Cancer Biology

Entosis is a process where a living cell launches an invasion into another living cell’s cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining.

Silver nanoparticles have been widely studied to possess antimicrobial as well as anticancer activity, and have found its applications in various fields including pharmaceutical industry, diagnostics, drug delivery, food industry, and others. For this purpose, several cell proliferation assays are widely used for the evaluation of anticancer activity of synthetic compounds as well as natural plant extracts. In general, a compound is said to possess an anticancer activity if it prevents the cancer cells to grow and divide actively, and indirectly activates the generic program of cell death. In this protocol, Alamar blue and MTT assay are described for the analysis of metabolic function and health of the cell. These procedures are generally used for the endpoint analysis. A549 cells are seeded in a 96-well plate, and after the adherence of the cells, they are treated with different concentrations of silver nanoparticles. Followed by 24 h of incubation, colorimetric dyes are added to the wells, and the absorbance is recorded to quantify the percentage cytotoxicity in the sample wells.

One fundamental property of the TNR receptor (TNFR) family relates to how ‘signal quality’ (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., agonistic anti-CD40 antibody), induces high level of pro-inflammatory cytokine secretion and causes extensive cell death (apoptosis) in malignant (but not normal) epithelial cells. In this article, we describe a co-culture system for the activation of CD40 by mCD40L and subsequent detection of various features of apoptosis (including cell membrane permeabilisation, DNA fragmentation, caspase activation) as well as detection of intracellular mediators of cell death (including adaptor proteins, pro-apoptotic kinases and reactive oxygen species, ROS).

In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death.

Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTracker^TM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.

This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

Detection of senescent cells using a cytochemical assay was first described in 1995 (Dimri et al., 1995). The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity (Kurz et al., 2000). Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized in the lysosome. The enzymatic activity can be detected at the optimal pH 4.0, using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Miller, 1972). In comparison, upon senescence, the lysosomal mass is increased, leading to production of a higher level of β-galactosidase, termed senescence-associated β-galactosidase (SA-β-gal) (Kurz et al., 2000). The abundant senescence-associated enzyme is detectable over background despite the less favorable pH conditions (pH 6.0) (Dimri et al., 1995). The SA-β gal positive cells stain blue-green, which can be scored under bright-field microscopy. In this assay it is best to avoid over-confluency of the cells, or cells that have undergone too many passages, as these conditions can cause false positive results.

This protocol utilizes PicoGreen 96-well plate technology. This method is applied to estimate the sensitivity of different tumor cell lines to chemodrugs.

Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version (Franken et al., 2006).