Pancreatic Tumor-initiating Cells Research Articles

Targeting autophagy in pancreatic cancer: The cancer stem cell perspective.

Pancreatic cancer is currently the seventh leading cause of cancer-related deaths worldwide, with the estimated death toll approaching half a million annually. Pancreatic ductal adenocarcinoma (PDAC) is the most common (>90% of cases) and most aggressive form of pancreatic cancer, with extremely poor prognosis and very low survival rates. PDAC is initiated by genetic alterations, usually in the oncogene KRAS and tumor suppressors CDKN2A, TP53 and SMAD4, which in turn affect a number of downstream signaling pathways that regulate important cellular processes. One of the processes critically altered is autophagy, the mechanism by which cells clear away and recycle impaired or dysfunctional organelles, protein aggregates and other unwanted components, in order to achieve homeostasis. Autophagy plays conflicting roles in PDAC and has been shown to act both as a positive effector, promoting the survival of pancreatic tumor-initiating cells, and as a negative effector, increasing cytotoxicity in uncontrollably expanding cells. Recent findings have highlighted the importance of cancer stem cells in PDAC initiation, progression and metastasis. Pancreatic cancer stem cells (PaCSCs) comprise a small subpopulation of the pancreatic tumor, characterized by cellular plasticity and the ability to self-renew, and autophagy has been recognised as a key process in PaCSC maintenance and function, simultaneously suggesting new strategies to achieve their selective elimination. In this review we evaluate recent literature that links autophagy with PaCSCs and PDAC, focusing our discussion on the therapeutic implications of pharmacologically targeting autophagy in PaCSCs, as a means to treat PDAC.

Calcium Channel α2δ1 is Essential for Pancreatic Tumor-Initiating Cells through Sequential Phosphorylation of PKM2

Tumor-initiating cells (TICs) drive pancreatic cancer tumorigenesis, therapeutic resistance, and metastasis. However, TICs are highly plastic and heterogenous, which impede the robust identification and targeted therapy of such a population. The aim of this study is to identify the surface marker and therapeutic target for pancreatic TICs. We isolated voltage-gated calcium channel α2δ1 subunit (isoform 5)-positive subpopulation from pancreatic cancer cell lines and freshly resected primary tissues by fluorescence-activated cell sorting and evaluated their TIC properties by spheroid formation and tumorigenic assays. Coimmunoprecipitation was used to identify the direct substrate of CaMKⅡδ. We demonstrate that the voltage-gated calcium channel α2δ1 subunit (isoform 5) marks a subpopulation of pancreatic TICs with the highest TIC frequency among the known pancreatic TIC markers tested. Furthermore, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca2+ influx, which activates CaMKⅡδ to directly phosphorylate PKM2 at T454 that results in subsequent phosphorylation at Y105 to translocate into nucleus, enhancing the stem-like properties. Interestingly, blocking α2δ1 with its specific antibody has remarkably therapeutic effects on pancreatic cancer xenografts by reducing TICs. α2δ1 promotes pancreatic TIC properties through sequential phosphorylation of PKM2 mediated by CaMKⅡδ, and targeting α2δ1 provides a therapeutic strategy against TICs for pancreatic cancer.

Proteins and Molecular Pathways Relevant for the Malignant Properties of Tumor-Initiating Pancreatic Cancer Cells.

Cancer stem cells (CSCs), a small subset of the tumor bulk with highly malignant properties, are deemed responsible for tumor initiation, growth, metastasis, and relapse. In order to reveal molecular markers and determinants of their tumor-initiating properties, we enriched rare stem-like pancreatic tumor-initiating cells (TICs) by harnessing their clonogenic growth capacity in three-dimensional multicellular spheroid cultures. We compared pancreatic TICs isolated from three-dimensional tumor spheroid cultures with nontumor-initiating cells (non-TICs) enriched in planar cultures. Employing differential proteomics (PTX), we identified more than 400 proteins with significantly different expression in pancreatic TICs and the non-TIC population. By combining the unbiased PTX with mRNA expression analysis and literature-based predictions of pro-malignant functions, we nominated the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14) as well as galactin-3-binding protein LGALS3BP (MAC-2-BP) as putative determinants of pancreatic TICs. In silico pathway analysis followed by candidate-based RNA interference mediated loss-of-function analysis revealed a critical role of S100A8, S100A9, and LGALS3BP as molecular determinants of TIC proliferation, migration, and in vivo tumor growth. Our study highlights the power of combining unbiased proteomics with focused gene expression and functional analyses for the identification of novel key regulators of TICs, an approach that warrants further application to identify proteins and pathways amenable to drug targeting.

Gemcitabine Enhances Kras-MEK-Induced Matrix Metalloproteinase-10 Expression Via Histone Acetylation in Gemcitabine-Resistant Pancreatic Tumor-initiating Cells.

Advanced pancreatic ductal adenocarcinoma is resistant to systemic chemotherapy, resulting in a poor prognosis. We previously isolated a human pancreatic tumor-initiating cell line, KMC07, from a patient with acquired resistance to gemcitabine chemotherapy. To improve the anticancer effects of gemcitabine, we investigated the molecular mechanism of KMC07 cells' resistance to gemcitabine. KMC07 cells were treated with gemcitabine, then gene expression and functional analyses performed using microarray, the quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, chromatin immunoprecipitation, and cell transplantation into nude mice. KMC07 cells, but not BxPC-3, PANC-1, MIA PaCa-2, or AsPC-1 cells, expressed matrix metalloproteinase-10 mRNA, the expression level of which was enhanced by gemcitabine. KMC07 cells were shown to carry a constitutively active Kras mutation, and a MEK inhibitor suppressed matrix metalloproteinase-10 mRNA expression. Gemcitabine enhanced histone H3 acetylation at the matrix metalloproteinase-10 promoter, and a histone acetyltransferase inhibitor reduced gemcitabine-enhanced matrix metalloproteinase-10 mRNA expression. Gemcitabine induced expression of matrix metalloproteinase-10 protein in KMC07-derived pancreatic tumors in vivo. We demonstrated constitutive activation of the Kras-MEK matrix metalloproteinase-10 signaling pathway in KMC07 cells that was enhanced by gemcitabine through histone acetylation. Our results may provide novel insights into gemcitabine-based treatment for gemcitabine-resistant pancreatic ductal adenocarcinoma.

Gemcitabine enhances Kras-MEK-induced matrix metalloproteinase-10 expression in gemcitabine-resistant pancreatic tumor-initiating cells

Gemcitabine enhances Kras-MEK-induced matrix metalloproteinase-10 expression in gemcitabine-resistant pancreatic tumor-initiating cells

Abstract B46: Low mitochondrial activity of CD133+ population enables evasion of apoptosis leading to increased chemo-resistance in tumor initiating cells

Abstract Background: Pancreatic ductal adenocarcinoma is currently the fourth most frequent cause of cancer-related death in the United States with poor survival. Tumor recurrence in this devastating disease adds to its grim survival. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC). Chemo-resistance is considered a hallmark of TICs. Standard chemotherapy typically targets rapidly growing cells as a result of which the quiescent TICs often evade cell death by these agents. However, the mechanism involved in conferring chemo-resistance to these cells is still unclear. Our study shows that CD133+ pancreatic cancer TICs have increased glycolysis and low mitochondrial activity compared to CD133- non TIC. Our study further shows that CD133+ cells have increased resistance to chemotherapeutic agents like gemcitabine, 5FU and paclitaxel resulting in decreased apoptotic cells and decreased accumulation of reactive oxygen species (ROS). This altered metabolic profile offers a survival advantage to the quiescent TICs. Methods: CD133+ pancreatic TICs were isolated from KPC tumor tissues , KPC cell lines isolated from primary tumors and patient tumor derived tumor xenografts using MACS beads (Miltnyi Biotech). Expression of genes controlling glucose metabolism and mitochondrial activity were evaluated using PCR array (SA biosciences). Activity assays for glycolytic enzymes (Sigma), Pentose phosphate pathway enzyme (Sigma) and mitochondrial complex I and Complex IV (AbCam) was done using kits from indicated vendors according to manufacturers instruction. Apoptosis assays were done using Caspase 3/7 assay kit (Promega). Cell viability was assessed using Trypan Blue exclusion method. Results: CD133+ pancreatic TIC (from KPC tumor, KPC cell lines and human patient derived xenografts) had increased glucose uptake (4 fold higher) compared to CD133- cells. These cells also showed increased expression of glycolytic enzymes compared to CD133- cells (4-20 fold higher). Consistent with this, the CD133+ cells had increased HK2 (3 fold) and LDH activity (8 fold). CD133+ pancreatic TIC showed 10-fold lower activity of mitochondrial enzyme complex 1 and mitochondrial enzyme complex IV compared to CD133- genes. Consistent with this, the expression of genes in these enzyme complexes was also significantly decreased. This indicated that CD133+ TICs had increased glycolysis but low mitochondrial activity Accumulation of ROS is one of the major inducers of apoptosis in cells. Treatment with standard chemotherapeutic agents results in generation of free radicals of oxygen, which trigger mitochondrial apoptotic pathways. Low mitochondrial complex activity prevents accumulation of ROS. Treatment of CD133+ population with standard chemotherapeutic agents like 5FU, Gemcitatbine and paclitaxel showed increased cell viability compared to CD133- cells. These treatments also resulted in decreased apoptosis in CD133+ population compared to CD133- population. Consistent with this, CD133+ cells also showed increased expression of anti-apoptotic proteins like Bcl2 and Survivin compared to CD133- cells. Further, CD133+ population showed decreased accumulation of ROS compared to CD133- cells. These results indicated that low mitochondrial activity in CD133+ TICs offered survival advantages to these cells, allowing them to be resistant to standard chemotherapeutic compounds. Conclusion: Understanding the mechanism behind persistence of TIC population in a tumor following chemotherapy is one of the prime thrust area of cancer research. Our study indicates that altered metabolic profile in CD133+ pancreatic TIC protect the cells from apoptosis induced by standard chemotherapeutic agents by reducing accumulation of ROS and decreasing apoptosis, thereby confering chemoresistance to these cells. Citation Format: Sulagna Banerjee, Alice Nomura, Olivia McGinn, Kelsey Jensen, Veena Sangwan, Vikas Dudeja, Ashok Saluja. Low mitochondrial activity of CD133+ population enables evasion of apoptosis leading to increased chemo-resistance in tumor initiating cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B46.

Abstract A102: Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts

Abstract Rationale: Using orthotopically-grown primary pancreatic cancer xenografts we recently identified an association between hypoxia and spontaneous metastasis formation, and we hypothesized that metastatic potential is conferred by the presence of tumor-initiating cells (TIC). We therefore investigated the relationship between hypoxia, tumor growth and TIC, and examined the effect of the hypoxia-activated prodrug TH-302 and ionizing radiation (IR) on pancreatic TIC and tumor growth. Methods: Eleven patient-derived pancreatic cancer xenograft models were used to examine tumor-initiation potential in relation to the tumor phenotype. To examine the effect of TH-302 and IR on TIC, tumor-bearing mice were treated with 50 mg/kg or 150 mg/kg TH-302, 10Gy of IR, or with the combination of TH-302 and IR. To examine the effect of TH-302 and IR on tumor growth, mice were treated with either TH-302 or IR alone, or with the combination treatment according to a clinically relevant schedule. Animals were treated with 50mg/kg TH-302 injected ip on days 1, 5 and 9 and/or 2Gy irradiation per day on days 2, 3 and 4. Tumor hypoxia was measured by EF5 administration and immunostaining. Results: TIC frequency varied across the patient-derived xenograft models and rapid tumor growth was strongly correlated with high TIC frequency but not hypoxia. Treatment with TH-302 in combination with IR led to a significant reduction in TIC frequency compared with either treatment alone in all models tested. Treatment-induced reduction in TIC frequency was independent of the magnitude of tumor hypoxia. Treatment-induced reduction of tumor growth was only observed in fast-growing hypoxic models. Additionally, TH-302 treatment also induced DNA damage in tumor tissue adjacent to the hypoxic zone which is consistent with the previously demonstrated bystander effect of TH-302. Conclusions: These results suggest that TIC can reside in both the oxic and the hypoxic microenvironment and that the combination of TH-302 and IR may present a novel therapeutic strategy to reduce the frequency of TIC and tumor growth rate of pancreatic tumors. Citation Format: Ines Lohse, Joanna Rasowski, Pinjian Cao, Melania Pintilie, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, Richard P. Hill, David W. Hedley. Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A102.

CD133+ Tumor Initiating Cells in a Syngenic Murine Model of Pancreatic Cancer Respond to Minnelide

Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer. We isolated CD133(+) cells from a spontaneous pancreatic ductal adenocarcinoma mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied tumor initiation properties by implanting low numbers of CD133(+) cells in immune competent mice. Effect of Minnelide, a drug currently under phase I clinical trial, was studied on the tumors derived from the CD133(+) cells. Our study showed for the first time that CD133(+) population demonstrated all the molecular markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed increased expression of prosurvival and proinvasive proteins compared to the CD133(-) non-TIC population. Our study further showed that Minnelide was very efficient in downregulating both CD133(-) and CD133(+) population in the tumors, resulting in a 60% decrease in tumor volume compared with the untreated ones. As Minnelide is currently under phase I clinical trial, its evaluation in reducing tumor burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy.

Identification of a Novel Subpopulation of Tumor-Initiating Cells from Gemcitabine-Resistant Pancreatic Ductal Adenocarcinoma Patients

Pancreatic ductal adenocarcinoma is highly resistant to systemic chemotherapy. Although there are many reports using pancreatic cancer cells derived from patients who did not receive chemotherapy, characteristics of pancreatic cancer cells from chemotherapy-resistant patients remain unclear. In this study, we set out to establish a cancer cell line in disseminated cancer cells derived from gemcitabine-resistant pancreatic ductal adenocarcinoma patients. By use of in vitro co-culture system with stromal cells, we established a novel pancreatic tumor-initiating cell line. The cell line required its direct interaction with stromal cells for its in vitro clonogenic growth and passaging. Their direct interaction induced basal lamina-like extracellular matrix formation that maintained colony formation. The cell line expressed CD133 protein, which expression level changed autonomously and by culture conditions. These results demonstrated that there were novel pancreatic tumor-initiating cells that required direct interactions with stromal cells for their in vitro cultivation in gemcitabine-resistant pancreatic ductal adenocarcinoma. This cell line would help to develop novel therapies that enhance effects of gemcitabine or novel anti-cancer drugs.

The Differentiation of Pancreatic Tumor-Initiating Cells by Vitronectin Can Be Blocked by Cilengitide

Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as most of the patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells (TICs) constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis, and chemo/radioresistance. Within pancreatic cancer, TICs have been identified based on the expression of specific cell surface markers. We use a sphere formation assay to enrich putative TICs and use human serum as a driver of differentiation. We demonstrate by using specific blocking reagents that we can inhibit the differentiation process and maintain TIC-associated markers and genes. We can induce differentiation of pancreatospheres with the addition of human serum, and we identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate-specific peptide, which is Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors. Overall, our studies further the definition of pancreatic TICs and provide further insight into both the maintenance and differentiation of this lethal population.

Abstract B83: Targeting tumor-infiltrating macrophages decreases pancreatic tumor-initiating cells and improves chemotherapeutic responses by relieving immune suppression.

Abstract Tumor-infiltrating immune cells can promote chemoresistance and metastatic spread in aggressive tumors. Consequently, the type and quality of immune responses present in the neoplastic stroma are highly predictive of patient outcome in several cancer types. In addition to host immune responses, intrinsic tumor cell activities that mimic stem cell properties have been linked to chemoresistance, metastatic dissemination, and the induction of immune suppression. Far from being a static cell population, the presence of cancer stem cells appears to be controlled by highly dynamic processes that are dependent on cues from the tumor stroma. However, the impact that immune responses have on the differentiation or expansion of tumor stem cells is not well understood. In this study, we demonstrate that targeting tumor-infiltrating macrophages and inflammatory monocytes by either inhibition of colony stimulating factor-1 receptor (CSF1R) or chemokine (C-C motif) receptor 2 (CCR2) decreases the number of tumor-initiating cells in pancreatic tumors. Targeting CCR2 or CSF1R improves chemotherapeutic efficacy, inhibits metastasis, and increases antitumor T-cell responses. We also found that tumor-educated macrophages could directly enhance the tumor-initiating capacity of pancreatic tumor cells through the activation of signal transducer and activator of transcription 3 (STAT3). In turn, these STAT3-activated tumor-initiating cells facilitate macrophage-mediated suppression of CD8+ T lymphocytes. These data suggest that targeting tumor-infiltrating macrophages is an effective strategy for overcoming therapeutic resistance due to the presence of tumor-initiating cells. Citation Format: Jonathan B. Mitchem, Donal J. Brennan, Dominic E. Sanford, Brett L. Knolhoff, Yu Zhu, Belt Brian, Andrea Wang-Gillam, Peter Goedegebuure, David C. Linehan, David G. DeNardo. Targeting tumor-infiltrating macrophages decreases pancreatic tumor-initiating cells and improves chemotherapeutic responses by relieving immune suppression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B83.

Abstract 5335: Identification and molecular characterization of pancreatic tumor-initiating cells

Abstract Pancreatic cancer has the worst prognosis of any major malignancy with complex etiology involving both environmental and genetic factors. Despite increasing knowledge in tumor biology, the treatment efficacy in pancreatic cancers has not improved significantly over the past decade. Thus, the identification of novel molecular pathways involved in pancreatic oncogenesis and the development of new and potent therapeutic options are highly desirable. The realization that tumors themselves function as complex organs birthed the concept that cancer cells with tumor-initiating properties of stem cells may be the key drivers of the complex machinery behind oncogenesis. Tumor-initiating cells (TICs) have been studied extensively in the hematopoietic system, however it is not known which subpopulation has the highest tumor-initiating potential in pancreatic cancer. We have identified that the CD133+/CD44+/ALDH1+ cell population derived from 31 pancreatic adenocarcinomas and 6 pancreatic cancer cell lines, has the highest tumorigenic potential, assessed by in vivo dilution tumor propagation assays in highly immunodeficient (NOD-SCID IL2Rγ-/-) mice. In addition, this population has the highest sphere-forming capacity and is highly resistant to gemcitabine treatment in comparison to the other subpopulations. RNA-sequencing analysis in CD133−/CD44−/ALDH1− and CD133+/CD44+/ALDH1+ subpopulations derived from BxPC3 pancreatic cells revealed the transcriptomic alterations between these cell types. Furthermore, gene network analysis revealed enrichment of inflammatory and lipid metabolism networks in pancreatic TICs, suggesting the potential role of inflammation and lipid metabolism in pancreatic TIC function. In addition, a gene network-based FDA-approved chemical library screen revealed that metformin perturbs the activated networks in pancreatic TICs leading to the suppression of their growth. Metformin suppressed pancreatic cancer growth, through suppression of pancreatic TICs, in different pancreatic cancer mouse models. Overall, we propose the identification of a novel pancreatic cancer subpopulation which harbors tumor-initiating properties and is regulated by inflammatory signals, contributing to our understanding of how inflammation is linked to pancreatic oncogenesis in the molecular level. Furthermore, by following a novel strategy, we have identified that metformin suppresses pancreatic tumor growth and relapse through inhibition of pancreatic TIC growth. 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 5335. doi:1538-7445.AM2012-5335

Holoclone Forming Cells from Pancreatic Cancer Cells Enrich Tumor Initiating Cells and Represent a Novel Model for Study of Cancer Stem Cells

BackgroundPancreatic cancer is one of the direct causes of cancer-related death. High level of chemoresistance is one of the major obstacles of clinical treatment. In recent years, cancer stem cells have been widely identified and indicated as the origin of chemoresistance in multi-types of solid tumors. Increasing evidences suggest that cancer stem cells reside in the cells capable of forming holoclones continuously. However, in pancreatic cancer, holoclone-forming cells have not been characterized yet. Therefore, the goal of our present study was to indentify the holoclone-forming pancreatic cancer stem cells and develop an in vitro continuous colony formation system, which will greatly facilitate the study of pancreatic cancer stem cells.Methodology/Principal FindingsPancreatic cancer cell line BxPC3 was submitted to monoclonal cultivation to generate colonies. Based on the morphologies, colonies were classified and analyzed for their capacities of secondary colony formation, long-term survival in vitro, tumor formation in vivo, and drug resistance. Flowcytometry and quantitative RT-PCR were performed to detect the expression level of cancer stem cells associated cell surface markers, regulatory genes and microRNAs in distinct types of colonies. Three types of colonies with distinct morphologies were identified and termed as holo-, mero-, and paraclones, in which only holoclones generated descendant colonies of all three types in further passages. Compared to mero- and paraclones, holoclones possessed higher capacities of long-term survival, tumor initiation, and chemoresistance. The preferential expression of cancer stem cells related marker (CXCR4), regulatory genes (BMI1, GLI1, and GLI2) and microRNAs (miR-214, miR-21, miR-221, miR-222 and miR-155) in holoclones were also highlighted.Conclusions/SignificanceOur results indicate that the pancreatic tumor-initiating cells with high level of chemoresistance were enriched in holoclones derived from BxPC3 cell line. Generation of holoclones can serve as a novel model for studying cancer stem cells, and attribute to developing new anti-cancer drugs.

Abstract 2449: Pancreatic cancer tumor initiating cells are marked by the presence of a primary cilium

Abstract Primary cilia are microtubule rich projections eminating from the apical surface of epithelial cells. Primary cilia are responsible for the transduction of growth factor and morphogenetic signaling pathways, including the hedgehog, wnt and platelet-derived growth factor pathways. Initial observations in the field of pancreatic cancer have suggested the absence of primary cilia in human pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma. While absent in the majority of pancreatic cancer cells, primary cilia have been identified in differentiated acinar cells undergoing an acinar to ductal metaplasia. This metaplasia is well characterized in pancreatic injury and regeneration models and accelerates tumor formation in the presence of acinar specific KrasG12D mutations. Thus, we are addressing the hypothesis that primary cilia, while not expressed in bulk tumor populations, are maintained in pancreatic tumor-initiating cells. To address the hypothesis, we have used confocal microscopy to quantify the percentage of cells within low grade human PanIN lesions that express a primary cilium. Performing confocal microscopy on a human PanIN tissue array (n=23), we have determined that in 15% of PanIN lesions, 13-30% of the cells are expressing a primary cilium, as is evidenced by staining for acetylated alpha tubulin. We have also developed a sorting strategy using an acetylated alpha tubulin antibody to isolate ciliated cells by Fluorescence Activated Cell Sorting (FACS) analysis. Using this protocol, we have discovered that human pancreatic cancer cells bearing a primary cilium are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of ciliated vs non-ciliated cells using tumor sphere experiments. In this assay, ciliated cells show a significant increase (100 fold) in tumor sphere-forming capacity, suggesting that the presence of a primary cilium may mark a pancreatic cancer tumor initiating population. These data are informative regarding the earliest initiating events in pancreatic cancer. 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 2449. doi:10.1158/1538-7445.AM2011-2449