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. 2017 Oct 16;8:947. doi: 10.1038/s41467-017-00983-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — TRPA1 expression level is significantly reduced upon LUAD brain metastasis. a IHC and H&E staining of lung cancer brain metastases. Upper panel: Normal brain tissue (green area), tumor tissue (black arrows), necrosis (blue arrows), and haemorrhage (red arrows). Middle panel: Higher magnification of normal brain (scale bar: 100 μm). Bottom panel: Higher magnification of metastatic lung cancer (scale bar: 200 μm) shows FGFR2-positive tumor cells with areas of negatively-stained fibrovascular connective tissue (black arrows). b Left panel: Images of IF staining at 20× magnification of mouse brain slices 5 days following injection with GFP-HCC-515. CD-34 (Alexa 555) was used as a blood vessel marker. Scale bars: 20 μm. Right panel: Percentage of cancer cells located inside vs. outside blood vessels 3 days (D3) and 5 days (D5) following injection. Error bars indicate s.d. *P ≤ 0.05. c Left panel: Two representative images from D5 (one with an additional zoom-in area). GFAP-positive reactive astrocytes (Alexa 647; pseudo colour: magenta) are shown in the vicinity of extravasating cancer cells (green) as indicated by the white arrows (arrow 1: cancer cell; arrow 2: blood vessel; and arrow 3: astrocyte). Scale bars: 20 μm. Right panel: Bar graph representation of the number of activated astrocytes present in the vicinity of cancer cells 3 days (D3) and 5 days (D5) following injection. Cancer cell-associated astrocytes are quantified within a distance of 150-μm from cancer cells. Error bars indicate s.d. *P ≤ 0.05. d Left panel: TRPA1 expression (Alexa 647; pseudo colour: cyan) in cancer cells 3 days (D3) and 5 days (D5) following injection. Scale bars: 20 μm. Right panel: Extravasated cancer cells were counted in 20 randomly chosen fields per brain followed by calculation of TRPA1-positive cells. Error bars indicate s.d. *P ≤ 0.05