Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in Phase I clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may only be efficacious in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. As such, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe “cytokine storm”. Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFNβ), tumor necrosis factor alpha (TNFα) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.
The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.
Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials are required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV refractive index, epitope abundance, size and concentration. Due to their predominant use among EV researchers, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.
Flow cytometers have been utilized for the analysis of submicron‐sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in‐turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron‐sized vesicles is becoming common‐place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry (FCM) data. In this proof‐of‐concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first‐time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized FCM data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between FCM platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross comparison in standard units. © 2020 International Society for Advancement of Cytometry
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