Fig. 1. In vitro TME model development and isolation of pancreatic cancer cells exhibiting direct cell-to-cell transfer.

a Schematic of the TME mimic model showing steps for collecting conditioned media (CM) from human pancreatic normal fibroblasts (n-fibroblasts) co-cultured with human pancreatic cancer-associated fibroblasts (CAFs) under hypoxic only or hypoxic/gemcitabine (gem)-induced stress conditions (upper panels); and for co-culturing pancreatic cancer cells with macrophages to isolate cells exhibiting direct cell-to-cell transfer (double fluorescent cells; DFCs) and to generate derivative cells. MØ: macrophage, CMTMR: 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine, CMFDA 5-chloromethylfluorescein diacetate, FACS fluorescence-activated cell sorting, STR short tandem repeat. b Morphological changes of total cultured cells with increased numbers of vacuoles, enlarged cytoplasm, and multiple TNTs at the end of the Seeding phase, prior to collection of the cells for FACS. Arrowheads denote TNTs. c Percentage of isolated Panc0203^CMTMR (red), MØ-U937^CMFDA (green), and DFCs (blue) detected via FACS following preparation of adherent and detached cells from total cultured cells. Detached cells represent dying cells. d, e Percentage of adherent double fluorescent dye-positive cells (DFCs) for FACS gating in the (d) gemcitabine-treated TME mimic model or (e) normal co-culture TME condition. The percentages in the graphs indicate the percentage of adherent DFCs. +Gem; gemcitabine-treated TME mimic model, no Gem; normal co-culture TME condition. f Confocal images of single-fluorescent dye-positive cells (SFCs) and DFCs (red box, a merged image of entire z-stack with orthogonal views in the same field). Green SFCs and red SFCs in the confocal images indicate MØ-U937^CMFDA and Panc0203^CMTMR, respectively (scale bar, 10 μm).