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Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning

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Science Advances  24 Jul 2020:
Vol. 6, no. 30, eaaz6197
DOI: 10.1126/sciadv.aaz6197
  • Fig. 1 DNA methylation heterogeneity reveals that MYO10 is differentially methylated and overexpressed in leader cells.

    (A) Heat map of MethylationEPIC array beta values for the 5000 most differentially methylated CpG probes (DMPs). (B) Venn diagram of differentially methylated regions (DMRs) with a beta difference ≥ 0.2 and adjusted P < 0.01. (C) Annotation of DMPs across genomic features. (D and E) Heat maps, z scores from log 2–normalized RNA-seq expression counts of most differentially expressed (DE) genes. (D) 98th percentile genes (N = 499) scaled by row and column. (E) Subset of the 15 most DE genes, without clustering. (F) Scatter plot of promoter CpG island (CGI) methylation beta differences and RNA-seq log 2 fold changes for all genes that are both differentially expressed (≥twofold difference, P < 0.01) and differentially methylated at the CGI (≥0.2 difference) between leaders and followers. (G) Violin plots of beta values for CpGs within the MYO10 TS1500 promoter (N = 18 probes) or MYO10 gene body (N = 95 probes). Kruskal-Wallis test with Dunn’s correction. (H) MYO10 expression by RNA-seq (left) or quantitative polymerase chain reaction (qPCR; right). Ordinary one-way analysis of variance (ANOVA) with Tukey’s correction. (I) Western blot, MYO10, actin as loading control. n = 5. (J and K) MYO10 immunofluorescence, follower and leader cells (J) or H1299, H1792, and H1975 NSCLC cells (K). Scale bars, 5 μm; representative images from n = 3, N ≥ 30 cells per cell type. (L and M) MYO10 immunofluorescence, 3D spheroid invasion of H1299 parental, follower, and leader cells (L) or of H1299, H1792, and H1975 NSCLC cells (M). Fire lookup table represents MYO10 signal intensity. Scale bars, 10 μm. (A to M) Unless noted, n = 3. Par, parental; F, followers. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 2 MYO10 regulates cell motility and collective invasion.

    (A) Violin plot of filopodia lengths during 3D invasion of followers, si-control (siCtrl) leaders, and siMYO10 leaders. Red line, median, dashed black lines, interquartile range. Kruskal-Wallis test with Dunn’s correction for multiple comparisons. n.s., not significant. (B) Representative images from live-cell imaging of LifeAct-RFP filopodia dynamics in leaders expressing siCtrl or siMYO10. Scale bars, 10 μm. (C) Violin plot of filopodia lifetimes during 3D spheroid invasion of siCtrl leaders and siMYO10 leaders. Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (D) Violin plot of filopodia lifetimes in 2D of siCtrl leaders and siMYO10 leaders. Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (E and F) Live-cell tracking analysis of 2D cell migration of leaders expressing siCtrl or siMYO10 (E) or in followers expressing a control mCherry vector or mCherry-MYO10 (F). (G) Representative images and quantification of 72-hour spheroid invasion assays H1299 leaders expressing siCtrl or one of two MYO10 siRNAs (siMYO10-1 and siMYO10-2). Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (H) Representative images and quantification of 72-hour spheroid invasion assays of parental H1299, H1792, and H1975 cells expressing siCtrl one of two MYO10 siRNAs (siMYO10-1 and siMYO10-2). Two-way ANOVA with Sidak’s multiple comparison test. Effect of the siRNA indicated to the right of the key. Representative images and quantification of 48-hour invasion assays in Matrigel of follower cells expressing a control empty vector or mCherry-MYO10. Mann-Whitney U test, two-tailed. For all panels: unless noted, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 JAG1 signaling is upstream of MYO10 expression in leader cells.

    (A) RNA-seq normalized gene counts for the canonical Notch receptors and ligands. Ordinary two-way ANOVA with Tukey’s correction. n = 3. (B) Western blots, H1299 parental, follower, and leader cells and H1299 cells FACS sorted for JAG1-low/IL13RA2+ (JLOWIL+) or JAG1-high/IL13RA2 (JHIGHIL). Probed for MYO10, JAG1 full-length (FL), JAG1 intracellular domain (ICD), and IL13RA2. n = 3. (C) JAG1 immunofluorescence of 3D invasion of H1299 parental, follower, or leader cell spheroids. Scale bars, 50 μm. n = 3. (D) qPCR, leader cell mRNA expressing shCtrl or shJAG1, normalized to actin. n = 3. (E) Western blots of JAG1 and MYO10 expressions in H1299 leaders expressing shCtrl or shJAG1. n = 3. (F) Western blots of JAG1 and MYO10 in H1299 leaders (L), followers expressing an empty vector (F Ctrl) or JAG1 (F JAG1). n = 4. (G) Quantification of MYO10 methylation status using qPCR. n = 3. (H and I) Violin plots quantifying 2D filopodia length with representative images of leader cell filopodia in leaders expressing shRNA-JAG1 and/or mCherry-MYO10 (H) or followers expressing JAG1 and/or mCherry-MYO10 (I), n = 2, Kruskal-Wallis test with Dunn’s multiple comparison test. Scale bars, 10 mm. (J) 3D spheroid invasion of leaders expressing shRNA-JAG1 and/or mCherry-MYO10. n = 4. (K) Representative images and quantification of 3D collective spheroid invasion follower cells expressing JAG1 and/or mCherry-MYO10. n = 3. (B, E, and F) Actin as loading control. (D, G, J, and K) Ordinary one-way ANOVA with Tukey’s correction. (A to K) n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 MYO10 regulates alignment of extracellular FN at the leading edge of 3D invasion.

    (A) RNA-seq gene counts of FN (FN1) in parental (P), follower (F), and leader (L) cells. Ordinary one-way ANOVA with Tukey’s correction. (B) Peptide spectra match (PSM) semiquantitative count for FN1 detected by liquid chromatography–mass spectrometry (LC-MS/MS) in media collected from parental, follower, and leader cells. n = 1 replicate per condition. (C) Scatter plot of PSM values for all peptides identified by LC-MS/MS secreted by leader (magenta) or follower (green) cells. Diagonal line indicates linear regression (R2 = 0.45); dashed lines indicate 95% prediction bands. PSMs shown are the sum of all isoforms for each protein minus the PSM for serum-free RPMI 1640 alone. n = 1 replicate per condition. (D) Immunofluorescence imaging of MYO10 and FN1 in spheroid invasion assays of leader cells expressing a control siRNA or MYO10 siRNA. Actin stained with phalloidin. Scale bars, 10 μm. (E) Quantification of extracellular FN1 immunofluorescence signal in (D). For siCtrl: n = 5. For siMYO10-1: n = 2. For siMYO10-2: n = 3. Kruskal-Wallis test with Dunn’s multiple comparison test. (F) Immunofluorescence imaging of leader cell spheroid invasion assays. Scale bar, 5 μm. (G) Violin plot of quantification of filopodia length for leader cell filopodia associated with MYO10 and/or FN1. n = 3. Kruskal-Wallis test with Dunn’s multiple comparison test. (A to G): *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 Follower cells lack the ability to align extracellular FN.

    (A) Representative FN1 immunofluorescence image and optical zooms of a H1299 leader cell spheroid invading through Matrigel. Actin was stained with phalloidin. Box indicates location of zoom. Arrowheads indicate locations of linear extracellular FN micropatterning (B) Representative FN1 immunofluorescence images and optical zoom of a representative H1299 follower cell spheroid cultured in Matrigel. (C) Representative FN1 immunofluorescence images and optical zoom of a H1299 follower cell spheroid cultured in Matrigel mixed with human plasma FN (10 μg/ml). (A to C) Box indicates location of zoom. n = 3, N = 18 spheroids imaged per condition. Scale bars, 20 μm.

  • Fig. 6 MYO10 drives leader cell filopodia persistence and FN micropatterning during 3D collective invasion.

    (A) Live-cell imaging, leader cell spheroid expressing membrane-bound Dendra2 (red) embedded into rBM + FN-rhodamine (10 μg/ml) (cyan). Arrows, linear FN micropatterning. Scale bar, 50 μm. (B and C) Representative kymograph (B) and time lapse (C) of a single filopodium. Arrow, filopodium tip pause; arrowhead, nascent FN fibril appears; asterisk, filopodia retraction. Scale bars, 2 μm. (A to C) n = 5, N = 112. (D) Live-cell image, H1299 cell spheroids expressing GFP-MYO10 (green) embedded into rBM + FN-rhodamine (10 μg/ml) (magenta). Cyan box, zoom in of (E); yellow box, zoom in of (F). Scale bar, 10 μm. (G) Time lapse of the filopodium in (E). Arrow, filopodium pause; arrowheads, boundaries of nascent FN puncta; asterisk, filopodia retraction; double asterisk, second extension of the filopodia. Scale bars, 2 μm. (H) Line plot of average fluorescence intensity of GFP-MYO10 and FN-rhodamine along H1299 GFP-MYO10 filopodia during invasion. (I) Violin plot of distance (in micrometer) between the peaks of MYO10-GFP and FN-rhodamine intensity. (J) Heat map of distance-normalized line plot intensity values. (D to J) n = 5, N = 92. (K and L) Violin plots of filopodia lifetimes from H1299 GFP-MYO10 spheroids, n = 4, N = 347 (K); or leader cell spheroids, n = 3, N = 344 (L), comparing filopodia associated with FN-rhodamine fibrillogenesis and filopodia that were not. Mann-Whitney U test, two-tailed. (M) Proportion of filopodia lifetimes, leader cells expressing siCtrl or siMYO10. Two-tailed Fisher’s exact test. n = 3, N = 824. (A to M) N = number of filopodia, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • Supplementary Materials

    Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning

    Emily R. Summerbell, Janna K. Mouw, Joshua S. K. Bell, Christina M. Knippler, Brian Pedro, Jamie L. Arnst, Tala O. Khatib, Rachel Commander, Benjamin G. Barwick, Jessica Konen, Bhakti Dwivedi, Sandra Seby, Jeanne Kowalski, Paula M. Vertino, Adam I. Marcus

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