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. 2017 Jun 26;36(43):5958–5968. doi: 10.1038/onc.2017.206

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Copyright © 2017 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Assessment of canonical Wnt activity and Ror2 expression uncovers the complex coexistence of Wnt pathways in TP53-null mouse models and human breast cancer. (a) The percentage of canonical Wnt-active cells in basal-like TP53-null tumors relative to luminal and claudin-low, based on fluorescence-activated cell sorting (FACS) analysis of tumors harboring a lentiviral Wnt reporter with a 7xTCF responsive promoter upstream of eGFP. FACS percentages for basal-like tumors: T1 (31.0±6.1%, n=3 tumors), T2 (50.9±2.5%, n=3 tumors) and 2225L (24.6±9.8%, n=3 tumors). (b) Western blot for Ror2 in a panel of TP53-null tumors representing luminal, basal-like and claudin-low subtypes. (c) Immunohistochemistry for Ror2 within T1, T2 and 2225L basal-like TP53-null models depicting a spectrum of Ror2 positivity within each basal-like model. Scale 50 μm. (d) Co-immunofluorescence for Ror2 (green) and 7xTCF-mCherry (red), demarcating active canonical Wnt activity, across three TP53-null basal-like models. Scale 50 μm. (e–g) Quantitation of immunofluorescence staining of mCherry positive (7xTCF responsive) Wnt populations within Ror2-negative and Ror2-positive populations within basal-like tumors (e) T1 (43.7±4.4% in Ror2-neg vs 7.3±2.1% in Ror2-pos tumor cells, n=6 tumors), (f) T2 (53.5±14.14% in Ror2-neg vs 10.2±1.7% in Ror2-pos tumor cells, n=6 tumors) and (g) 2225L (24±8.2% in Ror2-neg vs 2.2±1.3% in Ror2-pos tumor cells). (h) Scatter plot of individual human breast tumors within the TCGA database illustrating an inverse correlation between Ror2 expression and an active canonical Wnt signature (Spearman rank correlation, n=1095 cases, correlation coefficient: −0.26, one-sided P<1 × 10⁻¹⁷). (i) Scatter plot of human breast tumors of the basal-like subtype within TCGA demonstrating an inverse correlation between Ror2 expression and an active canonical Wnt signature (Spearman rank correlation, 122 cases, correlation coefficient: −0.32, one-sided P<0.0005).