Pancreatic Tumor Microenvironment Factor Promotes Cancer Stemness via SPP1-CD44 Axis.

ABSTRACT BACKGROUND & AIM Tumor-microenvironment (TME) factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which TME factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC. METHODS 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 2D and 3D sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice and human pancreatic tumors. PCR-array and gene knockdown were performed to identify the mechanism of stemness enrichment. RESULTS Long-term treatment of PC cells with CAF-CM enriched stemness, as demonstrated 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, CAF co-cultured with PC cells in the 3D model showed a substantial increase in stemness features. CD44 and α-SMA were positively correlated, and their expressions progressively increased from the early to late stages of KPC mice and human pancreatic tumors. OPN/SPP1 was identified as the top-differentially overexpressed gene in CAF-CM-treated PC cells, and knockdown of OPN/SPP1 significantly reduced stemness characteristics in CAF-CM-treated PC cells. CONCLUSIONS Our data uncover novel insight into the interplay between CAF and enrichment of stemness population through SPP1-CD44 axis in PC.