In vivo single-cell CRISPR uncovers distinct TNF-alphaprograms in clonal expansion and tumorigenesis

Renz, P. F.; Ghoshdastider, U.; Baghai Sain, S.; Valdivia-Francia, F.; Khandekar, A.; Ormiston, M.; Bernasconi, M.; Kretz, J. A.; Lee, M.; Hyams, K.; Forny, M.; Pohly, M.; Ficht, X.; Ellis, S. J.; Moor, A. E.; Sendoel, A.

doi:10.1101/2023.07.13.548697

bioRxiv · 10.1101/2023.07.13.548697

In vivo single-cell CRISPR uncovers distinct TNF-alphaprograms in clonal expansion and tumorigenesis

Renz, P. F.·Ghoshdastider, U.·Baghai Sain, S.·Valdivia-Francia, F.·Khandekar, A.·Ormiston, M.·Bernasconi, M.·Kretz, J. A.·Lee, M.·Hyams, K.·Forny, M.·Pohly, M.·Ficht, X.·Ellis, S. J.·Moor, A. E.·Sendoel, A.

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Abstract

The tumor evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions occur frequently in human epithelia and can remodel almost entire tissues, the mechanisms behind why only a small number of clones transform into malignant tumors remain enigmatic. Here, we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing, guide capture and spatial transcriptomics to longitudinally monitor cell type-specific clonal expansions, document their underlying gene programs and contrast clonal expansions from tumor initiation. We uncover a TNF- signaling module that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF- signaling module is downregulated, and instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF- gene program. By analyzing clonally expanded perturbations and their frequency in tumors, we demonstrate that the autocrine TNF- gene program is associated with epithelial-mesenchymal transition (EMT) and is preexistent in a subpopulation of expanded epidermal stem cells, contributing to the predisposition for tumor initiation. Finally, we provide in vivo evidence that the epithelial TNF- gene program is sufficient to mediate invasive properties of epidermal stem cells and show that the TNF- signature correlates with shorter overall survival in human squamous cell carcinoma patients. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues and unveils distinct TNF- programs in tumor evolution. Understanding the biology of clonal expansions in phenotypically normal epithelia and the mechanisms governing their transformation will guide the development of novel strategies for early cancer detection and therapy.

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Renz, P. F., Ghoshdastider, U., Baghai Sain, S., Valdivia-Francia, F., Khandekar, A., Ormiston, M., Bernasconi, M., Kretz, J. A., Lee, M., Hyams, K., Forny, M., Pohly, M., Ficht, X., Ellis, S. J., Moor, A. E., Sendoel, A.. 2023-07-14. In vivo single-cell CRISPR uncovers distinct TNF-alphaprograms in clonal expansion and tumorigenesis. https://doi.org/10.1101/2023.07.13.548697

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