Pharmacological inhibitors of glutathione synthesis and circulation, such as buthionine-sulfoximine, inhibit glutathione metabolism. These drugs decrease the aggressiveness of pancreatic cancer, inhibit tumor stem cell survival, and reduce chemotherapy resistance. Nevertheless, buthionine-sulfoximine also decreases the content of glutathione in normal cells, disrupts the balance between reactive oxygen species and glutathione, and eventually induces cell apoptosis. Pancreatic cancer is usually diagnosed at an advanced stage and has a poor prognosis. Consequently, the use of biomarkers to screen high-risk patients can be an effective method.

• Cancer stem cells
• Chemoresistance
• Pancreatic cancer
• Pancreatic ductal adenocarcinoma
• Redox

• cancer stem cells
• epithelial–mesenchymal transition
• metastasis
• microRNAs
• non-coding RNAs
• pancreatic ductal adenocarcinoma

• Biomarker
• Cancer stem cell
• GLRX3
• Glutaredoxin 3
• Pancreatic cancer
• Stemness

• Animals
• CA-19-9 Antigen/metabolism
• Carcinoma, Pancreatic Ductal/diagnosis
• Carcinoma, Pancreatic Ductal/metabolism
• Carcinoma, Pancreatic Ductal/mortality
• Carcinoma, Pancreatic Ductal/pathology
• Carrier Proteins/genetics
• Carrier Proteins/metabolism
• Cell Line, Tumor
• Cell Movement
• Cell Proliferation
• Disease-Free Survival
• Gene Silencing
• Humans
• Male
• Mice
• Mice, Inbred NOD
• Mice, SCID
• Neoplasm Proteins/metabolism
• Neoplastic Stem Cells/metabolism
• Pancreatic Neoplasms/diagnosis
• Pancreatic Neoplasms/metabolism
• Pancreatic Neoplasms/mortality
• Pancreatic Neoplasms/pathology
• Phosphatidylinositol 3-Kinases/metabolism
• Proto-Oncogene Proteins c-akt/metabolism
• Proto-Oncogene Proteins c-met/metabolism
• RNA, Small Interfering
• Secretome
• Sensitivity and Specificity
• Spheroids, Cellular/metabolism
• Spheroids, Cellular/pathology

• PDAC
• cancer stem cells
• lineage tracing
• pancreatic cancer
• stem cell dynamics
• stroma

• Anilides/pharmacology
• Animals
• Antineoplastic Agents/pharmacology
• Cancer-Associated Fibroblasts/metabolism
• Cancer-Associated Fibroblasts/pathology
• Carcinoma, Pancreatic Ductal/drug therapy
• Carcinoma, Pancreatic Ductal/genetics
• Carcinoma, Pancreatic Ductal/metabolism
• Carcinoma, Pancreatic Ductal/pathology
• Cell Communication
• Cell Line, Tumor
• Cell Lineage
• Cell Proliferation
• Female
• Hedgehog Proteins/antagonists & inhibitors
• Hedgehog Proteins/metabolism
• Humans
• Male
• Mice, Inbred NOD
• Mice, Nude
• Mice, SCID
• Neoplastic Stem Cells/drug effects
• Neoplastic Stem Cells/metabolism
• Neoplastic Stem Cells/pathology
• Pancreatic Neoplasms/drug therapy
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/metabolism
• Pancreatic Neoplasms/pathology
• Pyridines/pharmacology
• Signal Transduction
• Stromal Cells/metabolism
• Stromal Cells/pathology
• Time Factors
• Tumor Burden
• Tumor Microenvironment
• Xenograft Model Antitumor Assays
• Mice

• 14-1164 Worldwide Cancer Research

• CD24
• CD44
• Cancer stem cells
• HeLa cells
• chemoresistance
• radio-resistance
• stemness markers
• tumorigenicity

Pancreatic cancer is one of the most lethal malignancies. Hence, improved therapies are urgently needed. Recent research indicates that pancreatic cancers depend on cancer stem cells (CSCs) for tumor expansion, metastasis, and therapy resistance. However, the exact functionality of pancreatic CSCs is still unclear. CSCs have much in common with normal pancreatic stem cells that have been better, albeit still incompletely, characterized. In this literature review, we address how pancreatic stem cells influence growth, homeostasis, regeneration, and cancer. Furthermore, we outline which intrinsic and extrinsic factors regulate stem cell functionality during these different processes to explore potential novel targets for treating pancreatic cancer.

Cell generation and renewal are essential processes to develop, maintain, and regenerate tissues. New cells can be generated from immature cell types, such as stem-like cells, or originate from more differentiated pre-existing cells that self-renew or transdifferentiate. The adult pancreas is a dormant organ with limited regeneration capacity, which complicates studying these processes. As a result, there is still discussion about the existence of stem cells in the adult pancreas. Interestingly, in contrast to the classical stem cell concept, stem cell properties seem to be plastic, and, in circumstances of injury, differentiated cells can revert back to a more immature cellular state. Importantly, deregulation of the balance between cellular proliferation and differentiation can lead to disease initiation, in particular to cancer formation. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5-year survival rate of only ~9%. Unfortunately, metastasis formation often occurs prior to diagnosis, and most tumors are resistant to current treatment strategies. It has been proposed that a specific subpopulation of cells, i.e., cancer stem cells (CSCs), are responsible for tumor expansion, metastasis formation, and therapy resistance. Understanding the underlying mechanisms of pancreatic stem cells during homeostasis and injury might lead to new insights to understand the role of CSCs in PDAC. Therefore, in this review, we present an overview of the current literature regarding the stem cell dynamics in the pancreas during health and disease. Furthermore, we highlight the influence of the tumor microenvironment on the growth behavior of PDAC.

• microenvironment
• pancreas
• pancreatic cancer
• pancreatic stem cells
• pancreatitis
• stem cell dynamics

• Vidi 016.156.308 ZonMw
• 14-1164 Worldwide Cancer Research

Mining databases and data obtained from assays on human specimens had shown that Fzd7 is closely associated with Wnt7b, that Fzd7/Wnt7b expression is upregulated in pancreatic cancer tissues compared with normal tissues, and its expression is negatively correlated with survival. Fzd7/Wnt7b knockdown in Capan‐2 and Panc‐1 cells reduced the proliferative capacity of pancreatic cancer stem cells (PCSCs), reduced drug resistance, decreased the percentage of CD24⁺CD44⁺ subset of cells and the levels of ABCG2, inhibited cell‐sphere formation, and reduced gemcitabine (GEM) resistance. In contrast, Fzd7/Wnt7b overexpression increased the percentage of the CD24⁺CD44⁺ subset of cells, and increased the levels of ABCG2 detected in cell spheroids. The gem‐resistant cells exhibited higher levels of Fzd7/Wnt7b expression, an increased percentage of CD24⁺CD44⁺ cells, and higher levels of ABCG2 compared with the parental cells. Taken together, Fzd7/Wnt7b knockdown can reduce PDAC cell stemness and chemoresistance by reducing the percentage of CSCs. Mechanistically, Fzd7 binds with Wnt7b and modulates the levels of β‐catenin, and they may exert their role via modulation of the canonical Wnt pathway.

• Fzd7/Wnt7b
• Wnt signaling pathway
• chemoresistance
• pancreatic cancer
• stemness

• AC133 Antigen/metabolism
• ATP Binding Cassette Transporter, Subfamily B/metabolism
• Aldehyde Dehydrogenase 1 Family/metabolism
• Animals
• Carcinoma, Pancreatic Ductal/drug therapy
• Carcinoma, Pancreatic Ductal/metabolism
• Cell Line, Tumor
• Coculture Techniques
• Deoxycytidine/analogs & derivatives
• Deoxycytidine/pharmacology
• Deoxycytidine/therapeutic use
• Drug Resistance, Neoplasm
• Epithelial-Mesenchymal Transition
• Gene Expression Regulation, Neoplastic
• Glycolysis
• Humans
• L-Lactate Dehydrogenase/metabolism
• Liver Neoplasms/metabolism
• Liver Neoplasms/secondary
• Lung Neoplasms/metabolism
• Lung Neoplasms/secondary
• Metabolomics/methods
• Mice
• Neoplasm Metastasis
• Neoplasm Transplantation
• Neoplastic Stem Cells/metabolism
• Pancreatic Neoplasms/drug therapy
• Pancreatic Neoplasms/metabolism
• Retinal Dehydrogenase/metabolism
• Gemcitabine

• U01 CA200466 NCI NIH HHS
• R01 CA247471 NCI NIH HHS
• R01 CA228524 NCI NIH HHS
• R01 CA195586 NCI NIH HHS
• R01 CA201444 NCI NIH HHS
• U01 CA210240 NCI NIH HHS
• R01 CA183459 NCI NIH HHS
• R01 CA210637 NCI NIH HHS
• P01 CA217798 NCI NIH HHS

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related mortality. Cancer stem cells (CSCs) have been shown to be the drivers of pancreatic tumor growth, metastasis, and chemoresistance, but our understanding of these cells is still limited by our inability to efficiently identify and isolate them. While a number of markers capable of identifying pancreatic CSCs (PaCSCs) have been discovered since 2007, there is no doubt that more markers are still needed. The anthrax toxin receptor 1 (ANTXR1) was identified as a functional biomarker of triple-negative breast CSCs, and PDAC patients stratified based on ANTXR1 expression levels showed increased mortality and enrichment of pathways known to be necessary for CSC biology, including TGF-β, NOTCH, Wnt/β-catenin, and IL-6/JAK/STAT3 signaling and epithelial to mesenchymal transition, suggesting that ANTXR1 may represent a putative PaCSC marker. In this study, we show that ANTXR1⁺ cells are not only detectable across a panel of 7 PDAC patient-derived xenograft primary cultures but ANTXR1 expression significantly increased in CSC-enriched 3D sphere cultures. Importantly, ANTXR1⁺ cells also coexpressed other known PaCSC markers such as CD44, CD133, and autofluorescence, and ANTXR1⁺ cells displayed enhanced CSC functional and molecular properties, including increased self-renewal and expression of pluripotency-associated genes, compared to ANTXR1⁻ cells. Thus, this study validates ANTXR1 as a new PaCSC marker and we propose its use in identifying CSCs in this tumor type and its exploitation in the development of CSC-targeted therapies for PDAC.

• Cancer stem cell marker
• Cancer stem cell/CSC
• Pancreatic cancer
• Side population
• Sphere

• Biomarkers, Tumor/analysis
• Carcinoma, Pancreatic Ductal/diagnosis
• Carcinoma, Pancreatic Ductal/pathology
• Humans
• Neoplastic Stem Cells/pathology
• Pancreatic Neoplasms/diagnosis
• Pancreatic Neoplasms/pathology
• Pancreatic Neoplasms

• JP16K10613 JSPS KAKENHI

• ERK
• Nicotine Receptor Signaling
• PHF5A
• Pancreatic Carcinogenesis

Epithelial-to-mesenchymal transition is implicated in metastasis. Viotti et al. show that the histone methyltransferase SUV420H2 favors the mesenchymal identity in pancreatic tumor cells by silencing key drivers of the epithelial state. High levels of SUV420H2 also correlate with a loss of epithelial characteristics in invasive cancer.

Epithelial-to-mesenchymal transition is implicated in metastasis, where carcinoma cells lose sessile epithelial traits and acquire mesenchymal migratory potential. The mesenchymal state is also associated with cancer stem cells and resistance to chemotherapy. It might therefore be therapeutically beneficial to promote epithelial identity in cancer. Because large-scale cell identity shifts are often orchestrated on an epigenetic level, we screened for candidate epigenetic factors and identified the histone methyltransferase SUV420H2 (KMT5C) as favoring the mesenchymal identity in pancreatic cancer cell lines. Through its repressive mark H4K20me3, SUV420H2 silences several key drivers of the epithelial state. Its knockdown elicited mesenchymal-to-epithelial transition on a molecular and functional level, and cells displayed decreased stemness and increased drug sensitivity. An analysis of human pancreatic cancer biopsies was concordant with these findings, because high levels of SUV420H2 correlated with a loss of epithelial characteristics in progressively invasive cancer. Together, these data indicate that SUV420H2 is an upstream epigenetic regulator of epithelial/mesenchymal state control.

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

• DC-CIK
• adoptive transfer
• cancer stem cell
• slow-cycling tumor cells
• tumor dormancy

• PANC-1
• pancreatic adenocarcinoma
• radiosensitivity
• reactive oxygen species
• stem cell

• cancer stem cells
• curcumin
• metformin
• pancreatic cancer
• target

Cancer stem cells (CSCs) are important in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Agents with the ability to suppress CSCs are likely to provide novel opportunities for combating tumor proliferation and metastasis. The present study aimed to evaluate the effects of bufalin on pancreatic CSCs in vivo and in vitro. Using a serum-free suspension culture, tumor spheres were enriched in a gemcitabine-resistant human pancreatic cancer cell line, which had a higher percentage of CSCs, and western blotting, flow cytometry, and colony and tumor formation assays were used to demonstrate that these sphere cells exhibited CSC characteristics. Using these cancer stem-like cells as a model, the present study examined the effect of bufalin on pancreatic CSCs. It was demonstrated that bufalin inhibited the number of tumor spheres, and western blotting and immunohistochemical assays showed that the expression levels of CD24 and epithelial specific antigen (ESA) were downregulated by bufalin. Furthermore, in a subcutaneous xenograft model of implanted gemcitabine-resistant MiaPaCa2 cells, bufalin inhibited tumor growth and prolonged the duration of tumor formation. Additionally, the expression levels of CD24 and ESA were inhibited in the bufalin-treated mice. Notably, in another cancer model injected with tumor cells via the tail vein, fewer metastatic lesions were detected in the group in which tumor cells were pretreated with bufalin in vitro, compared with those without pretreatment. Of note, the Hedgehog (Hh) signaling pathway was found to be inhibited in the bufalin-treated cells. Taken together, these results suggested that bufalin suppressed pancreatic CSCs in gemcitabine-resistant MiaPaCa2 cells, and the Hh signaling pathway may be involved in this process.

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24⁻, CD44^+/++, CD326^−/+ and CD133/1⁻, while PANC-1 is CD24^−/+, CD44⁺, CD326^−/+ and CD133/1⁻. Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16^INK4A, but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

Recent studies suggest that latexin (Lxn) expression is involved in stem cell regulation and that it plays significant roles in tumor cell migration and invasion. The clinicopathological significance of Lxn expression and its possible correlation with CD133 expression in pancreatic ductal adenocarcinoma (PDAC) is currently unknown. In the present study, immunohistochemical analysis was performed to determine Lxn and CD133 expression in 43 PDAC patient samples and in 32 corresponding adjacent non-cancerous samples. The results were analyzed and compared with patient age, gender, tumor site and size, histological grade, clinical stage and overall mean survival time. Lxn expression was clearly decreased in the PDAC tissues compared with that in the adjacent non-cancerous tissues, while CD133 expression was increased. Low Lxn expression in the PDAC tissues was significantly correlated with tumor size (P=0.002), histological grade (P=0.000), metastasis (P=0.007) and clinical stage (P=0.018), but not with age (P=0.451), gender (P=0.395) or tumor site (P=0.697). Kaplan-Meier survival analysis revealed that low Lxn expression was significantly correlated with reduced overall survival time (P=0.000). Furthermore, Lxn expression was found to be inversely correlated with CD133 expression (r=−0.485, P=0.001). Furthermore, CD133-positive MIA PaCa-2 pancreatic tumor cells were sorted by magnetic-activated cell sorting (MACS), and those that overexpressed Lxn exhibited a significantly higher rate of apoptosis and lower proliferative activity. Our findings suggest that Lxn may function as a tumor suppressor that targets CD133-positive pancreatic cancer cells.

Breast cancer is a leading cause of death in women, and almost all complications are due to chemotherapy resistance. Drug-resistant cells with stem cell phenotypes are thought to cause failure in breast cancer chemotherapy. Dendritic cell (DC) therapy is a potential approach to eradicate these cells. This study evaluates the specificity of DCs for breast cancer stem cells (BCSCs) in vitro and in vivo. BCSCs were enriched by a verapamil-resistant screening method, and reconfirmed by ALDH expression analysis and mammosphere assay. Mesenchymal stem cells (MSCs) were isolated from allogeneic murine bone marrow. DCs were induced from bone marrow-derived monocytes with 20 ng/mL GC-MSF and 20 ng/mL IL-4. Immature DCs were primed with BCSC- or MSC-derived antigens to make two kinds of mature DCs: BCSC-DCs and MSC-DCs, respectively. In vitro ability of BCSC-DCs and MSC-DCs with cytotoxic T lymphocytes (CTLs) to inhibit BCSCs was tested using the xCELLigence technique. In vivo, BCSC-DCs and MSC-DCs were transfused into the peripheral blood of BCSC tumor-bearing mice. The results show that in vitro BCSC-DCs significantly inhibited BCSC proliferation at a DC:CTL ratio of 1:40, while MSC-DCs nonsignificantly decreased BCSC proliferation. In vivo, tumor sizes decreased from 18.8% to 23% in groups treated with BCSC-DCs; in contrast, tumors increased 14% in the control group (RPMI 1640) and 47% in groups treated with MSC-DCs. The results showed that DC therapy could target and be specific to BCSCs. DCs primed with MSCs could trigger tumor growth. These results also indicate that DCs may be a promising therapy for treating drug-resistant cancer cells as well as cancer stem cells.

• 4T1 cell line
• breast cancer stem cells
• breast tumor
• dendritic cells
• drug resistance
• verapamil

• Cancer stem cells
• Cancer stem cells signaling pathways
• Pancreatic cancer
• Targeted therapy
• miRNA

• Animals
• Antineoplastic Agents/therapeutic use
• Biomarkers, Tumor/genetics
• Biomarkers, Tumor/metabolism
• Drug Resistance, Neoplasm
• Humans
• Neoplastic Stem Cells/drug effects
• Neoplastic Stem Cells/metabolism
• Neoplastic Stem Cells/pathology
• Pancreatic Neoplasms/drug therapy
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/metabolism
• Pancreatic Neoplasms/pathology
• Signal Transduction

Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT.

Cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs.

Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs.

In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.

• pancreatic cancer
• cancer stem cells
• epithelial-to-mesenchymal transition
• developmental pathways
• tumor micro-environment
• chemoresistance

• Biomarkers, Tumor/analysis
• Carcinoma, Pancreatic Ductal/metabolism
• Carcinoma, Pancreatic Ductal/pathology
• Humans
• Immunohistochemistry
• Intermediate Filament Proteins/analysis
• Neoplasm Invasiveness
• Neoplasm Metastasis
• Neoplastic Stem Cells/metabolism
• Neoplastic Stem Cells/pathology
• Nerve Tissue Proteins/analysis
• Nestin
• Pancreatic Neoplasms/metabolism
• Pancreatic Neoplasms/pathology

Pancreatic carcinoma has a dismal prognosis as it often presents as locally advanced or metastatic. We have found that exposure to adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 resulted in growth inhibition and apoptosis induction in PANC-1, Capan-2, and MiaPaCa-2 pancreatic cancer cell lines. In addition, AHP3 and 3-Cl-AHPC inhibited growth and induced apoptosis in spheres derived from the CD44⁺/CD24⁺ (CD133⁺/EpCAM⁺) stem-like cell population isolated from the pancreatic cancer cell lines. 3-Cl-AHPC-induced apoptosis was preceded by decreasing expression of IGF-1R, cyclin D1, β-catenin, and activated Notch-1 in the pancreatic cancer cell lines. Decreased IGF-1R expression inhibited PANC-1 proliferation, enhanced 3-Cl-AHPC-mediated apoptosis, and significantly decreased sphere formation. 3-Cl-AHPC inhibited the Wnt/β-catenin pathway as indicated by decreased β-catenin nuclear localization and inhibited Wnt/β-catenin activation of transcription factor TCF/LEF. Knockdown of β-catenin using sh-RNA also induced apoptosis and inhibited growth in pancreatic cancer cells. Thus, 3-Cl-AHPC and AHP3 induce apoptosis in pancreatic cancer cells and cancer stem-like cells and may serve as an important potential therapeutic agent in the treatment of pancreatic cancer.

• pancreatic cancer
• biomarker
• tumor progression
• risk factor
• ca 19-9

• Animals
• Biomarkers/blood
• Biomarkers/metabolism
• Early Detection of Cancer
• Epigenesis, Genetic
• Humans
• MicroRNAs/blood
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Neoplasm Proteins/blood
• Neoplasm Proteins/genetics
• Neoplasm Proteins/metabolism
• Pancreatic Neoplasms/blood
• Pancreatic Neoplasms/diagnosis
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/metabolism

• P20 GM103505 NIGMS NIH HHS
• P20 RR020151 NCRR NIH HHS

• Bmi-1
• chemoresistance
• invasion
• pancreatic cancer
• tumorigenesis

Pancreatic adenocarcinoma has the worst prognosis of any major malignancy, with <5% of patients surviving five years. This can be contributed to the often late diagnosis, lack of sufficient treatment and metastatic spread. Heterogeneity within tumors is increasingly becoming a focus in cancer research, as novel therapies are required to target the most aggressive subpopulations of cells that are frequently termed cancer stem cells (CSCs). In the current study, we describe the identification of a slow-cycling cancer stem-like population of cells in vivo in BxPC-3 and Panc03.27 xenografts. A distinct slow-cycling label-retaining population of cells (DiI+/SCC) was found both at the edge of tumors, and in small circumscribed areas within the tumors. DiI+/SCC in these areas display an epithelial-to-mesenchymal transition (EMT) fingerprint, including an upregulation of the mesenchymal markers vimentin and N-cadherin and a loss of the epithelial marker E-cadherin. DiI+/SCC also displayed a critical re-localization of beta-catenin from the membrane to the nucleus. Additionally, the DiI+/SCC population was found to express the developmental signaling molecule sonic hedgehog. This study represents a novel step in defining the biological activities of a tumorigenic subpopulation within the heterogeneous tumor microenvironment in vivo. Understanding the interactions and functions of a CSC population within the context of the tumor microenvironment is critical to design targeted therapeutics.

• Animals
• Antibody Formation
• B-Lymphocytes/immunology
• Cancer Vaccines/genetics
• Cancer Vaccines/immunology
• Cancer Vaccines/pharmacology
• Epitopes/biosynthesis
• Epitopes/genetics
• Epitopes/immunology
• Humans
• Immunotherapy, Active/methods
• Mice
• Mice, Knockout
• Mucin-1/biosynthesis
• Mucin-1/genetics
• Mucin-1/immunology
• Neoplastic Stem Cells/immunology
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/immunology
• Pancreatic Neoplasms/metabolism
• Pancreatic Neoplasms/therapy
• Protein Engineering/methods
• Swine
• T-Lymphocytes/immunology
• Transfection
• Trisaccharides/biosynthesis
• Trisaccharides/genetics
• Trisaccharides/immunology

Evidence suggests that multiple tumors, including pancreatic adenocarcinoma, display heterogeneity in parameters that are critical for tumor formation, progression and metastasis. Understanding heterogeneity in solid tumors is increasingly providing a plethora of new diagnostic and therapeutic approaches. In this study, a particular focus was put on identifying a subpopulation of stem cell-like, slow cycling tumor cells in a pancreas adenocarcinoma cell lines. Using a label retention technique a subpopulation of slow cycling cells (DiI+/SCC) was identified and further evaluated in the BxPC-3 and Panc03.27 cell lines. These slowly cycling cells managed to retain the lipophilic labeling dye DiI, while the bulk of the cells (>94%) did not. The DiI+/SCC population, showed only a partial overlap with the CSC markers CD24⁺/CD44⁺, CD133⁺ and ALDH but they survived chemotherapeutic treatment, and were able to recreate the initial heterogeneous tumor cell population. DiI+/SCCs exhibited an increased invasive potential as compared with their non-label retaining, faster cycling cells (DiI−/FCC). They also had increased tumorigenic potential and morphological changes resembling cells that have undergone an epithelial to mesenchymal transition (EMT). Analysis of DiI+/SCC cells by real time PCR revealed a selective up-regulation of tell tale components of the Hedgehog/TGFβ pathways, as well as a down-regulation of EGFR, combined with a shift in crucial components implied in EMT. The presented findings offer an expanded mechanistic understanding that associates tumor initiating potential with cycling speed and EMT in pancreatic cancer cell lines.