Figure 2. CD31 expression in ASM angiosarcoma cells separates vasoformative ‘organotypic’ CD31^high cells from the more aggressive CD31^low cells with spindle cell morphology.

(A) Schematic summary of the experimental protocol used to isolate CD31^high and CD31^low fractions from the ASM cell line. Unsorted ASM_wt (wild-type) cells were stably transduced with the empty pLK0.1 vector under puromycin selection. Subsequently, surviving single clones with picked and immunoblotted (IB validation) for CD31 and classified as CD31^high or low CD31^low cells. Repetitive CD31-FACS analysis from CD31^high (clone #3) and CD31^low (clone #6) and Western blot demonstrated stable levels of CD31 up to 44 passages (data not shown). (B) Western blot analysis revealed only differential expression of CD31, but not of von-Willebrand factor (vWF), suggesting that both populations were endothelial cells. Longer exposure was needed to detect the presence of CD31 in CD31^low cells. (C) Microscopic images of CD31^high and CD31^low cells showing a polygonal cell shape in non-confluent CD31^high cells and cobblestone pattern upon confluence. In contrast, CD31^low cells had a more fibroblastic, spindle cell morphology. (D) HUVEC formed organized networks of tubular endothelial structures which matured after 6 hours and even presented signs of degradation after 18 hours. In CD31^high cells, pre-tubular structures could be detected after 6 hours and mature tubes after 18 hours. In strong contrast, CD31^low cells did not show tubular structures at any time point, even after extended observation for up to 24 hours (data not shown). (E) In a screening of 55 angiogenesis-associated proteins, 18 factors with both pro- and antiogenic properties were differentially expressed in CD31^low compared to CD31^high cells and HUVEC (n=2).