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Pharmacologic inhibition of SHP2 inhibits MBC.  A,  Schematic of the study ...
Published: 03 October 2022
FIGURE 1 Pharmacologic inhibition of SHP2 inhibits MBC. A, Schematic of the study combining SHP099 with PD-L1 antibodies to treat mice bearing D2.A1 pulmonary tumors. Elements in the scheme were created using BioRender. B, Representative bioluminescent images of pulmonary D2.A1 growth at day 8 and day 20 postinjection. C, Bioluminescent values from pulmonary regions of interest (ROI) quantified as the ratio of day 20 to day 8 postinjection (**, P < 0.01, n  =  5 mice per group). D, Plots comparing the wet lung weights of the mice at day 22 postinjection (**, P < 0.01, n  =  5 mice per group). E, Representative H&E staining of lung histologic sections at day 22 postinjection. More
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Pharmacologic inhibition of SHP2 relieves T-cell exhaustion and reprograms ...
Published: 03 October 2022
FIGURE 2 Pharmacologic inhibition of SHP2 relieves T-cell exhaustion and reprograms the tumor–immune microenvironment. A, Quantification of CD4+ population as a frequency of CD45+ cells in isolated spleens (left) and lung tissues (right) of each group. B, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD4+ cells in isolated spleens (left) and lung tissues (right) of each group. C, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD8+ cells (left) and CD45+CD8+PD-1+ cells (right) of in isolated lung tissues of each group. D, Quantification of F4/80+ population as a frequency of CD45+CD11b+ cells in isolated lung tissues of each group. E, Plots comparing the ratio of CD86+ and CD206+ in F4/80+ population as M1/M2 in isolated lung tissues of each group. F, Quantification of PD-L1+ population as a frequency of CD45 cells in isolated lung tissues of each group. In all panels. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3. More
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Depletion of tumor cell–autonomous SHP2 reduces pulmonary metastasis, alter...
Published: 03 October 2022
FIGURE 3 Depletion of tumor cell–autonomous SHP2 reduces pulmonary metastasis, alters immune profiles, and prevents T-cell exhaustion. A, Schematic of the study using doxycycline-inducible depletion of SHP2 in 4T1 cells. Elements in the scheme were created using BioRender. BALB/c mice (n = 7 mice per group for shScramble and shPTPN11 146, n = 6 mice per group for shPTPN11 369) were orthotopically engrafted with 4T1 cells (5 × 104) via intraductal injection. Primary tumors were surgically removed 2 weeks following the injection. Doxycycline was administrated in drinking water at 2 mg/mL 3 days following the removal of primary tumors. B, Plots comparing the primary tumor volume at day 14 postinjection. NS, no significance. C, Representative bioluminescent images of 4T1 pulmonary metastasis at day 17 and day 31 postinjection. D, Bioluminescent values from pulmonary ROI quantified as the ratio of day 31 to day 17 postinjection (*, P < 0.05; **, P < 0.01). More
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Depletion of tumor cell–autonomous SHP2 relieves immune suppression.  A,  P...
Published: 03 October 2022
FIGURE 4 Depletion of tumor cell–autonomous SHP2 relieves immune suppression. A, Plots comparing the ratio of the frequency of CD4+ and CD8+ as CD4/CD8 in isolated spleen (left) and lung tissues (right) of each group. B, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD4+ cells in isolated spleens of each group. C, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD4+PD-1+ cells in isolated lung tissues of each group. D, Quantification of TIM3+ population as a frequency of CD45+CD8+ cells in isolated spleens (left) and lung tissues (right) of each group. E, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD8+ cells in isolated spleens of each group. F, Quantification of TIM3+LAG3+ population as a frequency of CD45+CD8+PD-1+ cells in isolated lung tissues of each group. G, Quantification of F4/80+ population as a frequency of CD45+CD11b+ cells in isolated lung tissues of each group. H, Plots comparing the ratio of CD86+ and CD206+ in F4/80+ population as M1/M2 in isolated lung tissues of each group. In all panels. NS, no significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4 for shScramble and shPTPN11 146, n = 5 for shPTPN11 369 in pulmonary tumor panels, n = 4 for each group in spleen panels. More
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Phosphorylation of SHP2 predicts immune profiles in patients with MBC.  A, ...
Published: 03 October 2022
FIGURE 5 Phosphorylation of SHP2 predicts immune profiles in patients with MBC. A, B, Violin and box plots comparing the differential immune scores and stroma scores in patients grouped by phosphorylation levels of SHP2 at Y542 ( A ) or expression levels of SHP2 ( B ). C, D, Violin and box plots comparing the differential phosphorylation levels of SHP2 at Y542 and expression levels of SHP2 in patients grouped by CD4+ T-cell infiltration ( C ) and M1 Macrophage infiltration ( D ) levels. Heatmaps comparing the differential gene expression in patients grouped by phosphorylation levels of SHP2 at Y542 ( E ) or expression levels of SHP2 ( F ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. G, Volcano plots demonstrating pathways of differential ssGSEA scores in patients grouped by phosphorylation levels of SHP2 at Y542 with statistical significance. Specific pathways of interest are annotated. GSEA plots, Enrichment scores and P values of the key pathways from GO ( H ) and KEGG ( I ) enriched in patients with lower phosphorylation levels of SHP2 at Y542. More
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SHP2 facilitates growth factor–induced resistance to T-cell cytotoxicity vi...
Published: 03 October 2022
FIGURE 6 SHP2 facilitates growth factor–induced resistance to T-cell cytotoxicity via regulation of PD-L1. A, Schematic of T-cell cytotoxicity assays. Elements in the scheme were created using BioRender. CD8+ T cells are isolated from the spleens of tumor-bearing mice. The MBC cells are treated with growth factors and inhibitors, and cocultured with CD8+ T cells. The dead cells are quantified by with Incucyte imaging. B, Representative images of the MBC cells treated with FGF2 (20 ng/mL) and PDGF (100 ng/mL) at 1 hour following coculturing with T cells. The MBC cells without T cells served as background. C, Bar graph comparing the percentage of dead cell counts of FGF2 and PDGF groups to the no stimulation (NS) group. *, P < 0.05; n = 4 individual repeats. D, Representative images of the MBC cells treated with FGF2/PDGF and TNO155 (5 μmol/L) at 1 hour following coculture with T cells. E, Bar graph comparing the percentage of dead cell counts with different treatments. *, P < 0.05; ***, P < 0.001, n = 9. Histogram of cell surface PD-L1 using flow cytometry ( F ) and bar graph ( G ) comparing fold changes of PD-L1 MFIs in D2.A1 cells induced by PDGF and treated with different inhibitors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3. H, Histogram of cell surface PD-L1 using flow cytometry in D2.A1 cells induced by 3D culture on a fibronectin-coated scaffold and treated with the indicated inhibitors. I, Bar graph comparing fold change of PD-L1 mRNA of D2.A1 cells cultured and treated as in H . **, P < 0.01; n = 3. More
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SHP2 regulates the expression of MHC class I via a balance between MAPK and...
Published: 03 October 2022
FIGURE 7 SHP2 regulates the expression of MHC class I via a balance between MAPK and STAT1 signaling in MBC cells. A, Histogram of cell surface analysis of H-2 in D2.A1 cells treated with different growth factors, mouse IFNγ (200 ng/mL) and TNO155 (5 μmol/L). B, Bar graph comparing fold change of H-2 MFIs induced by different growth factors, IFNγ and TNO155 compared with DMSO+ IFNγ. NS, not significant; **, P < 0.01; ***, P < 0.001; n = 3. C, Immunoblotting showing differential STAT1 and ERK1/2 phosphorylation in D2.A1 cells treated with different growth factors, IFNγ, and TNO155. D, Bar graph comparing fold change of H-2 MFI induced by different growth factors and IFNγ compared with IFNγ alone with different inhibitors in D2.A1 cells. NS, not significant; **, P < 0.01; ***, P < 0.001; n = 3. More
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Tumor cell–autonomous SHP2 is a key signaling node in response to dynamic T...
Published: 03 October 2022
FIGURE 8 Tumor cell–autonomous SHP2 is a key signaling node in response to dynamic TME to induce immune suppression via regulating PD-L1 and MHC class I. SHP2 contributes to various downstream signaling pathways including PD-L1 and MHC class I to facilitate immune suppression in response to a vari... More
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Pathway-based analysis generates three distinct clusters based on enrichmen...
Published: 23 September 2022
FIGURE 1 Pathway-based analysis generates three distinct clusters based on enrichment profiles with clinical significance. Samples from TCGA were analyzed using either canonical pathways ( A ) or oncogenic pathways ( B ) from the GSEA to generate heatmaps based on the enrichment profile of each sample (column) with respect to each gene set (row) in both collections. C and D, Profiles from A and B , respectively, were used to generate PCA plots labeled by color and shape for each cluster. Circle lines represent the normal distribution of the samples in each cluster. E, TCGA samples were clustered on the basis of the original molecular subtypes described, and Kaplan–Meier curves were obtained. F and G , Samples clustered on the basis of enrichment profiles for canonical and oncogenic gene sets, respectively, were analyzed for survival using Kaplan–Meier curves. Tables at the bottom describe the distribution of the molecular subtypes for each cluster. Dotted lines represent median survival for each curve (also described in top tables). Time shown is in months. P values after post hoc analyses using Bonferroni–Hochberg correction. More
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Gene lists predict E2F1 as a main target in one of the clusters found in th...
Published: 23 September 2022
FIGURE 2 Gene lists predict E2F1 as a main target in one of the clusters found in the GS dataset. A, Enrichment profiles using gene lists were generated for GS samples. B, Each gene list was evaluated using IPA and top predicted activated (green arrows) and inhibited (red arrows) upstream regu... More
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Gene lists differentially correlate with cellular states and cell-specific ...
Published: 23 September 2022
FIGURE 3 Gene lists differentially correlate with cellular states and cell-specific markers. A, Scores generated for each cell in Supplementary Fig. S3 using gene lists were correlated with scores for cellular states and specific cell-type markers in development and adult brain. The presence o... More
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E2F1 silencing compromises self-renewal and proliferation <em>in vitro</em>...
Published: 23 September 2022
FIGURE 4 E2F1 silencing compromises self-renewal and proliferation in vitro and tumor formation in vivo. Samples from both clusters were treated with control (scrambled) or E2F1 siRNA ( A ) or were plated in regular media or media containing fulvestrant or calcitriol ( B ) under limiting dilution in a 96-well plate. Graphs depict the number of wells that did not form spheres after 10 days versus the number of cells plated (a vertical line implies all wells formed spheres). C, Cells treated with scrambled or E2F1 siRNA were plated at a density of 2,000 cells per well in a 96-well plate in quadruplicate, and their growth was evaluated using luminescence. Relative growth is the fold change compared with basal measurement. D–F , Cells treated with scrambled or E2F1 shRNA were intracranially injected in NSG mice. Kaplan–Meier survival curves for each group was calculated; dashed lines represent median survival and time shown is in weeks ( D ). Luminescence was assessed 2 weeks after transplantation ( E ). Quantification for each group is shown at 2, 5, and 8 weeks ( F ). Mice in both groups for HK408 did not reach the 8-week timepoint. Experiments in A–C were performed at least three times. Data are represented as mean ± SEM. **, P < 0.01 and ***, P < 0.001 as assessed by one-way ANOVA. More
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E2F1 silencing compromises DNA damage response induced after irradiation.  ...
Published: 23 September 2022
FIGURE 5 E2F1 silencing compromises DNA damage response induced after irradiation. A, Cells were treated with either C (control) or E (E2F1) shRNA, were subjected to irradiation (8 Gy) and fixed after 12 hours for γH2AX staining (red). Nuclei were counterstained using DAPI. B, Quantification for each group in A is shown. Experiment was performed at least two times. Data are represented as mean ± SEM. **, P < 0.01 as assessed by one-way ANOVA. More
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CDM-dependent CAFs generate sEVs with unique protein cargo.  A,  Representa...
Published: 19 September 2022
FIGURE 1 CDM-dependent CAFs generate sEVs with unique protein cargo. A, Representative transmission electron micrograph of sEVs isolated from CM collected from human pancreatic CAFs cultured within a CDM. Red box highlights a structure with canonical exosome morphology and size. Scale bar = 100 nm. B, Representative Western blot analysis of CAF lysate and assorted fractions collected following differential ultracentrifugation probing for enrichment of sEV markers. Lysate corresponding to 5 μg protein obtained from the EV-generating CAFs served as loading control. Fractions shown are: large EVs pellet (from the 10,000 × g centrifuge step); sEV supernatant; and sEV pelleted (collected from the 120,000 × g centrifuge step (see Materials and Methods for details). Note that the sEV pellet is enriched with canonical exosome markers and lacks organelle contaminants. C, Representative NTA histogram of an sEV pellet fraction; using the NanoSight platform. D, Control Western blot analysis showing sEVs isolated from media containing FBS (+FBS) and undetected in serum-depleted (−FBS) media. E, Enriched gene ontology clusters from proteomic analysis of CDM-producing human CAF isolated sEVs, n = 3. Note that full proteomics and enriched pathway analysis can be found in Supplementary Data S1—Proteomics (Tabs = Total proteins CAF.sEV, Fig. 1E Pathway Analysis) More
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NetG1<sup>+</sup> CAFs generate sEVs that rescue PDAC cells from nutrient d...
Published: 19 September 2022
FIGURE 2 NetG1+ CAFs generate sEVs that rescue PDAC cells from nutrient deprivation-induced apoptosis. A, Representative confocal immunofluorescent images obtained from NLFs and CAFs cultured to produce a CDM. Shown are nuclei (SYBR green) and corresponding fibronectin ECM fibers (white). Scale bars = 50 μm; see Supplementary Fig. S1 for measurements. B, Representative Western blot analysis of CAF and NLF cell lysates, probing for expression of myofibroblastic activation marker, αSMA. GAPDH as protein loading control. C, Relative differences in total particle concentrations found in sEV fractions isolated from NLFs and CAFs, normalized to the mean NLF value; using the NanoSight platform. Bars = standard error. Statistics: Unpaired t test using Welch correction. n = 4. Comprehensive statistical readouts provided in Supplementary Data S2—Statistical Analysis (Tab = Fig. 2C). D, Representative NTA histograms of sEV fractions from NLF and CAFs, using the NanoSight platform. E, Cell viability assay of PANC-1 cells at 48 hours posttreatment with CM or sEV from NLFs and CAFs. n = 3 biological replicates; each replicate consists of six technical repeats. All repeats per replicate were normalized to the mean of the corresponding PBS-treated condition. Bars = standard error. Statistics: one-way ANOVA, with multiple comparisons using Tukey correction. *, Compared with PBS (negative control), # comparing between conditions noted by connecting lines. A comprehensive list of statistical readouts is provided in Supplementary Data S2—Statistical Analysis (Tabs = Fig. 2E). F, Representative Western blots of PANC-1 cell lysates collected 48 hours posttreatment with PBS, CM, or sEVs from NLF or CAF cells. Probing for Akt activation via phosphorylation at Serine 473 and apoptosis occurring via PARP cleavage (lower band). Pan Akt as pAkt control, GAPDH as protein loading control. G, Apoptosis occurring in PANC-1 cells as measured by the ratio of cleaved PARP: full-length PARP, quantified from the optical density of digitized Western blots in F using the software ImageJ. n = 6. H, Akt activation as measured via the ratio of phosphorylated Akt at Serine 473: Pan Akt, quantified from the optical density of digitized Western blots in F using the software ImageJ. n = 3. G and H, Results were normalized to the PBS-treated condition (negative control) for each replicate blot, represented by dotted line. Bars = standard error. Statistics: one-way ANOVA, with multiple comparisons using Tukey correction. * Compared with PBS, # comparing between conditions noted by connecting lines. A comprehensive list of statistical readouts is provided in Supplementary Data S2—Statistical Analysis (Tabs = Fig. 2G, 2H). I, Representative Western blots of NLF and CAF cell lysate and sEV fractions, probing for NetG1 and Int.α5. GAPDH as protein loading control in cell lysate fraction. CD81 as loading control in sEV fraction. More