Introduction
Methods
Anchorage-independent culture technique
Quantitative reverse transcription-PCR (qrt-PCR)
Western blot analysis
Gene expression profiling and analysis
Chemotherapy resistance assays
Vorinostat and 5-azacytidine treatment
Orthotopic and non-orthotopic xenograft experiments
Results
Anoikis-resistant OS cells robustly maintain viability and exhibit sustained but down-regulated rates of growth
Anoikis-resistant OS cells have increased resistance to chemotherapy
Doxorubicin
|
Cisplatin
| ||||
---|---|---|---|---|---|
Cell type
|
Mean IC50 (uM)
|
Fold change
|
Mean IC50 (uM)
|
Fold change
| |
MS206-2 | Adherent | 0.062 ± 0.01 | 28.8^ | 4.38 ± 0.98 | 5.7^ |
AI | 1.79 ± 0.4 | 25.2 ± 6.9 | |||
143B | Adherent | 0.025 ± 0.01 | 13* | 1.97 ± 0.19 | 1.9* |
AI | 0.31 ± 0.1 | 3.12 ± 0.10 | |||
mHOS | Adherent | 0.085 ± 0.013 | 35* | 3.10 ± 0.53 | 6.9* |
AI | 2.97 ± 1.9 | 21.4 ± 10. | |||
MG-63 | Adherent | 0.13 ± 0.06 | >75# | 2.68 ± 1.6 | 6.2* |
AI | >50% survival | 16.9 ± 5.4 |
Anoikis-resistant OS cells are molecularly distinct
Upregulated genes
|
Downregulated genes
| ||
---|---|---|---|
Gene name
|
Fold increase
|
Gene name
|
Fold decrease
|
SOX5 | 11.30 | CCBE1 | 7.81 |
PTPRE | 5.59 | MGLL | 7.41 |
PARP14 | 3.15 | CTGF | 6.73 |
ZNF697 | 3.09 | ABI3BP | 5.76 |
SHC2 | 3.04 | ANXA1 | 5.50 |
RUNX2 | 3.00 | DDAH1 | 5.36 |
CHST2 | 2.45 | CRIM1 | 3.66 |
MTHFSD | 2.42 | TRNP1 | 3.64 |
SH3BP2 | 2.34 | KRT7 | 3.13 |
FHL2 | 2.92 |
Anoikis-resistant OS cells initiate tumors equal to adherent OS cells in both orthotopic and non-orthotopic in-vivo xenograft models, but maintain a lower growth rate
Number of cells injected
| |||||
---|---|---|---|---|---|
1 × 10
4
|
1 × 10
5
|
5 × 10
5
| |||
Subcutaneous | |||||
mHOS | |||||
Adherent | 0/5 | 3/5 | 4/5 | ||
AI | 0/5 | 3/5 | 3/5 | ||
143B | |||||
Adherent | 0/5 | 1/5 | 4/5 | ||
AI | 1/5 | 3/5 | 5/5 | ||
Intratibial | |||||
mHOS | |||||
Adherent | 6/7 | 7/7 | 7/7 | ||
AI | 5/7 | 6/7 | 7/7 | ||
143B | |||||
Adherent | 5/7 | 7/7 | 6/7 | ||
AI | 7/7 | 7/7 | 6/7 |