Alterations of circulating bone marrow-derived VEGFR-2+ progenitor cells in isolated limb perfusion with or without rhTNF-α

Abstract

Background: Circulating endothelial progenitor cells (cEPCs) as recruited to the angiogenic vascular system of malignant tumors have been proposed as a biomarker in malignancies. The effect of antitumor chemotherapy on cEPCs is not fully understood. We examined the level of cEPCs, vascular endothelial growth factor (VEGF), and angiopoietin-2 in the blood of sarcoma and melanoma patients before and after isolated limb perfusion (ILP) with or without recombinant human tumor necrosis factor-α (rhTNF-α).

Methods: Twenty-two patients, 11 each with soft tissue sarcoma or recurrent melanoma of the limb, were recruited. ILP was performed with rhTNF-α/melphalan (TNF) or melphalan only (no TNF). Fifteen healthy volunteers served as control subjects. Blood was sampled before and up to 6 weeks after ILP. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and annexin V-negative cells were characterized as cEPCs by triple staining for CD133(+), CD34, and VEGFR-2(+).

Results: Before treatment, cEPC numbers were significantly increased in sarcoma (0.179 ± 0.190 %) and melanoma patients (0.110 ± 0.073 %) versus healthy controls (0.025 ± 0.018 %; P < 0.01), but did not differ significantly between sarcoma and melanoma patients. cEPC decreased significantly after ILP in patients with no TNF compared to pretreatment values (P < 0.05) and were significantly lower at 4 h, 48 h, and 1 week compared to ILP with TNF (P < 0.05). Values 6 weeks after ILP were significantly lower than before ILP in both investigated groups (P < 0.01).

Conclusions: ILP with TNF results in activation of bone marrow-derived EPCs compared to ILP without TNF. Alteration of cEPCs and angiopoietin-2 by rhTNF-α might account for the cytotoxicity and hemorrhagic effects on tumor vessels during limb perfusion procedures.

Fig. 1

CD133- and CD34-positive cells in PBMC in healthy controls compared to sarcoma and melanoma patients before treatment. CD133- and CD34-positive cells in PBMC are significantly increased in sarcoma and in-transit metastasized melanoma patients compared to healthy controls (*P < 0.01 vs. healthy controls). Data are displayed as mean ± SD; P < 0.05 was considered to be statistically significant

Fig. 2

cEPC before and after ILP with or without rhTNF-α. cEPC did not differ significantly before ILP (basal) between the investigated groups. The amount of cEPC was significantly higher 4 and 48 h after ILP with rhTNF-α (TNF) compared to melphalan and cisplatin (no TNF) (*P < 0.05). Compared to basal values, cEPC were significantly lower in no TNF starting at 2 h after ILP and 1 and 6 weeks in TNF after ILP (#P < 0.05). Data are displayed as mean ± SEM; P < 0.05 was considered to be statistically significant

Fig. 3

VEGF in patient serum before and after ILP with TNF or without TNF (no TNF). VEGF in serum did not show significant differences between the investigated groups before and after ILP. In rhTNF-α (TNF)-treated patients, VEGF serum levels decreased significantly at 2 h after ILP compared to pretreatment values (basal; #P = 0.036). One week after ILP, a significant increase was observed compared to basal values in the TNF group (*P = 0.011). Data are displayed as mean ± SEM; P < 0.05 was considered to be statistically significant

Fig. 4

Ang-2 serum levels in patients before and after ILP with TNF or without TNF (no TNF). No significant differences in Ang-2 levels were observed before treatment within the tumor types melanoma and sarcoma and the treatment groups of ILP with rhTNF-α (TNF) compared to cisplatin and melphalan alone (no TNF). After ILP, Ang-2 differed significantly at all points of measurement between the two treatment groups, TNF and no TNF (#P < 0.021). A significant increase compared to basal values was found 24 h, 48 h, and 1 week after ILP in the TNF group (*P < 0.001). In no TNF patients were Ang-2 levels significantly higher 24 h after ILP compared to basal values (×P < 0.05). Data are displayed as mean ± SEM; P < 0.05 was considered to be statistically significant