Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: Antibodies for western blot (WB), Immunocytochemistry with immunofluorescent detection (ICC/IF), and Immunohistochemistry of paraffin sections with immunofluorescent detection (IHC-P/IF).

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Western Blot, Immunocytochemistry, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: List of IMC antibodies.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques:

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: Resistance to first- and second-generation EGFR inhibitors is associated with features of EMT. (A) Immunocytochemistry of HCC827 parental cells and the erlotinib-resistant clones ER3 and ER10 for the epithelial marker CDH1 (E-cadherin) and the mesenchymal marker VIM (vimentin) to examine markers of epithelial plasticity upon acquired drug resistance. Counterstain by DAPI. Scalebar = 30 μm. (B) TUBA1A (alpha-tubulin) immunocytochemistry of the cells described in (A) were applied to reveal the phenotypic shift in cell morphology. Counterstain by DAPI. Scalebar = 30 μm. (C) Western blots were prepared with lysates from the HCC827 parental cells and the erlotinib-resistant clones ER3, ER10, ER20, and ER30 H1975 parental cells and the clones COR1-1 and COR10-1 resistant to the second-generation EGFR inhibitor rociletinib. Immunodetection of epithelial marker CDH1 (E-cadherin) (135 kDa), mesenchymal markers CDH2 (N-cadherin) (135 kDa), VIM (vimentin) (54 kDa). Western blot analysis was repeated n = 3 times, and a representative experiment is presented in the figure. (D) Quantification of the western blot presented in (C) normalized against total protein presented in Supplementary Figure 1A (VIM and CDH1) and B (CDH2). Fold change values for the resistant clones ER3 and ER10 relative to their parental cell line HCC827 (E) Expression of transcripts encoding CDH1, CDH2, VIM , assessed by RT-qPCR on cDNA prepared from HCC827 parental, ER3, and ER10 cells. RT-qPCR analyses were repeated n = 3 times, and representative results from one experiment with n = 3 technical replicates are presented in the figure as mean fold change +/- SD calculated by the 2 –ΔΔCt method. Two-way ANOVA followed by Tukey’s multiple comparison test comparing ER3 and ER10 against the parental cell line showed that for all genes, the gene expression of all genes in both ER3 and ER10 were significantly different from the parental cells ( P < 0.0001). (F) Quantification of the western blot presented in (C) normalized against total protein presented in Supplementary Figure 1A (VIM and CDH1) and B (CDH2). Fold change values for the resistant clones COR1-1 and COR10-1 relative to their parental cell line H1975. ****P ≤ 0.0001.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Immunocytochemistry, Clone Assay, Marker, Western Blot, Immunodetection, Expressing, Quantitative RT-PCR, Comparison, Gene Expression

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: Characterization of spheroids using immunohistochemistry with fluorescent detection and imaging mass cytometry. HCC827-GFP monoculture, HCC827-GFP + SV80-dsRed co-culture, ER3-GFP + SV80-dsRed co-culture, and SV80-dsRed monoculture spheroids were stained with an imaging mass cytometry panel of 14 heavy metal-tagged antibodies ( Table 3 ). For each condition, five ROIs containing a single spheroid were ablated by a Hyperion imaging mass cytometer (Fluidigm, Inc.). Representative ROIs are displayed showing (A) GFP and RFP, (B) GFP, RFP and MKI67 (Ki67), (C) CDH1 (E-cadherin), and VIM (Vimentin), (D) GFP and EGFR, (E) and Collagen 1. DNA staining displayed in all images is a combination of Iridium intercalator stain (Ir191 and Ir193) and Histone H3. Images were pseudo-colored in MCD viewer software (Fluidigm) to enable visualization of multiple channels per ROI, and for each channel the minimum and maximum intensity display settings are manually set to be kept constant between the samples. Scalebar = 50 µm (F) Immunohistochemistry staining with the EGFRdel19 mutation-specific antibody and AF647 fluorescence tagged secondary antibody together with DAPI counterstain. Fluorescent images were taken with a Zeiss Collibri7 fluorescence microscope.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Immunohistochemistry, Imaging, Mass Cytometry, Co-Culture Assay, Staining, Cytometry, Software, Mutagenesis, Fluorescence, Microscopy

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: In-depth analysis of imaging mass cytometry single-cell data from mono- and co-culture spheroids. (A) Single-cell expression data obtained from segmentation of the imaging mass cytometry experiment are displayed as a heatmap of the ungated (left), GFP+ (middle) and RFP+ (right) populations. Single-cell data was first generated as the mean pixel intensity for each cell, and the median intensity of all cells within a given population is displayed in the heatmaps. (B) The tSNE algorithm was applied on the ungated population based upon expression of the three markers EGFR, CDH1 (E-cadherin), and VIM (vimentin). viSNE plots displaying the distribution of cells from the different samples. (C) Marker expression of EGFR, CDH1 (E-cadherin), VIM (vimentin), MKI67 (Ki67), and Collagen type 1 displayed on the viSNE plots.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Imaging, Mass Cytometry, Co-Culture Assay, Expressing, Generated, Marker

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: Characterization of erlotinib-treated heterotypic spheroids using H&E staining and imaging mass cytometry. (A) H&E-stained sections of heterotypic HCC827-GFP + SV80-dsRed co-culture, ER3-GFP + SV80-dsRed co-culture spheroids. (B–F) Paraffin sections of vehicle (DMSO) and erlotinib- treated heterotypic HCC827-GFP + SV80-dsRed co-culture, ER3-GFP + SV80-dsRed co-culture spheroids were stained with an imaging mass cytometry panel of 19 heavy metal-tagged antibodies ( Table 3 ). For each condition, five ROIs containing a single spheroid were ablated by a Hyperion imaging mass cytometer (Fluidigm, Inc.). Representative ROIs are displayed showing: (B) GFP and RFP, (C) epithelial marker CDH1 (E-cadherin) and MUC1 (Mucin1/CD227), (D) GFP, RFP and proliferation marker MKI67 (Ki67), (E) apoptosis marker Cleaved caspase 3 (CC3), and (F) Collagen 1. DNA staining by Iridium intercalator stain (Ir191 and Ir193). Images were pseudo-colored in MCD viewer software (Fluidigm) to enable visualization of multiple channels per ROI, and for each channel the minimum and maximum intensity display settings are kept constant between the samples. Scalebar = 50 µm.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Staining, Imaging, Mass Cytometry, Co-Culture Assay, Cytometry, Marker, Software

Journal: Frontiers in Oncology

Article Title: Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

doi: 10.3389/fonc.2022.818437

Figure Lengend Snippet: In-depth analysis of imaging mass cytometry data from erlotinib treated spheroids. Absolute cell counts for HCC827+SV80 and ER3+SV80 heterotypic spheroids treated with either vehicle control (DMSO) or erlotinib, respectively. Absolute cell counts for (A) GFP+ cells (cancer cells), and (B) RFP+ cells (fibroblasts) is shown. Individual values represent median expression of each ROIs, and the mean +/-SD is plotted. Statistical significance in absolute cell counts were calculated in cytobank using the Mann-Whithey U-test (C) The diameter of erlotinib treated versus vehicle control (DMSO) treated spheroids measured by quantifying H&E-stained images using the measuring tool in Fiji. Mean diameter +/- SD for each condition is shown, and the statistics is performed in GraphPad Prism using the Mann-Whithey U-test. (D–F) Median expression of proliferation marker MKI67 (Ki67) in the nuclei of the RFP+ population (D) . Median expression of ACTA2 (αSMA) (E) , and PDGFRB (PDGFRβ) (F) in the RFP+ (fibroblast) populations of HCC827+SV80 and ER3+SV80 heterotypic spheroids treated with either erlotinib or vehicle (DMSO) control. Individual values represent median expression for each ROIs, and the mean +/- SD is plotted. Statistical significance in channel expression were calculated in cytobank using the Mann-Whithey U-test (G–I) Distance to border measurements displayed as Violin plots for the (G) ungated (all) population, (H) GFP+ population, and (I) RFP+ population. (J) The tSNE algorithm was applied on the ungated population based upon expression of the markers EGFR, CDH1 (E-cadherin), VIM (vimentin), MET (c-Met), pan-cytokeratin, GFP and RFP. viSNE plots displaying the distribution of cells from the different samples. NS = P > 0.05, *P ≤ 0.05, **P ≤ 0.01.

Article Snippet: 158 Gd , CDH1 (E-cadherin) , 24E10 , 201505 , Fluidigm , 1:100.

Techniques: Imaging, Mass Cytometry, Control, Expressing, Staining, Marker