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.

SCLC is an aggressive cancer type with high metastatic potential and bad prognosis. CTCs are a valuable source of tumor cells in blood circulation and are among the major contributors to metastasis. In this study we evaluated the number of CTCs that express PD-L1 in treatment-naïve ES-SCLC patients receiving ICI in a front-line setting. Moreover, we explored the percentages of different immune T-cell subsets in circulation to assess their potential role in predicting responses. A total of 43 patients were enrolled—6 of them with LS-SCLC, and 37 with ES-SCLC disease. In addition, PBMCs from 10 healthy donors were used as a control group. Different T-cell subtypes were examined through multicolor FACS analysis and patients’ CTCs were detected using immunofluorescence staining. SCLC patients had higher percentages of PD-1-expressing CD3⁺CD4⁺ and CD3⁺CD8⁺ T-cells, as well as elevated PD-1 protein expression compared to healthy individuals. Additionally, in ES-SCLC patients, a positive correlation between CD3⁺CD8⁺PD-1⁺ T-cells and PD-L1⁺ CTCs was detected. Importantly, patients harboring higher numbers of CD3⁺CD8⁺PD-1⁺ T-cells together with PD-L1⁺CTCs had a survival advantage when receiving front-line immunotherapy. Thus, this study proposes, for first time possible, immune cell–CTCs interaction, as well as a potential novel clinical biomarker for ICI responses in ES-SCLC patients.

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 CK^lowpS6⁺ (p = 0.021) phenotypes after 1st cycle were related to significantly decreased one-year progression-free survival (PFS_12m) and PFS, respectively. CK⁺PD-L1⁺CD45⁻phenotype at baseline and after 1st cycle showed a trend for decreased PFS_12m. 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.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.