Using Pharmacokinetic Profiles and Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Early Signaling Changes Following Integrin-Linked Kinase Inhibition in an In Vivo Model of Cancer.
A small molecule inhibitor (QLT0267) targeting integrin-linked kinase is able to slow breast tumor growth in vivo; however, the mechanism of action remains unknown. Understanding how targeting molecules involved in intersecting signaling pathways impact disease is challenging. To facilitate this understanding, we used tumor tissue microarrays (TMA) and digital image analysis for quantification of immunohistochemistry (IHC) in order to investigate how QLT0267 affects signaling pathways in an orthotopic model of breast cancer over time. Female NCR nude mice were inoculated with luciferase-positive human breast tumor cells (LCC6(Luc)) and tumor growth was assessed by bioluminescent imaging (BLI). The plasma levels of QLT0267 were determined by LC-MS/MS methods following oral dosing of QLT0267 (200 mg/kg). A TMA was constructed using tumor tissue collected at 2, 4, 6, 24, 78 and 168 hr after treatment. IHC methods were used to assess changes in ILK-related signaling. The TMA was digitized, and Aperio ScanScope and ImageScope software were used to provide semi-quantitative assessments of staining levels. Using medium-throughput IHC quantitation, we show that ILK targeting by QLT0267 in vivo influences tumor physiology through transient changes in pathways involving AKT, GSK-3 and TWIST accompanied by the translocation of the pro-apoptotic protein BAD and an increase in Caspase-3 activity.
Experimental Therapeutics BC Cancer Agency, British Columbia, Canada (JK,WHD,MBB)|Langara College, Vancouver, British Columbia, Canada (JK)
Jessica
J
Kalra
Experimental Therapeutics BC Cancer Agency, British Columbia, Canada (JK,WHD,MBB)
Weislawa H
WH
Dragowska
Experimental Therapeutics BC Cancer Agency, British Columbia, Canada (JK,WHD,MBB)|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia (MBB)|Department of Biochemistry, University of British Columbia, Vancouver, British Columbia (MBB)|Faculty of Pharm. Sciences, University of British Columbia, Vancouver, British Columbia (MBB)|Center for Drug Research and Development Vancouver, British Columbia, Canada (MBB).
Marcel B
MB
Bally
D000818:Animals; D001391:Azo Compounds / Q000493:pharmacokinetics* / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism; D011720:Pyrazoles / Q000493:pharmacokinetics* / Q000494:pharmacology*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013194:Staining and Labeling; D046888:Tissue Array Analysis / Q000379:methods*; D051879:Twist-Related Protein 1 / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism; D000099200:Scaffold Protein ILK
D001391:Azo Compounds; D053148:Caspase 3; D038362:Glycogen Synthase Kinase 3; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011720:Pyrazoles; D012694:Serine; D051879:Twist-Related Protein 1; D051025:bcl-Associated Death Protein; D000099200:Scaffold Protein ILK; C552362:QLT 0267
Aperio; BAD; Caspase-3 pharmacokinetics; QLT0267; TWIST; apoptosis; breast cancer model; immunohistochemistry; integrin-linked kinase; pAKT; tissue microarray