Figure 7
From: A critical role for lymphatic endothelial heparan sulfate in lymph node metastasis
Genetic alteration of lymphatic endothelial heparan sulfate biosynthesis impairs tumor metastasis to regional lymph nodes. A. Cre reporter testing in the inducible Prox1+/CreERT2model: Bilateral subiliac lymph nodes (LN) were isolated from Prox1+/CreERT2Rosa26R reporter mice ("Prox1+/CreERT2", lower panel) or their Prox1-/CreERT2Rosa26R littermates ("Prox1-/CreERT2", upper panel) after intraperitoneal injection of Tamoxifen for 5 consecutive days; and stained with X-Gal (showing as deep blue stain in Cre positive lymph nodes). Right panels: subiliac LNs from A were sectioned and stained for LYVE-1 (brown) and X-Gal (blue); and imaged under 400 × magnification (Scale bar, 50 μm). For tumor establishment, LLC tumor cells were injected subcutaneously into the left caudal-medial inguinal region of Ndst1f/fProx1+/CreERT2mice (N = 10) and their Ndst1f/fProx1-/CreERT2wildtype littermates (N = 10) following a 5-day tamoxifen dosing schedule. Following 14 days of primary tumor growth, the left subiliac regional LN from each mouse was isolated. B. Comparison of primary tumor volumes from Cre postive vs Cre negative mice at time of experiment termination and lymph node harvesting. C. Representative photomicrographs of pan-keratin stained LN histologic sections are shown above plotted data on the left, which shows quantified pan-keratin signal per LN for each mouse. Graph to the right shows plotted data for degree of CCL21 immunoreactive signal per LN (and representative photomicrographs of CCL21 staining from Cre positive vs Cre negative animals above the graph). LN staining signal from both groups was imaged under 100× magnification, quantified as pixel values per LN using NIH Image J software, and normalized to the average pixel value/LN in Ndst1f/fProx1-/CreERT2control group. *P < 0.05, #P < 0.01, for comparison of mean mutant vs control group values. D. LNs from both groups were co-stained with CCL21 (blue) and LYVE1 (brown) (left two panels) or pan-keratin alone as a marker for metastatic tumor cells (blue signal, right panel). Scale bars, 100 μm for 100× and 50 μm for 400× magnification.