17 research outputs found

    Organoid-formation assay.

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    <p>(A) Organoid-forming capacity of sorted populations from villin-rtTA/TRE-H2B-GFP mice, chased for 72–78 days. Displayed are average percentages of organoids formed (±s.e.m.) from 5000 FACS-sorted and plated events in 5 independent experiments as scored 14 days after plating. (B–C) To assess the presence of doublets among the sorted LRCs, the different subpopulations were examined by confocal microscopy. 500 cells were sorted in each well of a 6-well plate, imaged by confocal microscopy (B) and counted (C). (B) Representative IF images showing the composition of the LRC CD24<sup>hi</sup>SSC<sup>hi</sup> population (left) and the H2B-GFP<sup>-</sup> CD24<sup>hi</sup>SSC<sup>hi</sup> population (right). (C) Quantification of the composition of FACS-sorted LRC CD24<sup>hi</sup>SSC<sup>hi</sup> (left) and H2B-GFP<sup>-</sup> CD24<sup>hi</sup>SSC<sup>hi</sup> (right) by morphology for single cells and aggregates of differently sized cells; displayed are percentages.</p

    Analysis of proliferation and differentiation markers in LRCs.

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    <p>(A–B) Progressive loss of expression of differentiation and proliferation markers in LRCs following doxycycline withdrawal. The enteroendocrine differentiation marker synaptophysin (A) and the proliferation marker Ki-67 (B) were detected by APC-A whereas FITC-A detects H2B-GFP. The size of each population is indicated as the percentage of epithelial cells. (C–D) Analysis of small intestinal sections from villin-rtTA/TRE-H2B-GFP mice chased for 5 weeks. (C) LRCs (GFP, green) and cycling cells (ki-67, red), the white arrowhead points to a cycling crypt base columnar cell (ki-67, red); (D) LRCs (GFP, arrowheads, brown) and goblet cell marker PAS (arrows, red).</p

    Characterization of LRCs for Paneth and stem cell markers.

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    <p>(A) Co-staining of small intestinal sections from villin-rtTA/TRE-H2B-GFP mice chased for 79 days of LRCs (arrow and arrowhead, GFP, green) and Paneth cells (lysozyme; red). Several (arrow) but not all (arrowhead) LRCs are positive for the Paneth cell marker lysozyme. Imaginary cell boundaries have been drawn (white dotted line). The number of lysozyme-positive and lysozyme-negative LRCs was quantified at chase day 41 and 70. Between 29% (chase 41 days) and 39.9% of LRCs are not expressing lysozyme. (B) Representative FACS image of mouse small intestinal epithelial crypt cells analyzed for the Paneth cell marker CD24. Paneth cells fall commonly within the CD24<sup>hi</sup>SSC<sup>hi</sup> gate. The size of each population is indicated as percentage of live epithelial cells. (C) Representative FACS image of label retaining cells (chase 75 days) analyzed for the Paneth cell marker CD24. The size of each population is indicated as percentage of live epithelial cells. The majority of LRCs falls directly into the CD24<sup>hi</sup>SSC<sup>hi</sup> gate, while others scatter just around the CD24<sup>hi</sup>SSC<sup>hi</sup> gate in close proximity. Some of these cells in close proximity to the CD24<sup>hi</sup>SSC<sup>hi</sup> gate fall within the upper edge of the CD24<sup>med</sup> gate. Of note, only a very small minority of LRCs falls outside of this cell cluster and is CD24<sup>-</sup> (5.54%). (D) Composition of LRCs according to CD24. Displayed are averages of five independent mice chase for 72 to 78 days (± s.d.). The percentage of CD24 subpopulations within the LRCs does not add up to 100%, as some cells fall outside of the drawn gates. (E) Plots of relative gene expression levels (qRT-PCR) of Defa1, Lgr5, Bmi1, Msi1, Tert, Dll1, Lrig1, and Prox1. CD24<sup>hi</sup>SSC<sup>hi</sup> LRCs (dark green) and their H2B-GFP-negative counterpart (grey) were sorted from five different animals and analyzed by qRT-PCR. Comparison of the marker expression at the two different chase time points (64 days and 83 days) did not show any significant differences (data not shown). Therefore, data from all five animals were analyzed together and shown here. Displayed are averaged β-actin normalized values (± s.e.m.) and the corresponding p-values obtained by two-sample t-test (* <0.02; ** <0.005; *** <2x10e-6). Analysis of Lgr5 was also performed in two populations sorted from 3 Lgr5-EGFP mice, namely Lgr5<sup>+</sup> (blue) cells and Lgr5<sup>-</sup>CD24<sup>hi</sup>SSC<sup>hi</sup> (black) cells, to compare the Lgr5 expression levels in Lgr5-EGFP cells (blue) with that of CD24<sup>hi</sup>SSC<sup>hi</sup> LRCs (dark green).</p

    Proliferative response of LRCs upon radiation-induced tissue injury.

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    <p>(A) Experimental scheme. (b–d) IF analysis for GFP (green, LRCs) and lysozyme (red, Paneth cells) relative to a non-irradiated control animal at four weeks of chase (B) and to two irradiated animals that were treated according to the scheme in (A) analyzed at 32 hours after irradiation (C,D). (E) Overlay of representative image of FACS analysis of intestinal epithelial cells of non-irradiated animals at four weeks of chase (black) compared to animals at 32 hours after whole-body irradiation (red, treated according to the scheme in 6A) for H2B-GFP intensity. Two GFP-positive populations, GFP<sup>hi</sup> and GFP<sup>med</sup> are resolved. Following radiation-induced tissue injury, a new GFP<sup>med</sup> population appears (red peak), which is almost absent in untreated control animals (black line). (F) BrdU uptake as a measure of the LRCs’ proliferative response to tissue injury. The GFP<sup>hi</sup> and GFP<sup>med</sup> populations were evaluated for the percentage of BrdU<sup>+</sup> cells at 28 and 32 hrs following radiation as depicted in A. The average BrdU levels (±s.d.) measured in 2 independent mice are displayed. (G) CyclinD1-expression as a measure of the LRCs’ proliferative response to tissue injury. Displayed is the percentage of CyclinD1-expressing cells from all Actb-positive ones as determined by single-cell RT-PCR. LRCs were obtained from three pulse chased animals; GFP<sup>hi</sup> and GFP<sup>med</sup> cells were obtained from three pulse-chased animals and isolated 36 hrs. after irradiation. Single cell RT-PCR was carried out on a total of 3×90 LRCs, 3×60 GFP<sup>hi</sup> and 3×120 GFP<sup>med</sup> cells. Single cells were only included in the analysis when the housekeeping gene Actb was expressed. The average percentages of CyclinD1-expressing cells from all Actb-positive ones (±s.d.) and the corresponding p-values obtained by two-sample t-test (* <0.02) are shown. (h) Percentage of GFP<sup>hi</sup> and GFP<sup>med</sup> cells expressing Defa1, Bmi1, and Lgr5 as determined by single cell RT-PCR. FACSorted GFP<sup>hi</sup> and GFP<sup>med</sup> cells were isolated 36 hrs. after radiation-induced tissue injury. Single-cell RT-PCR was carried out on a total of 2×30 GFP<sup>hi</sup> and 2×60 GFP<sup>med</sup> cells from each one male and one female mouse. Gene expression status of single cells was only included in the charts when the housekeeping gene Actb was expressed. The average proportion of cells expressing each gene (±s.d.) is displayed.</p

    β-catenin expression analysis in <i>Apc</i><sup>1638N/+</sup> and <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> intestinal tumours.

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    <p>Immuno-histochemistry (<b>a.</b>,<b>b.</b>) and western blot (<b>c.</b>) analysis of β-catenin in primary <i>Apc</i><sup>1638N/+</sup> intestinal adenomas (<b>a.</b>) and in FACSorted tumour populations from <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> intestinal tumours (<b>b.</b> and <b>c.</b>). The bars in <b>c.</b> represents the quantification of the bands obtained with an anti-active β-catenin Ab (anti-ABC; clone 8E7, #05–665, Millipore) by scanning and analyzing the western blot with the Odyssey scanner and after normalization with β-actin.</p

    FACS analysis of cell suspensions from <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> tumours.

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    <p><b>a.</b> Large panel: dot plot representative of the staining pattern obtained by staining with anti-CD24 APC-conjugated and anti-CD29 PE-conjugated antibodies. Lineage positive cells (Lin<sup>+</sup>) were excluded (gated out) by staining with biotinylated antibodies against lineage markers and Streptavidin-PerCPCy5.5. P1 (Lin<sup>−</sup>CD24<sup>low</sup>CD29<sup>+</sup>), P2 (Lin<sup>−</sup>CD24<sup>med</sup>CD29<sup>+</sup>), and P3 (Lin<sup>−</sup>CD24<sup>hi</sup>CD29<sup>+</sup>) populations are indicated in the plot. Small panels: dot plots representative of cells stained with isotypic control antibodies (left), compensation control stained only with anti CD24-APC antibodies (middle), compensation control stained only with anti CD29-PE antibodies (right). <b>b.</b> FACS analysis of the CD24/CD29 pattern of tumours obtained by serial transplantation of P3 cells suspensions from <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> intestinal tumours. Left: primary transplantation. Right: secondary transplantation. <b>c.</b> Immunohistochemistry analysis of tumors obtained by 3 rounds of serial transplantation of P3 cells suspensions from <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> intestinal tumours.</p

    Expression profiling analysis of tumor cell subpopulations from <i>Apc</i><sup>1638N/+</sup> and <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup> intestinal tumours.

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    <p>(a.) Hierarchical clustering and (b.) Principal Components Analysis (PCA) (both implemented in Partek) of Lin<sup>−</sup>CD24<sup>hi</sup>CD29<sup>+</sup> (P3), Lin<sup>−</sup>CD24<sup>med</sup>CD29<sup>+</sup>/Lin<sup>−</sup>CD24<sup>lo</sup>CD29<sup>+</sup> (P1+P2, merged gate) and Lin<sup>−</sup> (bulk) tumor cells from 5 individual mice of each genotype (<i>Apc</i><sup>1638N/+</sup> and <i>Apc</i><sup>1638N/+</sup>/<i>KRAS</i><sup>V12G</sup>). For better visualization individual colours were used for each group and in b. ellipsoids were drawn around the three tumour populations.</p

    Genomic exploration of distinct molecular phenotypes steering temozolomide resistance development in patient-derived glioblastoma cells

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    Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise. </p
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