Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A ) Depletion of SHP2 from B16F10 cells. ( B and C ) Tumors from s.c. inoculation of SHP2-silenced and p-LKO-control B16F10 cells (1 × 10 6 ). Tumor volume kinetics ( B ) and tumor weight at the endpoint ( C ). Mice: p-LKO, n = 6; shSHP2#1 n = 7; shSHP2#2, n = 7. Data indicate the mean ± SEM. ( D ) SHP2 depletion in SHP2-silenced B16F10 tumors at harvest. ( E and F ) Representative images of CD31 + vessels in tumors ( E ) and CD31 quantification ( F ). Scale bar: 100 μm. Gray circles: percentage of CD31 + area/unit of tumor area; colored dots: tumor mean, p-LKO tumors ( n = 3); shSHP2 tumors (mL1, n = 3; mL2, n = 4). * P < 0.05 by 1-way ANOVA. ( G ) Images of vessel perfusion by FITC-dextran; SHP2-silenced (shmL2). Scale bar: 100 μm. ( H – K ) p-LKO and SHP2-silenced MC38 colon carcinoma cells ( H ) generated s.c. tumors in mice (p-LKO n = 4; shSHP2 n = 8) ( I ) with similar tumor weights at harvest (day 24) ( J ). Harvested shSHP2-MC38 tumors expressed less Shp2 mRNA than did p-LKO control tumors ( K ). ** P < 0.01, by 2-tailed Student’s t test. ( L and M ) SHP2-depleted MC38 tumors ( n = 3) were less vascularized than p-LKO control tumors ( n = 3). Representative image ( L ) and quantification ( M ). Scale bar: 200 μm. Gray circles: percentage of CD31 + area/unit of tumor area; colored dots: tumor mean. * P < 0.05, by 2-tailed Student’s t test. ( N ) Tumor vessel perfusion by FITC-dextran visualization; representative images (left) and FITC quantification (right) in p-LKO control ( n = 3) and shSHP2-silenced tumors ( n = 3). Scale bar: 400 μm. Gray circles: percentage of FITC-dextran area/unit of tumor area; colored dots: tumor mean. ( O ) Tumor growth from s.c. inoculation of SHP2-silenced (shSHP2mL1 or mL2; n = 8) and p-LKO control ( n = 3) B16F10 cells (2 × 10 4 ). Individual mice were euthanized when the maximal (Max) tumor diameter was/approached 20 mm or at humane endpoints. On day 36: * P < 0.05, by 2-tailed Student’s t test. ( P ) Quantification of the percentage of CD31 + blood vessels; n = 3 mice/p-LKO; n = 6 mice/shSHP2 (day 36). Gray circles: percentage of CD31 + area/unit of tumor area; colored dots: tumor mean. ** P < 0.01, by 2-tailed Student’s t test. ( Q ) SHP2 was depleted (day 36) in cells of shSHP2#1- or shSHP2#2-silenced B16F10 tumors.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Control, Generated

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A and B ) Activity of signaling molecules in B16F10 cells propagated in culture after SHP2 silencing with 6 different shRNAs. Representative immunoblots ( A ) and quantification ( B ) of relative band intensities in control and SHP2-silenced cells. ( C and D ) Lysates from SHP2-silenced and p-LKO control B16F10 tumors harvested on day 21 after cell inoculation were evaluated by immunoblotting. Representative immunoblot results from individual tumors ( C ) and band quantification ( D ). ( E ) Signaling profile of SHP2-silenced and control B16F10 tumors harvested on day 21 after inoculation evaluated with a phospho-kinase assay kit. Lysates (900 mg) from pools of 4 p-LKO and 4 SHP2-silenced tumors were applied to the array. Visualization of the results and quantification of the relative pixel density in p-LKO control and SHP2-silenced tumor lysates are shown. The quantitative results are expressed relative to the control (identified by the dotted line). * P < 0.05 and ** P < 0.01, by 2-tailed Student’s t test. ( B and D ) The horizontal lines reflect the mean.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Activity Assay, Western Blot, Control, Kinase Assay

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A ) Control p-LKO and SHP2-silenced B16F10 cells cultured (14 days) in ultra-low attachment plates were visualized by bright-field imaging (top and mid panels show representative images). Confocal cluster images are shown in the bottom panels (p-ERK, green; DAPI/nuclei, blue). Scale bars: 500 μm (middle panel) and 50 μm (bottom panel). ( B ) Quantification of cell cluster size; gray circles reflect individual clusters; colored dots reflect the mean cluster size/culture ( n = 9); results are presented as mean (horizontal lines). ** P < 0.01 by 2-way Student’s t test. ( C ) Immunoblot results of p-ERK T202/Y204 in lysates of B16F10 cells cultured for 7 days on ultra-low attachment plates. ( D ) Schematic diagram illustrating the experimental in vitro culturing system; left: a suspension of tumor cells, Matrigel, and DMEM was introduced into a tight-sealed chamber to evenly occupy the space; right: the chamber was transferred into a Petri dish containing DMEM with 10% FBS, which entered the chamber only through the 2 holes. ( E ) Fluorescence images of chamber holes and surrounding Matrigel 3, 30, and 60 minutes after addition of the Alexa Fluor 594–Fab fragment, showing the time-dependent fluorescence diffusion from the hole. Scale bar: 200 μm. ( F ) Representative bright-field images of control and SHP2-silenced B16F10 cells surrounding the chamber holes after 1- and 48-hour incubations ( n = 3 experiments). The red arrows point to cell clustering around the hole. Scale bar: 500 μm. ( G ) Representative bright-field and fluorescence images of the indicated areas after staining with PI (48-hour incubation; n = 3 experiments). Scale bar: 500 μm (left panels) and 100 μm (middle and right panels). ( H ) Number of PI + /dead p-LKO and SHP2-silenced B16F10 cells as a function of distance from the corresponding hole ( n = 3 experiments). *** P < 0.001, by 2-tailed Student’s t test. Results are presented as means± SEM. ( I ) Representative images of chambers containing B16F10 cells stained with Hoechst (nuclei/blue), Ki67 (green), and p-ERK (red) (48-hour incubation; n = 3 experiments). Scale bar: 500 μm. ( J ) Magnified images of the areas outlined in I (area 1: p-LKO B16F10 cells; areas 2 and 3: SHP2-silenced B16F10 cells) showing the cell clusters. Scale bar: 100 μm.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Control, Cell Culture, Imaging, Western Blot, In Vitro, Suspension, Fluorescence, Diffusion-based Assay, Staining, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A and B ) Ki67 and CD31 immunostaining identified a distinct structural organization of SHP2-silenced B16F10 tumors. Nuclei are stained with DAPI. Representative confocal microscopy images ( A ) and quantification ( B ) of Ki67 fluorescence intensity in tumor vascular islands from SHP2-silenced tumors ( n = 3) and p-LKO control tumors ( n = 3). ( C and D ) p-ERK T202/Y205 and CD31 immunostaining detected p-ERK in representative tumor vascular islands of SHP2-silenced B16F10 tumors ( C ). Quantification of p-ERK fluorescence in live tissue from p-LKO tumors ( n = 5) and islands of SHP2-silenced tumors ( n = 5) ( D ). ( E and F ) p-CDK2 T160 and CD31 immunostaining identified the distinct structural organization of SHP2-silenced B16F10 tumors ( E ). p-CDK2 fluorescence quantification in p-LKO ( n = 3) and SHP2-silenced ( n = 3) tumor islands ( F ). ( G and H ) SHP2 and CD31 immunostaining detected strong SHP2 fluorescence intensity in representative tumor islands of control B16F10 tumors and faint SHP2 fluorescence in SHP2-silenced B16F10 tumor islands ( G ). SHP2 quantification in live tissue from p-LKO ( n = 3) and SHP2-silenced ( n = 3) tumor islands ( H ). In B , D , F , and H , the gray circles reflect fluorescence intensity/unit of tumor area; colored dots reflect the mean fluorescence intensity/tumor analyzed. ( I ) Representative confocal images of tumor islands from SHP2-silenced B16F10 tumors displaying the central CD31 + vessel surrounded by Ki67 + tumor cells limited at the periphery by a hypoxic zone (Hypoxyprobe + ) and further surrounded by areas lacking identifiable DAPI + cells. In B , D , F , and H , horizontal lines indicate the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test. Scale bars: 200 μm.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Immunostaining, Staining, Confocal Microscopy, Fluorescence, Control

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A ) Representative images of tumors harvested on days 9, 13, 17, and 21 following inoculation of p-LKO control and SHP2-silenced B16F10 cells. Top panels (scale bars: 200 μm) depict CD31 + vessels and distribution of hypoxia (Hypoxyprobe + ); bottom panels (scale bars: 50 μm) show the distribution CD45 + inflammatory cells, Ki67 + proliferating cells, and cell nuclei (DAPI + ). ( B ) Tumor weight of p-LKO and SHP2-silenced B16F10 tumors at the indicated time points; open circles represent individual tumors/mouse. n = 3 (days 9 and 13); n = 4 (days 17 and 21). ( C ) Quantification of tumor islands. A tumor island is defined as the assembly of live tumor cells around a blood vessel and surrounded by a zone of hypoxia. Quantification was performed by evaluating the entire tumor section through the maximum diameter; open circles represent individual tumors/mouse. n = 3 (days 9 and 13); n = 4 (days 17 and 21). * P < 0.05, by 2-tailed Student’s t test. ( D – G ) Quantification of CD31 + endothelial cells ( D ), hypoxic cells (Hypoxyprobe + ) ( E ), CD45 + inflammatory cells ( F ), and Ki67 + proliferating cells ( G ) at the indicated time points in control tumors ( n = 3, days 9 and 13; n = 4, days 17 and 21) and SHP2-silenced tumors ( n = 3, days 9 and 13; n = 4, days 17 and 21). Gray circles: percentage of relative positive area/unit of tumor area; colored dots/circles: relative mean percentage of positive area/tumor. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test. ( H and I ) Representative images (scale bar: 50 μm) from immunostaining of p-LKO ( n = 3) and SHP2 silenced ( n = 3) B16F10 tumor tissues harvested on day 13 for cleaved caspase 3 (cell death), CD31 endothelial cells, and nuclei (DAPI) ( H ) and quantification of cleaved caspase 3 + CD31 + endothelial cells at the indicated time points following tumor cell inoculation ( I ). Gray circles: relative cleaved caspase 3 + CD31 + cells/CD31 + cells analyzed; colored dots: tumor mean (control, n = 3; SHP2-silenced, n = 3). * P < 0.05 and ** P < 0.01, by 2-tailed Student’s t test ( I ) The results in panels B – G and I are presented as means± SEM.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Control, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A ) List of proteins that differed by more than 20% in relative pixel intensity between SHP2-silenced and p-LKO B16F10 cells. Cell lysates (900 mg) were applied to the array. ( B ) List of proteins with reduced pixel intensity of greater than 20% in TNO155-treated relative to control B16F10 cells. ( C ) Venn diagram of protein distribution. Some ( n = 18) proteins reduced by more than 20% by SHP2 silencing in B16F10 cells were also reduced by more than 20% by TNO155 treatment of B16F10 cells. ( D ) List of proteins reduced by more than 20% in SHP2-silenced B16F10 tumors compared with p-LKO tumors removed from mice 21 days after inoculation. Lysates (900 mg) from pools of 4 control and 4 SHP2-silenced tumors were applied to the array. ( E and F ) Venn diagrams of protein distribution. Some proteins reduced by more than 20% in SHP2-silenced B16F10 tumors compared with control were also reduced by more than 20% by SHP2 silencing ( E ) or TNO155 treatment ( F ) of B16F10 cells from culture. ( G ) Proteins reduced by more than 20% compared with controls in SHP2-silenced B16F10 cells, TNO155-treated B16F10 cells , and SHP2-silenced B16F10 tumors. ( H ) Proteins were selectively reduced by more than 20% in SHP2-silenced B16F10 tumors compared with the control, but not in B16F10 cells from culture (SHP2-silenced compared with control and TNO-treated compared with control).

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A ) Expression levels of Il1a , ccl2 , and ccl3 in sorted populations of EGFP + B16F10 cells, EGFP – VE-cadherin + endothelial cells, and EGFP – CD45 + hematopoietic cells as measured by qPCR. The results are expressed as the relative mean ± SEM of Gapdh mRNA levels ( n = 3 experiments). ( B and C ) Representative images (scale bar: 50 μm) ( B ) showing CD45 + cell infiltrates (green, indicated by arrowheads) in control and shSHP2-silenced B16F10 tumors and quantification ( C ). Gray circles: percentage of CD45 + area/unit of tumor area; colored dots: mean/tumor ( n = 5/group). ** P < 0.01, by 2-tailed Student’s t test. ( D and E ) Representative images (scale bar: 50 μm) ( D ) showing F4/80 (green) macrophage infiltration in control and shSHP2-silenced B16F10 tumors (indicated by arrowheads) and quantification ( E ). Gray circles: percentage of F4/80 + area/unit of tumor area; colored dots: mean/tumor ( n = 5/group). ** P < 0.01, by 2-tailed Student’s t test. ( F ) Proposed model of downstream effects from SHP2 silencing in B16F10 melanoma cells leading to the formation of vascular tumor islands and surrounding tumor cell death. Results in panels A , C and E are presented as means± SEM.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition

doi: 10.1172/JCI181609

Figure Lengend Snippet: ( A – D ) Tumor-bearing mice ( n = 5/group in A and B ; n = 10/group in C and D ) from s.c. inoculation of SHP2-silenced or control (p-LKO) B16F10 cells were treated with 30 mg/kg daily oral GDC-0623 (GDC) or buffer only. The experiment was terminated when the maximal tumor diameter reached 20 mm in any mouse. Tumor volumes ( A and C ) for shSHP2-GDC versus shSHP2-control (black asterisks) and shSHP2-GDC versus p-LKO–GDC (red asterisks). Tumor weights ( B and D ) at harvest. ( E and F ) Tumor-bearing mice ( n = 8/group) were s.c. inoculated with SHP2-silenced or control B16F10 cells and treated with 30 mg/kg daily oral GDC-0623 or buffer only. Each mouse was euthanized when the endpoint (20 mm maximum diameter or humane endpoint) was reached. ( E ) Tumor size measurements from treatment initiation to the day when the first mouse reached the endpoint; shSHP2 control versus shSHP2-GDC (black asterisks); p-LKO–GDC versus shSHP2-GDC (red asterisk). ( F ) Proportion of mice surviving as a function of time from the beginning of treatment (Kaplan-Meier curves). * P < 0.05, ** P < 0.01, and *** P < 0.001, by 1-way ANOVA ( B and D ) and 2-way ANOVA ( A , C , and E ) for multiple comparisons. ( G – O ) Tumors from inoculation of SHP2-silenced B16F10 cells were treated with GDC-0623 or buffer only. Tumor sections were visualized by confocal imaging after immunostaining (representative images in G : Ki67/CD31; H : p-ERK T202Y204 /DAPI; I : p-CDK2 T160 /DAPI; J : cleaved caspase 3/CD31/DAPI). Scale bars: 100 μm. The relative fluorescence intensity of specific markers/DAPI + cells was quantified ( K : Ki67; L : p-ERK T202Y204 ; M : p-CDK2 T160 ; N : cleaved caspase 3; O : CD31; gray circles, appearing as gray zones when numerous, reflect the positive area/unit of tumor area; green dots indicate the mean/tumor ( n = 4/group). ** P < 0.05 and *** P < 0.001, by 2-tailed Student’s t test. Horizontal lines indicate the mean. Results in panels A – E and panels K – O are presented as means± SEM.

Article Snippet: First, we profiled angiogenesis-related proteins in SHP2-silenced (shmL2) B16F10 cells from culture (Proteome Profiler Array for mouse angiogenesis, ARY015, R&D Systems).

Techniques: Control, Imaging, Immunostaining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation

doi: 10.3389/fimmu.2020.01098

Figure Lengend Snippet: STRING network analysis for modulated genes of transcription-related factors (TRFs) in DCs infected with Ab-opsonized amastigotes. Networks for down-modulated (green) (A) and up-modulated genes (red) (B) are shown. Interactions between TRFs and MHC II (1), DC markers/receptors (2), cytokine/chemokine receptors (3), TLR/Cytokine receptors and the NF-κB pathway (4), and inflammasome-related molecules (5) are depicted. Gray lines correspond to active interactions (settings used for STRING analysis) and black arrows exemplify key TRF – target gene interactions validated by publication. (C) Integrative network of modulated TRFs and their related target genes involved in regulation of the IL-1β gene. Dotted lines correspond to indirect interactions (mediated by RelA, related to the TLR/NF-κB axis, group 4) on the IL-1β gene.

Article Snippet: Cytokine/chemokine profiling was performed by the mouse XL cytokine array kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation

doi: 10.3389/fimmu.2020.01098

Figure Lengend Snippet: Multiparametric characterization of dendritic cell cultures. BALB/c-derived BMDCs alone (Ctrl) or incubated for 24 h with BCG or L. amazonensis amastigotes without ( L. am ) or with Ab-epsonization ( L. am -IS) were analyzed. (A) Confocal microscopy imaging of DCs stained for MHC Class II and H2-M molecules. Images of representative DCs from the different conditions are displayed. (1) Merged images of the three detection channels showing MHC Class II molecules (green), H2-M molecules (red), and nuclear DNA (blue). (2) Intensity distribution of the three detected fluorescence signals along the transect line indicated in (1). (3) Percentage of DCs showing dissociated localization for MHC Class II and H-2M ( n = 3 independent experiments). (B) Expression levels of co-stimulatory and adhesion molecules. Flow cytometric analysis was performed on DCs from the different culture conditions ( n = 3–6 independent experiments). Mean (+ SD) fluorescence intensity (MFI) signals obtained for the corresponding stainings are shown. (C) Chemokine and cytokine determination in culture supernatants. The level of secretion was analyzed at 24 h post-infection in culture supernatants using the proteome profiler mouse XL cytokine array kit. Heat maps of background-corrected Raw Integrated Density (RID) values for technical duplicates are represented for chemokines (1) and cytokines (2). Scanned membranes of protein arrays are shown in . P -values for inter-group differences are indicated. NS, not significant.

Article Snippet: Cytokine/chemokine profiling was performed by the mouse XL cytokine array kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Derivative Assay, Incubation, Confocal Microscopy, Imaging, Staining, Fluorescence, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation

doi: 10.3389/fimmu.2020.01098

Figure Lengend Snippet: Expression analysis of genes related to the maturation process in Leishmania -infected DCs. (A,B) Affymetrix analyses were performed on sorted DCs infected with non-opsonized ( L. am ) or Ab-opsonized ( L. am -IS) Ds Red2-transgenic amastigotes. Transcriptional modulation of genes involved in the DC maturation process are displayed as the mean fold change values calculated using uninfected sorted DCs as a calibrator. (A) Expression of genes related to maturation and antigen presentation. Heatmaps representing the expression modulation of genes coding for costimulatory molecules and surface receptors (1), MHC I (2), and MHC II (3) molecules. (B) Expression of chemokine and cytokine genes. Heatmaps representing the modulation of genes coding for cytokines (1), chemokines (2), and chemokine receptors (3). (C) FACS analysis of CCR-5 expression levels. DCs from unsorted cultures were analyzed by FACS for the expression of CCR5, a marker that is rapidly lost during the maturation process. Infected cells were detected using the amastigote-specific, Alexa Fluor-488 conjugated mAb 2A3-26.

Article Snippet: Cytokine/chemokine profiling was performed by the mouse XL cytokine array kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Expressing, Infection, Transgenic Assay, Immunopeptidomics, Marker