bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 124: P21-activated protein kinase 1 high expression is a positive prognostic factor in human primary and metastatic pancreatic cancer
Authors: Yuan Shuqiang , Wang Feng , Juan Han , Pu Hengying , Zhou Zhiwei , Xu Rui-hua
Published: August 2015

Background: P21-activated protein kinase 1 (PAK1), a main downstream effector of small Rho GTPases, is overexpressed in many malignancies. Its overexpression was associated with poor prognosis in some tumor types including breast cancer, gastric cancer, colorectal cancer and so on. However, the expression and clinical relevance of PAK1 expression in human pancreatic cancer remain unknown.

Methods: The present study was designed to investigate the clinical and prognostic significance of PAK1 expression in pancreatic carcinoma. We examined the expression of PAK1 by immunohistochemistry in 72 primary pancreatic carcinoma samples and 20 liver metastatic samples. The relationships between PAK1 and clinicopathological parameters and prognosis in primary and metastatic pancreatic cancer were analyzed.

Results: Among the total 92 cases, primary pancreatic cancer samples had significantly higher rate (38/72, 52.8%) of high PAK1 expression than liver metastatic samples (5/20, 25.0%) (p = 0.028). In 72 primary pancreatic cancer patients, high PAK1 expression was associated with younger age (p = 0.038) and moderately or well differentiated tumor (p = 0.007). Moreover, a positive relationship was found between high PAK1 expression and overall survival (OS) (p<0.005). Patients with high PAK1 expression had a better OS than those with low PAK1 expression. Univariate and multivariate analysis by Cox regression including PAK1 and other prognostic pathological markers demonstrated high PAK1 immunostaining as an independent prognostic factor for survival in pancreatic cancer patients (P<0.005).

Conclusions: For the first time, we report that PAK1 is a novel prognostic marker for pathologically confirmed human pancreatic cancer. Reduced expression of p21-activated protein kinase 1 correlates with poor histological differentiation in pancreatic cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Shuqiang Yuan, Feng Wang, Han Juan, Hengying Pu, Zhiwei Zhou, Rui-hua Xu. P21-activated protein kinase 1 high expression is a positive prognostic factor in human primary and metastatic pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 124. doi:10.1158/1538-7445.AM2015-124
Abstract 127: Overexpression of neurotrophic tyrosine receptor kinases (NTRKs) as a potential therapeutic target for cancer
Authors: Wei Ge (Gina) , Cam Nicholas , Patel Roopal , Shoemaker Robert , Wild Robert , Li Gang
Published: August 2015

Neurotrophic tyrosine receptor kinases (NTRKs), or Tropomyosin receptor kinases (Trks), belong to a family of tyrosine kinases that regulate synaptic strength and plasticity during neural development. Deregulated kinase activity of Trk family members due to chromosomal rearrangements, gene mutations, splice variants and overexpression has been shown to be associated with cancer cell proliferation, survival, invasion and chemo-resistance in a number of cancer types. For example, chromosomal rearrangements involving NTRK1-3 have been reported in lung, colorectal, papillary thyroid, glioblastoma, melanoma and other cancers, and have been demonstrated to function as oncogenic drivers in these tumors. Moreover, overexpression of TrkA, TrkB and/or their corresponding ligands has been observed in a number of solid tumors such as breast, lung, prostate and pancreatic cancers. However, the functional implication of Trk overexpression has not been well defined or fully explored as a potential therapeutic strategy.

To characterize the expression, signaling and function of full-length Trks in cancer, a series of stable cell lines with full-length NTRK genes were established. The engineered cell lines were characterized for Trk expression, Trk phosphorylation and their response to ligand stimulation. Moreover, cell proliferation, cell transformation and growth inhibition by RXDX-101, a clinical stage, potent and selective pan-Trk inhibitor, were assessed. Trk downstream effectors, such as PLCγ, AKT and ERK, were also evaluated in the relevant settings. Furthermore, several cancer cell lines with endogenous overexpression of full-length Trks or rearranged Trks were identified and functionally characterized.

Our studies highlight the similarities and differences between Trk rearrangement and overexpression as potential oncogenic drivers and support further exploration of RXDX-101 in cancers with Trk overexpression.

Citation Format: Ge (Gina) Wei, Nicholas Cam, Roopal Patel, Robert Shoemaker, Robert Wild, Gang Li. Overexpression of neurotrophic tyrosine receptor kinases (NTRKs) as a potential therapeutic target for cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2015-127
Abstract 1276: CD169-positive sinus macrophages in regional lymph nodes are associated with better survival in patients with malignant melanoma
Authors: Saito Yoichi , Ohnishi Koji , Miyashita Azusa , Nakahara Satoshi , Fujiwara Yukio , Horlad Hasita , Motoshima Takanobu , Fukushima Satoshi , Jinnin Masatoshi , Ihn Hironobu , Takeya Motohiro , Komohara Yoshihiro
Published: August 2015

CD169 (sialoadhesin) is a sialic acid receptor that is expressed on the particular macrophages such as lymph node sinus macrophages. Animal studies suggested that CD169+ macrophages in lymph nodes have tumor-preventing properties, but the role of these cells in human tumors has not been clarified. Therefore, we speculated that CD169+ macrophages in regional lymph nodes (RLNs) are suggested to be essential to the anti-tumor immunity and involved in the prognosis of cancer patients. To determine the significance of CD169+ macrophages in patients with malignant melanoma (MM), we examined the expression of CD169 in paraffin-embedded tissue samples from 93 patients by immunohistochemistry and their relationship with overall survival and clinicopathological factors. A high density of CD169+ cells was significantly associated with longer overall survival in patients with MM (P = 0.001). Multivariate analysis showed that the density of CD169+ cells was an independent prognostic factor. We also found that the density of CD169+ macrophage was positively correlated with the number of CD8+ cytotoxic T cells infiltrating into tumor tissues. Furthermore, interferon (IFN)-α induced strong CD169 expression on human macrophages in vitro, and IFN-α-producing cells were indeed detected surrounding CD169+ cells in lymph node. Our data support that IFN-α-producing cells may be plasmacytoid dendritic cells (pDCs) known to produce IFN-α. These results suggest that CD169+ macrophages in RLNs are intimately involved in T-cell-mediated antitumor immunity and may be a helpful marker for assessing clinical prognosis and monitoring antitumor immunity in patients with MM.

Citation Format: Yoichi Saito, Koji Ohnishi, Azusa Miyashita, Satoshi Nakahara, Yukio Fujiwara, Hasita Horlad, Takanobu Motoshima, Satoshi Fukushima, Masatoshi Jinnin, Hironobu Ihn, Motohiro Takeya, Yoshihiro Komohara. CD169-positive sinus macrophages in regional lymph nodes are associated with better survival in patients with malignant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1276. doi:10.1158/1538-7445.AM2015-1276
Abstract 1295: Role of glycine N-methyltransferase in the regulation of T cell responses in experimental autoimmune encephalomyelitis
Authors: Li Chung-Hsien , Lin Ming-Hong , Chen Yen-Fu , Chu Shih-Han , Tu Pang-Hsien , Fang Cheng-Chieh , Yen Chia-Hung , Liang Peir-In , Huang Jason C. , Su Yu-Chia , Sytwu Huey-Kang , Chen Yi-Ming A.
Published: August 2015

Glycine N-methyltransferase (GNMT) is known for its function as a tumor suppressor gene. Since 100% of female Gnmt−/− mice developed hepatocellular carcinoma, we hypothesized that Gnmt−/− mice may have defective immune surveillance. In this study, we examined the immune modulation of GNMT in T cell responses using experimental autoimmune encephalomyelitis (EAE). The results showed that EAE severity was significantly reduced in Gnmt−/− mice. Pathological examination of the spinal cords revealed that Gnmt−/- mice had significantly lower level of mononuclear cell infiltration and demyelination than the wild-type mice. In addition, quantitative real-time PCR showed that expression levels of pro-inflammatory cytokines, including IFN-gamma and IL-17A, were much lower in the spinal cord of Gnmt−/- than in that of wild-type mice. Accordingly, myelin oligodendrocyte glycoprotein (MOG)-specific T cell proliferation and induction of T helper (Th) 1 and Th17 cells were markedly suppressed in MOG35-55 induced Gnmt−/- mice. Moreover, the amount of regulatory T cells was significantly increased in these mice. When the T cell receptor was stimulated, the proliferative capacity and the activation status of mTOR-associated downstream signaling were significantly decreased in GNMT−/- CD4+ T cells via an IL-2 and CD25-independent manner. Moreover, GNMT deficiency enhanced the differentiation of regulatory T cells without affecting the differentiation of Th1 and Th17 cells. Furthermore, the severity of EAE in mice adoptive transferred with GNMT-deficient CD4+ T cells was much milder than in those with wild-type CD4+ T cells. In summary, our findings suggest that GNMT is involved in the pathogenesis of EAE and plays a crucial role in the regulation of CD4+ T cell functions.

Citation Format: Chung-Hsien Li, Ming-Hong Lin, Yen-Fu Chen, Shih-Han Chu, Pang-Hsien Tu, Cheng-Chieh Fang, Chia-Hung Yen, Peir-In Liang, Jason C. Huang, Yu-Chia Su, Huey-Kang Sytwu, Yi-Ming A. Chen. Role of glycine N-methyltransferase in the regulation of T cell responses in experimental autoimmune encephalomyelitis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1295. doi:10.1158/1538-7445.AM2015-1295
Abstract 1298: The effect of adipocyte-derived factors on lung cells: Exploring the protective nature of excess weight on lung cancer risk
Authors: Penney Rosalind B. , Sappington Daniel , Siegel Eric , Boysen Gunnar , Kadlubar Susan
Published: August 2015

Lung cancer is the second most common cancer in men and in women (not including skin cancer) and the most common cause of cancer mortality. According to several epidemiologic papers, excess weight, identified as the BMI categories of overweight and obese, lowers lung cancer risk. Despite the numerous studies describing this anomaly, the underlying mechanisms for the protective effects are still unknown. Adipose tissue is known to secrete inflammatory molecules such as IL6, IL1β, and IL12. Activated IL6- and IL1β-associated inflammatory pathways are known to promote tumorigenesis, while the activated IL12-associated pathway inhibits tumorigenesis. We hypothesize that adipocytes create an environment that prevents lung carcinogenesis through activating inflammatory pathways that inhibit tumor growth and through blocking inflammatory pathways that promote tumor growth.

To study the effect of adipocyte-derived factors on lung cells, alveolar fibroblasts (MRC5) and lung carcinoma (A549) cells were cultured in standard medium and in a 50:50 standard: adipocyte-conditioned medium (CM). In addition, cells were treated with benzo[a]pyrene (B[a]P), a known lung carcinogen. After 48 hours, mRNA expression levels of inflammatory genes were determined by RT-PCR. The data demonstrate that B[a]P treatment induced expression of IL1β, IL6 and IL12 in both cell lines. CM treatment decreased expression of IL1β and IL6 in MRC5 cells and increased their expression in A549 cells. In contrast, CM treatment decreased expression of IL12 in both cells lines. Cells treated with B[a]P plus CM had increased expression of IL12 in both cell lines, and reduced expression of IL1β and IL6 in MRC5 (2.3-fold and 6.5-fold, respectively) and A549 cells (40-fold and 11.5-fold, respectively) over B[a]P-treated cells. It was demonstrated that combined treatment increased NFκB (a transcription factor for IL1β, IL6 and IL12) mRNA expression 4-fold over control. The data suggest that conditioned medium inhibits B[a]P-induced and NFκB-mediated expression of IL1β and IL6 but increases NFκB-mediated expression IL12. This suggests that excreted factors from an adipocyte-rich environment may lead to the activation of growth-prohibiting inflammatory pathways, as opposed to growth-promoting pathways. Understanding these mechanisms and the possible discovery of biomarkers can be the foundation for new preventive techniques and treatments for lung cancer.

Citation Format: Rosalind B. Penney, Daniel Sappington, Eric Siegel, Gunnar Boysen, Susan Kadlubar. The effect of adipocyte-derived factors on lung cells: Exploring the protective nature of excess weight on lung cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1298. doi:10.1158/1538-7445.AM2015-1298
Abstract 1311: The frequency of circulating Vδ1-positive but not Vδ2-positive γ/δ T cells correlates negatively with survival in late-stage melanoma and may be increased by ipilimumab treatment
Authors: Wistuba-Hamprecht Kilian , Weide Benjamin , Martens Alexander , Pawelec Graham P.
Published: August 2015

Recently, there has been a resurgence of interest in the possibility of using γ/δ-T cells in cancer immunotherapy, because these cells can kill melanoma cells in vitro, and IL-2 and zoledronate treatment can expand some human γδ cells in vitro and in animal models in vivo. The presence of the main subsets of γ/δ-T cells in late-stage melanoma patients and relationship with survival has not been extensively explored, although relatively lower percentages of Vδ2-positive cells has been reported in melanoma. Here, we present preliminary data on associations between the major γ/δ-T cell subsets and survival in melanoma patients, and whether baseline data are informative for predicting responses to ipilimumab treatment. We used a carefully validated set of monoclonal antibodies to assess γ/δ-phenotypes, because we found that certain commercial reagents were not behaving as specified (Wistuba-Hamprecht et al. OMIP-020: phenotypic characterization of human γδ T-cells by multicolor flow cytometry. Cytometry A. 2014; 85:522-4). Comparing a total of 39 stage IV melanoma patients with 27 controls, we found no overall differences in Vδ1 or Vδ2 T-cell frequencies, or in proportions of naïe (CD27, CD28-double positive, CD45RA+)-vs -late-differentiated (CD27,CD28-double negative, CD45RA+) γ/δ cells. However, Kaplan-Meier plots stratifying individual patients into groups with Vδ1 or Vδ2 frequencies above or below the median showed that the presence of Vδ1 but not Vδ2 cells negatively correlated with longer survival (p = 0.033 log-rank; p = 0.047 Breslow). In a subgroup of 9 patients, ipilimumab treatment resulted in an increase of peripheral Vδ1+ T-cells (p = 0.0391, Wilcoxon) but not of Vδ2+ T-cells (p = 0.375, Wilcoxon). We are currently analyzing data from a larger prospective study of ipilimumba treatment with over 100 patients, to confirm or refute these results.

Citation Format: Kilian Wistuba-Hamprecht, Benjamin Weide, Alexander Martens, Graham P. Pawelec. The frequency of circulating Vδ1-positive but not Vδ2-positive γ/δ T cells correlates negatively with survival in late-stage melanoma and may be increased by ipilimumab treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1311. doi:10.1158/1538-7445.AM2015-1311
Abstract 1343: Abcc3 up-regulation confers protection from chemotherapy to NK cells in a murine model of malignant glioma
Authors: Pellegatta Serena , Pessina Sara , Cantini Gabriele , Cazzato Emanuela , Kapetis Dimos , Finocchiaro Gaetano
Published: August 2015

Growing evidence suggests that chemotherapy can influence the immune response by inducing lymphopenia or enhancing immunogenicity of dying tumor cells. In a clinical trial currently active in our Institution, patients with first diagnosis of glioblastoma are treated with dendritic cells (DC) loaded with autologous tumor lysate together with standard radio and chemotherapy with temozolomide. Peripheral blood lymphocytes from 22 patients were analyzed by flow cytometry to identify immune cell activation before and after DC vaccines. The ratio of vaccine/baseline frequencies (V/B ratio) was correlated with the survival of each patient. Increased V/B ratio for NK cells was significantly associated with prolonged PFS and OS (median 15.0 vs 8.0 mo, p = 0.003; 22.0 vs 12.0 mo, p = 0.02, respectively). Using the murine malignant glioma GL261, we investigated the molecular mechanisms induced by TMZ on NK and CD8 T cells. We treated mice 9 days after intracranial implantation of GL261 cells with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed at different time points and tumor and peripheral blood harvested and analyzed by flow cytometry. Gene expression profiling on peripheral blood lymphocytes revealed an up-regulation of multidrug resistance genes in NK cells, but not in CD8 T lymphocytes, in TMZ-treated mice compared to controls. The increased expression of the ATP transporter gene Abcc3 was confirmed by real time PCR (4.2-fold higher than controls, P = 0.006). Using eFluxx-ID multidrug resistance (MDR) assay based on specific inhibitors, we also demonstrated that Abcc3 was functionally active during TMZ treatment. NK cell resistance to chemotherapy was accompanied by an increase in migration and homing ability into the brain at early time point and in cytotoxicity at later phases (beyond the end of TMZ treatment).

Our data show that murine NK cells are resistant to and activated by TMZ chemotherapy. Further investigations in patients treated by radio-chemotherapy with or without DC immunotherapy are warranted.

Note: This abstract was not presented at the meeting.

Citation Format: Serena Pellegatta, Sara Pessina, Gabriele Cantini, Emanuela Cazzato, Dimos Kapetis, Gaetano Finocchiaro. Abcc3 up-regulation confers protection from chemotherapy to NK cells in a murine model of malignant glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2015-1343
Abstract 1358: Generation of induced pluripotent stem cells from tumor-infiltrating lymphocytes
Authors: Saito Hidehito , Okita Keisuke , Fusaki Noemi , Sabel Michael , Chang Alfred , Ito Fumito
Published: August 2015

Human induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) were found to have tumor-reactive T cells and have been used for adoptive cell therapy for metastatic melanoma. The objective in this study is to generate iPSCs from TILs and assess their pluripotency. Under the approved protocol by the Institutional Review Board and the Human Pluripotent Stem Cell Research Oversight Committee, we obtained melanomas from a patient who provided informed consent for tissue procurement and generation of iPSCs. TILs were reprogrammed by Sendai virus (SeV) vector encoding four pluripotency transcription factors (Oct3/4, Klf4, Sox2 and c-Myc). TIL-derived iPSCs (TIL-iPSCs) were characterized by their pluripotency. Within 3 weeks of infection with SeV to deliver pluripotency transcription factors, we identified colonies that resembled embryonic stem cells (ESCs) among the T cell derivatives. TIL-iPSCs displayed typical stem cell morphology and had a normal karyotype. Alkaline phosphatase and immunofluorescence staining with stem cell surface markers, OCT3/4, SSEA3, SSEA 4, TRA1-81 and TRA1-60 proteins suggested pluripotency of colonies. RT-PCR analyses revealed that TIL-iPSCs expressed pluripotent stem cell markers such as NANOG, OCT3/4 at levels comparable to those in ESCs. T cell receptor (TCR) gene rearrangement analysis in TIL-iPSCs confirmed that they originated from mature T cells and retained diversity of TCR gene rearrangement. In conclusion, human iPSCs with diverse TCR gene rearrangement can be generated from TILs efficiently by SeV encoding four pluripotency transcription factors. Future work will explore their differentiation potential to T cells for cancer immunotherapy. We acknowledge in DNAVEC Corporation for kindly providing Sendai virus vectors. This study was supported by the University of Michigan Start-up fund, American College of Surgeons and Central Surgical Association.

Note: This abstract was not presented at the meeting.

Citation Format: Hidehito Saito, Keisuke Okita, Noemi Fusaki, Michael Sabel, Alfred Chang, Fumito Ito. Generation of induced pluripotent stem cells from tumor-infiltrating lymphocytes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1358. doi:10.1158/1538-7445.AM2015-1358
Abstract 1361: The decoy receptor interleukin-1 receptor type 2 acts as an angiogenic factor in human colorectal cancer
Authors: Mar Ai-Chung
Published: August 2015

We previously demonstrated that interleukin-1 receptor type 2 (IL1R2), an IL-1 decoy receptor that inhibits exogenous IL-1 signaling, intracellularly activates the expression of several pro-inflammatory cytokines. In this study, we observed that IL1R2 was overexpressed in human colon cancer cells (CRC) compared to normal colon cells. In addition, we observed that the IL1R2 level was higher in late-stage clinical CRC patients and that a high level of IL1R2 was associated with a poor 5-year survival rate in CRC patients. Furthermore, we observed that the levels of IL1R2 and IL-6 were strongly correlated in 40 CRC patients. To elucidate the oncogenic roles of intracellular IL1R2, we modulated its expression levels in human CRC cell lines. We verified that the intracellular level of IL1R2 was correlated with the migration ability of CRC, cells. We observed that the VEGF-A and IL-6 levels were significantly increased in conditioned media harvested from IL1R2-overexpressing cells, and these elevated VEGF-A and IL-6 levels enhanced the proliferation, migration, and tube formation of cultured endothelial cells. We further demonstrated that intracellular IL1R2 levels were positively associated with tumor growth and microvessel density in xenograft mouse models. Furthermore, we observed that IL1R2 formed a complex with c-Fos. This complex bound to the AP-1 site at the IL-6 and VEGF-A promoters. Together, these results reveal that intracellular IL1R2 acts with c-Fos to enhance the transcription of IL-6 and VEGF-A, which promotes tumor malignancy and angiogenesis.

Citation Format: Ai-Chung Mar. The decoy receptor interleukin-1 receptor type 2 acts as an angiogenic factor in human colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2015-1361
Abstract 1390: Combination of carbon ion beam and gemcitabine causes unrepairable DNA damage and death of radioresistant pancreatic cancer stem-like cells in vitro and in vivo
Authors: Sai Sei , Wakai Toshifumi , Vares Guillaumes , Yamada Shigeru , Kamijo Takehiko , Kim Eun Ho , Kamada Tadashi , Shirai Toshiyuki
Published: August 2015

Purpose

To elucidate whether carbon ion beam alone or in combination with gemcitabine has advantages over X-ray irradiation in targeting putative human pancreatic cancer stem cells (CSCs).

Materials and Methods

Human pancreatic CSCs sorted from PANC1 and PK45 cells were treated with carbon ion beam, X-ray alone or in combination with gemcitabine, and then colony, spheroid and tumor formation assays, RT Profiler PCR Array assay, immunofluorescence γH2AX foci assay as well as in vivo tumor control analysis were performed.

Results

The colony, spheroid formation as well as tumorigenicity assays confirmed that CD44+/ESA+ cells exactly have CSC properties compared to CD44-/ESA- cells. CSCs were more highly enriched after X-ray or gemcitabine compared to carbon ion beam alone, but extremely enhanced either X-ray or carbon ion beam in combination with gemcitabine. The number of colony and spheroid formed from CSCs after carbon ion beam was significantly reduced compared to that of X-ray, and it was extremely suppressed when combined with gemcitabine. The relative biological effectiveness (RBE) values for the carbon ion beam relative to X-ray at the D10 levels for CSCs were 2.13-2.78. RT Profiler PCR Array analysis showed that expressions of apoptosis-related genes (Bax, Cytochrome c), autophagy-related genes (LC3, p62), and senescence-related gene p21 were remarkably induced after carbon ion beam combined with gemcitabine compared to carbon ion beam alone or X-ray combined with gemcitabine. Immunofluorescence assay showed that not only the number but also the size of γH2AX foci in CSCs were lager 24 h after carbon ion beam combined with gemcitabine compared to carbon ion beam alone or X-ray combined with gemcitabine. Xenograft tumor control analysis showed that the tumors were not completely controlled even treated with 60 Gy of X-ray, but it was destroyed with 25 Gy of carbon ion beam combined with 50 mg/kg gemcitabine.

Conclusions

Carbon ion beam combined with gemcitabine has superior potential to kill pancreatic CSCs via unrepairable clustered DSB, and multiple cell death pathways at relatively lower doses compared to carbon ion alone or X-ray combined ith gemcitabine.

Citation Format: Sei Sai, Toshifumi Wakai, Guillaumes Vares, Shigeru Yamada, Takehiko Kamijo, Eun Ho Kim, Tadashi Kamada, Toshiyuki Shirai. Combination of carbon ion beam and gemcitabine causes unrepairable DNA damage and death of radioresistant pancreatic cancer stem-like cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2015-1390
Abstract 1407: Identification of sialylated glycoproteins in doxorubicin-treated hepatoma cells with glycoproteomic analyses
Authors: Miyoshi Eiji , Azuma Kanako , Takamatsu Shinji , Terao Naoko , Serada Satoshi , Naka Tetsuji , Kamada Yoshihiro
Published: August 2015

Sialylation is one of the most important types of glycosylation involved in carcinogenesis and establishment of cancer stemness. We previously showed that increased sialylation is a characteristic glycan change in cancer stem cells (CSCs) from hepatocellular carcinoma (Cancer Science 2011, Moriwaki et al). However, the identities of glycoproteins targeted for sialylation remain unknown. CSC fraction is too small to identify target proteins with glycol-proteomics analyses. In the present study, we identified glycoproteins targeted for sialylation in doxorubicin (DXR)-treated hepatocarcinoma cell line, Huh7, using glycoproteomic analyses. Since CSCs constitute a small subset of cells within carcinoma cell lines, it is difficult to identify sialylated proteins using general glycoproteomic strategies. It is known that treatment with anti-cancer drug can condense CSCs, we used DXR to concentrate CSCs. In DXR-treated Huh7 cells, isobaric tag for relative and absolute quantitation (iTRAQ) analysis identified 17 sialylated glycoproteins. Most of the identified glycoproteins were cancer-associated proteins. Furthermore, two proteins of approximately 70 kDa were detected using Sambucus sieboldoana agglutinin (SSA) blot analysis and identified as beta-galactosidase and alpha-2-HS-glycoprotein (fetuin-A) by SSA precipitation followed by liquid chromatography-tandem mass spectrometry analyses. Sialylation levels of fetuin-A were increased in DXR-treated Huh7 cell lysates. Furthermore, intracellular localization of fetuin A with sialylation was changed in DXR-treated Huh7 cells under observation with confocal microscopy These changes in sialylation of glycoproteins might be involved in the establishment of cancer stemness.

Citation Format: Eiji Miyoshi, Kanako Azuma, Shinji Takamatsu, Naoko Terao, Satoshi Serada, Tetsuji Naka, Yoshihiro Kamada. Identification of sialylated glycoproteins in doxorubicin-treated hepatoma cells with glycoproteomic analyses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1407. doi:10.1158/1538-7445.AM2015-1407
Abstract 1409: Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells
Authors: Alipour Amina , Burdick Monica M. , Tees David F.J.
Published: August 2015

It has been shown that the mechanical properties of pathological cells are different from healthy ones. Therefore, mechanical characterization of cells may be useful for investigating mechanisms and progression of diseases. In the case of cancer, the finding that cancerous cells are considerably more deformable than normal ones provides the motivation for investigating rheological differences between stem-like and non-stem-like cancer cells. In this study, the viscoelastic properties of six breast cancer cell lines, three of which have been regarded (based on surface markers) as stem-like and three considered less stem-like, were measured using micropipette aspiration and particle tracking microrheology. In micropipette experiments, cancer cells were aspirated one-by-one at controlled pressures into small glass micropipettes and the length of the aspirated section of the cells was tracked as a function of time. The applied pressure was either increased linearly or increased suddenly to different levels and then held constant. The homogeneous elastic half-space model was used to interpret the viscoelastic properties of the different cell lines. The effect of pressure loading rate on cell viscoelasticity was examined by applying three different aspiration pressure rates of 9, 6, and 3 Pa/s. The apparent Young's modulus was found to depend on pressure loading rate, which suggests a more complex rheology than a standard viscoelastic solid. Above a threshold force level, cells were also found to enter the micropipette continuously in a fashion that is analogous to white blood cells, i.e. as a liquid drop with constant cortical tension. A comparison between the Young's moduli showed that at least some of the stem-like breast cancer cell lines were less stiff than less stem-like cell lines and more likely to exhibit the liquid drop-like behavior. The reduction in stiffness and change in rheological regime might be the result of lower actin filament density which allowed the stem-like cancer cells to be more deformable while undergoing extravasation and intravasation during the metastatic process. Regional rheological differences were also found using particle tracking microrheology. Our hypothesis is that the differences in the rheological properties might help to distinguish between stem-like cancer cells and non-stem-like ones, which could eventually lead to a new detection and diagnostic method in cancer research.

Citation Format: Amina Alipour, Monica M. Burdick, David F.J. Tees. Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2015-1409
Abstract 1425: FBXO11 suppresses epithelial plasticity and proliferation by ubiquitinating the Snail family of transcription factors
Authors: Lu Jianrong , Jin Yue , Shenoy Anitha K. , Chen Hao , Luo Huacheng , Wu Lizi , Mohammed Kamal A.
Published: August 2015

Epithelial-to-mesenchymal transition (EMT) is central to embryonic development and carcinoma progression. The cell adhesion molecule E-cadherin critically maintains epithelial property and restricts epithelial cell proliferation and motility. The Snail family of transcription factors are core inducers of EMT in part through direct repression of E-cadherin transcription. The Snail factors are normally under tight control to safeguard epithelial identity and homeostasis. However, their physiological regulation remains largely elusive. In the present study, we show that the F-box protein FBXO11 binds to Snail in a phosphorylation-independent manner and targets it for ubiquitin-mediated proteasomal degradation. FBXO11 promotes the degradation of other Snail family members Slug and SCRT1 as well. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and activates E-cadherin transcription. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion. Human cancer-derived FBXO11 missense mutants are impaired to degrade Snail. In human cancer, FBXO11 expression correlates with E-cadherin, and decreased expression of FBXO11 is robustly associated with adverse clinical outcomes. Inactivation of FBXO11 in mice results in neonatal lethality, increased Snail and Slug protein levels, decreased E-cadherin expression, and epidermal hyperplasia, which phenotypically resemble epidermis-specific transgenic overexpression of Snail or deletion of E-cadherin. These findings establish FBXO11 as a physiological ubiquitin ligase of Snails that is indispensable for suppressing epithelial hyperproliferation and plasticity during embryonic development and carcinoma progression.

Note: This abstract was not presented at the meeting.

Citation Format: Jianrong Lu, Yue Jin, Anitha K. Shenoy, Hao Chen, Huacheng Luo,Lizi Wu, Kamal A. Mohammed. FBXO11 suppresses epithelial plasticity and proliferation by ubiquitinating the Snail family of transcription factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1425. doi:10.1158/1538-7445.AM2015-1425
Abstract 1439: RANK expression by osteosarcoma cells increases lung metastasis in Nude mouse while has no effect in immune-competent mouse
Authors: Navet Benjamin , Ando Kosei , Yagita Hideo , Mueller Christopher G. , Heymann Dominique , Lezot Frederic
Published: August 2015

Abstract

Background: Osteosarcoma, the most frequent malignant primary bone tumor, is characterized by an osteoid tumor tissue formation associated to a tumor-induced osteolysis. Lung metastasis occurs in some osteosarcomas with dramatic consequence for patient lifespan. Expression of RANK has been reported in some osteosarcoma cells and the question of the potential repercussion on tumor growth, tumor-induced osteolysis and lung metastasis was raised.

Methods: Human HOS, human MG63 and mouse MOS-J osteosarcoma cell-lines were transfected to stably over-express RANK and injected in para-tibia muscle of Nude mice and syngeneic C57BL/6 mice for MOS-J cells. Tumor growth was follow and associated osteolysis analyzed by micro-CT and histology comparatively to injections of native cells. Lung metastases were numbered in each group.

Results: In Nude mouse RANK over-expression, whatever the cell-line considered (human or mouse), induces a significant increase of the number of lung metastasis while the tumor growth and the induced osteolysis are not affected. Injections of a RANKL blocking antibody reverse the impact of RANK over-expression on lung metastasis. In the C57BL/6, RANK over-expression has no impact on the lung metastasis and tumor-induced osteolysis. The tumor growth was slightly reduced.

Conclusions: Reported data evidence that RANK expression by osteosarcoma cells has no significant effect on the tumor growth and tumor-induced osteolysis but in immune-deficient Nude mouse induces an important increase of the lung metastases dependently of RANKL stimulation.

Citation Format: Benjamin Navet, Kosei Ando, Hideo Yagita, Christopher G. Mueller, Dominique Heymann, Frederic Lezot. RANK expression by osteosarcoma cells increases lung metastasis in Nude mouse while has no effect in immune-competent mouse. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2015-1439
Abstract 1523: Prostaglandin E2 promotes colorectal cancer stem-like cell expansion and metastasis
Authors: Wang Dingzhi , Fu Lingchen , Sun Haiyan , DuBois Raymond N.
Published: August 2015

Emerging evidence suggests that chronic inflammation and inflammatory mediators contribute to cancer stem-like cell (CSLC) formation, maintenance, and expansion. However, the mechanisms underlying their contribution to CSLC regulation are poorly understood. Here we demonstrate that an inflammatory mediator, prostaglandin E2 (PGE2), induces colorectal CSLC expansion in vivo and in vitro. PGE2 also accelerates liver metastatic tumor incidence and numbers in an orthotopic model. Furthermore, we show that NF-κB is required for PGE2 induction of CSLCs and colorectal tumor metastatic spread. PI3K and MAPK pathways mediate PGE2 activation of NF-κB in induction of CSLCs. These results demonstrate that PGE2 induces colorectal CSLC expansion by activating NF-κB via PI3K-Akt and MAPK pathways. Our findings not only uncover a previously unrecognized role of PGE2 in induction of CSLCs and metastasis, but also may delineate a novel therapeutic approach for CRC patients by targeting CSLCs via inhibition of the PGE2 signaling pathway.

Citation Format: Dingzhi Wang, Lingchen Fu, Haiyan Sun, Raymond N. DuBois. Prostaglandin E2 promotes colorectal cancer stem-like cell expansion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2015-1523
Abstract 1524: Establishing long-term circulating tumor cells (CTCs) in a 3D culture system in vitro
Authors: Cabrera Marina C. , Hurte Elaine
Published: August 2015

Circulating tumor cells (CTCs) are cells that have detached from the primary tumor and entered the bloodstream with the potential to seed metastatic tumors in distal sites. High CTC numbers correlate with aggressive disease, increased metastasis, and decreased time to relapse. It has also been shown that cancer stem cells (CSCs) represent a proportion of the CTCs present in patients. Given that CSCs are resistant to chemotherapy and are responsible for tumor initiation, it is posited that the CSCs are the seeds of metastasis. However, direct evidence for this hypothesis is limited due to the fact that there are few methods for culturing and studying these rare cells. We used a 3D culture chamber system (RealBio D4 ™) to establish long-term cultures of human-derived pancreatic tumors and to assess the effect of gemcitabine treatment on the CSC population of these cultures. We observed that the system's 3D matrix supported culture development and incorporation of tissue organization and microenvironment. The chamber design allowed CTCs generated within the cultures to migrate out of the cell mass into the circulating nutrient medium where they were collected for characterization. Further, tumor tissue grown on the matrix showed an enrichment of CSCs after gemcitabine treatment analogous to the enrichment observed in vivo. Future studies will focus on characterizing isolated CTCs and circulating CSCs from pancreas and breast cancer models, determine their response to therapy, and elucidate the relationship of different CTC populations on tumor spread. Ultimately, these studies may support the use of CTCs and circulating CSCs as biomarkers in clinical trials.

Citation Format: Marina C. Cabrera, Elaine Hurte. Establishing long-term circulating tumor cells (CTCs) in a 3D culture system in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1524. doi:10.1158/1538-7445.AM2015-1524
Abstract 1546: Immunosuppressive activity of cancer-associated fibroblasts in head and neck squamous cell carcinoma
Authors: Takahashi Hideyuki , Sakakura Koichi , Chikamatsu Kazuaki
Published: August 2015

Introduction:

Evidence has accumulated indicating that cancer-associated fibroblasts (CAFs) play an important role in angiogenesis, invasion, and metastasis. Simultaneously, CAFs might actively participate in inducing immunosuppressive ability and promoting evasion from immunosurveillance. In the present study, we investigated whether CAFs within the tumor microenvironment contribute as one of key components of tumor immune evasion in head and neck squamous cell carcinoma (HNSCC).

Materials and methods:

CAFs and normal fibroblasts (NFs) were prepared from surgical tissue of HNSCC patients. Flow cytometric analysis was performed for the expression of A-smooth muscle actin (A-SMA) and co-stimulatory molecules. Inhibition of T-cell proliferation, T-cell apoptosis and induction of regulatory T cells (Treg) were also examined using flow cytometry. The mRNA expression of various cytokines was investigated by real-time qRT-PCR.

Results:

Generated CAFs and NFs were negative for CD11b, CD34, and CD45, and were positive for CD90 and FAP. The A-SMA expression of CAFs was higher than that of NFs. CAFs efficiently suppressed T cell proliferation as compared with NFs. Of note, this suppressive activity was found under co-cultivation with not only CAFs, but also supernatant from CAFs. CAFs, but not NFs expressed co-regulatory molecules, B7H1 and B7DC and CAFs showed increased expression levels of cytokine genes including IL-6, IL-8, TNF-A, TGF-β, and VEGF. Moreover, PBMCs co-cultured with supernatant from CAFs preferentially induced T-cell apoptosis and Treg as compared with those from NFs.

Conclusion:

Our findings suggest that, in the tumor microenvironment, CAFs might play a pivotal role in establishing an immunosuppressive network along with tumor cells, and subsequent facilitation to activate the immunosuppressive pathway.

Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Immunosuppressive activity of cancer-associated fibroblasts in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1546. doi:10.1158/1538-7445.AM2015-1546
Abstract 1565: Magnetic bead-based serum peptidome profiling for identifying potential biomarkers in esophageal squamous cell carcinoma
Authors: Jia Kun , Li Wei , Wang Feng , Liu Fang , Liu Xiaofei , Qiao Yuanyuan , Xu Yang , Zhou Lanping , Sun Yulin , Ma Qingwei , Zhao Xiaohang
Published: August 2015

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide, especially in China. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Therefore, there is an urgent need to develop novel serological biomarkers for ESCC. Here, we performed a two-stage serum peptide profiling in 477 unique samples using weak cationic exchange magnetic beads coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The three significantly differentially expressed peaks were used to establish a genetic algorithm model for ESCC. The sensitivity and specificity were 97% and 95.92% in the training set, and 97.03% and 100% in the validation set, respectively. It was significantly better than the other well studied serum proteins such as squamous cell carcinoma antigen (SCC-Ag) and cytokeratin 19 fragment 21-1 (Cyfra 21-1). Notably, the diagnostic capability of this model was superior for early stage ESCC with a sensitivity of 96.94% and a specificity of 100%. Furthermore, the three diagnostic peptides were identified as fragments of alpha-2-HS-glycoprotein, thrombospondin-1, and fibrinogen alpha, respectively. The immunohistochemical staining assay showed that TSP1 was significantly overexpressed in tumor tissues and associated with lymph node metastasis and poor prognosis. Overall, we constructed a novel diagnostic model which will benefit the screening, early detection and diagnosis of ESCC. And the identified proteins and their derivative peptides will deepen our understanding of tumorigenesis and serve as potential serological biomarkers for ESCC.

Note: This abstract was not presented at the meeting.

Citation Format: Kun Jia, Wei Li, Feng Wang, Fang Liu, Xiaofei Liu, Yuanyuan Qiao, Yang Xu, Lanping Zhou, Yulin Sun, Qingwei Ma, Xiaohang Zhao. Magnetic bead-based serum peptidome profiling for identifying potential biomarkers in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1565. doi:10.1158/1538-7445.AM2015-1565