CAM-Delam – an in vivo assay to visualize and quantify the delamination and invasion capacity of human cancer cells

Abstract

Objectives. 1. To develop and optimise CAM-Delam assay. 2. To induce metastatic and nonmetastatic conditions regarding cancer cells used. 3. To induce ectopic delamination using non-metastatic cell lines followed by recovery using broad spectrum MMP inhibitor. Materials and methods. Chicken embryos were incubated ex ovo on day 3 using 3 conditions to access the best survival: internal humidified chamber (HC), non-humidified chamber (N-HC) and Petri dish (PD) were used. Cancer cells were then seeded on embryonic day 10 (E10) on chicken embryo chorioallantoic membrane (CAM). 1×106 PC-3U-GFP and U251-GFP cells were seeded in the silicon ring on the CAM. After 14h, 1.5 days, 2.5 days and 3.5 days were collected together with CAM. Additionally, non-metastatic U251-GFP were pretreated by hypoxia inducible CoCl2 followed by broad spectrum MMP inhibitor GM6001. Then immunohistochemistry staining was performed using an anti-Laminin-111 antibody and stained sections were scored for delamination according to 4 categories: intact, altered, damaged and invasion. Results. Survival of the eggs on day 10 in HC was double compared to N-HC and PD (p < 0.05 and p < 0.01, respectively). On E13, survival in HC compared to N-HC and PD was significantly higher (p < 0.001). PC-3U cells induced minimal alteration of laminin after 14h, followed by the invasion on 3.5 days. U251 cells cultured alone caused minor alterations during the period of 1.5–3.5 days. U251 cells cultured together with CoCl2 induced laminin damage and cell invasion. Pretreatment with GM6001 (1h) followed by CoCl2 exposure (24 h) suppressed the effect of the CoCl2 treatment. Conclusions. The advantage of the CAM-delam assay is obtaining informative results regarding delamination within a few days to estimate the aggressiveness of human cancer cells and the potential risk for metastasis. It can also be used to study the molecular mechanism, as seen in the study with CoCl2 and GM6001, that regulate delamination, invasion and formation of micro-metastases.