Radiogenic lymphangiogenesis in the skin

Abstract

The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin(+) lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 microm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68(+) and vascular endothelial growth factor-C(+) cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis.

Figure 1

Microphotographs of the upper dermal plexus of irradiated and naive control skin. a–f: H&E staining. a: Four weeks after radiation, sparse perivascular mononuclear infiltrate (arrow) and shrunken sebaceous glands (asterisk) were evident. b and c: Details of a (compare with asterisk or arrow). d: Greater enlargement shows microvessel degeneration (arrows). e: Twelve months after radiation, lymphatic spaces (arrow, inset), and disfigured microvessels were visible. f: Similar microvessel changes and, in this case, sweat duct changes (inset) could be found 23 months after radiotherapy. g–k: Podoplanin immunostaining (hematoxylin counterstained). g: In periaxillar normal control skin, small and large lymphatic vessels (arrows) can be found regularly in horizontal orientation. h: Four weeks after radiation, lymphatic vessels (asterisk) showed vertical enlargement of the lumina, but the number of small lymphatics (arrow) was comparable with the controls. i: One year after radiotherapy, the number of small horizontally oriented lymphatics (>10 μm diameter, arrows) was increased dramatically. j and k: Higher enlargement of podoplanin staining 11 months (j) or 17 months (k) after radiation: Only clearly identifiable podoplanin⁺ longitudinal structures were evaluated as lymphatics (arrows; E, epidermis). l–n: CD68 immunostaining (red). l: Low density of CD68⁺ cells (macrophages, arrows) in the normal control. m and n: High densities of CD68⁺ cells were characteristic 4 weeks after radiation. o and p: VEGF-C immunostaining (brown). Normal control (o) and irradiated skin (4 weeks after radiation) (p) showed significant difference in the densities of VEGF-C⁺ cells (arrows). Scale bars: 100 μm (a, e–i, l, and m); 50 μm (b–d, j, n–p, and insets in e and f); 25 μm (k).

Figure 2

Ratios of “treated/control” densities of different microvessel calibers during different time stages of the postradiation period. a: Lymphatics with four groups of microvessel diameter and three time groups after radiotherapy, respectively. b: Analogous figure of blood microvessel densities with three groups of microvessel diameter ranges. Intertime group significance analysis by U-test. Boxes include all values from 25th to 75th percentile; –, median; 𝕋⊥, largest to smallest observed values that are not outliers; ○, outlier more than 1.5 box lengths from the 25 or 75 percentile; and asterisk, outlier value >2 × SD from mean.

Figure 3

Densities of CD68⁺ cells (a), VEGF-C⁺ (b), or VEGF-D⁺ (c) cells. Dependence on the time after radiation and comparison with the data of unirradiated control skin. Wilcoxon test, comparison of controls with irradiated samples; U-test, between irradiated samples. Boxes include all values from 25th to 75th percentile; –, median; 𝕋⊥, largest to smallest observed values that are not outliers; ○, outlier more than 1.5 box lengths from the 25 or 75 percentile.