Single-cell and spatial transcriptomics reveal the interaction between PRRX2-driven epithelial cells and SPP1+ macrophages in mediating gemcitabine resistance in pancreatic ductal adenocarcinoma.

[BACKGROUND] The development of gemcitabine resistance significantly diminishes treatment efficacy and worsens prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). A comprehensive understanding of the molecular and cellular mechanisms driving gemcitabine resistance is thus essential for optimizing treatment strategies and improving survival outcomes in PDAC.

[METHODS] An integrated analysis of bulk RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), and spatial transcriptomics was performed to elucidate the molecular mechanisms of gemcitabine resistance. Tumor-associated epithelial and myeloid cell subsets were identified, and their interactions were characterized using clustering, pseudotime analysis, and cell-to-cell communication networks. Findings were validated through molecular and cellular experiments in PDAC cell lines, as well as multicolor immunofluorescence staining in clinical cohort samples.

[RESULTS] The findings revealed that PRRX2+ epithelial cells (EC) and SPP1+ tumor-associated macrophages (TAM) were enriched in gemcitabine-resistant samples, with robust cell-to-cell communication observed between these two populations. PRRX2+ EC cells and SPP1+ TAM cells interacted through the TGFB1/TGFBR2 axis, promoting epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, thereby contributing to drug resistance. The transcription factor PRRX2 was identified as a central regulator of this resistance mechanism, amplifying TGF-β signaling through TGFB1 to induce EMT and activating Fibronectin 1 to remodel the ECM, thereby enhancing an immunosuppressive tumor microenvironment. Furthermore, experimental downregulation of PRRX2 in pancreatic cancer cell lines resulted in reduced tumor cell migration. High expression levels of PRRX2+EC/SPP1+TAM were associated with poor prognosis and may indicate susceptibility to Dasatinib and antibody-drug conjugates (ADCs) targeting TROP2, MUC1, and CEACAM5.

[CONCLUSIONS] The results revealed the critical role of PRRX2+EC/SPP1+TAM cell communication in mediating gemcitabine resistance in PDAC. These findings provide new insights into potential therapeutic strategies, highlighting the importance of targeting the PRRX2+EC/SPP1+TAM axis and supporting the use of multidrug regimens to overcome resistance and improve patient outcomes in PDAC.