenvironment. 4) Targeting IL-8 may be reasonable therapeutic strategy. Citation Format: Thiruvengadam Arumugam, Takahiko Fujii, Huang Haojie, Rosa F. Hwang, Vijaya Ramachandran, Craig D. Logsdon. Gemcitabine-induced interleukin-8 expression is a surrogate marker for effects on pancreatic ductal adenocarcinoma cells and is associated with tumor microenvironment remodeling. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B51.