Fig. 3: Cell-collagen interaction supports the suppressive role of HLF in cell invasion.

a RNA-seq analysis of 786-O cells with HLF overexpression (OE) or knockout (KO), n = 3 biological replicates. b GO analysis of overlapped genes in both HLF OE and KO group. c GSEA analysis of the HLF-OE seq data in terms of the collagen binding pathway. d RT-qPCR of the critical genes enriched in the “collagen binding” pathway from GSEA analysis, n = 3 biological replicates. e Schematic showing spheroid formation followed by embedding in collagen to monitor cell invasion. Created in BioRender. Zhou, J. (2025) https://BioRender.com/x5scj3g. f, g RT-qPCR (n = 3 biological replicates) of HLF mRNA level (left) and quantification of 3D spheroid invasion (n = 6 independent cell cultures) (Right) (f), and corresponding representative images (g). Scale bar, 100 µm. h, i Representative images (h) and quantification (i) of 3D spheroid invasion in cells with HLF KO after 2 days (786-O) or 6 days (UMRC6) of collagen culturing, n = 6 independent cell cultures. Scale bar, 200 µm. j, k Representative images (j) and quantification (k) of 3D spheroid invasion after 3 days (786-O) or 6 days (Caki-1, A498) of collagen culturing, n = 6 independent cell cultures. Scale bar, 100 µm. l, m Scheme of 3D co-culture in collagen followed by fluorescence imaging of the live cell spheres (l), and quantification (m) of cell leading protrusions after 3 days. (n = 3 spheres from three independent cell cultures). Scale bar, 100 µm. Created in BioRender. Zhou, J. (2025) https://BioRender.com/x5scj3g. n Scheme of the orthotopic injection of mixed sgCtrl (red) and sgHLF (green) cells into NSG mice. Primary tumors and lung metastases were dissected and subjected to immunofluorescence staining. Scale bar, 20 µm. Consistent results were observed across all five mice. Created in BioRender. Zhou, J. (2025) https://BioRender.com/x5scj3g. Data are mean ± s.e.m. (d, f_left), box plots show the median and interquartile range, and whiskers show the data range (f_right, i, k). DESeq2’s Wald test (two-sided test) and False Discovery Rate (FDR) correction was applied (a), Fisher’s exact test and FDR correction (b), One-way ANOVA followed by a post hoc Dunnett-t-test (f_ left, I) or unpaired two-tailed t-test (d, f_ right, k, m). Source data are provided as a Source Data file.