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: Nutrients

Article Title: In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells

doi: 10.3390/nu13124227

Figure Lengend Snippet: Osteogenic Differentiation Assay—ALP and HA. Osteogenic differentiation at 14 days ( A ) and 35 days ( B ) of induction by cytochemical staining for ALP with Fast Red Violet B and for HA with Von Kossa staining. The ALP+ cells are in red and the grainy deposits are in black. Nuclei are counterstained in green. Observation in brightfield (AxioVision, ZEISS). Original magnification: 20×.

Article Snippet: The presence of multiple intracellular lipid droplets was observed in brightfield (AxioVision, ZEISS) ( A).

Techniques: Differentiation Assay, Staining

Journal: Nutrients

Article Title: In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells

doi: 10.3390/nu13124227

Figure Lengend Snippet: Adipogenic Differentiation Assay. Adipogenic differentiation at 35 days ( A ) and after 0 days ( B ) of induction by cytochemical staining with Oil Red O. In red the lipidic vesicles and in violet the nuclei counterstained by Toluidine Blue. Observation in brightfield. Original magnification: 20×.

Article Snippet: The presence of multiple intracellular lipid droplets was observed in brightfield (AxioVision, ZEISS) ( A).

Techniques: Differentiation Assay, Staining