VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites

Abstract

The mechanisms by which tumors metastasize to sentinel and distant lymph nodes, and beyond, are poorly understood. We developed transgenic mice that overexpress vascular endothelial growth factor-C (VEGF-C) and green fluorescent protein specifically in the skin and studied the effects of chemically-induced skin carcinogenesis in this model. We found that in contrast to VEGF-A, VEGF-C does not increase the growth of primary tumors, but instead induces expansion of lymphatic networks within sentinel lymph nodes, even before the onset of metastasis. Once the metastatic cells arrived at the sentinel lymph nodes, the extent of lymphangiogenesis at these sites increased. Of importance, in mice with metastasis-containing sentinel lymph nodes, tumors that expressed VEGF-C were more likely to metastasize to additional organs, such as distal lymph nodes and lungs. No metastases were observed in distant organs in the absence of lymph node metastases. These findings indicate an important role of VEGF-C-induced lymph node lymphangiogenesis in the promotion of cancer metastasis beyond the sentinel lymph nodes. VEGF-C is therefore a good target to slow or even prevent the onset of metastasis.

Figure 1

Transgenic overexpression of VEGF-C does not affect the number or frequency of papillomas and squamous-cell carcinomas that form in mice. (A) Incidence of skin papilloma formation, over time (weeks), in VEGF-C transgenic mice (n = 31; ■), compared with control mice (n = 32; ○). Incidence is expressed as the percentage of mice with detectable papillomas (> 1 mm) during the 20 weeks of topical PMA application. (B) No significant increase in the frequency (average number of papillomas per mouse) of papilloma formation was observed in VEGF-C transgenic mice. (C-D) No differences were observed in incidence or number of large papillomas more than 3 mm that formed in VEGF-C transgenic mice, compared with control mice, over the 20-week period of carcinogen application. (E-F) When mice were observed for an extended time period (33 weeks), squamous-cell carcinomas (SCCs) developed in both control and VEGF-C transgenic mice with the same incidence and numbers. (G) A comparable percentage of large papillomas underwent malignant conversion into SCCs in VEGF-C transgenic mice and control mice.

Figure 2

Transgenic VEGF-C expression is maintained during skin carcinogenesis. In situ hybridization demonstrates strong expression of human VEGFC mRNA in squamous-cell carcinomas (SCCs) of VEGF-C transgenic mice. (A) In primary tumors, SCC cells express high levels of VEGFC mRNA, as indicated by hybridization to an antisense human VEGFC probe. (B) In SCC metastases that form in sentinel lymph nodes (LNs), this expression of VEGF-C mRNA is maintained. (C-D) A human VEGF-C sense control probe did not hybridize with tumor samples. (A-D) Scale bars represent 100 μm. (E) ELISA analysis of human VEGF-C protein expression in skin and tumor lysates (n = 5 per group) revealed significant increases in the levels of VEGF-C protein in SCCs that form in VEGF-C transgenic mice, compared with the normal skin of these mice. No human VEGF was detected in skin or tumor lysates of control mice. Data are expressed as mean ± SD; ***P < .001.

Figure 3

Increased tumor lymphangiogenesis and angiogenesis in VEGF-C transgenic mice. Immunofluorescence analyses of CD31 (green) and LYVE-1 (red) expression in normal skin (A-B), early papillomas (C-D), and SCCs (E-F) of control (WT; A,C,E) and VEGF-C transgenic (VEGFC TG; B,D,F) mice revealed increased vascularization of papillomas and SCCs in VEGF-C transgenic mice and in control mice, compared with their PMA-treated skin. Tumor lymphangiogenesis was more prominent in VEGFC transgenic mice (D,F) than in control mice (C,E), with increased numbers of enlarged lymphatic vessels (red). Slight increases in the amount of tumor angiogenesis (green) in SCCs of VEGFC transgenic mice (F) were also observed, compared with that of control mice (E). Nuclei are labeled blue (Hoechst stain). (A-F) Scale bars represent 200 μm. (G-H) Computer-assisted morphometric analysis of normal cutaneous vessels and of tumor-associated lymphatic and blood vessels was performed. A significant increase in the relative area occupied by blood vessels in the peritumoral area of SCCs (Peri SCC), as well as within SCCs (Intra SCC), was observed in VEGFC transgenic mice (TG; ■), compared with that of the control mice (WT, □) (G). A significant increase of the relative area occupied by lymphatic vessels was observed in the VEGFC transgenic mice, throughout all stages of skin carcinogenesis (H). Skin indicates PMA-treated normal skin (n = 7); SP, small papillomas (1-3 mm; n = 6); LP, large papillomas (> 3 mm; n = 6); and SCC, squamous-cell carcinoma (n = 7). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001; NS = not significant.

Figure 4

Increased expression of VEGFR-3 in tumor-associated lymphatic vessels of VEGFC transgenic mice. Immunofluorescence analysis of LYVE-1 (red) and VEGFR-3 (green) expression was performed in SCC samples from control (A,C,E) and VEGFC transgenic (TG; B,D,F) mice. Based on LYVE-1 expression, the SCCs of VEGFC transgenic mice demonstrated prominent lymphangiogenesis (B), whereas tumor-associated lymphatic vessels were less pronounced in SCCs of control mice (A). Expression of the VEGF-C receptor (VEGFR-3) was strongly up-regulated in the tumor-associated lymphatic vessels of VEGF-C–overexpressing mice (D). Merging of images revealed that VEGFR-3 expression completely colocalized with that of LYVE-1 (F, yellow to orange), whereas VEGFR-3 was only weakly expressed in the tumor-associated lymphatic vessels of control mice (C,E). (A-F) Scale bars represent 200 μm.

Figure 5

Increased tumor metastasis to sentinel lymph nodes in VEGFC transgenic mice. (A-D) Flow cytometry was used to calculate the percentage of GFP-expressing tumor cells in primary squamous-cell carcinomas (SCC; A-B) and in sentinel lymph nodes (LN; C-D) of control and VEGFC transgenic mice. Of all SCC-associated cells, 25.5% in the control GFP-transgenic mice and 23.8% in the VEGFC transgenic mice were observed to be GFP positive (A-B). Eight weeks after the first cutaneous SCCs were detected, the number and percentage of GFP-expressing tumor cells was significantly higher in metastases that formed in the sentinel lymph nodes of the VEGFC transgenic mice (44.5%) than of the control GFP-transgenic mice (0.3%; C). Data are expressed as mean ± SEM (n = 5 per group). (E) Fluorescence microscopy analysis revealed an increased incidence of sentinel lymph node metastasis in VEGFC transgenic mice, compared with control mice (n = 12). (B,D,E) ***P < .001; *P < .05; NS = no significance.

Figure 6

Prominent lymph node lymphangiogenesis in VEGFC transgenic mice. (A-B) Routine H&E stains of lymph nodes of non–tumor-bearing mice. (C-D) Double immunofluorescence staining of lymph nodes of non–tumor-bearing mice demonstrated a comparable pattern of CD31⁺/LYVE-1–negative blood vessels (green) and LYVE-1–positive sinusoids (red) in wild-type mice (C) and in VEGFC transgenic (TG; D) mice. Nonmetastatic sentinel lymph nodes of SCC-bearing VEGFC transgenic mice have increased numbers of enlarged LYVE-1–positive sinusoids (red; F), compared with control mice (E). An increased number of enlarged LYVE-1–positive lymphatic vessels (red) was also found in the metastatic sentinel lymph nodes of VEGFC transgenic mice (H), compared with control mice (G). The tumor-associated LYVE-1–positive vessels (red) in VEGFC transgenic mice showed high levels of BrdU staining in lymphatic endothelial cells (J; green), indicating active lymphatic proliferation (arrowheads) within sentinel lymph nodes. These cells also expressed Prox1 (I; green). Nuclei are stained blue (Hoechst stain). (A-F, I-J) Scale bars represent 100 μm; (G-H) scale bars represent 200 μm.