The sialomucin CD34 is a marker of lymphatic endothelial cells in human tumors

Abstract

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.

Figure 1

CD31 and CD34 expression in tumor LECs. Tissue sections from human colon tumors were stained with antibodies against LYVE-1 (red; A–I), CD31 (green; A–C), and CD34 (green; D–I). A low-magnification overview of CD34-positive lymphatic vessels is shown in G–I. Intratumoral tumor lymphatics are marked by an arrow, peritumoral lymphatics are marked by an arrowhead. Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar: 50 μm (A–F); 200 μm (G–I).

Figure 2

Podoplanin, LYVE-1, and Prox1 are reliable markers for the same CD31⁺/CD34⁺ tumor lymphatic vessels. A–F: Serial sections from human colon tumors were stained with antibodies against CD34 (green; A and D) and Prox1 (red; B) or LYVE-1 (red; E). Merged images are shown in C and F. G–I: Serial sections from human colon tumors were stained with antibodies against CD31 (green; G and J) and LYVE-1 (red; H) or podoplanin (K). Merged images are shown in I and L. Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar: 20 μm (A–F); 50 μm (G–L).

Figure 3

Expression of CD34 by LECs in human colon tumors, but not by resting organ LECs. Matched pairs of human colon tumors (A–D) and adjacent normal tissue (E–H) from four different patients were stained with antibodies against LYVE-1 (red) and CD34 (green). Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar, 20 μm.

Figure 4

Expression of CD34 by LECs in different human tumors. Tissue sections of human lung (A–C), colon (D–F), and breast tumors (G–I) were double-stained with antibodies against CD34 (green; A, D, G) and LYVE-1 (red; B, E, H). Merged images are shown in C, F, and I. Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar, 50 μm.

Figure 5

Lack of CD34 expression in normal resting organ LECs. Tissue sections of normal human breast (A–C), skin (D–F), and small intestine (G–I) were double-stained with antibodies against CD34 (A, D, G) and LYVE-1 (B, E, H). Merged images are shown in C, F, and I. Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar, 50 μm.

Figure 6

Expression of CD34 intratumoral and peritumoral LECs of human melanomas. Serial sections of human melanoma were stained with antibodies against CD34 (A) and podoplanin (D). Staining was visualized by light microscopy of diaminobenzidine staining. The arrowhead marks a podoplanin-positive peritumoral lymphatic that co-expresses CD34. The arrows mark CD34-positive blood vessels that are podoplanin-negative. Immunofluorescent staining of human melanoma (B) and adjacent normal tissue (E) with podoplanin (red) and CD34 (green). One lymphatic vessel each (insets) is shown in a higher magnification in D and F. Nuclei were counterstained with DAPI. Scale bar: 200 μm (A, B, D, E); 50 μm (C, F).

Figure 7

Lack of CD34 expression in mouse tumor LECs. Tissue sections from the pancreas of Rip1VEGF-C mice (A, D), Rip1Tag2 mice (B, E), and Rip1VEGF-C × Rip1Tag2 double-transgenic mice (C, F) were stained with antibodies against LYVE-1 (red, A–F), CD31 (green, A–C), and CD34 (green, D–F). Antibodies were detected with fluorescent-labeled secondary antibodies. Nuclei were counterstained with DAPI. Scale bar, 200 μm.