title: Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma.
abstract: 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.
authors.affiliation: Department of Surgery, Division of Surgical Oncology, University of California Davis Medical Center, 4501 X Street, Sacramento, CA 95817, USA. Robert.canter@ucdmc.ucdavis.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Canter
authors.forename: Erik
authors.initials: E
authors.lastname: Ames
authors.forename: Stephanie
authors.initials: S
authors.lastname: Mac
authors.forename: Steven K
authors.initials: SK
authors.lastname: Grossenbacher
authors.forename: Mingyi
authors.initials: M
authors.lastname: Chen
authors.forename: Chin-Shang
authors.initials: CS
authors.lastname: Li
authors.forename: Dariusz
authors.initials: D
authors.lastname: Borys
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Smith
authors.forename: Joe
authors.initials: J
authors.lastname: Tellez
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Sayers
authors.forename: Arta M
authors.initials: AM
authors.lastname: Monjazeb
authors.forename: William J
authors.initials: WJ
authors.lastname: Murphy
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D020533:Angiogenesis Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D007191:Indazoles; D007527:Isoenzymes / Q000378:metabolism; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000556:secondary; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000556:secondary; D016688:Mice, Inbred NOD; D020360:Neoadjuvant Therapy; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D010671:Phenylurea Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D011743:Pyrimidines / Q000494:pharmacology / Q000627:therapeutic use; D050697:Retinal Dehydrogenase / Q000378:metabolism; D012509:Sarcoma / Q000188:drug therapy* / Q000556:secondary; D000077157:Sorafenib; D013449:Sulfonamides / Q000494:pharmacology / Q000627:therapeutic use; D046888:Tissue Array Analysis; D023041:Xenograft Model Antitumor Assays
chemical_list: D020533:Angiogenesis Inhibitors; D015703:Antigens, CD; D007191:Indazoles; D007527:Isoenzymes; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013449:Sulfonamides; D009536:Niacinamide; C516667:pazopanib; D000077157:Sorafenib; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase