mouse recombinant wisp 1  (R&D Systems)

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: Digital cytometry deconvolutes a bulk transcriptomic profile using gene signatures that correspond to different stromal, malignant, and immune cell types. The results estimate the prevalence of the different cell types within the tissue sample, that is the digital cytometry features. By using bulk transcriptomic profiles of defined patient populations, underlying variation in the inferred cellular composition coupled with features associated with a patient sample, such as over-expression of a secreted gene product by malignant cells, can be used to estimate how the heterocellular network is impacted by a genetic alteration intrinsic to the malignant cell using Bayesian Network inference. To illustrate the approach, we focused on malignant cell expression of Cell Communication Network factor 4 (CCN4), a secreted matricellular protein. The resulting directed acyclic graph represents the collective conditional independence among the modeled features, or nodes, of the network.

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Cytometry, Over Expression, Expressing

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Expressing, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. B A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). C The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. D TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO pairs using two-sided Student’s t test.

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Isolation, Generated, Comparison, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). B The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. C A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. D – F TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets ( D : MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets ( E : CD11c- (top) and CD11c+ (bottom) macrophages, F : neutrophils (top) and dendritic cells (bottom)) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO variants using two-sided Student’s t test.

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A Expression of genes for transcription factors (left panel - Snai1: red triangle, Snai2: blue diamond, Zeb1: black circle, and Zeb2: gray square) and adhesion proteins (right panel - Cdh1: blue triangle, Cdh2: black circle, Fn1: red square) associated with the epithelial-mesenchymal transition were assayed as a function of time following addition of rmCCN4 to CCN4 KO YUMM1.7 (top row) and CCN4 KO B16F0 cells (bottom row). Colored asterisks indicate whether gene at a particular time point was significantly different than untreated cells, where n = 3 biological independent samples. B The distribution in cell trace staining among live CD4 + (left panel) and CD8 + (right panel) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. C Bivariate projection of the weights of genes within the resting (y-axis) and activated (x-axis) NK cell signatures. D Using spleens from C57BL/6 mice that were challenged with YUMM1.7 cells, isolated CD8+ T cells were assayed by in vitro ELISpot for IFN γ expression using variants of the YUMM1.7 cell line as targets (CCN4 KO YUMM1.7 with a blank inducible expression vector and CCN4 KO YUMM1.7 with a CCN4 inducible expression vector). To induce CCN4 expression, these YUMM1.7 variants were also cultured in the absence (−) or presence of doxycycline (+) and quantified following 24 h co-culture. Statistical significance between WT and CCN4 KO variants was assessed using two-way ANOVA followed by Tukey’s multiple comparison ad hoc post-test, where n = 6 biologically independent samples. Results summarized as mean ± s.d.

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Expressing, Staining, Recombinant, Isolation, In Vitro, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Co-Culture Assay, Comparison

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: Digital cytometry deconvolutes a bulk transcriptomic profile using gene signatures that correspond to different stromal, malignant, and immune cell types. The results estimate the prevalence of the different cell types within the tissue sample, that is the digital cytometry features. By using bulk transcriptomic profiles of defined patient populations, underlying variation in the inferred cellular composition coupled with features associated with a patient sample, such as over-expression of a secreted gene product by malignant cells, can be used to estimate how the heterocellular network is impacted by a genetic alteration intrinsic to the malignant cell using Bayesian Network inference. To illustrate the approach, we focused on malignant cell expression of Cell Communication Network factor 4 (CCN4), a secreted matricellular protein. The resulting directed acyclic graph represents the collective conditional independence among the modeled features, or nodes, of the network.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 protein (rCCN4, R&D) at a final concentration of 10 ng/mL, which was selected based on the concentration of CCN4 observed in medium conditioned by WT cells.

Techniques: Cytometry, Over Expression, Expressing

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 protein (rCCN4, R&D) at a final concentration of 10 ng/mL, which was selected based on the concentration of CCN4 observed in medium conditioned by WT cells.

Techniques: Gene Expression, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. B A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). C The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. D TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO pairs using two-sided Student’s t test.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 protein (rCCN4, R&D) at a final concentration of 10 ng/mL, which was selected based on the concentration of CCN4 observed in medium conditioned by WT cells.

Techniques: Isolation, Generated, Comparison, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). B The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. C A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. D – F TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets ( D : MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets ( E : CD11c- (top) and CD11c+ (bottom) macrophages, F : neutrophils (top) and dendritic cells (bottom)) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO variants using two-sided Student’s t test.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 protein (rCCN4, R&D) at a final concentration of 10 ng/mL, which was selected based on the concentration of CCN4 observed in medium conditioned by WT cells.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A Expression of genes for transcription factors (left panel - Snai1: red triangle, Snai2: blue diamond, Zeb1: black circle, and Zeb2: gray square) and adhesion proteins (right panel - Cdh1: blue triangle, Cdh2: black circle, Fn1: red square) associated with the epithelial-mesenchymal transition were assayed as a function of time following addition of rmCCN4 to CCN4 KO YUMM1.7 (top row) and CCN4 KO B16F0 cells (bottom row). Colored asterisks indicate whether gene at a particular time point was significantly different than untreated cells, where n = 3 biological independent samples. B The distribution in cell trace staining among live CD4 + (left panel) and CD8 + (right panel) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. C Bivariate projection of the weights of genes within the resting (y-axis) and activated (x-axis) NK cell signatures. D Using spleens from C57BL/6 mice that were challenged with YUMM1.7 cells, isolated CD8+ T cells were assayed by in vitro ELISpot for IFN γ expression using variants of the YUMM1.7 cell line as targets (CCN4 KO YUMM1.7 with a blank inducible expression vector and CCN4 KO YUMM1.7 with a CCN4 inducible expression vector). To induce CCN4 expression, these YUMM1.7 variants were also cultured in the absence (−) or presence of doxycycline (+) and quantified following 24 h co-culture. Statistical significance between WT and CCN4 KO variants was assessed using two-way ANOVA followed by Tukey’s multiple comparison ad hoc post-test, where n = 6 biologically independent samples. Results summarized as mean ± s.d.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 protein (rCCN4, R&D) at a final concentration of 10 ng/mL, which was selected based on the concentration of CCN4 observed in medium conditioned by WT cells.

Techniques: Expressing, Staining, Recombinant, Isolation, In Vitro, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Co-Culture Assay, Comparison

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I fibrils in lattices from A (n ¼ 15; three independent experiments; five images/lattice). C, Binding of WISP1 to fibrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to fibrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to fibrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to fibrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to fibrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsignificant, P > 0.05; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Electron Microscopy, Recombinant, Binding Assay, Negative Control, Inhibition

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsignificant, P > 0.05; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Gene Expression, Control, Migration, Membrane, MANN-WHITNEY

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to fibrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to fibrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I fibrils in lattices from D (n ¼ 15, three independent experiments, five images/lattice). Statistical significance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsignificant, P > 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Binding Assay, Construct, Stable Transfection, Transduction, Plasmid Preparation, Control, Negative Control, Electron Microscopy, Migration

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsignificant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Blocking Assay, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Cell Culture, Control, Protein Concentration, CRISPR, Knock-Out

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of fibrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-field images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of fibrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-field images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsignificant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Staining, Light Microscopy, Recombinant, Injection

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of fibrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- field images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsignificant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Control, Migration, Staining, Light Microscopy

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: Using the TCGA BRCA dataset, a consensus seed network was generated multiple times using a blacklist that was progressively increased from an empty list (A) to a list that included cancer (B-E) and CCN4 (C-E) as tail nodes and CD8 T cells (D,E) and CD4 T cells and neutrophils (E) as head nodes. As the number of arcs included in the blacklist was increased, the number of edges with unclear direction was decreased. Seventeen edges had an unclear direction without specifying a blacklist (A), while only 6 edges had an unclear direction in the final blacklist (E). Edges were ordered based on the number of algorithms that detected either arc were enriched (bar graph - left axis) and the strength of enrichment (dotted lines - right axis). The lines associated with the strength of enrichment represent the minimum (dashed line) and maximum (dotted line) values obtained by the different algorithms for each edge. Coloring of bar graph indicates whether the direction associated with an edge was clear (gray - i.e., only one arc was found to be significant among the algorithms) or unclear (yellow - i.e., both arcs were found to be significant among the algorithms).

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Generated

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Gene Expression, Cytometry

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: (A) The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. (B) A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). (C) The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. (D) TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) (n = 7 for YUMM1.7 and n = 4 for B16F0 variants and mean ± s.d.). p-values calculated between WT and CCN4 KO pairs using Student’s t-test.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Isolation, Generated, Comparison, Cytometry

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: Distribution in digital cytometry features extracted from the BRCA dataset stratified into quantiles based on CCN4 expression. The mean value for quantile 1 was used to represent CCN4 KO phenotype while quantile 5 was used to represent WT phenotype.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Cytometry, Expressing

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: Distribution in digital cytometry features extracted from the SKCM dataset stratified into quantiles based on CCN4 expression. The mean value for quantile 1 was used to represent CCN4 KO phenotype while quantile 5 was used to represent WT phenotype.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Cytometry, Expressing

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: (A) A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells. (B) The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. (C) A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. (D - F) TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets (D: MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets (E: CD11c- (top) and CD11c+ (bottom) macrophages, F: neutrophils (top) and dendritic cells (bottom)) (n = 7 for YUMM1.7 and n = 4 for B16F0 variants and mean ± s.d.). p-values calculated between WT and CCN4 KO pairs using Student’s t-test.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: The distribution in cell trace staining among live CD4 + (A) and CD8 + (B) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. (C) CD8+ T cells isolated from the spleens of C57BL/6 mice that rejected YUMM1.7 tumors were cultured in an in vitro ELISpot assay using variants of the YUMM1.7 cell line as targets (WT YUMM1.7 - yellow, CCN4 KO YUMM1.7 - light green, CCN4 KO YUMM1.7 with a blank inducible expression vector - dark green and blue, CCN4 KO YUMM1.7 with a CCN4 inducible expression vector - purple and red). Variants containing the inducible expression vector were also cultured in the absence (dark green and purple) or presence of doxycycline (blue and red). CD8+ T cells expressing IFN γ and TNF α were quantified following 24 hour co-culture (bar graph). Statistical significant between pairs was assessed using a Student’s t-test.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Staining, Recombinant, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunospot, Expressing, Plasmid Preparation, Co-Culture Assay

Journal: bioRxiv

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1101/2020.05.04.077107

Figure Lengend Snippet: (A) CCN4 secretion, measured with ELISA, from CCN4-inducible cells in conditioned media in the presence or absence of 0.5 μ g/ml doxycycline (n.d. = not detected). (B) ELISpot for IFN γ release with different target cells and different amount of effector CD8 + T cells (In vivo activated CD8 + T cells against YUMM1.7 (Ym1.7)). (C) ELISpot for IFN γ with CCN4-inducible cells as targets using in vivo activated CD8 + T cell against YUMM1.7. Results shown as mean ± S.D. for three biological replicates.

Article Snippet: In addition, splenocytes were either left unstimulated or stimulated with AP beads alone, or stimulated in the presence of recombinant mouse CCN4 (rCCN4, R&D) at a final concentration of 10 ng/mL.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, In Vivo