anti dr tfap2e  (Cell Signaling Technology Inc)

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: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

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: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

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: a . Schematic of experiment. Prior to flow cytometry and 10X single-cell RNA-seq, ZMEL1-PRO and -INV cells were either grown in vitro (individual or co-culture) or isolated from zebrafish orthotopically transplanted with a 1:1 mixture of the two subpopulations (primary tumors or metastases). b . Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). c . Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of 40,293 ZMEL1 cells sequenced as in (a). Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET). d . PRO and INV scores based on ZMEL1 bulk RNA-seq are plotted for all cells in gray. ZMEL1-PRO (purple) and ZMEL1-INV (green) for the indicated condition are colored. Diagonal line represents the classifier used in (e). e . Confusion matrices comparing initial cell identity with observed cell classification based on a linear classifier trained on in vitro individual culture samples. f . ZMEL1-INV cells plotted as in (d) colored according to tfap2e mRNA expression reveal re-activation of tfap2e upon metastatic dissemination.

Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

Techniques: Flow Cytometry, RNA Sequencing Assay, In Vitro, Co-Culture Assay, Isolation, Expressing, Activation Assay

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 . Single-cell expression of tfap2e in ZMEL1-PRO and -INV cells. Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET).

Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

Techniques: Expressing, Co-Culture Assay