RESUMO
Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.
Assuntos
Antraciclinas/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Bioensaio/métodos , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Impedância Elétrica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Microscopia Intravital/métodos , Miócitos Cardíacos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Imagem com Lapso de TempoRESUMO
Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.