Darbepoetin-α Accelerates Neovascularization and Engraftment of Extrahepatic Colorectal Metastases

Abstract

Purpose: Erythropoietin and its analogue darbepoetin (DPO)-α have been shown to improve liver function and regeneration after partial hepatectomy (Phx). However, previous experimental studies have also shown that DPO significantly enhances Phx-induced engraftment of colorectal liver metastases by increasing neovascularization and tumor cell proliferation. Therefore, the present study analyzed whether DPO affects engraftment and neovascularization of extrahepatic colorectal metastases after major hepatectomy. Methods: Green fluorescent protein-transfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Animals received a single dose of DPO (10 µg/kg body weight) at the day of tumor cell implantation (day 0). Phosphate-buffered saline-treated animals served as controls. To study whether the effect of DPO is influenced by Phx, additional animals with and without DPO treatment underwent 70% Phx at day 0. Tumor vascularization and growth as well as tumor cell migration, proliferation and apoptosis were studied repetitively over 14 days using intravital fluorescence microscopy, histology and immunohistochemistry. Results: In nonhepatectomized animals, DPO significantly accelerated tumor cell engraftment and slightly enhanced tumor neovascularization. Tumor cell migration and host tissue infiltration were not affected by DPO. In hepatectomized animals, DPO slightly enhanced tumor growth and significantly accelerated tumor neovascularization, but did not affect tumor cell migration and infiltration. Conclusion: The present study indicates that DPO accelerates extrahepatic engraftment of colorectal cancer cells, most probably by stimulating the process of neovascularization.

Darbepoetin-α Promotes Cell Proliferation in Established Extrahepatic Colorectal Tumors after Major Hepatectomy

“…Accordingly, in intrahepatic metastases, intraportal application of DPO has been shown to significantly stimulate angiogenesis and neovascularization [13,14]. In contrast, at extrahepatic sites, systemic application of DPO did not stimulate angiogenesis during tumor cell engraftment [15]. In line with this, DPO also did not stimulate angiogenesis or neovascularization in the present study.…”

“…Herein, we also observed a significantly increased tumor cell proliferation in the extrahepatic tumors in hepatectomized animals after DPO treatment. This increased tumor cell proliferation is remarkable, because our previous study on extrahepatic tumor cell engraftment showed that DPO does not increase cell proliferation in nonestablished metastases [15]. This difference may be due to the fact that established metastases already have a vascular network, which facilitates drug delivery to the tumor cells.…”

“…Then, either DPO (Aranesp®; Amgen GmbH, Munich, Germany), a hyperglycosylated recombinant human EPO analogue, or PBS solution was administered as a single shot in a dose of 5 µg/kg BW s.c. and 5 µg/kg BW i.v. [15]. The study consisted of two experimental parts: (1) to study DPO effects on established extrahepatic tumors, animals received DPO at d5 after tumor cell implantation (DPO); additional animals received an equal amount of PBS and served as controls (CON); all animals underwent a sham operation, i.e.…”

“…This was documented and scored 0-4, with 0 indicating angiogenesis in none of the ROIs and 4 indicating angiogenesis in all of the ROIs. Neovascularization was determined by measuring the functional vessel density, defined as the length of newly formed red blood cell-perfused microvessels per observation area (cm/cm 2 ) [15,16,18]. Functional vessel density was analyzed in the 4 ROIs within the tumor margin.…”

“…Recently published data from our group indicate that DPO accelerates the extrahepatic engraftment of nonestablished colorectal cancer cells [15]. Because the effect of DPO treatment on established extrahepatic colorectal metastasis is still unknown, we herein studied the combined effects of systemic DPO treatment on established extrahepatic colorectal metastases in nonhepatectomized animals and after major hepatectomy.…”