The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells.

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells.

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC

Characterization of ABCG2 activity in PDAC cell lines.

<p>Inhibition of the SP phenotype. Cells were treated with blocking antibodies for ABCG2 (5D3) and MDR-1 (MRK-16), and then fractionated by FACS, as described in the Materials and Methods. H295, top row; Panc-1 monolayer, middle row; and BxPC3 spheroids, bottom row. Columns 1–4 are as labeled in the figure: untreated, verapamil (VP), 5D3 antibody, and MRK-16 antibody.</p

The SP phenotype in monolayers and spheroid cultures of human PDAC cell lines.

<p>(A) Panc-1 and BxPC3 cells were stained with Hoechst 33342 dye (5μg/ml) and analyzed with a FACSaria flow cytometer, as described in the Materials and Methods. The gate for SP cells was determined by treatment with verapamil (100μM). The fraction of cells in the SP gate is given in each panel. Profile of Panc-1 cells, upper row; and BxPC3 cells, lower row; verapamil control (VP), right panels. (B) Top row: BxPC3 cells were grown either in standard medium on tissue culture dishes (TC), in serum-free DMEM with B27 (SFDB) on TC dishes, or as spheroids in SFDB on uncoated bacteriological (non-TC) petri dishes. Bottom row: quantification of SP by FACS sorting for each culture condition, verapamil control (VP), far right panel.</p

Comparison of ABCG2 and MDR-1 expression in PDAC cell lines.

<p>Microarray analysis of ABCG2 and MDR-1 in 22 pancreatic cancer cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148807#pone.0148807.ref036" target="_blank">36</a>]. The log₂ expression data for ABCG2 and MDR-1 from three non-malignant human pancreatic ductal epithelial cell lines (hPDEC; #904, #916, #515) were averaged and subtracted from the log₂ from each pancreatic cancer cell line and plotted as a Waterfall and Manhattan plot, Student’s <i>t</i> test = <i>P</i><0.001.</p

Tumor initiating potential of SP and NSP cells.

<p>SP and NSP cells were obtained by fractionation using the gates shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148807#pone.0148807.g001" target="_blank">Fig 1</a>. The tumor initiating potential of SP and NSP was tested by orthotopically injecting 500, 5000, or 50000 cells of luciferase-labeled cells from each population, using cohorts of five NSG mice for each dose of cells. After two weeks, the relative amount of tumor volume was detected using real-time <i>in vivo</i> bioluminescence imaging. (A) Panc-1 monolayer cells (B) BxPC3 spheroids cells. (C) Histogram of tumor incidence for each cohort of five NSG mice, Student’s <i>t</i> test = <i>P</i><0.002.</p

ABCG2 expression in PDAC cell lines and primary tumors.

<p>(A) Cell surface staining of ABCG2 (5D3 antibody) and ABCB1/MDR-1 (MRK-16 antibody) on Panc-1 monolayer cells, BxPC3 spheroid cells and H295 cells. (B) Immunohistochemistry staining of ABCG2 on SP and NSP generated primary tumors (positive DAB stain, brown). (C) Immunohistochemistry staining of ABCG2 on primary PDAC tumors from four patients (positive DAB stain, brown).</p

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient’s macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis

Progenitor Cell Line (hPheo1) Derived from a Human Pheochromocytoma Tumor

<div><p>Background</p><p>Pheochromocytomas are rare tumors generally arising in the medullary region of the adrenal gland. These tumors release excessive epinephrine and norepinephrine resulting in hypertension and cardiovascular crises for which surgery is the only definitive treatment. Molecular mechanisms that control tumor development and hormone production are poorly understood, and progress has been hampered by the lack of human cellular model systems. To study pheochromocytomas, we developed a stable progenitor pheochromocytoma cell line derived from a primary human tumor.</p><p>Methods</p><p>After IRB approval and written informed consent, human pheochromocytoma tissue was excised, minced, dispersed enzymatically, and cultured in vitro. Primary pheochromocytoma cells were infected with a lentivirus vector carrying the catalytic subunit of human telomerase reverse transcriptase (hTERT). The hTERT immortalized cells (hPheo1) have been passaged >300 population doublings. The resulting cell line was characterized morphologically, biochemically and for expression of neuroendocrine properties. The expression of marker enzymes and proteins was assessed by immunofluorescence staining and immunoblotting. Telomerase activity was determined by using the telomeric repeat amplification protocol (TRAP) assay.</p><p>Results</p><p>We have established a human pheochromocytoma precursor cell line that expresses the neuroendocrine marker, chromogranin A, when differentiated in the presence of bone morphogenic protein 4 (BMP4), nerve growth factor (NGF), and dexamethasone. Phenylethanolamine N-methyltransferase (PNMT) expression is also detected with this differentiation regimen. CD-56 (also known as NCAM, neural cell adhesion molecule) is expressed in these cells, but CD31 (also known as PECAM-1, a marker of endothelial cells) is negative.</p><p>Conclusions</p><p>We have maintained hTERT-immortalized progenitor cells derived from a pheochromocytoma (hPheo1) in culture for over 300 population doublings. This progenitor human cell line is normal diploid except for a deletion in the p16 region and has inducible neuroendocrine biomarkers. These cells should be a valuable reagent for studying mechanisms of tumor development and for testing novel therapeutic approaches.</p></div