The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus that predominantly spreads through cell-to-cell contact due to the limited infectivity of cell-free viruses. Among various modes of intercellular transmission, HTLV-1 biofilms emerge as adhesive structures, polarized at the cell surface, which encapsulate virions within a protective matrix. This biofilm is supposed to facilitate simultaneous virion delivery during infection. Yet, the molecular and functional intricacies of viral biofilms remain largely unexplored, despite their pivotal role in understanding retroviral pathogenesis. In this study, we optimized a protocol to isolate HTLV-1 biofilms from chronically infected T cells, facilitating their structural and molecular characterization using proteomic and super-resolution microscopy analyses. This protocol involves cultivating HTLV-1 chronically infected T cells at high density to facilitate the natural detachment of viral biofilms into the supernatant. Then, employing successive centrifugations, the cells are separated from the detached biofilms, and these structures are pelleted at medium speed (10,000× g). This method circumvents the need for mechanical, chemical, or enzymatic biofilm detachment, bypasses the use of ultracentrifugation, and enables us to resuspend the biofilms in the appropriate buffer for subsequent analyses such as western blotting or super-resolution microscopy imaging as presented.

The quality of cellular products used in biological research can impact the accuracy of results. Epstein–Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. Existing tests to detect EBV can be divided into three categories: nucleic acid assays, serological assays, and in situ hybridization assays. However, most methods are time-consuming, expensive, and not conducive to high-volume clinical screening. Therefore, a simple system that allows for the rapid detection of EBV in multiple contexts, including both cell culture and tissue samples, remains necessary. In our research, we developed EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 10³ copy numbers for the RPA-based and RPA-LFA systems and 1 × 10⁴ copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those of the assays specified in industry standards. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet–tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet–tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTracker^TM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet–tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader–based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet–tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well.

Key features

• Analysis of platelet–tumor cell binding dynamics.

• In vitro methods developed for measuring platelet–tumor cell binding to enable rapid testing of antiplatelet and other compounds.

• Complementary analysis of platelet–tumor cell binding by imaging and fluorimetry-based readings.

• Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet–tumor cell binding using the protocol.

• Validation results were presented with both a TPr agonist and ifetroban (antagonist).

Graphical overview

Representative overview of the process to isolate and label platelets, incubate platelets and tumor cells in the presence of antiplatelet agents, and image and/or quantify platelet–tumor cell interactions.