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.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. There are few targeted therapies for these patients, leading to an unmet need for new biomarkers. The present study aimed to investigate the expression of PD-L1, CTLA-4, GLU, and VIM in CTCs of TNBC patients. Ninety-five patients were enrolled in this study: sixty-four TNBC and thirty-one luminal. Of these patients, 60 were in the early stage, while 35 had metastatic disease. Protein expression was identified by immunofluorescence staining experiments and VyCAP analysis. All the examined proteins were upregulated in TNBC patients. The expression of the GLU⁺VIM⁺CK⁺ phenotype was higher (50%) in metastatic TNBC compared to early TNBC patients (17%) (p = 0.005). Among all the BC patients, a significant correlation was found between PD-L1⁺CD45⁻CK⁺ and CTLA-4⁺CD45⁻CK⁺ phenotypes (Spearman test, p = 0.024), implying an important role of dual inhibition in BC. Finally, the phenotypes GLU⁺VIM⁺CK⁺ and PD-L1⁺CD45⁻CK⁺ were associated with shorter OS in TNBC patients (OS: log-rank p = 0.048, HR = 2.9, OS: log-rank p < 0.001, HR = 8.7, respectively). Thus, PD-L1, CTLA-4, GLU, and VIM constitute significant biomarkers in TNBC associated with patients’ outcome, providing new therapeutic targets for this difficult breast cancer subtype.

Metastasis, rather than the growth of the primary tumor, accounts for approximately 90% of breast cancer patient deaths. Microtentacles (McTNs) formation represents an important mechanism of metastasis. Triple-negative breast cancer (TNBC) is the most aggressive subtype with limited targeted therapies. The present study aimed to isolate viable circulating tumor cells (CTCs) and functionally analyze them in response to drug treatment. CTCs from 20 TNBC patients were isolated and maintained in culture for 5 days. Biomarker expression was identified by immunofluorescence staining and VyCap analysis. Vinorelbine-induced apoptosis was evaluated based on the detection of M30-positive cells. Our findings revealed that the CTC absolute number significantly increased using TetherChips analysis compared to the number of CTCs in patients’ cytospins (p = 0.006) providing enough tumor cells for drug evaluation. Vinorelbine treatment (1 h) on live CTCs led to a significant induction of apoptosis (p = 0.010). It also caused a significant reduction in Detyrosinated α-tubulin (GLU), programmed death ligand (PD-L1)-expressing CTCs (p < 0.001), and disruption of McTNs. In conclusion, this pilot study offers a useful protocol using TetherChip technology for functional analysis and evaluation of drug efficacy in live CTCs, providing important information for targeting metastatic dissemination at a patient-individualized level.

BRCA1 and PARP are involved in DNA damage repair pathways. BRCA1 mutations have been linked to higher likelihood of triple negative breast cancer (TNBC). The aim of the study was to determine PARP-1 expression and BRCA1 mutations in circulating tumor cells (CTCs) of BC patients. Fifty patients were enrolled: 23 luminal and 27 TNBC. PARP expression in CTCs was identified by immunofluorescence. Genotyping was performed by PCR-Sanger sequencing in the same samples. PARP-1 expression was higher in luminal (61%) and early BC (54%), compared to TNBC (41%) and metastatic (33%) patients. In addition, PARP-1 distribution was mostly cytoplasmic in luminal patients (p = 0.024), whereas it was mostly nuclear in TNBC patients. In cytokeratin (CK)-positive patients, those with the CK⁺PARP⁺ phenotype had longer overall survival (OS, log-rank p = 0.046). Overall, nine mutations were detected; M1 and M2 were completely new and M4, M7 and M8 were characterized as pathogenic. M7 and M8 were predominantly found in metastatic TNBC patients (p = 0.014 and p = 0.002). Thus, PARP-1 expression and increased mutagenic burden in TNBC patients’ CTCs, could be used as an indicator to stratify patients regarding therapeutic approaches.

Background Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with a worse outcome, highlighting the need for novel biomarkers to treat patients accordingly. This study aimed to evaluate the presence of prostate-specific membrane antigen (PSMA) and programmed death-ligand 1 (PD-L1) on DTCs in the bone marrow of TNBC patients before and after neo-adjuvant chemotherapy, to provide critical insights into their prognostic and therapeutic potential. Methods We investigated the expression of PSMA and PD-L1 in disseminated, cytokeratin (CK)-positive tumor cells (DTCs) from the bone marrow of TNBC patients before (55 patients) and after (47 patients) neo-adjuvant treatment using triple immunofluorescence stainings and VyCAP platform analysis. A weighted scoring system was developed to quantify PSMA and PD-L1 expression on DTCs, categorizing patients into negative, low/intermediate, and high expression groups. Statistical analyses included Spearman’s correlation, Wilcoxon signed-rank, chi-square, and McNemar’s tests, with significance set at p < 0.05. Results At baseline, 69% of patients exhibited CK + PSMAhighCD45- DTCs, which significantly decreased to 9% post-therapy ( p < 0.001). Similarly, CK + PD-L1highCD45- DTCs declined from 43–27% after therapy ( p = 0.045). Based on our scoring system, most patients initially classified as PSMA-high transitioned to PSMA-negative (n = 20, p < 0.001), while only one patient remained PSMA-high post-therapy. Post-NACT, a strong inverse correlation emerged between CK + PD-L1highCD45- and CK + PSMA-CD45- cells ( p = 0.010), and dual-positive phenotypes decreased to 4% ( p < 0.001). Presence of PSMA + DTCs at diagnosis was associated with shorter progression-free ( p = 0.002, HR = 16.1) and overall survival ( p = 0.016, HR = 2.1). Patients transitioning from PSMA-high to PSMA-negative post-therapy had improved overall survival compared to those remaining PSMA-high ( p < 0.001, HR = 1). Conclusions PSMA and PD-L1 are frequently overexpressed on DTCs of TNBC patients, predominantly before the administration of NACT. PSMA-positive DTCs emerged as a significantly poor prognostic factor, holding promise as a biomarker for identifying individuals at higher risk of relapse. It also provides a potential therapeutic target. Similarly, the presence and persistence of PD-L1-positive DTCs suggests their utility as biomarkers to stratify patients for ICI therapies.