Journal: Cancer letters

Article Title: Pharmacological targeting of GLI1 inhibits proliferation, tumor emboli formation and in vivo tumor growth of inflammatory breast cancer cells

doi: 10.1016/j.canlet.2017.09.033

Figure Lengend Snippet: GANT61 disrupts formation of SUM149 tumor spheroids and tumor emboli. For the tumor spheroid assay, cells were seeded in ultralow attachment 384-well plates and treated 24 h later with either vehicle control (DMSO), staurosporine, or GANT61 at the indicated concentrations. Cells were grown for a further 72 h, spheroids stained with Hoechst/YOYO-1 and high content 3D images obtained using the CellInsight HCS platform. (A) representative images are shown for each treatment. Three independent experiments were carried out. (B) Data represent mean ± SD percent normalized to DMSO vehicle control for spheroid area (Hoechst) and spheroid YOYO-1 comprising a minimum of five replicate wells. Dose response curves were generated using non-linear regression and IC50 values determined in GraphPad Prism 6. (C) Tumor emboli: Representative images (10 × – scale bar = 100 microns) of SUM149 cells grown under tumor emboli conditions. Cells were treated at the time of plating with vehicle, staurosporine or GANT61 and cells grown for 7d. Images from at least two independent experiments comprising a minimum of three replicate wells.

Article Snippet: HCS Screen software (ThermoFisher) collected data using a modified “Cell health profiling” algorithm which was compiled in HCS View software, normalized against DMSO controls and plotted in ScreenAble (ScreenAble, Chapel Hill, NC) for analysis.

Techniques: Staining, Generated

Journal: Cell reports

Article Title: Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

doi: 10.1016/j.celrep.2019.11.056

Figure Lengend Snippet: (A) Representative qmIF composite image of a pancreas section stained with antibodies specific for CD3 (purple), the ductal marker CK19 (green), DAPI nuclear counterstain (gray), and the neuroendocrine marker chromogranin (white) are shown (left) adjacent to a representative single color CD3 image (middle). Acinar, ductal, and endocrine areas were defined based on CK19 and chromogranin staining. White bar, 100 µm for scale. Right: densities of CD3 + T cells were quantified in the three regions of pancreas using inForm software. Plots show mean ± SEM from 13 donors. (B) T cells were analyzed in cell suspensions of pancreas (Panc), jejunum (Jej), pancreas-draining lymph node (PLN), and mesenteric lymph node (MLN). Shown are representative (left) and the compiled (right) CD4 and CD8 T cell frequencies (gated on DAPI lo CD45 + CD3 + cells) from the four tissue sites. Bars indicate comparisons for CD8 + T cells. (C) Expression of CD69 in conjunction with TRM signature markers CD103, CD49a, and PD-1 on CD8 + TEM cells (CD45RA − CCR7 − ) subsets isolated from indicated sites shown as representative flow cytometry plots (left) with the compiled frequencies ±SEM of the indicated subsets from three to eight donors (right). Bars indicate comparisons of the CD69 + CD103 + (top), CD69 + CD49a + (middle), and CD69 + PD-1 hi (bottom) subsets. (D) Expression of intracellular granzyme B (GZMB) in CD8 + CD69 + TEM cells isolated from pancreas, jejunum, and PLN shown as representative flow cytometry plots (left), and compiled frequencies ± SEM of GZMB + cells from three to six donors for each tissue (right). Bars indicate comparisons of the GZMB + frequencies within the indicated subsets. **p < 0.001 as calculated by two-way ANOVA with Dunnett’s multiple comparisons test. See also .

Article Snippet: Descriptive statistics of compiled flow cytometry data, graphs and statistical testingwere performed using GraphPad Prism (Graphpad software, San Diego, CA).

Techniques: Staining, Marker, Software, Expressing, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

doi: 10.1016/j.celrep.2019.11.056

Figure Lengend Snippet: CD8 + TRMs (CD8 + CD69 + TEM) were sorted from pancreas, PLN, and jejunum along with blood (BL) CD8 + CD69 − TEM cells for whole-transcriptome profiling by RNA-seq. (A) Principal-component analysis (PCA) of gene expression profiles of tissue CD8 + TRMs compared to blood CD8 + CD69 − TEM cells. (B) Gene set enrichment analysis (GSEA) comparing expression of genes in pancreas TRMs versus blood CD8 + TEM cells to a core human TRM gene signature . (C) Biplots of all genes showing log FC of pancreas/blood versus log FC of jejunum/blood (top) and log FC of pancreas/PLN versus log FC of pancreas/jejunum (bottom). Significantly differentially expressed genes (DEGs) for each comparison were identified using DeSeq and functionally classified and are indicated as colored dots based on gene pathway. (D) Heatmaps showing representative DEG from annotated gene sets T cell activation, proliferation, migration (left) and mitochondrion (right) which significantly overlap with DEGs in pancreas versus PLN and pancreas versus jejunum. (E) Determination of mitochondrial mass using MitoTracker green staining in verapamil-treated CD8 + TRMs from matched donor sites pancreas, jejunum, and PLN. Representative histogram of MitoTracker green signal in matched sites from one donor is shown (top) with the compiled frequencies ±SEM from three donors for each tissue (bottom). (F) Sorted CD8 + CD69 + TEM subsets isolated from pancreas, jejunum, PLN, and sorted blood CD8 + CD69 − TEM cells and naive CD8 + T cells were stimulated with PMA and ionomycin, and intracellular staining was used to assess IFN-γ, IL-2, and TNF-α production. Profiles of IFN-γ and IL-2 are shown in representative flow cytometry plots for the indicated tissues (left). Graphs of compiled frequencies of IFN-γ -producing cells and the polyfunctionality index (see ) ±SEM from three to six donors for each tissue (right). **p < 0.01 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. See also .

Article Snippet: Descriptive statistics of compiled flow cytometry data, graphs and statistical testingwere performed using GraphPad Prism (Graphpad software, San Diego, CA).

Techniques: RNA Sequencing, Gene Expression, Expressing, Comparison, Activation Assay, Migration, Staining, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

doi: 10.1016/j.celrep.2019.11.056

Figure Lengend Snippet: TCR sequences of CD8 + TRMs from pancreas, jejunum, and PLN were extracted from RNA-seq data using MIXR (see ). (A) Representative clone tracking analysis showing the distribution and degree of expansion of PLN clonotypes across tissues. Each line represents a tracked clone present in PLN (far left, arrow) and at least one of the other tissues. Line thickness indicates degree of clonal expansion at the indicated site and line color indicates the tissue site in which that clone exhibits the greatest degree of clonal expansion. (B) The percentage of the total TCR sequences (±SEM from three to five donors for each tissue) detected in pancreas and jejunum TRM that are either shared or not shared with PLN clonotypes is shown (top). The diversity of the TCR repertoires in TRMs isolated from indicated tissues is depicted as the Chao1 index (bottom). (C) The percent contribution of expanded clonotypes from TRMs isolated from pancreas (top) and jejunum (bottom) to the total TCR repertoire extracted from pancreas, jejunum, and PLN (±SEM from three to five donors for each tissue). **p < 0.01, *p < 0.05 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Coordinate expression of CD127 and CD28 by CD8 + CD69 + TEM cells isolated from pancreas, jejunum, PLN, and MLN shown as representative flow cytometry plots (left), with the compiled frequencies (±SEM) of indicated subsets from 9 to 20 donors for each tissue (right). **p < 0.01 as calculated by two-way ANOVA with Sidak’s multiple comparisons test. Bars indicate comparisons of the CD127 + CD28 − subset. See also .

Article Snippet: Descriptive statistics of compiled flow cytometry data, graphs and statistical testingwere performed using GraphPad Prism (Graphpad software, San Diego, CA).

Techniques: RNA Sequencing, Isolation, Expressing, Flow Cytometry

Journal: Cell reports

Article Title: Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

doi: 10.1016/j.celrep.2019.11.056

Figure Lengend Snippet: (A) Light scatter—forward scatter (FSC) and side scatter (SSC)—profile of DAPI lo CD45 + cells from a pancreas cell suspension after dissociation in a Ricordi chamber (left). The proportion of these cells expressing the myeloid lineage markers CD14 and CD64 is shown in a representative flow cytometry plot (center) with the compiled percentages of the indicated CD14 + CD64 + subset (right) (± SEM from 12 to 16 donors for each tissue). (B and C) Expression of tissue macrophage markers CD163 and CD206 (B), as well as MHC class II and CD86 (C), on the DAPI lo CD45 + CD64 + CD14 + myeloid cells isolated from pancreas, jejunum, PLN, and spleen. Shown are representative flow cytometry plots (left) with the compiled percentages of the indicated subsets (right) (±SEM from 12 to 16 donors for each tissue). **p < 0.01 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Density of pancreas macrophages in endocrine and exocrine pancreas. Shown is a representative qmIF image with the single-marker CD163 (left) and the corresponding composite image (right) of CD163 (yellow), the ductal marker CK19 (green), and the neuroendocrine marker chromogranin (white). Acinar, ductal, and endocrine areas were defined using inForm software, and the densities of CD163 + macrophages were quantified in the indicated pancreatic tissue areas (right) (± SEM from 13 donors). **p < 0.01 as calculated by one-way ANOVA with Sidak’s multiple comparisons test. White bar, 100 µm for scale. See also .

Article Snippet: Descriptive statistics of compiled flow cytometry data, graphs and statistical testingwere performed using GraphPad Prism (Graphpad software, San Diego, CA).

Techniques: Suspension, Expressing, Flow Cytometry, Isolation, Marker, Software

Journal: Cell reports

Article Title: Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

doi: 10.1016/j.celrep.2019.11.056

Figure Lengend Snippet: Sorted pancreas TRMs (CD8 + CD69 + TEM cells) were stimulated with monomeric TCR-activating anti-CD3 antibody (OKT3) in the presence or absence of sorted pancreas macrophages (MF; CD14 + CD64 + CD163 + ) (1:1 ratio) or with microbeads coated with anti-CD3 and exogenous co-stimulation (activating anti-CD2 and anti-CD28 antibodies). (A) Cytokine production (IFN-γ and IL-2) with the indicated stimulation conditions is shown in representative flow cytometry plots (left) with compiled data for each cytokine and the cytokine polyfunctionality index (right) (±SEM from three to five donors). **p < 0.01, as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (B and C) Representative histograms of PD-L1 (B) and CD58 (C) expression on pancreatic macrophages (CD45 + CD14 + CD163 + , red), non-myeloid immune cells (CD45 + CD14 − , blue), and CD45 − cells (gray) with graphs of compiled frequencies (±SEM from four to seven donors) expressing the indicated marker (right). **p < 0.01 macrophages versus both other cells types as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Pancreas TRMs in macrophage co-cultures were stimulated with anti-CD3 in the presence or absence of PD1 blocking antibody (nivolumab) or CD58 blocking antibody followed by intracellular cytokine staining. Shown are flow cytometry profiles of TRM IFN-γ and IL-2 production (top) with compiled data showing the frequencies of TRMs producing each cytokine and the cytokine polyfunctionality index (bottom) (±SEM from four donors). **p < 0.01, *p < 0.05 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. See also .

Article Snippet: Descriptive statistics of compiled flow cytometry data, graphs and statistical testingwere performed using GraphPad Prism (Graphpad software, San Diego, CA).

Techniques: Flow Cytometry, Expressing, Marker, Blocking Assay, Staining