Journal: bioRxiv

Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

doi: 10.1101/2020.08.24.265140

Figure Lengend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

Techniques: CRISPR, Labeling, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

doi: 10.1101/2020.08.24.265140

Figure Lengend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

Techniques: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

doi: 10.1101/2020.08.24.265140

Figure Lengend Snippet: (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.

Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

Techniques: CRISPR, Expressing, RNA Sequencing Assay, Western Blot

Journal: bioRxiv

Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

doi: 10.1101/2020.08.24.265140

Figure Lengend Snippet: a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).

Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

Techniques: RNA Sequencing Assay, Expressing, Immunofluorescence, CRISPR

Journal: bioRxiv

Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

doi: 10.1101/2020.08.24.265140

Figure Lengend Snippet: (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).

Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

Techniques: CRISPR