Abstract In vitro monitoring cancer cells response to treatment often involves laborious sample processing and collects single data points that are against the dynamic nature of cancer cells. Here we present the adaptation of an impedance-based methodology to dynamically monitor cancer cell behavior and therapeutic response. The technology detects cell death, proliferation or migration by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. It avoids sample labeling and processing and allows continuous monitoring of cell response. Our data shows validation with cancer cell lines and primary cells monitored for cell invasion/migration through transwells or after a scratch has been performed with a dedicated device, anticancer drug response and receptor signaling activation, with similar results over end point assays. Easy experiment set up and minimal sample processing make the technology ideal for applications in large screening campaigns, while label-free technology allows less artifacts and further analysis on the same samples through orthogonal assays. Citation Format: Fabio Cerignoli, Biao Xi, Brandon Lamarche, Leyna Zhao, Yama Abassi. Label-free impedance analysis as a versatile tool for dynamic screening of cancer molecular therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2159.
In this paper we report the fabrication of a multivalent, cell-type specific and cytoplasmic delivery system based on single-walled carbon nanotubes. The latter were functionalized through adsorption of phospholipids terminated by biotinylated PEG chains functionalized with fluorochrome-coupled neutravidin, and subsequently with antibodies (anti-CD3 ε and anti-CD28) for T cell receptor post-signaling endocytosis and a synthetic fusogenic polymer for disruption of lysosomal compartments. The biomimetic nanoassemblies were composed by PEGylated individual/very small bundles of carbon nanotubes having an average length and a standard deviation of 176 nm and 77 nm, respectively. The nanoassemblies were stably dispersed under physiological conditions, visible by conventional optical and confocal microscopy and specifically targeted to T cells both in vitro and in living animals. The addition of a fusogenic polymer to the nanoassemblies did not affect the cellular uptake and allowed the release into the cytosol of the targeted cells both in vitro and in the animals. The present manuscript is the first report about the cytoplasmic delivery of carbon nanotubes in a specific cell type in intact animals and paves the way for their use as in vivo intracellular delivery systems.
Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response. To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor). To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector. Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.
Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.
Abstract In vitro characterization of reagents efficacy in the context of cancer immunotherapy for solid tumors is a necessary step before moving to more expensive animal models and clinical studies. However, current in vitro assays are difficult to implement in high throughput environment and are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent cell lines and primary tumor cells with different expression levels of the EpCAM antigen, which is currently under evaluation as a target for cancer immunotherapy. Using PBMCs as effector cells and combinations of EpCAM/CD3 BiTE we demonstrated the suitability of such impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint inhibitors were also effective when evaluated with the same approach. Finally, we validated the technology with engineered Car-T cells directed against common antigens expressed in tumors. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays like Chromium 51 release or ATP-based luminescence. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in personalized medicine, like therapeutic protocol validation directly on patient samples. Citation Format: Fabio Cerignoli, Biao Xi, Garret Guenther, Trent Rector, Lincoln Muir, Leyna Zhao, Yama Abassi. In vitro testing solid cancers immunotherapy protocols through impedance technology allows dynamic and label-free evaluation of immune response and reagent efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B093.
The bone marrow is a frequent location of primary relapse after conventional cytotoxic drug treatment of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Because stromal cells have a major role in promoting chemotherapy resistance, they should be included to more realistically model in vitro drug treatment. Here we validated a novel application of the xCELLigence system as a continuous co-culture to assess long-term effects of drug treatment on BCP-ALL cells. We found that bone marrow OP9 stromal cells adhere to the electrodes but are progressively displaced by dividing patient-derived BCP-ALL cells, resulting in reduction of impedance over time. Death of BCP-ALL cells due to drug treatment results in re-adherence of the stromal cells to the electrodes, increasing impedance. Importantly, vincristine inhibited proliferation of sensitive BCP-ALL cells in a dose-dependent manner, correlating with increased impedance. This system was able to discriminate sensitivity of two relapsed Philadelphia chromosome (Ph) positive ALLs to four different targeted kinase inhibitors. Moreover, differences in sensitivity of two CRLF2 -drivenBCP-ALL cell lines to ruxolitinib were also seen. These results show that impedance can be used as a novel approach to monitor drug treatment and sensitivity of primary BCP-ALL cells in the presence of protective microenvironmental cells.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.
