Figure 7. Periostin-expressing CAFs promote lymphatic metastasis by remodeling the extracellular matrix and directing lymphovascular invasion along organized collagen fibers.

(A) Study design in Postn^DTA mice. (B) Volume measurements of EO771.LMB tumors in control versus periostin⁺ cell-depleted mice. Each line represents an individual mouse (n = 8–10 mice per group). Statistics shown for multiple Mann-Whitney tests. (C) SHG images of intratumoral collagen fibers (pseudo-colored in cyan) in control versus periostin⁺ cell-depleted mice. Scale bars: 150 μm, insets: 3x zoom. (D) Tissue tilescans of axillary lymph nodes from control versus periostin⁺ cell-depleted mice bearing EO771.LMB mammary tumors. Tumor cells labelled with mCherry and nuclei counterstained with DAPI. Scale bars: 200 μm, insets: 3x zoom. (E) Percentage of tissue area positive for mCherry in sections of axillary lymph nodes from control versus periostin⁺ cell-depleted mice bearing highly metastatic EO771.LMB mammary tumors. Each data point represents an individual histological section (n = 8–10 mice per group). Statistics shown for Mann-Whitney test. (F) Experimental setup for in vitro lymphovascular invasion assay using MDA-MB-231^mCherry human breast cancer cells, primary human breast CAFs, and primary human lymphatic endothelial cells. (G) Representative images of MDA-MB-231^mCherry tumor cells that have invaded across the lymphatic endothelial cell barrier in the in vitro lymphovascular invasion assay. Scale bar: 100 μm. (H) Quantification of MDA-MB-231^mCherry tumor cells that have invaded across the lymphatic endothelial cell (LEC) barrier or blood endothelial cell (BEC) barrier in the lymphovascular invasion assay. Spheroids consisted of either MDA-MB-231^mCherry tumor cells alone (mono-spheroids) or co-cultures of MDA-MB-231^mCherry tumor cells with primary human breast CAFs treated with either non-targeting control siRNA (si-Control) or periostin-targeting siRNA (si-POSTN). Each data point represents an individual spheroid. Experiment performed in triplicate. Statistics shown for 2-way ANOVA. (I) RT-qPCR analysis of periostin expression in paired primary breast cancer specimens (Primary BrCa) and lymph node metastases (Paired LNmet) from human breast cancer patients (n = 28 patients). qPCR performed in triplicate. (J) Kaplan-Meier plot of overall survival in lymph node positive breast cancer patients stratified by periostin protein expression (n = 27 patients). (K) Summary model. (Schematics created with BioRender.com).