癌症生物学

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.

The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca²⁺ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca²⁺ entry through the plasma membrane leading to Ca²⁺-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca²⁺ flux, and cell viability elicited by HK2pep treatment of tumor cells.

Graphical abstract:

Flowchart for the analysis of the effects of HK2 detachment from MAMs.

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.

Natural killer (NK) cells are the major effectors of the innate immune system when activated resulting in modulation of immune response of the host defense through target cell lysis and secretion of cytokines. Precise functions of NK cells are essential for the treatment outcome of different virus infections and malignant diseases. NK cells impart cytotoxic effect to the target cells lacking MHC class I molecules and thus the final readout of the activity is death of target cells. The NK cell function is evaluated by the ⁵¹Cr-release and/or flow cytometry-based assays. In the present protocol, we have determined the activation of NK cells by the liberation of IL-10 and IFNγ, and subsequently its function by enumerating the number of dead tumor cells originally isolated from the ascitic fluid of ovarian cancer patients. The entire assay is based on cells of the healthy donors and patients. Besides determining function, this method is able to demarcate between NK-cell sensitive and insensitive tumor cells. This technique enables researchers to study NK cell functions in healthy donors or in patients to reveal their impact on different malignancies and to further discover new therapeutic strategies.

Planarians are freshwater flatworms, well known for their ability to regenerate a complete organism from any piece of their body. Furthermore, planarians are constantly growing and degrowing throughout their lives, maintaining a functional and proportioned body. These properties rely on the presence of a population of adult stem cells and on the tight control of their cell renewal, which is based on the balance between the proliferation of new cells and their differentiation, and the death of unnecessary cells. Due to the importance of these two processes in planarian biology, over the years, researchers have optimized molecular techniques to detect both cell proliferation and cell death in planarians. Here, we present the two main protocols currently used for cell death detection and quantification in the planarian field: Caspase-3 activity quantification and TUNEL assay.

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.

Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy is regulated provides an opportunity to explore druggable targets and subsequently develop treatment strategies for these diseases. To identify novel autophagy regulators, we developed an image-based phenotypic RNAi screening approach using autophagic marker proteins at endogenous levels (Jung et al., 2017). In contrast to previously performed autophagy screens, this approach does not use overexpressed, tagged autophagy marker proteins but rather detects autophagic structures at endogenous levels. Furthermore, we monitored early and late phases of autophagy in parallel while other screens employed only a single autophagosome marker mostly GFP-LC3B. Here, we describe this multiplex screening protocol in detail and discuss general considerations about how to establish image-based siRNA screens.

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.

Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or Annexin V. CALR exposure on the cell membrane can be detected by flow cytometry. ATP and HMGB1 release can be quantified by luminescence assay and ELISA assay respectively.