A Transcriptomics comparison of melanoma cells reveals increased expression of genes regulating cell migration and decreased expression of lysosome-associated genes in metastatic cells, quadruplicate. Differentially expressed genes (pAdj<0.01) were analyzed using gene ontology (GO). Left: Selected GO terms are listed, showing their fold enrichment and log10 FDR in paired comparisons, Right: Heatmaps for two main pathways identified, white = low expression, blue/red = high expression. B, C Lysosome positioning in non-constrained cells, live cell imaging. B Representative images: NucBlue (nucleus), green lysotracker (lysosomes). WM1862 n = 51, WM983A n = 40, WM983B n = 108 cells, in triplicate. C Spreading index, CellProfiler (Mean ± SD) = 3.390 ± 1.597, 4.477 ± 1.180, 4.111 ± 1.225, respectively. p values (from top) = 0.0069, 0.0011, 0.6009, one dot= 1 cell. D, E Lysosome positioning in melanoma cells on micropatterns. D Left: Immunofluorescence (max z-projection), Right: overlay of LAMP1 signal and 2D density maps showing LAMP1 distribution (R software), displaying the smallest area occupied by 25% (red), 50% (orange) and 75% (yellow) of all compartments. E Left: Inter-organelle distance (IOD)= average of distances between all lysosomes. IOD (Mean ± SD) = 10.30 ± 0.94, 11.56 ± 1.15, 13.99 ± 1.40, respectively. One dot= 1 cell, p < 0.0001 in all conditions. Right: Distance to barycenter (DB)= distance from each lysosome to the geometric center (lysosomes). DB (Mean ± SD) = 7.40 ± 0.69, 8.21 ± 0.93, 10.03 ± 1.07, respectively. One dot= 1 cell, p < 0.0001 in all conditions. WM1862, n = 182 cells, WM983A, n = 231 cells, WM983B, n = 82 cells, in triplicate. F, G Electron microscopy reveals that the presence of endolysosomal compartments in protrusions scales with melanoma aggressiveness. F Representative images, endolysosome-rich regions in protrusions are circled in dashed lines, color-coded per melanoma stage. G Area occupied by endo-lysosomes in protrusions: WM1862 n = 14, WM983A n = 24, WM983B n = 15, Area ± SD = 0.1075 ± 0.05614, 1.219 ± 1.474, 2.106 ± 1.457, respectively. One dot= 1 field of view, p values (from top): <0.0001, 0.1672, 0.0001. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All statistics are done using Kruskal-Wallis with Dunn’s multiple comparison post-hoc test. Source data are provided as a Source Data file.

A Samples of patient biopsies were obtained from healthy skin donors (n = 4) and from patients with benign tissue (nevus, n = 7), primary melanoma (n = 6) and metastatic melanoma (skin metastasis, n = 6). Silhouettes (Servier medical art CC:BY 4.0 license) are color-coded according to melanoma stage. B Representative images of patient biopsies sections: samples were labeled for SOX10 (red), LAMP1 (cyan) and nuclei (blue) by immunofluorescence, full tissue section was imaged by slide scanner. C Quantification of lysosome spreading. Ten random regions were analyzed in a blinded setup for lysosome positioning. Lysosome spreading score, range 1–4: (Mean ± SD) = 1.184 ± 0.1276, 1.929 ± 0.3450, 1.656 ± 0.2373, 2.793 ± 0.6094, respectively. One dot represents 1 patient, p values (from top, left) = <0.0001, 0.1544, 0.0087, 0.0218, 0.2298, 0.0010, ordinary one-way ANOVA with Holm-Sidak’s test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. D, E Lysosome spreading shows a spatial heterogeneity within the tumor mass. D Representative images of biopsies from patient 1 (benign tissue, nevus) and patient 2 (metastatic melanoma). Low magnification image mapping the tissue section, stained for LAMP1 (cyan), SOX10 (red) by immunohistochemistry, 2 patients per group. E Zoomed regions from panel (D), showing the progressive change in lysosome spreading with increasing distance to the epidermis, single channel: LAMP1 antibody staining. Red arrows point to clustered lysosomes. Source data are provided as a Source Data file.

A, B Rapalog-mediated perinuclear clustering of lysosomes. A Lysosomes in WM983B cells stably expressing LAMP1-mCherry-FKBP and BicD2-FRB can be clustered using Rapalog compound (Rap) which induces FKBP-FRB rapid heterodimerization. B Representative images of lysosome perinuclear clustering 24 h after 5 nM Rapalog treatment. C Correlative light and electron microscopy of clustered perinuclear LAMP1 positive compartments. Representative immunofluorescent images and a high magnification overlay of fluorescent and electron microscopy images (LAMP1 in red/magenta, nucleus in blue) are shown for each condition, in duplicate. D Melanoma secretome is altered by lysosome perinuclear clustering. Differential quantitative mass spectrometry analysis of proteins secreted to the cell medium by WM983B (FKBP-FRB) cells in the absence or presence of 5 nM Rapalog. Proteins known to be lysosome-associated are labeled with their name. Magenta = proteins upregulated in cells with spread lysosomes, blue = proteins upregulated in cells with clustered lysosomes (p < 0.05). Differential analysis was done using Limma moderated t test with adaptive Benjamini-Hochberg procedure to adjust the p values. E–I Rapalog-induced perinuclear clustering inhibits lysosome exocytosis. E Lysosome secretion was assessed by TIRF microscopy using VAMP7-pHluorin probe, which is quenched in lysosome acidic environment and brightly fluorescent once exposed to the alkaline pH of the extracellular space. F Representative images of co-cultured WM983B-LAMP1-mCherry cells expressing either single heterodimerizing domain (FKBP only, magenta) or both the domains (FKBP-FRB, blue) treated with 25 nM Rapalog, TIRF microscopy. G TIRF movie was divided into three time-segments (black: 0–25 s, green: 25–50 s, dark red: 50–75 s) and displayed as maximum projection showing the number of events per each time-segment. Two representative examples: ROI 1= spread lysosomes, ROI 2= clustered lysosomes. H, I Quantification of the TIRF movie. H Number of lysosome secretion events in 1 min, one dot= 1 cell. I Event duration, one dot= 1 event, n = 5 cells. Events per cell (Mean ± SD): spread = 27.80 ± 7.19, clustered = 14.40 ± 8.23, p value = 0.0397. Event duration (Mean ± SD): spread = 2.46 ± 2.36, clustered = 2.56 ± 2.20 s, p value = 0.3518. Mann–Whitney test, two-tailed with Dunn’s multiple comparison post hoc test. Source data are provided as a Source Data file.

A–C Peripherally positioned lysosomes promote gelatin degradation. A Representative images of WM983B cells (LAMP1-mCherry-FKBP-FRB) with spread lysosomes (medium) and clustered lysosomes (25 nM Rapalog), cultured on FITC-gelatin (gray), stained for cortactin (green), LAMP1 (red) and actin (cyan), in triplicate. B Degradation Area (Mean ± SD) = 24.75 ± 23.29 n = 73 cells, 9.41 ± 9.94 n = 50 cells, respectively, p value: <0.0001, Mann–Whitney test, two-tailed. One dot represents 1 cell. C Degradation frequency (Mean ± SD) = 10.14 ± 10.45 n = 138 cells, 4.43 ± 6.04 n = 102 cells, respectively, p value: <0.0001, Mann–Whitney test, two-tailed. D, E Lysosome perinuclear clustering decreases collagen invasion, live cell imaging. D Bottom: Representative images of WM983B cells with spread lysosomes (FKBP-FRB control and FKBP only with Rapalog 5 nM) and clustered lysosomes (FKBP-FRB with 5 nM Rapalog), nucleus (blue), lysosomes (red). Top: Representative images of cell invasion after 24 h, nucleus (cyan). E Quantification of the invasion index: FKBP-FRB (spread) n = 20, FKBP only (spread) n = 25, FKBP-FRB (clustered) n = 25, (Mean ± SD) = 0.1526 ± 0.094, 0.1592 ± 0.079, 0.0836 ± 0.053, respectively, in triplicate, p value (from top, left) = 0.0067, 0.7728, 0.0023, ordinary one-way ANOVA, Holm-Sidak multiple comparison test. One dot represents 1 field of view. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a Source Data file.

A–C Lysosome clustering prevents tumor cell invasion in mice. Cells with spread (FKBP only, 5 nM Rap) and clustered (FKBP-FRB, 5 nM Rap) lysosomes were injected intradermally in mouse ear. B Representative Immunofluorescence examples of mouse ear sections. Red = tumor cells, green = ear (autofluorescence). C Quantification of cell mass in the ears when lysosomes are spread and clustered (n = 3 mice, 4 tumors per group), calculated from cell area per each section and distance in tumor: (Mean ± SD) = 55.06 ± 14.47, 27.55 ± 17.34, respectively, p value 0.0286, Mann–Whitney test, two-tailed. D–H Perinuclear lysosome clustering prevents cell invasion in zebrafish. D Illustration of the invasion assay in zebrafish. Fli1a:GFP (green endothelium) zebrafish embryos (48 hpf) were injected intra-vascularly with WM983B cells and imaged at day 0 and day 3 post injection to assess the post-extravasation invasion potential of cancer cells with different lysosomal positioning. E Representative confocal images of WM983B cells (stably expressing LAMP1-mCherry= black, and LifeActin-miRFP670= magenta) with spread (FKBP-FRB control; FKBP only 5 nM Rapalog) and clustered (FKBP-FRB, 5 nM Rapalog) lysosomes at 3 h post-injection shown as maximum z projection, in triplicate. F Top: IMARIS segmentation, representative examples at day 0 (3 h after cell injection) and at day 3 (72 h post injection). Bottom: Zoomed regions showing confocal images corresponding to the IMARIS segmentation at day 3. Images are shown as a maximum z projection (LAMP1-mCherry= black, LifeActin-miRFP670= magenta, vasculature= green). G Quantification of cell extravasation at day 0 and day 3. Negative axis= intravascular cells, positive axis= extravascular cells. H Post-extravasation invasion (PEI) potential (day 3) was calculated as a ratio: area of cells that migrated outside of the vasculature region to the total area of cells. FKBP-FRB (spread) n = 23 embryos, FKBP only (spread) n = 12 embryos, FKBP-FRB (clustered) n = 15 embryos, PEI (Mean ± SD) = 0.9616 ± 0.3284, 1.060 ± 0.3316, 0.7085 ± 0.2568, respectively, in triplicate, p values (from top, left) = 0.0317, >0.9999, 0.0172, Kruskal-Wallis with Dunn’s test. One dot= 1 fish, normalized to spread condition. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a Source Data file.