Subpopulation Of Pancreatic Cancer Cells Research Articles

The Significance of Targeting Poly (ADP-Ribose) Polymerase-1 in Pancreatic Cancer for Providing a New Therapeutic Paradigm.

Genome-wide studies focusing on elucidating the effects on cancer progression have enabled the consequent identification of a distinct subpopulation of pancreatic cancer cells with unstable genomic characteristics. Based on this background, deleterious changes by poly (adenosine diphosphate (ADP)-ribose) polymerase-1 (PARP)-1 have been concentrated in oncology. One of the critical functions of PARP-1 is the response to DNA damage, which plays a pivotal role in DNA repair in cancers. PARP-1 also has widespread functions that are essential for the survival and growth of cancer cells. It regulates oxidative stress in mitochondria through the regulation of superoxide and oxidation. PARP-1 is in charge of regulating mitosis, which is a crucial role in tumorigenesis and remodels histones and chromatin enzymes related to transcriptional regulation, causing alterations in epigenetic markers and chromatin structure. Given the significance of these processes, it can be understood that these processes in cancer cells are at the frontline of the pathogenetic changes required for cancer cell survival, and these contributions can result in malignant transformation. Therefore, this review addresses the current molecular biological features for understanding the multifactorial function of PARP-1 in pancreatic cancer related to the aforementioned roles, along with the summary of recent approaches with PARP-1 inhibition in clinical studies targeting pancreatic cancer. This understanding could help to embrace the importance of targeting PARP-1 in the treatment of pancreatic cancer, which may present the potential to find out a variety of research topics that can be both challenged clinically and non-clinically.

Abstract PO-043: Interception of pancreatic cancer progression

Abstract Pancreatic Cancer (PC) is projected to be one of the leading causes of cancer related death by 2030. Although other cancers have made significant strides towards improved treatment, PC still remains at a 9% fiver year survival rate due to end-stage diagnosis, aggressive metastasis, and a protective stromal wall. Therefore, to identify novel therapeutic targets, our lab focuses on studying cancer stem cells, a highly chemo-resistant sub-population of pancreatic cancer cells. RNA-seq analysis of these cancer stem cells has revealed a number of genes that are enriched compared to non-therapy resistant cells and normal pancreas. We hypothesize that genes turned on through pancreatic cancer progression contribute to therapy resistance. If PC could be targeted at earlier stages, it would be possible for therapeutics to bypass the wealth of issues that occur as it advances. By cross referencing our RNA-seq analysis to sequencing performed at various stages of pancreatic cancer development in mice, a subset of genes is not only up-regulated in therapy resistant cells, but increases in expression as normal pancreas progresses to an end stage tumor. Preliminary studies have shown that inhibition of these select genes reduces growth of mouse pancreatic cancer in vitro and in vivo. Ongoing studies are to (1) test if inhibition of these genes reduces early stage PC growth in vitro and in vivo, and (2) test how gene knockdown affects human pancreatic cancer cell growth in vitro and in vivo. Citation Format: Matthew L. McDermott, Lesley P. Ferguson, Kendall Chambers, Nirakar Rajbhandari, Tannishtha Reya. Interception of pancreatic cancer progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-043.

Abstract B30: Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a propensity for metastasis and drug resistance. To overcome this and improve the outcome of patients with PDAC, there is an urgent need to elucidate the molecular mechanisms of metastasis in which cancer stem cells (CSCs) may play a role. Prrx1, a homeobox transcription factor has been identified by us, as a key molecule involved in pancreatic development, regeneration and early carcinogenesis (Reichert et al. Genes and Development 2013). Furthermore, we have reported recently that Prrx1 isoform switching governs EMT-MET plasticity to regulate PDAC invasion and metastasis (Takano et al. Genes and Development 2016). Herein, we examined that the correlation between the two Prrx1 isoforms and putative CSC markers to investigate the role of Prrx1 on pancreatic CSC functions. Materials and Methods: Mouse pancreatic ductal cell lines were isolated and established from Pdx1Cre mice (normal pancreatic ductal cells), Pdx1Cre;KrasG12D/+ mice (PanIN cells), Pdx1Cre;KrasG12D/+;p53R175H/+ mice (primary and metastatic PDAC cells). Either Prrx1a and Prrx1b was overexpressed by lentiviral transduction in each cell line with controls. Prrx1 knockdown was done in primary PDAC cells and their matched liver metastatic cells by two isoform specific siRNAs. We immunophenotyped putative pancreatic CSCs using antibodies against CSC markers (CD133, CD44, CD24 and Epcam) by flow cytometry. These subpopulations of cells were evaluated in 3D cultures, invasion assays and single-pancreatosphere formation assays. Additionally, we evaluated the expression of pancreatic CSC markers in Prrx1a and Prrx1b overexpressing and knockdown cells by qRT-PCR and flow cytometry. Results: CD133High cells and CD44HighCD24High cells are expressed highly in metastatic PDAC cells compared to primary PDAC cells. CD133High cells form spheroid cysts and CD133Low cells form spindle-shaped cells in 3D culture. CD133High cells demonstrate more invasiveness and higher self-renewal capacity compared to CD133 Low cells. The percentage of CD133High cells, CD44HighCD24High cells and EpCAMHigh cells are increased significantly in Prrx1a overexpressing primary PDAC cells. However, these cells are decreased in Prrx1a knockdown metastatic cells. Contrary to this, these subpopulation of cells are significantly decreased in Prrx1b overexpressing primary PDAC cells, and increased in Prrx1b knockdown metastatic cells. These data suggest that Prrx1a might regulate pancreatic CSC functions in metastatic PDAC cells. Conclusion: Prrx1a may be associated with a subpopulation of pancreatic cancer cells that have features of cancer stem cells. This may influence metastatic properties of pancreatic cancer cells to the liver. Citation Format: Shigetsugu Takano, Maximilian Reichert, Hideyuki Yoshitomi, Basil Bakir, Koushik K. Das, Steffen Heeg, Shingo Kagawa, Hiroaki Shimizu, Masayuki Ohtsuka, Katsunori Furukawa, Masaru Miyazaki, Anil K. Rustgi.{Authors}. Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B30.

Abstract 2489: Therapeutic implication of identifying pancreatic cancer stem cells possessing fructose metabolic signature

Abstract The emergence of the cancer stem-cell theory in the last 20 years has shed light on many of the unresolved challenges. For example, the current cancer chemotherapy can mainly cause cancer remission but often fails to cure cancer due to the existence of cancer stem cells. Therefore, development of methods targeting cancer stem cells could facilitate the improvement of therapeutics for pancreatic ductal adenocarcinomas (PDACs). However, the key characteristics of cancer stem cells (CSCs) remain to be determined. Accumulating evidences revealed the similar features shared by pluripotent stem cells and cancer stem cells, we therefore hypothesize acquisition of stemness characteristics during the reprogramming process is superficially reminiscent of the dysplastic transformation proposed in cancers. In the current work, comparative genome-wide profiling analysis was initially conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and different subpopulation of pancreatic cancer cells. We initially revealed expression level of pluripotent transcriptional factors and surface markers such as Oct4, Sox2, Nanog, Lin28, CD133, and ABCG2 was significantly higher in ABCG2+CD44+ subpopulation of pancreatic cancer cells correlated with their drug resistance and high metastatic potentials suggesting ABCG2+CD44+ possess the features of CSCs. The further analysis was performed on metabolic pathway and revealed, very similar to ESCs/iPSCs, the metastatic ABCG2+CD44+ subpopulations had evaluated level of GLUT5 & KHK and retain the expression of critical enzymes involved in Krebs cycle suggesting ABCG2+ CD44+ CSC subpopulations can utilize fructose efficiently and possess the capability to be grown in different nutrient-supplying and high-oxygen environment. Utilizing tumor-engraft mice and spontaneous pancreatic cancer mice, we actually revealed fructose replacement enhanced cancer drug resistance and CSC-mediated cancer progression possibly via up-regulating ST6Gal1 as knockdown of ST6Gal1 or treatment of ST6Gal1 inhibitors can suppress self-renewal and metastasis capability of ABCG2+CD44+ CSC subpopulations. In summary, Otto Warburg proposed in 1920s that tumor cells product energy by glycolysis followed by lactic acid fermentation in cytosol rather than by oxidation pyruvate in mitochondria. However, accumulating evidence from recent work actually showed that many cancers exhibit the Warburg effect while retaining mitochondrial respiration. The current work actually elucidated that pancreatic CSCs possess metabolic features of pluripotent stem cells which can utilize fructose efficiently and possess the capability to be grown in different nutrient-supplying and high-oxygen environment. Importantly, these key metabolic features are critical in CSC-mediated cancer drug resistance and metastasis. Citation Format: Chia-Ning Shen, Chi-Che Hsieh, Wen-Shan Li, Michael Hsiao. Therapeutic implication of identifying pancreatic cancer stem cells possessing fructose metabolic signature. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2489.

Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

Abstract 1511: Identification of metastatic subsets of pancreatic cancer stem cells possessing metabolic features of pluripotent stem cells

Abstract The current cancer chemotherapy can mainly cause pancreatic cancer remission but often fails to cure cancer due to the existence of cancer stem cells in pancreatic ductal adenocarcinomas (PDACs). Therefore, development of methods targeting pancreatic cancer stem cells could lead to improvement in PDAC therapeutic. However, the key characteristics of pancreatic cancer stem cells remain to be determined. Accumulating evidences revealed the similar features shared by pluripotent stem cells and cancer stem cells, we therefore hypothesize acquisition of pluripotent characteristics during the reprogramming process is superficially reminiscent of the dysplastic transformation proposed in cancers. In the current work, comparative genome-wide profiling analysis was conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and different subpopulation of pancreatic cancer cells. We initially revealed expression of pluripotent transcriptional factors and surface markers such as Oct4, Sox2, Lin28, CD133, ABCG2 and CD44 was significantly higher in ABCG2+ CD44+ pancreatic CSC subpopulation which was correlated with their drug resistance and high metastatic potentials. Metabolic pathway analysis was further performed and identified that, very similar to ESCs/iPSCs, the metastatic ABCG2+CD44+ subpopulations had evaluated levels of of GLUT5 and KHK and expressed higher levels of ST6Gal1. Utilizing tumor-engraft mice and spontaneous pancreatic cancer mice, we actually revealed fructose diet enhanced cancer drug resistance and CSC-mediated cancer metastasis possibly via up-regulating ST6Gal1 as knockdown of ST6Gal can affect self-renewal, invasion potential and ABCG2 efflux ability of ABCG2+CD44+ CSC subpopulations. In summary, the current work elucidated that pancreatic ABCG2+CD44+ CSC subpopulations possess metabolic features of pluripotent stem cells including the capability to efficiently to utilize fructose and to perform 2,6 sialylation via sialyltransferase ST6Gal1 which critically mediated cancer drug resistance and metastasis. Hopefully, the findings can lead to developing efficient treatments for patients of PDAC in the near future. Citation Format: Chi-Che Hsieh, Wen-Ying Liao, Chih-Lung Chen, Pei-Yu Lin, Chia-Ning Shen. Identification of metastatic subsets of pancreatic cancer stem cells possessing metabolic features of pluripotent stem 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 1511. doi:10.1158/1538-7445.AM2015-1511

The Notch Pathway Is Important in Maintaining the Cancer Stem Cell Population in Pancreatic Cancer

BackgroundPancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.MethodsExpression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.ResultsNotch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.ConclusionThe Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.

Tu1759 A CD166 Negative Subpopulation of Pancreatic Cancer Cells Has Strong Invasive and Migratory Activity

BACKGROUND: CD166 expression is correlated with prognosis in several cancers. However, its significance in pancreatic cancer is not clear. The aim of this study is to clarify the significance of CD166 expression in pancreatic cancer. METHODS: We performed flow cytometry to analyze expression of CD166 in pancreatic cancer cell lines. We also analyzed the functional differences between CD166+ and CD166cells using invasion, migration and proliferation assays. We performed immunohistochemistry to investigate CD166 expression in surgically resected pancreatic cancer tissues. RESULTS: In flow cytometry, CD166 was expressed in pancreatic cancer cells in wide range (0-99.5%). In invasion assay, the invasiveness of CD166cells was greater than that of CD166+ cancer cells (p ,0.05). .In migration assay, the migratory activity of CD166cells was greater than that of CD166+ cancer cells (p,0.05). In proliferation assay, there was no significant difference between CD166+ pancreatic cancer cells and CD166cells. The analysis of real-time quantitative RT-PCR revealed that epithelial-mesenchymal transition activator Zeb1 mRNA was over-expressed in CD166cells (p,0.001 compared with that in CD166 + cells). In immunohistochemistry, there was no significant difference in prognosis between CD166 high staining group (15 patients; 48.4%) and CD166 low staining group (16 patients; 51.6%). CONCLUSION: Our findings suggest that CD166cells exhibit more aggressive behavior and activation of Zeb1 may play a role in this behavior, although further investigation is needed.

Combination of salinomycin and gemcitabine eliminates pancreatic cancer cells

Previous research has documented that a subpopulation of pancreatic cancer cells, named cancer stem cells (CSCs), harbor stem cell-like properties. Here, we examined the efficacy of combined treatments of salinomycin and gemcitabine in human pancreatic cancer cells. Salinomycin inhibited the growth of CSCs, while gemcitabine suppressed the viability of non-CSCs. Consistently, in vivo studies showed that salinomycin combined with gemcitabine could eliminate the engraftment of human pancreatic cancer more effectively than the individual agents. These data indicated that administration of salinomycin, which targets CSCs, may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate pancreatic cancer.

Use of a Magnetically Coupled Camera and Novel Mini Laparoscopic Instruments to Perform Minimally Invasive Sigmoid Colon Resection

The stem cell spheroids were infected with NV1066, a third-generation herpesvirus, or NDV-F3aa-GFP, a Newcastle Disease virus mutant. Both viruses carried the marker gene green fluorescent protein (GFP), which allowed monitoring by fluorescent microscopy. Cell cycle analysis and cell migration assay were also performed. Results: Viral infection of cancer stem cells was rapid (GFP expression was seen by 24 hours). The viruses from both families each produced efficient infection and killing of cancer. At doses of multiplicity of infection (MOI, number of viruses per tumor cell) of 0.5 or 1, >95% of cells were dead by day 6. Infection with virus also produced decreased migratory capacity of the cancer stem cells and shifted the population to a higher fraction in S phase. Conclusion: Multiple types of oncolytic viruses effectively target the stem cell subpopulation of pancreatic cancer cells. Infection decreases metastatic potential and effects killing of such stem cells. These data support clinical studies of oncolytic viruses in the treatment of chemoand radioresistant tumors.

Transesophageal Endoscopic Myotomy (TEEM) for the Treatment of Achalasia – The United States Human Experience

The stem cell spheroids were infected with NV1066, a third-generation herpesvirus, or NDV-F3aa-GFP, a Newcastle Disease virus mutant. Both viruses carried the marker gene green fluorescent protein (GFP), which allowed monitoring by fluorescent microscopy. Cell cycle analysis and cell migration assay were also performed. Results: Viral infection of cancer stem cells was rapid (GFP expression was seen by 24 hours). The viruses from both families each produced efficient infection and killing of cancer. At doses of multiplicity of infection (MOI, number of viruses per tumor cell) of 0.5 or 1, >95% of cells were dead by day 6. Infection with virus also produced decreased migratory capacity of the cancer stem cells and shifted the population to a higher fraction in S phase. Conclusion: Multiple types of oncolytic viruses effectively target the stem cell subpopulation of pancreatic cancer cells. Infection decreases metastatic potential and effects killing of such stem cells. These data support clinical studies of oncolytic viruses in the treatment of chemoand radioresistant tumors.

Abstract 2444: STAT3 is necessary for proliferation and survival in pancreatic cancer-initiating cells

Abstract In recent years, cancer-initiating cells or cancer stem cells have been demonstrated in a variety of solid tumors including pancreatic cancer. This subpopulation of cancer cells has the ability to initiate and perpetuate tumor growth, and may be highly resistant to radiation or chemotherapy. Therefore, this subpopulation could be a very important and novel target for cancer therapy. Persistent activation of STAT3 is detected in many types of cancer including in pancreatic cancer. However, whether STAT3 is activated in pancreatic cancer-initiating cells and what role STAT3 signaling may play in them is still unknown. It has been reported that pancreatic cancer stem cells can be identified and isolated with several cancer stem cell markers, such as ALDH1, CD44, and CD24. Therefore, we examined STAT3 phosphorylation and ALDH1, CD44, CD24 expression in 30 cases of human pancreatic cancer tissues using tissue microarray slides. We observed there is a significant association between the nuclear staining of STAT3 phosphorylation and ALDH1 (p<0.01). In addition, the tumor samples express elevated levels of phosphorylated STAT3 also associated with CD44+ and CD24+ (p<0.01). These results indicate constitutive STAT3 signaling may be a novel therapeutic target in pancreatic cancer initiating cells. We next separated pancreatic cancer-initiating cells from human pancreatic cancer cell lines using ALDH1, CD44, and CD24 markers by flow cytometry. We found ALDH+ or CD44+/CD24+ pancreatic cells had more potent tumorspheres formation ability than ALDH− or CD44−/CD24− cells. Interestingly, we observed the ALDH+ and CD44+/CD24+ subpopulation of pancreatic cancer cells expressed higher levels of phosphorylated STAT3, compared to ALDH− or CD44−/CD24− subpopulation of pancreatic cancer cells. These results are consistent to the data using pancreatic cancer tissues from cancer patients suggesting that STAT3 is activated in pancreatic cancer-initiating cells. Furthermore, we demonstrated that three small molecular STAT3 inhibitors, LLL12, FLLL32, and Stattic inhibited STAT3 phosphorylation, cell viability, tumorsphere growth and induced apoptosis in pancreatic cancer-initiating cells. The inhibitory efficacy of the novel STAT3 inhibitor on pancreatic cancer-initiating cells in mouse tumor model will be examined. In conclusion, this is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in pancreatic cancer-initiating cells. Our study is also the first attempt to target STAT3 in pancreatic cancer-initiating cells and we demonstrated for the first time that pancreatic cancer stem cells are indeed sensitive to the inhibition by three small molecular STAT3 inhibitors. Our results suggest STAT3 is a novel therapeutic target in pancreatic cancer-initiating cells and inhibition of activated STAT3 in cancer-initiating cells may offer an effective therapy approach for 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 2444. doi:10.1158/1538-7445.AM2011-2444

2-Methoxyestradiol mediated signaling network in pancreatic cancer

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17 beta-estradiol, is known to be a potent inhibitor of neovascularization. Our previous studies have shown that 2-ME can suppress growth of pancreatic tumor cells in vitro and in vivo by the induction of apoptosis (Cancer Res 66: 4309-18, 2006). In order to better comprehend the signaling modulators of 2-ME in pancreatic cancer, we employed a PowerBlot Western Array screening system. Our proteomic profiling has provided framework to define the novel mechanisms of actions of 2-ME in pancreatic cancer. Interestingly, this high-throughput analysis identified proteins such as Rac1, Gelsolin, Glucocorticoid receptor (GR), Smad 2/3, Smad 4, IRS-1, which were not previously reported with 2-ME response. Interestingly, 2-ME modulated down regulation of GR level is accompanied by NF-k B activation in 2-ME responsive but not in resistant pancreatic cancer cells. In view of this observation, possible reciprocal relationship between GR and NF-kappaB activation might be an important regulatory factor in 2-ME mediated demise of a subpopulation of pancreatic cancer cells.

Identification of Pancreatic Cancer Stem Cells

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.