Journal: Translational Lung Cancer Research

Article Title: Development of a 3D-3 co-culture microbead consisting of cancer-associated fibroblasts and human umbilical vein endothelial cells for the anti-tumor drug assessment of lung cancer

doi: 10.21037/tlcr-2025-525

Figure Lengend Snippet: Characterization of the biological properties of sodium Alg-HA 3D micro-beads. (A) Bright-field microscopy images showing the morphology of CRLCs, CAFs, and HUVECs, as well as the spheroid formation of cancer cells within the micro-beads. The red arrow indicates 3T3-J2 mouse fibroblasts, and the black arrows indicate CRLCs. Scale bar: 250 µm. (B) Expression of CK7, α-SMA, and CD31 in CRLCs, CAFs, and HUVECs. The cells were stained with DAPI (blue) to indicate the nuclei, anti-CK7 antibody (green) for CRLCs, anti-α-SMA antibody (violet) for CAFs, and anti-CD31 antibody (red) for HUVECs. Scale bar: 50 µm (upper) and 20 µm (lower). (C,D) The relationship among oscillatory strain, deformation percentage, and energy storage modulus of sodium Alg micro-beads at varying concentrations. (E) The effect of the F+Y to ECM medium ratio on the proliferation of CRLC, CAF, and HUVEC co-cultures in 2D environments. (F) The impact of irradiated cancer fibroblasts on the proliferation of lung cancer progenitor cells. (G) Proliferation levels of CRLCs, CAFs, and HUVECs within micro-beads measured from culture days 1 to 7. (H) Visualization of live cells (green) and dead cells (red) using calcein-AM and PI staining. Scale bar: 250 µm. 3D-2-LC_1: CRLCs-CAFs co-culture; 3D-2-LC_2: CRLCs-HUVECs co-culture; 3D-3-LC: CRLCs-CAFs-HUVECs co-culture. 2D, two-dimensional; 3D, three-dimensional; α-SMA, alpha-smooth muscle actin; Alg, alginate; CAFs, cancer-associated fibroblasts; CK7, cytokeratin 7; CRLCs, conditionally reprogrammed lung cancer cells; DAPI, 4',6-diamidino-2-phenylindole; ECM, extracellular matrix; HA, hyaluronic acid; HUVECs, human umbilical vein endothelial cells; LC, lung cancer; PI, propidium iodide.

Article Snippet: The HUVECs cell line was purchased from iCell Inc., China, and maintained in an ECM (ScienCell).

Techniques: Microscopy, Expressing, Staining, Irradiation, Co-Culture Assay

Journal: Translational Lung Cancer Research

Article Title: Development of a 3D-3 co-culture microbead consisting of cancer-associated fibroblasts and human umbilical vein endothelial cells for the anti-tumor drug assessment of lung cancer

doi: 10.21037/tlcr-2025-525

Figure Lengend Snippet: Transcriptomic correlation analysis of CRLCs in Alg-HA micro-beads across different groups. (A) Bright-field microscopy images showing the structure of 3D-3 micro-beads. Histological stain showing the structure and status in 3D-3 micro-beads and primary tumors. The arrows point to spherical cell clusters. Scale bar: 100 µm. (B) Immunofluorescence images showing the ADC related markers (CK7 and TTF-1) in primary tumors and 3D-3 micro-beads. Scale bar: 50 µm. (C) PCA of RNA-seq data comparing tumor tissue, 2D-LC, Mono-LC, 3D-2-LC_1, 3D-2-LC_2, and 3D-3-LC conditions. (D) Volcano plots illustrating upregulated (red) and downregulated (blue) genes in pooled 3D-3-LC relative to 3D-2-LC_1 and 3D-2-LC_2. (E) Volcano plots and intersectional clustering were used to analyze the DEGs related to tumor stem cells in primary lung cancer cells under both 3D-3 co-culture and 3D mono-culture microbeads. (F) Dot plot shows the up-regulated Reactome pathways (FDR <0.1) enriched for different contrasts. The size of the dot is based on gene count enriched in the pathway, and the color of the dot shows the pathway enrichment significance. (G) Differences in gene expression between lung cancer tissues and the 3D-3-LC model. The heatmap shows the differences in gene expression levels related to the extracellular matrix-receptor interaction pathway between lung cancer tissues and the 3D-3-LC model. Tissue: lung cancer tissue; 2D-LC: 2D cultured lung cancer cells; Mono-LC: lung cancer cells cultured in 3D conditions; 3D-2-LC_1: CRLCs-CAFs co-culture; 3D-2-LC_2: CRLCs-HUVECs co-culture; 3D-3-LC: CRLCs-CAFs-HUVECs co-culture. 2D, two-dimensional; 3D, three-dimensional; ADC, adenocarcinoma; Alg, alginate; BF, bright-field microscopy; CAFs, cancer-associated fibroblasts; CK7, cytokeratin 7; CRLCs, conditionally reprogrammed lung cancer cells; DEGs, differentially expressed genes; ECM, extracellular matrix; FDR, false discovery rate; H&E, hematoxylin and eosin; HA, hyaluronic acid; HUVECs, human umbilical vein endothelial cells; LC, lung cancer; PC, principal component; PCA, principal component analysis; RNA-seq, RNA sequencing; TTF-1, thyroid transcription factor-1.

Article Snippet: The HUVECs cell line was purchased from iCell Inc., China, and maintained in an ECM (ScienCell).

Techniques: Microscopy, Staining, Immunofluorescence, RNA Sequencing, Co-Culture Assay, Gene Expression, Cell Culture

Journal: Translational Lung Cancer Research

Article Title: Development of a 3D-3 co-culture microbead consisting of cancer-associated fibroblasts and human umbilical vein endothelial cells for the anti-tumor drug assessment of lung cancer

doi: 10.21037/tlcr-2025-525

Figure Lengend Snippet: The influence of co-culturing with CAFs and HUVECs on lung cancer cell stemness. (A) A heatmap displaying the fold changes in expression levels of lung CSC-specific markers across co-cultured, mono-cultured, and lung tissue samples. The categories include: tissue, 2D-LC, Mono-LC, 3D-2-LC1, 3D-2-LC2, and 3D-3-LC. (B) A comparison of expression levels (FPKM) of lung CSC-specific marker genes among tissue, 2D-LC, Mono-LC, 3D-2-LC1, 3D-2-LC2, and 3D-3-LC samples (n=7). (C) Immunofluorescence analysis of lung cancer tissue and 3D-3 microbeads. Cells were stained with DAPI (blue) to indicate cell nuclei and target proteins in CRLCs were labeled with antibodies against SOX2 (red), SOX9 (red), ALDH1A1 (red), NANOG (red), CD44 (red) and CD24 (red). All scale bars represent 100 µm. (D) Quantitative analysis of western blot results depicting the expression of stemness markers in lung cancer cells under different co-culture conditions. *, P<0.05; **, P<0.005; ***, P<0.001; ****, P<0.0001. Tissue: lung cancer tissue; 2D-LC: 2D cultured lung cancer cells; Mono-LC: lung cancer cells cultured in 3D conditions; 3D-2-LC_1: CRLCs-CAFs co-culture; 3D-2-LC_2: CRLCs-HUVECs co-culture; 3D-3-LC: CRLCs-CAFs-HUVECs co-culture. 2D, two-dimensional; 3D, three-dimensional; CAFs, cancer-associated fibroblasts; CRLCs, conditionally reprogrammed lung cancer cells; CSC, cancer stem cell; DAPI, 4',6-diamidino-2-phenylindole; FPKM, fragments per kilobase of transcript per million mapped reads; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells.

Article Snippet: The HUVECs cell line was purchased from iCell Inc., China, and maintained in an ECM (ScienCell).

Techniques: Expressing, Cell Culture, Comparison, Marker, Immunofluorescence, Staining, Labeling, Western Blot, Co-Culture Assay