Fig. 2: ɑSMA⁺ fibroblasts directly bind to tumor cells.

a The schematic procedure of cell cluster isolation. Pancreases were isolated from 6 to 7 weeks old PdKP53 transgenic mice. The whole pancreas was cut into pieces of <1 mm³ and underwent mechanical dissociation to cell clusters <40 μm. EpCAM⁺/PDGFRα⁺ cell clusters were captured by FACS. b Representative immunofluorescence (IF) images of CK19/αSMA/DAPI expression in EpCAM⁺/PDGFRα⁺ clusters. Scale bar, 10 μm. c Representative IF images of CK19/αSMA/CD45/DAPI expression in circulating tumor microemboli (CTM) in 6 to 7 weeks old PdKP53 transgenic mice. Scale bar, 10 μm. d Number of CTM in 6 to 7 weeks old Pdx1-Cre; LSL-Kras^+/+; Trp53^flox/flox (WT) and PdKP53 mice. Each sample was pooled by three mice. Thus, total of 9 PdKP53 mice were used for triplicate data. Values were presented as mean ± SD (n = 3). e–g 3D-Matrigel mono-culture or co-culture assay of mouse pancreatic stellate cells labeled with RFP (mPSC-RFP) and mouse pancreatic cancer cells labeled with GFP (PdKP53-GFP). The culture condition e and representative images f. Scale bar, 100 μm. g After 2 h and 24 h, the percentage of RFP⁺-RFP⁺, GFP⁺-GFP⁺, or RFP⁺-GFP⁺ cells with distance ≤ 1 cell diameter were determined by Imaris software at indicated time points. Values were presented as mean ± SD (n = 4). h–j 3D-Matrigel mono-culture or co-culture assay of human pancreatic stellate cells labeled with GFP (PSC-GFP) and human pancreatic cancer cells labeled with RFP (BxPC-3-RFP). The cultural condition h and representative images i. Scale bar, 100 μm. j After 2 h and 24 h, the percentage of RFP⁺-RFP⁺, GFP⁺-GFP⁺, or RFP⁺-GFP⁺ cells with distance ≤ 1 cell diameter were determined by Imaris software at indicated time points. Values were presented as mean ± SD (n = 4). **p < 0.01, ***p < 0.001 (two-tailed t-test). Panel a, e, h were created with BioRender.com.