Circulating tumour cells and PD-L1-positive small extracellular vesicles: the liquid biopsy combination for prognostic information in patients with metastatic non-small cell lung cancer.

BACKGROUND: Circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and extracellular vesicles (EVs) are minimally invasive liquid biopsy biomarkers. This study investigated whether they predict prognosis, alone or in combination, in heterogenous unbiased non-small cell lung cancer (NSCLC) patients. METHODS: Plasma samples of 54 advanced NSCLC patients from a prospective clinical trial. CtDNA mutations were identified using the UltraSEEK™ Lung Panel (MassARRAY® technology). PD-L1 expression was assessed in small EVs (sEVs) using an enzyme-linked immunosorbent assay. RESULTS: At least one ctDNA mutation was detected in 37% of patients. Mutations were not correlated with overall survival (OS) (HR = 1.1, 95% CI = 0.55; 1.83, P = 0.980) and progression-free survival (PFS) (HR = 1.00, 95% CI = 0.57-1.76, P = 0.991). High PD-L1+ sEV concentration was correlated with OS (HR = 1.14, 95% CI = 1.03-1.26, P = 0.016), but not with PFS (HR = 1.08, 95% CI = 0.99-1.18, P = 0.095). The interaction analysis suggested that PD-L1+ sEV correlation with PFS changed in function of CTC presence/absence (P interaction = 0.036). The combination analysis highlighted worse prognosis for patients with CTCs and high PD-L1+ sEV concentration (HR = 7.65, 95% CI = 3.11-18.83, P < 0.001). The mutational statuses of ctDNA and tumour tissue were significantly correlated (P = 0.0001). CONCLUSION: CTCs and high PD-L1+ sEV concentration correlated with PFS and OS, but not ctDNA mutations. Their combined analysis may help to identify patients with worse OS. TRIAL REGISTRATION: NCT02866149, Registered 01 June 2015, https://clinicaltrials.gov/ct2/show/study/NCT02866149 .

Early detection of pancreatic cancer by liquid biopsy "PANLIPSY": a french nation-wide study project.

BACKGROUND: Pancreatic cancer, predominantly characterized by ductal adenocarcinoma (PDAC) accounts for 90% of cases and is the fourth leading cause of cancer-related deaths globally. Its incidence is notably increasing. This poor prognosis is primarily due to late-stage diagnosis (approximately 70% to 80% of patients are diagnosed at an advanced stage), aggressive tumor biology, and low sensitivity to chemotherapy. Consequently, it is crucial to identify and develop a simple, feasible and reproducible blood-based signature (i.e., combination of biomarkers) for early detection of PDAC. METHODS: The PANLIPSY study is a multi-center, non-interventional prospective clinical trial designed to achieve early detection of PDAC with high specificity and sensitivity, using a combinatorial approach in blood samples. These samples are collected from patients with resectable, borderline or locally advanced, and metastatic stage PDAC within the framework of the French Biological and Clinical Database for PDAC cohort (BACAP 2). All partners of the BACAP consortium are eligible to participate. The study will include 215 PDAC patients, plus 25 patients with benign pancreatic conditions from the PAncreatic Disease Cohort of TOuLouse (PACTOL) cohort, and 115 healthy controls, totaling 355 individuals. Circulating biomarkers will be collected in a total volume of 50 mL of blood, divided into one CellSave tube (10 mL), two CELL-FREE DNA BCT® preservative tubes (18 mL), and five EDTA tubes (22 mL in total). Samples preparation will adhere to the guidelines of the European Liquid Biopsy Society (ELBS). A unique feature of the study is the AI-based comparison of these complementary liquid biopsy biomarkers. Main end-points: i) to define a liquid biopsy signature that includes the most relevant circulating biomarkers, ii) to validate the multi-marker panel in an independent cohort of healthy controls and patients, with resectable PDAC, and iii) to establish a unique liquid biopsy biobank for PDAC study. DISCUSSION: The PANLIPSY study is a unique prospective non-interventional clinical trial that brings together liquid biopsy experts. The aim is to develop a biological signature for the early detection of PDAC based on AI-assisted detection of circulating biomarkers in blood samples (CTCs, ctDNA, EVs, circulating immune system, circulating cell-free nucleosomes, proteins, and microbiota). TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT06128343 / NCT05824403. Registration dates: June 8,2023 and April 21, 2023.

Detection of Circulating Tumor Cells in Cerebrospinal Fluid of Patients with Suspected Breast Cancer Leptomeningeal Metastases: A Prospective Study.

BACKGROUND: The diagnosis of breast cancer (BC)-related leptomeningeal metastases (LM) relies on the detection of tumor cells in cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, the sensitivity of this technique is low. Our goal was to evaluate whether circulating tumor cell (CTC) detection in CSF using the CellSearch® system could be used for LM diagnosis. METHODS: This prospective, monocentric study included adult patients with suspected BC-related LM. The clinical sensitivity and specificity of CTC detection in CSF for LM diagnosis were calculated relative to conventional CSF cytology. RESULTS: Forty-nine eligible patients were included and 40 were evaluable (CTC detection technical failure: n = 8, eligibility criteria failure: n = 1). Cytology was positive in 18/40 patients. CTCs were detected in these 18 patients (median: 5824 CTC, range: 93 to 45052) and in 5/22 patients with negative cytology (median: 2 CTC, range: 1 to 44). The detection of ≥1 CSF CTC was associated with a clinical sensitivity of 100% (95% CI, 82.4-100) and a specificity of 77.3% (95% CI, 64.3-90.3) for LM diagnosis. HER2+ CTCs were detected in the CSF of 40.6% of patients with HER2- BC (median: 500 CTC, range: 13 to 28 320). CONCLUSIONS: The clinical sensitivity of CTC detection in CSF with the CellSearch® system for LM diagnosis is higher than that of CSF cytology. CTC detection in patients with negative cytology, however, must be further investigated. The finding of HER2+ CTCs in patients with HER2- BC suggests that the HER2 status of LM should be evaluated to increase the treatment opportunities for these patients.

Selective treatment pressure in colon cancer drives the molecular profile of resistant circulating tumor cell clones.

The characterization of circulating tumor cells (CTCs) holds promises for precision medicine because these cells are an important clinical indicator of treatment efficacy. We established the first and still only nine permanent colon CTC lines from peripheral blood samples of a patient with metastatic colon cancer collected at different time points during treatment and cancer progression. The study objectives were (i) to compare the gene expression profiles of these CTC lines, and (ii) to determine the main features acquired during treatment. The number of upregulated genes was higher in the CTC lines obtained after treatment, indicating that they acquired properties to escape treatment pressure. Among these upregulated genes, some are involved in the mTOR and PI3K/AKT signaling pathways. Moreover, cytidine deaminase expression was significantly increased in the CTC lines obtained after failure of the first- and second-line 5-fluorouracile-based treatments, suggesting that these CTCs can eliminate this specific drug and resist to therapy. Several enzymes involved in xenobiotic metabolism also were upregulated after treatment, suggesting the activation of detoxification mechanisms in response to chemotherapy. Finally, the significant higher expression of aldolase B in four of the six CTC lines obtained after treatment withdrawal and cancer progression indicated that these clones originated from liver metastases. In conclusion, these CTC lines generated at different time points during treatment of metastatic colon cancer in a single patient are characterized by the deregulation of different genes that promote (i) drug resistance, (ii) xenobiotic and energy metabolism, and (iii) stem cell properties and plasticity.

Programmed Cell Death Ligand 1-Expressing Circulating Tumor Cells: A New Prognostic Biomarker in Non-Small Cell Lung Cancer.

BACKGROUND: In non-small cell lung cancer (NSCLC), analysis of programmed cell death ligand 1 (PD-L1) expression in circulating tumor cells (CTCs) is a potential alternative to overcome the problems linked to the tumor biopsy spatiotemporal heterogeneity. However, the prognostic significance of PD-L1-positive [PD-L1(+)] CTCs remains controversial. METHODS: We prospectively evaluated the correlation with clinicopathological variables and prognostic value of PD-L1(+) CTCs, detected with the FDA-cleared CellSearch® system, in 54 patients with advanced NSCLC. RESULTS: We detected CTCs and PD-L1(+) CTCs in 43.4% and 9.4% of patients with NSCLC. PD-L1 expression concordance between tumor tissue and CTCs was low (54%). The presence of PD-L1(+) CTC correlated with the absence of gene alterations in tumor tissue and with poor prognosis-related biological variables (anemia, hyponatremia, increased lactate dehydrogenase). In univariate analysis, absence of gene alterations, number of metastatic sites, prior systemic therapies, and presence of CTCs and PD-L1(+) CTCs were associated with worse overall survival, whereas PD-L1 expression in tumor tissue was not. In multivariate analysis, squamous cell carcinoma histology, number of prior systemic treatments, and the presence of CTC were significantly associated with overall survival. Survival was worse in patients with PD-L1(+) CTCs than in patients with PD-L1-negative CTC or without any CTC. CONCLUSIONS: Our study suggests that the presence of PD-L1(+) CTCs is associated with poor prognosis in patients with advanced NSCLC. Studies with larger samples are needed to confirm our results and to determine how PD-L1(+) CTC detection could help to predict the response or resistance to anti-PD-1/PD-L1 therapies.Clinical trial registration NCT02866149.

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

BACKGROUND: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. METHODS: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. RESULTS: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. CONCLUSIONS: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Tumor-Associated Release of Prostatic Cells into the Blood after Transrectal Ultrasound-Guided Biopsy in Patients with Histologically Confirmed Prostate Cancer.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS) is a standard procedure for prostate cancer diagnosis. Because prostate cancer is a multifocal disease in many patients, multiple sampling (n ≥ 10) is required, which may bear the risk of systemic spread of cancer cells. DESIGN: Using the standardized CellSearch® system that allows for the detection of single epithelial cell adhesion molecule-positive circulating tumor cells (CTCs) in blood, we investigated whether prostate biopsy is associated with release of prostatic tumor cells into the circulation. Peripheral blood was obtained before and within 30 min after performing prostate biopsy from 115 men with increased serum prostate-specific antigen. RESULTS: The number of CTCs significantly increased after biopsy in men with histologically confirmed prostate cancer (odds ratio, 7.8; 95% CI, 4.8-12.8), whereas no biopsy-related changes could be detected in men without confirmed prostate cancer. Multivariable analysis showed that biopsy-related increase of CTCs was significantly correlated with a worse progression-free survival (hazard ratio, 12.4; 95% CI, 3.2-48.6) within the median follow-up of 41 months. CONCLUSIONS: Prostate biopsies may lead to a tumor-associated release of CTCs into the blood circulation. Larger confirmatory trials with longer follow-up periods are required before any change in clinical practice can be recommended.

Clinical Correlations of Programmed Cell Death Ligand 1 Status in Liquid and Standard Biopsies in Breast Cancer.

BACKGROUND: Data regarding the prognostic value of programmed cell death ligand 1 (PD-L1) expression on circulating tumor cells (CTCs) are lacking. However, CTCs could represent an alternative approach to serial biopsies, allowing real-time monitoring of cancer phenotype. METHODS: We evaluated, in a dedicated prospective clinical trial, the clinicopathological correlations and prognostic value of PD-L1(+)-CTCs in 72 patients with metastatic breast cancer (MBC). RESULTS: Eighteen of 56 patients with available archival tissue presented at least one positive (≥1%) PD-L1 tumor sample. Baseline CTCs and PD-L1(+)-CTCs were detected in 57 (79.2%) and 26 (36.1%) patients. No significant correlation was found between PD-L1 tumors and CTC expression. In univariate analysis, triple negative (TN) phenotype, number of metastatic treatments, >2 metastatic sites, ≥5 CTCs and PD-L1(+)-CTCs were significantly associated with progression-free survival, while tissue PD-L1 expression was not. In multivariate analysis, TN phenotype, number of metastatic treatments and of metastatic sites were the only 3 variables independently associated with progression-free survival. Progesterone receptor negativity, TN phenotype, >2 metastatic sites and ≥5 CTCs were significantly associated with overall survival in univariate analysis. In multivariable analysis, TN phenotype and >2 metastatic sites were the only 2 independent variables. CONCLUSIONS: Unlike PD-L1(+)-tumor, PD-L1(+)-CTCs correlate to survival in MBC. Reappraisal of the role of PD-L1 expression by tumor tissue and by CTCs under anti-PD-1/PD-L1 treatment is necessary to evaluate its predictive value and potential role as a stratifying factor in strategies and trials for MBC patients with MBC. CLINICAL TRIAL REGISTRATION: NCT02866149.

Circulating Tumor Cells as a Prognostic Factor in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma: The CIRCUTEC Prospective Study.

BACKGROUND: This prospective multicenter study evaluated the prognostic value of circulating tumor cells (CTCs) in relapsing nonoperable or metastatic head and neck squamous cell carcinoma (rHNSCC) treated by chemotherapy and cetuximab. METHODS: In 65 patients suitable for analyses, peripheral blood was taken at day 0 (D0) D7, and D21 of treatment for CTC detection by CellSearch®, EPISPOT, and flow cytometry (FCM). Progression-free survival (PFS) was assessed with the Kaplan-Meier method and compared with the log-rank test (P < 0.05). RESULTS: At D0, CTCs were detected with EPISPOT, CellSearch, and FCM in 69% (45/65), 21% (12/58), and 11% (7/61) of patients, respectively. In the patients tested with all 3 methods, EPISPOT identified 92% (36/39), 92% (35/38), and 90% (25/28) of all positive samples at D0, D7, and D21, respectively. Median PFS time was significantly lower in (a) patients with increasing or stable CTC counts (36/54) from D0 to D7 with EPISPOTEGFR (3.9 vs 6.2 months; 95% CI, 5.0-6.9; P = 0.0103) and (b) patients with ≥1 CTC detected with EPISPOT or CellSearch® (37/51) (P = 0.0311), EPISPOT or FCM (38/54) (P = 0.0480), and CellSearch or FCM (11/51) (P = 0.0005) at D7. CONCLUSIONS: CTCs can be detected before and during chemotherapy in patients with rHNSCC. D0-D7 CTC kinetics evaluated with EPISPOTEGFR are associated with the response to treatment. This study indicates that CTCs can be used as a real-time liquid biopsy to monitor the early response to chemotherapy in rHNSCC. CLINICALTRIALSGOV IDENTIFIER: NCT02119559.

Molecular Portrait of Metastasis-Competent Circulating Tumor Cells in Colon Cancer Reveals the Crucial Role of Genes Regulating Energy Metabolism and DNA Repair.

BACKGROUND: Unraveling the molecular mechanisms that regulate the biology of metastasis-competent circulating tumor cells (CTCs) is urgently needed to understand metastasis formation and tumor relapse. Our group previously established the first cell line (CTC-MCC-41) derived from metastasis-competent CTCs of a patient with colon cancer. METHODS: In this study, we analyzed the transcriptome of CTC-MCC-41 cells using Human Genome U133 Plus 2.0 microarrays with the aim of unraveling the molecular basis of their special features (stem cell properties and ability to initiate and support metastasis formation). RESULTS: Comparison of the transcriptome data of metastasis-competent CTC-MCC-41 cells and of HT-29 cells (derived from a primary colon cancer) highlights the differential expression of genes that regulate energy metabolism [peroxisome proliferator-activated receptor γ coactivator 1A (PPARGC1A), peroxisome proliferator-activated receptor γ coactivator 1B (PPARGC1B), fatty acid binding protein 1 (FABP1), aldehyde dehydrogenase 3 family member A1 (ALDH3A1)], DNA repair [BRCA1 interacting protein C-terminal helicase 1 (BRIP1), Fanconi anemia complementation group B (FANCB), Fanconi anemia complementation group M (FANCM)], and stemness [glutaminase 2 (GLS2), cystathionine-beta-synthase (CBS), and cystathionine gamma-lyase (CTH)]. The differential expression of 20 genes was validated by quantitative reverse transcription PCR. CONCLUSIONS: This study gives a comprehensive outlook on the molecular events involved in colon cancer progression and provides potential CTC biomarkers that may help develop new therapies to specifically target CTCs with stem cell properties that cause metastases and tumor relapse in patients with colon cancer.

Prognostic relevance of viable circulating tumor cells detected by EPISPOT in metastatic breast cancer patients.

BACKGROUND: Detection of circulating tumor cells (CTC) in breast cancer patients is currently performed in many clinical trials, using different technologies, in particular the EpCAM-dependent CellSearch® system. The purpose of this study was to investigate the incidence and prognostic relevance of viable CTC in a large cohort of metastatic breast cancer (MBC) patients. METHODS: A total of 254 MBC patients were enrolled in a prospective multicenter study at first diagnosis of metastatic disease or disease progression (before the start of a new treatment regimen). After EpCAM-independent enrichment, viable CTC releasing cytokeratin-19 as an epithelial cell marker were detected in the peripheral blood by an EPISPOT assay, and the Food and Drug Administration cleared CellSearch was used as the reference method. RESULTS: Using the EPISPOT assay, CTC were detected in 59% of MBC patients. The overall survival (OS) was linked with the CTC status measured by EPISPOT (P = 0.0191), which allowed stratification of MBC patients in low- and high-risk groups. This stratification could be improved by addition of the CTC status assessed by the CellSearch system. In multivariate Cox proportional-hazards regression analysis, the 3 methods used to determine the level of CTC (EPISPOT, CellSearch, and combination of EPISPOT/CellSearch) were compared by the Bayesian information criterion method. Interestingly, the combination of the EPISPOT and CellSearch assays was the strongest predictor of OS (hazard ratio, 22.6; 95% CI, 2.8-184.08). CONCLUSIONS: This is the first study in which CTC detection using the EPISPOT assay was evaluated on a large cohort of MBC patients, showing prognostic relevance of the presence of viable CTC.

Liquid biopsy: paving a new avenue for cancer research.

The current constraints associated with cancer diagnosis and molecular profiling, which rely on invasive tissue biopsies or clinical imaging, have spurred the emergence of the liquid biopsy field. Liquid biopsy involves the extraction of circulating tumor cells (CTCs), circulating free or circulating tumor DNA (cfDNA or ctDNA), circulating cell-free RNA (cfRNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs) from bodily fluid samples. Subsequently, these components undergo molecular characterization to identify biomarkers that are critical for early cancer detection, prognosis, therapeutic assessment, and post-treatment monitoring. These innovative biosources exhibit characteristics analogous to those of the primary tumor from which they originate or interact. This review comprehensively explores the diverse technologies and methodologies employed for processing these biosources, along with their principal clinical applications.

Analysis of the Plasticity of Circulating Tumor Cells Reveals Differentially Regulated Kinases During the Suspension-to-Adherent Transition.

BACKGROUND: Research on circulating tumor cells (CTCs) offers the opportunity to better understand the initial steps of blood-borne metastasis as main cause of cancer-related deaths. Here, we have used the colon cancer CTC-MCC-41 and breast cancer CTC-ITB-01 lines, which were both established from human CTCs as permanent cell lines as models to further study CTC biology with special emphasis on anchorage-independent survival and growth. METHODS AND RESULTS: Both cell lines showed a marked intrinsic plasticity to switch between suspension and adherent in vitro growth, in 2D adherent culture conditions, and established an equilibrium of both growth patterns with predominant adherent cells in the CTC-MCC-41 line (77%) and suspension cells in the CTC-ITB-01 line (85%). Western blot analysis revealed a higher expression of pERK1/2 in CTC-ITB-01 adherent cells compared to the suspension counterpart that suggested the involvement of kinases in this process. Subsequent functional kinome profiling identified several serine/threonine as well as tyrosine kinases that were differentially regulated in adherent and suspension CTCs. In the adherent cells of the breast cancer line CTC-ITB-01 the activity of MSK1, Src family kinases and the PKG family was increased compared to the suspension counterpart. In adherent cells of the colorectal CTC-MCC-41 line, an increased activity of TYRO3 and JAK2 was detected, whereas p38 MAPK was strongly impaired in the suspension CTC-MCC-41 cells. Some of the regulated kinases, which include the Src family, TYRO3, MSK1, JAK2 and p38 MAPK, have been associated with crucial cellular processes including proliferation, migration and dormancy in the past. CONCLUSIONS: The investigated CTC lines exhibit a high plasticity, similar to the concept of 'adherent-to-suspension transition (AST)' that was recently suggested as a new hallmark of tumor biology by Huh et al. Moreover, we identified differentially regulated kinome profiles that may represent potential targets for future studies on therapeutic interventions.

First evidence of AXL expression on circulating tumor cells in metastatic breast cancer patients: A proof-of-concept study.

BACKGROUND: For several years, the AXL tyrosine kinase receptor, a member of the Tyro3-Axl-Mer (TAM) family, has been considered a new strategic target in oncology. AXL overexpression is common in solid tumors and is associated with poor prognosis. In this context, the detection of a subset of circulating tumor cells (CTCs) that express AXL (AXL+ CTCs) could be clinically relevant. METHODS: Immunostaining was performed to assess AXL expression in human breast cancer cell lines. The optimal conditions were established using flow cytometry. Spiking experiments were carried out to optimize the parameters of the CellSearch® system detection test. CTC enumeration and AXL expression were evaluated in patients with metastatic breast cancer (mBC) before treatment initiation. RESULTS: An innovative AXL+ CTC detection assay to be used with the CellSearch® system was developed. In a prospective longitudinal clinical trial, blood samples from 60 patients with untreated mBC were analyzed to detect AXL+ CTCs with this new assay. CTCs were detected in 35/60 patients (58.3%) and AXL+ CTCs were identified in 7 of these 35 patients (11.7% of all patients). CONCLUSION: This newly established AXL+ CTC assay is a promising tool that can be used for liquid biopsy in future clinical trials to stratify and monitor patients with cancer receiving anti-AXL therapies.

Circulating Tumor Cell Detection by Liquid Biopsy during Early-Stage Endometrial Cancer Surgery: A Pilot Study.

The recurrence of non-metastatic endometrial carcinoma (EC) (6 to 21%) might be due to disseminated tumor cells. This feasibility study investigated whether circulating tumor cells (CTCs) were detectable in blood samples from the peripheral and ovarian veins of 10 patients undergoing laparoscopic resection of stage I-II EC between July 2019 and September 2021. CTCs were detected using the CellSearch® system (i) preoperatively (T0) in peripheral blood, (ii) after ovary suspensory ligament pediculation in ovarian vein blood (T1), and (iii) before colpotomy in peripheral blood (T2). CTCs were detected only in ovarian vein samples in 8/10 patients. The CTC median number did not differ with patient age (37 (min-max: 0-91) in <70-year-old vs. 11 (0-65) in ≥70 year-old women, p = 0.59), tumor grade (15 (0-72) for grade 1 vs. 15 (0-91) for grade 2, p = 0.97), FIGO stage (72 (27-91) vs. 2 (0-65) vs. 3 (0-6]) for stage IA, B, and II, respectively; p = 0.08), and tumor size (40 (2-72) for size < 30 mm vs. 4 (0-91) for size ≥ 30 mm, p = 0.39). Estrogen receptor-positive CTCs and CTC clusters were identified. The prognostic and therapeutic values of CTCs released during EC surgery need to be determined.

Epigenomic reprogramming of therapy-resistant circulating tumor cells in colon cancer.

Therapy resistance is a major challenge in colorectal cancer management. Epigenetic changes, such as DNA methylation, in tumor cells are involved in the development of acquired resistance during treatment. Here, we characterized the DNA methylation landscape of colon circulating tumor cells (CTCs) during cancer progression and therapy resistance development. To this aim, we used nine permanent CTC lines that were derived from peripheral blood samples of a patient with metastatic colon cancer collected before treatment initiation (CTC-MCC-41) and during treatment and cancer progression (CTC-MCC-41.4 and CTC-MCC-41.5 [A-G]). We analyzed the DNA methylome of these nine CTC lines using EPIC arrays and also assessed the association between DNA methylation and gene expression profiles. We confirmed DNA methylation and gene expression results by pyrosequencing and RT-qPCR, respectively. The global DNA methylation profiles were different in the pre-treatment CTC line and in CTC lines derived during therapy resistance development. These resistant CTC lines were characterized by a more hypomethylated profile compared with the pre-treatment CTC line. Most of the observed DNA methylation differences were localized at CpG-poor regions and some in CpG islands, shore regions and promoters. We identified a distinctive DNA methylation signature that clearly differentiated the pre-treatment CTC line from the others. Of note, the genes involved in this signature were associated with cancer-relevant pathways, including PI3K/AKT, MAPK, Wnt signaling and metabolism. We identified several epigenetically deregulated genes associated with therapy resistance in CTCs, such as AP2M1. Our results bring new knowledge on the epigenomic landscape of therapy-resistant CTCs, providing novel mechanisms of resistance as well as potential biomarkers and therapeutic targets for advanced CRC management.

Inhibition of Wnt Signaling in Colon Cancer Cells via an Oral Drug that Facilitates TNIK Degradation.

We have synthesized an oxetane derivative of the benzimidazole compound mebendazole (OBD9) with enhanced solubility and strong anticancer activity in multiple types of cancer cells, especially colorectal cancer. In this report, we provide evidence that OBD9 suppresses colorectal cancer growth by interfering with the Wnt signaling pathway, a main driver of cell growth in colorectal cancer. Specifically, we find that OBD9 induces autophagic degradation of TNIK (traf2 and Nck-interacting kinase), which promotes T-cell factor-4 (TCF4)/beta-catenin-mediated gene expression. Thus, OBD9 as a TNIK inhibitor blocks Wnt/beta-catenin signaling at the final step of transcriptional activation. We suggest that OBD9 provides a potential novel autophagy-mediated, Wnt-damping therapeutic strategy for the treatment of colorectal cancer.

In vitro cross-talk between metastasis-competent circulating tumor cells and platelets in colon cancer: a malicious association during the harsh journey in the blood.

Background: Platelets are active players in hemostasis, coagulation and also tumorigenesis. The cross-talk between platelets and circulating tumor cells (CTCs) may have various pro-cancer effects, including promoting tumor growth, epithelial-mesenchymal transition (EMT), metastatic cell survival, adhesion, arrest and also pre-metastatic niche and metastasis formation. Interaction with CTCs might alter the platelet transcriptome. However, as CTCs are rare events, the cross-talk between CTCs and platelets is poorly understood. Here, we used our established colon CTC lines to investigate the colon CTC-platelet cross-talk in vitro and its impact on the behavior/phenotype of both cell types. Methods: We exposed platelets isolated from healthy donors to thrombin (positive control) or to conditioned medium from three CTC lines from one patient with colon cancer and then we monitored the morphological and protein expression changes by microscopy and flow cytometry. We then analyzed the transcriptome by RNA-sequencing of platelets indirectly (presence of a Transwell insert) co-cultured with the three CTC lines. We also quantified by reverse transcription-quantitative PCR the expression of genes related to EMT and cancer development in CTCs after direct co-culture (no Transwell insert) with platelets. Results: We observed morphological and transcriptomic changes in platelets upon exposure to CTC conditioned medium and indirect co-culture (secretome). Moreover, the expression levels of genes involved in EMT (p < 0.05) were decreased in CTCs co-cultured with platelets, but not of genes encoding mesenchymal markers (FN1 and SNAI2). The expression levels of genes involved in cancer invasiveness (MYC, VEGFB, IL33, PTGS2, and PTGER2) were increased. Conclusion: For the first time, we studied the CTC-platelet cross-talk using our unique colon CTC lines. Incubation with CTC conditioned medium led to platelet aggregation and activation, supporting the hypothesis that their interaction may contribute to preserve CTC integrity during their journey in the bloodstream. Moreover, co-culture with platelets influenced the expression of several genes involved in invasiveness and EMT maintenance in CTCs.

Epigenomic analysis reveals a unique DNA methylation program of metastasis-competent circulating tumor cells in colorectal cancer.

Circulating tumor cells (CTCs) and epigenetic alterations are involved in the development of metastasis from solid tumors, such as colorectal cancer (CRC). The aim of this study was to characterize the DNA methylation profile of metastasis-competent CTCs in CRC. The DNA methylome of the human CRC-derived cell line CTC-MCC-41 was analyzed and compared with primary (HT29, Caco2, HCT116, RKO) and metastatic (SW620 and COLO205) CRC cells. The association between methylation and the transcriptional profile of CTC-MCC-41 was also evaluated. Differentially methylated CpGs were validated with pyrosequencing and qMSP. Compared to primary and metastatic CRC cells, the methylation profile of CTC-MCC-41 was globally different and characterized by a slight predominance of hypomethylated CpGs mainly distributed in CpG-poor regions. Promoter CpG islands and shore regions of CTC-MCC-41 displayed a unique methylation profile that was associated with the transcriptional program and relevant cancer pathways, mainly Wnt signaling. The epigenetic regulation of relevant genes in CTC-MCC-41 was validated. This study provides new insights into the epigenomic landscape of metastasis-competent CTCs, revealing biological information for metastasis development, as well as new potential biomarkers and therapeutic targets for CRC patients.

Epithelial-to-Mesenchymal Plasticity in Circulating Tumor Cell Lines Sequentially Derived from a Patient with Colorectal Cancer.

Metastasis is a complicated and only partially understood multi-step process of cancer progression. A subset of cancer cells that can leave the primary tumor, intravasate, and circulate to reach distant organs are called circulating tumor cells (CTCs). Multiple lines of evidence suggest that in metastatic cancer cells, epithelial and mesenchymal markers are co-expressed to facilitate the cells' ability to go back and forth between cellular states. This feature is called epithelial-to-mesenchymal plasticity (EMP). CTCs represent a unique source to understand the EMP features in metastatic cascade biology. Our group previously established and characterized nine serial CTC lines from a patient with metastatic colon cancer. Here, we assessed the expression of markers involved in epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transition in these unique CTC lines, to define their EMP profile. We found that the oncogenes MYC and ezrin were expressed by all CTC lines, but not SIX1, one of their common regulators (also an EMT inducer). Moreover, the MET activator GRHL2 and its putative targets were strongly expressed in all CTC lines, revealing their plasticity in favor of an increased MET state that promotes metastasis formation.