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Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma., Significance: This work describes a readily implementable method that allows for the isolation of individual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus. At the synapse, dynein-derived forces induce negatively curved membrane pockets to which granules are transported around the nucleus. These highly concave degranulation pockets are located directly opposite positively curved bulges on the target cell membrane. We identify a curvature bias in the action of perforin, which preferentially perforates positively curved tumor cell membrane. Together, these findings demonstrate murine and human T cell-mediated cytotoxicity to be a highly tuned mechano-biochemical system, in which the forces that polarize lytic granules locally bend the synaptic membrane to favor the unidirectional perforation of the target cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes., Competing Interests: JG, SP, ST, FC, DK, JH, JM, JC, MG, LD, KH, SR, JZ, GR, VP, GO, RW, DN, BK, AW, FL, MR, MB No competing interests declared, (© 2020, Galeano Niño et al.)