Induction of potassium channel regulator KCNE4 in a submandibular lymph node metastasis model

Figures

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  Figure 1. Selection of SLNs before melanoma metastasis for the analysis of early metastasis. (A) Schedule of sample preparation for microarray analysis. (B) Macroscopic images of the tongue and SLNs on day 3 after transplantation. Ctrl is a mouse injected with PBS and #1–8 are mice injected with B16–F10 into the tongue. LNs in which melanoma had metastasized turned black, as indicated by the arrowheads. (C) LNs #1, 3, and 8 with no metastasis and #4 with metastasis in macroscopic images were stained with Giemsa. Melanin pigment was detected as shown by the arrowhead in #4. Bars: 100 µm and 50 µm. (D) Mlana expression was examined by qRT-PCR in the right-hand LNs of mice #1–8 transplanted with B16–F10. Relative expression levels were adjusted for Actb expression. One-way ANOVA, ***p < 0.001 versus ctrl.

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  Figure 2. Genes with altered expression in SLNs during early metastasis. (A) Annotation analysis of genes whose expression had changed (B16–F10 vs control, fold change of < − 2 and > 2, respectively; p < 0.05). (B) Volcano plot of genes with altered expression. (C) Verification of Kcne4, Slc7a11, Fscn1, and Gadd45b expression by qRT-PCR. The expression levels of target genes in SLNs of control mice and SLNs of mice implanted with B16–F10 (right and left) were analyzed. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. ns; not significant. (D) LECs were co-cultured with B16–F10 in transwell plates for 24 h, and the expression levels of Kcne4, Slc7a11, Fscn1 and Gadd45b were analyzed by qRT-PCR. ***p < 0.001. ns; not significant.

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  Figure 3. KCNE4 is upregulated by melanoma transplantation and is expressed on podoplanin-positive cells in SLNs. Expression of KCNE4 in SLN of mice transplanted with B16–F10 was examined by immunohistochemistry. Double staining was performed with anti-KCNE4 antibody and anti-CD45 antibody (A), anti-podoplanin antibody (B), and anti-PNAd antibody (C). Lymph nodes from mice injected with PBS were used as controls. Bars: 200 µm and 50 µm. (D) The percentages of KCNE4-positive areas in SLNs of ctrl or B16–F10 transplanted mice were measured by ImageJ software and divided by the area of the SLNs. Student’s t test, **p < 0.01.

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  Figure 4. Expression of KCNQ1 and KCNE4 in SLNs. (A) Expression of Kcnq family members and Kcna1 and Kcna3 in primary cultured LECs were examined by qRT-PCR. The expression of KCNQ1 in SLNs was analyzed by immunostaining. Double staining of KCNQ1 and CD45 (B), Podoplanin (C), and KCNE4 (D) was performed. Bars: 200 µm and 50 µm (B,D), 200 µm and 10 µm (C).

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  Figure 5. Effects of KCNE4 on the expression of chemokines and adhesion factors in primary cultured LECs. (A) Kcne4 was suppressed by siRNA, and expression levels of Ccl17, Ccl19, Fn1, Mmp2, Mmp3, and Mmp14 were analyzed by qRT-PCR. (B) LECs were transfected with KCNE4 expression plasmid, and expression levels of Ccl17, Ccl19, Fn1, Mmp2, Mmp3, and Mmp14 were analyzed by qRT-PCR. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. ns; not significant.

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  Figure 6. Expression of KCNE4 in human melanoma lymph node metastasis. (A) HE staining of human LNs with melanoma metastasis. (B) Human LNs were examined by immunostaining using anti-KCNE4 and anti-podoplanin antibodies. ctrl C3_03; normal LN. meta B9_02, A4_01, C6_01 and B3_01: metastatic malignant melanoma from the neck, groin, and neck and groin, respectively. Nuclei were stained with DAPI. Bars: 200 µm and 50 µm.