Lung cancer is the leading cause of cancer-related mortality globally. Among the types of lung cancer, non-small-cell lung cancer (NSCLC) is more common, while small-cell lung cancer (SCLC) is less frequent yet more aggressive. Circulating tumor cells (CTCs), albeit rare, have been portrayed as essential players in the progression of lung cancer. CTCs are considered to adopt an epithelial-to-mesenchymal transition (EMT) phenotype and characteristics of cancer stem cells (CSCs). This EMT (or partial) phenotype affords these cells the ability to escape from the primary tumor, travel into the bloodstream, and survive extremely adverse conditions, before colonizing distant foci. Acquisition of CSC features, such as self-renewal, differentiation, and migratory potential, further reflect CTCs’ invasive potential. CSCs have been identified in lung cancer, and expression of EMT markers has previously been correlated with poor clinical outcomes. Thus far, a vast majority of studies have concentrated on CTC detection and enumeration as a prognostic tools of patients’ survival or for monitoring treatment efficacy. In this review, we highlight EMT and CSC markers in CTCs and focus on the clinical significance of these phenotypes in the progression of both non-small- and small-cell lung cancer.

In the present study, we evaluated the expression of JUNB and CXCR4 in circulating tumor cells (CTCs) of lung cancer patients and investigated whether these proteins have prognostic clinical relevance. Peripheral blood from 30 patients with non-small-cell lung cancer (NSCLC) was filtered using ISET membranes, and cytospins from 37 patients with small-cell lung cancer (SCLC) were analyzed using confocal and VyCAP microscopy. Both JUNB and CXCR4 were expressed in the vast majority of lung cancer patients. Interestingly, the phenotypic patterns differed between NSCLC and SCLC patients; the (CK+/JUNB+/CXCR4+) phenotype was present in 50% of NSCLC vs. 71% of SCLC patients. Similarly, the (CK+/JUNB+/CXCR4–) was present in 44% vs. 71%, the (CK+/JUNB–/CXCR4+) in 6% vs. 71%, and the (CK+/JUNB–/CXCR4–) phenotype in 38% vs. 84%. In NSCLC, the presence of ≥1 CTCs with the (CK+/JUNB+/CXCR4+) phenotype was associated with worse progression-free survival (PFS) (p = 0.007, HR = 5.21) while ≥2 with poorer overall survival (OS) (p < 0.001, HR = 2.16). In extensive stage SCLC patients, the presence of ≥4 CXCR4-positive CTCs was associated with shorter OS (p = 0.041, HR = 5.01). Consequently, JUNB and CXCR4 were expressed in CTCs from lung cancer patients, and associated with patients’ survival, underlying their key role in tumor progression.

The PD-1/PD-L1 axis provides CTCs an escape route from the immune system. Phosphorylation of the ribosomal protein S6 is implicated in the same pathway, following mTOR activation. The aim of the study was to investigate the expression of PD-L1 and pS6 in CTCs from NSCLC patients under Osimertinib treatment at a single cell level. CTCs were isolated using ISET from NSCLC patients’ blood [37 at baseline, 25 after the 1st cycle, and 23 at the end of treatment (EOT)]. Staining was performed using immunofluorescence. Cytokeratin-positive (CK+) CTCs were detected in 62% of patients. CK+PD-L1+CD45− and CK+pS6+ phenotypes were detected in 38% and 41% of the patients at baseline, in 28% and 32% after 1st cycle, and in 30% and 35% at EOT, respectively. Spearman’s analysis revealed statistically significant correlations between PD-L1 and pS6 phenotypes at all time points. Survival analysis revealed that CK+pS6+ (p = 0.003) and CKlowpS6+ (p = 0.021) phenotypes after 1st cycle were related to significantly decreased one-year progression-free survival (PFS12m) and PFS, respectively. CK+PD-L1+CD45−phenotype at baseline and after 1st cycle showed a trend for decreased PFS12m. Increased expression of PD-L1/pS6 in CTCs of Osimertinib-treated NSCLC patients implies the activation of the corresponding pathway, which is potentially associated with poor clinical outcomes.

Background: According to recent publications of our group, JUNB and CXCR4 were overexpressed in CTCs and DTCs isolated from breast cancer patients. This expression was related with patients’ clinical outcome. Current study investigates for the first time, the expression of JUNB and CXCR4 in CTCs from patients with Non-Small Cell Lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). Methods: Forty four patients were enrolled in this study (30 NSCLC and 14 SCLC patients, before the initiation of 1st line treatment). ISET system was used for NSCLC patients' samples preparation, while the CTCs from SCLC patients were isolated using Ficoll density gradient. Triple immunofluorescence experiments were performed, using CK, JUNB, and CXCR4 antibodies. Results conducted using confocal laser scanning microscopy for NSCLC samples and the VyCAP system for SCLC samples. Results: Sixteen out of 30 NSCLC patients (53.33%), were positive for CTCs, while all SCLC patients harbored CK-positive cells. Most common phenotypes in CK-positive NSCLC patients were the [(CK+/JUNB+/CXCR4+): 50% (8/16patients)], [(CK+/JUNB+/CXCR4-): 43.75%, (7/16)] and the [(CK+/JUNB-/CXCR4-): 37.5% (6/16)]. Less frequent phenotype was the [(CK+/JUNB-/CXCR4+): 6.25%, (1/16)]. Analysis of the mean isolated CTCs/patient revealed that most of the isolated CTCs belonged to the (CK+/JUNB+/CXCR4+) phenotype (42.19%), while less frequent were the (CK+/JUNB+/CXCR4-): 33.13%, (CK+/JUNB-/CXCR4-): 18.44% and the (CK+/JUNB-/CXCR4+): 6.25% phenotypes. Survival analysis revealed that the presence of (CK+/JUNB+/CXCR4+) was related to poorer OS (cox regression: p=0.008) and PFS (Log Rank, p=0.014) All SCLC patients had detectable CTCs in their blood with 13 out of 14 of them having the (CK+/JUNB-/CXCR4-) phenotype (92.86%). The rest of CTC’s phenotypes (CK+/JUNB+/CXCR4+), (CK+/JUNB+/CXCR4-) and (CK+/JUNB-/CXCR4+) where found in 10 out of 14 patients (71.43%) each. Examination of the mean percentage of the total isolated CTCs/patient indicated that the (CK+/JUNB-/CXCR4-) phenotype was the most frequent (49.71%), while the percentages for the rest phenotypes were [(CK+/JUNB+/CXCR4-): 25.94%], [(CK+/JUNB-/CXCR4+): 12.65%] and (CK+/JUNB+/CXCR4+): 11.70%]. Conclusion: JUNB and CXCR4 were upregulated in CTCs from NSCLC and SCLC patients. However, in NSCLC the most frequent phenotype (CK+/JUNB+/CXCR4+) was also related to patients’ outcome, underlying the key role of these molecules in metastatic dissemination. Further examination will determine the role of this expression in all lung cancer subtypes. Acknowledge: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2ΕΔΚ-01562). Citation Format: Argyro Roumeliotou, Evangelia Pantazaka, Anastasia Xagara, Thomas Makatsoris, Angelos Koutras, Vassilis Georgoulias, Athanasios Kotsakis, Galaktia Kallergi. Dr [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1966.