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. 2018 Jul 11;37:146. doi: 10.1186/s13046-018-0796-1

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 1 — E-cadherin membrane expression contributes to EOC MCA formation. a Real-time RT-PCR showing the levels of E-cadherin transcript in freshly isolated matched solid peritoneal tumor masses (st) and ascites-derived MCAs (Asc) from eight HG-SOC patients. Results are presented as relative expression normalized to GAPDH mRNA levels. b Western blotting on total cell lysates from two pairs of MCAs (Asc) and solid tumors (st) of PDXs obtained from two different HG-SOC patients as reported in the Methods. β-actin was used as control of gel loading. c Representative images of MCAs from two HG-SOC patients. Left panel: staining with anti-E-cadherin and β-catenin Abs on MCAs from the same HG-SOC patients. Higher magnification images, corresponding to the highlighted white box are reported. Right panel: phase contrast microscopy performed on MCAs from two (#15 and #16) HG-SOC patients. d Upper panel: phase contrast and IF with anti-E-cadherin Ab (green) of 3D of SKOV3, OVCAR5, and OAW42 cells. Lower panel: IF with anti-E-cadherin Ab performed on confluent SKOV3, OVCAR5, and OAW42 cells (2D). e OAW42 cells transiently transfected with a control (CO) siRNA or with two E-cadherin siRNAs, separately (E-cadh-1, E-cadh-2) or pooled (E-cadh-1/2), and then grown as MCAs for 9 days. Upper panel: cell viability assay performed on silenced OAW42 MCAs. Lower panel: western blotting for evaluation of E-cadherin levels in OAW42 MCAs after 9 days of culture. A representative experiment is shown. Immunoblottings were performed with Abs against the proteins reported on the left. β-actin was used as a control for gel loading. f Left upper panel: representative phase contrast images of OAW42 MCAs obtained as above; bar, 100 μm. Left lower panel: phase contrast images of OAW42 MCAs obtained as above, dissociated from Algimatrix, and stain with LIVE/DEAD™ Viability/Cytotoxicity Kit. Merge of phase contrast, green (live cells) and red (dead cells) fluorescent images are shown. Single images are shown in Additional file 2: Figure S2a. Bar, 50 μm. Right panel: number of OAW42 MCAs (named spheres) obtained as above. Asterisks indicate statistically significant values by Student’s t test (p < 0.01)