Journal: Cancers

Article Title: Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids

doi: 10.3390/cancers13246158

Figure Lengend Snippet: Effect of drug alone on organoid growth and viability. ( a ) Brightfield images and quantification of organoid area using cultures treated with Nivolumab, Mubritinib and Cabozantinib alone. ( b ) Brightfield images and quantification of organoid area using organoid/CTL/MDSC co-cultures treated with Cabozantinib alone, Nivolumab plus Cabozantinib, or Mubritinib plus Cabozantinib. * p < 0.05 compared to Cabozantinib alone. Scale bar = 100 μm.

Article Snippet: Organoid-immune cell co-cultures were treated for 48 h and observed using brightfield microscopy (Nikon Spinning disk Ti 2 Eclipse inverted confocal microscope, Nikon Corporation, Tokyo, Japan).

Techniques:

Journal: Cancers

Article Title: Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids

doi: 10.3390/cancers13246158

Figure Lengend Snippet: HER2 expression is significantly correlated with PD-L1 expression in gastric cancer tissues. Brightfield images showing morphological differences among huTGOs derived from intestinal ( a , e ) and diffuse ( i , m ) subtypes of gastric cancer tissues. Immunofluorescence staining of these organoids showed a high expression of ( b – d , j – l ) HER2 (red) and PD-L1 (green) in two of the organoid lines while others showed a negative expression ( f – h , n – p ) for both. Western blot ( v ) analysis of gastric cancer tissues showed a similar expression pattern. ( q ) shRNA knockdown of HER2 showed a downregulation of PD-L1 expression which was confirmed by ( u ) Western blot analysis and quantification. ( r , s ) Quantification of HER2 and PD-L1 positive cells showed a significant reduction in the expression of both genes after HER2 shRNA knockdown; * p < 0.0001. ( t ) Linear regression analysis showing a positive correlation of HER2 and PD-L1; Immunohistochemistry of PD-L1 ( w ) and HER2 ( x ) retained high expression levels among gastric cancer tissues, tissue-derived organoids and orthotopically transplanted organoids. Scale bar = 100 μm and 50 μm.

Article Snippet: Organoid-immune cell co-cultures were treated for 48 h and observed using brightfield microscopy (Nikon Spinning disk Ti 2 Eclipse inverted confocal microscope, Nikon Corporation, Tokyo, Japan).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Western Blot, shRNA, Knockdown, Immunohistochemistry

Journal: Cancers

Article Title: Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids

doi: 10.3390/cancers13246158

Figure Lengend Snippet: Inhibition of HER2 regulates PD-L1 expression by suppressing HER2/AKT/MTOR signaling in gastric cancer-derived organoids. The expression of the HER2/AKT/MTOR signaling pathway components were determined by ( a ) Western blot analysis. ( b ) Violin plots representing densitometric values of protein bands measured from 8a. Brightfield images and the calculated organoid area in cultures treated with ( c ) vehicle (control), or ( d ) Everolimus. n = 15 ROI from 3 different experiments, * p < 0.05 in comparison to vehicle. Immunofluorescences staining using antibodies specific for HER2 (green) and PD-L1 (red) using ( e ) vehicle (control), or ( f ) Everolimus treated organoid cultures. ( g ) Quantification of fluorescence intensity for PD-L1 expression in the control or Everolimus treated cultures. ( h ) Proposed mechanisms of anti-PD1 inhibition and suppression of MDSCs in a combination immunotherapy strategy for gastric cancer patients. Within the gastric TME, PMN-MDSCs override the checkpoint inhibition by releasing Arg1, iNOS and ROS. Pharmacological inhibition of ERBB2/HER2 could diminish PD-L1 +ve cells from the TME, improving patient responses to immunotherapy. Scale bar = 100 μm.

Article Snippet: Organoid-immune cell co-cultures were treated for 48 h and observed using brightfield microscopy (Nikon Spinning disk Ti 2 Eclipse inverted confocal microscope, Nikon Corporation, Tokyo, Japan).

Techniques: Inhibition, Expressing, Derivative Assay, Western Blot, Control, Comparison, Staining, Fluorescence

Journal: PLoS Pathogens

Article Title: Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

doi: 10.1371/journal.ppat.1003056

Figure Lengend Snippet: Huh7 cells were infected with HCV (strain Jc1) using 30 TCID 50 /cell and 48 h later cells were fixed and processed for fluorescence microscopy. In case of samples shown in panels B–D, cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above. Samples were analyzed with a Nikon TE2000-E inverted confocal microscope at 60× magnification. (A)–(D) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase (PDI; an ER marker), GFP-Rab21 (marker for early endosomes), GFP-Rab7 (marker for late endosomes) or βCOP-YFP (marker for COP I vesicles). The upper panels represent a low magnification overview; boxed areas are shown as enlargement in the corresponding panel below. The nucleus was stained with DAPI (blue). Scale bars represent 10 µm (top panels) and 2 µm (lower panels). The quantification of the degree of colocalization (Pearson's correlation coefficient) is given at the top of the enlarged pictures.

Article Snippet: For image analysis we used a Perkin Elmer Ultraview ERS spinning disk on a Nikon TE2000-E inverted confocal microscope equipped with a Plan-Apochromat VC 60× objective (NA 1.20) and the Volocity 5.3 software package.

Techniques: Infection, Fluorescence, Microscopy, Transfection, Expressing, Construct, Marker, Staining