Table 1.
Expression analyses of chordoma specific and candidate genes in MUG-Chor1 cells.
Table 2.
Expression analyses of MUG-Chor1 candidate genes in U-CH1 cells.
Figure 1.
Morphological and immunohistochemical characterization of the chordoma tumor giving rise to MUG-Chor1 cell line
. A) Hematoxylin/eosin stained section show lobulated myxoid tumor tissue with cords, strands and nests of tumor cells with pale/eosinophilic to vacuolated cytoplasm. B, C) In detail, the tumor is composed of small cells with eosinophilic cytoplasm and partly spindle cell morphology and large vacuolated/physaliferous tumor cells including “signet ring” shaped cells. D) All cell phenotypes yield the chordoma-specific nuclear staining for brachyury. Arrowheads: small cells; asterisks: large vacuolated/physaliferous cells; arrows: “signet ring” cells. Scale bars: 500 µm (A), 50 µm (B, C); D: original magnification X20.
Figure 2.
Morphological characteristics of vacuoles in large cells.
Large physaliferous cells consisting of equal vacuole content may present with either few very large (A, C, E) or numerous smaller vacuoles (B, D, F). Both morphological phenotypes were detected throughout all samples ranging from tumor tissue (A, B), cell culture (C, D) to sample preparations just before being micromanipulated (E) or microdissected (F) indicating stable characteristics of this chordoma tumor. C, D: original magnification X10; F: original magnification X40.
Figure 3.
Ultrastructural analysis of small and intermediate cells.
A) Small MUG-Chor1 cells show typical ultrastructural features of a diploid cell including nucleus (N), mitochondria (M), and endoplasmic reticulum (ER) in a dense cytoplasm. B) Stitched image of two intermediate cells tightly connected alongside their cell membranes (arrow) present with nucleus (N, only visible in one cell), ER, and mitochondria. Both cells already contain prominent vacuoles (V1, V2) and show highly organized cytoskeleton in close proximity to the nucleus and vacuoles (asterisk). Scale bars: 1 µm (A), 5 µm (B).
Figure 4.
Morphological data analysis of MUG-Chor1 cells.
Cells were tracked over a period of seven days with images taken every 30-vacuolated cells (n = 175; sm), intermediate cells containing at least one vacuole (n = 209; int), and large cells containing an estimated total vacuole compartment at least the size of the nucleus (n = 35; l). Cells leaving or entering the monitored areas or undergoing cell division at the very beginning or end of the time lapse were excluded from investigation. A) Intermediate cells significantly divided at a higher rate than small or large cells whereas no difference was seen between small and large cells (p = 0.57). B) Development of small into intermediate cells was significantly higher than intermediate to large cells. No backward-development was detected. C) Cell death rates did not differ significantly. D) The fraction of cells remaining in their phenotype without developing or dividing was found to be highest in large physaliferous cells followed by intermediate and small cells. p-Values as indicated by asterisks: p<0.01 (*); p<0.001 (**), p<0.0001 (***).
Figure 5.
Cell fate of different cell phenotypes suggesting a one-way development.
Analysis of the activities of the cells draws a picture of small cells (left) developing into large cells (right) via a mainly proliferative intermediate cell phenotype (middle). As observed in the time lapse experiments the respective cell phenotypes could also emerge through cell division. These cells also fed into the developmental process as depicted (e. g. intermediate cell dividing into one intermediate and one small cell that subsequently develops into an intermediate cell). The developmental process is highly directed as reduction of vacuolization (“backwards development”) was solely observed in dying cells. Due to a proliferation rate comparable to the small cells the large cell phenotype is not representing senescent cells but rather the end of this developmental process. Proportions of cells that undergo proliferation, development, cell death or remain in their original phenotype are given in percentage of the amount of cells allocated to the respective phenotype.
Figure 6.
Array-CGH profiles of MUG-Chor1 phenotypes.
Comparative genome hybridization of 100 large cells (red line) and small cells (blue line) each yielded identical chromosomal profiles. Both populations show gains at chromosomes 2q, 5q, 7, 17q and losses at 2q, 6p, 9p, 10p, 10q, 12p, 17p and 22. Small gains at chromosomes 2q as well as a small loss at chromosome 17q were detected in the large cell phenotype. This indicates that both morphologically different cell types evolved from a common clonal origin. Bars on the left of the moving average indicate losses of DNA. Bars on the right of the moving average indicate gains of DNA. Both profiles are in line with previously published data [8].