Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression.

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient's survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

The role of galectins in mediating the adhesion of circulating cells to vascular endothelium.

Vascular cell adhesion is a complex orchestration of events that commonly feature lectin-ligand interactions between circulating cells, such as immune, stem, and tumor cells, and endothelial cells (ECs) lining post-capillary venules. Characteristically, circulating cell adherence to the vasculature endothelium is initiated through interactions between surface sialo-fucosylated glycoprotein ligands and lectins, specifically platelet (P)- or endothelial (E)-selectin on ECs or between leukocyte (L)-selectin on circulating leukocytes and L-selectin ligands on ECs, culminating in circulating cell extravasation. This lectin-ligand interplay enables the migration of immune cells into specific tissue sites to help maintain effective immunosurveillance and inflammation control, the homing of stem cells to bone marrow or tissues in need of repair, and, unfortunately, in some cases, the dissemination of circulating tumor cells (CTCs) to distant metastatic sites. Interestingly, there is a growing body of evidence showing that the family of ß-galactoside-binding lectins, known as galectins, can also play pivotal roles in the adhesion of circulating cells to the vascular endothelium. In this review, we present contemporary knowledge on the significant roles of host- and/or tumor-derived galectin (Gal)-3, -8, and -9 in facilitating the adhesion of circulating cells to the vascular endothelium either directly by acting as bridging molecules or indirectly by triggering signaling pathways to express adhesion molecules on ECs. We also explore strategies for interfering with galectin-mediated adhesion to attenuate inflammation or hinder the metastatic seeding of CTCs, which are often rich in galectins and/or their glycan ligands.

Circulating tumor cells are a good predictor of tumor recurrence in clinical patients with gastric cancer.

Circulating tumor cells (CTCs) as a liquid biopsy have great potential in clinical applications and basic cancer research, but their clinical use in gastric cancer remains unclear. This study investigated whether CTCs could be used as a potential prognosis predictor in patients with gastric cancer. A total of 120 patients with pathologically confirmed gastric cancer were enrolled from January 1, 2015, to December 1, 2019. All patients were initially diagnosed without previous treatment, and then the number of CTCs was detected using the NEimFISH method before radical surgical resection. Regular follow-up was performed in all patients, and the correlations between the number of CTCs and clinical endpoints, such as disease-free survival (DFS) and overall survival (OS), were evaluated. The univariate and multivariate hazard ratios were calculated using the Cox proportional hazard model. Based on the number of CTCs, we defined CTCs ≥ 2 per 7.5 mL of whole blood as the positive group and CTCs < 2 as the negative group. Among the 120 patients who underwent CTC detection before surgery, the rate of CTC-positive patients was 64.17% (77/120) of which stage I and II patients accounted for 22.50% and stage III patients accounted for 41.67% (P = 0.014). By detecting CTCs before surgery and at the time of recurrence, the number of CTCs tends to increase concomitantly with disease progression (median: 2 VS 5 per 7.5 mL). Multivariate analysis showed that age (HR, 0.259; 95% CI, 0.101-0.662; P = 0.005), D-dimer (HR, 3.146; 95% CI, 1.169-8.461; P = 0.023), and lymph node metastasis (HR, 0.207; 95% CI, 0.0071-0.603; P = 0.004) were factors correlated with CTCs. In addition, the median follow-up of all the patients was 38.0 months (range of 28-80 months); the DFS in CTC-positive patients was significantly shorter than that of the CTC-negative patients, and a significant difference was found based on the Cox proportional hazard regression model analysis (44.52 ± 2.83 m vs. 74.99 ± 2.78 m, HR = 4.550, P = 0.018). The OS was shorter in the CTC-positive group than in the CTC-negative group before the operation, but the result was not significant based on the Cox proportional hazard regression model analysis (47.58 ± 2.46 m vs. 70.68 ± 3.53 m, HR = 2.261, P = 0.083). The number of CTCs tends to increase concomitantly with disease progression. In addition, the detection of CTCs was an independent predictor of shorter DFS in gastric cancer. However, the relationship between CTCs and OS needs to be determined in future studies.

High-throughput viable circulating tumor cell isolation using tapered-slit membrane filter-based chipsets in the differential diagnosis of ovarian tumors.

OBJECTIVE: To evaluate the diagnostic performance of circulating tumor cells (CTCs) using tapered-slit membrane filter (TSF)-based chipsets for the differential diagnosis of adnexal tumors. METHODS: A total of 230 women with indeterminate adnexal tumors were prospectively enrolled. The sensitivity, specificity, and accuracy of the CTC-detecting chipsets were analyzed according to postoperative pathological results and compared with those of cancer antigen (CA)-125 and imaging tests. RESULTS: Eighty-one (40.3%) benign tumors, 31 (15.4%) borderline tumors, and 89 (44.3%) ovarian cancers were pathologically confirmed. The sensitivity, specificity, and accuracy of CTC-detecting chipsets (75.3%, 58.0%, and 67.1%) for differentiating ovarian cancer from benign tumors were similar to CA-125 (78.7%, 53.1%, and 66.5%), but lower than CT/MRI (94.2%, 77.9%, and 86.5%). "CTC or CA125" showed increased sensitivity (91.0%) and "CTC and CA-125" revealed increased specificity (77.8%), comparable to CT/MRI. CTC detection rates in stage I/II and stage III/IV ovarian cancers were 69.6% and 81.4%, respectively. The sensitivity to detect high-grade serous (HGS) cancer from benign tumors (84.6%) was higher than that to detect non-HGS cancers (68.0%). CONCLUSION: Although the diagnostic performance of the TSF platform to differentiate between ovarian cancer and benign tumors did not yield significant results, the combination of CTC and CA-125 showed promising potential in the diagnostic accuracy of ovarian cancer.

Layer-by-layer fabrication of alginate/polyethyleneimine multilayer on magnetic interface with enhanced efficiency in immuno-capturing circulating tumor cells.

BACKGROUND: The technology of capturing circulating tumor cells (CTCs) plays a crucial role in the diagnosis, evaluation of therapeutic efficacy, and prediction of prognosis in lung cancer. However, the presence of complex blood environment often results in severe nonspecific protein adsorption and interferences from blood cells, which negatively impacts the specificity of CTCs capture. There is a great need for development of novel nanomaterials for CTCs capture with prominent anti-nonspecific adsorptions from proteins or blood cells. RESULTS: We present a novel immune magnetic probe Fe3O4@(PEI/AA)4@Apt. The surface of Fe3O4 particles was modified with four layers of PEI/AA composite by layer-by-layer assembly. Furthermore, aptamers targeting epithelial marker EpCAM (SYL3C) and mesenchymal marker CSV (ZY5C) were simultaneously connected on Fe3O4@(PEI/AA)4 to improve the detection of different phenotypic CTCs and reduce false negatives. The results demonstrated that the (PEI/AA)4 coatings not only minimized non-specific protein adsorptions, but also significantly reduced the adsorption rate of red blood cells to a mere 1 %, as a result of which, the Fe3O4@(PEI/AA)4@Apt probe achieved a remarkably high capture efficiency toward CTCs (95.9 %). In the subsequent validation of clinical samples, the probe was also effective in capturing rare CTCs from lung cancer patients. SIGNIFICANCE AND NOVELTY: A (PEI/AA) polymerized composite with controllable layers was fabricated by layer-by-layer self-assembly technique, which displayed remarkable anti-nonspecific adsorption capabilities toward proteins and cells. Importantly, Fe3O4@(PEI/AA)4@Apt probe significantly improved CTCs capture purity in lung cancer patients to 89.36 %. For the first time, this study combined controllable (PEI/AA) layers with magnetic separation to innovatively build a resistant interface that significantly improves the specific capture performances of CTCs, broadening the application of this polymerized composite.

Circulating tumor cell detection may offer earlier diagnosis in patients suspected of asbestos-related lung cancer.

Asbestos-Related Lung Cancer (ARLC) presents ongoing diagnostic challenges despite improved imaging technologies. The long latency period, coupled with limited access to occupational and environmental data along with the confounding effects of smoking and other carcinogens adds complexity to the diagnostic process. Compounding these challenges is the absence of a specific histopathologic or mutational signature of ARLC. A correlation between PD-L1 expression and response to immune checkpoint inhibition has not yet been proven. Thus, new biomarkers are needed to allow accurate diagnoses of ARLC, to enable prognostication and to offer personalized treatments. Liquid biopsies, encompassing circulating DNA and circulating tumor cells (CTCs), have gained attention as novel diagnostic methods in lung cancer to screen high-risk populations including those exposed to asbestos. CTCs can be enumerated and molecularly profiled to provide predictive and prognostic information. CTC studies have not been undertaken in populations at risk of ARLC to date. The potential of CTCs to provide real-time molecular insight into ARLC biology may significantly improve the diagnosis and management of ARLC patients.

EpCAM-CD24+ circulating cells associated with poor prognosis in breast cancer patients.

Following the discovery of circulating tumor cells (CTCs) in the peripheral blood of cancer patients, CTCs were initially postulated to hold promise as a valuable prognostic tool through liquid biopsy. However, a decade and a half of accumulated data have revealed significant complexities in the investigation of CTCs. A challenging aspect lies in the reduced expression or complete loss of key epithelial markers during the epithelial-mesenchymal transition (EMT). This likely hampers the identification of a pathogenetically significant subset of CTCs. Nevertheless, there is a growing body of evidence regarding the prognostic value of such molecules as CD24 expressing in the primary breast tumor. Herewith, the exact relevance of CD24 expression on CTCs remains unclear. We used two epithelial markers (EpCAM and cytokeratin 7/8) to assess the count of CTCs in 57 breast cancer patients, both with (M0mts) and without metastasis (M0) during the follow-up period, as well as in M1 breast cancer patients. However, the investigation of these epithelial markers proved ineffective in identifying cell population expressing different combinations of EpCAM and cytokeratin 7/8 with prognostic significance for breast cancer metastases. Surprisingly, we found CD24+ circulating cells (CCs) in peripheral blood of breast cancer patients which have no epithelial markers (EpCAM and cytokeratin 7/8) but was strongly associated with distant metastasis. Namely, the count of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs was elevated in both groups of patients, those with existing metastasis and those who developed metastases during the follow-up period. Simultaneously, an elevation in these cell counts beyond the established threshold of 218.3 cells per 1 mL of blood in patients prior to any treatment predicted a 12-fold risk of metastases, along with a threefold decrease in distant metastasis-free survival over a 90-month follow-up period. The origin of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs remains unclear. In our opinion their existence can be explained by two most probable hypotheses. These cells could exhibit a terminal EMT phenotype, or it might be immature cells originating from the bone marrow. Nonetheless, if this hypothesis holds true, it's worth noting that the mentioned CCs do not align with any of the recognized stages of monocyte or neutrophil maturation, primarily due to the presence of CD45 expression in the myeloid cells. The results suggest the presence in the peripheral blood of patients with metastasis (both during the follow-up period and prior to inclusion in the study) of a cell population with a currently unspecified origin, possibly arising from both myeloid and tumor sources, as confirmed by the presence of aneuploidy.

High-throughput enrichment of portal venous circulating tumor cells for highly sensitive diagnosis of CA19-9-negative pancreatic cancer patients using inertial microfluidics.

The carbohydrate antigen 19-9 (CA19-9) is commonly used as a representative biomarker for pancreatic cancer (PC); however, it lacks sensitivity and specificity for early-stage PC diagnosis. Furthermore, some patients with PC are negative for CA19-9 (<37 U/mL), which introduces additional limitations to their accurate diagnosis and treatment. Hence, improved methods to accurately detect PC stages in CA19-9-negative patients are warranted. In this study, tumor-proximal liquid biopsy and inertial microfluidics were coupled to enable high-throughput enrichment of portal venous circulating tumor cells (CTCs) and support the effective diagnosis of patients with early-stage PC. The proposed inertial microfluidic system was shown to provide size-based enrichment of CTCs using inertial focusing and Dean flow effects in slanted spiral channels. Notably, portal venous blood samples were found to have twice the yield of CTCs (21.4 cells per 5 mL) compared with peripheral blood (10.9 CTCs per 5 mL). A combination of peripheral and portal CTC data along with CA19-9 results showed to greatly improve the average accuracy of CA19-9-negative PC patients from 47.1% with regular CA19-9 tests up to 87.1%. Hence, portal venous CTC-based microfluidic biopsy can be used with high sensitivity and specificity for the diagnosis of early-stage PC, particularly in CA19-9-negative patients.

An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer.

BACKGROUND: Hepatoblastoma and HCC are the most common malignant hepatocellular tumors seen in children. The aim of this study was to develop a liquid biopsy test for circulating tumor cells (CTCs) for these tumors that would be less invasive and provide real-time information about tumor response to therapy. METHODS: For this test, we utilized indocyanine green (ICG), a far-red fluorescent dye used clinically to identify malignant liver cells during surgery. We assessed ICG accumulation in cell lines using fluorescence microscopy and flow cytometry. For our CTC test, we developed a panel of liver tumor-specific markers, including ICG, Glypican-3, and DAPI, and tested it with cell lines and noncancer control blood samples. We then used this panel to analyze whole-blood samples for CTC burden with a cohort of 15 patients with hepatoblastoma and HCC and correlated with patient characteristics and outcomes. RESULTS: We showed that ICG accumulation is specific to liver cancer cells, compared to nonmalignant liver cells, non-liver solid tumor cells, and other nonmalignant cells, and can be used to identify liver tumor cells in a mixed population of cells. Experiments with the ICG/Glypican-3/DAPI panel showed that it specifically tagged malignant liver cells. Using patient samples, we found that CTC burden from sequential blood samples from the same patients mirrored the patients' responses to therapy. CONCLUSIONS: Our novel ICG-based liquid biopsy test for CTCs can be used to specifically detect and quantify CTCs in the blood of pediatric patients with liver cancer.

Investigating Radiotherapy Effects on PD-L1 Expression in Circulating Tumor Cells: An Exploratory Study.

INTRODUCTION: Circulating tumor cells (CTCs) and Programmed death-ligand 1 (PD-L1) play pivotal roles in cancer biology and therapy response. This exploratory study aimed to elucidate the influence of neoadjuvant radiotherapy on PD-L1 expression in tumor tissues and CTCs of patients with inoperable locally advanced breast cancer. METHODS: We conducted a prospective cohort study at Universitas Andalas Hospital Padang from January to December 2022 with 27 patients. Biopsies and blood draws were executed before and after the tenth fractions of neoadjuvant radiotherapy. Following radiotherapy, CTCs were isolated using magnetic beads enrichment, followed by an RT-PCR analysis for PD-L1 expression. Correlations between PD-L1 expression and tumor response, evaluated via local response and RECIST criteria before and after radiotherapy breast CT scan, were examined using Fisher's exact and chi-square tests. RESULTS: Our data revealed no significant alterations in PD-L1 expression in either tumor tissues or CTCs during radiotherapy (p=0.848 for tissue, p=0.548 for CTCs). Notably, PD-L1 expression in tumor tissue before treatment was significantly associated with RECIST (p=0.021), while other correlations with local response and RECIST were not statistically significant. CONCLUSION: The study implies radiotherapy may not significantly influence PD-L1 expression in tumor tissue and CTCs. However, pre-treatment PD-L1 expression in tumor tissue correlates with RECIST criteria. These findings highlight the need for additional, comprehensive studies to elucidate further the interplay between PD-L1, CTCs, and radiotherapy response.

Prognostic relevance of circulating lymphoma cells at diagnosis in newly diagnosed follicular lymphoma patients.

Follicular lymphoma (FL) is the most common indolent B-cell non-Hodgkin lymphoma. Circulating lymphoma (CL) cells can be seen at diagnosis in some FL patients, however, previous studies evaluating this have shown mixed results. Therefore, we sought to evaluate the impact of CL at diagnosis on outcomes in patients with newly diagnosed FL using data from a single center. Patients were divided into CL+ and CL- based on immunophenotyping via peripheral blood (PB) flow cytometry. CL was defined as detectable clonally restricted B-cells that matched the actual or expected B-cell immunophenotype of FL. The primary endpoint was progression-free survival (PFS) after first-line treatment and secondary endpoints included overall response rate (ORR), overall survival (OS), diagnosis to treatment interval (DTI), progression of disease within 2 years of diagnosis (POD24), and cumulative incidence of transformation between the two groups. Among the 541 patients with FL, 204 had PB flow cytometry performed at diagnosis, and after excluding patients not meeting the eligibility criteria, 147 cases remained with 24 (16%) CL+ at diagnosis. Patients in the CL+ group were younger (53 vs. 58 years, p = 0.02), had more extranodal involvement (83% vs. 44%, p < 0.01), follicular lymphoma international prognostic index 3-5 (55% vs. 31%, p = 0.01), and a higher proportion received first-line immunochemotherapy (75% vs. 43%, p = 0.01) compared to the CL-group. The median PFS was not significantly different between CL+ (6.27 years, 95% CI = 3.61-NR) and CL- (6.61 years, 95% CI = 5.10-9.82) cohorts regardless of the first-line treatment or level of absolute PB CL cells. There was no significant difference in ORR, median OS, DTI, POD24, and cumulative incidence of transformation between the two groups. In our study, we found that the presence of CL cells at diagnosis in FL in the contemporary era did not impact outcomes and survival.

Prognostic Significance of Excision Repair Cross-Complementation Group 1 on Circulating Tumor Cells for Nasopharyngeal Carcinoma.

BACKGROUND: Liquid biopsy, including the detection of circulating tumor cells (CTCs), has emerged as a promising tool for cancer diagnosis and monitoring. However, the prognostic value of CTCs in nasopharyngeal carcinoma (NPC) remains unclear due to the lack of phenotypic characterization. The expression of Excision Repair Cross-Complementation Group 1 (ERCC1) and CTCs epithelial-mesenchymal transition (EMT) have been associated with treatment efficacy. In this study, we aimed to evaluate the prognostic significance of ERCC1 expression on CTCs and their EMT subtypes before treatment in NPC. METHODS: We retrospectively analyzed 108 newly diagnosed locally advanced NPC patients who underwent CanPatrol™ CTC testing between November 2018 and November 2021. CTCs were counted and classified into epithelial, epithelial-mesenchymal hybrid, and mesenchymal subtypes. ERCC1 expression was divided into negative and positive groups. Clinical features and survival outcomes were analyzed. RESULTS: The positive rate of CTCs was 92.6% (100/108), with an ERCC1 positivity rate of 74% (74/100). Further analysis of the subtypes showed that positive ERCC1 on mesenchymal CTCs was associated with a later N stage (P = .01). Positive ERCC1 expression was associated with poor overall survival (OS; P = .039) and disease-free survival (DFS; P = .035). Further analysis of subtypes showed that the positive ERCC1 on mesenchymal-type CTCs was associated with poor OS (P = .012) and metastasis-free survival (MFS; P = .001). CONCLUSION: Our findings suggest that ERCC1 expression on CTCs may serve as a new prognostic marker for NPC patients. Evaluating CTCs subtypes may become an auxiliary tool for personalized and precise treatment.

BackgroundLiquid biopsy, including the detection of circulating tumor cells (CTCs), has emerged as a promising tool for cancer diagnosis and monitoring. However, the prognostic value of CTCs in nasopharyngeal carcinoma (NPC) remains unclear due to the lack of phenotypic characterization. The expression of Excision Repair Cross-Complementation Group 1 (ERCC1) and CTCs epithelial-mesenchymal transition (EMT) have been associated with treatment efficacy. In this study, we aimed to evaluate the prognostic significance of ERCC1 expression on CTCs and their EMT subtypes before treatment in NPC.MethodsWe retrospectively analyzed 108 newly diagnosed locally advanced NPC patients who underwent CanPatrol™ CTC testing between November 2018 and November 2021. CTCs were counted and classified into epithelial, epithelial-mesenchymal hybrid, and mesenchymal subtypes. ERCC1 expression was divided into negative and positive groups. Clinical features and survival outcomes were analyzed.ResultsThe positive rate of CTCs was 92.6% (100/108), with an ERCC1 positivity rate of 74% (74/100). Further analysis of the subtypes showed that positive ERCC1 on mesenchymal CTCs was associated with a later N stage (P = .01). Positive ERCC1 expression was associated with poor overall survival (OS; P = .039) and disease-free survival (DFS; P = .035). Further analysis of subtypes showed that the positive ERCC1 on mesenchymal-type CTCs was associated with poor OS (P = .012) and metastasis-free survival (MFS; P = .001).ConclusionOur findings suggest that ERCC1 expression on CTCs may serve as a new prognostic marker for NPC patients. Evaluating CTCs subtypes may become an auxiliary tool for personalized and precise treatment.

Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs. METHODS: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. RESULTS: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. CONCLUSIONS: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.

Heterogeneity and molecular landscape of melanoma: implications for targeted therapy.

Uveal cancer (UM) offers a complex molecular landscape characterized by substantial heterogeneity, both on the genetic and epigenetic levels. This heterogeneity plays a critical position in shaping the behavior and response to therapy for this uncommon ocular malignancy. Targeted treatments with gene-specific therapeutic molecules may prove useful in overcoming radiation resistance, however, the diverse molecular makeups of UM call for a patient-specific approach in therapy procedures. We need to understand the intricate molecular landscape of UM to develop targeted treatments customized to each patient's specific genetic mutations. One of the promising approaches is using liquid biopsies, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), for detecting and monitoring the disease at the early stages. These non-invasive methods can help us identify the most effective treatment strategies for each patient. Single-cellular is a brand-new analysis platform that gives treasured insights into diagnosis, prognosis, and remedy. The incorporation of this data with known clinical and genomics information will give a better understanding of the complicated molecular mechanisms that UM diseases exploit. In this review, we focused on the heterogeneity and molecular panorama of UM, and to achieve this goal, the authors conducted an exhaustive literature evaluation spanning 1998 to 2023, using keywords like "uveal melanoma, "heterogeneity". "Targeted therapies"," "CTCs," and "single-cellular analysis".

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

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.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

Detection of circulating tumor cells by means of machine learning using Smart-Seq2 sequencing.

Circulating tumor cells (CTCs) are tumor cells that separate from the solid tumor and enter the bloodstream, which can cause metastasis. Detection and enumeration of CTCs show promising potential as a predictor for prognosis in cancer patients. Furthermore, single-cells sequencing is a technique that provides genetic information from individual cells and allows to classify them precisely and reliably. Sequencing data typically comprises thousands of gene expression reads per cell, which artificial intelligence algorithms can accurately analyze. This work presents machine-learning-based classifiers that differentiate CTCs from peripheral blood mononuclear cells (PBMCs) based on single cell RNA sequencing data. We developed four tree-based models and we trained and tested them on a dataset consisting of Smart-Seq2 sequenced data from primary tumor sections of breast cancer patients and PBMCs and on a public dataset with manually annotated CTC expression profiles from 34 metastatic breast patients, including triple-negative breast cancer. Our best models achieved about 95% balanced accuracy on the CTC test set on per cell basis, correctly detecting 133 out of 138 CTCs and CTC-PBMC clusters. Considering the non-invasive character of the liquid biopsy examination and our accurate results, we can conclude that our work has potential application value.

Recent advances in the applications of DNA frameworks in liquid biopsy: A review.

Cancer is one of the serious threats to public life and health. Early diagnosis, real-time monitoring, and individualized treatment are the keys to improve the survival rate and prolong the survival time of cancer patients. Liquid biopsy is a potential technique for cancer early diagnosis due to its non-invasive and continuous monitoring properties. However, most current liquid biopsy techniques lack the ability to detect cancers at the early stage. Therefore, effective detection of a variety of cancers is expected through the combination of various techniques. Recently, DNA frameworks with tailorable functionality and precise addressability have attracted wide spread attention in biomedical applications, especially in detecting cancer biomarkers such as circulating tumor cells (CTCs), exosomes and circulating tumor nucleic acid (ctNA). Encouragingly, DNA frameworks perform outstanding in detecting these cancer markers, but also face some challenges and opportunities. In this review, we first briefly introduced the development of DNA frameworks and its typical structural characteristics and advantages. Then, we mainly focus on the recent progress of DNA frameworks in detecting commonly used cancer markers in liquid-biopsy. We summarize the advantages and applications of DNA frameworks for detecting CTCs, exosomes and ctNA. Furthermore, we provide an outlook on the possible opportunities and challenges for exploiting the structural advantages of DNA frameworks in the field of cancer diagnosis. Finally, we envision the marriage of DNA frameworks with other emerging materials and technologies to develop the next generation of disease diagnostic biosensors.