Lymphangiotropism of mammary carcinomas of the cat: role of VEGF-C and CD-44

Introduction. Tumour dissemination through the lymphatics is well known in feline mammary carcinomas in which regional lymph node is the site of early metastases. VEGF-C increases vascular permeability and facilitates transendothelial neoplastic migration. CD-44 promotes cell migration inducing small gaps in the endothelial wall. This study is focused on the expression of VEGF-C and CD-44 in a series of feline mammary samples including normal gland (NMG), benign (BT), and malignant (MT) tumours, and to ascertain any correlation with the quantitative analysis of lymphatics. Materials and Methods. Immunohistochemistry (IHC) was applied to 4μm-thick sections of 48 samples of NMG, BT and MT, using anti-VEGF-C (clone Z-CVC7) and anti-CD44var (v5) (clone VFF-8) antibodies. The IHC expression of both VEGF-C and CD44 was analyzed with a 40x objective in five intratumoral(IT)(intramammary-IM) and 10 extratumoral(ET)(extramammary-EM) fields, in areas with major expression (Hot Spots) at low magnification, and then evaluated in the epithelial adenomatous or carcinomatous component (absent=0; low=10; intermediate=100; high=1000). Results.VEGF-C stained positive in the cytoplasm of endothelial and epithelial (normal, benign or malignant) cells, and CD44 only in epithelial cells. Comparing IT(IM) and ET(EM) VEGF-C expression, there was no difference in NMG, BT and non-infiltating MT, whereas a significant lower expression emerged in invasive MT (P<0.05 Spearman Test). Again, no difference was found in VEGF-C expression in NMG, BT and MT respectively in ET(EM) and IT(IM) fields. Comparing IT(IM) vs ET(EM), CD44 expressed a significant difference (lower values in ET) in BT and invasive MT (P<0.01 Spearman Test), but not in non-invasive MT and NMG. CD44 expression was increased only in the ET(EM) from NMG to BT to MT (P<0.05 Spearman Test).Comparing mean CD44 or VEGF-C expression with lymphatics assessment from previous data, a significant inverse correlation was found between CD44 expression in the epithelial cells and VEGFR-3 expression in lymphatic vessels in BT and invasive MT. Discussion. Intratumoural VEGF-C expression reflected the stronger need for angiogenesis in infiltrating MT than non-infiltrating MT and BT, but showed no correlation with lymphatics assessment. CD-44 expression, instead, was similar to VEGF-C in MT, but inversely and significantly correlated to lymphatics expressing VEGFR-3. Both molecules were not strictly associated to malignancy, but showed major differences in infiltrating MT. CD44, which can interact with vessel endothelium and has also a role in promoting cell-cell adhesion, is inversely associated with VEGFR-3 expressed in lymphatics of in invasive MT. This may indicate that extratumoural fields, characterized by low CD44 expression and high numbers of VEGFR-3 positive lymphatics, are the site of neoplastic-endothelial cells interaction. Conclusion. From the present results, it seems that a chemical CD44-mediated interaction with lymphatic vessels leads invasive malignant neoplastic cells into the lymphatic circulation, even if true lymphangiogenesis was not demonstrated.