Fig. 3: Direct contact with fibroblasts promotes EMT of tumor cells.

a, b Sphere-formation assay of mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) tumor cells (BxPC-3-RFP and SU.86.86-RFP) with fibroblasts (PSC-GFP). Representative images a and quantification results b of RFP⁺ spheroids. Scale bar, 100 μm. Values were presented as mean ± SD (n = 3). c–f 3D spheroid invasion assay of mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) tumor spheroids (BxPC-3-RFP spheroids) with fibroblasts (PSC-GFP cells). The culture condition c and representative images d. Scale bar, 200 μm. The extensive rate of each tumor spheroid was calculated by normalizing RFP⁺ area at 72 h to RFP⁺ area at 1 h (n = 4). Panel c was created with BioRender.com. e and RFP⁺ maximal diameter at 72 h to RFP⁺ diameter at 1 h f. Values were presented as mean ± SD (n = 4). g The differential gene expression analysis with RNA-seq. Overlap of the commonly up-regulated genes (464) in tumor cells (BxPC-3-RFP and SU.86.86-RFP) after direct (D) co-cultured with fibroblasts compared to mono-cultured (−) or indirect co-cultured (ID) conditions were used for Ingenuity Pathway Analysis (IPA). h Top 6 canonical pathways with the largest -log (p-value) calculated by Fisher’s Exact Test in IPA were shown. i Quantitative real-time PCR analyses of CLDN1, VIM, Snail, Twist1, ZEB1, and ZEB2 expression were performed to compare gene expression levels in mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) tumor cells (BxPC-3-RFP) with fibroblasts. FACS was performed to collect RFP⁺ tumor cells after 24 h of mono- or co-cultured conditions. Values were presented as mean ± SD (n = 3). j Western blotting analysis of proteins harvested from mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) tumor cells (BxPC-3-RFP) with fibroblasts. FACS was performed to collect RFP⁺ tumor cells after 48 h of mono- or co-cultured conditions. k BxPC-3-GFP-Luc cells with and without direct contact with fibroblasts were injected into the pancreas of NSG mice (n = 5). l PdKP53-GFP-Luc cells with and without direct contact with fibroblasts were injected into the pancreas of C57BL/6 mice (n = 6). m, n The effect of direct contact with fibroblasts on liver metastasis. m BxPC-3-GFP-Luc cells with and without direct contact with fibroblasts were injected into the spleen of NSG mice (n = 6). n PdKP53-GFP-Luc cells with and without direct contact with fibroblasts were injected into spleen of C57BL/6 mice (n = 6). Tumor growth and liver metastasis were assessed by IVIS bioluminescent analysis (Lumina, Perkin Elmer) after 2 weeks of orthotopic or splenic injection. The bioluminescent signal (pseudocolor) was recorded as photons per second (p/s). Values were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed t-test).