human plk1 cdna carcer  (Addgene inc)

Journal: Cell reports

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer.

doi: 10.1016/j.celrep.2023.113266

Figure Lengend Snippet: Figure 1. Plk1 overexpression drives inflammatory monocyte recruitment as a consequence of the SASP (A) Schematic showing the mouse models used in this study, where Her2 mammary tumors show low levels of CIN and Her2-Plk1 overexpression results in high CIN tumors. (B) Average SCNAs in 10 Her2 and 10 Her2-Plk1 tumors; unpaired t test, p = 0.0004. (C) SCNAs in 10 Her2 and 10 Her2-Plk1 mammary tumors. Shown are whole-chromosome gain (WCG) and whole-chromosome loss (WCL), focal amplification (AMP), deletion (DEL) and gross chromosomal rearrangement (GCR). (D) Total transcriptome of Her2 and Her2-Plk1 tumor tissues was analyzed by bulk RNA-seq. Heatmap showing differentially expressed cell cycle and SASP- related genes (fold change [FC] > 1.5 or < 1.5, p < 0.05, n = 3 tumors per genotype).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Gene expression data Breast cancer METABRIC cohort, Pereira et al.50 https://cbioportal-datahub.s3. amazonaws.com/brca_metabric.tar.gz Experimental models: Cell lines MCF-7 Obtained from Guillermo de Cárcer Laboratory N/A Cal51 Obtained from Uri-Ben David Laboratory N/A Experimental models: Organisms/strains Mouse: TetO-Her2/Plk1/MMTV-rtTA, FVB Carcer et al.7 https://doi.org/10.1038/s41467-018-05429-5 Oligonucleotides Mycoplasma antisense primer MGSO (50-TGCACCATCTGTCACTCTGTTA ACCTC-30), (10 mM) Integrated DNA Technologies https://doi.org/10.1038/nprot.2010.43 Mycoplasma sense primer GPO-3 (50-GGGAGCAAACAGGATTAGATA CCCT-30), (10 mM) Integrated DNA Technologies https://doi.org/10.1038/nprot.2010.43 Recombinant DNA pLenti CMVtight Hygro DEST Addgene #26433 Human PLK1 cDNA Carcer et al.7 https://doi.org/10.1038/s41467-018-05429-5 Software and algorithms GraphPad Prism 8 and 9 GraphPad Software https://www.graphpad.com/ scientific-software/prism/ FlowJo ImageJ software FlowJo https://imagej.net/plugins/flowj StrataQuest software TissueGnostics https://tissuegnostics.com/products/ contextual-image-analysis/strataquest R, Version 4.3.0 R Project for Statistical Computing https://www.r-project.org/ Seurat Hao et al.58; https://satijalab.org/seurat/ https://cloud.r-project.org/web/ packages/Seurat/index.html SingleR Aran et al.29 https://bioconductor.org/packages/ release/bioc/html/SingleR.html clusterProfiler Yu et al.59 https://bioconductor.org/packages/ release/bioc/html/clusterProfiler.html Python Python Software Foundation https://www.python.org/ matplotlib Hunter et al.60 https://matplotlib.org/

Techniques: Over Expression, RNA Sequencing

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic representation showing the mouse models used in this study, where Her2 mammary tumors show low levels of CIN and Her2-Plk1 overexpression results in high CIN tumors. B ) Average SCNAs in Her2 and Her2/Plk1 tumors; Unpaired t test, p = 0.012. C ) SCNAs in 10 Her2 and 10 Her2/Plk1 mammary tumors. Shown are whole chromosome gain (WCG) and loss (WCL), partial chromosome gain (PCG) and loss (PCL), focal amplification (AMP), deletion (DEL) and gross chromosomal rearrangement (GCR). D) Total transcriptome of Her2 and Her2-Plk1 tumor tissues was analyzed by bulk RNA-sequencing. Heatmap showing differentially expressed cell cycle and SASP-related genes (Fold change (FC) > 1.5 or < −1.5, p <0.05, n=3 tumors per genotype). E) GSEA using gene sets from KEGG pathways as analyzed by cluster profiler. Pathways overrepresented in Her2-Plk1 tumors are shown in yellow, whereas pathways overrepresented in Her2 tumors are displayed in blue. F) Fluorometric measurement of ß-galactosidase activity in Her2 and Her2-Plk1 tumor lysates (Mann Whitney test p<0.005**, n=10 (Her2) and n=16 (Her2-Plk1)). G) Cytokine array of tumor lysates of Her2 and Her2-Plk1 showing increased CXCL1, IL16 and IL1RN in the high CIN group (p<0.05* p<0.0005***, 2-way ANOVA, Sidak’s multiple comparison test, n=3). H) Characterization of TAM’s and monocytes. Live CD45 + CD11b + cells were divided into two groups based on Ly6C and MHCII. Flow cytometric analysis of inflammatory monocytes (Ly6c high MHCII low ) and M1 TAMs (Ly6c low MHCII high) in endpoint tumors (p<0.05*, n=5 (Her2) and n=8 (Her2-Plk1)).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Over Expression, Amplification, RNA Sequencing, Activity Assay, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Expression of P38a and NF-kB-p65 in breast tumors from Her2 and Her2-Plk1 mice. Tumor cell lysates were used for immunodetection by using anti-PLK1, anti-NFkB-p65, anti-RELB, anti-P38, anti-pP38 and anti-vinculin as a loading control. Bar graphs represent the relative intensity of the signal from immunodetection (*, p<0.05, **, p<0.005, Mann-Whitney test, n=11 (Her2), n=14 (Her2-Plk1); numbers represent different tumors). B) Immunohistochemistry of PLK1, PD-L1 and CD206 in Her2 and Her2-Plk1 tumor sections and their corresponding bar graphs representing total sum area (μm 2 ). Positive regions per sample was calculated by dividing DAB positive area in each ROI/total area of the ROI (region of interest) and total sum area (μm 2 ) and adding positive regions in all the individual samples. Scale bar: 100μm. (*, p<0.05, **** p< 0.0001, Mann-Whitney test, n=5 (Her2), n=8 (Her2-Plk1)). C) Quantification of tumor-infiltrating lymphocytes in Her2 and Her2-Plk1 tumors and spleens. Representative two-parameter dot plots of CD45 + gated Tregs (CD25 + CD4 + Foxp3 + ), dendritic cells (CD11c + MHCII high ), NK cells (CD11c − Nkp46/Nk1.1 + )) and T cells (CD3 + CD4 + , CD3 + CD8 + ) in both spleen (n=5 (Her2), n=7 (Her2-Plk1)) and tumors (*, p<0.05, ** p<0.005, ***, p< 0.0005, Mann-Whitney test n=6 (Her2), n=10 (Her2-Plk1)) and bar graphs represented as % of CD45 positive cells. The numbers depicted in the boxes refer to the percentage of each cell type in different samples (Her2 and Her2-Plk1).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Expressing, Immunodetection, Control, MANN-WHITNEY, Immunohistochemistry

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic depiction of isolation of hyperplastic mammary glands and FACS sorting of individual cells to obtain CD45 hematopoietic cells at early stage Her2 (16 days on doxycycline) and Her2-Plk1 (22 days on doxycycline) samples. Samples were extracted from three different mice per genotype from two mammary glands each. B) Clustered immune cell populations displayed by UMAP in all samples. The cell types in the clusters were annotated by using SingleR and gene expression information from the ImmGen reference database and displayed in a color-coded fashion. C) Volcano graph showing differentially expressed genes in the CD11b + CD11c − macrophage subtype with log2-fold change in gene expression versus -log10 of adjusted p-values. Genes that were found to be downregulated in Her2-Plk1 with respect to Her2 are shown in blue, while genes that were upregulated are shown in yellow (by a fold change of at least ±0.6 and an adjusted p-value of ≤0.05). D) Violin graphs showing the normalized expression levels of CD11b + CD24 − macrophages for genes related to antigen presentation, EMT and anti-inflammatory wound-healing phenotypes. Her2 samples are in blue and Her2-Plk1 in yellow. p-values adjusted for multiple testing are shown above each graph. Each dot represents the gene expression level of an individual cell.

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Isolation, Gene Expression, Expressing, Immunopeptidomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A ) Survival analysis of Her2 (left) and Her2-Plk1 (right) tumors of mice treated with NK1.1 blocking antibody. Survival probability over time (upper graphs) and number and percentage of mice alive over time (lower table). The curves in light blue and light yellow depict untreated mice, whereas the curves in dark blue and dark yellow show mice treated with NK block, respectively. B) Normalized gene expression of CD27 versus Itgam ( CD11b ) in NK cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. C) Violin graphs showing the log-normalized gene expression values of NK cells in Her2 (blue) and Her2-Plk1 (yellow) tumors exemplarily shown for genes involved in cytotoxic response and surface receptors of NK cells. D) Heatmap showing normalized gene expression levels of differentially expressed effector genes, chemokines and their receptors, and activating receptor genes in NK cells. Each line represents a single cell. E) Gene set enrichment analysis for identification of pathways with enrichment of differentially expressed genes for two macrophage subsets and NK cells. Y-axis: hallmark signatures and X-axis: normalized enrichment scores with adjusted p-values, color-coded by the direction of the effect (yellow: upregulation in Her2-Plk1, blue: upregulation in Her2).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Blocking Assay, Gene Expression, Quantitative Proteomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Volcano graph with reduced axes to focus on selected differentially expressed genes (labeled) in the B cell subset of Her2 and Her2-Plk1 tumors displayed as log2 fold change versus - log10 of the adjusted p-value. Genes that were found to be downregulated in Her2-Plk1 with respect to Her2 are shown in blue, while genes that were overexpressed are shown in yellow (by a fold change of at least ±0.4 and an adjusted p-value of ≤0.05). B) UMAP graph of T cells clustered into different T cell subtypes. Seven clusters with different cell types were identified after cell type annotation. C ) Relative distribution of the number of cells in each of the T cell subtypes with Her2 in blue and Her2-Plk1 in yellow. D) Normalized gene expression of Sell ( Cd62L ) versus Ccr7 in regulatory T cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. E) Volcano graph with reduced axes to focus on selected differentially expressed genes in the cluster of regulatory T cells displayed as log2 fold change versus -log10 of the adjusted p-value. Genes downregulated in Her2-Plk1 compared to Her2 are shown in blue, genes upregulated in Her2-Plk1 are displayed in yellow (by a fold change of at least ±0.4 and an adjusted p-value of ≤0.05).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Labeling, Gene Expression, Quantitative Proteomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic description of sorting PLK1 high versus PLK1 low tumors from the TCGA-BRCA cohort. A total of 186 (PLK1 high) and 165 (PLK1 low) tumors with a Z-score of +1 and –1, respectively, were identified. B) Unsupervised clustering of PLK1 high versus PLK1 low tumors using principal component analysis. Samples color-coded based on the groups (PLK1 low: blue, PLK1 high: yellow). C) Cellular deconvolution of PLK1 high and PLK1 low tumors using relative CIBERSORT to characterize the heterogeneity in immune cell composition of tumors with different levels of CIN. D) Heatmap showing gene expression data (Log 2 norm_count+1) and clustered into three different signatures associated with senescence, T-cell exhaustion and immune suppression from both PLK1 high (yellow) and PLK1 low (blue) tumor cohorts.

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet:

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: