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Supplementary Materials and Methods.
Culture of ovarian and other cancer cell lines and RAS-transformed ovarian and
fallopian tubal cell lines. RAS-transformed ovarian epithelial cell lines T29H and T80H
were described in our previous study (1), RAS-transformed fallopian cell lines
FTE187SV40hT and FTE187SV40hTHras were from our paper described previously (2).
All RAS-transformed cancer cells were maintained in Media 199/MCDB 205 (1:1;
Sigma-Aldrich) supplemented with 10% fetal bovine serum (Intergen) and 100 U/mL
penicillin/streptomycin (Sigma-Aldrich). HEY and SKOv3 were maintained in Eagle’s
minimum essential medium, and MDA-MB-231, MCF-7 cancer cells, phoenix, and 293T
cells were purchased from the American Type Culture Collection and cultured in
Dulbecco's modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS),
100 U/mL penicillin, and 100 g/mL streptomycin.
Induction of PGCCs by Low Concentration of Oxygen
When HEY cells reached 90% confluence, they were washed with phosphate-buffered
saline (PBS) and were then continuously exposed 0.1% oxygen with O2 Control Cabinets
(The Coy InVitro Cabinet System) for at least 8 days.
Assay for Chemosensitivity
To test the cells’ resistance to cisplatin, 10 µg/ml cisplatin was added into a flask for 48
hours. The number of and size of survived cells were examined under the microscope.
STR DNA fingerprinting
Cell lines were validated by STR DNA fingerprinting using the Promega, Powerplex
16HS system according to manufacturer's instructions (Promega DC2100). The STR
profiles were compared to known ATCC fingerprints (ATCC.org), and to the Cell Line
Integrated Molecular Authentication database (CLIMA) version 0.1.200808
(http://bioinformatics.istge.it/clima/) (Nucleic Acids Research 37:D925-D932 PMCID:
PMC2686526). The STR profiles matched known DNA fingerprints or were unique.
1.
Liu J, Yang G, Thompson-Lanza JA, et al. A genetically defined model for
human ovarian cancer. Cancer Res 2004; 64: 1655-63.
2.
Shan W, Mercado-Uribe I, Zhang J, et al. Mucinous adenocarcinoma developed
from human fallopian tube epithelial cells through defined genetic modifications. Cell
Cycle 2012; 11: 2107-13.
Figure Legends.
Figure S1. Morphologic characteristics of regular cells and PGCCs. A. PGCCs in a
total of 10 additional ovarian cancer cell lines, OVCA433, OVCA432, SKOv3ip,
HEYA8, two primary human ovarian cancers, RAS-transformed ovarian epithelial cell
line T29H, T80H, or fallopian tubal line, FTE187SV40hTHras, or SV40T/hTERTimmoralized fallopian tubal line, breast cancer cell line MCF-7, phoenix and 293T.
Compared with the control cells without CoCl2 treatment, the more PGCCs were
observed (Black arrow points). B. Increased PGCCs can be observed in the presence of
0.1% oxygen for 8 days (original magnification x 100 for all figures).
Figure S2. Time-lapse imaging analysis on HEY PGCCs. A. Time-lapse observation on
budding of small daughter cancer cell from the body of PGCC every 30 min for 240 min.
B. Time-lapse observation of budding from the body of PGCC every 30 min for 150 min.
Figure S3. Slow-cycling nature of HEY PGCCs. A. HEY PGCC (black arrows) and
daughter cells (red arrows) stained with PKH26 and observed at day 1, day 3, and day 5
(10×) and with loss of fluorescence in the regular cell lines at day 5. B. The PKH26
florescent dye remains at day 10 in the presence of cell cycle inhibitor nocodazole
(original magnification x 100 for all figures).
Figure S4. A. Survival of regular HEY cells and HEY cells derived from PGCCderived tumor. Total number of cells was examined following 300 µM CoCl2 treatment
for 72 hours. (a) Regular HEY cells; (b) PGCCs from (a) treated with 300 µM CoCl2 for
72 hours; (c) Primary culture derived from single PGCC injection tumor. (d) PGCCs
from (c) treated with 300 µM CoCl2 for 72 hours. B. PGCCs confer resistance to
cisplatin treatment. (a) Regular HEY cancer cells as a control. (b) Regular HEY cancer
cells treated with 10 µg/ml cisplatin for 48 hours. (c) Presence of predominantly PGCCs
from PGCCs-derived primary culture in the presence of CoCl2. (d). No significant change
of PGCCs from (c) following treatment of 10µg/ml cisplatin for 48 hours. C. Pictures in
human ovarian cancer tissue before chemotherapy and after chemotherapy. a and c.
H&E pictures (10x) and (20x) before chemotherapy, b and d. H&E pictures after
chemotherapy. The number and size of PGCCs are markedly increased in ovarian cancer
after chemotherapy (black arrow points) (original magnification x 100 for all figures
except x 200 for C-c).