Abstract
In vitro three dimensional (3D) cancer models were developed to observe the invasive capacity of melanoma cell spheroids co-cultured with the vascular-formed endothelial cell network. An array-like multicellular pattern of mouse melanoma cell line B16F1 was developed by magnetic cell labeling using a pin-holder device for allocation of magnetic force. When the B16F1 patterned together with a vascular network of human umbilical vein epithelial cells (HUVEC), spreading and progression were observed along the HUVEC network. The B16F1 cells over 80 µm distance from HUVEC remain in a compact spheroid shape, while B16F1 in the proximity of HUVEC aggressively changed their morphology and migrated. The mRNA expression levels of IL-6, MDR-1 and MMP-9 in B16F1 increased along with the distance the HUVEC network, and these expressions were increased by 5, 3 and 2-fold in the B16F1 close to HUVEC (within 80 µm distance) as compared to that far from HUVEC (over 80 µm distance). Our results clearly show that malignancy of tumor cells is enhanced in proximity to vascular endothelial cells and leads to intravasation.
Highlights
Cancer invasion and metastasis are the hallmarks that transform a locally growing tumor into a systematic, metastatic, and lifethreatening disease [1]
human umbilical vein epithelial cells (HUVEC) were utilized as a model for human endothelial cells, and cultured on 10 cm dishes in HuMedia-EB2 (Kurabo, Osaka, Japan) consisting of 2% fetal bovine serum, 10 ng/ml human epidermal growth factor, 1.34 mg/mL hydrocortisone hemisuccinate, 50 mg/mL Gentamicin, 50 ng/mL Amphotericin B, 5 ng/mL hEGF-B, and 10 mg/ mL heparin, all supplied by Kurabo
Effects of extracellular matrix (ECM) mimetic gels on cell morphology To construct the in vitro 3D cell culture array to observe the invasive capacity of B16F1 melanoma cell spheroids co-cultured with the HUVEC network, cell morphological behaviors of HUVEC and B16F1 were investigated
Summary
Cancer invasion and metastasis are the hallmarks that transform a locally growing tumor into a systematic, metastatic, and lifethreatening disease [1]. Since vascular endothelial cells release numerous cytokines, hormones, and growth factors such as TNF-a [4] and VEGF [5], cultured media of vascular endothelial cells including these secretory factors significantly enhanced proliferation, migration, and invasion of hepatocellular carcinoma cells in vitro via activation of PI3K/Akt and ERK1/2 pathways [3] These pathways stimulate the overexpression of invasion/ metastasis associated genes such as MMPs and interleukins (ILs), and these genes promote ECM degradation [6,7], inflammation [8], angiogenesis [9], and proliferation [10]. These interactions of tumor cells with vascular endothelial cells via direct contact and paracrine signaling have been investigated
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