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. 2017 Feb;214(2):339–358. doi: 10.1084/jem.20160596

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© 2017 Mescher et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 1. — Epidermal Par3 deletion results in elevated melanoma multiplicity and lung metastasis. (A) Epidermis of DMBA/TPA-treated control (ctrl) and Par3^eKO mice, showing MC hyperplasia and melanoma in DMBA/TPA-treated Par3^eKO mice. H&E staining of back skin cross sections is shown. Bars: (top) 10 µm; (bottom left) 50 µm; (bottom right)100 µm. Data are representative of one skin tumor experiment with control (n = 25) and Par3^eKO (n = 27) mice. (B) Skin cross sections of mTmG dual-fluorescence reporter mice with and without K14Cre allele and simultaneous immunostaining for the MC marker TRP2. Note that K14Cre-mediated recombination as evident by switch from membranous red (mTomato) to green (EGFP) fluorescence does not occur in MCs. Bar, 25 µm. Data are represented from two independent experiments. n = 3 mice per group. (C) HGF;Cdk4^R24C mice: an autochthonous mouse melanoma model for spontaneous melanoma. (Top) Single topical DMBA treatment (or single UV exposure) serves to accelerate and synchronize melanoma formation. (Bottom) Representative photographs of control and Par3^eKO mice crossed to the HGF;Cdk4^R24C melanoma model (Ctrl;HGF⁺;Cdk4^R24C/R24C and Par3^eKO;HGF⁺;Cdk4^R24C/R24C) 16 wk after DMBA. (D) Tumor multiplicity in the melanoma model shown in C. Pooled data are depicted as mean ± SEM, with in total n (Ctrl;HGF⁺;Cdk4^R24C/R24C) = 18 mice, n (Par3^eKO; HGF⁺;Cdk4^R24C/R24C) = 22 mice, n (control) = 5 mice, and n (Par3^eKO) = 11 mice at the start of the experiment. P-values weeks 8–15 (left to right): *, P = 0.01242; **, P = 0.00931; **, P = 0.00125; **, P = 0.00456; *, P = 0.03295; NS, 0.07363; **, P = 0.00142; *, P = 0.01482; Ctrl;HGF⁺;Cdk4^R24C/R24C vs. Par3^eKO;HGF⁺;Cdk4^R24C/R24C; multiple Student’s t tests. (E) Par3 and GAPDH immunoblots of protein extracts isolated from primary melanoma that developed in Ctrl;HGF⁺;Cdk4^R24C/R24C and Par3^eKO;HGF⁺;Cdk4^R24C/R24C mice, demonstrating robust Par3 expression in melanoma that is unaffected by K14Cre-mediated recombination. Data are representative of three independent experiments. n = 3 biological replicates per group. (F) H&E staining of primary melanoma in control and Par3^eKO melanoma mice. TRP2 immunohistochemistry of primary melanoma and macroscopically visible vascularization close to melanoma formed in these mice are shown. Bars: (left) 1 mm; (middle) 60 µm. Data are representative of at least three independent experiments (TRP-2 immunohistochemistry) or analyses of in total n (Ctrl;HGF⁺;Cdk4^R24C/R24C) = 18 mice and n (Par3^eKO; HGF⁺;Cdk4^R24C/R24C) = 22 mice (H&E and tumor vascularization). (G) Lung metastasis in Ctrl;HGF⁺;Cdk4^R24C/R24C and Par3^eKO;HGF⁺;Cdk4^R24C/R24C melanoma mice. Arrows indicate macroscopically visible metastases. (H) Quantification of macrometastases per lung in Ctrl;HGF⁺;Cdk4^R24C/R24C (n = 12 mice) and Par3^eKO;HGF⁺;Cdk4^R24C/R24C (n = 7 mice) during melanomagenesis <15 wk after DMBA. The mean includes all mice analyzed irrespective of metastasis status. Incidence of lung metastasis: Ctrl;HGF⁺;Cdk4^R24C/R24C, 16.7%; Par3^eKO;HGF⁺;Cdk4^R24C/R24C, 57.1%. Pooled data are depicted as mean ± SEM. *, P = 0.0436; Mann Whitney U test. (I) H&E (left) and TRP2 (right) immunostaining of melanoma metastases in lung. Bars: (H&E)100 µm; (immunofluorescence) 20 µm. Data are representative of two independent experiments. n = 3 biological replicates.