Lies Gremeaux

Pituitary Progenitor Cells Tracked Down by Side Population Dissection

The pituitary gland represents the endocrine core, governing the bodys hormonal landscape by adapting its cellular composition to changing demands. It is assumed that stem/progenitor cells are involved in this remodeling. Recently, we uncovered a candidate stem/progenitor cell population in the anterior pituitary. Here, we scrutinized this “side population” (SP) and show that, unexpectedly, not the subset expressing high levels of “stem cell antigen‐1” (Sca1high) but the remainder non‐Sca1high fraction clusters the pituitary progenitor cells. Transcriptomal interrogation revealed in the non‐Sca1high SP upregulated expression of the pituitary stem/progenitor cell markers Sox2 and Sox9, and of multiple factors critically involved in pituitary embryogenesis. The non‐Sca1high SP encloses the cells that generate spheres and display multipotent hormone differentiation capacity. In culture conditions selecting for the non‐Sca1high subset within the SP, stem cell growth factors that induce SP expansion, affect transcription of embryonic factors, suggesting impact on a developmental program that unfolds within this SP compartment. Non‐Sca1high SP cells, revealed by Sox2 expression, are observed in the postulated periluminal stem/progenitor cell niche, but also in small groups scattered over the gland, thereby advocating the existence of multiple niches. In early postnatal mice undergoing a pituitary growth wave, Sox2+ cells are more abundant than in adults, concordant with a larger SP and higher non‐Sca1high proportion. Together, we tracked down pituitary progenitor cells by SP phenotype, and thus provide a straightforward method to isolate and scrutinize these cells from the plastic pituitary ex vivo, as well as a culture system for in‐depth exploration of their regulatory network. Stem Cells 2009;27:1182–1195

Activated phenotype of the pituitary stem/progenitor cell compartment during the early-postnatal maturation phase of the gland

The rodent pituitary gland undergoes prominent maturation during the first weeks after birth, including a well-known increase in hormone-producing cells. In the past, it has frequently been postulated that stem cells are involved in this early-postnatal growth phase. This hypothesis can now be explored, as pituitary stem/progenitor cells were recently identified. Here, we analyzed in detail the mouse pituitary stem/progenitor cell compartment during the first postnatal week and compared its phenotype with that at the end of the first pituitary growth wave and at adult age. Stem/progenitor cells, as assessed by both side population phenotype and Sox2 expression, are most abundant at birth and gradually decline toward adulthood. The neonatal stem/progenitor cell compartment is clearly more active in terms of proliferation, stemness gene expression, and stem cell-related functional activity including sphere formation and multipotent differentiation capacity. In situ examination of pituitary sections reveals peculiar topographical arrangements of Sox2+ cells, again more pronounced at the neonatal age. Sox2+ cells are particularly prominent at the wedge junction of the anterior and intermediate lobe, and clusters of Sox2+ cells appear to sprout from this and other cleft-lining, marginal zone regions. Colocalization of Sox2 and hormones is generally not observed, thus suggesting mutually exclusive expression. Together, the neonatal pituitary stem/progenitor cell compartment displays an activated phenotype, thus supporting its involvement in the early-postnatal maturation process of the gland.

Human pancreatic cancer contains a side population expressing cancer stem cell-associated and prognostic genes

In many types of cancers, a side population (SP) has been identified based on high efflux capacity, thereby enriching for chemoresistant cells as well as for candidate cancer stem cells (CSC). Here, we explored whether human pancreatic ductal adenocarcinoma (PDAC) contains a SP, and whether its gene expression profile is associated with chemoresistance, CSC and prognosis. After dispersion into single cells and incubation with Hoechst dye, we analyzed human PDAC resections specimens using flow cytometry (FACS). We identified a SP and main population (MP) in all human PDAC resection specimens (n = 52) analyzed, but detected immune (CD45+) and endothelial (CD31+) cells in this fraction together with tumor cells. The SP and MP cells, or more purified fractions depleted from CD31+/CD45+ cells (pSP and pMP), were sorted by FACS and subjected to whole-genome expression analysis. This revealed upregulation of genes associated with therapy resistance and of markers identified before in putative pancreatic CSC. pSP gene signatures of 32 or 10 up- or downregulated genes were developed and tested for discriminatory competence between pSP and pMP in different sets of PDAC samples. The prognostic value of the pSP genes was validated in a large independent series of PDAC patients (n = 78) using nCounter analysis of expression (in tumor versus surrounding pancreatic tissue) and Cox regression for disease-free and overall survival. Of these genes, expression levels of ABCB1 and CXCR4 were correlated with worse patient survival. Thus, our study for the first time demonstrates that human PDAC contains a SP. This tumor subpopulation may represent a valuable therapeutic target given its chemoresistance- and CSC-associated gene expression characteristics with potential prognostic value.

Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine.

BackgroundTherapy resistance remains one of the major challenges to improve the prognosis of patients with pancreatic cancer. Chemoresistant cells, which potentially also display cancer stem cell (CSC) characteristics, can be isolated using the side population (SP) technique. Our aim was to search for a SP in human pancreatic ductal adenocarcinoma (PDAC) and to examine its chemoresistance and CSC(−like) phenotype.MethodsHuman PDAC samples were expanded in immunodeficient mice and first-generation xenografts analyzed for the presence of a Hoechst dye-effluxing SP using flow cytometry (FACS). To investigate chemoresistance of the SP, mice bearing PDAC xenografts were treated with gemcitabine and SP proportion determined. In addition, the SP and the main tumour cell population (MP) were sorted by FACS for RNA extraction to profile gene expression, and for culturing under sphere-forming conditions.ResultsA SP was identified in all PDAC samples, analyzed. This SP was more resistant to gemcitabine than the other tumour cells as examined in vivo. Whole-genome expression profiling of the SP revealed upregulation of genes related to therapy resistance, apoptotic regulation and epithelial-mesenchymal transition. In addition, the SP displayed higher tumourigenic (CSC) activity than the MP as analyzed in vitro by sphere-forming capacity.ConclusionWe identified a SP in human PDAC and uncovered a chemoresistant and CSC-associated phenotype. This SP may represent a new therapeutic target in pancreatic cancer.Trial registrationClinicaltrials.gov NCT00936104

The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.

Increased Glucocorticoid Receptor Expression and Activity Mediate the LPS Resistance of SPRET/EI Mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.