Mapping Cellular Interactions between Cancer and Normal Cells Via Synthetic Notch Ligand/Receptor Pair

Understanding how cancer cells interact within its surroundings is critical for elucidating fundamental mechanisms of cancer biology and metastasis. We utilized the synthetic Notch ligand/receptor pair system (synNotch – Morsut L., Roybal KT., et al. Cell 2016) to track the physical interactions between normal and cancer cells with an ultimate goal of investigating the changes in gene expression profile of normal cells before and after the interactions occur.

Here we use normal epithelial cells (RPE1 hTERT) and adenoid cystic carcinoma (ACC), immortalized patient derived salivary gland cancer cell line). In our study, cell lines express synthetic Notch ligand/receptor pairs to measure the dynamics of touch-based interactions between normal-normal, cancer-cancer and normal-cancer cells. We find that, when co-cultured with ligand-presenting cancer cells, syn-Notch receptor carrying normal cells (1) can internalize the ligand molecules (trans-endocytosis) on average ~500 (+/- 280) times more when compared to receptor negative cells, (2) have detectable ligand molecules in their cytoplasm up to 24 hours post termination of interactions, (3) can be programmed for activation/inactivation of fluorescent reporters for longer term tracking. Moreover, by performing RNA-seq analysis on fluorescently sorted receptor positive normal cells, we compare the gene expression profiles of co-cultured cells with or without trans-endocytosis. Pairwise pathway comparison analysis shows differential regulation of genes with roles in axon signaling, ECM organization, steroid metabolism and Interleukin-10 signaling, providing a basis for physical interaction dependent gene signatures in the cancer microenvironment.

Ultimately, we seek to integrate our system into 3D spheroid/organoid and animal models to identify changes in gene expression levels of non-tumor host cells upon interactions with cancer cells. We are particularly interested with perineural invasion in cancer during the metastatic processes. We envision the same sensor mice will be valuable model to study other processes, such as neurogenesis or development of the immune system.