Abstract
[Background/Aims] In anti-cancer drug (candidate) screening, the demand exists for evaluation at physiological concentrations similar to in vivo. This is often performed by three-dimensionally (3D) cultured cells. It necessitates a long culture period of 2-4 weeks with tedious experimental procedures based on endpoint assay and labeling agents causing low reliability. The previous device methods depend on the pharmaceutical mode of action, little related to the conventional method. Furthermore, a separate set of experiments is required to obtain both efficacy and toxicity. Here, we report on a high precision surface plasmon resonance-3D system to overcome all these problems sensing dynamic cellular reaction against target compound(s) by laser. [Results] We developed the system with average fluctuation of 50 ndeg/s combined with two-dimensionally cultured cells attached onto a sensor chip by applying collagen on the top. The 3D cell activity was shortly obtained by this without cell division. New system gave real-time monitoring of mitochondrial membrane potential (MMP) within live cells without both labeling and invasion. It allowed in vivo-like phenotypic screening for anti-cancer drugs within 1h of drug addition. The data were collected as the stable linear signal change parts for at least 5 min after 25 min following drug addition. The results provided compatibility to clinically related chemosensitivity test (P<0.001) using two cell lines of pancreatic cancer and three anti-cancer drugs to represent differences in individual gene expression and drug mode of action. Early MMP change rate is concluded as a key to quantitatively predict the efficacy and toxicity.
Highlights
Steps for efficacy and toxicity prediction of bioactive compoundsIn vitro cell-based test Animal test Clinical testAnimal test should be avoided based on opinions.Rapid and reliable in vitro cell-based test is demanded for discovery of bioactive compoundsKey is efficacy and toxicity prediction technology extremely before endpoint of test!
We report on the newly proposed HP-SPRHigh Precision-Surface Plasmon Resonance (HP-SPR)-3D system, which evaluates the efficacy of an anti-pancreatic cancer drug at physiological concentrations
SPR angle trend in MIA PaCa-2 cancer cells with 2D and 3D conditioned status exposed to 50nM doxorubicin
Summary
Steps for efficacy and toxicity prediction of bioactive compoundsIn vitro cell-based test Animal test Clinical testAnimal test should be avoided based on opinions.Rapid and reliable in vitro cell-based test is demanded for discovery of bioactive compoundsKey is efficacy and toxicity prediction technology extremely before endpoint of test!. Steps for efficacy and toxicity prediction of bioactive compounds. In vitro cell-based test Animal test Clinical test. Rapid and reliable in vitro cell-based test is demanded for discovery of bioactive compounds. Key is efficacy and toxicity prediction technology extremely before endpoint of test!. In vivo-like label-free cell-based assay is demanded. Measurement of cellular statuses such as extracellular fluxes of H+, O2, and cell morphology changes using impedance and semiconductor during [1-4] days of cocultivation of target compounds with live cells. In vivo-like label-free and non-invasive cell-based assay is demanded. − One of laser spectroscopic methods with non-label and real-time analysis to detect refractive index change derived from interaction between molecules etc. Cell reaction = dielectric constant of a few 100 n/s Detection of live cell reaction − Apply to efficacy prediction of target compounds −. Mitochondrial polarization change rate after addition of a target compound
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