Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumo
Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological process
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Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells Mariela Navarrete, Flavio Salazar-Onfray, and Andre´s Tittarelli Abstract Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells. Key words Intercellular communications, Gap junctions, Cytotoxic T lymphocytes, Tumor cells, Immunological synapse, Calcein transfer, Flow cytometry, Melanoma
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Introduction Cancer cells isolate themselves from their cellular environment by downregulating gap junction-mediated intercellular communications (GJIC) [1]. The study of GJIC and the proteins that built them, the connexins (Cx), in cancer tumorigenesis, progression, and therapy is an exciting and promising biomedical research topic [2]. Gap junctions (GJs) are clusters of intercellular channels that allow the direct communication between the cytoplasm of adjacent cells. Each GJ channel is formed by two hexameric connexons, which are hemichannels of Cx proteins (a family of 21 members in humans and 20 in mice), one provided by each of the two contacting cells [3]. Once functional GJs are established, they allow the bidirectional transfer of small (up to 1.4 nm) molecules of varied nature, including inositol triphosphate, Ca2+, peptides, microRNAs, and cyclic guanosine monophosphate-adenosine (cGAMP) [4–6]. The molecular permeability of Cx channels is usually assessed using several classes of non-biological fluorescent molecular tracers, such as calcein, ethidium iodide (bromide), propidium iodide, and Lucifer yellow, among others [7]. GJICs suppress tumor progression by different mechanisms that impact the cancer-immunity cycle, such as (1) spreading and
Mariela Navarrete et al.
amplification of tumor-associated antigen (TAA) crosspresentation pathways [8–12], (2) increasing tumor immunogenicity through the propagation of cGAMP-mediated signaling [13], (3) potentiating dendritic cell (DC)-mediated activation of TAA-specific T cells and natural killer (NK) cells [14–16], and (4) contributing to granzyme B-mediated tumor cell killing by NK cells and cytotoxic T lymphocytes (CTLs) [16–18]. We identified that GJs are important components of the immunological synapses and allow the cell-to-cell communication between cytolytic immune cells (NK cells, CTLs) and target tumor cells [6]. Here, we adapted a previously reported method [19], to quantitatively measure GJ coupling among target tumor cells and CTL during the formation of cytotoxic immunological synapse by flow cytometry. This method could be easily adapted to evaluate multiple variables in the interacting cells given that current fluorescen
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