Cell-type-specific signaling networks in heterocellular organoids.

Xiao Qin,Petra Vlckova,Sophie E Acton,Pelagia Kyriakidou,Vivian S W Li,Christopher J Tape,Jahangir Sufi,Mark Nitz

doi:10.1038/s41592-020-0737-8

Abstract

Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc, KrasG12D, and Trp53R172H cell-autonomously mimic signalling 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 signalling analysis of healthy and cancerous organoids.

Highlights

Organoids are self-organising 3D tissue models comprising stem and differentiated cells [1]
Amine-reactive probes bind diffusely to Matrigel with very poor binding to organoids, whereas thiol-reactive probes bypass Matrigel and bind directly to organoids. b) Small intestinal organoids stained with either amine-reactive NHS ester-DOTA-157Gd or thiol-reactive C2 maleimide-DOTA-157Gd in situ or ex situ and analysed by MC. While both probes bind organoid cells ex situ, only thiol-reactive C2 maleimide-DOTA-157Gd bind organoids in situ. c) Model of amine- and thiol-reactive barcodes in organoid culture. d) Thiol-reactive tellurium maleimide (TeMal) (124Te, 126Te, 128Te, 130Te) and Cisplatin (196Pt, 198Pt) isotopologs combined to form a 20-plex (6-choose-3) doublet-filtering barcoding strategy. e) Thiol-Reactive Organoid Barcoding in situ (TOBis) workflow
We showed how Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks across different organoid mono- and co-cultures

Summary

INTRODUCTION

Organoids are self-organising 3D tissue models comprising stem and differentiated cells [1]. Given MC’s capacity to measure PTMs in mixtures of fixed cells, we theorised that MC workflows typically applied to immunophenotyping could be modified to study cell-type specific signalling networks in organoids. We report the development of a custom multivariatebarcoded MC method to measure single-cell signalling in epithelial organoids and organoids co-cultured with stromal and immune cells This method reveals that intestinal organoids display cell-type specific signalling networks that are intimately linked with cell-state. When applied to colorectal cancer (CRC) tumour microenvironment (TME) organoid co-cultures, we discovered that epithelial oncogenic mutations mimic signalling networks normally induced by stromal cells. These results demonstrate how a modified MC method can enable powerful multivariate single-cell analysis of celltype specific signalling in heterocellular organoids

RESULTS

Million Small Intestinal Organoid Cells Enterocyte

DISCUSSION