Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.
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We are grateful to L. Dow (Cornell University) for sharing CRC organoids, X. Lu (University of Oxford) for sharing murine intestines and O. Ornatsky (Fluidigm) for providing monoisotopic cisplatin (195Pt and 196Pt). We thank the UCL CI Flow-Core for MC support and L. McInnes for UMAP advice. Graphical organoid renderings were designed by J. Claus (Phospho Biomedical Animation). This work was supported by Cancer Research UK (grant no. C60693/A23783, C.J.T.), UCLH Biomedical Research Centre (grant no. BRC422, C.J.T.), The Royal Society (grant no. RSG\R1\180234, C.J.T.) and Rosetrees Trust (grant no. A1989, C.J.T.).
M.N. has pending intellectual property on the use of tellurium reagents for mass cytometry applications which has been licensed to Fluidigm Corporation.
Peer review information Nina Vogt and Nicole Rusk were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Rare-Earth Metal-Conjugated Antibodies for Organoid Cell-Type Identification.
a) Summary of reagents used in organoid directed differentiation. b) Fuzzy logic diagram of organoid directed differentiation. c) Confocal immunofluorescence (IF) of small intestinal organoids stained with rare-earth metal-conjugated mass cytometry (MC) antibodies showing individual cell-type markers (red), F-Actin (white), and DAPI (blue) following directed differentiation, scale bars = 50 μm. Images are representative of three independent experiments. d) UMAP (Uniform Manifold Approximation and Projection) distributions of ~20,000 single organoid cells analyzed by MC following directed differentiation. e) Force-directed Scaffold maps of directedly differentiated small intestinal organoids. Cell-type landmark nodes are identified from untreated small intestinal organoids. f) Earth Mover’s Distance (EMD) quantification of cell-type identification markers from MC analysis of Lgr5-EGFP-ires-CreERT2 small intestinal crypt-cells. Source data
a) Strategy for organoid cell-type identification. b) Strategy for organoid cell-state classification. c) EMD quantification of cell-type identification markers across all small intestinal organoid cell-types from Figs. 1 and 2. d) Cell-state quantification of all small intestinal organoid cell-types from Figs. 1 and 2. Source data
Supplementary Figure 3 Cell-Type and Cell-State Specific Signaling Networks in Intestinal Organoids.
Cell-type and cell-state specific signaling networks of 27 PTMs from ~1 million single organoid cells analyzed by MC, with nodes colored by PTM-EMD scores quantifying PTM intensity (relative to all organoid cells), and edges colored by DREMI scores quantifying PTM-PTM connectivity. Source data
a) De-barcoded cell counts from small intestinal organoids barcoded using the Maxpar Cell-IDTM Barcoding Kit either after removal from Matrigel (ex situ) or while still in Matrigel (in situ) (n = 2 independent samples). b) Mean dual counts of 110Pd from Matrigel incubated in PBS, Maxpar Cell-IDTM barcode #20 (106Pd, 108Pd, and 110Pd) resuspended in PBS (PBS + BC), or Maxpar Cell-IDTM barcode #20 resuspended in Maxpar Cell Staining Buffer (CSB + BC) (two-tailed unpaired t-test, p < 0.0001, n = 3 independent samples). Error bars represent standard deviation (SD). c) Maximum-gain confocal IF images of NHS ester-/C2 maleimide-Alexa 647 fluorescent probes shown in Fig. 3a to highlight background Matrigel staining. Each IF image is representative of three independent experiments. d) Small intestinal organoids concurrently stained in situ with Maxpar Cell-IDTM (102Pd, 104Pd, 105Pd, 106Pd, 108Pd, and 110Pd) and TOBis (124Te, 126Te, 128Te, 130Te, 196Pt, and 198Pt) barcodes using analogous 20-plex 6-choose-3 matrices. Data is representative of three independent experiments.
a) Example 10-plex barcoding workflow for organoid MC using Maxpar Cell-IDTM Barcoding Kit. b) Example 10-plex barcoding workflow for organoid MC using TOBis. c) Dissociation and d) centrifugation steps (s) required for multiplexed organoid experiments using Maxpar or TOBis barcoding. e) Single-cell recovery of serially-titrated small intestinal organoids barcoded with either Maxpar (ex situ) or TOBis (in situ) (two-tailed ratio-paired t-test, p < 0.001, n = 2 independent set of serially-titrated samples). f) Cell-type recovery (cell count) from equally seeded small intestinal organoids analyzed by MC and de-barcoded by Maxpar or TOBis. g) Cell-type recovery (population percentage) from equally sampled small intestinal organoid cells analyzed by MC and de-barcoded by Maxpar or TOBis (linear regression of correlation, R2 = 0.86, n = 2 independent samples). h) Mean debarcoded cell counts in the absence or presence of glutathione (GSH) in PBS washes following TOBis barcoding (two-tailed unpaired t-test, p < 0.0001, n = 3 independent samples). Error bars represent SD.. Source data
a) Confocal IF of CRC organoid genotypes (wild-type (WT), shApc (A), shApc and KrasG12D/+ (AK), and shApc, KrasG12D/+ and Trp53R172H/- (AKP)) stained with Pan-CK, LRIG1, CD44, and C-MYC (red), F-Actin (white), and DAPI (blue), scale bars = 50 μm. Images are representative of three independent IF experiments. Pan-CK and LRIG1 (in combination with CEACAM1) were subsequently used for epithelial and stem cell identification in MC. b) Confocal IF of colonic fibroblasts cultured in 2D (plastic) and 3D (Matrigel) demonstrates positive staining for colonic fibroblast markers PDPN, Vimentin, PDGFRɑ, FOXL1, and GLI1 (red), F-Actin (white), and DAPI (blue), scale bars = 50 μm. Images are representative of three independent IF experiments. PDPN and RFP were subsequently used for fibroblast identification in MC. c) Confocal IF of primary bone marrow derived macrophages cultured in 2D (plastic) and 3D (Matrigel) demonstrates positive staining for macrophages markers CD68, F4/80, CD45, CD11b, and CX3CR1 (red), F-Actin (white), and DAPI (blue), scale bars = 50 μm. Images are representative of three independent IF experiments. CD68 and F40/80 were subsequently used for macrophage identification in MC.
Signaling networks of 28 PTMs in colonic organoid cells from each genotype (wild-type (WT), shApc (A), shApc and KrasG12D/+ (AK), or shApc, KrasG12D/+, and Trp53R172H/- (AKP)) and microenvironment condition in the CRC TME model analyzed by MC. Signaling nodes are connected by DREMI edges and PTM activity is displayed as EMD relative to all cells across all conditions. Source data
a) and b) Signaling networks of 28 PTMs in macrophages and colonic fibroblasts from each genotype and microenvironment condition in the CRC TME model analyzed by MC. Signaling nodes are connected by DREMI edges and PTM activity is displayed as EMD relative to all cells across all conditions. c) and d) PCA of 28 PTM-EMDs for macrophages and colonic fibroblasts across all genotype/microenvironment combinations (10 conditions each). e) and f) PCA of 756 PTM-DREMIs for macrophages and colonic fibroblasts across all genotype/microenvironment combinations (10 conditions each). Source data
a) Force-directed Scaffold maps were constructed from WT colonic organoids alone or co-cultured with macrophages and/or colonic fibroblasts. b) Force-directed Scaffold maps were constructed from WT, A, AK, and AKP colonic organoid monocultures.
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Qin, X., Sufi, J., Vlckova, P. et al. Cell-type-specific signaling networks in heterocellular organoids. Nat Methods 17, 335–342 (2020). https://doi.org/10.1038/s41592-020-0737-8
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