The phenotypic heterogeneity of tumor-derived cells in the bloodstream of pancreatic cancer patients

Abstract

Introduction Pancreatic cancer (PC) remains an aggressive malignancy with poor clinical outcomes, underscoring the need for novel biomarkers to monitor patients. Immune checkpoint molecules [programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)] have been detected in CTCs, and their expression is associated with poor prognosis. Overexpression of STIM1 and ORAI1, core regulators of calcium signaling, and the amino acid transporter chaperone CD98hc, have been shown to enhance cancer cell survival and migration. This study evaluated the expression of these molecules in PC-CTCs, alongside cytokeratin (CK, tumor cell marker) and CD45 (pan leukocyte marker). Methods CTCs were isolated from 40 patients using Ficoll density gradient centrifugation and characterized by immunofluorescence. Additionally, 15 were analyzed using the ISET platform. PBMCs from 10 healthy donors were also evaluated. Biomarker expression was assessed using the automated VyCAP platform, followed by ACCEPT software evaluation and statistical analysis of adjunct clinical data. Results CTCs were detected in 20% of patients, whereas all displayed (CK-/CD45-) cells with tumor characteristics and nucleus size of ≥10 μm, designated as circulating tumor-associated cells (CTACs). CTACs were significantly elevated in patients compared to healthy donors, and ROC analysis demonstrated strong discriminative capacity (AUC = 0.9083). Similarly, using the ISET platform, CTCs were detected in 26.7% (4/15), whereas CTACs were detected in 93.3% (14/15). PD-L1-positive CTCs were identified in 2.5% of patients, while PD-L1 and CTLA-4 expression on CTACs was detected in 30% and 17.5% of patients, respectively. STIM1-positive and ORAI1-positive CTCs were observed in 15% and 2.5% of patients, while expression on CTACs was detected in 70% and 10% of patients. Concerning CD98hc, 32.5% of patients harbored (CD98hc+/CD45-) cells. Interestingly, metastatic patients with PD-L1-positive CTCs exhibited shorter overall survival (OS; p = 0.025), while the presence of ≥2 (CD98hc+/CD45-) cells among patients who progressed within 1 year was associated with shorter progression-free survival (PFS; p = 0.007). Conversely, the presence of STIM1-positive CTCs correlated with improved OS (p = 0.021). Discussion These findings provide a comprehensive characterization of CTCs and CTACs in PC using a multiplex biomarker panel associated with calcium signaling and immune checkpoint pathways, highlighting the pronounced heterogeneity and potential clinical relevance of these circulating cell populations.