BACKGROUND: Increasing studies implicate cancer stem cells (CSCs) as the source 
of resistance and relapse following conventional cytotoxic therapies. Few 
studies have examined the response of CSCs to targeted therapies, such as 
tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have 
differential effects on CSC populations depending on their mechanism of action 
(anti-proliferative vs. anti-angiogenic).
METHODS: We exposed human sarcoma cell lines to sorafenib, regorafenib, and 
pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, 
CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring 
sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma 
tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray 
(TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC 
markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and 
non-parametric statistical analyses were performed as appropriate.
RESULTS: After functionally validating the CSC phenotype of ALDHbright sarcoma 
cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell 
lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright 
cells from baseline (P < 0.05). In contrast, we observed negligible effects on 
viability and CSC sub-populations with pazopanib. At low doses, there was 
progressive CSC enrichment in vitro after longer term exposure to sorafenib 
although the anti-proliferative effects were attenuated. In vivo, sorafenib 
improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 
2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed 
direct cytotoxicity following exposure to sorafenib and regorafenib with a 
corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects 
from pazopanib were observed. TMA analysis of archived specimens from sarcoma 
patients treated with sorafenib demonstrated significant enrichment for 
ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas 
clinical specimens obtained longitudinally from a patient treated with pazopanib 
showed no enrichment for ALDHbright cells (P > 0.05).
CONCLUSIONS: Anti-proliferative TKIs appear to enrich for sarcoma CSCs while 
anti-angiogenic TKIs do not. The rational selection of targeted therapies for 
sarcoma patients may benefit from an awareness of the differential impact of 
TKIs on CSC populations.