Articleshttps://doi.org/10.1038/s41551-020-0576-z

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsiesNobuyuki Tanaka   1,2,12 ✉, Shigeaki Kanatani   1,12, Dagmara Kaczynska1,12, Keishiro Fukumoto   1,2, Lauri Louhivuori   1, Tomohiro Mizutani   3, Oded Kopper3, Pauliina Kronqvist4, Stephanie Robertson   5,6, Claes Lindh5,6, Lorand Kis5,6, Robin Pronk   7, Naoya Niwa   2, Kazuhiro Matsumoto   2, Mototsugu Oya2, Ayako Miyakawa1,8, Anna Falk   7, Johan Hartman   5,6, Cecilia Sahlgren   9,10,11, Hans Clevers   3 and Per Uhlén   1 ✉

1Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 2Department of Urology, Keio University School of Medicine, Tokyo, Japan. 3Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands. 4Department of Pathology, University of Turku, Turku, Finland. 5Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden. 6Department of Clinical Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden. 7Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. 8Department of Molecular Medicine and Surgery, Karolinska University Hospital, Stockholm, Sweden. 9Turku Bioscience Centre, Åbo Akademi University and University of Turku, Turku, Finland. 10Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland. 11Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands. 12These authors contributed equally: Nobuyuki Tanaka, Shigeaki Kanatani, Dagmara Kaczynska. ✉e-mail: urotanaka@keio.jp; per.uhlen@ki.se

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Table of Contents Supplementary Figures ........................................................................................................... 2

Supplementary Fig. 1 ..................................................................................................................................................... 2 Supplementary Fig. 2 ..................................................................................................................................................... 3 Supplementary Fig. 3 ..................................................................................................................................................... 4 Supplementary Fig. 4 ..................................................................................................................................................... 5 Supplementary Fig. 5 ..................................................................................................................................................... 6 Supplementary Fig. 6 ..................................................................................................................................................... 7 Supplementary Fig. 7 ..................................................................................................................................................... 8 Supplementary Fig. 8 ..................................................................................................................................................... 9 Supplementary Fig. 9 ................................................................................................................................................... 10 Supplementary Fig. 10 ................................................................................................................................................. 11

Supplementary Tables........................................................................................................... 12 Supplementary Table 1 ................................................................................................................................................ 12 Supplementary Table 2 ................................................................................................................................................ 12

Supplementary Videos .......................................................................................................... 13 Supplementary Video 1 ............................................................................................................................................... 13 Supplementary Video 2 ............................................................................................................................................... 13 Supplementary Video 3 ............................................................................................................................................... 13 Supplementary Video 4 ............................................................................................................................................... 13 Supplementary Video 5 ............................................................................................................................................... 13 Supplementary Video 6 ............................................................................................................................................... 13

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Supplementary Figures

Supplementary Fig. 1 | RNA Retention in Fixed Tissue Sections. a,b, Confocal microscopy images of 100-μm-thick mouse cortical sections fixed with 4% PFA, KINFix, PAXgene, zinc-based fixative Z7 or ZBF, with EDC ((EDC(+), a) or without EDC (EDC(-), b), stained for Pvalb mRNA (red) and DAPI (blue). c, Pvalb mRNA levels in mouse cortex treated with different fixatives. ++, high level. +, intermediate level. -, low level. NA, not applicable due to tissue disruption. Scale bars, 200 μm (white).

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Supplementary Fig. 2 | RNA Detection in Mouse Tissue Volumes. a, Representative protocol for RNA detection with DIIFCO. b-h, Optical sections at indicated z-depths (left) and volume rendering (right) of 2-mm-thick adult mouse brain sagittal sections that were stained for RNAs Sst (b), Gad1 (c), Meg3 (d), Slc17a7 (e), Th (f), Kit (g), and Gapdh (h). Middle, Allen Brain Atlas images. Image credit: Allen Institute. Insets, magnified views of indicated boxes. i-p, Volume rendering of E11.5 mouse embryos that were whole-mount stained for RNAs: Meg3 (i), Mcm2 (j), Kit (k), Tubb3 (l), and Gapdh (m), and E12.5-15.5 mouse brains that were whole-mount stained for RNAs: Th (n), Slc17a7 (o), and Sst (p). Scale bars and x,y,z-indicators, 1000 μm (white) and 2500 μm (magenta).

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Supplementary Fig. 3 | RNA Detection in Fresh-Frozen Mouse Tissue Volumes. a, Simplified flowchart of detecting RNA in fresh-frozen samples using DIIFCO. b,c, Volume rendering of an immediately fixed (b) or fresh-frozen (c) mouse bladder cancer sample that was whole-mount stained for Mcm2 mRNA. Right and below, magnified optical sections of cancerous and non- cancerous regions at indicated z-depths. Bounding boxes, 3100 x 2200 x 2200 μm (b) and 4300 x 3100 x 2000 μm (c). d, Analysis of the signal-to-background ratio for Mcm2 mRNA in immediately fixed (n = 4) and fresh-frozen (n = 4) mouse bladder cancers. Signal-to-background ratios were calculated as the ratio of the mean intensity of the Mcm2 fluorescence to the rest of the image. Two images per mouse bladder tumour from two independent tumours were randomly selected for each condition. The line within the box is the median, the upper and lower ends of the box are the upper and lower quartiles, and the bars are the minimum and maximum values. n.s., not significant by Student’s t-test. e, Optical sections 500 μm, 685 μm, 1145 μm, and 1045 μm into immediately fixed, fresh frozen for < 1 month, fresh frozen for > 6 months, and control, respectively, mouse placenta samples that were whole-mount stained for Pecam1 mRNA or EGFP (Control). Below, magnified views of indicated boxes. Autofluorescence was used as background (grey). Scale bars, 1000 μm (white) and 50 μm (yellow).

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Supplementary Fig. 4 | Degradation of Nuclear DNA Binding in Intact Tissue Volumes. a, Optical sections from 850 μm, 950 μm, 900 μm, and 900 μm into 2-mm-thick mouse cortical sections treated with dichloromethane for 0 h, 0.5 h, 3.5 h, and >12 h, respectively, stained with YO-PRO-1. b, Analysis of the signal-to-background ratio for YO-PRO-1 in mouse cortex treated with dichloromethane for 0 h (n = 4), 0.5 h (n = 4), 3.5 h (n = 4), and >12 h (n = 4). Signal-to-background ratios were calculated as the ratio of the mean intensity of the YO-PRO-1 fluorescence to the rest of the image. Two images per mouse brain from two independent brains were randomly selected for each condition. The line within the box is the median, the upper and lower ends of the box are the upper and lower quartiles, and the bars are the minimum and maximum values. *P < 0.05, **P < 0.01 by Student’s t-test. c, Light-sheet microscopy images of human ovarian cancer samples treated with dichloromethane for 0.5 h (above) and >12 h (below), stained with YO-PRO-1. d, Light-sheet microscopy images of 2-mm-thick mouse cortical sections treated with dichloromethane for 0.5 h (above) or >12 h (below) that were immunostained for NeuN. Scale bars, 100 μm (white).

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Supplementary Fig. 5 | Protein Detection in Intact Fresh-Frozen Human Tissues with DIIFCO. a, Representative protocol for protein detection using DIIFCO. b, Simplified flowchart of detecting protein in fresh-frozen samples using DIIFCO. c, Optical section from 585 μm into fresh-frozen Cervical cancer sample 1, which was stored at -80°C after pre-treatment with RNAlater, whole-mount stained for E-cadherin protein (pseudo colour) and YO-PRO-1 (grey). Right, magnified view of indicated box (above) and bright-field image of cleared tumour (below). The grid lines are 3 mm apart. d-f, Volume rendering of fresh-frozen Cervical cancer sample 2, which was stored at -80°C after pre-treatment with RNAlater, whole-mount stained for CD34 protein (red) and YO-PRO-1 (blue). Bounding box, 5200 x 4600 x 1600 μm. e, Magnification views of indicated boxes depict hypervascular (left) and hypovascular (right) regions. f, Segmentation of blood vasculature network. The pseudo-colours indicate thin (blue) and thick (red) blood vessels. g, Volume rendering of fresh-frozen Ovarian cancer sample 1, stored at -80°C after pre-treatment with RNAlater, whole-mount stained for N-cadherin protein. The pseudo-colours indicate low (blue) and high (red) protein levels. Bounding box, 3902 x 4471 x 1700 μm. h, Quantitative cell-by-cell analysis of YO-PRO-1 labelled nuclei in Ovarian cancer sample 1, yielded 610,920 individual cells. Scale bars and x,y,z-indicators, 1000 μm (white) and 200 μm (yellow).

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Supplementary Fig. 6 | RNA Detection in FFPE Samples with DIIFCO. a, Optical sections from 700 μm, 1000 μm, and 1300 μm into a 21-months old mouse FFPE bladder cancer sample that was whole-mount stained for Mcm2 mRNA. The pseudo-colours indicate low (blue) and high (red) mRNA levels. b, Magnified views of indicated boxes in a. c, Surface plot of the in situ hybridization chain reaction signal of indicated box in a. The surface plot was generated by MATLAB’s mesh function. d, Optical sections from 600 μm, 900 μm, and 1200 μm into a >9-years old mouse FFPE bladder cancer sample that was whole-mount stained for Mcm2 mRNA. The pseudo-colours indicate low (blue) and high (red) mRNA levels. e, Analysis of the RNA yield following RNA extraction of mouse FFPE bladder cancer samples stored for <1-year (n = 5), 2-years (n = 3), 5-years (n = 3), and 10-years (n = 3). The line within the box is the median, the upper and lower ends of the box are the upper and lower quartiles, and the bars are the minimum and maximum values. *P < 0.05, **P < 0.01 by Student’s t-test. Storage time of FFPE samples are displayed in images. Scale bars, 1000 μm (white) and 100 μm (yellow).

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Supplementary Fig. 7 | RNA and Protein Detection in Intact Human Tissue Volumes. a, Confocal microscopy image of a colon cancer sample that was stained for PROM1 mRNA (green) and DAPI (blue). Insert, magnified view of indicated box. b, Multimodal image of a kidney cancer sample that was stained for NES mRNA (green) and N-cadherin protein (red). Insert, magnified view of indicated box. c, Multimodal images of 14 cancer types (#1 vulva, #2 ovary, #3 stomach, #4 esophagus, #5 skin, #6 testis, #7 thyroid, #8 lung, #9 liver, #10 kidney, #11 skeletal muscle, #12 colon, #13 breast, #14 rectum) that were stained for UCA1 lncRNA (green), E-cadherin protein (red), and DAPI (blue). d, Magnified view of #5 skin melanoma. Right, magnified views of indicated boxes depicting high expression of UCA1 lncRNA (above, green frame) and E-cadherin protein (below, red frame). e-h, Histological assessment of FFPE renal pelvic cancer samples, 8 months old (e,f) and 7 months old (g,h), whole-mount stained for CD274 mRNA (e,g) and serial sections of the same blocks as in e,g stained for PD-L1 protein (f,h). Inserts, magnified views of indicated boxes showing the same regions for CD274 mRNA and PD-L1 protein. i, High-magnification image of a 12 months old FFPE ureteral cancer sample that was whole-mount stained for MALAT1 lncRNA. j, Multimodal image of a 12 months old FFPE renal pelvic cancer sample that was whole-mount stained for CD274 mRNA (red) and KRT5/6 protein (green). Storage time of FFPE samples are displayed in images. Scale bars, 300 μm (white), 300 μm (black), 100 μm (yellow) and 50 μm (magenta).

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Supplementary Fig. 8 | Validation of the RNA and Protein Detection-Specificity of DIIFCO. a, Workflow for the validation of DIIFCO. b,c, Confocal microscopy images of 100-μm-thick mouse brain sections that were stained for tyrosine hydroxylase (Th) mRNA (red), without staining (green), and Histone (blue) (b) and after stripping (red, see methods), immunostained for Th protein (green), and Histone (blue) (c). Magnified view of indicated box. d, Allen Brain Atlas image of P56 mouse brain coronal section in situ hybridized for Th mRNA. Magnified views of indicated boxes. Image credit: Allen Institute. e, Merged image showing Th mRNA (red), Th protein (green) and colocalized signals (yellow). Note, Th mRNA is expressed only in nuclei and Th protein is expressed all over the cell, including neurite extensions. Right, image showing yellow nuclei in cells with colocalized singles. f, Quantification of cells with overlapping Th mRNA and Th protein fluorescence signal in tissue sections (n = 405 cells, N = 3 separate experiments). g, Light-sheet microscopy image of a 2-mm-thick mouse brain volume that was whole-mount stained for Th mRNA (red), Th protein (green), and Histone (blue). Magnified view of indicated box. h, Quantification of cells with overlapping Th mRNA and Th protein fluorescence signal in tissue volumes (n = 1180 cells, N = 4 separate experiments). i, Segmentation of individual cells based on the signal from Th mRNA (multi-colour and red), Th protein (green) and their colocalized signal (yellow). Scale bars and x,y,z-indicators, 100 μm (white), 500 μm (yellow) and 1000 μm (black).

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Supplementary Fig. 9 | Cell-by-Cell RNA and Protein Analysis in Intact Tissue Volumes. a, Volume rendering of human Bladder cancer sample 4 that was whole-mount stained for UCA1 lncRNA (green), E-cadherin protein (red), and YO-PRO-1 (blue). Below, optical sections from 690 μm into Bladder cancer sample 4. Right, pseudo-coloured point cloud images, 5 μm thick, of UCA1 lncRNA and E-cadherin protein. The pseudo-colours indicate low (blue) and high (red) RNA and protein levels. Magnified views of indicated boxes. The dashed line indicates boundaries between tumour cells and stroma. Bounding box, 3700 x 7600 x 2900 μm. b, Representative overview of the workflow for separating individual cells. c, Magnified optical section from 180 μm into Bladder cancer sample 5 that was whole-mount stained for UCA1 lncRNA (green), E-cadherin protein (red), and YO-PRO-1 (blue). Inset, volume rendering of Bladder cancer sample 5. Box indicate magnified region. Bounding box, 3900 x 5400 x 1000 μm. d, Volume rendering of Thyroid cancer sample 1 that was whole-mount stained for MALAT1 lncRNA (green), CD34 protein (red), and YO-PRO-1 (blue). Bounding box, 4200 x 4700 x 2000 μm. Right, optical section from 850 μm into Thyroid cancer sample 1. Below, volume rendering of optical sections 700-950 μm into Thyroid cancer sample 1 showing tumour periphery and tumour stroma. Storage time of FFPE samples are displayed in images. Scale bars and x,y,z-indicators, 1000 μm (white) and 100 μm (yellow).

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Supplementary Fig. 10 | Normal Human Breast Tissue Whole-Mount Stained for PROM1 mRNA. a,b, Volume rendering of a fresh-frozen normal human breast tissue sample that was whole-mount stained for PROM1 mRNA (red) and Histone (green) (a) and solely PROM1 mRNA (red) (b). Bounding box, 3520 x 4490 x 2620 μm. c, Optical section at indicated z-depth of the same normal human breast tissue as in a. Magnified views of indicated boxes show mammary gland, stroma, and fat regions. Scale bars and x,y,z-indicators, 500 μm (white) and 100 μm (yellow).

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Supplementary Tables Supplementary Table 1 | Key resources table (pdf file 66.8 kB) Supplementary Table 2 | List of probe sequences and accession numbers of the targeted RNAs. (pdf file 117.7 kB)

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Supplementary Videos Supplementary Video 1 | Volume Rendering of Mouse Embryo. Volume rendering of mouse embryo at embryonic day 13.5 (E13.5) that was whole-mount stained for Gad1 mRNA. The outer layer of the embryo is generated by volume rendering the auto fluorescence signal. Bounding box, 6210 x 8860 x 4120 μm. (mp4 file 50.2 MB) Supplementary Video 2 | Cell-by-Cell Analysis of Human Brest Cancer. Volume rendering of human breast cancer that was whole-mount stained for PROM1 mRNA and CD34 protein. Cell-by-cell analysis of the shortest distance from each individual PROM1+ cell to the nearest blood vessel (red, short distances; blue, long distances). Bounding box, 3800 x 5800 x 2700 μm. (mp4 file 24.2 MB) Supplementary Video 3 | Parvalbumin RNA and Protein Staining in Mouse Cortex. Volume rendering of a 100-μm-thick adult mouse cortical section that was stained for Pvalb mRNA in red and Parvalbumin protein in green. (mp4 file 1.4 MB) Supplementary Video 4 | Volume Rendering of Human Organoid. Volume rendering of 5 weeks old human forebrain organoid that was whole-mount stained for mRNAs NES in red and DCX in green. Bounding box, 1200 x 1200 x 1600 μm. (mp4 file 16.7 MB) Supplementary Video 5 | Cell-by-Cell Analysis of Human Colon Cancer. Volume rendering of human colon cancer that was whole-mount stained for LGR5 mRNA and CD34 protein. Cell-by-cell analysis of the shortest distance from each individual LGR5+ cell to the nearest blood vessel (red, short distances; blue, long distances). Bounding box, 793 x 984 x 805 μm. (mp4 file 69.9 MB) Supplementary Video 6 | Spatial Niche Analysis of Human Brest Cancer. Volume rendering of human breast cancer that was whole-mount stained for PROM1 mRNA. Distinct spatial niches of PROM1+ cells are highlighted in separate colours. Bounding box, 2860 x 2540 x 1750 μm. (mp4 file 49.0 MB)