Figure 1. E4-ECs promote BCCs self-renewal and survival in a contact-dependent manner.

A) Schematic representation of the co-culture system developed for assessing tumor cell proliferation in direct contact with GFP⁺E4-ECs. BCCs and GFP⁺E4-ECs were co-cultivated at 1∶5 ratio without serum and cytokine supplementation and proliferation and survival of BCCs was evaluated 2, 4, and 7 days post co-culture by counting the GFP^-BCCs using a fluorescent microscope. B) Phase contrast and fluorescent microscopy images showing higher BCC proliferation (dark gray cells) in co-culture with GFP⁺E4-ECs as compared with BCCs grown without ECs. C) Quantitative analysis of breast cancer cells MDA-231 and MCF-7 proliferation cultured with or without E4-ECs (***p<0.001, mean ± SEM). D) Schematic representation of a transwell system used for co-culturing BCCs and E4-EC without any direct contact. BCCs and E4-ECs were separately grown as monolayers in multi-well culture plates and transwell inserts respectively. Then, inserts were positioned in the multi-well plates and both cell types were continued to grow in a serum- and cytokine-free medium (conditioned medium) and the proliferation of BBCs was evaluated at 2, 4, and 7day intervals by manual counting. E) Phase contrast and fluorescent microscopy images demonstrating BCCs grown under starvation either alone or in E4-EC conditioned media (CM). F) Quantitative analysis of proliferation of breast cancer cells MDA-231 and MCF-7 with or without direct physical contact with E4-ECs demonstrating the importance of contact (***p<0.001, mean ± SEM).