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Confinement promotes drug tolerance by downregulating proliferation. a, Schematic detailing the influence of confinement on melanoma phenotype switching. b, Enrichment scores for GO:BP pathways related to cell division from RNA-seq of confined cells relative to unconfined cells. FDR is indicated. c, Stills from confocal imaging of A375 cells stably expressing the FastFUCCI reporter and stained with SiR-tubulin. d, Quantification of FUCCI signal over time in confined cells; n = 37 cells from four videos. Error bars, s.e.m. e, Schematic of in vitro invasion assay workflow. f,h, Western blot for HMGB2 (top) and tubulin (f; loading control; bottom) or β-actin (h; loading control; bottom) for A375 cells transfected with the indicated siRNAs (f) or plasmids (h). siHMGB2, HMGB2-targeting siRNA. g,i, Quantification of in vitro invasion assay results for A375 transfected with the indicated siRNAs (g) or plasmids (i); n = 3 biological replicates. P value is indicated (two-sample t-test; two-sided). EV, empty vector; OE, overexpression (of HMGB2). j, Representative images of adult zebrafish with melanomas generated by means of Transgene Electroporation in Adult Zebrafish (TEAZ), 12 weeks after electroporation. NT, non-targeting; sgRNA, single-guide RNA. k, Tumour surface area over time for melanomas induced in zebrafish using TEAZ. Error bars, s.e.m. l, Tumour surface area at 12 weeks after TEAZ. P value is indicated (two-sample t-test; two-sided). Horizontal line, median; box edges, 25th and 75th percentiles; whiskers, data range. NT sgRNA, n = 10 fish; sgRNA targeting HMGB2 (sgHMGB2), n = 8 fish (j–l). m, Representative haematoxylin and eosin (H&E) images of tumour invasion in the indicated conditions at 12 weeks after TEAZ. n, Schematic of the drug treatment experiment workflow. o, Tumour volume over time for the indicated conditions; n = 11 mice per condition from two biological replicates. Error bars, s.e.m. *P < 0.05. P value calculated using likelihood ratio tests with a fitted exponential; two-sided (P = 0.043). Scale bars, 25 µm (c), 2 mm (j), 250 µm (m). Illustrations in e were created using BioRender (https://biorender.com). Mouse cartoons in n were adapted from Wikimedia, under a Creative Commons licence CC BY-SA 3.0. Source Data
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