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Objective: Chemoresistance associating CD44-positive cancer stem cells may presents a crucial problem in treating pancreatic cancer. Among the various isoforms of CD44, valiant 9 (CD44v9) have revealed especially implicated in tumor growth, invasion, and metastasis. Interaction of CD44v9 with the cystine transporter subunit xCT provide the ability of cancer cells to defend themselves against reactive oxygen species, mediating chemoresistance. Sulfasalazine (SSZ), which is a specific inhibitor of xCT-mediated cystine transport,could be a new therapeutic weapon fight against cancer stem cells. However, the extent of this remains unclear, suggesting the need for a histological evaluation of CD44v9 expression in clinical pancreatic cancer and the anti-tumor effect on pancreatic cancer of SSZ. Therefore, In this study, we analyze the expression of CD44v9 in a pancreatic cancer surgical specimen and examine the anti-tumor effect of SSZ and altered expression of CD44v9 by administration of GEM (GEM) with tumorgrafts. Methods: Experiment 1: A total of 70 clinical primary ductal adenocarcinoma of the pancreas were applied for immunohistochemical evaluation of CD44v9 expression. Randomly picked up 10 fields for each cases were analyzed for intensity of membrane staining and the percentage of stained cancer cells per field, followed by summing the scores using our own scoring system. Experiment 2: Three lines of created tumorgrafts from clinical pancreatic cancer. We administered GEM to the tumorgrafts and also scored the clinical specimens’ immunohistochemical evaluation by CD44v9. In addition, using one line, we divided the mice into two groups, a GEM group and a GEM + SSZ group, and repeated as above to determine the effect on GEM of SSZ. Results: Experiment 1: There was no correlation for prognosis. It was found that CD44v9-positive cells are present in high numbers. Experiment 2: In 1 line, which increased CD44v9-positive cell after GEM treatment,the GEM group showed tumor regression of 85% and the GEM+SSZ group showed tumor regression of 90%. Discussion: Result in Experiment 1 is reasonable, therefore, to consider the significance of the treatment target CD44vv9 as likely to be very high. Further, there was one line of tumorgrafts used, but not all percentages of CD44v9-positive cells increased markedly by GEM administration. This suggests the possibility that CD44v9 may be regarded as a marker of GEM resistance. In addition, a small but insignificant tumor regression effect on GEM of SSZ was found. We present evidence that for pancreatic cancer as well as other carcinomas, CD44v9-positive cells may be regarded as one of the markers of chemo resistance. Furthermore, since SSZ shows the antitumor effects of plus for GEM, we suggest that SSZ treatment is a possibility for a new treatment option. Citation Format: Tomohiro Kurokawa, Tatsuya Oda, Yuki Inagaki, Ryoichi Miyamoto, Yoshimasa Akashi, Nobuhiro Ohkohchi. CD44v9 expression in clinical pancreatic cancer and the gemcitabine plus sulfasalazine therapy against chemoresistant pancreatic cancer murine model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 195. doi:10.1158/1538-7445.AM2014-195

Objective: Histopathological characteristics of pancreatic carcinoma are the presence of abundant stroma that prevent mAb diffusion and consequently hampers mAb from direct attacking cancer cells. With a strong conviction that additional tool that improve mAb penetration and diffusion in dense fibroblastic solid cancer should indispensable for better therapeutic effect, we focused and employed the simple but strongest modalities, heat. Heat might increase tumor blood flow and tumor vessel permeability, leading enhanced accumulation of anticancer mAb. In this study, we aimed the increased accumulation of mAb in mouse pancreatic subcutaneous models and also boosted antitumor effects by applying moderate heat. Methods: Heat generation was completed by hot bathtub method for subcutaneous tumor in nude mice leg. Three human pancreatic cell of different stromal amount were employed (abundant: Capan-1, moderate: BxPC-3, and scant: MIAPaCa-2). At the timing of each tumors growth up to 70mm3, Cetuximab (1 mg/kg) was systematically administrated via caudal vein, subsequently applying heat to tumors for 30 min at three different temperatures of 25°C (control temperature), 37°C (intraabdominal organs), or 41°C (hyperthermia) (n = 5 each). The amount of accumulated Cetuximab were quantified by the fluorescent intensity of Alexa 488 against anti-human IgG at 24 hr, followed by figuring up the intensity of one cancer cell by dividing the total intensity value by the number of a cancer cell nuclei stained with DAPI. Enhanced antitumor effects, among irradiating various heat doses, were evaluated by the tumor volume over time. Results: In Capan-1, the fluorescent intensity per a cancer cell at 25°C, 37°C, 41°C were 675, 1130, 2332 respectively. Similarly, those in BxPC-3 were 673, 1347, 1573, and in MIAPaCa-2 were 1632, 1921, 1949, respectively. In Capan-1 and BxPC-3, the mean tumor growth on day 40 were significantly inhibited at 37°C group (934, 189 mm3), and 41°C group (491, 121 mm3) in compared with 25°C group (1385, 470 mm3) respectively. In MIAPaCa-2, however, that on day 50 was not inhibited at 37 degrees group (109 mm3), and 41 group (151 mm3) as compared with 25 group (123 mm3). Discussion: We demonstrated that hyperthermia actually contributed to measurable add-on antitumor effect for two among three pancreatic cancer models. Intending to provide more add-on effect, we are now evaluating the combined effects of hyperthermia plus applying a cyclic tumor-penetrating peptide (iRGD), which improved anticancer mAb penetration to cancer cells by binding to integrin αv/β3 or β5 and to neuropillin-1 on cancer cells. We suggested that this novel combined strategy would remarkably contribute to the enhanced accumulation of anticancer mAb in tumors and to the enhanced antitumor effects. Citation Format: Ryoichi Miyamoto, Tatsuya Oda, Shinji Hashimoto, Yoshimasa Akashi, Tomohiro Kurokawa, Yuki Inagaki, Nobuhiro Ohkohchi. Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2014-2663

[Objectives] Crosstalk between cancer cells and Cancer-Associated Fibroblast (CAF) plays a crucial role that comprises 3D organization of solid cancers. It seems general understanding that the main origin of CAF is circulating bone marrow derived Mesenchymal Stem Cell (BM-MSC), though locally pre-existing resident MSC of adipose tissue (AD-MSC) should partly contribute to CAF formation. The role of CAF and MSC to tumorigenesis remains controversial,since some studies reported cancer stimulating effect by externally added MSC or its potential differentiating into CAF, while others showed its suppressive effect. The capability of MSC's innate tropism for cancer, which enables us to apply it for the cellular delivery of anti-cancer molecules, has attracted much attention. One of the key issues should be whether externally added MSC would accepted as a member of cancer 3D organization, and would contribute to the cancer morphology. We tried to reveal whether AD-MSC, which is much convenient source as engineered MSC than BM-MSC, stimulates or inhibit tumor growth, and how MSC contribute to tumor stromal morphogenesis. [Materials & Methods] Experiment: To evaluate the growth advantage by adding MSC, and how MSC contribute to tumor stromal morphogenesis, two differently-originated ADSC cell lines transfected with GFP was mixed with human pancreatic-cancer cell line (Capan-1) at a rate of 3×106 /5×106 , and inoculated into the back subcutaneous resion of BALB/cAJcl-nu/nu mice. The tumor volume was calculated at Day4, Day7 and Day10 to assess the growth advantage of tumor mixed with ADSC. The mice was sacrificed at Day 10 and the histology of the resected tumor was evaluated with fluorescence microscopy and immunostaining procedure with anti-GFP antibody. [Results] Capan-1 formed significantly larger subcutaneous tumor by adding with both two AD-MSC cell lines(352mm3,265mm3) compared to control (70mm3) at Day10. In the tumor mixed with one ADSC cell line, cancer cells formed ductal stractures and contained fiber rich stroma with GFP positive fibroblasts among cancer ducts, mimicked the similar distribution to that in clinical solid cancer specimen. However, in the graft with the other ADSC cell line, cancer cells presented cord-like structures or deregulated cell proliferation, not formed ductal stractures. Though GFP positive cells could be observed, they thinly and rondamly distributed in the stroma. [Discussion] Both two AD-MSC plays promotive role in tumor growth, and one of which strongly contribute to the tumor stromal morphogenesis. Our AD-MSC could be regarded as a case which obviously differentiated into CAF, and would be a good candidate source for forecomming CAF therapy. Further studies revealing CAFs specific signature would helpful for providing stable AD-MSC source. Citation Format: Yuki Inagaki, Tatsuya Oda, Tomohiro Kurokawa, Ryoichi Miyamoto, Yasuyuki Kida, Nobuhiro Ohkohchi. Adipose-derived mesenchymal stem cell (ADSC) has the differentiation capacity toward cancer associated fibroblast (CAF) and reproduce the morphology of the clinical tumor stroma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 171. doi:10.1158/1538-7445.AM2014-171