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Introduction This is an investigation of the capability of FAIMS (Field Asymmetric Ion Mobility Spectrometry) technology as a tool for non-invasive detection of Colorectal Cancer (CRC) through urinary volatile organic compound analysis. It expands on "Detection of Colorectal Cancer (CRC) by Urinary Volatile Organic Compound Analysis" (Ramesh P. Arasaradnam et al, 2014) with an increased sample population and local to Japan, also comparing in-house data of CA19-9 and carcinoembryonic antigen (CEA) markers. Experiment Conducted at Nippon Medical School Chiba Hokusoh Hospital, urine samples were collected and frozen at -80°C from 139 patients at various stages of CRC and 78 healthy control samples. The samples were thawed in batches and placed in ice hours prior to testing. 2 ml of each urine sample was aliquoted into 10 ml vials for processing with the commercial FAIMS device (Lonestar, Owlstone, UK). Each vial was heated in the device to 40°C to create headspace with sufficient VOCs then a carrier gas (clean dry air) delivered the headspace (0.5 L/min) diluted with a make-up flow (2 L/min). The FAIMS device was set to scan at 0 to 100% electric dispersion field in 51 steps and compensation voltage between -6 V and +6 V in 512 steps, producing data matrices for each sample's analysis. Principal Component Analysis (PCA) followed by Partial Least Squares Discriminant Analysis (PLS-DA) of each sample was conducted using SIMCA 13 (Umetrics, Sweden). See Table Conclusions FAIMS technology achieved a high rate of separation between the CRC and healthy control urine samples with 64.7% sensitivity and 82.1% specificity overall. As the CRC stage advances the sensitivity increased from 27.3% to 100%. Results show excellent potential to use FAIMS technology as an early screening tool for CRC, particularly impressive compared to in-house sensitivity data of CA19-9 and CEA markers. Further research into FAIMS screening of other cancer types through VOC biomarker analysis of urine, breath, and feces is recommended. >Detection ResultsCRC STAGEFAIMSOTHER MARKERSCA19-9CEATrue PositiveFalse NegativeSensitivityFalse PositiveTrue NegativeSpecificityNo. of SamplesSensitivitySensitivityI123227.3%146482.1%434.7%18.6%II181260.0%146482.1%3116.1%32.3%III45590.0%146482.1%5219.2%36.5%IV150100%146482.1%1735.3%64.7%ALL904964.7%146482.1%14316.1%33.6% Citation Format: Christopher Psutka, Marina Yamada, Akihisa Matsuda, Kazuya Yamahatsu, Satoshi Matsumoto, Toshihiko Kitayama, Nobuo Nakano, Jyunichi Koyano, Tohru Mikoshiba, Masao Miyashita. FAIMS technology in urinary volatile organic compound analysis to detect colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2017-5303

Background: Recent studies have shown that epithelial splicing regulatory protein 1 (ESRP1) plays important roles in the epithelial mesenchymal transition of cancer cells via regulating the splicing of several RNAs, including fibroblast growth factor receptor 2 (FGFR2). Previously we reported that the expression of FGFR2IIIb correlates with vascular invasion and liver metastasis of pancreatic ductal adenocarcinoma (PDAC) (Cho, et al. Am J Pathol. 2008), while the expression of FGFR2IIIc correlates with the growth and invasion of PDAC cells (Ishiwata, et al. AACR. 2010). However, the expression and roles of ESRP1 in PDAC have not been well-defined. Methods: We performed immunohistochemical analysis of ESRP1, FGFR2IIIb, and FGFR2IIIc using pancreatic tissue specimens of PDAC (N=52) and non-tumorous pancreatic tissue (N=8). The ESRP1 expression vector and small interference RNA (siRNA) targeting ESRP1 were used to transfect human PDAC cells, and cell growth, morphology, migration, and invasion were analyzed. To clarify the ESRP1-regulating molecules, we performed proteomic analysis of ESRP1-gene-transfected PDAC cells using mass spectrometry. Results: In immunohistochemical analysis, ESRP1 was strongly expressed in the nuclei of PDAC cells in well- and moderately differentiated adenocarcinoma, while weakly expressed in poorly differentiated adenocarcinoma and normal pancreatic ducts. Well-moderately differentiated adenocarcinomas showed high-FGFR2IIIb/low-FGFR2IIIc pattern, with opposite for poorly differentiated adenocarcinoma. The overall and disease-free survival rates of the high-ESRP1 group were significantly than those of the low-ESRP1 group. In vitro, transient transfection of PDAC cells with ESRP1 expression vector led to an increase in FGFR2IIIb expression and decrease in migration and invasion. Stable transfection of PDAC cells with the ESRP1 expression vector resulted in an increase of FGFR2IIIb expression and decrease in migration and invasion. In contrast, ESRP1 siRNA-transfected PDAC cells induced FGFR2IIIc expression and increased cell growth, migration, and invasion. In proteomic analysis, transiently ESRP1 transfected PDAC cells showed a decreased expression of vimentin, 14-3-3 protein epsilon, heat shock protein 70, and the IQ motif containing GTPase activating protein 1, but an increased expression of filamin alpha. Several proteins as determined by proteomic analysis are involved in cell growth, migration, and invasion. Conclusion: The expression of ESRP1 correlates with the differentiation and prognosis of PDAC, and it modulates cell growth, migration, and invasion in PDAC cells via the alternation of several proteins including FGFR2. 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 2412. doi:1538-7445.AM2012-2412

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis. Recent studies have shown that cancer stem cells (CSCs) are important in cancer cell growth, invasion, metastasis, and recurrence. Several studies have revealed some CSC-specific markers for PDAC, such as CD133, CD24, CD44, CXCR4, ESA, and nestin. Some evidence also suggests that PDAC progresses through a multistep process comprised of noninvasive precursor lesions known as pancreatic intraepithelial neoplasias (PanINs). The reports of CSCs in PanINs are limited. We performed a comprehensive analysis of the expression of CSC markers in PDACs, PanINs, and normal pancreatic tissues. Materials & Methods: CD24, CD44, ESA, CD133, CXCR4, and nestin were used as CSC markers. Human PDACs (n=67), PanIN-1 (n=35), PanIN-2 (n=52), PanIN-3 (n=18), and normal pancreatic tissues (n=54), which were obtained from patients who underwent surgical operations in the surgical department of Nippon Medical School, were used for immunohistochemical analysis. Human PDAC cell lines, PANC-1, KLM-1, and MIA PaCa-2, were used for flow cytometric assays and quantitative RT-PCR analysis. Results: Regarding the clinicopathological features, the positivity of CD44 and CD133 showed significant correlations for UICC classifications and histological features In the survival analysis, CD133-positive PDACs led to a significantly poorer prognosis. In the immunohistochemical analysis, CD24, CD44, ESA, CXCR4, and nestin showed a gradual increase of positivity along with the grades of PanINs. Flow cytometric assays and RT-PCR analysis confirmed the expression profile in PDAC. Conclusion: CSC markers were expressed to various extents in PDAC, and the expression of CD133 and CD44 was correlated with the prognosis and stage. Furthermore, CSC markers showed a gradual increase along with the grades of PanINs; therefore, CSC may be involved in the carcinogenesis of PDAC. 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 412. doi:1538-7445.AM2012-412

BACKGROUND: Pancreatic cancer is associated with extremely high mortality rates due to rapid progression and a high incidence of metastases. Liver and lung metastases in the early postoperative period are one of the causes for the poor prognosis of patients with resected pancreatic cancer. Information on the pathobiology of metastatic pancreatic cancer and elucidation of the underlying molecular mechanisms are urgently needed in order to develop novel and effective therapeutic approaches to treatment. Injection of human pancreatic cancer cells in the spleens of nude mice is one of the major liver metastasis models. However, this model yields few metastatic tumors in the livers and none in the lungs. NOG mice have severe immunodeficiency, including defects of T, B and NK cell functions, and dysfunction of dendritic cells. Human pancreatic cancer cells are reported to easily proliferate and metastasize to other organs in NOG mice. In this study, we established and characterized human pancreatic cancer cell lines from liver and lung metastases in NOG mice. METHODS: PANC-1 cells (1×105 cells) were injected into the spleens of NOG mice. The mice were euthanized 8 weeks after injection, and the livers and lungs were removed. Metastatic tumors of the liver and lung were cut into small pieces and dispersed into single cells in a medium containing antibiotics. Biological and morphological characteristics, and gene expression patterns of mesenchymal markers of the liver and lung metastatic cells, were compared with parental cells. RESULTS: These metastatic cells were confirmed as being of human-cell origin using PCR with primer pair of human mitochondrial DNA. There were no morphological changes between liver and lung metastatic cells and parental cells, but the metastatic cells proliferated more slowly than parental cells as determined by MTT assays and cell counts. By contrast, the metastatic cells exhibited greater migration than the parental cells, as determined in Boyden chamber assays and by time lapse analysis. Attachment assays revealed that adhesion of metastatic cells to the type I and IV collagen, laminin and fibronectin was stronger than that of parental cells. Nestin, a cancer stem cell marker, was highly expressed in the liver and lung metastatic cells. CONCLUSION: These findings suggest that human pancreatic cancer cell lines derived from liver and lung metastases in NOG mice have different characteristics from parental cells. These new cell lines may contribute to resolving the mechanisms of metastasis of pancreatic cancer and the roles of cancer stem cells in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2011-5240

Background: Tumor angiogenesis is an important factor in the proliferation and metastasis of pancreatic ductal adenocarcinoma (PDAC). CD34, CD31 and factor VIII-related antigen are commonly used as endothelial cell markers for tumor vessels. However, these markers identify not only newly formed small tumor vessels, but also pre-existing large blood vessels. Nestin, a class VI intermediate filament protein, has been reported to be up-regulated in endothelial cells accompanying the process of angiogenesis. In an acute pancreatitis murine model, we have previously reported that nestin is strongly expressed in proliferating endothelial cells. Furthermore, we have reported that nestin-positive blood vessels correlated with a poor prognosis in colorectal cancer. In this study, we examined the effectiveness of nestin as an angiogenic marker and potent target for anti-angiogenic therapy in PDAC. Methods: Tissues from 45 patients with PDAC were immunostained with nestin and other common vascular endothelial markers including CD34, CD31 and factor VIII-related antigen. We measured the number and dimension of the nestin- and CD34-positive blood vessels using image analyzing software. In addition, we compared proliferation activity between nestin- and CD34-positive vessels as determined by PCNA-labeling indices. To clarify the roles of nestin in endothelial cells, we transfected siRNA targeting nestin transcripts to mouse endothelial TKD2cells, and performed cell growth and migration assays. Results: Immunohistochemically, CD34, CD31 and factor VIII-related antigen were localized in blood vessels of all sizes, while nestin was localized only in the small blood vessels in PDAC tissues. Nestin was also expressed in myofibroblasts and nerve fibers, but not in lymphatic vessels. In image analyzing software, nestin-positive vessels were small in number, and they formed small lumen compare with CD34-positive blood vessels. Nestin-positive vessels showed higher PCNA-labeling indices than those of CD34-positive vessels. Nestin was expressed in small and proliferating blood vessels in PDAC tissues, suggesting that nestin plays important roles in tumor angiogenesis in cancer. Knock down of nestin in mouse endothelial cells using siRNA inhibited the cell growth, but not cell migration in vitro. Conclusion: Nestin was specifically expressed in small and proliferating blood vessels in pancreatic cancer tissues, indicating that nestin is a useful angiogenic marker in cancer. Furthermore, nestin may be a novel therapeutic target for inhibition of tumor angiogenesis in pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5145. doi:10.1158/1538-7445.AM2011-5145

Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of K-ras in the nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. To examine cancer stem cell (CSC) functions, floating cultures in plates with a low-attachment surface have been widely employed. CSCs are reported to be able to grow in floating spheres in serum-free culture medium supplemented with epidermal growth factor (EGF) and/or fibroblast growth factor-2 (FGF-2), whereas non-CSCs, more differentiated non-CSCs cannot form spheres. In this study, we examined nestin expression in spheres and clarified whether nestin regulates sphere forming activity. Methods: Human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1, KLM-1, MIAPaCa-2 and PK-45H were used to perform sphere-forming assay in ultra-low attachment plates supplemented with EGF and FGF-2. Expression of CSC markers including CD133, CD44, CD24, epithelial specific antigen (ESA), and nestin in spheres were determined by real time RT-PCR and immunocytochemistry. Sphere-forming cells were dispersed with trypsin, and then cell growth and migration were assessed. Next, short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H, human PDAC cells, which express high levels of nestin, in order to assess the effects of decreased nestin expression on sphere-forming ability. Conversely, KLM-1 cells which express low level of nestin were stably transfected with a nestin expression vector and their sphere forming ability was then determined. Results: All the PDAC cell lines formed spheres, and all CSC markers examined were expressed in the spheres. The sphere cells showed low cell growth rates and increased cell migration as compared to non-sphere cells. Expression levels of nestin in the spheres of PDAC cells were higher than those of non-sphere cells. Nestin shRNA transfected PANC-1 and PK-45H cells showed a marked decrease in sphere number and diameter. By contrast, nestin-gene-transfected KLM-1 cells exhibited increased sphere formation. Conclusion: Nestin is a pancreatic CSC marker that promotes the sphere-forming ability of CSCs. Therefore, nestin may represent a novel therapeutic target for CSCs in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2011-2457

Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of Kras in the Nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. We have reported that Nestin was expressed in 30% of human pancreatic ductal adenocarcinoma (PDAC) cases, and Nestin expression in PDAC positively correlated with nerve involvement and invasion of the peripancreatic tissue margin. Therefore, we hypothesized that Nestin may play an important role in the migration and invasive potential of PDAC cells. In this study, we used a silencing strategy to clarify the roles of Nestin in human pancreatic cancer cells. Methods: An expression vector carrying a short hairpin RNA (shRNA) targeting Nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high levels of Nestin. Changes in the morphology and alignment of actin filaments were analyzed using phase-contrast images and immunocytochemistry. Effects of decreased expression of Nestin on cell growth, migration in scratch and Boyden chamber assays, invasion through matrigel, and cell adhesion on extracellular matrices were examined. Differences in mRNA levels of selected signaling molecules were examined by PCR arrays. Results: Targeting Nestin with shRNA caused a marked decrease in Nestin mRNA and protein levels. Nestin shRNA-transfected cells (Nes-sh cells) exhibited a sheet-like appearance with tight cell-cell adhesion, and increased expression of filamentous (F)-actin by comparison with sham-transfected cells, whereas anchorage-dependent and -independent cell growth and cell-extracellular matrix adhesion of Nes-sh cells did not differ from those of sham cells. By contrast, migration and invasion of Nes-sh cells were markedly attenuated. To clarify the mechanisms underlying this observation, we analyzed differences in selected signaling molecules by PCR arrays that could determine the expression of mRNA closely related to tumor metastasis. The gene expressing the greatest value in Nes-sh cells was E-cadherin. Real-time PCR and western blotting confirmed that E-cadherin expression was increased in Nes-sh cells by comparison with sham cells. Conclusion: Nestin participates in the regulation of pancreatic cancer cell migration and invasion, in part, by altering F-actin and E-cadherin expression. These observations suggest that Nestin may modulate the migration of Nestin-positive progenitor cells during pancreatic development, and may serve as a novel target for suppression of invasion and metastasis in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 408.

Background: Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level, and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we clarified the role of lumican in pancreatic cancer. Methods: Immunohistochemical analysis using anti-lumican antibody was performed in 65 pancreatic cancer patients. Lumican mRNA and protein were examined in pancreatic cancer cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1) by real-time PCR and western blot. Lumican stably transfected PANC-1 cells were prepared. Then, the growth rates in vitro and in vivo were compared with mock cells. The lumican expression of PANC-1 cell was knocked down using siRNA. Then, the growth rate in vitro was compared with the control cell. Results: Immunohistochemically, lumican was localized in the cytoplasm of cancer cells in 33 of 65 patients (51%). Lumican expression in cancer cells did not correlated with clinicopathological factors. However, the survival rate of patients with lumican-positive cancer cells was significantly lower than those with lumican-negative cancer cells in the surgery-only group. Lumican mRNA and the protein were detected in all pancreatic cancer cell lines and PANC-1 cells expressed median level of lumican in the cancer cell lines examined. The growth rate of lumican-transfected PANC-1 cells was significantly higher than that of mock cells both in vitro and in vivo. Moreover, the growth rate of lumican-knocked down PANC-1 cell was significantly lower than that of the control cell. Conclusion: Lumican might regulate pancreatic cancer cell growth and become a new target to develop anti-cancer drug for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2264.