Evangelia Pantazaka

Extracellular vesicles (EVs) are a diverse population of membrane nanoparticles secreted by nearly all cell types, playing a key role in intercellular communication by transferring bioactive macromolecular cargo. In cancer, EVs shape both the local tumour microenvironment and distant premetastatic niches. They are essential regulators of tumour initiation, progression, immune modulation, angiogenesis and metastatic dissemination. Due to their abundance in biofluids, EVs have attracted attention as diagnostic and prognostic biomarkers for early detection and assessment of therapeutic response. Additionally, EVs represent a promising therapeutic platform for delivering chemotherapeutic agents, nucleic acids and gene‐editing tools, with enhanced specificity and reduced systemic toxicity. This review summarises current therapeutic applications of EVs across breast, lung, colorectal, prostate and pancreatic cancers and glioblastoma. Key findings are presented for each formulation, with emphasis on the EV physicochemical properties and engineered modifications, their cargo and mechanisms underlying their biological effects. Although the clinical translation of EV‐based advances remains limited and challenging, this review highlights significant preclinical findings and examples of clinical trials where EVs have been used as therapeutic agents.

Background: Monocarboxylate transporters (MCTs) facilitate lactate transfer and support cancer cell survival and metastasis. MCT1 and MCT4 expression has been associated with poor prognosis and resistance to therapy with tyrosine kinase inhibitors. Objectives: This pilot study examined the relevance of MCT1 and MCT4 expression in circulating tumour cells (CTCs) isolated from non-small cell lung cancer (NSCLC) patients during osimertinib treatment through single-cell analysis. Design: Fifty-three NSCLC patients were enrolled at three different time points: baseline ( n = 53), post-first cycle ( n = 20) and progressive disease (PD; n = 21), to evaluate MCT1 and MCT4 expression in patients’ samples. Methods: CTCs were isolated with the ISET platform. MCT1/MCT4 expression was assessed using immunofluorescence triple staining experiments and confocal laser scanning microscopy. Results: CTCs were detected in 75% (40/53), 40% (8/20) and 29% (6/21) of patients at baseline, post-first cycle and PD, respectively. Among cytokeratin (CK)-positive patients, MCT1 was overexpressed at all time points in a significant percentage: 67% (18/27) at baseline, 57% (4/7) at post-first cycle and 50% (2/4) at PD. Similarly, MCT4 was overexpressed in 55% (16/29), 50% (2/4) and 60% (3/5) of cases, respectively. Statistical analysis revealed that the (CK+MCT1−CD45−) phenotype was associated with worse progression-free survival [PFS; log rank, p = 0.041, hazard ratio (HR) = 1.971] and overall survival (log rank, p = 0.028; HR = 2.288) of the patients. Conversely, the presence of ⩾3 MCT4+ CTCs was correlated with poorer PFS (log rank, p = 0.042, HR = 4.189). Significant inverse correlations were observed between MCT1 and MCT4 expression, implying their distinct biological roles. Conclusion: MCT1 and MCT4 are overexpressed in CTCs from NSCLC patients, supporting their potential as prognostic biomarkers and therapeutic targets.

Objectives: Circulating tumor cells (CTCs) drive metastasis and exhibit resistance to conventional therapies, making them crucial therapeutic targets. Artesunate (AS), a derivative of artemisinin, displays anticancer activity, including inhibition of JunB proto-oncogene (JUNB) and programmed death ligand-1 (PD-L1) and upregulation of Vimentin (VIM), markers related to poor prognosis in CTCs. This study aimed to evaluate the effects of AS on adherent and non-adherent cancer cell lines (breast, lung, colon), the patient-derived colon cancer CTC-MCC-41 line, and CTCs from small-cell lung cancer (SCLC) patients. Methods: AS’s effect was evaluated using TetherChip technology. Cell viability was measured using MTT assay, while immunofluorescence staining and the VyCAP platform were applied to characterize and quantify CTCs. Results: AS significantly reduces viability in all tested cell lines in a time- and concentration-dependent manner, with non-adherent cells showing higher resistance. Notably, CTC-MCC-41 cells are the most sensitive to AS treatment. AS demonstrates stronger cytotoxicity than 5-fluorouracil (5-FU) in most cancer models. In SCLC patient samples, AS reduces total CTC counts (p < 0.001), eliminates aggressive phenotypes such as (CK+/CXCR4+/JUNB–) and (CK+/VIM+/GLU+), and increases apoptotic (M30+) CTCs (p = 0.021). AS additionally impairs structural features like microtentacles, which facilitate CTC reattachment. Conclusions: These findings underscore AS’s ability to target metastasis-competent and anoikis-resistant tumor cells, reducing their viability, invasiveness, and survival mechanisms. AS emerges as a promising candidate for anti-metastatic therapy and warrants further investigation in precision oncology.

G protein-coupled receptors (GPCRs) are the largest family of diverse receptors in eukaryotic organisms, playing a critical role in modulating human physiology. It therefore comes as no surprise that about 36% of all currently available drugs target this superfamily. When an agonist binds to a GPCR, it induces conformational changes in the receptor that allow it to interact with intracellular proteins. This interaction triggers downstream signaling cascades that alter the cell’s activity. GPCR signaling is complex, as GPCRs transmit signals through coupling with G proteins, arrestins, and numerous other intracellular effectors. Different ligands, receptor subtypes, and cellular environments can result in the activation of distinct signaling pathways. Biased signaling through GPCRs has emerged as a frontier area in pharmacological research efforts towards designing targeted therapeutic interventions and enhancing drug efficacy and safety. This review presents the types of bias associated with GPCRs and the mechanisms underlying biased signaling. Examples of biased ligands and their therapeutic implications will be discussed. In addition, the inherent challenges in measuring signaling bias, and especially the translational gap between in vitro and in vivo assays and clinical outcomes, will be outlined.

Isolation of cells from tissues can be as daunting a task as it is satisfying. Primary cells closely resemble the tissue they originate from, recapitulating its properties. Here, the procedure for isolating smooth muscle cells (SMCs) from the rat aorta using enzymatic digestions is outlined. Notwithstanding the difference in size and thickness of the aortic wall, the protocol can also be applied to isolate cells from the mouse aorta, which is advantageous considering the plethora of transgenic mice models. A single-cell suspension of cells staining positive for smooth muscle α-actin is obtained.

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca²⁺, regulated by the pore-forming proteins ORAI and the Ca²⁺ sensing stromal interaction molecules (STIM), via store-operated Ca²⁺ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca²⁺ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45−) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

Background: Despite the expanding use of orthopedic devices and the application of strict pre- and postoperative protocols, the elimination of postoperative implant-related infections remains a challenge. Objectives: To identify and assess the in vitro and in vivo properties of antimicrobial-, silver- and iodine-based implants, as well as to present novel approaches to surface modifications of orthopedic implants. Methods: A systematic computer-based review on the development of these implants, on PubMed and Web of Science databases, was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results: Overall, 31 in vitro and 40 in vivo entries were evaluated. Regarding the in vitro studies, antimicrobial-based coatings were assessed in 12 entries, silver-based coatings in 10, iodine-based in 1, and novel-applied coating technologies in 8 entries. Regarding the in vivo studies, antimicrobial coatings were evaluated in 23 entries, silver-coated implants in 12, and iodine-coated in 1 entry, respectively. The application of novel coatings was studied in the rest of the cases (4). Antimicrobial efficacy was examined using different bacterial strains, and osseointegration ability and biocompatibility were examined in eukaryotic cells and different animal models, including rats, rabbits, and sheep. Conclusions: Assessment of both in vivo and in vitro studies revealed a wide antimicrobial spectrum of the coated implants, related to reduced bacterial growth, inhibition of biofilm formation, and unaffected or enhanced osseointegration, emphasizing the importance of the application of surface modification techniques as an alternative for the treatment of orthopedic implant infections in the clinical settings.

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.

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.