Adrenomedullin gene dosage correlates with tumor and lymph node lymphangiogenesis

Abstract

Adrenomedullin (AM) is a potent lymphangiogenic factor that promotes lymphatic endothelial cell (LEC) proliferation through a pharmacologically tractable G-protein-coupled receptor. Numerous types of human cancers have increased levels of AM; however, the functional consequences of this fact have not been characterized. Therefore, we evaluated whether modulating adrenomedullin (Adm) gene dosage within tumor cells affects lymphangiogenesis. Murine Lewis lung carcinoma (LLC) cells that overexpress or underexpress Adm were injected subcutaneously into C57BL/6 mice, and tumors were evaluated for growth and vascularization. A dosage range from ∼10 to 200% of wild-type Adm expression did not affect LLC proliferation in vitro or in vivo, nor did it affect angiogenesis. Notably, the dosage of Adm markedly and significantly influenced tumor lymphangiogenesis. Reduced Adm expression in tumors decreased the proliferation of LECs and the number of lymphatic vessels, while elevated tumor Adm expression led to enlarged lymphatic vessels. Moreover, overexpression of Adm in tumors induced sentinel lymph node lymphangiogenesis and led to an increased incidence of Ki67-positive foci within the lung. These data show that tumor-secreted AM is a critical factor for driving both tumor and lymph node lymphangiogenesis. Thus, pharmacological targeting of AM signaling may provide a new avenue for inhibition of tumor lymphangiogenesis.

Figure 1.

In vitro characterization of the LLC cell line. A–C) qRT-PCR analysis amplifying murine Adm and Calcrl mRNA. Mouse embryo normalized to 1 was used as a calibrator in panels A and B. Scrambled RNAi and empty vector conditions were normalized to 100 in panel C. Graphs in panels A and C depict 1 representative experiment, while the data in panel B are an average of 3 independent experiments. D) Growth curve for the Adm RNAi, Adm OExp, and control LLC cells showed no differences in cell growth over 6 d. E) No change in AM-dosed LLC cell viability was detected by trypan blue exclusion over 6 d.

Figure 2.

In vivo tumor analysis. A) Measurements of tumor volume showed no differences in growth of Adm RNAi (n=8), Adm OExp (n=7), and control (n=13) tumors during a 14 d time course. B, C) qRT-PCR analysis revealed that Adm dosage is maintained in the tumors (B); no statistical change was found in Calcrl expression among the tumor groups (C).

Figure 3.

Adm expression does not affect angiogenesis. PECAM-1 (green)-, Ki67 (red)-, and DAPI (blue)-stained tumor sections revealed that dosage of Adm did not affect the average number of blood vessels (A), and the total number (B) or proliferation (C) of PECAM-1⁺ cells. Dashed line delineates tumor margin. Data are expressed as the average ± sem of 2 fields/step section/tumor (6 total fields), with no statistical differences among the tumor groups; 3 step sections/tumor were analyzed. Scale bars = 50 μm.

Figure 4.

Adm expression is necessary for tumor lymphangiogenesis. A) Representative H&E-stained tumor sections. B) LYVE-1 (green) and podoplanin (red) costain confirmed lymphatic identity of the vessels and showed that the average number of lymphatic vessels per tumor section increased with Adm dosage. Dashed line delineates tumor margin. Data represent the average number of lymphatic vessels within the tumor but at the tumor margin per tumor section; 3 step sections/tumor were analyzed. C, D) Dosage of Adm affected the total number of LYVE-1⁺ cells (C), and proliferation (Ki67, red) of LYVE-1⁺ (green) cells correlated with increased Adm tumor expression (D). Data are expressed as the average of 2 fields/step-section/tumor (6 fields total) ± sem (from 3 sections). Scale bars = 50 μm.

Figure 5.

Adm dosage does not affect macrophage or Vegf levels. A) LYVE-1 (green)-, F4/80 (red)-, and DAPI (blue)-stained tumor sections revealed the presence of LYVE-1⁺ and F4/80+ cells. B) No colocalization of LYVE-1 and F4/80 was found in lymphatic vessels. C) Dosage of Adm did not affect F4/80 expression or Mac-1 levels (graph). D) Dosage of Adm did not affect Vegf-a (left panel), Vegf-c (center panel), or Vegf-d (right panel) levels. Scale bars = 50 μm.

Figure 6.

Elevated Adm expression increases the diameter of blood and lymphatic vessels. Immunofluorescence analyses revealed that overexpression of Adm in LLC tumors caused a statistically significant increase in the area of PECAM-1⁺ blood vessels (A) and LYVE-1+ lymphatic vessels (B). Dashed line delineates tumor margin. Data are expressed as means ± se, with an average of 40 vessels/genotype analyzed. Scale bars = 50 μm.

Figure 7.

Tumor Adm expression influences sentinel LN lymphangiogenesis. Statistically significant increase in the density of lymphatic (LYVE-1) staining in the LNs was observed when mice harbored an Adm OExp tumor. Data are expressed as means ± se for 4–6 mice/group. Scale bars = 200 μm.

Figure 8.

Overexpression of AM promotes distant metastasis. A) Scans showed and quantitative evaluation confirmed that lung lobes from mice that harbored an Adm OExp (n=6) tumor had more Ki67⁺ foci as compared to lobes from Adm RNAi (n=7) and control mice (n=12). B, C) H&E (B) and Ki67 (C) staining verified OPT scans and showed scattered Ki67 staining in lung sections from mice bearing Adm RNAi or control tumors. By contrast, proliferative (Ki67⁺) foci were detected in lungs harboring an Adm OExp tumor. Arrows indicate individual proliferating cells; arrowhead points to proliferative foci. D) Model illustrates how tumor levels of AM affect local vascular changes, which then may affect distant metastasis. Tumors with low AM levels have fewer lymphatic vessels (green), no blood (red) or lymphatic vessel area enlargement, reduced LN lymphangiogenesis, and fewer Ki67⁺ spots in the lungs as compared to a tumor with high levels of AM. Notably, while expression of tumor AM is necessary for lymphangiogenesis, levels of angiogenesis remain unaffected with changes in AM dosage. Scale bars = 800 μm (A); 20 μm (B, C).