Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC.

PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous Kras^G12D/+; Trp53^R172H/+; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment.

Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44⁺, ALDH⁺, and AF⁺ populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM–treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α–smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of Kras^G12D/+; Trp53^R172H/+; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM–treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM–treated PC cells.

Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1–CD44 axis in PC.