Investigation of intratumoural and peritumoural lymphatics expressed by podoplanin and LYVE-1 in the hybridoma-induced tumours

Abstract

Tumour-associated lymphatics contribute to a key component of metastatic spread, however, the biological interaction of tumour cells with intratumoural and peritumoural lymphatics (ITLs and PTLs) has remained unclear. To address this important issue, we have focused on the morphological and molecular aspects of newly formed lymphatics (lymphangiogenesis) and pre-existing lymphatics in the intratumoural and peritumoural tissues by using a hybridoma-induced tumour model. In the present study, ITLs with very high vessel density within the tumour mass showed small and flattened contours that varied from non-solid-to-solid tumours, whereas PTLs were relatively disorganized and tortuous, and packed with a cluster of tumour cells at the tumour periphery. Lymphatic endothelial cells (LECs) both in ITLs and PTLs were expressed with LYVE-1 and podoplanin in various tumour tissues, in which initial lymphatics were extremely extended and dilated. The tumour cells were frequently detected adhering to or penetrating lymphatic walls, especially near the open junctions. In the metastatic tissues, lymphangiogenic vasculatures occurred within the tumour matrix, and collecting PTLs represented abnormal twisty valve leaflets. The Western blot and RT-PCR analysis showed local variations of LEC proliferating potentials and lymphatic involvement in metastasis by a distinct profile of the protein and mRNA expression by LYVE-1, podoplanin, Prox-1 and vascular endothelial growth factor-3 (VEGFR-3). These findings indicated that both ITLs and PTLs, including enlarged pre-existing and newly formed lymphatics, may play a crucial role in metastasis with an active tumour cell adhesion, invasion, migration and implantation.

Figure 1

In the hybridoma-induced mouse model, the lymphatic size (μm², a and d), lymphatic vessel density (counts/mm², b and e), and lymphatic area fraction (%, c and f) show significant differences between the intratumoural and peritumoural tissues, and between non-solid and solid tumour tissues.

Figure 2

Photomicrographs of the hybridoma-induced tumour model. In serial cryosections of the stomach, 5′-Nase-positive lymphatics in the mucosa and submucosa show flattened and irregular contour, in comparison with ALPase-positive blood vessels (a). The lymphatics represent weaker CD31 immunoreactivity than the blood vessels (b), and strongly express VEGFR-3 (c) but not VEGF-C (d). L, lymphatic vessel; B, blood vessel. Bars = 50μm.

Figure 3

Photomicrographs of the sections through hybridoma-induced-tumour tissues. (a–c) 5′-Nase-positive intratumoural lymphatics (ITLs) and peritumoural lymphatics (PTLs) are obviously clustered within the tumour mass that show very strong ALPase activity or distributed in the pancreas (Pa)-tumour boundary zone (a). The numerous podoplanin-expressing lymphatics filled with a cluster of tumour cells, extend into the pancreatic parenchyma (b), and PTLs expressed for podoplanin are extremely enlarged due to squeeze of tumour cells (c). (d) Prox-1 is expressed by the enlarged lymphatics, and up-expressed by the blood vessels (arrow) in the tumour–uterus (Ut) boundary zone. (e) Podoplanin is specifically expressed in the lymphatic vessel with a thin endothelial wall and irregular contour, and in some tumour cells adjacent to the hepatic tissues (He). (f) In the hepatic capsule, ITLs expressed for podoplanin show different sizes due to the low density of tumour cells and loose intratumoural matrix, in which some lymphatics are filled with tumour cells. (g and h) In the skin, tumour cells are accumulated in both LYVE-1-expressing peritumoural (g) and intratumoural (h) lymphatics. ITLs show uncollapsed although the tumour tissue appears a high-density mass (h). (i and j) In the intestine (Int)-tumour boundary zone, 5′-Nase-positive ITLs are small and flattened, and PTLs are accumulated in the tumour periphery (i), and the lymphatics with LYVE-1 immunoreactivity become obviously enlarged (j). L, lymphatic vessel. Bars = 100μm.

Figure 4

Expression of VEGF-C (a), Prox-1 (b), vWF (c) and podoplanin (d) in lymphatics and tumour cells. (a) VEGF-C-expressing tumour cells (arrowheads) are located near the intestinal muscular layer. (b) Numerous Prox-1-expressing cells (arrowheads) are clearly defined within the tumour tissues. (c, d) In the metastatic lymph node, a high-endothelial venule (HEV) shows strong vWF immunoreactivity (c), and a podoplanin-expressing cell (arrowhead) is seen within the medullary cords (MC) (d), whereas the medullary sinus (MS) contains a cluster of tumour cells (c, d). Bars = 50μm.

Figure 5

Transmission electron microscopic (TEM) views of 5′-Nase-positive lymphatics. Dense 5′-Nase-cerium precipitates are evenly distributed on the luminal and abluminal surfaces of the endothelial cells, and on the surface of the valves. (a and b) In the pancreas-tumour boundary zone, the lymphatics become collapsed (a, intratumoural) or enlarged (b, peritumoural) due to squeezing or over-filling of crowded tumour cells in the diaphragm. (c and d) The collecting lymphatic vessel shows typical two-flap valves (arrows) in the pancreas (c) and abnormally twisty valves (arrows) in the abdominal skin (d). (e and f) The open junction (asterisk) in the initial lymphatics of intestinal walls is adjacent to tumour cells (e). Within tumour tissues of the skin, the lymphatic-like structure is attached with 5′-Nase-cerium product on its incomplete luminal surface (f). (a–d, f) bars = 5μm; (e) bar = 2μm.

Figure 6

The tumour cells and their metabolic products stick to or penetrate 5′-Nase-positive lymphatic walls, especially near the intercellular junctions in the diaphragm. Panels (b) and (g) are further magnification of the area indicated by the asterisks of (a) and (f) respectively. Dense 5′-Nase-cerium precipitates extend into the typical interdigitating (b, arrows) and overlapping (d, arrowhead) junctions. L, lymphatic vessel. a, e, f, bars = 5μm; c, g, bars = 1μm; b,d, bars = 0.5μm.

Figure 7

Immunocytochemistry on the lymphatic endothelial cells (LECs) (a–d) and tumour cell (e). (a) Prox-1-expressing lymphatic vessel in the intestine shows a nuclear deposition of the product. (b)–(d) LECs in the diaphragm display immunoreactivities for podoplanin (b), JC815 (c) and VEGFR-3 (d), which are mainly detected as a granular deposition on cell surfaces (b, c) and valve tips (d). (e) The podoplanin-positive cell is distinctly deposited with reaction product in the organelles in intestine-tumour boundary tissues. Bars = 0.2μm.

Figure 8

Western blot (a, b) and RT-PCR (c) analysis in hybridoma-induced tumour tissues. (a) and (b) The immunoblotting sheet transferred from the SDS-PAGE gels is specifically stained for VEGFR-3, whose positive bands on the nitrocellulose strip are noted at approximately 70 and 130 kDa. Note the non-metastatic lymph node (a) shows extremely lower VEGFR-3 signals than the metastatic one (b). (c) RT-PCR expression patterns of mRNAs for LYVE-1, podoplanin, Prox-1, VEGFR-3 and vWF are shown in each panel separately with different molecular sizes of the amplified fragments. GAPDH is used as an internal control.