High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)--mediated inhibition of platelet production.
Proplatelet production represents a terminal stage of megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel high-content, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse megakaryocytes, inhibits megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias.
Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Jonathan N
JN
Thon
Matthew T
MT
Devine
Antonija
A
Jurak Begonja
Jay
J
Tibbitts
Joseph E
JE
Italiano
D000080044:Ado-Trastuzumab Emtansine; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D001681:Biological Assay; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D005333:Fetus; D005434:Flow Cytometry; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008453:Maytansine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008533:Megakaryocytes / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure; D057054:Molecular Imaging; D010976:Platelet Count; D061251:Primary Cell Culture; D013921:Thrombocytopenia / Q000139:chemically induced* / Q000517:prevention & control; D034061:Thrombopoiesis / Q000187:drug effects / Q000502:physiology; D000068878:Trastuzumab; D014404:Tubulin / Q000378:metabolism
D061067:Antibodies, Monoclonal, Humanized; D014404:Tubulin; D008453:Maytansine; D000068878:Trastuzumab; D000080044:Ado-Trastuzumab Emtansine