Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas

Abstract

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

Pituitary stem cell regulation: who is pulling the strings?

“…showed self-renewal and also limited differentiation capacity in another study (Würth et al 2016). However, these cells were sensitive to the antiproliferative effect of drugs (in this case, dopamine/somatostatin chimeric agonists), which does not fit with the general concept of therapy resistance of TSC.…”

“…Pronounced expression of DLL1, JAG1, HES1, NOTCH1 and NOTCH2 was found in the pituitary tumor SP (Mertens et al 2015) (Table 1) while other candidate TSC populations (i.e. tumorspheres and CD133 + cell populations) showed upregulation of DLL1, JAG2, NOTCH1 and NOTCH4 (Xu et al 2009b, Würth et al 2016, the latter NOTCH receptor subtype in general is more associated with endothelial cells.…”

Animal models of MEN1

“…A hallmark of tumors is the ability to engraft after transplantation into an appropriate recipient animal. While xenografts in mice are still the gold standard, xenografting in zebrafish is increasing in popularity and examples of transplanting human cancer cell lines (Stoletov et al 2007, Lara et al 2011, patient-derived cancer cells (Gaudenzi et al 2016) or tissues (Marques et al 2009) have been reported. Tumor cells can be engrafted in zebrafish embryos, juveniles or adults, and working protocols have been established that outline the advantages and disadvantages of each approach.…”

“…Wurth and coworkers transplanted human pituitary adenoma stem cells (hPASCs) in zebrafish and in NOD/SCID mice (Wurth et al 2016). While there was no 24:10 proliferation of hPASCs in murine hosts up to 8 months after injection, these cells readily engrafted into zebrafish embryos.…”

“…This approach has also been tested for NETs. In a proof-ofconcept study, using eight different human primary NET samples, Gaudenzi and coworkers evaluated angiogenesis and cell migration in zebrafish xenotransplants (Gaudenzi et al 2016). The tumors engrafted in six of eight cases and tumor originating from metastases showed a higher migration capacity, thereby proving the general applicability of the concept.…”