Establishment of FU-MFH-2 cell line and doubling time
Four weeks after initial cultivation in primary culture, spindle-shaped, round, or polygonal tumor cells reached sub-confluence with some piled-up foci of cells. These cells were collected after a 5-minute digestion at 37°C with a 0.1% trypsin solution, and replated in two 25-cm2 plastic flasks containing fresh medium. Once confluent they were serially subcultured at a dilution of 1:2. Approximately 2 months later, at passages 4 to 5, the cells began to grow rapidly, and thereafter could be serially subcultured at a dilution of 1:2 every week. This new cell line was designated FU-MFH-2, and has been maintained in vitro for more than 80 passages (a period of more than 12 months). The population-doubling time of FU-MFH-2 cells in logarithmic growth phase was approximately 56 hours.
Tumor formation in vivo
Small elastic hard nodules became palpable in all SCID mice at approximately 4 weeks after inoculation of FU-MFH-2 cells. Two months later, the tumors had grown up to 2.2 cm in diameter. The cut surfaces of these tumors were solid and white with no secondary changes. The mice were sacrificed humanely, and no metastatic lesions were identified at autopsy.
Morphologic characterization in vitro and in vivo
As assessed by light microscopy, FU-MFH-2 cells growing in chamber slides were spindle-shaped, round or polygonal with extended slender cytoplasmic processes. The cells proliferated loosely or in a storiform pattern accompanied by irregularly piled up foci. The nuclei were oval with distinct nucleoli (Figure ). As shown by immunocytochemistry (Table ), these cells were positive for vimentin (Figure ) and CD68 (Figure ). The other antibodies tested in vitro were negative. On the other hand, the histological features of the heterotransplanted tumors were essentially similar to those of the original tumor. Namely, the tumors were composed of a mixture of atypical spindle cells, round cells, and bizarre giant cells arranged in a storiform pattern (Figure ). Mitotic figures were frequently found. Immunohistochemically (Table ), the tumor cells were positive for vimentin and focally for CD68, but were negative for the other antibodies tested in vivo.
Figure 2 Light microscopic findings of FU-MFH-2 cells in vitro. (A) FU-MFH-2 cells are spindle, round or polygonal in shape with oval nuclei and extension of slender cytoplasmic processes. Most FU-MFH-2 cells exhibit immunopositive reaction for vimentin (B) and (more ...)
Reactivity of FU-MFH-2 cells, in vitro and in vivo, including the original tumor cells with various antibodies.
Light microscopic finding of FU-MFH-2 cells in vivo. A representative portion of the tumor in a SCID mouse, essentially resembling the original tumor.
A representative karyotype is shown in Figure . FU-MFH-2 displayed a highly complex karyotype with numerous marker chromosomes. The composite karyotype was as follows: 55-61,XY,-X,add(X)(p22.1),add(1)(q11),der(1)add(1)(p13)del(1)(q42),-2,-2,add(2)(p11.1), -3,add(3)(q21),-4,add(4)(q31.1),-5,add(5)(q11.1),del(6)(q11) × 2,del(7)(p11.1), del(7)(q11.1),der(7)add(7)(p22)add(7)(q22),-8,add(9)(p11) × 2, der(9)del(9)(p11)add(9)(q22),-10,add(10)(p13),-11,add(11)(q23),-12,-13,-14,add(14)(p11.1),add(15)(p11.1),add(15)(p11.1),-17,-17,-18,-19,-20,add(20)(q13.1),+add(21)(p11.1),-22,-22, +mar1,+mar2,+mar3,+mar4,+mar5,+mar6,+mar7,+mar8,+mar9,+mar10,+mar11,+mar12 [cp20]. Precisely the same karyotype was recognized in the original tumor cells (data not shown).
A representative G-banded karyotype of a metaphase FU-MFH-2 cell, including 12 marker chromosomes. Arrows indicate the structural chromosome aberrations.
Molecular cytogenetic findings
An M-FISH analysis identified 19 structural rearrangements in the FU-MFH-2 cell (Figure ). Chromosomes 3, 6, 8, 9, 10, and 16 were frequently involved in rearrangements.
Multicolor FISH of FU-MFH-2 cell line. Aberrant chromosomes are displayed in classified color image.
Urovysion™ FISH revealed homozygous deletions of the 9p21 locus containing the tumor suppressor gene p16INK4A in all analyzed metaphase and interphase cells (Figure ).
Figure 6 Multitarget FISH analysis performed on metaphase cells of FU-MFH-2 cell line with the Urovysion™ probe set reveals loss of gold signals indicating homozygous deletions of the 9p21 locus. Centromeric signals (arrows) of chromosomes 3 (red), 7 (green), (more ...)
CGH analysis showed similar profiles in the original tumor and FU-MFH-2 cell line. A high-level amplification of 9q31-q34 was observed. Significant gains of DNA sequences were detected in the 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X regions. Significant losses of DNA sequences were detected in the 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22 regions. This CGH profile is represented in Figure .
Figure 7 CGH profile of FU-MFH-2 cell line showing high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, (more ...)