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. 2021 Mar 5;4:294. doi: 10.1038/s42003-021-01786-y

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© The Author(s) 2021

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. 1 — a Scheme of whole-organ profiling of the tumour microenvironment using tissue clearing, image acquisition, and automated image analysis. LSFM light-sheet fluorescence microscope. b Protocol of one-day whole-organ clearing (top). Bright-field images of organs (lung, brain, heart, liver, kidney, pancreas, and spleen) after fixation (RI = 1.33) and clearing (RI = 1.52). Fixed organs were stocked in PBS buffer after PFA fixation. c Visualization of the tumour microenvironment in the experimental lung metastasis model. A549-mCherry cells were intravenously injected in mice (day 0). Then, the lung was subjected to whole-organ clearing protocol and immunostained with FITC-conjugated anti-α-SMA antibody (day 7), anti-VEGFR3 antibody, Alexa 546-conjugated anti-goat IgG antibody (day 1), Red Fluorochrome (635)-conjugated anti-Iba1 antibody (day 14), or FITC-conjugated anti-Ki67 antibody (day 14). d Visualization of the platelets in the experimental lung metastasis model. A549-mCherry cells were intravenously injected in mice (hour 0). Mice were administered with DyLight 649-conjugated anti-CD42c antibody immediately. Then, the lung was subjected to whole-organ clearing protocol, followed by 3D imaging (hour 1). e Visualization of TGF-β in the experimental lung metastasis model. A549-mCherry cells were intravenously injected in mice (day 0). Mice were administered with Alexa 647-conjugated anti-TGF-β antibody (day 6). Then, the lung was subjected to whole-organ clearing protocol, followed by 3D imaging (day 7). Representative images are shown. 3D image (whole), scale bar = 2000 μm. 3D image (enlarged), scale bar = 200 μm. 2D image, scale bar = 200 μm. Figure schematic created with biorender.com.