High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)–mediated inhibition of platelet production

Abstract

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 highcontent, 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. IntroductionPlatelets are formed and released into the bloodstream by specialized precursor cells called megakaryocytes. During the final stages of development, megakaryocytes remodel their cytoplasm into long, microtubule-based proplatelet extensions from which platelets are produced. Each day, an adult human produces approximately 100 billion platelets 1 to maintain a circulating concentration of 1.5-4 ϫ 10 8 platelets/mL. Platelets are essential for blood clotting, and the rate of platelet production can increase by a factor of 10 or more when the demand for platelets suddenly increases. Conversely, when platelet numbers are very low, a patient is at serious risk of death from hemorrhage. Recent efforts to generate useful numbers of clinically viable platelets for infusion by reproducing elements of the BM vascular niche have had limited success. 2,3 Significant improvements in platelet quality and yield will require the ability to assay multiple individual components of the BM microenvironment empirically in high throughput for quantitative improvements in megakaryocyte maturation and proplatelet production before they are incorporated in scaled biomimetic systems.Drug-induced thrombocytopenia can be triggered by a wide range of medications through the inhibition of megakaryocyte development and platelet biogenesis and/or the clearance of platelets in the blood. 4,5 Approximately 10 persons per million in the United States and Europe are affected by drug-induced thrombocytopenia per year, which can range from mild to lifethreatening. 6 The slow recovery of platelet levels...

Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity

“…However, the mechanism of DM1 release remains elusive as the authors were unable to detect colocalization between the internalized T-DM1 and lysosomal LAMP1 by immunofluorescence. Thon and colleagues reported that T-DM1-induced thrombocytopenia occurred via a mechanism that is both HER2-and FcgRIIa-independent due to the fact that mouse MKs and platelets do not express HER2 and FcgRIIa and that both take up T-DM1 (41). This study suggests that T-DM1 is endocytosed through an alternative pathway (41).…”

“…Thon and colleagues reported that T-DM1-induced thrombocytopenia occurred via a mechanism that is both HER2-and FcgRIIa-independent due to the fact that mouse MKs and platelets do not express HER2 and FcgRIIa and that both take up T-DM1 (41). This study suggests that T-DM1 is endocytosed through an alternative pathway (41). Although it is still a matter of debate whether trastuzumab associates with mouse HER2, many laboratories have been using mouse model to investigate the mechanisms of trastuzumab-induced cardiotoxicity, and data from these studies suggest that trastuzumab-…”

Platelet bioreactor-on-a-chip

“…ProPLTs in shear cultures were uniformly extended into the lower channel and aligned in the direction of flow against the vascular channel wall, recapitulating physiological proPLT production 11,30,32 (supplemental Movie 2). The percent of proPLT-producing primary mouse MKs under physiological shear stress were doubled over static cultures 26 to ;90% ( Figure 3D).…”

“…Although second-generation cell culture approaches have provided further insight into the physiological drivers of PLT release, they have been unable to recreate the entire BM microenvironment, exhibiting limited individual control of extracellular matrix (ECM) composition, 19,20 BM stiffness, 21 endothelial cell contacts, 22,23 and vascular shear stresses, 24,25 and have been unsuccessful in synchronizing proPLT production, resulting in nonuniform PLT release over a period of 6 to 8 days. 26 Moreover, the inability to reproduce the BM microenvironment ex vivo, and resolve physiological proPLT extension and release by high-resolution live-cell microscopy, has significantly hampered efforts to study the cytoskeletal and signaling mechanics of PLT production. Biomimetic platforms that model human BM are needed to support drug development and establish new treatments for thrombocytopenia.…”

“…Under static conditions, primary mouse MKs begin producing proPLTs ;6 hours postisolation and reach maximal proPLT production at 18 hours ( Figure 3B). 26 By comparison, primary mouse MKs under physiological shear stress (;600 mPa) began producing proPLTs within seconds of trapping, reaching maximal proPLT production and bioreactor saturation within the first 2 hours of culture ( Figure 3C). Primary mouse MKs cultured under physiological shear stress produced fewer, longer proPLTs that were less highly branched relative to static cultures (supplemental Movies 1-2).…”