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The increased metastatic ability of small-cell lung cancer (SCLC) necessitates the identification of new prognostic biomarkers for clinical evaluation during the disease course. Our previous research highlighted the clinical relevance of transcription factor JunB (JUNB), C-X-C chemokine receptor type 4 (CXCR4), and programmed cell death 1 ligand 1 (PD-L1) in breast and non-small cell lung cancer (NSCLC) patients. In the current study, we examined these biomarkers in circulating tumor cells (CTCs) and plasma-derived exosomes from 100 treatment-naïve SCLC patients. CTCs were analyzed using the VyCAP system, whereas exosomes were characterized molecularly and transcriptomically. JUNB, CXCR4, and PD-L1 were highly prevalent in CTCs. Patients exhibited significantly increased protein exosomal expression of JUNB and CXCR4 compared to healthy individuals. Overexpression of JUNB and CXCR4 in exosomes can distinguish patients from normal donors, offering an interesting tool for early diagnosis. The presence of JUNB and/or CXCR4 in CTCs correlated with significantly poorer overall survival. CXCR4 exosomal overexpression was associated with CTC presence and their phenotypes. Conclusively, a comprehensive analysis of CTCs and exosomes provides useful prognostic and potential diagnostic tools for SCLC patients., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Gastric cancer (GC) is an asymptomatic malignancy in early stages, with an invasive and cost-ineffective diagnostic toolbox that contributes to severe global mortality rates on an annual basis. Ectopic expression of the lineage survival transcription factors (LS-TFs) GATA4 and 6 promotes stomach oncogenesis. However, LS-TFs also govern important physiological roles, hindering their direct therapeutic targeting. Therefore, their downstream target genes are particularly interesting for developing cancer-specific molecular biomarkers or therapeutic agents. In this work, we couple inducible knockdown systems with chromatin immunoprecipitation and RNA-seq to thoroughly detect and characterize direct targets of GATA-mediated transcriptional regulation in gastric cancer cells. Our experimental and computational strategy provides evidence that both factors regulate the expression of several coding and non-coding RNAs that in turn mediate for their cancer-promoting phenotypes, including but not limited to cell cycle, apoptosis, ferroptosis, and oxidative stress response. Finally, the diagnostic and prognostic potential of four metagene signatures consisting of selected GATA4/6 target transcripts is evaluated in a multi-cancer panel of ~7000 biopsies from nineteen tumor types, revealing elevated specificity for gastrointestinal tumors. In conclusion, our integrated strategy uncovers the landscape of GATA-mediated coding and non-coding transcriptional regulation, providing insights regarding their molecular and clinical function in gastric cancer.

T-cell-mediated anti-tumoral responses may have significant clinical relevance as a biomarker for response to immunotherapy. The value of peripheral blood pre-existing tumor antigen-specific T cells (PreI + ) as a predictive immunotherapy biomarker in NSCLC patients was investigated, along with the frequency of various circulating immune cells. Fifty-two treatment-naïve, stage III/IV NSCLC patients, treated with front-line immune checkpoint inhibitors (ICI)-containing regimens were enrolled. PreI was calculated as the percentages of CD3 + IFNγ + cells after in vitro co-cultures of PBMCs with peptides against four different Tumor-Associated Antigens (TAA). Immunophenotyping of peripheral blood immune cells was performed using multicolor flow cytometry. PreI + T cells were detected in 44% of patients. Median overall survival (OS) was significantly higher in PreI + patients compared to PreI - patients (not reached vs. 321 days, respectively; p = 0.014). PreI + patients had significantly higher numbers of possible exhausted CD3 + CD8 + PD-1 + cells and lower percentages of immunosuppressive Tregs compared to PreI - patients. Additionally, patients with PreI + and low numbers of peripheral blood M-MDSCs had a significant survival advantage compared to the rest of the patients. Thus, combining pre-existing tumor antigen-specific immunity before initiation of ICI in NSCLC patients with selected immune-suppressive cells could identify patients who have a favorable clinical outcome when treated with ICI-containing regimens.

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.

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.

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.

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.

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 Hippo pathway regulates and contributes to several hallmarks of prostate cancer (PCa). Although the elucidation of YAP function in PCa is in its infancy, emerging studies have shed light on the role of aberrant Hippo pathway signaling in PCa development and progression. YAP overexpression and nuclear localization has been linked to poor prognosis and resistance to treatment, highlighting a therapeutic potential that may suggest innovative strategies to treat cancer. This review aimed to summarize available data on the biological function of the dysregulated Hippo pathway in PCa and identify knowledge gaps that need to be addressed for optimizing the development of YAP-targeted treatment strategies in patients likely to benefit.

Several lines of research are being investigated to better understand mechanisms implicated in response or resistance to immune checkpoint blockade in prostate cancer (PCa). Myeloid-derived suppressor cells (MDSCs) have emerged as a major mediator of immunosuppression in the tumor microenvironment that promotes progression of various tumor types. The main mechanisms underlying MDSC-induced immunosuppression are currently being explored and strategies to enhance anti-tumor immune response via MDSC targeting are being tested. However, the role of MDSCs in PCa remains elusive. In this review, we aim to summarize and present the state-of-the-art knowledge on current methodologies to phenotypically and metabolically characterize MDSCs in PCa. We describe how these characteristics may be linked with MDSC function and may influence the clinical outcomes of patients with PCa. Finally, we briefly discuss emerging strategies being employed to therapeutically target MDSCs and potentiate the long-overdue improvement in the efficacy of immunotherapy in patients with PCa.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8 + T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8 + T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity., Competing Interests: Declaration of Interests T.R.B. is a cofounder and SAB member of Haplogen GmbH and a cofounder and director of Scenic Biotech BV., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For hundreds of thousands of years, the human genome has extensively evolved, resulting in genetic variations in almost every gene. Immunological reflections of these genetic variations become clearly visible after an allogeneic stem cell transplantation (allo-SCT) as minor Histocompatibility (H) antigens. Minor H antigens are peptides cleaved from genetically encoded variable protein regions after which they are presented at the cell surface by HLA molecules. After allo-SCT with minor H antigen mismatches between donor and recipient, donor T cells recognize the minor H antigens of the recipient as foreign, evoking strong alloreactive immune responses. Studies in the late eighties have discovered that a subset of minor H antigens are encoded by hematopoietic system-specific genes. After allo-SCT, this subset is strictly expressed on the hematopoietic malignant cells and was therefore the first well-defined highly immunogenic group of tumor-specific antigens. In the last decade, neoantigens derived from genetic mutations in tumors have been identified as another group of immunogenic tumor-specific antigens. Therefore, hematopoietic minor H antigens and neoantigens are therapeutic equivalents. This review will connect our current knowledge about the immune biology and identification of minor H antigens and neoantigens leading to novel conclusions on their prediction., (Copyright © 2020 Mutis, Xagara and Spaapen.)