Mass Cytometry for Multivariate Organoid Signalling Analysis CURRENT STATUS: POSTED

Organoids are powerful biomimetic tissue models. Despite their increasing popularity, no existing methods are suitable for cell-type specific analysis of post-translational modification (PTM) signalling networks in organoids. Here we report a multivariate mass cytometry (MC) protocol for single-cell analysis of cell-type specific PTM signalling in organoid monocultures and organoids co-cultured with stromal and immune cells. Thiol-reactive Organoid Barcoding in situ (TOB is ) was developed to facilitate high-throughput comparison of signalling networks between organoid cultures. Taken together, our protocol enables high-throughput multivariate PTM signalling analysis of healthy and cancerous organoids at the single-cell level.


Introduction
Organoids are self-organising 3D tissue models comprising stem and differentiated cells 1 . Organoid monocultures typically contain one major cell class (e.g. epithelial) and can be co-cultured with heterotypic cell-types (e.g. mesenchymal 2

or immune cells 3 ) to model cell-cell interactions in vitro.
When compared with traditional 2D cell culture, organoids more accurately represent their parental tissue and are powerful models for studying multicellular diseases such as cancer 4 .
While culturing organoids has become increasingly widespread, methods to analyse cell-type specific phenotypes in organoids are limited. Mass cytometry (MC, also known as cytometry time-of-flight (CyTOF)) uses heavy metal-conjugated antibodies to measure >35 proteins in single cells 5 . Although MC is traditionally used for high-dimensional immunophenotyping, MC can also measure PTMs in heterocellular systems (e.g. peripheral blood mononuclear cells (PBMCs) 6 and tissue 7 ). Here we report a protocol to perform cell-type specific analysis of post-translational modification (PTM) signalling networks in organoids and organoid co-cultures using MC.
In addition to high dimensional single-cell PTM measurements, a major advantage of MC is its ability to perform multiplexed barcoding of experimental variables. Unfortunately, commercially available Palladium-based barcodes cannot bind organoids in situ as they react with Matrigel proteins commonly used in organoid culture. This means organoids must be removed from Matrigel and dissociated separately before using Pd barcoding. To overcome this, we developed Thiol-reactive Organoid Barcoding in situ (TOBis) to enable high-throughput multivariate single-cell organoid signalling analysis in a single tube.
Taken together, our protocol demonstrates how traditional MC workflows can be modified to measure cell-type and cell-state specific signal transduction in organoids in a high-throughput multiplexed manner ( Figure 1).

Rare-Earth Metal Conjugated Antibodies
We advise users to develop custom heavy-metal conjugated antibody panels specifically for their biological questions. In our experience, cell-state (e.g. proliferating, quiescent, and apoptotic) has a huge influence on PTM signalling. We therefore strongly advise users to include cell-cycle (e.g. pRB [S807/S811], Cyclin B1, and pHistone H3 [S28]) and apoptosis (e.g. cCaspase3 [D175]) markers in their panels. Particular care should also be taken to validate cell-type specific antibodies that have not been used in MC before. The antibody panels used when developing this protocol can be found in Qin et al., bioRxiv, 2019 10 .

Phosphatase & Protease Inhibitor Treatment
2) Add the protease inhibitor cocktail (100 ) and PhosSTOP (40 ) directly to culture media, rotate the plate, and incubate for 5 mins at 37 ºC, 5 % CO 2 . In order for organoids to be sufficiently dissociated with the gentleMACS Octo Dissociator, we suggest users not to overload the gentleMACS C-Tubes. In our experience, up to ~ 5 10 6 cells per C-Tube can be dissociated sufficiently using our custom dissociation program. We also recommend users to prepare fresh dissociation solution before each use.

Antibody staining is not working.
Antibody panels for MC experiments need to be carefully designed and titrated in accordance with known monoisotopic impurities 11 and antigen abundance. We also encourage users to test alternative fixation and permeabilisation conditions for their specific experimental system.

Cells are lost during staining.
Cell loss is inevitable during staining due to the multiple washing steps, and it is more striking with fewer cells. We recommend users to start with sufficient materials, barcode the cells, and pool different conditions as early as possible during the protocol (that is part of the motivation of the development of TOBis). In addition, during optimisation we observed that coating polypropylene FACS tubes with CSB prior to centrifugation of cells resuspended in PBS also facilitates the cells to spin down properly and thereby reduces cell loss.

Time Taken
The pre-treatment and fixation of organoids takes around 2 hours. Live / dead discrimination staining takes around 0.5 hour. TOBis barcoding can be performed in 1-2 hours but an overnight incubation will give superior debarcoding efficiencies. Depending on the number of samples, single-cell dissociation takes 2 to 2.5 hours. The MC staining protocol (extracellular staining, permeabilisation, and intracellular staining) takes around 4 hours. DNA intercalation is performed for 1 hour at room temperature or overnight at 4 ºC. The MC run takes 1 to 2 hours depending on the number of cells to be analysed.

Anticipated Results
The data generated from the workflow reported here is single-cell measurements of up to 50 channels (cell-type identification antibodies, cell-state antibodies / probes, and PTM antibodies etc.) across multiple organoid culture conditions. TOBis barcoded conditions can be resolved into individual FCS files using: https://github.com/zunderlab/single-cell-debarcoder. All FCS files can be uploaded to the Cytobank platform (http://www.cytobank.org/) and proceed to single-cell signalling analysis 10 . All selection of tools to analyse organoid CyTOF data can be found at: https://github.com/TAPE-Lab.