mRNA sequencing SUM159PT WT, Kod and KOs cells cultured in adherent or suspension (i.e. secondary tumorspheres) conditions.

The cellular heterogeneity within a tumor can be determined by core genetic and epigenetic programs that operate in certain cells (tumor initiating cells – TICs), providing them with the degree of plasticity needed for induction of metastasis and resistance to therapy. Here we show that the metabolic enzyme nicotinamide N-methyl transferase (NNMT) promotes TIC plasticity in Estrogen Receptor (ER) alpha negative breast cancer. NNMT downregulation delays tumor formation and its full depletion impairs metastasis formation in mice. At the transcriptomic level, NNMT loss results in dowregulation of stem cell and mesenchymal gene signatures, and in the upregulation of luminal features and oxidative metabolism related genes. SUM159PT cells were cultured in adherent conditions until they reached 80% confluency. At this time point, the cells were collected for mRNA extraction with the Qiagen RNeasy Plus Mini Kit. For breast cancer cell line tumorsphere culture, cells were plated at 10’000-30'000 cells per mL density in ultra-low attachment plates for 6 days. Primary tumorspheres were dissociated with 0.05% trypsin and replated at the same density for additional 6 days for secondary tumorsphere formation. RNA was extracted with the Qiagen RNeasy micro kit. NNMT was knocked out SUM159PT using two different strategies: one single guide (sg) RNA targeting the transcription start site (KOs) and two sgRNAs that generate a deletion of exon 1 (KOd). Several clones from each strategy were mixed to generate two oligoclonal pools of cells, KOd and KOs.