Cancer Stemness in Apc- vs. Apc/KRAS-Driven Intestinal Tumorigenesis

Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (®-catenin intracellular stabilization

Identification of quiescent, stem-like cells in the distal female reproductive tract

In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are in

Ectopic activation of WNT signaling in human embryonal carcinoma cells and its effects in short- and long-term in vitro culture

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4−/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4−/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells

Spheroids differentiation assay.

<p>20 day old spheroids derived from oviducts of 12 week chased mice were pulsed for one day with doxycycline and were subsequently allowed to differentiated for 0 (A), 1 (B and C), 2 (D and E), 3 (F) or 6 days (G) in the presence of 10% FCS. The two columns of images on the left hand side of the figure represent confocal images taken at two different planes (bottom and middle). The phase-contrast images in the third colum from the left represents a detail from the confocal image. C represents a spheroid derived from a different animal which was induced to differentiate for 1 day, displaying formation of multiple cell layers as indicated by nuclear DAPI staining and immunofluorescence for CK8. In E attachment of the spheroid to the culture dish is shown. H and I represent spheroids isolated from different animals that were allowed to differentiate for 9 days (H) and for 20 days (I) showing complex structures (nuclei were stained with Hoechst 34580).</p

Differentiation of spheroids towards specific cell lineages of the female reproductive tract.

<p>Early spheroids (2 days of culture, A), late spheroids (2 weeks of culture, B) and spheroids which were stimulated to differentiate (10% FCS added to the culture medium for 2 days, C) were stained for ERα, CD44, PR and PAEP expression and compared to stained sections from mice distal oviduct (D), proximal oviduct (E) and endometrium (F). The red arrows indicate ERα positive cells, the black arrows indicate ERα negative cells. In panel A, 4 different spheroids were used, in panel B two (ER and PR, CD44 and PEAP stainings were performed on the same spheroid, respectively) and stainings in panel C are on consecutive sections from one spheroid. Images are representative for three different experiments containing more than 10 early and late spheroids and 2 – 3 differentiated spheroids.</p

Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.

<p>A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.</p

Characterization of identified LT-LRCs

<p>(<b>12 week chase</b>) <b>in mouse distal oviduct.</b> Immunofluorescent double-labeling was performed for GFP and ERα (A), GFP and PR (B), GFP and Ki67 (C), GFP and CD44 (D) and CD44 and ERα (E). DAPI staining was performed to display all cell nuclei. White arrowheads indicate Ki67 positive cells, the yellow arrowhead indicates a cell which is positive for CD44 as well as GFP.</p

Isolated LT-LRC can form self-renewing spheroids in culture.

<p>Oviducts from 12 week chased mice were dissected and a single cell suspension was analyzed by FACS. From three different 12 week chased mice, GFP⁺ and GFP-negative (GFP−) cells were isolated by FACSorting and cultured in Matrigel under serum-free conditions to assay their capacity to form spheroids (A). Self-renewal was assayed by dissociating spheroids and plating the unsorted cell suspension to form new spheroids as indicated in the Materials and Methods section. In short, primary spheroids (i.e. obtained directly from the chased animals) were reduced to single cell suspensions and plated again (unsorted) in matrigel/serum-free culture conditions. The corresponding secondary organoids were then dissociated and plated to obtain tertiary ones (B). Using limiting dilutions, individual GFP⁺ cells were cultured for 0 to 20 days in Matrigel under serum-free conditions, thus forming a polarized epithelial organoid. The bars represent 50 μM (C).</p