staining panck  (Ultivue Inc)

Journal: Frontiers in Oncology

Article Title: Digital Spatial Profiling identifies distinct patterns of immuno-oncology-related gene expression within oropharyngeal tumours in relation to HPV and p16 status

doi: 10.3389/fonc.2024.1428741

Figure Lengend Snippet: GeoMx DSP ROI selection and segmentation approach. (A) Tumour sections were stained with three morphology markers: PanCK (tumour; green), CD3e (T cell marker; yellow), CA-IX (surrogate marker for regions of hypoxia; red), plus a DNA stain to identify all cells (blue). ROIs were placed to identify regions within tumour nests, tumour-stroma interface and peri-tumoural stroma. In this illustrative example from patient 11, ROIs were placed to capture tumour and adjacent stroma in regions of high (ROIs 1, 2, 4, 5) and low (ROIs 3, 6) hypoxia. (B) Segmentation strategy – PanCK staining was used for identification of tumour (PanCK+) and stromal regions (PanCK-), enabling separate analysis. (C) Comparison of keratin gene expression between stromal and tumour compartments. Graph shows combined normalised KRT gene expression for all PanCK- versus PanCK+ segments within each of the 12 patient samples. P values for comparison of PanCK- versus PanCK+ were calculated using unpaired t test, adjusted p values reported as * <0.05, ** <0.005.

Article Snippet: ROIs were segmented into multiple regions representing tumour and stromal (non-tumour) tissue, using a threshold classifier on a fluorescently labelled pan-cytokeratin (PanCK) stain (clone AE1/AE3 594 Novus, 1:200 dilution).

Techniques: Selection, Staining, Marker, Comparison, Gene Expression

Journal: Frontiers in Oncology

Article Title: Digital Spatial Profiling identifies distinct patterns of immuno-oncology-related gene expression within oropharyngeal tumours in relation to HPV and p16 status

doi: 10.3389/fonc.2024.1428741

Figure Lengend Snippet: Expression of selected immuno-oncology-related genes within the three p16/HPV subgroups. Violin plots showing log2-transformed normalised gene expression for p16+/HPV+ (white), p16+/HPV- (pale grey) and p16-/HPV- (dark grey) OPC samples. First and third columns represent tumour (PanCK+), second and fourth columns represent stroma (PanCK-). Dashed and dotted lines represent the median and quartiles respectively. Data were analysed using Kruskal–Wallis test, and pair-wise comparisons were conducted with the Wilcoxon rank-sum test. Adjusted P values (see Supplementary Table 1 ) are reported as: ns, nonsignificant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: ROIs were segmented into multiple regions representing tumour and stromal (non-tumour) tissue, using a threshold classifier on a fluorescently labelled pan-cytokeratin (PanCK) stain (clone AE1/AE3 594 Novus, 1:200 dilution).

Techniques: Expressing, Transformation Assay, Gene Expression

Journal: Nature Medicine

Article Title: Autologous T cell therapy for MAGE-A4 + solid cancers in HLA-A*02 + patients: a phase 1 trial

doi: 10.1038/s41591-022-02128-z

Figure Lengend Snippet: a , Digital image quantification of T cell markers (total CD3 + ), CD4 (CD3 + CD4 + ), cytotoxic (CD3 + CD8 + ), regulatory (CD3 + CD4 + FoxP3 + ) and co-staining for phenotypes (proliferating, Ki67 + ; activated, GrazB + ; PD-L1 + ) and combination of these phenotypes for the four patients referenced in Extended Data Fig. . Baseline biopsies were taken 2–5 weeks before afami-cel infusion. b , A multiparametric analysis of T cell infiltration in tumor biopsies using IHC, MAGE-A4 SPEAR RNAscope (Advanced Cell Diagnostics) and multiplex immunofluorescence. Images of post-infusion biopsy (liver) from Patient 4: A, spatial plot generated using spatial analysis module in HALO (Indica Labs) showing malignant cells (PanCK + (red), regulatory (CD3 + CD4 + FoxP3 + ) T cells (yellow) and cytotoxic (CD3 + CD8 + ) T cells (blue/cyan)). Scale bar is not applicable as this is not a raw image; B, CD3 IHC/SPEAR + T cell RNAscope duplex (CD3 IHC staining (blue); MAGE-A4 SPEAR T cell staining (purple)); C, Ultivue 8-plex multiplex dataset showing CD4 (orange), PD-L1 (red), CD8 (green), Ki67 (purple), FoxP3 (blue), GrazB (white), CD3 (yellow) and PanCK (teal); D, PanCK immunofluorescence staining from Ultivue 8-plex panel (displayed in absorption mode in HALO for clarity); E, MAGE-A4 IHC stain (DAB (brown)); and F, hematoxylin (purple) and eosin (pink) stain. c , Heat map of log 2 -transformed normalized counts of genes associated with ‘T cell exhaustion’ and ‘Negative regulation of T cell-mediated immunity’ in baseline and post-infusion biopsies from Patients 1–4. Patients 1–3 were patients with SS; Patient 4 was a patient with ovarian cancer. DAB, 3,3′-diaminobenzidine; FoxP3, forkhead box protein 3; GrazB, granzyme B; Ov, ovarian; PanCK, pancytokeratin; PD-L1, programmed death ligand 1.

Article Snippet: Scale bar is not applicable as this is not a raw image; B, CD3 IHC/SPEAR + T cell RNAscope duplex (CD3 IHC staining (blue); MAGE-A4 SPEAR T cell staining (purple)); C, Ultivue 8-plex multiplex dataset showing CD4 (orange), PD-L1 (red), CD8 (green), Ki67 (purple), FoxP3 (blue), GrazB (white), CD3 (yellow) and PanCK (teal); D, PanCK immunofluorescence staining from Ultivue 8-plex panel (displayed in absorption mode in HALO for clarity); E, MAGE-A4 IHC stain (DAB (brown)); and F, hematoxylin (purple) and eosin (pink) stain. c , Heat map of log 2 -transformed normalized counts of genes associated with ‘T cell exhaustion’ and ‘Negative regulation of T cell-mediated immunity’ in baseline and post-infusion biopsies from Patients 1–4.

Techniques: Staining, Multiplex Assay, Immunofluorescence, Generated, Immunohistochemistry, Transformation Assay

Journal: Cell Death & Disease

Article Title: Inhibition of mPGES-1 attenuates efficient resolution of acute inflammation by enhancing CX3CL1 expression

doi: 10.1038/s41419-021-03423-2

Figure Lengend Snippet: Starting 24 h post i.p. zymosan (5 mg/kg) injection, mice received daily i.p. injections of the mPGES-1 inhibitor compound III (CIII) (25 mg/kg) or the appropriate vehicle control (VEH). Cx3cr1 mRNA expression in A F4/80 lo and B F4/80 hi macrophages (Mϕ) was analyzed by RT-qPCR analysis. C Concentrations of CX3CL1 in the peritoneal lavage were determined by ELISA. D Representative pictures of peritoneal membranes from VEH- and CIII-treated animals at day 6 using multiplexed IHC staining for the epithelial marker pan-cytokeratin (PanCK), CX3CL1, and DAPI. E Quantification of CX3CL1 staining intensity in PanCK-expressing epithelial cells using the InForm-software. F Epithelial E0771 cells were treated with medium supplemented with peritoneal lavages obtained from VEH- or CIII-treated mice at day 6 of the peritonitis model for 4 h. Cx3cl1 mRNA expression was analyzed by RT-qPCR. Data are represented as means ± SEM ( n ≥ 4 per group). For statistical analysis within each treatment, t test or one-way ANOVA with Holm–Sidak posthoc test was used for parametric data, otherwise, Kruskal–Wallis test was used. Analysis between the treatments for parametric data was done via two-way ANOVA with Holm–Sidak posthoc test, non-parametric data were log-transformed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The relative abundance of CX3CL1 expressing cells in the epithelium was scored upon tissue segmentation based on panCK staining with the InForm software (Akoya Biosciences).

Techniques: Injection, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Marker, Staining, Software, Transformation Assay