Incucyte zoom or sx5 imaging system

For flow cytometry and FACS, cells (~0.5–2 × 10⁶) were stained with anti-human CD44 APC (clone BJ18; #338806; BioLegend) and anti-human CD24 PE (clone ML5; #311106; BioLegend). Cells were then either sorted using the FACS-ARIA or analyzed using a BD LSRII and FACSDiva software. Migration assays were performed using the IncuCyte ZOOM or Sx5 imaging system (Essen BioScience) as indicated in the figure legends. Briefly, cells (1000 cells/well) were suspended in their base media (DMEM/F-12, RPMI, DMEM) containing 0.5% FBS and seeded onto 96-well ClearView-Chemotaxis plates with 8-mm pores. HPAC cells were stained with live cell NIR Nuclight Dye (Sartorius; #4804) before the plates were incubated and imaged over the indicated time points. Cells migrating to the bottom chamber across the pores were imaged and quantified. gemcitabine outgrowth assays were performed using the live cell IncuCyte ZOOM or Sx5 imaging system (Essen BioScience) after cells were stained with Incucyte Nuclight Rapid Red Dye, which marks live Cells (#4717; Sartorius) and plated at 2000 cells/well in 96-well flat-bottom plates (#3599; Corning), prior to treatment with gemcitabine (#G6423–50Mg; Sigma Aldrich) at the dose indicated in the figure legends. After gemcitabine addition, cells were imaged at regular intervals and quantified with Incucyte software.