Extremely proud to share my lab’s first paper: ‘Cell-Type Specific Signaling Networks in Heterocellular Organoids’ out today in @naturemethods (1/14) nature.com/articles/s4159…
#Organoids are amazing biomimetic tissue models that contain multiple cell-types (e.g. stem, differentiated), each in their own cell-state (e.g. proliferating, quiescent, apoptotic). (2/14) 
While this complexity is what makes organoids so useful, it also makes them tricky to study. My lab wanted to use organoids to study cell-signalling in cancer – but bulk methods (e.g. LC-MS/MS) couldn’t easily provide cell-type and cell-state specific signalling data. (3/14)
To overcome this, we developed a #CyTOF protocol to measure 28 post-translational modifications (PTMs) in millions of single-organoid cells. This technology allowed us to study cell-type and cell-state specific signalling in intestinal organoids. (4/14)
We found that organoid cell-types have distinct signalling networks and that PTM signalling is hugely influenced by cell-state. (5/14)
For example, here is an enterocyte PTM network during proliferation and quiescence: (6/14) (thanks @phospho!)
As the standard CyTOF multiplexing kit doesn’t work in Matrigel, we also developed a new barcoding strategy for organoids (w’ @NitzUofT). This Thiol-reactive Organoid Barcoding in situ (TOBis) method facilitates high-throughput single-cell organoid multiplexing by CyTOF. (7/14)
We used TOBis to study cell-type specific PTM networks during intestinal organoid development. This revealed stem and Paneth cells maintain stable PTM networks throughout organoid culture, whereas goblet cells and enterocytes switch signalling before organoids collapse. (8/14)
We then used TOBis CyTOF to compare how oncogenes and stromal cells deregulate PTM signalling in colorectal cancer (CRC) tumour microenvironment organoid co-cultures. (9/14)
This study revealed oncogenic driver mutations (Apc, Kras, and p53) mimic the epithelial signalling profiles normally provided by stromal cells. Moreover, stromal cells can activate PI3K signalling nodes beyond oncogenic mutations in CRC organoids. (10/14)
Collectively, TOBis CyTOF enables multiplexed single-cell signalling network analysis of organoids and organoid co-cultures. If you’re interested in what’s happening inside your heterogenous little organoids – check out the paper! (11/14)
This paper is a huge team effort. The study was led by @QinXiao1990, Jahangir Sufi developed TOBis, @Petra47715998 built CRC TME organoids, and @PelagiaKyr analysed data. I’d also really like to thank @VivianLiLab, @ActonLab, and @dow_lab for generously sharing reagents. (12/14)
I’d also like to thank @CRUKresearch, @UCLHresearch, @RosetreesT, and @royalsociety for funding this work. (13/14)
Finally, all raw and processed data can be found on @cytobank (id=1271) and a step-by-step protocol to perform organoid TOBis CyTOF is available on our website: tape-lab.com/resources. (14/14)
